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PMID:4377 | Cephacetrile, a new cephalosporin: in vitro, pharmacological and clinical evaluation. | Cephacetrile, a parenteral cephalosporin, was evaluated for in vitro antibacterial activity, clinical pharmacology and effectiveness in the treatment of severe infections. The antibacterial activity against 187 isolates was determined by an agar-dilution technique. The MICs were 0.06 to 0.5 mug/ml for Group A Streptococcus, D. pneumoniae, and Staph. aureus, 4-6 mug/ml for E. coli and Klebsiella-Enterobacter 8-32 mug/ml for Pr. mirabilis and more than 500 mug/ml for Ps. aeruginosa. A few strains of Klebsiella and E. coli had MICs of more than 125 mcg/ml. Serum levels after 0.5 and 1 g of i.m. cephacetrile were respectively 14.6 and 18.6 mug/ml after 1 hr, and 1.5 and 2.5 mug/ml after 6 hr. Serum levels after i.v. infusion of 0.5 and 1 g were respectively 16 and 25 mug/ml after 1 hr., and 1 and 2 mug/ml after 6 hr. Urine levels after 0.5 and 1 g i.m. cephacetrile were respectively 500 and 650 mug/ml in the 0-3 hr period, and 250 and 300 mug/ml in the 3-6 hr period. Renal clearance was 166 +/- 5 ml/min/1.73 m2; renal excretion was about 20% of the dose 6 hr after i.m. injection. Cephacetrile was well tolerated when administered i.m. with lidocaine. Mild phlebitis occurred sometimes after i.v. infusions. The clinical response, evaluated in 36 patients with severe systemic, respiratory and urinary infections, was good in all but two cases. | Cephacetrile, a new cephalosporin: in vitro, pharmacological and clinical evaluation. Cephacetrile, a parenteral cephalosporin, was evaluated for in vitro antibacterial activity, clinical pharmacology and effectiveness in the treatment of severe infections. The antibacterial activity against 187 isolates was determined by an agar-dilution technique. The MICs were 0.06 to 0.5 mug/ml for Group A Streptococcus, D. pneumoniae, and Staph. aureus, 4-6 mug/ml for E. coli and Klebsiella-Enterobacter 8-32 mug/ml for Pr. mirabilis and more than 500 mug/ml for Ps. aeruginosa. A few strains of Klebsiella and E. coli had MICs of more than 125 mcg/ml. Serum levels after 0.5 and 1 g of i.m. cephacetrile were respectively 14.6 and 18.6 mug/ml after 1 hr, and 1.5 and 2.5 mug/ml after 6 hr. Serum levels after i.v. infusion of 0.5 and 1 g were respectively 16 and 25 mug/ml after 1 hr., and 1 and 2 mug/ml after 6 hr. Urine levels after 0.5 and 1 g i.m. cephacetrile were respectively 500 and 650 mug/ml in the 0-3 hr period, and 250 and 300 mug/ml in the 3-6 hr period. Renal clearance was 166 +/- 5 ml/min/1.73 m2; renal excretion was about 20% of the dose 6 hr after i.m. injection. Cephacetrile was well tolerated when administered i.m. with lidocaine. Mild phlebitis occurred sometimes after i.v. infusions. The clinical response, evaluated in 36 patients with severe systemic, respiratory and urinary infections, was good in all but two cases. | [
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PMID:4378 | Pituitary gonadotropin response to luteinizing hormone-releasing hormone (LH-RH) in males with azo- and oligospermia. | Twenty-one males with azo- or oligospermia presenting with infertility and with no evidence of organic disease were studied with luteinizing hormone - releasing hormone (LH-RH). A pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) response was present in all cases and was normal in the majority of the subjects. Serum testosterone and 17 beta estradiol levels were normal in all cases studied. No significant correlations were found between the gonadotropin estimations and sperm count, basal serum testosterone or testosterone response to human chorionic gonadotropin. It is concluded that LH-RH is of limited diagnostic use in the investigation of this group of patients with male infertility and provides no further insight into the pathogenesis of this condition. | Pituitary gonadotropin response to luteinizing hormone-releasing hormone (LH-RH) in males with azo- and oligospermia. Twenty-one males with azo- or oligospermia presenting with infertility and with no evidence of organic disease were studied with luteinizing hormone - releasing hormone (LH-RH). A pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) response was present in all cases and was normal in the majority of the subjects. Serum testosterone and 17 beta estradiol levels were normal in all cases studied. No significant correlations were found between the gonadotropin estimations and sperm count, basal serum testosterone or testosterone response to human chorionic gonadotropin. It is concluded that LH-RH is of limited diagnostic use in the investigation of this group of patients with male infertility and provides no further insight into the pathogenesis of this condition. | [
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PMID:4380 | Age at the menopause and onset of the climacteric in women of Martin District, Czechoslovkia. Statistical survey and some biological and social correlations. | In this study, 6877 women were analysed whose ages ranged between 38 and 58 (born between 1909 and 1929) and who had had no artificial menopause. This is 88.04% of the total female population in this actual period of life, living in Martin District in 1967. The mean age at the menopause was found, by status quo method, to be 51.21 years (standard deviation 4.4), and by the method of weighted arithmetical means, 48.81 years, (standard deviation 3.9). The mean age at the onset of the climacteric, calculated by the same methods, was 47.55 years, or 46.74 years, respectively. The mean age at menarche was 14.6 years. The average birth-rate was 2.8. The mean period of fertility for the series as a whole was 36.6 years. Women with menstrual disturbances had their menopause about 1 year earlier. We have noted a similar tendency in nulliparas and primiparas. We could find no great difference in the age at menopause between those who had had an early or a late menarche. Menstrual disturbances also influenced the onset of the climacteric. It was at least one year earlier than with regular menstruation. Age at menarche and parity had no effect on the age at the onset of climacteric. Women working in agriculture and housewives had their menopause a little later than mean age of the series, whereas manual workers and those in other occupational categories had their menopause and onset of the climacteric about 1 year earlier. Furthermore, single women had their menopause about one year earlier than the married ones. Widows had their menopause twice so often as the married women and they got it very soon after the husbands's death. | Age at the menopause and onset of the climacteric in women of Martin District, Czechoslovkia. Statistical survey and some biological and social correlations. In this study, 6877 women were analysed whose ages ranged between 38 and 58 (born between 1909 and 1929) and who had had no artificial menopause. This is 88.04% of the total female population in this actual period of life, living in Martin District in 1967. The mean age at the menopause was found, by status quo method, to be 51.21 years (standard deviation 4.4), and by the method of weighted arithmetical means, 48.81 years, (standard deviation 3.9). The mean age at the onset of the climacteric, calculated by the same methods, was 47.55 years, or 46.74 years, respectively. The mean age at menarche was 14.6 years. The average birth-rate was 2.8. The mean period of fertility for the series as a whole was 36.6 years. Women with menstrual disturbances had their menopause about 1 year earlier. We have noted a similar tendency in nulliparas and primiparas. We could find no great difference in the age at menopause between those who had had an early or a late menarche. Menstrual disturbances also influenced the onset of the climacteric. It was at least one year earlier than with regular menstruation. Age at menarche and parity had no effect on the age at the onset of climacteric. Women working in agriculture and housewives had their menopause a little later than mean age of the series, whereas manual workers and those in other occupational categories had their menopause and onset of the climacteric about 1 year earlier. Furthermore, single women had their menopause about one year earlier than the married ones. Widows had their menopause twice so often as the married women and they got it very soon after the husbands's death. | [
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PMID:4381 | Fibrinolytic activity of the rat ovum, appearance during tubal passage and disappearance at implantation. | Fertilized rat ova were histochemically examined for their fibrinolytic activity. Activity was found during tubal passage, which disappeared at implantation. Simultaneously the fibrinolytic activity of the endometrium disappeared at deciduation. These changes might be a prerequisite for implantation of the zygote. | Fibrinolytic activity of the rat ovum, appearance during tubal passage and disappearance at implantation. Fertilized rat ova were histochemically examined for their fibrinolytic activity. Activity was found during tubal passage, which disappeared at implantation. Simultaneously the fibrinolytic activity of the endometrium disappeared at deciduation. These changes might be a prerequisite for implantation of the zygote. | [
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PMID:4382 | Juvenile diabetes and human sperm quality. | Spermiological findings in 25 juvenile diabetics in the age range 16 to 22 years (average 18.5) have been compared with normal spermiological findings in 24 individuals of the same age (16 to 22 years, average 18.7). A drift to lower sperm values in the group of the juvenile diabetics became evident in all parameters observed. As concerns motility and morphology, the differences between the groups reached statistically significant values. | Juvenile diabetes and human sperm quality. Spermiological findings in 25 juvenile diabetics in the age range 16 to 22 years (average 18.5) have been compared with normal spermiological findings in 24 individuals of the same age (16 to 22 years, average 18.7). A drift to lower sperm values in the group of the juvenile diabetics became evident in all parameters observed. As concerns motility and morphology, the differences between the groups reached statistically significant values. | [
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PMID:4383 | On round-headed human spermatozoa. | In an infertile man and his only, likewise infertile, brother the sperm contained only round-headed spermatozoa. Electron microscopic examination of embedded ejaculate revealed a malformation, viz. absence of acrosomes in all the spermatozoa with consequent lack of normal development of the heads of the spermatozoa. Acrosomeless spermatozoa were also found in the testicular tubuli. No disturbance of the endocrine functions could be demonstrated. The blood cells were of normal male karyotype. Investigation of meiosis, on the other hand, showed rudimentary second division. Two other unrelated infertile men showed the same isolated disturbance in their spermiogram. Treatment with chlomiphene had no effect on the structure of the spermatozoa or on the infertility. The rare disorder, which seems to be a special syndrome, the first hitherto distinguishable among the various types of teratospermia, and its probable background are discussed. | On round-headed human spermatozoa. In an infertile man and his only, likewise infertile, brother the sperm contained only round-headed spermatozoa. Electron microscopic examination of embedded ejaculate revealed a malformation, viz. absence of acrosomes in all the spermatozoa with consequent lack of normal development of the heads of the spermatozoa. Acrosomeless spermatozoa were also found in the testicular tubuli. No disturbance of the endocrine functions could be demonstrated. The blood cells were of normal male karyotype. Investigation of meiosis, on the other hand, showed rudimentary second division. Two other unrelated infertile men showed the same isolated disturbance in their spermiogram. Treatment with chlomiphene had no effect on the structure of the spermatozoa or on the infertility. The rare disorder, which seems to be a special syndrome, the first hitherto distinguishable among the various types of teratospermia, and its probable background are discussed. | [
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PMID:4384 | Assessment of the therapeutic effect of epimestrol and epimestrol associated with clomiphene in female sterility. | In 24 women with disturbances of ovulation treated for sterility with Epimestrol, ovulation was achieved in 3 patients but none of these became pregnant after therapy. Since it has been suggested that the association of Clomiphene with a weak estrogen might improve the pregnancy rate, we decided to administer Clomiphene associated with Epimestrol. Using this combined therapy in 58 patients, 32 out of the 58 women ovulated and 17 conceived. The overall rate of pregnancy using the combined therapy was no better than that obtained when Clomiphene alone is administered. From this study it is concluded that: (1) Epimestrol is not an effective method for the induction of ovulation, and (2) addition of Epimestrol to Clomiphene is of no clinical benefit. | Assessment of the therapeutic effect of epimestrol and epimestrol associated with clomiphene in female sterility. In 24 women with disturbances of ovulation treated for sterility with Epimestrol, ovulation was achieved in 3 patients but none of these became pregnant after therapy. Since it has been suggested that the association of Clomiphene with a weak estrogen might improve the pregnancy rate, we decided to administer Clomiphene associated with Epimestrol. Using this combined therapy in 58 patients, 32 out of the 58 women ovulated and 17 conceived. The overall rate of pregnancy using the combined therapy was no better than that obtained when Clomiphene alone is administered. From this study it is concluded that: (1) Epimestrol is not an effective method for the induction of ovulation, and (2) addition of Epimestrol to Clomiphene is of no clinical benefit. | [
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PMID:4385 | Improvement of fertility and semen quality in men treated with a combination of anticongestive and antibiotic drugs. | The effects of a combined therapy of antibiotics and antiinflammatory drugs was evaluated in 344 men referred to our clinic for treatment of infertility. Physical signs of congestion, usually not severe, were detected in 244 men. The treatment caused significant improvement in semen quality, especially in sperm concentration, morphology and motility. Forty percent of the wives became pregnant. There was a striking relationship between increase in morphologically normal spermatozoa and incidence of pregnancy. It is speculated that the therapy had its major effect at the level of the epididymis and/or testis. | Improvement of fertility and semen quality in men treated with a combination of anticongestive and antibiotic drugs. The effects of a combined therapy of antibiotics and antiinflammatory drugs was evaluated in 344 men referred to our clinic for treatment of infertility. Physical signs of congestion, usually not severe, were detected in 244 men. The treatment caused significant improvement in semen quality, especially in sperm concentration, morphology and motility. Forty percent of the wives became pregnant. There was a striking relationship between increase in morphologically normal spermatozoa and incidence of pregnancy. It is speculated that the therapy had its major effect at the level of the epididymis and/or testis. | [
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PMID:4386 | Genital organs. Auto and homotransplantation in forty dogs. | Auto and homo transplantation of the uterus and ovaries was studied in 40 bitches divided into four groups. Group A served as a control. In group B the omentopexy technique of auto-transplantation was used. This method did not prove very rewarding and all 12 grafts were found to have degenerated and were non-functioning when relaparotomy was performed, 3 months after transplantation. In group C, auto transplantation with vascular anastomosis was performed in 12 dogs, yielding satisfactory results including one successful pregnancy and the delivery of 3 healthy puppies. In group D homotransplantation of the internal genital organs was performed in two sets of twin dogs. No immunosuppressive treatment was used. These grafts survived for a limited period as proved by the functional tests and X-ray examination but after 3 months were found to be necrotic and degenerated. Graft function and viability was established by: X-ray examination, cytology of vaginal smears, the effect of gonadotropins on the grafted ovaries and finally relaparotomy undertaken after 3 weeks and 3 months. | Genital organs. Auto and homotransplantation in forty dogs. Auto and homo transplantation of the uterus and ovaries was studied in 40 bitches divided into four groups. Group A served as a control. In group B the omentopexy technique of auto-transplantation was used. This method did not prove very rewarding and all 12 grafts were found to have degenerated and were non-functioning when relaparotomy was performed, 3 months after transplantation. In group C, auto transplantation with vascular anastomosis was performed in 12 dogs, yielding satisfactory results including one successful pregnancy and the delivery of 3 healthy puppies. In group D homotransplantation of the internal genital organs was performed in two sets of twin dogs. No immunosuppressive treatment was used. These grafts survived for a limited period as proved by the functional tests and X-ray examination but after 3 months were found to be necrotic and degenerated. Graft function and viability was established by: X-ray examination, cytology of vaginal smears, the effect of gonadotropins on the grafted ovaries and finally relaparotomy undertaken after 3 weeks and 3 months. | [
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PMID:4387 | Crossed immunoelectrophoresis of sperm antibodies in human serum and cervical mucus. | Sperm agglutinating antibodies are purified from sera and cervical mucus of women with unexplained causes of infertility which were positive in the FD-test. Fractionation was performed by affinity-chromatography in a batch device and the sperm agglutinating activity controlled by the Franklin and Dukes test. This sperm antibody fraction was determined via crossed immunoelectrophoresis by migration into an anti-human serum containing gel. In all cases only one big peak resulted. The negative control serum and mucus samples demonstrated no precipitation peaks. By absorption studies it was shown that the sperm agglutinating antibodies in sera were IgM and in cervical mucus IgA. The concentration of IgA and IgM was determined by comparison with standard human IgA and IgM. Thus only one serum- and one cervical mucus antibody seems to be responsible for agglutination. The number of experiments, however, is still too small for general conclusions. This method is easily and quickly performed and can therefore be used as a routine method for the determination of sperm agglutinating antibodies. Its application for sperm-immobilizing or cytotoxic activity remains to be tested. | Crossed immunoelectrophoresis of sperm antibodies in human serum and cervical mucus. Sperm agglutinating antibodies are purified from sera and cervical mucus of women with unexplained causes of infertility which were positive in the FD-test. Fractionation was performed by affinity-chromatography in a batch device and the sperm agglutinating activity controlled by the Franklin and Dukes test. This sperm antibody fraction was determined via crossed immunoelectrophoresis by migration into an anti-human serum containing gel. In all cases only one big peak resulted. The negative control serum and mucus samples demonstrated no precipitation peaks. By absorption studies it was shown that the sperm agglutinating antibodies in sera were IgM and in cervical mucus IgA. The concentration of IgA and IgM was determined by comparison with standard human IgA and IgM. Thus only one serum- and one cervical mucus antibody seems to be responsible for agglutination. The number of experiments, however, is still too small for general conclusions. This method is easily and quickly performed and can therefore be used as a routine method for the determination of sperm agglutinating antibodies. Its application for sperm-immobilizing or cytotoxic activity remains to be tested. | [
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PMID:4388 | Microbiopsy of the fallopian tube as a method for clinical investigation of tubal function in infertility. | Microbiopsy of the fimbrial end of the fallopian tube may prove to be a valuable method for investigating the tubal pick-up and transport mechanism in infertility patients. Counting of the percentage of ciliated cells on semithin sections shows that in normal fertile women a percentage of such cells is present that is higher than in abnormal conditions such as peritubal adhesions, ectopic pregnancy and amenorrhoea following prolonged progestogen treatment. It is also suggested that ultrastructural examination of the biopsy may provide additional valuable information. | Microbiopsy of the fallopian tube as a method for clinical investigation of tubal function in infertility. Microbiopsy of the fimbrial end of the fallopian tube may prove to be a valuable method for investigating the tubal pick-up and transport mechanism in infertility patients. Counting of the percentage of ciliated cells on semithin sections shows that in normal fertile women a percentage of such cells is present that is higher than in abnormal conditions such as peritubal adhesions, ectopic pregnancy and amenorrhoea following prolonged progestogen treatment. It is also suggested that ultrastructural examination of the biopsy may provide additional valuable information. | [
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PMID:4389 | Improvement of sperm motility in patients with asthenozoospermia by kallikrein treatment. | Parenteral and oral application of kallikrein (EC 3.4.21.8)--a kinin-releasing proteinase from porcine pancreatic tissue--significantly stimulates quantitative and qualitative sperm motility in subfertile males with semen criteria of asthenozoospermia. Improvement of sperm motility was found to exist for at least 3 months following termination of the 7 week duration kallikrein treatment. Additionally, a significant increase in the number of spermatozoa was observed 3 and 5 months after starting parenteral application of kallikrein. | Improvement of sperm motility in patients with asthenozoospermia by kallikrein treatment. Parenteral and oral application of kallikrein (EC 3.4.21.8)--a kinin-releasing proteinase from porcine pancreatic tissue--significantly stimulates quantitative and qualitative sperm motility in subfertile males with semen criteria of asthenozoospermia. Improvement of sperm motility was found to exist for at least 3 months following termination of the 7 week duration kallikrein treatment. Additionally, a significant increase in the number of spermatozoa was observed 3 and 5 months after starting parenteral application of kallikrein. | [
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PMID:4390 | Oligozoospermia: a seven-year survey of the incidence, chromosomal aberrations, treatment and pregnancy rate. | No sperm count should be regarded too low to consider extensive treatment in order to improve semen or to correct any possible abnormalities in femal partners. The infertile couple should be given devoted care and advice to improve their sexual relations and their psychological attitude towards the problem of infertility. A pregnancy rate of 51.9% where the husband's count was less than 10 million/ml, offers adequate support for this statement. In cases of mental trauma inflicted on patients by a verdict of inability to achieve parenthood--on people who have already suffered severe psychological shock and tension resulting from a period of infertility--discouragement lessens the already doubtful chances of achieving pregnancy. In no circumstance should oligozoospermic patients receive treatment for infertility unless a chromosomal analysis has been completed and found normal, since our rate of chromosomal anomalies (11.4%) in a group of oligozoospermic patients is considered to be too high. | Oligozoospermia: a seven-year survey of the incidence, chromosomal aberrations, treatment and pregnancy rate. No sperm count should be regarded too low to consider extensive treatment in order to improve semen or to correct any possible abnormalities in femal partners. The infertile couple should be given devoted care and advice to improve their sexual relations and their psychological attitude towards the problem of infertility. A pregnancy rate of 51.9% where the husband's count was less than 10 million/ml, offers adequate support for this statement. In cases of mental trauma inflicted on patients by a verdict of inability to achieve parenthood--on people who have already suffered severe psychological shock and tension resulting from a period of infertility--discouragement lessens the already doubtful chances of achieving pregnancy. In no circumstance should oligozoospermic patients receive treatment for infertility unless a chromosomal analysis has been completed and found normal, since our rate of chromosomal anomalies (11.4%) in a group of oligozoospermic patients is considered to be too high. | [
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PMID:4391 | Further studies on the fertility promoting factor from human seminal plasma. | A small molecular weight substance from human seminal plasma has been further purified by chromatography. The fertility promoting action of this factor on epididymal sperm has been confirmed in mouse in vitro and in vivo. Experimental evidence indicates that the factor acts on the sperm and not on the eggs. Its possible mode of action is by improving the motility and survival of the epididymal sperm. | Further studies on the fertility promoting factor from human seminal plasma. A small molecular weight substance from human seminal plasma has been further purified by chromatography. The fertility promoting action of this factor on epididymal sperm has been confirmed in mouse in vitro and in vivo. Experimental evidence indicates that the factor acts on the sperm and not on the eggs. Its possible mode of action is by improving the motility and survival of the epididymal sperm. | [
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PMID:4392 | The presence of paternal H-2 antigens on hybrid mouse blastocysts during experimental delay of implantation and the disappearance of these antigens after onset of implantation. | The presence of paternal H-2 antigens on hybrid mouse blastocysts before and during implantation was investigated by means of the isotope anti-globulin technique. It was found that experimentally delayed blastocysts possess paternal H-2 antigens whereas these antigens can no longer be detected 14 hours after estradiol activation of delayed blastocysts. | The presence of paternal H-2 antigens on hybrid mouse blastocysts during experimental delay of implantation and the disappearance of these antigens after onset of implantation. The presence of paternal H-2 antigens on hybrid mouse blastocysts before and during implantation was investigated by means of the isotope anti-globulin technique. It was found that experimentally delayed blastocysts possess paternal H-2 antigens whereas these antigens can no longer be detected 14 hours after estradiol activation of delayed blastocysts. | [
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PMID:4394 | Effect of copper and plastic intra-uterine devices on the fibrinolytic activity of the endometrium in the rat. | The effect of copper and plastic intrauterine devices (IUD) on the fibrinolytic activity of the endometrium was studied in the rat. A copper or a plastic device was placed in one of the uterine horns, while the other horn served as a control. Biopsy specimens were obtained from both horns and examined histochemically. The copper concentration was determined by atomic absorption spectroscopy. The fibrinolytic activity of the control horn was found to be localized to small vessels in the outer layer of the uterine wall, while that of the endometrium was low. Plastic as well as copper IUDs increased the fibrinolytic activity which, in contrast with what was seen in the controls, was localized to the endometrium. Compared with the effect of the plastic device, the increase in the fibrinolytic activity induced by the copper device was more widespread in the endometrial area and was accompanied by an increase in the concentration of copper. These findings might help to explain why the contraceptive effect of IUDs is more reliable when they are partly coated with copper. | Effect of copper and plastic intra-uterine devices on the fibrinolytic activity of the endometrium in the rat. The effect of copper and plastic intrauterine devices (IUD) on the fibrinolytic activity of the endometrium was studied in the rat. A copper or a plastic device was placed in one of the uterine horns, while the other horn served as a control. Biopsy specimens were obtained from both horns and examined histochemically. The copper concentration was determined by atomic absorption spectroscopy. The fibrinolytic activity of the control horn was found to be localized to small vessels in the outer layer of the uterine wall, while that of the endometrium was low. Plastic as well as copper IUDs increased the fibrinolytic activity which, in contrast with what was seen in the controls, was localized to the endometrium. Compared with the effect of the plastic device, the increase in the fibrinolytic activity induced by the copper device was more widespread in the endometrial area and was accompanied by an increase in the concentration of copper. These findings might help to explain why the contraceptive effect of IUDs is more reliable when they are partly coated with copper. | [
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PMID:4395 | Participation of vitamin A in the maturation of rabbit spermatozoa. | Vitamin A concentration was fluorometrically measured in epididymal and ejaculated rabbit spermatozoa and in some of the sperm cells subcellular components. The concentration of vitamin A in the epididymal cells was about one-half that observed in the ejaculated spermatozoa (2.68 as against 1.05 mug/10(8) cells) and seemed to be the same in the sperms obtained from both the head and the tail of the epididymus. The concentration of vitamin A was also found to be significantly higher in the seminal plasma than in the epididymal secretion (0.06 as against 0.039 mug/mg protein respectively). Practically all the vitamin A was found in the fractions obtained by treatment with hypotonic MgCl2 (acrosomal region) and/or with hyamine and dithiothreitol (plasma membrane). It was concluded that the sudden increase in the sperm concentration of vitamin A that occurs upon ejaculation may be required for the stabilization of the acrosomal and plasma membranes. | Participation of vitamin A in the maturation of rabbit spermatozoa. Vitamin A concentration was fluorometrically measured in epididymal and ejaculated rabbit spermatozoa and in some of the sperm cells subcellular components. The concentration of vitamin A in the epididymal cells was about one-half that observed in the ejaculated spermatozoa (2.68 as against 1.05 mug/10(8) cells) and seemed to be the same in the sperms obtained from both the head and the tail of the epididymus. The concentration of vitamin A was also found to be significantly higher in the seminal plasma than in the epididymal secretion (0.06 as against 0.039 mug/mg protein respectively). Practically all the vitamin A was found in the fractions obtained by treatment with hypotonic MgCl2 (acrosomal region) and/or with hyamine and dithiothreitol (plasma membrane). It was concluded that the sudden increase in the sperm concentration of vitamin A that occurs upon ejaculation may be required for the stabilization of the acrosomal and plasma membranes. | [
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PMID:4396 | Evaluation of d-norgestrel 1.0 mg as a post-coital contraceptive. | Two hundred and ninety-eight women were followed for 2578 months (2739 'bleeding intervals') of treatment with d-Norgestrel 1.0 mg given as a post-coital oral contraceptive. Fourteen pregnancies were recorded (general failure rate, 6.5 per 100 woman/years); at least 6 of these patients did not miss any tablet (corrected failure rate, 2.8). The acceptability rates (life table method) were 0.58 and 0.40 after 6 and 12 months of follow-up. The most important medical reason for drop-out was cycle irregularities. The cycle pattern is deeply disturbed by this method of oral contraception. | Evaluation of d-norgestrel 1.0 mg as a post-coital contraceptive. Two hundred and ninety-eight women were followed for 2578 months (2739 'bleeding intervals') of treatment with d-Norgestrel 1.0 mg given as a post-coital oral contraceptive. Fourteen pregnancies were recorded (general failure rate, 6.5 per 100 woman/years); at least 6 of these patients did not miss any tablet (corrected failure rate, 2.8). The acceptability rates (life table method) were 0.58 and 0.40 after 6 and 12 months of follow-up. The most important medical reason for drop-out was cycle irregularities. The cycle pattern is deeply disturbed by this method of oral contraception. | [
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PMID:4397 | "Genital dyscrinism" as a cause of subfertility in mice of the CBA strain. | Following four generations of inbred mating (brother-sister) in three direct lineages of CBA strain mice, sterility appeared which from that generation forward became more frequent. The genital organs in animals of both sexes were altered. There was a noticeable occurrence of cysts in the ovaries of female animals already following the third month; in mice approximately one year of age this condition was followed by cystic glandular hyperplasia of the endometrium, sometimes complicated by disturbances in blood circulation, inflammation or even malignancy. In some female animals, manifesting, due to cysts, completely degenerated ovaries, the rest of the genital system was severely atrophic. Male animals frequently showed severe atrophy of the seminiferous epithelium along with preserved interstitial cells and hypertrophic seminal vesicles. These pathological changes represent an independent nosologic unit, for which the label "genital dyscrinism" has been proposed. The authors have considered an endocrine mechanism as the possible cause of these pathological changes which are presumed to be genetically conditioned. | "Genital dyscrinism" as a cause of subfertility in mice of the CBA strain. Following four generations of inbred mating (brother-sister) in three direct lineages of CBA strain mice, sterility appeared which from that generation forward became more frequent. The genital organs in animals of both sexes were altered. There was a noticeable occurrence of cysts in the ovaries of female animals already following the third month; in mice approximately one year of age this condition was followed by cystic glandular hyperplasia of the endometrium, sometimes complicated by disturbances in blood circulation, inflammation or even malignancy. In some female animals, manifesting, due to cysts, completely degenerated ovaries, the rest of the genital system was severely atrophic. Male animals frequently showed severe atrophy of the seminiferous epithelium along with preserved interstitial cells and hypertrophic seminal vesicles. These pathological changes represent an independent nosologic unit, for which the label "genital dyscrinism" has been proposed. The authors have considered an endocrine mechanism as the possible cause of these pathological changes which are presumed to be genetically conditioned. | [
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PMID:4398 | Motility of the rat oviductal tract isolated in different stages of the sex cycle. Effects of catecholamines. | Physiological and pharmacological characteristics of the spontaneous motility of rat oviductal tracts (the coiled oviduct plus its mesosalpinx), isolated in proestrus, estrus or metestrus, are described. The initial contractile tension (recorded following isolation) was comparable in the three stages of the cycle, but its decrement with time was greater in metestrus than in proestrus; the opposite being observed regarding the rate of contractions. Norepinephrine and phenylephrine depress motility in proestrus and metestrus, but not in estrus. The inhibition of motility during proestrus, produced by added norepinephrine, phenylephrine or isoproterenol was not modified by phenotolamine but was abolished by propranolol. During estrus, norepinephrine and phenylephrine inhibited tubal contractions of preparations incubated with phentolamine, whereas it produced a distinct stimulation in the presence of propranolol. It is concluded that: (a) the rat mesosalpinx might play some role in the motility of the whole isolated oviductal tract; (b) there are variations in the decrement with time of contractile tension and frequency of contractions in different stages of the sex cycle; (c) the effects of catecholamines upon the rat oviductal tract also varies within the cycle, probably due to influences imposed by sex hormones. | Motility of the rat oviductal tract isolated in different stages of the sex cycle. Effects of catecholamines. Physiological and pharmacological characteristics of the spontaneous motility of rat oviductal tracts (the coiled oviduct plus its mesosalpinx), isolated in proestrus, estrus or metestrus, are described. The initial contractile tension (recorded following isolation) was comparable in the three stages of the cycle, but its decrement with time was greater in metestrus than in proestrus; the opposite being observed regarding the rate of contractions. Norepinephrine and phenylephrine depress motility in proestrus and metestrus, but not in estrus. The inhibition of motility during proestrus, produced by added norepinephrine, phenylephrine or isoproterenol was not modified by phenotolamine but was abolished by propranolol. During estrus, norepinephrine and phenylephrine inhibited tubal contractions of preparations incubated with phentolamine, whereas it produced a distinct stimulation in the presence of propranolol. It is concluded that: (a) the rat mesosalpinx might play some role in the motility of the whole isolated oviductal tract; (b) there are variations in the decrement with time of contractile tension and frequency of contractions in different stages of the sex cycle; (c) the effects of catecholamines upon the rat oviductal tract also varies within the cycle, probably due to influences imposed by sex hormones. | [
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PMID:4399 | The effect of prostaglandins and prostaglandin inhibitors on spermatogenesis. | The effect of the prostaglandin inhibitors, aspirin and indomethacin and of prostaglandins PGE1 and PGE2 on spermatogenesis in the mature male mouse has been studied. Aspirin at 100 mg/kg and at 200 mg/kg, and indomethacin at 1.0 mg/kg given orally twice a day for fifteen days produced a marked increase in spermatogenesis. The number of step 7 spermatids increased significantly over controls at about the same rate in all three groups. No significant changes in seminal vesicle weight or testicular weight was noted, although testicular weight did show an increase. Administration of prostaglandins E1 and E2 subcutaneously in doses of either 2 mg/kg or 3 mg/kg once a day for fifteen days produced a marked decrease in spermatogenesis. Step 7 spermatids decreased significantly at both dosage levels of PGE2 and at the higher dosage level of PGE1. Spermatocyte showed a significant decrease at the higher dose of PGE2. Testicular weight showed a significant decrease at the higher dose of PGE2. Seminal vesicle weight showed a significant decrease at the lower dose of PGE1 and at the higher dose of PGE2. Epididymal weight decreased at the higher dose of PGE2. Increased numbers of exfoliated immature germ cells and mature spermatozoa were observed in the epididymus of both the PGE1 and PGE2 treated animals. | The effect of prostaglandins and prostaglandin inhibitors on spermatogenesis. The effect of the prostaglandin inhibitors, aspirin and indomethacin and of prostaglandins PGE1 and PGE2 on spermatogenesis in the mature male mouse has been studied. Aspirin at 100 mg/kg and at 200 mg/kg, and indomethacin at 1.0 mg/kg given orally twice a day for fifteen days produced a marked increase in spermatogenesis. The number of step 7 spermatids increased significantly over controls at about the same rate in all three groups. No significant changes in seminal vesicle weight or testicular weight was noted, although testicular weight did show an increase. Administration of prostaglandins E1 and E2 subcutaneously in doses of either 2 mg/kg or 3 mg/kg once a day for fifteen days produced a marked decrease in spermatogenesis. Step 7 spermatids decreased significantly at both dosage levels of PGE2 and at the higher dosage level of PGE1. Spermatocyte showed a significant decrease at the higher dose of PGE2. Testicular weight showed a significant decrease at the higher dose of PGE2. Seminal vesicle weight showed a significant decrease at the lower dose of PGE1 and at the higher dose of PGE2. Epididymal weight decreased at the higher dose of PGE2. Increased numbers of exfoliated immature germ cells and mature spermatozoa were observed in the epididymus of both the PGE1 and PGE2 treated animals. | [
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PMID:4400 | Low fertility rate in vasovasostomized males and its possible immunologic mechanism. | Contradictory views have been expressed about the role of the various antisperm antibodies which develop after vasoligation. The present study was conducted in 50 normal fertile males, 50 vasectomized subjects and 25 subjects after recanalization of their vas deferens in order to investigate the development of various anti-sperm antibodies after vasectomy, along with their incidence, their persistence after successful relief of vaso-obstruction by vasovasostomy and their role in the causation of infertility in vasoanatomized normospermia males. Sperm agglutinating, immobilizing and haemagglutinating antibodies showed rises in titres with increase during the post-vasectomy period, indicating continuous antigenic stimulus. Age, post-operative complications and blood group did not seem to alter the results. 86% of subjects developed antisperm agglutinins, mostly tail-to-tail type (54.5%), 1-12 years after vasoligation, while only 2% of fertile men had circulating spermagglutinins. A lower incidence of positive sperm in the immobilization test than in the agglutination test suggests either that different antibodies are detected by these two tests or these tests have differing sensitivities. Of the 25 vasovasostomized subject, 13 (52%) cases became normospermic and 4 (16%) oligospermic while 8 (32%) remained azoospermics. Except for 3 oligospermic subjects, all had circulating spermagglutinins. Among the 13 normospermic vasovasostomized persons, a significant correlation was found between the titres of circulating antisperm agglutinins and autoagglutination of spermatozoa in their ejaculates; and also between the sperm immobilization values of their sera and the degree of their sperm motility. Three normospermic recanalized men, having low levels of sperm agglutinins and haemagglutinins with normal seminogram and no sperm immobilizing antibody, successfully impregnated their wives. Another 10 vasovasotomized infertile subjects had sperm agglutinins in significant titre; 5 showed positive sperm immobilization values, a similar number showed autoagglutination of sperm, while a decreased degree of motility of sperms was noted in 6 cases. Thus there was a significant correlation between the titres of anti-sperm antibodies and autoagglutination of spermatozoa, which might be an important cause of male infertility after successful anatomic relief of vasoobstruction. Histological studies of testicular biopsy showed normal spermatogenesis in azoospermic recanalized subjects, although they had high levels of antisperm antibodies. This suggests that these antibodies do not affect normal spermatogenesis, and sperm counts. | Low fertility rate in vasovasostomized males and its possible immunologic mechanism. Contradictory views have been expressed about the role of the various antisperm antibodies which develop after vasoligation. The present study was conducted in 50 normal fertile males, 50 vasectomized subjects and 25 subjects after recanalization of their vas deferens in order to investigate the development of various anti-sperm antibodies after vasectomy, along with their incidence, their persistence after successful relief of vaso-obstruction by vasovasostomy and their role in the causation of infertility in vasoanatomized normospermia males. Sperm agglutinating, immobilizing and haemagglutinating antibodies showed rises in titres with increase during the post-vasectomy period, indicating continuous antigenic stimulus. Age, post-operative complications and blood group did not seem to alter the results. 86% of subjects developed antisperm agglutinins, mostly tail-to-tail type (54.5%), 1-12 years after vasoligation, while only 2% of fertile men had circulating spermagglutinins. A lower incidence of positive sperm in the immobilization test than in the agglutination test suggests either that different antibodies are detected by these two tests or these tests have differing sensitivities. Of the 25 vasovasostomized subject, 13 (52%) cases became normospermic and 4 (16%) oligospermic while 8 (32%) remained azoospermics. Except for 3 oligospermic subjects, all had circulating spermagglutinins. Among the 13 normospermic vasovasostomized persons, a significant correlation was found between the titres of circulating antisperm agglutinins and autoagglutination of spermatozoa in their ejaculates; and also between the sperm immobilization values of their sera and the degree of their sperm motility. Three normospermic recanalized men, having low levels of sperm agglutinins and haemagglutinins with normal seminogram and no sperm immobilizing antibody, successfully impregnated their wives. Another 10 vasovasotomized infertile subjects had sperm agglutinins in significant titre; 5 showed positive sperm immobilization values, a similar number showed autoagglutination of sperm, while a decreased degree of motility of sperms was noted in 6 cases. Thus there was a significant correlation between the titres of anti-sperm antibodies and autoagglutination of spermatozoa, which might be an important cause of male infertility after successful anatomic relief of vasoobstruction. Histological studies of testicular biopsy showed normal spermatogenesis in azoospermic recanalized subjects, although they had high levels of antisperm antibodies. This suggests that these antibodies do not affect normal spermatogenesis, and sperm counts. | [
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PMID:4403 | Studies on urinary arylsulphatase activity in vitamin A deficient rats. | Urinary arylsulphatases (E.C.3.1.6.1) A and B were increased in male rats fasted for 24 hours. Excretion of non dialysable protein nitrogen decreased whereas creatinine excretion increased. On refeeding diet arylsulphatase A activity was restored to normal whereas arylsulphatase B was not normalised. A single oral supplementation of vitamin A acetate (20 000 IU) to rats fasted for 24 hours resulted in a significant reduction of both arylsulphatase A and B eventhough no further reduction of protein nitrogen excretion was evident. In vitamin A deficient male rats significant reduction in urinary excretion of both arylsulphatases A and B occured. In a smaller number of female rats depression of only arylsulphatase A was observed. This effect of vitamin A deficiency leading to reduced urinary arylsulphatase activity was evident even at the "weight plateau" stage when no reduction in food intake or growth had occurred. These results suggest a possible direct or indirect role for vitamin A on urinary excretion pattern of arylsulphatases presumably released from lysosomes of tissues. | Studies on urinary arylsulphatase activity in vitamin A deficient rats. Urinary arylsulphatases (E.C.3.1.6.1) A and B were increased in male rats fasted for 24 hours. Excretion of non dialysable protein nitrogen decreased whereas creatinine excretion increased. On refeeding diet arylsulphatase A activity was restored to normal whereas arylsulphatase B was not normalised. A single oral supplementation of vitamin A acetate (20 000 IU) to rats fasted for 24 hours resulted in a significant reduction of both arylsulphatase A and B eventhough no further reduction of protein nitrogen excretion was evident. In vitamin A deficient male rats significant reduction in urinary excretion of both arylsulphatases A and B occured. In a smaller number of female rats depression of only arylsulphatase A was observed. This effect of vitamin A deficiency leading to reduced urinary arylsulphatase activity was evident even at the "weight plateau" stage when no reduction in food intake or growth had occurred. These results suggest a possible direct or indirect role for vitamin A on urinary excretion pattern of arylsulphatases presumably released from lysosomes of tissues. | [
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PMID:4404 | Effect of long-term starvation on the rat liver lysosomes. | The effect of 120- and 240-h starvation on rats hepatocytes ultrastructure and particularly the changes of the lysosomes were studied. Eelectronmicroscopically and cytochemically there have been observed diminution of the number of mitochondria and degranulation and vacuolzation of the ER. At the same time Golgi complex was hypertrophied and the number of lysosomes was much increased, mainly those of the autophagic type. Biochemically was shown, that the activity of some acid hydrolases (beta-glucosidase, alpha- and beta-galactosidases, beta-N-acetylglucosaminidase, beta-glucuronidase and arylsulphatases A and B) in the liver of starved rats was markedly expressed. The sedimentation properties of the lysosomes and the lysosomal membrane stability was damaged as well. The data received have been discussed in the light of the reconstructive role of lysosomes. | Effect of long-term starvation on the rat liver lysosomes. The effect of 120- and 240-h starvation on rats hepatocytes ultrastructure and particularly the changes of the lysosomes were studied. Eelectronmicroscopically and cytochemically there have been observed diminution of the number of mitochondria and degranulation and vacuolzation of the ER. At the same time Golgi complex was hypertrophied and the number of lysosomes was much increased, mainly those of the autophagic type. Biochemically was shown, that the activity of some acid hydrolases (beta-glucosidase, alpha- and beta-galactosidases, beta-N-acetylglucosaminidase, beta-glucuronidase and arylsulphatases A and B) in the liver of starved rats was markedly expressed. The sedimentation properties of the lysosomes and the lysosomal membrane stability was damaged as well. The data received have been discussed in the light of the reconstructive role of lysosomes. | [
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PMID:4405 | Hypogastric carotid bypass for Takayasu's disease. | Two cases of Takayasu's disease are presented which were successfully resolved by a Dacron graft from the right hypogastric artery to the right internal carotid artery. We recommend this bypass procedure of connecting the hypogastric artery to one of the aortic branches. Medical treatment with antibiotics, steroids and anti-coagulants has not been satisfactory. | Hypogastric carotid bypass for Takayasu's disease. Two cases of Takayasu's disease are presented which were successfully resolved by a Dacron graft from the right hypogastric artery to the right internal carotid artery. We recommend this bypass procedure of connecting the hypogastric artery to one of the aortic branches. Medical treatment with antibiotics, steroids and anti-coagulants has not been satisfactory. | [
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PMID:4406 | Psychopharmacology research ward. Ten years' experience. | The differences between the results of controlled clinical research and the experiences of the practitioners can be attributed to a certain extent to the artificiality of the setting in which controlled studies are performed. The system developed in the wards of the Department of Psychopharmacology of the Institute of Psychiatry in Prague is an attempt to overcome some of these difficulties. Certain similarities exist between this system called continuous controlled trial and 'silent' trial proposed by Ross et al. The main characteristics of the system are: (1) all patients admitted to the ward are assigned to the trial without exception; (2) the trial goes on continuously without interruption for more than 10 years; (3) except that the patients do not know the quality of drugs administered they do not realize any difference between this research setting and the usual routine treatment. The advantages of this system are: reduction of artificiality; the possibility to perform early clinical trials in controlled conditions and to shorten the usual three-phase testing of a new drug; the possibility to perform long-term, longitudinal studies with individual patients admitted several times in double-blind conditions. | Psychopharmacology research ward. Ten years' experience. The differences between the results of controlled clinical research and the experiences of the practitioners can be attributed to a certain extent to the artificiality of the setting in which controlled studies are performed. The system developed in the wards of the Department of Psychopharmacology of the Institute of Psychiatry in Prague is an attempt to overcome some of these difficulties. Certain similarities exist between this system called continuous controlled trial and 'silent' trial proposed by Ross et al. The main characteristics of the system are: (1) all patients admitted to the ward are assigned to the trial without exception; (2) the trial goes on continuously without interruption for more than 10 years; (3) except that the patients do not know the quality of drugs administered they do not realize any difference between this research setting and the usual routine treatment. The advantages of this system are: reduction of artificiality; the possibility to perform early clinical trials in controlled conditions and to shorten the usual three-phase testing of a new drug; the possibility to perform long-term, longitudinal studies with individual patients admitted several times in double-blind conditions. | [
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PMID:4407 | [Comparative study of three psychotropics for treatment of depressions]. | The effect of three anti-depressive psychotropes (Clorimipramine, Doxepine and Dibenzepine) was studied in 107 depressed patients. In each patient the mean value of twelve symptoms was evaluated and compared weekly (for 4 weeks), by statistical methods. In addition, the effect of each drug was analysed in personality stratus. A thymeretic and thymoanaleptic rapid action on 'corporality' and 'endotimic-vital' layer was found with Clorimipramine. Doxepine acts rapidly with sedative and anxiolitic actions on reactive symptoms related with personality super-structures having long term anti-depressive effects. Dibenzepine has a thymeretic rapid and intensive action and a slow thymoanaleptic effect on the same personality stratus of Clorimipramine. | [Comparative study of three psychotropics for treatment of depressions]. The effect of three anti-depressive psychotropes (Clorimipramine, Doxepine and Dibenzepine) was studied in 107 depressed patients. In each patient the mean value of twelve symptoms was evaluated and compared weekly (for 4 weeks), by statistical methods. In addition, the effect of each drug was analysed in personality stratus. A thymeretic and thymoanaleptic rapid action on 'corporality' and 'endotimic-vital' layer was found with Clorimipramine. Doxepine acts rapidly with sedative and anxiolitic actions on reactive symptoms related with personality super-structures having long term anti-depressive effects. Dibenzepine has a thymeretic rapid and intensive action and a slow thymoanaleptic effect on the same personality stratus of Clorimipramine. | [
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PMID:4411 | Properties of rat lens phosphofructokinase. | Two interconvertible forms of phosphofructokinase (PFK) have been eluted from a DEAE-cellulose column from the supernatant fraction of rat lens homogenates centrifuged at 96,000 x g for 1 hour at 0 to 4 degrees C. The interconversion can be manipulated by a change in the pH of the extracting and eluting buffers. PFK-I is the dominant form at pH between 7.4 to 7.05, while PFK-II dominates at pH 7.4 to 8.2. PFK-II is believed to be the functional form; it is inhibited by high concentrations of ATP and the inhibitory effect is enhanced by more acidic pH. Fructose-6-phosphate counteracts ATP inhibition, but the most potent de-inhibitors are ADP and AMP. Among the inorganic ions tested, sulfate, phosphate, ammonium, and potassium also de-inhibit, whereas calcium further inhibits the enzyme. The behavior of PFK under physiologic conditions and the significance of the presence of two forms of PFK in the lens are discussed. | Properties of rat lens phosphofructokinase. Two interconvertible forms of phosphofructokinase (PFK) have been eluted from a DEAE-cellulose column from the supernatant fraction of rat lens homogenates centrifuged at 96,000 x g for 1 hour at 0 to 4 degrees C. The interconversion can be manipulated by a change in the pH of the extracting and eluting buffers. PFK-I is the dominant form at pH between 7.4 to 7.05, while PFK-II dominates at pH 7.4 to 8.2. PFK-II is believed to be the functional form; it is inhibited by high concentrations of ATP and the inhibitory effect is enhanced by more acidic pH. Fructose-6-phosphate counteracts ATP inhibition, but the most potent de-inhibitors are ADP and AMP. Among the inorganic ions tested, sulfate, phosphate, ammonium, and potassium also de-inhibit, whereas calcium further inhibits the enzyme. The behavior of PFK under physiologic conditions and the significance of the presence of two forms of PFK in the lens are discussed. | [
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PMID:4412 | Effects of pregnancy on the development of acute uremic syndrome in the rat. | We evaluated the contributory role which gestational metabolic adaptions may play in the development of acute uremic syndrome in rats by studying the effects of nephrectomy in pregnant and nonpregnant animals. Whereas no significant differences in creatinine and uric acid concentrations were found between control and pregnant rats, urea concentrations were significantly lower in the pregnant animals before, as well as well as 6, 12 and 24 h after, bilateral nephrectomy. It is suggested that creatinine production, which is related to muscle mass, is not altered during late gestation, whereas urea synthesis, which is affected by catabolic and anabolic factors, is significantly reduced. | Effects of pregnancy on the development of acute uremic syndrome in the rat. We evaluated the contributory role which gestational metabolic adaptions may play in the development of acute uremic syndrome in rats by studying the effects of nephrectomy in pregnant and nonpregnant animals. Whereas no significant differences in creatinine and uric acid concentrations were found between control and pregnant rats, urea concentrations were significantly lower in the pregnant animals before, as well as well as 6, 12 and 24 h after, bilateral nephrectomy. It is suggested that creatinine production, which is related to muscle mass, is not altered during late gestation, whereas urea synthesis, which is affected by catabolic and anabolic factors, is significantly reduced. | [
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PMID:4414 | Blood-gas analysis and the assessment of acid-base status. | In this article the more common kinds of acid-base disorders have been discussed. To accurately assess the kind of acid-base disturbance found in a patient, the clinician must know the arterial pH, PaCO2, and the [HCO-3] in arterial blood. Acid-base disturbances are associated with fluid and electrolyte disturbances. Potassium balance is often upset in acid-base disturbances. Accurate determination of K+ balance requires serial or repeated determinations of plasma K+ concentration as well as careful clinical monitoring. | Blood-gas analysis and the assessment of acid-base status. In this article the more common kinds of acid-base disorders have been discussed. To accurately assess the kind of acid-base disturbance found in a patient, the clinician must know the arterial pH, PaCO2, and the [HCO-3] in arterial blood. Acid-base disturbances are associated with fluid and electrolyte disturbances. Potassium balance is often upset in acid-base disturbances. Accurate determination of K+ balance requires serial or repeated determinations of plasma K+ concentration as well as careful clinical monitoring. | [
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PMID:4418 | Intracellular distinction between peroxidase and catalase in exocrine cells of rat lacrimal gland: a biochemical and cytochemical study. | The lacrimal gland (Glandula orbitalis externa) of rat contains both peroxidase and catalase and was used as a model for biochemical and cytochemical distinction between peroxidase and catalase. Both enzymes were isolated by ammonium sulfate precipitation from tissue homogenates, and the effects of fixation with glutaraldehyde and various conditions of incubation were investigated colorimetrically using DAB as hydrogen donor. The lacrimal gland peroxidase is strongly inhibited by glutaraldehyde treatment. In contrast, for catalase the fixation with glutaraldehyde is the prerequistie for demonstration of its peroxidatic activity. The maximal peroxidatic activity was obtained after treatment of catalase with 3% glutaraldehyde, higher concentrations being inhibitory. For lacrimal gland peroxidase, the maximal rate of oxidation of DAB is at pH 6.5, whereas for catalase it is at pH 10.5. The optimal concentration of H2O2 for lacrimal gland peroxidase is at 10(-3)M and for peroxidatic activity of catalase at 10(-1)M. These optimal conditions obtained biochemically were applied to tissue sections of rat lacrimal gland. After the fixation of tissue with a low concentration of glutaraldehyde and incubation in the DAB medium at neutral pH containing 10(-3)M H2O2 (Peroxidase medium), the reaction product was localized in the cisternae of the rough endoplasmic reticulum, in elements of the Golgi apparatus, and in secretory granules. After the fixation of tissue with 3% glutaraldehyde and incubation in the DAB-medium containing 10(-1)M H2O2 and at pH 10.5 (catalase medium), the staining in the endoplasmic reticulum, the Golgi-apparatus and in secretory granules was completely inhibited and reaction product was localized exclusively in small (0.2-0.5 mu) particles similar to small peroxisomes described in various other cell-types. | Intracellular distinction between peroxidase and catalase in exocrine cells of rat lacrimal gland: a biochemical and cytochemical study. The lacrimal gland (Glandula orbitalis externa) of rat contains both peroxidase and catalase and was used as a model for biochemical and cytochemical distinction between peroxidase and catalase. Both enzymes were isolated by ammonium sulfate precipitation from tissue homogenates, and the effects of fixation with glutaraldehyde and various conditions of incubation were investigated colorimetrically using DAB as hydrogen donor. The lacrimal gland peroxidase is strongly inhibited by glutaraldehyde treatment. In contrast, for catalase the fixation with glutaraldehyde is the prerequistie for demonstration of its peroxidatic activity. The maximal peroxidatic activity was obtained after treatment of catalase with 3% glutaraldehyde, higher concentrations being inhibitory. For lacrimal gland peroxidase, the maximal rate of oxidation of DAB is at pH 6.5, whereas for catalase it is at pH 10.5. The optimal concentration of H2O2 for lacrimal gland peroxidase is at 10(-3)M and for peroxidatic activity of catalase at 10(-1)M. These optimal conditions obtained biochemically were applied to tissue sections of rat lacrimal gland. After the fixation of tissue with a low concentration of glutaraldehyde and incubation in the DAB medium at neutral pH containing 10(-3)M H2O2 (Peroxidase medium), the reaction product was localized in the cisternae of the rough endoplasmic reticulum, in elements of the Golgi apparatus, and in secretory granules. After the fixation of tissue with 3% glutaraldehyde and incubation in the DAB-medium containing 10(-1)M H2O2 and at pH 10.5 (catalase medium), the staining in the endoplasmic reticulum, the Golgi-apparatus and in secretory granules was completely inhibited and reaction product was localized exclusively in small (0.2-0.5 mu) particles similar to small peroxisomes described in various other cell-types. | [
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PMID:4415 | Arterial blood-gas interpretations in the respiratory intensive-care unit. | The role of the nurse in the respiratory intensive-care unit requires increased sophistication as our knowledge of the patient becomes more complex. This expanded role should include a thorough understanding of disturbances in acid-base balance, the relationship of PaCO2 to ventilation, the difference in acute and chronic respiratory problems, and the causes and treatment of hypoxemia. The ability to analyze and evaluate blood-gas determinations is simply one more important tool the nurse may utilize in the care and treatment of the critically ill patient. | Arterial blood-gas interpretations in the respiratory intensive-care unit. The role of the nurse in the respiratory intensive-care unit requires increased sophistication as our knowledge of the patient becomes more complex. This expanded role should include a thorough understanding of disturbances in acid-base balance, the relationship of PaCO2 to ventilation, the difference in acute and chronic respiratory problems, and the causes and treatment of hypoxemia. The ability to analyze and evaluate blood-gas determinations is simply one more important tool the nurse may utilize in the care and treatment of the critically ill patient. | [
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PMID:4419 | Antimycin A fermentation. II. Fermentation in aerated-agitated fermenters. | Fermentation characteristics, previously studied in shake flasks, were reproduced in aerated-agitated fermenters, using three strains of Streptomyces sp. which had been selected for their high antimycin A productivity in shake flasks. Fermentation in fermenters was run in three stages. The medium consisted of soy flour, glucose, ammonium sulfate and calcium carbonate; initial pH was 7.2 approximately 7.5, and temperature 25 degrees C. The course of fermentation was then modified to encourage maximal growth and eliminate the intermediate lag period observed in shake flasks. Useful corrections included continuous addition of soybean oil at 1.25 %/day and maintenance of pH at 6 by addition of ammonium hydroxide on demand. The ammonium hydroxide added also served as a rapidly utilized nitrogen source and could not be replace by NaOH or KOH. Under optimal conditions antimycin A was produced at constant rate from the second to the sixth day, when maximum yields of more than 9 g/liter were attained. A procedure for antimycin A extraction is described. | Antimycin A fermentation. II. Fermentation in aerated-agitated fermenters. Fermentation characteristics, previously studied in shake flasks, were reproduced in aerated-agitated fermenters, using three strains of Streptomyces sp. which had been selected for their high antimycin A productivity in shake flasks. Fermentation in fermenters was run in three stages. The medium consisted of soy flour, glucose, ammonium sulfate and calcium carbonate; initial pH was 7.2 approximately 7.5, and temperature 25 degrees C. The course of fermentation was then modified to encourage maximal growth and eliminate the intermediate lag period observed in shake flasks. Useful corrections included continuous addition of soybean oil at 1.25 %/day and maintenance of pH at 6 by addition of ammonium hydroxide on demand. The ammonium hydroxide added also served as a rapidly utilized nitrogen source and could not be replace by NaOH or KOH. Under optimal conditions antimycin A was produced at constant rate from the second to the sixth day, when maximum yields of more than 9 g/liter were attained. A procedure for antimycin A extraction is described. | [
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PMID:4421 | Nutritionally defined conditions for germination of Streptomyces viridochromogenes spores. | Spores of Streptomyces viridochromogenes were removed from the surface of solid media with glass beads and suspended in a buffer-detergent solution. Addition of yeast extract and glucose resulted in rapid loss of refractility of the spores. Appearance of germ tubes followed. Germination was accompanied by a decrease in the optical density (OD) of the suspension. The OD decrease was used as an assay for germination. A defined germination medium (DGM) comprised of L-alanine, L-glutamic acid, adenosine, para-aminobenzoic acid, and calcium and magnesium ions provided a germination rate nearly equal to that of complex media. The germination rate was essentially the same if D-alanine and D-glutamate replaced the L-isomers. The optimum pH and temperature for germination were 7.0 and 35 C. Germination was absolutely dependent on the presence of CO2. Spores harvested after growth for longer periods than the usual time (10 days) became less germinable in DGM. The same was observed for spores grown at 37 C as compared with 30 C. Spores incubated in DGM for various time periods before being transferred to a buffer solution did not continue to germinate. Spores harvested after growth of eight species of Streptomyces did not show a decrease in OD when incubated in yeast extract medium. Another strain of S. viridochromogenes did exhibit an OD decrease in the medium. Comparative properties of spores of streptomycetes, fungi, and bacilli are discussed. | Nutritionally defined conditions for germination of Streptomyces viridochromogenes spores. Spores of Streptomyces viridochromogenes were removed from the surface of solid media with glass beads and suspended in a buffer-detergent solution. Addition of yeast extract and glucose resulted in rapid loss of refractility of the spores. Appearance of germ tubes followed. Germination was accompanied by a decrease in the optical density (OD) of the suspension. The OD decrease was used as an assay for germination. A defined germination medium (DGM) comprised of L-alanine, L-glutamic acid, adenosine, para-aminobenzoic acid, and calcium and magnesium ions provided a germination rate nearly equal to that of complex media. The germination rate was essentially the same if D-alanine and D-glutamate replaced the L-isomers. The optimum pH and temperature for germination were 7.0 and 35 C. Germination was absolutely dependent on the presence of CO2. Spores harvested after growth for longer periods than the usual time (10 days) became less germinable in DGM. The same was observed for spores grown at 37 C as compared with 30 C. Spores incubated in DGM for various time periods before being transferred to a buffer solution did not continue to germinate. Spores harvested after growth of eight species of Streptomyces did not show a decrease in OD when incubated in yeast extract medium. Another strain of S. viridochromogenes did exhibit an OD decrease in the medium. Comparative properties of spores of streptomycetes, fungi, and bacilli are discussed. | [
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PMID:4422 | Effect of pH on competence development and deoxyribonucleic acid uptake in Streptococcus sanguis (Wicky). | Streptococcus sanguis (Wicky) cells, strain WE4, developed little or no competence and failed to autolyze in permissive conditions when treated with competence factor (CF) below PH 7.0. This lack of activity was directly correlated with the inability of the cells to bind or take up CF at pH values of 5.5, 6.0, and 6.5. On the other hand, competent cells bound deoxyribonucleic acid molecules maximally below pH 7.0 and transformed maximally at pH 6.5. Deoxyribonucleic acid was optimally bound to cells in a deoxyribonuclease-resistant form at pH values between 7.0 and 8.5. Concomitant with this binding, undefined acid-soluble DNA fragments appeared in the culture menstrua. CF binding and uptake by cells was not only influenced by low pH but also by low temperature. At 0 C, WE4 cells bound only 4% of the input CF and took up less than 1% into a trypsin-insensitive state compared to cells treated at 37 C. Cells treated with CF at 0 C did not autolyze when transferred to permissive conditions. The results presented in this report extend earlier findings that showed that competence development and autolysis are related to the uptake of CF. | Effect of pH on competence development and deoxyribonucleic acid uptake in Streptococcus sanguis (Wicky). Streptococcus sanguis (Wicky) cells, strain WE4, developed little or no competence and failed to autolyze in permissive conditions when treated with competence factor (CF) below PH 7.0. This lack of activity was directly correlated with the inability of the cells to bind or take up CF at pH values of 5.5, 6.0, and 6.5. On the other hand, competent cells bound deoxyribonucleic acid molecules maximally below pH 7.0 and transformed maximally at pH 6.5. Deoxyribonucleic acid was optimally bound to cells in a deoxyribonuclease-resistant form at pH values between 7.0 and 8.5. Concomitant with this binding, undefined acid-soluble DNA fragments appeared in the culture menstrua. CF binding and uptake by cells was not only influenced by low pH but also by low temperature. At 0 C, WE4 cells bound only 4% of the input CF and took up less than 1% into a trypsin-insensitive state compared to cells treated at 37 C. Cells treated with CF at 0 C did not autolyze when transferred to permissive conditions. The results presented in this report extend earlier findings that showed that competence development and autolysis are related to the uptake of CF. | [
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PMID:4423 | Control of inositol biosynthesis in Saccharomyces cerevisiae: properties of a repressible enzyme system in extracts of wild-type (Ino+) cells. | Inositol biosynthesis was studied in soluble, cell extracts of a wild-type (Ino) strain of Saccharomyces cerevisiae. Two reactions were detected: (i) conversion of D-glucose-6-phosphate to a phosphorylated form of inositol, presumably inositol-1-phosphate (IP synthethase, EC5.5.1.4), and (ii) conversion of phosphorylated inositol to inositol (IP phosphatase, EC3.1.3.25). The in vitro rate of conversion of glucose-6-phosphate to inositol was proportional to incubaion time and enzyme concentration. The pH optimum was 7.0. The synthesis of inositol required oxidized nicotinamide adenine dinucleotide (NAD) and was stimulated byNH4C1 and MgC12. NADP substituted poorly for NAD, and NADH inhibitedthe reaction. Phosphorylated inositol accumulated in the absence of MgC12, suggesting that inositol-phosphate is an intermediate in the pathway and that Mg ions stimulate the dephosphorylation of inositol-phosphate. IP synthetase was inhibited approximately 20% in the presence of inositol in the reaction mixture at concentrations exceeding 1 mM. The enzyme was repressed approximately 50-fold when inositol was present in the growth medium at concentrations exceeding 50 muM. IP synthetase reached the fully repressed level approximately 10 h after the addition of inositol to logarithmic cultures grown in the absence of inositol. The specific activity of the enzyme increased with time in logarithmically growing cultures lacking inositol andapproached the fully depressed level as the cells entered stationary phase. | Control of inositol biosynthesis in Saccharomyces cerevisiae: properties of a repressible enzyme system in extracts of wild-type (Ino+) cells. Inositol biosynthesis was studied in soluble, cell extracts of a wild-type (Ino) strain of Saccharomyces cerevisiae. Two reactions were detected: (i) conversion of D-glucose-6-phosphate to a phosphorylated form of inositol, presumably inositol-1-phosphate (IP synthethase, EC5.5.1.4), and (ii) conversion of phosphorylated inositol to inositol (IP phosphatase, EC3.1.3.25). The in vitro rate of conversion of glucose-6-phosphate to inositol was proportional to incubaion time and enzyme concentration. The pH optimum was 7.0. The synthesis of inositol required oxidized nicotinamide adenine dinucleotide (NAD) and was stimulated byNH4C1 and MgC12. NADP substituted poorly for NAD, and NADH inhibitedthe reaction. Phosphorylated inositol accumulated in the absence of MgC12, suggesting that inositol-phosphate is an intermediate in the pathway and that Mg ions stimulate the dephosphorylation of inositol-phosphate. IP synthetase was inhibited approximately 20% in the presence of inositol in the reaction mixture at concentrations exceeding 1 mM. The enzyme was repressed approximately 50-fold when inositol was present in the growth medium at concentrations exceeding 50 muM. IP synthetase reached the fully repressed level approximately 10 h after the addition of inositol to logarithmic cultures grown in the absence of inositol. The specific activity of the enzyme increased with time in logarithmically growing cultures lacking inositol andapproached the fully depressed level as the cells entered stationary phase. | [
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PMID:4424 | Heat activation of Streptomyces viridochromogenes spores. | The lag period preceding germination of Streptomyces viridochromogenes spores during incubation in a defined germination medium was completely eliminated by a gentle heat shock. The rate of germination was not affected. The optimum pH for activation extended from 6.0 to 9.6. The time of heating required for maximum activation was 1 min at 60 C, 2 to 5 min at 55 C, 20 min at 50 C, and 40 to 50 min at 45 C. Activated spores had the same temperature and pH optima and nutritional requirements for germination as unactivated spores. Activated spores deactivated during incubation for 8 h at 25 C and were activated again by a second heat shock. Spores that had been aged for 4 weeks or longer did not germinate in the defined germination medium unless they were first heat activated. | Heat activation of Streptomyces viridochromogenes spores. The lag period preceding germination of Streptomyces viridochromogenes spores during incubation in a defined germination medium was completely eliminated by a gentle heat shock. The rate of germination was not affected. The optimum pH for activation extended from 6.0 to 9.6. The time of heating required for maximum activation was 1 min at 60 C, 2 to 5 min at 55 C, 20 min at 50 C, and 40 to 50 min at 45 C. Activated spores had the same temperature and pH optima and nutritional requirements for germination as unactivated spores. Activated spores deactivated during incubation for 8 h at 25 C and were activated again by a second heat shock. Spores that had been aged for 4 weeks or longer did not germinate in the defined germination medium unless they were first heat activated. | [
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PMID:4425 | Glucose transport in isolated prosthecae of Asticcacaulis biprosthecum. | Active transport of glucose in prosthecae isolated from cells of Asticcacaulis biprosthecum was stimulated by the non-physiological electron donor N, N, N', N'-tetramethyl-p-phenylenediamine dihydrochloride. Glucose uptake was mediated by two transport systems; the apparent Km of the high-affinity system was 1.8 muM and that of the low-affinity system was 34 muM. Free glucose accumulated within prosthecae at a concentration 60 to 200 times above that present externally, depending on the Km of the system being observed. The glucose transport system in prosthecae was stereospecific for D-glucose, and neither methyl alpha-D-glucopyranoside nor 2-deoxyglucose was transported. Uptake of glucose was inhibited by N-ethylmaleimide (NEM) and p-chloromercuribenzoate (PCMB), and the inhibition by PCMB but not by NEM was reversed by dithiothreitol. Glucose uptake was also inhibited by the uncoupling agents 5-chloro-3-t-butyl-2'-nitrosalicylanilide (S-13), 5-chloro-3-(p-chlorophenyl)-4'-chlorosalicylanilide (S-6), and carbonyl-cyanide m-chlorophenylhydrazone (CCCP) and by the respiratory inhibitor KCN. Efflux of glucose from preloaded prosthecae was induced by PCMB and KCN, but not by S-13 or CCCP. Glucose uptake was not affected by arsenate or an inhibitor of membrane-bound adenosine triphosphatases, N, N'-dicyclohexylcarbodiimide. The lack of inhibition by these two compounds, combined with the extremely low levels of adenosine 5'-triphosphate present in prosthecae, indicates that adenosine 5'-triphosphate is not involved in the transport of glucose by prosthecae. | Glucose transport in isolated prosthecae of Asticcacaulis biprosthecum. Active transport of glucose in prosthecae isolated from cells of Asticcacaulis biprosthecum was stimulated by the non-physiological electron donor N, N, N', N'-tetramethyl-p-phenylenediamine dihydrochloride. Glucose uptake was mediated by two transport systems; the apparent Km of the high-affinity system was 1.8 muM and that of the low-affinity system was 34 muM. Free glucose accumulated within prosthecae at a concentration 60 to 200 times above that present externally, depending on the Km of the system being observed. The glucose transport system in prosthecae was stereospecific for D-glucose, and neither methyl alpha-D-glucopyranoside nor 2-deoxyglucose was transported. Uptake of glucose was inhibited by N-ethylmaleimide (NEM) and p-chloromercuribenzoate (PCMB), and the inhibition by PCMB but not by NEM was reversed by dithiothreitol. Glucose uptake was also inhibited by the uncoupling agents 5-chloro-3-t-butyl-2'-nitrosalicylanilide (S-13), 5-chloro-3-(p-chlorophenyl)-4'-chlorosalicylanilide (S-6), and carbonyl-cyanide m-chlorophenylhydrazone (CCCP) and by the respiratory inhibitor KCN. Efflux of glucose from preloaded prosthecae was induced by PCMB and KCN, but not by S-13 or CCCP. Glucose uptake was not affected by arsenate or an inhibitor of membrane-bound adenosine triphosphatases, N, N'-dicyclohexylcarbodiimide. The lack of inhibition by these two compounds, combined with the extremely low levels of adenosine 5'-triphosphate present in prosthecae, indicates that adenosine 5'-triphosphate is not involved in the transport of glucose by prosthecae. | [
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PMID:4426 | Defective synthesis of lipid intermediates for peptidoglycan formation in a stabilized L-form of Streptococcus pyogenes. | Membrane preparations obtained from a stabilized L-form of Streptococcus pyogenes are incapable of synthesizing peptidoglycan from uridine-5'-diphospho-N-acetyl-D-muramyl-L-Ala-D-iso-Glu-L-Lys-D-Ala-D-Ala and uridine-5'-diphospho-N-acetyl-D-glucosamine, in contrast with similar preparations from the parental streptococcus. Furthermore, 50-fold higher levels of lipid intermediates which serve as membrane-bound substrates for peptidoglycan synthesis are synthesized in reaction mixtures containing streptococcal membranes than with similar preparations from the L-form. These observations suggest that the inability of this stabilized L-form to form a cell wall in vivo lies, at least in part, in its failure to synthesize significant quantities of the lipid substrates for peptidoglycan synthesis. | Defective synthesis of lipid intermediates for peptidoglycan formation in a stabilized L-form of Streptococcus pyogenes. Membrane preparations obtained from a stabilized L-form of Streptococcus pyogenes are incapable of synthesizing peptidoglycan from uridine-5'-diphospho-N-acetyl-D-muramyl-L-Ala-D-iso-Glu-L-Lys-D-Ala-D-Ala and uridine-5'-diphospho-N-acetyl-D-glucosamine, in contrast with similar preparations from the parental streptococcus. Furthermore, 50-fold higher levels of lipid intermediates which serve as membrane-bound substrates for peptidoglycan synthesis are synthesized in reaction mixtures containing streptococcal membranes than with similar preparations from the L-form. These observations suggest that the inability of this stabilized L-form to form a cell wall in vivo lies, at least in part, in its failure to synthesize significant quantities of the lipid substrates for peptidoglycan synthesis. | [
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PMID:4427 | Protonmotive force as the source of energy for adenosine 5'-triphosphate synthesis in Escherichia coli. | Net synthesis of adenosine 5'-triphosphate (ATP) in energy-depleted cells of Escherichia coli was observed when an inwardly directed protonmotive force was artificially imposed. In wild-type cells, ATP synthesis occurred whether the protonmotive force was dominated by the membrane potential (negative inside) or the pH gradient (alkaline inside). Formation of ATP did not occur unless the protonmotive force exceeded a value of 200 mV. Under these conditions, no ATP synthesis was found when cells were exposed to an inhibitor of the membrane-bound Ca2+- and Mg2+- stimulated adenosine triphosphatase (EC 3.6.1.3), dicyclohexylcarbodiimide, or to a proton conductor, carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone. Adenosine triphosphatase-negative mutants failed to show ATP synthesis in response to either a membrane potential or a pH gradient. ATP synthesis driven by a protonmotive force was observed in a cytochrome-deficient mutant. These observations are consistent with the chemiosmotic hypothesis of Mitchell (1961, 1966, 1974). | Protonmotive force as the source of energy for adenosine 5'-triphosphate synthesis in Escherichia coli. Net synthesis of adenosine 5'-triphosphate (ATP) in energy-depleted cells of Escherichia coli was observed when an inwardly directed protonmotive force was artificially imposed. In wild-type cells, ATP synthesis occurred whether the protonmotive force was dominated by the membrane potential (negative inside) or the pH gradient (alkaline inside). Formation of ATP did not occur unless the protonmotive force exceeded a value of 200 mV. Under these conditions, no ATP synthesis was found when cells were exposed to an inhibitor of the membrane-bound Ca2+- and Mg2+- stimulated adenosine triphosphatase (EC 3.6.1.3), dicyclohexylcarbodiimide, or to a proton conductor, carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone. Adenosine triphosphatase-negative mutants failed to show ATP synthesis in response to either a membrane potential or a pH gradient. ATP synthesis driven by a protonmotive force was observed in a cytochrome-deficient mutant. These observations are consistent with the chemiosmotic hypothesis of Mitchell (1961, 1966, 1974). | [
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PMID:4428 | Inhibition of dimethyl ether and methane oxidation in Methylococcus capsulatus and Methylosinus trichosporium. | Metal-chelating or -binding agents inhibited the oxidation of dimethyl ether and methane, but not methanol, by cell suspensions of Methylococcus capsulatus and Methylosinus trichosporium. Evidence suggests that the involvement of metal-containing enzymatic systems in the initial step of oxidation of dimethyl ether and methane. | Inhibition of dimethyl ether and methane oxidation in Methylococcus capsulatus and Methylosinus trichosporium. Metal-chelating or -binding agents inhibited the oxidation of dimethyl ether and methane, but not methanol, by cell suspensions of Methylococcus capsulatus and Methylosinus trichosporium. Evidence suggests that the involvement of metal-containing enzymatic systems in the initial step of oxidation of dimethyl ether and methane. | [
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PMID:4429 | Effect of colicin K on a membrane-associated, energy-linked function. | The purpose of this work was in investigate the capability of cell extracts of Escherichia coli and E. coli treated with colicin K to catalyze the following energy-dependent reverse transhydrogenase reaction: NADP + NADH + ATP in equilibrium NADPH + NAD +ADP + Pi. Under anaerobic conditions this reaction requires the presence of a specific portion of the electron transport chain, a functional energy coupling system, including an adenosine triphosphatase, enzyme, and ATP as energy source. The ATP-linked reaction was partially inhibited in French press extracts of E. coli K-12 C600 cells that had been pretreated with colicin K but not in extracts from similarly treated cells of a colicin-tolerant mutant. Ultracentrifugation of extracts yielded particulate fractions competent in catalyzing the reaction; this reaction is substantially inhibited in fractions from colicin-treated cells. The extent of inhibition increased with increasing concentration of colicin. Supernatants also supported ATP-linked formation of NADPH, but this reaction was insensitive to the colicin effect. A comparison between the requirement of the reaction in supernatant and particulate fractions suggests that the reaction in the supernatant is different from the one inhibited by colicin. The ATP-hydrolyzing ability of particulate fractions from the control or treated bacteria was identical. Likewise, the electron transport chain was not affected by colicin treatment, as evidenced from lack of effect on NADH oxidase, succinic dehydrogenase, and NADPH-NAD transhydrogenase. It is concluded that colicin K interferes with the coupling of ATP the utilization of the intermediate for the ATP-linked transdehydrogenase reaction. | Effect of colicin K on a membrane-associated, energy-linked function. The purpose of this work was in investigate the capability of cell extracts of Escherichia coli and E. coli treated with colicin K to catalyze the following energy-dependent reverse transhydrogenase reaction: NADP + NADH + ATP in equilibrium NADPH + NAD +ADP + Pi. Under anaerobic conditions this reaction requires the presence of a specific portion of the electron transport chain, a functional energy coupling system, including an adenosine triphosphatase, enzyme, and ATP as energy source. The ATP-linked reaction was partially inhibited in French press extracts of E. coli K-12 C600 cells that had been pretreated with colicin K but not in extracts from similarly treated cells of a colicin-tolerant mutant. Ultracentrifugation of extracts yielded particulate fractions competent in catalyzing the reaction; this reaction is substantially inhibited in fractions from colicin-treated cells. The extent of inhibition increased with increasing concentration of colicin. Supernatants also supported ATP-linked formation of NADPH, but this reaction was insensitive to the colicin effect. A comparison between the requirement of the reaction in supernatant and particulate fractions suggests that the reaction in the supernatant is different from the one inhibited by colicin. The ATP-hydrolyzing ability of particulate fractions from the control or treated bacteria was identical. Likewise, the electron transport chain was not affected by colicin treatment, as evidenced from lack of effect on NADH oxidase, succinic dehydrogenase, and NADPH-NAD transhydrogenase. It is concluded that colicin K interferes with the coupling of ATP the utilization of the intermediate for the ATP-linked transdehydrogenase reaction. | [
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PMID:4430 | Ribonucleic acid synthesized in meiotic cells of Saccharomyces cerevisiae: effect of culture medium pH. | Pulse-labeled ribonucleic acid (RNA) was extracted from polysomes of sporulating cells of Saccharomyces cerevisiae and characterized in sucrose gradients and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Transfer RNA, ribosomal RNA, and heterodisperse RNA, presumed to be messenger RNA, were synthesized during a 20-min pulse at T4 and T6 when labeling was performed in sporulation medium adjusted to pH 6.0. Furthermore, ribosomal RNA was processed into functional ribosomes during the pulse. The specific activity of pulse-labeled RNA of cells labeled in sporulation medium where the pH was unadjusted at T4 (pH 7.8) and T9 (pH 8.6) was 20- to 50-fold lower than RNA from cells labeled at pH 6.0. The low specific activity resulted from a 50-fold reduction in uptake of labeled precursors when the medium pH was greater than 7.2. However, heterodisperse RNA ranging from 4-17S in size and transfer RNA were synthesized during the pulse at T4 (pH 7.8),but the low specific activity of ribosomal RNA prevented a thorough analysis of its synthesis. Cellular impermeability at T9 (pH 8.6) resulted in minimal uptake of label, and an analysis of pulse-labeled transcripts was impossible. A comparison of the percantage of polysomal material indicate, however, that these cells were at least as active in translation as cells pulse-labeled at pH 6.0. | Ribonucleic acid synthesized in meiotic cells of Saccharomyces cerevisiae: effect of culture medium pH. Pulse-labeled ribonucleic acid (RNA) was extracted from polysomes of sporulating cells of Saccharomyces cerevisiae and characterized in sucrose gradients and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Transfer RNA, ribosomal RNA, and heterodisperse RNA, presumed to be messenger RNA, were synthesized during a 20-min pulse at T4 and T6 when labeling was performed in sporulation medium adjusted to pH 6.0. Furthermore, ribosomal RNA was processed into functional ribosomes during the pulse. The specific activity of pulse-labeled RNA of cells labeled in sporulation medium where the pH was unadjusted at T4 (pH 7.8) and T9 (pH 8.6) was 20- to 50-fold lower than RNA from cells labeled at pH 6.0. The low specific activity resulted from a 50-fold reduction in uptake of labeled precursors when the medium pH was greater than 7.2. However, heterodisperse RNA ranging from 4-17S in size and transfer RNA were synthesized during the pulse at T4 (pH 7.8),but the low specific activity of ribosomal RNA prevented a thorough analysis of its synthesis. Cellular impermeability at T9 (pH 8.6) resulted in minimal uptake of label, and an analysis of pulse-labeled transcripts was impossible. A comparison of the percantage of polysomal material indicate, however, that these cells were at least as active in translation as cells pulse-labeled at pH 6.0. | [
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PMID:4431 | An enzyme common to histidine and aromatic amino acid biosynthesis in Bacillus subtilis. | Two transaminases exist for tyrosine and phenylalanine synthesis in Bacillus subtilis. One enzyme is also responsible for the transamination of imidazole acetol phosphate to histidinol phosphate, an obligatory reaction in the synthesis of histidine. The gene involved in the synthesis of this enzyme lies in the middle of a cluster of genes, all of which are concerned with the synthesis of the aromatic amino acids. The other gene has not yet been mapped. Mutants have been isolated that lack one or the other enzyme activity. These mutants are prototrophic for tyrosine and phenylalanine. However, both classes of mutants are more sensitive than the wild-type strain to the phenylalanine analogue, fluorophenylalanine, suggesting that each of these mutants synthesizes less phenylalanine than does the wild-type strain. The two enzymes can be separated from one another by ion-exchange chromatography and glycerol-gradient centrifugation. The significance of the observation that an enzyme of histidine synthesis also plays a role in the synthesis of the aromatic acids is considered in light of cross-pathways regulation between the two pathways. | An enzyme common to histidine and aromatic amino acid biosynthesis in Bacillus subtilis. Two transaminases exist for tyrosine and phenylalanine synthesis in Bacillus subtilis. One enzyme is also responsible for the transamination of imidazole acetol phosphate to histidinol phosphate, an obligatory reaction in the synthesis of histidine. The gene involved in the synthesis of this enzyme lies in the middle of a cluster of genes, all of which are concerned with the synthesis of the aromatic amino acids. The other gene has not yet been mapped. Mutants have been isolated that lack one or the other enzyme activity. These mutants are prototrophic for tyrosine and phenylalanine. However, both classes of mutants are more sensitive than the wild-type strain to the phenylalanine analogue, fluorophenylalanine, suggesting that each of these mutants synthesizes less phenylalanine than does the wild-type strain. The two enzymes can be separated from one another by ion-exchange chromatography and glycerol-gradient centrifugation. The significance of the observation that an enzyme of histidine synthesis also plays a role in the synthesis of the aromatic acids is considered in light of cross-pathways regulation between the two pathways. | [
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PMID:4432 | Regulation of Chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from alcaligenes eutrophus. | Highly purified enzymes from Alcaligenes eutrophus H 16 were used for kinetic studies. Chorismate mutase was feedback inhibited by phenylalanine. In the absence of the inhibitor, the double-reciprocal plot was linear, yielding a Km for chorismate of 0.2 mM. When phenylalanine was present, a pronounced deviation from the Michaelis-Menten hyperbola occurred. The Hill coefficient (n) was 1.7, and Hill plots of velocity versus inhibitor concentrations resulted in a value of n' = 2.3, indicating positive cooperativity. Chorismate mutase was also inhibited by prephenate, which caused downward double-reciprocal plots and a Hill coefficient of n = 0.7, evidence for negative cooperativity. The pH optimum of chorismate mutase ranged from 7.8 to 8.2; its temperature optimum was 47 C. Prephenate dehydratase was competitively inhibited by phenylalanine and activated by tyrosine. Tyrosine stimulated its activity up to 10-fold and decreased the Km for prephenate, which was 0.67 mM without effectors. Tryptophan inhibited the enzyme competitively. Its inhibition constant (Ki = 23 muM) was almost 10-fold higher than that determined for phenylalanine (Ki = 2.6 muM). The pH optimum of prephenate dehydratase was pH 5.7; the temperature optimum was 48 C. Prephenate dehydrogenase was feedback inhibited by tyrosine. Inhibition was competitive with prephenate (Ki = 0.06 mM) and noncompetitive with nicotinamide adenine dinucleotide. The enzyme was further subject to product inhibition by p-hydroxyphenylpyruvate (Ki = 0.13 mM). Its Km for prephenate was 0.045 mM, and that for nicotinamide adenine dinucleotide was 0.14 mM. The pH optimum ranged between 7.0 and 7.6; the temperature optimum was 38 C. It is shown how the sensitive regulation of the entire enzyme system leads to a well-balanced amino acid production. | Regulation of Chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from alcaligenes eutrophus. Highly purified enzymes from Alcaligenes eutrophus H 16 were used for kinetic studies. Chorismate mutase was feedback inhibited by phenylalanine. In the absence of the inhibitor, the double-reciprocal plot was linear, yielding a Km for chorismate of 0.2 mM. When phenylalanine was present, a pronounced deviation from the Michaelis-Menten hyperbola occurred. The Hill coefficient (n) was 1.7, and Hill plots of velocity versus inhibitor concentrations resulted in a value of n' = 2.3, indicating positive cooperativity. Chorismate mutase was also inhibited by prephenate, which caused downward double-reciprocal plots and a Hill coefficient of n = 0.7, evidence for negative cooperativity. The pH optimum of chorismate mutase ranged from 7.8 to 8.2; its temperature optimum was 47 C. Prephenate dehydratase was competitively inhibited by phenylalanine and activated by tyrosine. Tyrosine stimulated its activity up to 10-fold and decreased the Km for prephenate, which was 0.67 mM without effectors. Tryptophan inhibited the enzyme competitively. Its inhibition constant (Ki = 23 muM) was almost 10-fold higher than that determined for phenylalanine (Ki = 2.6 muM). The pH optimum of prephenate dehydratase was pH 5.7; the temperature optimum was 48 C. Prephenate dehydrogenase was feedback inhibited by tyrosine. Inhibition was competitive with prephenate (Ki = 0.06 mM) and noncompetitive with nicotinamide adenine dinucleotide. The enzyme was further subject to product inhibition by p-hydroxyphenylpyruvate (Ki = 0.13 mM). Its Km for prephenate was 0.045 mM, and that for nicotinamide adenine dinucleotide was 0.14 mM. The pH optimum ranged between 7.0 and 7.6; the temperature optimum was 38 C. It is shown how the sensitive regulation of the entire enzyme system leads to a well-balanced amino acid production. | [
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PMID:4433 | Role of deoxyribonucleic acid ligase in a doxyribonucleic acid membrane fraction extracted from pneumococci. | Deoxyribonucleic acid (DNA) ligase has been detected in a DNA membrane fraction extracted from Pneumococcus. The specific activity of the enzyme in this fraction is 10-fold greater than in the remaining cell extract. It remains firmly bound (with other enzymes) to the complex after a purification procedure in which a considerable percentage of the macromolecules are dissociated. The ligase acts in two ways in the DNA membrane fraction in vitro. One, it catalyzes the linkage of small-molecular-weight pieces of newly synthesized DNA into heavier-molecular-weight DNA strands as shown by others (M Gellert, 1976; R. Okazaki, A. Sugino, S. Hirose, T. Okazaki, Y. Imae, R. Kainuma-Kuroda, T. Ogawa, M. Arisawa, and Y. Kurosowa, 1973; B. Olivera and I. Lehman, 14; and A. Sugino, S. Hirose, and R. Okazaki, 1972) and, two, it protects DNA from degradation by deoxyribonucleases. This latter effect is due to a competition between the ability of the nucleases to degrade DNA and the ability of DNA ligase to seal the nicks produced by these degradative enzymes. The ligase acts cooperatively with other enzymes in the DNA membrane fraction to synthesize DNA. | Role of deoxyribonucleic acid ligase in a doxyribonucleic acid membrane fraction extracted from pneumococci. Deoxyribonucleic acid (DNA) ligase has been detected in a DNA membrane fraction extracted from Pneumococcus. The specific activity of the enzyme in this fraction is 10-fold greater than in the remaining cell extract. It remains firmly bound (with other enzymes) to the complex after a purification procedure in which a considerable percentage of the macromolecules are dissociated. The ligase acts in two ways in the DNA membrane fraction in vitro. One, it catalyzes the linkage of small-molecular-weight pieces of newly synthesized DNA into heavier-molecular-weight DNA strands as shown by others (M Gellert, 1976; R. Okazaki, A. Sugino, S. Hirose, T. Okazaki, Y. Imae, R. Kainuma-Kuroda, T. Ogawa, M. Arisawa, and Y. Kurosowa, 1973; B. Olivera and I. Lehman, 14; and A. Sugino, S. Hirose, and R. Okazaki, 1972) and, two, it protects DNA from degradation by deoxyribonucleases. This latter effect is due to a competition between the ability of the nucleases to degrade DNA and the ability of DNA ligase to seal the nicks produced by these degradative enzymes. The ligase acts cooperatively with other enzymes in the DNA membrane fraction to synthesize DNA. | [
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PMID:4434 | Relationship between hemagglutinin and sialidase from Clostridium perfringens CN3870: chromatographic characterization of the biologically active proteins. | Biochemical characterization of hemagglutinin and sialidase activities from Clostridium perfringens strain CN3870 revealed that this strain produced three sialidase enzymes that were separable to gel filtration, ion exchange chromatography, and polyacrylamide gel electrophoresis. The molecular weights of sialidase I, II, and III activities were 310,000 +/- 10,000, 105,000 +/- 4,000 and 64,000 +/- 2,000, respectively, the first figure being an approximate value only. | Relationship between hemagglutinin and sialidase from Clostridium perfringens CN3870: chromatographic characterization of the biologically active proteins. Biochemical characterization of hemagglutinin and sialidase activities from Clostridium perfringens strain CN3870 revealed that this strain produced three sialidase enzymes that were separable to gel filtration, ion exchange chromatography, and polyacrylamide gel electrophoresis. The molecular weights of sialidase I, II, and III activities were 310,000 +/- 10,000, 105,000 +/- 4,000 and 64,000 +/- 2,000, respectively, the first figure being an approximate value only. | [
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PMID:4435 | Relationship between hemagglutinin and sialidase from Clostridium perfringens CN3870: gel filtration of mutant and reverant activities. | Gel filtration of supernatant fluids, from the wild-type Clostridium perfringens, strain CN3870, and several of the mutants and reverants derived from this strain, showed that these mutants failed to product detectable amounts of still produced sialidase III activity. The reverants tested had regained the ability to produce approximately wild-type levels of the I and II forms of both activities. These results showthat there is a direct relationship between the production of the I form and hemagglutinin and sialidase activities and the production of the II form of these biologically active proteins. Models that explain the genetic basis for these results are discussed. | Relationship between hemagglutinin and sialidase from Clostridium perfringens CN3870: gel filtration of mutant and reverant activities. Gel filtration of supernatant fluids, from the wild-type Clostridium perfringens, strain CN3870, and several of the mutants and reverants derived from this strain, showed that these mutants failed to product detectable amounts of still produced sialidase III activity. The reverants tested had regained the ability to produce approximately wild-type levels of the I and II forms of both activities. These results showthat there is a direct relationship between the production of the I form and hemagglutinin and sialidase activities and the production of the II form of these biologically active proteins. Models that explain the genetic basis for these results are discussed. | [
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PMID:4436 | Development of Microbodies in the yeast Kloeckera growing on methanol. | A number of microbodies appear regularly in methanol-grown yeast cells, but rarely in ethanol- or glucose-grown cells. When one of representative methanol-utilizing yeasts, Kloeckera sp.no. 2201 (also known as Candida bodinii), was cultured on glucose and then transferred into a methanol medium, microbodies of small size could be observed in 2-h old cells. The number of microbodies per sectioned cell reached five to six after 4 h of cultivation. Though the number of microbodies did not change during prolonged cultivation, their size became larger with the passage of cultivation time. The activities of catalase and alcohol oxidase were confirmed in the particulate fractions throughout the cultivation period, whereas the activities of formaldehyde dehydrogenase and formate dehydrogenase were not detected in the particles. The activity of isocitrate lyase was detected in the particulate fractions only at the early growth phase. | Development of Microbodies in the yeast Kloeckera growing on methanol. A number of microbodies appear regularly in methanol-grown yeast cells, but rarely in ethanol- or glucose-grown cells. When one of representative methanol-utilizing yeasts, Kloeckera sp.no. 2201 (also known as Candida bodinii), was cultured on glucose and then transferred into a methanol medium, microbodies of small size could be observed in 2-h old cells. The number of microbodies per sectioned cell reached five to six after 4 h of cultivation. Though the number of microbodies did not change during prolonged cultivation, their size became larger with the passage of cultivation time. The activities of catalase and alcohol oxidase were confirmed in the particulate fractions throughout the cultivation period, whereas the activities of formaldehyde dehydrogenase and formate dehydrogenase were not detected in the particles. The activity of isocitrate lyase was detected in the particulate fractions only at the early growth phase. | [
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PMID:4437 | Phosphatase of Chlamydomonas reinhardi: biochemical and cytochemical approach with specific mutants. | The unicellular alga Chlamydomonas reinhardi produces two constitutive acid phosphatases and three depressible phosphatases (a neutral and two alkaline ones) that can utilize napthyl phosphate as a substrate. Specific mutants depressible phosphatase were used to investigate biochemical properties and the cytochemical localization of these enzymes. The two constitutive phosphatases show similar pH optima (about 5.0) and Km values (2 x 10(-3) to 3.3 x 10(-3) M) but differ in their heat sensitivity and affinity for glycerophosphate. | Phosphatase of Chlamydomonas reinhardi: biochemical and cytochemical approach with specific mutants. The unicellular alga Chlamydomonas reinhardi produces two constitutive acid phosphatases and three depressible phosphatases (a neutral and two alkaline ones) that can utilize napthyl phosphate as a substrate. Specific mutants depressible phosphatase were used to investigate biochemical properties and the cytochemical localization of these enzymes. The two constitutive phosphatases show similar pH optima (about 5.0) and Km values (2 x 10(-3) to 3.3 x 10(-3) M) but differ in their heat sensitivity and affinity for glycerophosphate. | [
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PMID:4438 | Autolysis of Neisseria gonorrhoeae. | Autolysis of Neisseria gonorrhoeae was studied under different conditions. It was found that low pH and temperature, as well as the presence of divalent cations, spermine, sucrose, and polyvinylpyrrolidone, stabilized nongrowing gonococci. Ethylenediaminetetraacetic acid alone promoted lysis, whereas lysozyme had only a limited additive effect. The autolytic behavior of gonococci appears to be connected with their prolonged cell division process. The relative dependence on the outer membrane and the peptidoglycan layer for the mechanical stability of gonococci is discussed. | Autolysis of Neisseria gonorrhoeae. Autolysis of Neisseria gonorrhoeae was studied under different conditions. It was found that low pH and temperature, as well as the presence of divalent cations, spermine, sucrose, and polyvinylpyrrolidone, stabilized nongrowing gonococci. Ethylenediaminetetraacetic acid alone promoted lysis, whereas lysozyme had only a limited additive effect. The autolytic behavior of gonococci appears to be connected with their prolonged cell division process. The relative dependence on the outer membrane and the peptidoglycan layer for the mechanical stability of gonococci is discussed. | [
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PMID:4439 | Glucose-6-phosphate dehydrogenase. Purification and partial characterization. | Glucose-6-phosphate dehydrogenase has been purified 1000-fold from pig liver. This enzyme exists as an active dimer of molecular weight 133,000 and an inactive monomer of molecular weight 67,500. The pH of maximum activity is 8.5 and the ionic strength maximum is 0.1 to 0.5 M. Glucose-6-phosphate dehydrogenase is highly specific for NADP+ and glucose 6-phosphate. Apparent Km values of 3.6 muM and 5.4 muM were obtained for glucose 6-phosphate and NADP+. This enzyme is located almost entirely within the soluble portion of the cellular cytoplasm. | Glucose-6-phosphate dehydrogenase. Purification and partial characterization. Glucose-6-phosphate dehydrogenase has been purified 1000-fold from pig liver. This enzyme exists as an active dimer of molecular weight 133,000 and an inactive monomer of molecular weight 67,500. The pH of maximum activity is 8.5 and the ionic strength maximum is 0.1 to 0.5 M. Glucose-6-phosphate dehydrogenase is highly specific for NADP+ and glucose 6-phosphate. Apparent Km values of 3.6 muM and 5.4 muM were obtained for glucose 6-phosphate and NADP+. This enzyme is located almost entirely within the soluble portion of the cellular cytoplasm. | [
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PMID:4440 | A kinetic study of glucose-6-phosphate dehydrogenase. | The steady state kinetics of pig liver glucose-6-phosphate dehydrogenase is consistent with an ordered, sequential mechanism in which NADP is bound first and NADPH released last. Kia is 9.0 muM, Ka is 4.8 muM, and Kb is 36 muM. Glucosamine 6-phosphate, a substrate analogue and competitive inhibitor, is used to help rule out a possible random mechanism. ADP is seen to form a complex with the free form of the enzyme whereas ATP forms a complex with both the free and E-NADP forms of the enzyme. The KI for the E-ADP complex is 1.9 mM, while the Ki values for the E-ATP and E-NADP-ATP complexes are 7.2 and 4.5 mM, respectively. | A kinetic study of glucose-6-phosphate dehydrogenase. The steady state kinetics of pig liver glucose-6-phosphate dehydrogenase is consistent with an ordered, sequential mechanism in which NADP is bound first and NADPH released last. Kia is 9.0 muM, Ka is 4.8 muM, and Kb is 36 muM. Glucosamine 6-phosphate, a substrate analogue and competitive inhibitor, is used to help rule out a possible random mechanism. ADP is seen to form a complex with the free form of the enzyme whereas ATP forms a complex with both the free and E-NADP forms of the enzyme. The KI for the E-ADP complex is 1.9 mM, while the Ki values for the E-ATP and E-NADP-ATP complexes are 7.2 and 4.5 mM, respectively. | [
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PMID:4441 | alpha-Aminomethylglutarate, a beta-amino analog of glutamate that interacts with glutamine synthetase and the enzymes that catalyze glutathione synthesis. | The glutamate analog, alpha-aminomethylglutaric acid, was synthetized by Michael addition of ammonia to 2-methylene glutaronitrile followed by hydrolysis of the intermediate alpha-aminomethylglutaryl nitrile; the analog cyclizes readily on heating to 2-piperidone-5-carboxylic acid. Sheep brain glutamine synthetase utilizes one isomer of DL-alpha-aminomethylglutarate at about 10% of the rate with L-glutamate. gamma-Glutamylcysteine synthetase uses both isomers of DL-alpha-aminomethylglutarate, preferentially acting on the same isomer used by glutamine synthetase. gamma-(alpha-Aminomethyl)glutaryl-alpha-aminobutyrate, prepared enzymatically with gamma-glutamylcysteine synthetase, was found to be a substrate and an inhibitor of glutathione synthetase. alpha-Aminomethylglutarate does not inhibit gamma-glutamyl cyclotransferase and gamma-glutamyl transpeptidase appreciably. When alpha-aminomethylglutarate was administered to mice, there were substantial decreases in the levels of glutamine, glutathione, glutamate, and glycine in the kidney, and of glutamine and glutamate in the liver, indicating that this glutamate analog is effective as an inhibitor of glutamine and glutathione synthesis in vivo, and suggesting that it may also inhibit other enzymes. | alpha-Aminomethylglutarate, a beta-amino analog of glutamate that interacts with glutamine synthetase and the enzymes that catalyze glutathione synthesis. The glutamate analog, alpha-aminomethylglutaric acid, was synthetized by Michael addition of ammonia to 2-methylene glutaronitrile followed by hydrolysis of the intermediate alpha-aminomethylglutaryl nitrile; the analog cyclizes readily on heating to 2-piperidone-5-carboxylic acid. Sheep brain glutamine synthetase utilizes one isomer of DL-alpha-aminomethylglutarate at about 10% of the rate with L-glutamate. gamma-Glutamylcysteine synthetase uses both isomers of DL-alpha-aminomethylglutarate, preferentially acting on the same isomer used by glutamine synthetase. gamma-(alpha-Aminomethyl)glutaryl-alpha-aminobutyrate, prepared enzymatically with gamma-glutamylcysteine synthetase, was found to be a substrate and an inhibitor of glutathione synthetase. alpha-Aminomethylglutarate does not inhibit gamma-glutamyl cyclotransferase and gamma-glutamyl transpeptidase appreciably. When alpha-aminomethylglutarate was administered to mice, there were substantial decreases in the levels of glutamine, glutathione, glutamate, and glycine in the kidney, and of glutamine and glutamate in the liver, indicating that this glutamate analog is effective as an inhibitor of glutamine and glutathione synthesis in vivo, and suggesting that it may also inhibit other enzymes. | [
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PMID:4442 | Studies of human kidney gamma-glutamyl transpeptidase. Purification and structural, kinetic and immunological properties. | gamma-Glutamyl transpeptidase, present in various mammalian tissues, transfers the gamma-glutamyl moiety of glutathione to a variety of acceptor amino acids and peptides. This enzyme has been purified from human kidney cortex about 740-fold to a specific activity of 200 units/mg of protein. The purification steps involved incubation of the homogenate at 37 degrees followed by centrifugation and extraction of the sediment with 0.1 M Tris-HCl buffer, pH 8.0, containing 1% sodium deoxycholate; batchwise absorption on DEAE-cellulose; DEAE-cellulose (DE52) column chromatography; Sephadex G-200 gel filtration; and affinity chromatography using concanavalin A insolubilized on beaded Agarose. Detergents were used throughout the purification of the enzyme. The purified enzyme separated into three protein bands, all of which had enzyme activity, on polyacrylamide disc electrophoresis in the presence of Triton X-100. The enzyme has an apparent molecular weight of about 90,000 as shown by Sephadex G-200 gel filtration, and appears to be a tetramer with subunits of molecular weights of about 21,000. The Km for gamma-glutamyl transpeptidase using the artificial substrate, gamma-glutamyl-p-nitroanilide, with glycylglycine as the acceptor amino acid was found to be about 0.8 mM. The optimum pH for the enzyme activity is 8.2 and the isoelectric point is 4.5. Both GSH and GSSG competitively inhibited the activity of gamma-glutamyl transpeptidase when gamma-glutamyl-p-nitroanilide was used as the substrate. Treatment of the purified enzyme with papain has no effect on the enzyme activity or mobility on polyacrylamide disc electrophoresis. The purified gamma-glutamyl transpeptidase had no phosphate-independent glutaminase activity. The ratio of gamma-glutamyl transpeptidase to phosphate-independent glutaminase changed significantly through the initial steps of gamma-glutamyl transpeptidase purification. These studies indicate that the transpeptidase and phosphate-independent glutaminase activities are not exhibited by the same protein in human kidney. | Studies of human kidney gamma-glutamyl transpeptidase. Purification and structural, kinetic and immunological properties. gamma-Glutamyl transpeptidase, present in various mammalian tissues, transfers the gamma-glutamyl moiety of glutathione to a variety of acceptor amino acids and peptides. This enzyme has been purified from human kidney cortex about 740-fold to a specific activity of 200 units/mg of protein. The purification steps involved incubation of the homogenate at 37 degrees followed by centrifugation and extraction of the sediment with 0.1 M Tris-HCl buffer, pH 8.0, containing 1% sodium deoxycholate; batchwise absorption on DEAE-cellulose; DEAE-cellulose (DE52) column chromatography; Sephadex G-200 gel filtration; and affinity chromatography using concanavalin A insolubilized on beaded Agarose. Detergents were used throughout the purification of the enzyme. The purified enzyme separated into three protein bands, all of which had enzyme activity, on polyacrylamide disc electrophoresis in the presence of Triton X-100. The enzyme has an apparent molecular weight of about 90,000 as shown by Sephadex G-200 gel filtration, and appears to be a tetramer with subunits of molecular weights of about 21,000. The Km for gamma-glutamyl transpeptidase using the artificial substrate, gamma-glutamyl-p-nitroanilide, with glycylglycine as the acceptor amino acid was found to be about 0.8 mM. The optimum pH for the enzyme activity is 8.2 and the isoelectric point is 4.5. Both GSH and GSSG competitively inhibited the activity of gamma-glutamyl transpeptidase when gamma-glutamyl-p-nitroanilide was used as the substrate. Treatment of the purified enzyme with papain has no effect on the enzyme activity or mobility on polyacrylamide disc electrophoresis. The purified gamma-glutamyl transpeptidase had no phosphate-independent glutaminase activity. The ratio of gamma-glutamyl transpeptidase to phosphate-independent glutaminase changed significantly through the initial steps of gamma-glutamyl transpeptidase purification. These studies indicate that the transpeptidase and phosphate-independent glutaminase activities are not exhibited by the same protein in human kidney. | [
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PMID:4443 | Kinetics of the hemerythrin-oxygen interaction. | The kinetics of the reaction of Golfingia gouldii hemerythrin with O2 have been studied by stopped flow spectrophotometry. For the second order oxygenation process, k1 = 7.4 X 10(6) M-1 s-1, deltaH1++ = 8.2 kcal-mol-1 and deltaS1++ = +1 e.u. at 25 degrees, pH 8.2, and I = 0.015 M. The rate constant is unchanged when protein concentration is varied from 3 to 25 muM, the ionic strength is increased to 0.07 M, and the pH moved to 6.8. The deoxygenation of oxyhemerythrin is studied with stopped flow by scavenging liberated O2 with S2O4(2-). For the first order dissociation, k-1 = 51 s-1, deltaH-1++ = 20.6 kcal-mol-1 and deltaS-1++ = +19 e.u. at 25 degrees, pH 8.2, and I = 0.015 M. The value of k-1 is independent of [protein] = 50 to 200 muM, [S2O4(2-)] = 5 to 100 mM I = 0.015 to 0.30 M and pH 6.8 to 9.0. Using myoglobin instead of S2O4(2-) as scavenger gives similar results. Combination of activation parameters for the oxygenation and deoxygenation processes gives K1 = 1.5 X 10(5) M-1, deltaH = -12.4 kcal-mol-1, and deltaS = -18 e.u., values in good agreement with independent thermodynamic data. Perchlorate ion (0.05 M) enhances k-1 about 3-fold and hardly effects k1. There is no sign of other than a single reaction in either direction, and octameric hemerythrin apparently behaves kinetically as eight single units. | Kinetics of the hemerythrin-oxygen interaction. The kinetics of the reaction of Golfingia gouldii hemerythrin with O2 have been studied by stopped flow spectrophotometry. For the second order oxygenation process, k1 = 7.4 X 10(6) M-1 s-1, deltaH1++ = 8.2 kcal-mol-1 and deltaS1++ = +1 e.u. at 25 degrees, pH 8.2, and I = 0.015 M. The rate constant is unchanged when protein concentration is varied from 3 to 25 muM, the ionic strength is increased to 0.07 M, and the pH moved to 6.8. The deoxygenation of oxyhemerythrin is studied with stopped flow by scavenging liberated O2 with S2O4(2-). For the first order dissociation, k-1 = 51 s-1, deltaH-1++ = 20.6 kcal-mol-1 and deltaS-1++ = +19 e.u. at 25 degrees, pH 8.2, and I = 0.015 M. The value of k-1 is independent of [protein] = 50 to 200 muM, [S2O4(2-)] = 5 to 100 mM I = 0.015 to 0.30 M and pH 6.8 to 9.0. Using myoglobin instead of S2O4(2-) as scavenger gives similar results. Combination of activation parameters for the oxygenation and deoxygenation processes gives K1 = 1.5 X 10(5) M-1, deltaH = -12.4 kcal-mol-1, and deltaS = -18 e.u., values in good agreement with independent thermodynamic data. Perchlorate ion (0.05 M) enhances k-1 about 3-fold and hardly effects k1. There is no sign of other than a single reaction in either direction, and octameric hemerythrin apparently behaves kinetically as eight single units. | [
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PMID:4444 | Guanylate cyclase and cyclic guanosine 3':5'-monophosphate phosphodiesterase activities and cyclic guanosine 3':5'-monophosphate levels in normal and transformed fibroblasts in culture. | To investigate the role of guanosine 3':5'-monophosphate (cyclic GMP) in cultured cells we have measured guanylate cyclase and cyclic GMP phosphodiesterase activities and cyclic GMP levels in normal and transformed fibroblastic cells. Guanylate cyclase activity is found almost exclusively in the particulate fraction of normal rat kidney (NRK) and BALB 3T3 cells. Enzyme activity is stimulated 3- to 10-fold by treatment with the detergent Lubrol PX. However, enhancement of guanylate cyclase by fibroblast growth factor could not be demonstrated under a variety of assay conditions. In both NRK and BALB 3T3 cells guanylate cyclase activity is low during logarithmic growth and increases as the cells crowd together and growth slows. Guanylate cyclase activity is undetectable in homogenates of NRK cells transformed by the Kirsten sarcoma virus (KNRK cells) either in the presence or absence of Lubrol PX. Guanylate cyclase activity is also greatly decreased in NRK cells transformed by Moloney, Schmidt-Ruppin, or Harvey viruses. BALB 3T3 cells transformed by RNA viruses (Kirsten, Harvey, or Moloney), by a DNA virus (SV40), by methylcholanthrene, or spontaneously, all have diminished but readily detectable guanylate cyclase activity. Cyclic GMP phosphodiesterase activity is found predominately in the soluble fraction of NRK cells. This activity increases slightly as NRK cells enter the stationary growth phase. Cyclic GMP phosphodiesterase activity is undetectable in two clones of KNRK cells under a variety of assay conditions, and is decreased relative to the level present in NRK cells in a third KNRK clone. However, both Moloney- and Schmidt-Ruppin-transformed NRK cells have a phosphodiesterase activity similar to that found in NRK cells. Boiled supernatant from both NRK and KNRK cells is observed to appreciably enhance the activity of activator-deficient phosphodiesterase from bovine heart. This result indicates that the absence of cyclic GMP phosphodiesterase activity in KNRK cells is not due to a loss of the phosphodiesterase activator. The intracellular concentration of cyclic GMP is found to be very low in transformed NRK cells when compared to levels measured in confluent NRK cells. The low levels of cyclic GMP in transformed NRK cells reflect the greatly decreased guanylate cyclase activity observed in these cells. These results do not appear to support the suggestion that cyclic GMP promotes the growth of fibroblastic cells. | Guanylate cyclase and cyclic guanosine 3':5'-monophosphate phosphodiesterase activities and cyclic guanosine 3':5'-monophosphate levels in normal and transformed fibroblasts in culture. To investigate the role of guanosine 3':5'-monophosphate (cyclic GMP) in cultured cells we have measured guanylate cyclase and cyclic GMP phosphodiesterase activities and cyclic GMP levels in normal and transformed fibroblastic cells. Guanylate cyclase activity is found almost exclusively in the particulate fraction of normal rat kidney (NRK) and BALB 3T3 cells. Enzyme activity is stimulated 3- to 10-fold by treatment with the detergent Lubrol PX. However, enhancement of guanylate cyclase by fibroblast growth factor could not be demonstrated under a variety of assay conditions. In both NRK and BALB 3T3 cells guanylate cyclase activity is low during logarithmic growth and increases as the cells crowd together and growth slows. Guanylate cyclase activity is undetectable in homogenates of NRK cells transformed by the Kirsten sarcoma virus (KNRK cells) either in the presence or absence of Lubrol PX. Guanylate cyclase activity is also greatly decreased in NRK cells transformed by Moloney, Schmidt-Ruppin, or Harvey viruses. BALB 3T3 cells transformed by RNA viruses (Kirsten, Harvey, or Moloney), by a DNA virus (SV40), by methylcholanthrene, or spontaneously, all have diminished but readily detectable guanylate cyclase activity. Cyclic GMP phosphodiesterase activity is found predominately in the soluble fraction of NRK cells. This activity increases slightly as NRK cells enter the stationary growth phase. Cyclic GMP phosphodiesterase activity is undetectable in two clones of KNRK cells under a variety of assay conditions, and is decreased relative to the level present in NRK cells in a third KNRK clone. However, both Moloney- and Schmidt-Ruppin-transformed NRK cells have a phosphodiesterase activity similar to that found in NRK cells. Boiled supernatant from both NRK and KNRK cells is observed to appreciably enhance the activity of activator-deficient phosphodiesterase from bovine heart. This result indicates that the absence of cyclic GMP phosphodiesterase activity in KNRK cells is not due to a loss of the phosphodiesterase activator. The intracellular concentration of cyclic GMP is found to be very low in transformed NRK cells when compared to levels measured in confluent NRK cells. The low levels of cyclic GMP in transformed NRK cells reflect the greatly decreased guanylate cyclase activity observed in these cells. These results do not appear to support the suggestion that cyclic GMP promotes the growth of fibroblastic cells. | [
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PMID:4445 | Specificity of alpha-chymotrypsin with exposed carboxyl groups blocked. | The 15 exposed carboxyl groups of alpha-chymotrypsin were modified with glycine ethyl ester at low pH using barbodiimide reagent. The specificity of the modified enzyme (Chy-15) was studied over the pH range of 4 to 9 with both N-acylated and non-N-acylated amino acid esters. The modified enzyme had lower reactivity toward N-acylated esters than non-N-acylated esters compared to the native enzyme. Typical substances such as acetyl- and benzoyl-L-tyrosine ethyl esters retained 4 and 9% activity, whereas phenylalanine ethyl ester was slightly more reactive with the modified than with the native enzyme. The pH-rate profiles of acetyl-L-phenylalanine ethyl ester and tryptophan ethyl and benzyl esters were investigated in detail. Analysis of these profiles revealed three pKa values of approximately 5, 7, and 9 related to a functional carboxyl, imidazoyl, and an amino group, respectively. Since similar pKa values occur for the native enzyme, modification did not block the carboxyl corresponding to pKa 5. A mechanism is proposed for catalysis which includes both the protonated and unprotonated form of the imidazoyl (His-57) and utilizes water rather than a carboxyl (Asp-102) as the proton sink. | Specificity of alpha-chymotrypsin with exposed carboxyl groups blocked. The 15 exposed carboxyl groups of alpha-chymotrypsin were modified with glycine ethyl ester at low pH using barbodiimide reagent. The specificity of the modified enzyme (Chy-15) was studied over the pH range of 4 to 9 with both N-acylated and non-N-acylated amino acid esters. The modified enzyme had lower reactivity toward N-acylated esters than non-N-acylated esters compared to the native enzyme. Typical substances such as acetyl- and benzoyl-L-tyrosine ethyl esters retained 4 and 9% activity, whereas phenylalanine ethyl ester was slightly more reactive with the modified than with the native enzyme. The pH-rate profiles of acetyl-L-phenylalanine ethyl ester and tryptophan ethyl and benzyl esters were investigated in detail. Analysis of these profiles revealed three pKa values of approximately 5, 7, and 9 related to a functional carboxyl, imidazoyl, and an amino group, respectively. Since similar pKa values occur for the native enzyme, modification did not block the carboxyl corresponding to pKa 5. A mechanism is proposed for catalysis which includes both the protonated and unprotonated form of the imidazoyl (His-57) and utilizes water rather than a carboxyl (Asp-102) as the proton sink. | [
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PMID:4446 | Evidence for the coordinate control of activity of liver glycogen synthase and phosphorylase by a single protein phosphatase. | Homogeneous rabbit liver phosphorylase phosphatase (Brandt, H., Capulong, Z. L., and Lee, E. Y. C. (1975) J. Biol. Chem. 250, 8038-8044) also dephosphorylates glycogen synthase b. During purification, phosphorylase phosphatase and glycogen synthase phosphatase co-purified with a constant ratio of activities. The two activities co-migrated on disc gel electrophoresis. Both substrates competed with each other for the phosphatase, and both phosphatase activities were inhibited by lysine ethyl ester. It is concluded that liver phosphorylase phosphatase and glycogen synthase phosphatase have a common identity and that coordinate regulation of the phosphatase-catalyzed activation of glycogen synthase and inactivation of phosphorylase occurs in vivo. This provides a parallel and opposing mechanism to that mediated by adenosine 3':5'-monophosphate-dependent protein kinase, which coordinately inactivates glycogen synthase and, via phosphorylase kinase, activates phosphorylase. Maximal glycogen synthase phosphatase activity was observed near neutrality. Mg2+ and glucose-6-P activated the glycogen synthase phosphatase reaction and this activation was pH-dependent. The Km for glycogen synthase b was 0.12 muM. | Evidence for the coordinate control of activity of liver glycogen synthase and phosphorylase by a single protein phosphatase. Homogeneous rabbit liver phosphorylase phosphatase (Brandt, H., Capulong, Z. L., and Lee, E. Y. C. (1975) J. Biol. Chem. 250, 8038-8044) also dephosphorylates glycogen synthase b. During purification, phosphorylase phosphatase and glycogen synthase phosphatase co-purified with a constant ratio of activities. The two activities co-migrated on disc gel electrophoresis. Both substrates competed with each other for the phosphatase, and both phosphatase activities were inhibited by lysine ethyl ester. It is concluded that liver phosphorylase phosphatase and glycogen synthase phosphatase have a common identity and that coordinate regulation of the phosphatase-catalyzed activation of glycogen synthase and inactivation of phosphorylase occurs in vivo. This provides a parallel and opposing mechanism to that mediated by adenosine 3':5'-monophosphate-dependent protein kinase, which coordinately inactivates glycogen synthase and, via phosphorylase kinase, activates phosphorylase. Maximal glycogen synthase phosphatase activity was observed near neutrality. Mg2+ and glucose-6-P activated the glycogen synthase phosphatase reaction and this activation was pH-dependent. The Km for glycogen synthase b was 0.12 muM. | [
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PMID:4447 | Solubilization and characterization of the beta-adrenergic receptor binding sites of frog erythrocytes. | Specific beta-adrenergic receptors present in membrane preparations of frog erythrocytes were identified by binding of (-)-[3H]dihydroalprenolol, a potent competitive beta-adrenergic antagonist. The (-)-[3H]dihydroalprenolol binding sites could be solubilized by treatment of a purified erythrocyte membrane fraction with the plant glycoside digitonin but not by treatment with a wide variety of other detergents. The binding sites appeared to be soluble by several independent experimental criteria including (a) failure to sediment of 105,000 X g for 2 hours; (b) passage through 0.22-mu Millipore filters; (c) chromatography on Sepharose 6B gels; and (d) electron microscopy. The soluble receptor sites retained all of the essential characteristics of the membrane-bound sites, namely rapid and reversible binding of beta-adrenergic agonists and antagonists; strict stereospecificity toward both beta-adrenergic agonists and antagonists; appropriate structure-activity relationships; saturability of the sites at low concentrations of ligand; no affinity for alpha-adrenergic drugs, nonphysiologically active catechol compounds, and catecholamine metabolites. Based on gel chromatography in the presence of detergent, the molecular weight of the soluble receptor is estimated to be no greater than 130,000 to 150,000. Equilibrium binding studies indicated a KD for the soluble receptor of 2 nM. Hill coefficients (nH) of 0.77 and curved Scatchard plots suggested the presence of negatively cooperative interactions among the solubilized receptors in agreement with previous findings with the membrane-bound sites. Kinetic studies indicated an association rate constant K1 = 3.8 X 10(6) M-1 min-1 and a reverse rate constant k2 = 2.3 X 10(-3) min-1 at 4 degrees. The kinetically derived KD (k2/k1) of 0.6 nM is in reasonable agreement with that determined by equilibrium studies. The soluble receptors were labile at temperature greater than 4 degrees but could be stabilized with high concentrations of EDTA. Guanidine hydrochloride and urea produced concentration-dependent losses of binding activity which were partially reversible upon dialysis. Trypsin and phospholipase A both degraded the soluble receptors but a variety of other proteases and phospholipases as well as DNase and RNase were without effect. Experiments with group-specific reagents indicated that free lysine, tryptophan, serine, and sulfhydryl groups may be important for receptor binding. These studies suggest that the receptor is probably a protein which requires lipids for functional integrity. Data obtained with the solubilized binding sites are consistent with the contention that these sites represent the physiologically relevant beta-adrenergic receptors which have been extracted from the membranes with full retention of their properties. | Solubilization and characterization of the beta-adrenergic receptor binding sites of frog erythrocytes. Specific beta-adrenergic receptors present in membrane preparations of frog erythrocytes were identified by binding of (-)-[3H]dihydroalprenolol, a potent competitive beta-adrenergic antagonist. The (-)-[3H]dihydroalprenolol binding sites could be solubilized by treatment of a purified erythrocyte membrane fraction with the plant glycoside digitonin but not by treatment with a wide variety of other detergents. The binding sites appeared to be soluble by several independent experimental criteria including (a) failure to sediment of 105,000 X g for 2 hours; (b) passage through 0.22-mu Millipore filters; (c) chromatography on Sepharose 6B gels; and (d) electron microscopy. The soluble receptor sites retained all of the essential characteristics of the membrane-bound sites, namely rapid and reversible binding of beta-adrenergic agonists and antagonists; strict stereospecificity toward both beta-adrenergic agonists and antagonists; appropriate structure-activity relationships; saturability of the sites at low concentrations of ligand; no affinity for alpha-adrenergic drugs, nonphysiologically active catechol compounds, and catecholamine metabolites. Based on gel chromatography in the presence of detergent, the molecular weight of the soluble receptor is estimated to be no greater than 130,000 to 150,000. Equilibrium binding studies indicated a KD for the soluble receptor of 2 nM. Hill coefficients (nH) of 0.77 and curved Scatchard plots suggested the presence of negatively cooperative interactions among the solubilized receptors in agreement with previous findings with the membrane-bound sites. Kinetic studies indicated an association rate constant K1 = 3.8 X 10(6) M-1 min-1 and a reverse rate constant k2 = 2.3 X 10(-3) min-1 at 4 degrees. The kinetically derived KD (k2/k1) of 0.6 nM is in reasonable agreement with that determined by equilibrium studies. The soluble receptors were labile at temperature greater than 4 degrees but could be stabilized with high concentrations of EDTA. Guanidine hydrochloride and urea produced concentration-dependent losses of binding activity which were partially reversible upon dialysis. Trypsin and phospholipase A both degraded the soluble receptors but a variety of other proteases and phospholipases as well as DNase and RNase were without effect. Experiments with group-specific reagents indicated that free lysine, tryptophan, serine, and sulfhydryl groups may be important for receptor binding. These studies suggest that the receptor is probably a protein which requires lipids for functional integrity. Data obtained with the solubilized binding sites are consistent with the contention that these sites represent the physiologically relevant beta-adrenergic receptors which have been extracted from the membranes with full retention of their properties. | [
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PMID:4448 | Reconstitution and characterization of the adenine nucleotide transporter derived from bovine heart mitochondria. | 1. Adenine nucleotide exchange-transport was reconstituted in vesicles prepared from phospholipids and protein fractions derived from bovine heart submitochondrial particles. The transport, which was specific for ATP and ADP was measured either as ADP/ADP, ATP/ATP, or ADP/ATP exchange. The highest specific activity (370 nanomoles of ADP/ADP exchange/min/mg of protein at room temperature) was obtained with a protein fraction prepared by cholate extraction of partly resolved submitochondrial particles followed by ammonium sulfate fractionation. 2. At 200 muM external nucleotide, the exchange reactions were inhibited by low concentrations of bongkrekate, atractyloside, and palmitoyl-CoA, with Ki values of 1.8, 3.0, and 7.5 muM, respectively. The ADP/ADP nucleotide exchange was stimulated about 5-fold by 500 muM MgCl2 or MnCl2(km of 40 muM) and about 3-fold by 500 muM CaCl2(Km of 90 muM). It was optimal between pH 6.0 and 7.0 and decreased rapidly above pH 7.5. Arrhenius plots between 0 degrees and 40 degrees showed a break point at 15 degrees with soybean phospholipids and an activation energy of 29.5 kcal/mole from 0 degrees-15 degrees and 9.0 kcal/mole from 15 degrees-40 degrees. With mitochondrial phospholipids the break point was at 9 degrees and activation energies were 42.4 kcal/mole from 0 degrees-9 degrees and 7.6 kcal/mole from 9 degrees-40 degrees. 3. The phospholipid requirements for adenine nucleotide exchange were similar to those of oxidative phosphorylation. Optimal rates were observed with a phosphatidylethanolamine to phosphatidylcholine ratio of 4:1. Cardiolipin had a slight stimulatory effect. 4. The uptake of ADP into vesicles containing ATP was stimulated by KCl or by KPi as well as by hexafluoracetonylacetone, and uncoupler of oxidative phosphorylation. The uptake of ATP into vesicles containing ADP was inhibited by KCl or by KPi, but was also stimulated by hexafluoracetonylacetone. In both cases valinomycin reversed the effects of KCl, while mersalyl or N-ethylmaleimide prevented the effects of KPi. In contrast, none of these salts nor hexafluoracetonylactone affected the ADP/ADP or ATP/ATP exchange. These findings suggest that in the reconstituted system the ADP/ATP exchange is electrogenic. | Reconstitution and characterization of the adenine nucleotide transporter derived from bovine heart mitochondria. 1. Adenine nucleotide exchange-transport was reconstituted in vesicles prepared from phospholipids and protein fractions derived from bovine heart submitochondrial particles. The transport, which was specific for ATP and ADP was measured either as ADP/ADP, ATP/ATP, or ADP/ATP exchange. The highest specific activity (370 nanomoles of ADP/ADP exchange/min/mg of protein at room temperature) was obtained with a protein fraction prepared by cholate extraction of partly resolved submitochondrial particles followed by ammonium sulfate fractionation. 2. At 200 muM external nucleotide, the exchange reactions were inhibited by low concentrations of bongkrekate, atractyloside, and palmitoyl-CoA, with Ki values of 1.8, 3.0, and 7.5 muM, respectively. The ADP/ADP nucleotide exchange was stimulated about 5-fold by 500 muM MgCl2 or MnCl2(km of 40 muM) and about 3-fold by 500 muM CaCl2(Km of 90 muM). It was optimal between pH 6.0 and 7.0 and decreased rapidly above pH 7.5. Arrhenius plots between 0 degrees and 40 degrees showed a break point at 15 degrees with soybean phospholipids and an activation energy of 29.5 kcal/mole from 0 degrees-15 degrees and 9.0 kcal/mole from 15 degrees-40 degrees. With mitochondrial phospholipids the break point was at 9 degrees and activation energies were 42.4 kcal/mole from 0 degrees-9 degrees and 7.6 kcal/mole from 9 degrees-40 degrees. 3. The phospholipid requirements for adenine nucleotide exchange were similar to those of oxidative phosphorylation. Optimal rates were observed with a phosphatidylethanolamine to phosphatidylcholine ratio of 4:1. Cardiolipin had a slight stimulatory effect. 4. The uptake of ADP into vesicles containing ATP was stimulated by KCl or by KPi as well as by hexafluoracetonylacetone, and uncoupler of oxidative phosphorylation. The uptake of ATP into vesicles containing ADP was inhibited by KCl or by KPi, but was also stimulated by hexafluoracetonylacetone. In both cases valinomycin reversed the effects of KCl, while mersalyl or N-ethylmaleimide prevented the effects of KPi. In contrast, none of these salts nor hexafluoracetonylactone affected the ADP/ADP or ATP/ATP exchange. These findings suggest that in the reconstituted system the ADP/ATP exchange is electrogenic. | [
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PMID:4449 | Solubilization of thyroid peroxidase by nonionic detergents. | We have examined the ability of nonionic detergents to solubilize thyroid peroxidase from a porcine thyroid particulate fraction, as measured by the release of peroxidase activity into the supernatant fraction after centrifugation at 105,000 X g for 1 hour and the retardation of the supernatant peroxidase of Sepharose 6B. The parameters of peroxidase solubilization by Triton X-100 have been investigated in detail. Under optimum conditions, 60 to 95% of the thryoid peroxidase and about 50% of the total protein is released into the 105,000 X g, 1-hour supernatant. Under the optimum conditions established with Triton X-100, a series of Brij detergents of different chemical structure were equally effective in releasing peroxidase and protein. The protein patterns of the supernatants obtained with these detergents were similar on sodium dodecyl sulfate-polyacrylamide electrophoresis gels, suggesting that the detergents studied release similar membrane proteins. The Triton X-100 and Brij 58 supernatants were chromatographed separately on Sepharose 6B equilibrated with 0.1% Triton X-100 or Brij 58, respectively. In both cases, 75 to 80% of the peroxidase activity was retarded, thereby indicating that the nonionic detergents effect solubilization of the peroxidase rather than dispersal of nonsedimentable membrane fragments. These studies report the first successful solubilization of thyroid peroxidase by nonionic detergents. Together with previous evidence from our laboratory, these experiments indicate that thyroid peroxidase is an integral membrane protein. | Solubilization of thyroid peroxidase by nonionic detergents. We have examined the ability of nonionic detergents to solubilize thyroid peroxidase from a porcine thyroid particulate fraction, as measured by the release of peroxidase activity into the supernatant fraction after centrifugation at 105,000 X g for 1 hour and the retardation of the supernatant peroxidase of Sepharose 6B. The parameters of peroxidase solubilization by Triton X-100 have been investigated in detail. Under optimum conditions, 60 to 95% of the thryoid peroxidase and about 50% of the total protein is released into the 105,000 X g, 1-hour supernatant. Under the optimum conditions established with Triton X-100, a series of Brij detergents of different chemical structure were equally effective in releasing peroxidase and protein. The protein patterns of the supernatants obtained with these detergents were similar on sodium dodecyl sulfate-polyacrylamide electrophoresis gels, suggesting that the detergents studied release similar membrane proteins. The Triton X-100 and Brij 58 supernatants were chromatographed separately on Sepharose 6B equilibrated with 0.1% Triton X-100 or Brij 58, respectively. In both cases, 75 to 80% of the peroxidase activity was retarded, thereby indicating that the nonionic detergents effect solubilization of the peroxidase rather than dispersal of nonsedimentable membrane fragments. These studies report the first successful solubilization of thyroid peroxidase by nonionic detergents. Together with previous evidence from our laboratory, these experiments indicate that thyroid peroxidase is an integral membrane protein. | [
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PMID:4450 | Carbon 13 magnetic resonance studies of DL-2-(alpha-hydroxyethyl) thiamin and related compounds. Relation of kinetic acidity to electronic factors in thiamin catalysis. | Carbon 13 NMR spectra have been obtained for aqueous solutions of DL-2-(alpha-hydroxyethyl)thiamin, DL-2-(alpha-hydroxybenzyl)thiamin, DL-2-(alpha-hydroxybenzyl)oxythiamin, and related N-3 methyl and N-3 benzyl analogs. The unusually large downfield shift of the 13C resonance of C-2 of hydroxyethylthiamin suggests that this carbon bears a partial positive charge. This result stands in contrast to results of x-ray crystallographic studies of hydroxyethylthiamin, which place a partial negative charge on C-2 (Pletcher, J., and Sax, M. (1974) J. Am. Chem. Soc. 96, 155-165). A partial positive charge on C-2 helps to explain the facility of carbanion formation at the alpha carbon both enzymatically and in model systems. The rates of proton-deuteron exchange of (C-alpha)-H with solvent deuterium, and of release of aldehyde to regenerate thiamin have been measured for hydroxyethylthiamin and analogs. The differences in kinetic acidity of (C-alpha)-H and of rates of aldehyde release are rationalized in terms of differing electron-withdrawing abilities of the substituents attached to N-3, and appear not to be related to intramolecular basic catalysis of these processes by the C-4' amino group. | Carbon 13 magnetic resonance studies of DL-2-(alpha-hydroxyethyl) thiamin and related compounds. Relation of kinetic acidity to electronic factors in thiamin catalysis. Carbon 13 NMR spectra have been obtained for aqueous solutions of DL-2-(alpha-hydroxyethyl)thiamin, DL-2-(alpha-hydroxybenzyl)thiamin, DL-2-(alpha-hydroxybenzyl)oxythiamin, and related N-3 methyl and N-3 benzyl analogs. The unusually large downfield shift of the 13C resonance of C-2 of hydroxyethylthiamin suggests that this carbon bears a partial positive charge. This result stands in contrast to results of x-ray crystallographic studies of hydroxyethylthiamin, which place a partial negative charge on C-2 (Pletcher, J., and Sax, M. (1974) J. Am. Chem. Soc. 96, 155-165). A partial positive charge on C-2 helps to explain the facility of carbanion formation at the alpha carbon both enzymatically and in model systems. The rates of proton-deuteron exchange of (C-alpha)-H with solvent deuterium, and of release of aldehyde to regenerate thiamin have been measured for hydroxyethylthiamin and analogs. The differences in kinetic acidity of (C-alpha)-H and of rates of aldehyde release are rationalized in terms of differing electron-withdrawing abilities of the substituents attached to N-3, and appear not to be related to intramolecular basic catalysis of these processes by the C-4' amino group. | [
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PMID:4451 | Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin. | Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin have been studied using laser photolysis, air flash, and stopped flow techniques. The reactions of this hemoglobin with both ligands were found to be more rapid than the corresponding reactions involving myoglobin and were also biphasic in nature, the rate constants being approximately an order of magnitude different for the fast and slow phases in each case. No pH or hemoglobin concentration dependence of the pseudo-first order rate constants was apparent between pH 6 and 9 and in the concentration range of 1.25 to 40 muM heme. Both fast and slow pseudo-first order oxygen combination rate constants varied linearly with oxygen concentration between 16 and 1300 muM. A first order slow relaxation was also noted which was linearly dependent on heme concentration and inversely dependent on oxygen concentration. This reaction has been shown to be due to a replacement of oxygen by carbon monoxide. The presence of this reaction is a result of the high affinity of Glycera monomer for carbon monoxide as shown by the partition coefficient Mr = approximately 20,000 ana an equilibrium dissociation constant of the order L = 1.1 X 10(-9) M. | Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin. Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin have been studied using laser photolysis, air flash, and stopped flow techniques. The reactions of this hemoglobin with both ligands were found to be more rapid than the corresponding reactions involving myoglobin and were also biphasic in nature, the rate constants being approximately an order of magnitude different for the fast and slow phases in each case. No pH or hemoglobin concentration dependence of the pseudo-first order rate constants was apparent between pH 6 and 9 and in the concentration range of 1.25 to 40 muM heme. Both fast and slow pseudo-first order oxygen combination rate constants varied linearly with oxygen concentration between 16 and 1300 muM. A first order slow relaxation was also noted which was linearly dependent on heme concentration and inversely dependent on oxygen concentration. This reaction has been shown to be due to a replacement of oxygen by carbon monoxide. The presence of this reaction is a result of the high affinity of Glycera monomer for carbon monoxide as shown by the partition coefficient Mr = approximately 20,000 ana an equilibrium dissociation constant of the order L = 1.1 X 10(-9) M. | [
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PMID:4452 | Analysis of phosphate metabolites, the intracellular pH, and the state of adenosine triphosphate in intact muscle by phosphorus nuclear magnetic resonance. | 31P nuclear magnetic resonance spectra recorded from intact muophosphate, and the sugar phosphates. Quantitation of these metabolites by 31P nuclear magnetic resonance was in good agreement with values obtained by chemical analyses. The spectra obtained from various muscles showed considerable variation in their phosphorus profile. Thus, differences could be detected between (a) normal and diseased muscle; (b) vertebrates and invertebrates; (c) different species of the same animal. The time course of change in phosphate metabolites in frog muscle showed that ATP level remains unchanged until phosphocreatine is nearly depleted. Comparative studies revealed that under anaerobic conditions the Northern frog maintains its ATP content for 7 hours, while other types of amphibian, bird, and mammalian muscles begin to show an appreciable decay in ATP after 2 hours. Several lines of evidence indicated that ATP forms a complex with magnesium in the muscle water: (a) the phosphate resonances of ATP in the muscle were shifted downfield as compared to those in the alkaline earth metal-free perchloric acid extract of the muscle; (b) the coupling constants of ATP measured in various live muscles closely corresponded to those for MgATP in a solution resembling the composition of the muscle water; (c) in the muscle the gamma-phosphate group of ATP exhibited no shift change over a period of 10 hours under conditions where resonances of other phosphate compounds could be titrated. This behavior is similar to that of MgATP in model solutions in the physiological pH range, and it is different from that of CaATP. The chemical shifts of the phosphate metabolites were determined in several relevant solutions as a function of pH. Under all conditions only inorganic orthophosphate showed an invariant titration curve. From the chemical shift of inorganic phosphate observed during aging of intact muscle the intracellular pH of frog muscle was estimated to be 7.2. | Analysis of phosphate metabolites, the intracellular pH, and the state of adenosine triphosphate in intact muscle by phosphorus nuclear magnetic resonance. 31P nuclear magnetic resonance spectra recorded from intact muophosphate, and the sugar phosphates. Quantitation of these metabolites by 31P nuclear magnetic resonance was in good agreement with values obtained by chemical analyses. The spectra obtained from various muscles showed considerable variation in their phosphorus profile. Thus, differences could be detected between (a) normal and diseased muscle; (b) vertebrates and invertebrates; (c) different species of the same animal. The time course of change in phosphate metabolites in frog muscle showed that ATP level remains unchanged until phosphocreatine is nearly depleted. Comparative studies revealed that under anaerobic conditions the Northern frog maintains its ATP content for 7 hours, while other types of amphibian, bird, and mammalian muscles begin to show an appreciable decay in ATP after 2 hours. Several lines of evidence indicated that ATP forms a complex with magnesium in the muscle water: (a) the phosphate resonances of ATP in the muscle were shifted downfield as compared to those in the alkaline earth metal-free perchloric acid extract of the muscle; (b) the coupling constants of ATP measured in various live muscles closely corresponded to those for MgATP in a solution resembling the composition of the muscle water; (c) in the muscle the gamma-phosphate group of ATP exhibited no shift change over a period of 10 hours under conditions where resonances of other phosphate compounds could be titrated. This behavior is similar to that of MgATP in model solutions in the physiological pH range, and it is different from that of CaATP. The chemical shifts of the phosphate metabolites were determined in several relevant solutions as a function of pH. Under all conditions only inorganic orthophosphate showed an invariant titration curve. From the chemical shift of inorganic phosphate observed during aging of intact muscle the intracellular pH of frog muscle was estimated to be 7.2. | [
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PMID:4453 | 5' leads to 3'-Exonucleases of bacteriophage T4. | Two enzyme activities which release nucleotides preferentially from the 5' termini of DNA were found in T4-infected Escherichia coli. Since no corresponding activities were found in uninfected cells, the activities appeared to be induced by T4. Both activities are capable of excising pyrimidine dimers from ultraviolet-irradiated DNA which has been treated with T4 endonuclease V. One of the activities , referred to as T4 exonuclease B, was purified 400-fold from an extract of T4v 1- infected cells. The enzyme initiates hydrolysis of DNA specifically at the 5' termini to yield products which are mainly oligonucleotides of varying length. The hydrolysis reaction proceeds in a limited manner. The enzyme shows optimal activity at pH 7.0 and absolutely requires Mg2+. The molecular weight of the enzyme , as estimated by gel filtration, is approximately 35,000. Another activity, referred to as T4 exonuclease C, was purified 240-fold from the extract. This activity also excises pyrimidine dimers from ultraviolet-irradiated, incised DNA and releases nucleotides at 5' termini. It has a pH optimum at 7.5 and requires Mg2+. The molecular weight of the enzyme is approximately 20,000. | 5' leads to 3'-Exonucleases of bacteriophage T4. Two enzyme activities which release nucleotides preferentially from the 5' termini of DNA were found in T4-infected Escherichia coli. Since no corresponding activities were found in uninfected cells, the activities appeared to be induced by T4. Both activities are capable of excising pyrimidine dimers from ultraviolet-irradiated DNA which has been treated with T4 endonuclease V. One of the activities , referred to as T4 exonuclease B, was purified 400-fold from an extract of T4v 1- infected cells. The enzyme initiates hydrolysis of DNA specifically at the 5' termini to yield products which are mainly oligonucleotides of varying length. The hydrolysis reaction proceeds in a limited manner. The enzyme shows optimal activity at pH 7.0 and absolutely requires Mg2+. The molecular weight of the enzyme , as estimated by gel filtration, is approximately 35,000. Another activity, referred to as T4 exonuclease C, was purified 240-fold from the extract. This activity also excises pyrimidine dimers from ultraviolet-irradiated, incised DNA and releases nucleotides at 5' termini. It has a pH optimum at 7.5 and requires Mg2+. The molecular weight of the enzyme is approximately 20,000. | [
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PMID:4454 | Nuclear magnetic resonance titration curves of histidine ring protons. A direct assignment of the resonances of the active site histidine residues of ribonuclease. | One of the four titrating histidine ring C-2 proton resonances of bovine pancreatic ribonuclease has been assigned to histidine residue 12. This was accomplished by a direct comparison of the rate of tritium incorporation into position C-2 of histidine 12 of S-peptide (residues 1 to 20) derived from ribonuclease S, with the rates of deuterium exchange of the four histidine C-2 proton resonances of ribonuclease S under the same experimental conditions. The same assignment was obtained by a comparison of the NMR titration curves of ribonuclease S, the noncovalent complex of S-peptide and S-protein (residues 21 to 124) with the results for the recombined complex in which position C-2 of histidine 12 was fully deuterated. The second active site histidine resonance was assigned to histidine residue 119 by consideration of the NMR titration results fro carboxymethylated histidines and 1-carboxymethylhistidine 119 ribonuclease. This assignment is a reversal of that originally reported, and has important implications for the interpretation of NMR titration data of ribonuclease. | Nuclear magnetic resonance titration curves of histidine ring protons. A direct assignment of the resonances of the active site histidine residues of ribonuclease. One of the four titrating histidine ring C-2 proton resonances of bovine pancreatic ribonuclease has been assigned to histidine residue 12. This was accomplished by a direct comparison of the rate of tritium incorporation into position C-2 of histidine 12 of S-peptide (residues 1 to 20) derived from ribonuclease S, with the rates of deuterium exchange of the four histidine C-2 proton resonances of ribonuclease S under the same experimental conditions. The same assignment was obtained by a comparison of the NMR titration curves of ribonuclease S, the noncovalent complex of S-peptide and S-protein (residues 21 to 124) with the results for the recombined complex in which position C-2 of histidine 12 was fully deuterated. The second active site histidine resonance was assigned to histidine residue 119 by consideration of the NMR titration results fro carboxymethylated histidines and 1-carboxymethylhistidine 119 ribonuclease. This assignment is a reversal of that originally reported, and has important implications for the interpretation of NMR titration data of ribonuclease. | [
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PMID:4455 | Nuclear magnetic resonance titration curves of histidine ring protons. Ribonuclease S-peptide and S-proteins. | The histidine C-2 proton NMR titration curves of ribonuclease S-peptide (residues 1 to 20) and S-protein (residues 21 to 124) are reported. Although S-protein contains 3 histidine residues, four discrete resonances are observed to titrate. One of these arises from the equivalent histidine residues of unfolded S-protein. The variation in area of the four resonances indicate that there is a reversible pH-dependent equilibrium between the folded and unfolded forms of S-protein, with some unfolded material being present at most pH values. Two of the resonances of the folded S-protein can be assigned to 2 of the histidine residues, 48 and 105, from the close similarity of their titration curves to those in ribonuclease. These similarities indicate a homology of portions of the folded conformation of S-protein to that of ribonuclease in solution. These results indicate that the complete amino acid sequence is not required to produce a folded conformation similar to the native globular protein, and they appear to eliminate the possibility that proteins fold from their NH2 terminus during protein synthesis. The low pH inflection present in the titration curve assigned to histidine residue 48 in ribonuclease is absent from this curve in S-protein. This is consistent with our previous conclusion that this inflection arises from the interaction of histidine 48 with aspartic acid residue 14, which is also absent in S-protein. The third titrating resonance of native S-protein is assigned to the remaining histidine residue at position 119. The properties of this resonance are not identical with either of the titration curves of the active site histidine residues 12 and 119 of ribonuclease. The resonance assigned to histidine 119 is the only one significantly affected on the addition of sodium phosphate to S-protein, indicating that some degree of phosphate binding occurs. In both the absence and presence of phosphate this curve also lacks the low pH inflection observed in the histidine 119 NMR titration curve in ribonuclease. This difference presumably arise from a conformational between ribonuclease and the folded S-protein involving a carboxyl group. | Nuclear magnetic resonance titration curves of histidine ring protons. Ribonuclease S-peptide and S-proteins. The histidine C-2 proton NMR titration curves of ribonuclease S-peptide (residues 1 to 20) and S-protein (residues 21 to 124) are reported. Although S-protein contains 3 histidine residues, four discrete resonances are observed to titrate. One of these arises from the equivalent histidine residues of unfolded S-protein. The variation in area of the four resonances indicate that there is a reversible pH-dependent equilibrium between the folded and unfolded forms of S-protein, with some unfolded material being present at most pH values. Two of the resonances of the folded S-protein can be assigned to 2 of the histidine residues, 48 and 105, from the close similarity of their titration curves to those in ribonuclease. These similarities indicate a homology of portions of the folded conformation of S-protein to that of ribonuclease in solution. These results indicate that the complete amino acid sequence is not required to produce a folded conformation similar to the native globular protein, and they appear to eliminate the possibility that proteins fold from their NH2 terminus during protein synthesis. The low pH inflection present in the titration curve assigned to histidine residue 48 in ribonuclease is absent from this curve in S-protein. This is consistent with our previous conclusion that this inflection arises from the interaction of histidine 48 with aspartic acid residue 14, which is also absent in S-protein. The third titrating resonance of native S-protein is assigned to the remaining histidine residue at position 119. The properties of this resonance are not identical with either of the titration curves of the active site histidine residues 12 and 119 of ribonuclease. The resonance assigned to histidine 119 is the only one significantly affected on the addition of sodium phosphate to S-protein, indicating that some degree of phosphate binding occurs. In both the absence and presence of phosphate this curve also lacks the low pH inflection observed in the histidine 119 NMR titration curve in ribonuclease. This difference presumably arise from a conformational between ribonuclease and the folded S-protein involving a carboxyl group. | [
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PMID:4456 | Effects of formylation of vinyl side chains of heme on optical and ligand binding properties of horse heart ferric myoglobin. | Effects of substitution of vinyl groups of hemin with formyl groups on the optical and ligand binding properties of horse heart ferric myoglobin were investigated. The peak positions as well as the line shapes of the absorption spectra of the ferric derivatives of three kinds of formylmyoglobin, 2-vinyl-4-formyl-, 2-formyl-4-vinyl-, and 2,4-diformylmyoglobins depend on the number and the position of the formyl groups. Absorption maxima in the Soret region of the acid forms of these ferric formylmyoglobins in 0.1 M potassium phosphate buffer, pH 6.0, at 20 degrees were 415.2, 422, and 429 nm, respectively. The acid forms of these formylmyoglobins exhibit absorption spectra of the mixture of high- and low spin states at ambient temperature. Since proto-, deutero- and mesomyoglobins have a high spin state under the same condition, the increase of the low spin iron in these formylmyoglobins may be due to the strong electron withdrawal by the formyl groups toward the periphery of the porphyrin ring. The affinities of these ferric formylmyoglobins and protomyoglobin for N3-, F-, OCN-, and SCN- increased in the order of proto-, monoformyl-monovinyl-, 2,4-diformyl-myoglobin, which corresponds to the increasing order of electron-withdrawing power of the porphyrin side chains. The pKa values of the acid-alkaline transition decreased in the same order. Although the ferric forms of the two isomeric monoformyl-monovinylmyoglobins exhibited different optical spectra, the dissociation constants of the complexes of these isomers for various ligands were similar to each other. The pKa values of the acid-alkaline transition were also similar. These results indicate that affinities of ferric myoglobin for ligands, in contrast to those of the ferrous form for oxygen and carbon monoxide (Sono, M., and Asakura, T. (1975) J. Biol. Chem. 250, 5527-5232 and Sono, M., Smith, P.D., McCray, J.A., and Asakura, T. (1976) J. Biol. Chem 251, 1418-1426), are not affected by the position of modifications at the two vinyl groups, but are determinedby the number of the formyl groups and that two vinyl groups at position 2 and 4 are equivalent in the binding of various ligands by ferric myoglobin. The electron density of the ferric iron appears to be similar for the two isomeric monoformyl-monovinylmyoglobins. | Effects of formylation of vinyl side chains of heme on optical and ligand binding properties of horse heart ferric myoglobin. Effects of substitution of vinyl groups of hemin with formyl groups on the optical and ligand binding properties of horse heart ferric myoglobin were investigated. The peak positions as well as the line shapes of the absorption spectra of the ferric derivatives of three kinds of formylmyoglobin, 2-vinyl-4-formyl-, 2-formyl-4-vinyl-, and 2,4-diformylmyoglobins depend on the number and the position of the formyl groups. Absorption maxima in the Soret region of the acid forms of these ferric formylmyoglobins in 0.1 M potassium phosphate buffer, pH 6.0, at 20 degrees were 415.2, 422, and 429 nm, respectively. The acid forms of these formylmyoglobins exhibit absorption spectra of the mixture of high- and low spin states at ambient temperature. Since proto-, deutero- and mesomyoglobins have a high spin state under the same condition, the increase of the low spin iron in these formylmyoglobins may be due to the strong electron withdrawal by the formyl groups toward the periphery of the porphyrin ring. The affinities of these ferric formylmyoglobins and protomyoglobin for N3-, F-, OCN-, and SCN- increased in the order of proto-, monoformyl-monovinyl-, 2,4-diformyl-myoglobin, which corresponds to the increasing order of electron-withdrawing power of the porphyrin side chains. The pKa values of the acid-alkaline transition decreased in the same order. Although the ferric forms of the two isomeric monoformyl-monovinylmyoglobins exhibited different optical spectra, the dissociation constants of the complexes of these isomers for various ligands were similar to each other. The pKa values of the acid-alkaline transition were also similar. These results indicate that affinities of ferric myoglobin for ligands, in contrast to those of the ferrous form for oxygen and carbon monoxide (Sono, M., and Asakura, T. (1975) J. Biol. Chem. 250, 5527-5232 and Sono, M., Smith, P.D., McCray, J.A., and Asakura, T. (1976) J. Biol. Chem 251, 1418-1426), are not affected by the position of modifications at the two vinyl groups, but are determinedby the number of the formyl groups and that two vinyl groups at position 2 and 4 are equivalent in the binding of various ligands by ferric myoglobin. The electron density of the ferric iron appears to be similar for the two isomeric monoformyl-monovinylmyoglobins. | [
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PMID:4457 | An essential residue at the active site of aspartate transcarbamylase. | Reaction of phenylglyoxal with aspartate transcarbamylase and its isolated catalytic subunit results in complete loss of enzymatic activity. This modification reaction is markedly influenced by pH and is partially reversible upon dialysis. Carbamyl phosphate or carbamyl phosphate with succinate partially protect the catalytic subunit and the native enzyme from inactivation by phenylglyoxal. In the native enzyme complete protection from inactivation is afforded by N-(phosphonacetyl)-L-aspartate. The decrease in enzymatic activity correlates with the modification of 6 arginine residues on each aspartate transcarbamylase molecule, i.e. 1 arginine per catalytic site. The data suggest that the essential arginine is involved in the binding of carbamyl phosphate to the enzyme. Reaction of the single thiol on the catalytic chain with 2-chloromercuri-4-nitrophenol does not prevent subsequent reaction with phenylglyoxal. If N-(phosphonacetyl)-L-aspartate is used to protect the active site we find that phenylglyoxal also causes the loss of activation of ATP and inhibition by CTP. The rate of loss of heterotropic effects is exactly the same for both nucleotides indicating that the two opposite regulatory effects originate at the same location on the enzyme, or are transmitted by the same mechanism between the subunits, or both. | An essential residue at the active site of aspartate transcarbamylase. Reaction of phenylglyoxal with aspartate transcarbamylase and its isolated catalytic subunit results in complete loss of enzymatic activity. This modification reaction is markedly influenced by pH and is partially reversible upon dialysis. Carbamyl phosphate or carbamyl phosphate with succinate partially protect the catalytic subunit and the native enzyme from inactivation by phenylglyoxal. In the native enzyme complete protection from inactivation is afforded by N-(phosphonacetyl)-L-aspartate. The decrease in enzymatic activity correlates with the modification of 6 arginine residues on each aspartate transcarbamylase molecule, i.e. 1 arginine per catalytic site. The data suggest that the essential arginine is involved in the binding of carbamyl phosphate to the enzyme. Reaction of the single thiol on the catalytic chain with 2-chloromercuri-4-nitrophenol does not prevent subsequent reaction with phenylglyoxal. If N-(phosphonacetyl)-L-aspartate is used to protect the active site we find that phenylglyoxal also causes the loss of activation of ATP and inhibition by CTP. The rate of loss of heterotropic effects is exactly the same for both nucleotides indicating that the two opposite regulatory effects originate at the same location on the enzyme, or are transmitted by the same mechanism between the subunits, or both. | [
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PMID:4458 | Physical and kinetic properties of homogenous bovine lens aldose reductase. | Aldose reductase from calf lens was purified 15,000-fold. The homogeneity of the final preparation was demonstrated by molecular sieve chromatography, analytical ultracentrifugation, sodium dodecyl sulfate gel electrophoresis, Ouchterlony immunodiffusion, and polyacrylamide gel electrophoresis at three pH values. The monomeric nature of the enzyme is suggested by the molecular weight of 37,000 from both molecular sieve chromatography and sodium dodecyl sulfate-gel electrophoresis with beta-mercaptoethanol. This closely corresponds with a molecular weight of 40,400 estimated by using calculate physical constants in the Svedberg equation. The S20,w was 3.6 to 3.7 as determined from ultracentrifuge and sucrose density gradient data. The Stokes radius was found to be 2.5 +/- 0.2 nm and 2.75 +/- 0.15 nm by two different methods. The diffusion constant D20,w is (7.8 +/- 10(-7) +/- 0.45 X 10(-7) cm2/s). The molecule is nearly spherical as indicated by a frictional ratio f/fo = 1.14. The alpha-helical content was estimated from circular dichroism data to be 5% and did not change in the presence of added substrates, products, and some enzyme inhibitors. Homotropic cooperative effects were observed as shown by the concave downward curvature of the reciprocal plots. | Physical and kinetic properties of homogenous bovine lens aldose reductase. Aldose reductase from calf lens was purified 15,000-fold. The homogeneity of the final preparation was demonstrated by molecular sieve chromatography, analytical ultracentrifugation, sodium dodecyl sulfate gel electrophoresis, Ouchterlony immunodiffusion, and polyacrylamide gel electrophoresis at three pH values. The monomeric nature of the enzyme is suggested by the molecular weight of 37,000 from both molecular sieve chromatography and sodium dodecyl sulfate-gel electrophoresis with beta-mercaptoethanol. This closely corresponds with a molecular weight of 40,400 estimated by using calculate physical constants in the Svedberg equation. The S20,w was 3.6 to 3.7 as determined from ultracentrifuge and sucrose density gradient data. The Stokes radius was found to be 2.5 +/- 0.2 nm and 2.75 +/- 0.15 nm by two different methods. The diffusion constant D20,w is (7.8 +/- 10(-7) +/- 0.45 X 10(-7) cm2/s). The molecule is nearly spherical as indicated by a frictional ratio f/fo = 1.14. The alpha-helical content was estimated from circular dichroism data to be 5% and did not change in the presence of added substrates, products, and some enzyme inhibitors. Homotropic cooperative effects were observed as shown by the concave downward curvature of the reciprocal plots. | [
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PMID:4459 | L-Asparaginase of Klebsiella aerogenes. Activation of its synthesis by glutamine synthetase. | An L-asparaginase has been purified some 250-fold from extracts of Klebsiella aerogenes to near homogeneity. The enzyme has a molecular weight of 141,000 as measured by gel filtration and appears to consist of four subunits of molecular weight 37,000. The enzyme has high affinity for L-asparagine, with a Km below 10(-5) M, and hydrolyzes glutamine at a 20-fold lower rate, with a Km of 10(-3) M. Interestingly, the enzyme exhibits marked gamma-glutamyltransferase activity but comparatively little beta-aspartyl-transferase activity. A mutant strain lacking this asparaginase has been isolated and grows at 1/2 to 1/3 the rate of the parent strain when asparagine is provided in the medium as the sole source of nitrogen. This strain grows as well as the wild type when the medium is supplemented with histidine or ammonia. Glutamine synthetase activates the formation of L-asparaginase. Mutants lacking glutamine synthetase fail to produce the asparaginase, and mutants with a high constitutive level of glutamine synthetase also contain the asparaginase at a high level. Thus, the formation of asparaginase is regulated in parallel with that of other enzymes capable of supplying the cell with ammonia or glutamate, such as histidase and proline oxidase. Formation of the asparaginase does not require induction by asparaginase and is not subject to catabolite repression. | L-Asparaginase of Klebsiella aerogenes. Activation of its synthesis by glutamine synthetase. An L-asparaginase has been purified some 250-fold from extracts of Klebsiella aerogenes to near homogeneity. The enzyme has a molecular weight of 141,000 as measured by gel filtration and appears to consist of four subunits of molecular weight 37,000. The enzyme has high affinity for L-asparagine, with a Km below 10(-5) M, and hydrolyzes glutamine at a 20-fold lower rate, with a Km of 10(-3) M. Interestingly, the enzyme exhibits marked gamma-glutamyltransferase activity but comparatively little beta-aspartyl-transferase activity. A mutant strain lacking this asparaginase has been isolated and grows at 1/2 to 1/3 the rate of the parent strain when asparagine is provided in the medium as the sole source of nitrogen. This strain grows as well as the wild type when the medium is supplemented with histidine or ammonia. Glutamine synthetase activates the formation of L-asparaginase. Mutants lacking glutamine synthetase fail to produce the asparaginase, and mutants with a high constitutive level of glutamine synthetase also contain the asparaginase at a high level. Thus, the formation of asparaginase is regulated in parallel with that of other enzymes capable of supplying the cell with ammonia or glutamate, such as histidase and proline oxidase. Formation of the asparaginase does not require induction by asparaginase and is not subject to catabolite repression. | [
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PMID:4460 | Binding and degradation of insulin by human peripheral granulocytes. Demonstration of specific receptors with high affinity. | The interaction of insulin with human circulating granulocytes was studied with the use of 125I-insulin. Human granulocytes, isolated from blood by the Böyum technique, showed high insulin-degrading activity in vitro which almost obscured the presence of specific, high affinity binding sites. Degradation, measured by trichloroacetic acid precipitation and by binding to well characterized insulin receptors on cultured human lymphocytes (IM-9 line), was due to extracellular as well as cell-bound enzymes. Degradation was enhanced by Ca2+ and thiols and inhibited by various protease inhibitors and sulfhydryl-blocking reagents. Phenylmethylsulfonyl fluoride (5 X 10(-4) M), a serine protease inhibitor, was the most potent and inhibited 125I-insulin degradation by 80 to 90%. Tert-butyl hydroperoxide (2 X 10(-3) M), a glutathione-oxidizing reagent, inhibited degradation by 35 to 50%, possibly due to an effect on a glutathione-insulin transhydrogenase. Neither of the inhibitors affected cell viability. In the presence of inhibitors of degradation, binding sites for insulin with high affinity were detected, which by multiple criteria were true insulin receptors. Binding to these sites was rapid, saturable, and reversible with about 1000 sites/cell. The Hill coefficient for binding was 0.7, and the Scatchard plot of B/F versus B was curvilinear, due to site-site interactions of the negative cooperative type; the latter were demonstrated directly by kinetic studies. As shown previously for all other insulin receptors, binding was highly pH-dependent, and insulin analogues had affinities for these sites that closely correlated with their biological potencies. | Binding and degradation of insulin by human peripheral granulocytes. Demonstration of specific receptors with high affinity. The interaction of insulin with human circulating granulocytes was studied with the use of 125I-insulin. Human granulocytes, isolated from blood by the Böyum technique, showed high insulin-degrading activity in vitro which almost obscured the presence of specific, high affinity binding sites. Degradation, measured by trichloroacetic acid precipitation and by binding to well characterized insulin receptors on cultured human lymphocytes (IM-9 line), was due to extracellular as well as cell-bound enzymes. Degradation was enhanced by Ca2+ and thiols and inhibited by various protease inhibitors and sulfhydryl-blocking reagents. Phenylmethylsulfonyl fluoride (5 X 10(-4) M), a serine protease inhibitor, was the most potent and inhibited 125I-insulin degradation by 80 to 90%. Tert-butyl hydroperoxide (2 X 10(-3) M), a glutathione-oxidizing reagent, inhibited degradation by 35 to 50%, possibly due to an effect on a glutathione-insulin transhydrogenase. Neither of the inhibitors affected cell viability. In the presence of inhibitors of degradation, binding sites for insulin with high affinity were detected, which by multiple criteria were true insulin receptors. Binding to these sites was rapid, saturable, and reversible with about 1000 sites/cell. The Hill coefficient for binding was 0.7, and the Scatchard plot of B/F versus B was curvilinear, due to site-site interactions of the negative cooperative type; the latter were demonstrated directly by kinetic studies. As shown previously for all other insulin receptors, binding was highly pH-dependent, and insulin analogues had affinities for these sites that closely correlated with their biological potencies. | [
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PMID:4461 | Triiodothyronine effects on some electrogenic properties of frog sartorius. | At 21 degrees C in vitro, 0.2 and 2.0 muM of triiodothyronine (T3) produced an increase in resting membrane potential (RMP) of Rana pipens sartorius when the pH of the external solution was 7.4. The RMP was increased by 2.0 muM T3 in the presence of 10(-4) and 10(-3) M ouabain but not in 10(-3) M of 2,4 dinitrophenol. Small increases in RMP were observed with 2.0 muM T3 in solutions with low external Na. At pH 7.1 0.2 muM T3 produced a small transient increase in RMP. Membrane resistance (Rm) was found to decline gradually during exposure to 0.2 muM at a pH of 7.4. Treatment with 2.0 muM T3 at pH 7.4 was accompanied by a transient reduction in Rm. Similar transient changes in Rm were produced by 0.2 and 2.0 muM T3 at pH of 7.1 T3 reduced membrane resistance in isotonic K2SO4 and tris-buffered Mn (20 mM) solutions indicating that T3 increases potassium permeability. Direct action potentials were studied at pH 7.1. Overshoot, amplitude and rate of rise of the action potential underwent a gradual decrease in the presence of 0.2 muM T3 while thresholds remained unchanged. Thresholds were increased during exposure to 2.0 muM T3 whereas overshoot, amplitude and rate of rise underwent transient decreases followed by a return toward control levels. | Triiodothyronine effects on some electrogenic properties of frog sartorius. At 21 degrees C in vitro, 0.2 and 2.0 muM of triiodothyronine (T3) produced an increase in resting membrane potential (RMP) of Rana pipens sartorius when the pH of the external solution was 7.4. The RMP was increased by 2.0 muM T3 in the presence of 10(-4) and 10(-3) M ouabain but not in 10(-3) M of 2,4 dinitrophenol. Small increases in RMP were observed with 2.0 muM T3 in solutions with low external Na. At pH 7.1 0.2 muM T3 produced a small transient increase in RMP. Membrane resistance (Rm) was found to decline gradually during exposure to 0.2 muM at a pH of 7.4. Treatment with 2.0 muM T3 at pH 7.4 was accompanied by a transient reduction in Rm. Similar transient changes in Rm were produced by 0.2 and 2.0 muM T3 at pH of 7.1 T3 reduced membrane resistance in isotonic K2SO4 and tris-buffered Mn (20 mM) solutions indicating that T3 increases potassium permeability. Direct action potentials were studied at pH 7.1. Overshoot, amplitude and rate of rise of the action potential underwent a gradual decrease in the presence of 0.2 muM T3 while thresholds remained unchanged. Thresholds were increased during exposure to 2.0 muM T3 whereas overshoot, amplitude and rate of rise underwent transient decreases followed by a return toward control levels. | [
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PMID:4462 | [Acute amebic abscess of the liver followed by colonic ulcers. Report of 2 cases]. | Two unusual cases of ruptured acute amebic liver abcess are presented one with inferior vena caval compression. Surgical drainage was followed by the development of colonic complications (haemorrhage and perforation) although the patients were under specific therapy. After a new surgical operation both cases were cured. The relation with amebiasis is discussed. The possibility of such an association must always be kept in mind. The value of immunofluorescence test in the diagnosis is emphasised. | [Acute amebic abscess of the liver followed by colonic ulcers. Report of 2 cases]. Two unusual cases of ruptured acute amebic liver abcess are presented one with inferior vena caval compression. Surgical drainage was followed by the development of colonic complications (haemorrhage and perforation) although the patients were under specific therapy. After a new surgical operation both cases were cured. The relation with amebiasis is discussed. The possibility of such an association must always be kept in mind. The value of immunofluorescence test in the diagnosis is emphasised. | [
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PMID:4463 | High-performance ion-pair partition chromatography of sulfa drugs. Study and optimization of chemical parameters. | High-performance ion-pair partition chromatography is shown to be a versatile, efficient method for separating sulfonamides. For a group of fourteen sulfa drugs varying widely in pKA and hydrophobicity, the effect of mobile phase composition, counterion composition, pH, and ionic strength on their ion-pair partition chromatographic separation using tetrabutylammonium as the counterion and n-butanol-n-heptane as the mobile phase is shown. Wide variation in k' and a is possible by changing these parameters. Silica columns coated with buffered aqueous solutions of tetrabutylammonium sulfate resulted in efficiencies of 4000-6000 theoretical plates per 25 cm. These columns are stable for long periods of time, and can be stripped and re-used in the adsorption mode with little or no loss in efficiency. Several chromatograms are presented in order to illustrate the performance of ion-pair partition chromatography. | High-performance ion-pair partition chromatography of sulfa drugs. Study and optimization of chemical parameters. High-performance ion-pair partition chromatography is shown to be a versatile, efficient method for separating sulfonamides. For a group of fourteen sulfa drugs varying widely in pKA and hydrophobicity, the effect of mobile phase composition, counterion composition, pH, and ionic strength on their ion-pair partition chromatographic separation using tetrabutylammonium as the counterion and n-butanol-n-heptane as the mobile phase is shown. Wide variation in k' and a is possible by changing these parameters. Silica columns coated with buffered aqueous solutions of tetrabutylammonium sulfate resulted in efficiencies of 4000-6000 theoretical plates per 25 cm. These columns are stable for long periods of time, and can be stripped and re-used in the adsorption mode with little or no loss in efficiency. Several chromatograms are presented in order to illustrate the performance of ion-pair partition chromatography. | [
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PMID:4464 | Gas chromatographic determination of carboxylic acid chlorides and residual carboxylic acid precursors used in the production of some penicillins. | An improved gas chromatographic method is described for the simultaneous determination of carboxylic acid chlorides and related carboxylic acids used in the production of some commercial semisynthetic penicillins. The acid chloride reacts with diethylamine to form the corresponding diethylamide. Carboxylic acid impurities are converted to trimethylsilyl esters. The two derivatives are separated and quantitated in the same chromatographic run. This method, an extension of the earlier procedure of Hishta and Bomstein (1), has been applied to the acid chlorides used to make oxacillin, cloxacillin, dicloxacillin, and methicillin (Figure 1); it shows promise of application to other acid chlorides. The determination is more selective than the usual titration methods, which do not differentiate among acids with similar pK's. Relative standard deviations of the acid chloride determination are 1.0-2.5%. Residual carboxylic acid can be repetitively determined within a range of 0.6% absolute. | Gas chromatographic determination of carboxylic acid chlorides and residual carboxylic acid precursors used in the production of some penicillins. An improved gas chromatographic method is described for the simultaneous determination of carboxylic acid chlorides and related carboxylic acids used in the production of some commercial semisynthetic penicillins. The acid chloride reacts with diethylamine to form the corresponding diethylamide. Carboxylic acid impurities are converted to trimethylsilyl esters. The two derivatives are separated and quantitated in the same chromatographic run. This method, an extension of the earlier procedure of Hishta and Bomstein (1), has been applied to the acid chlorides used to make oxacillin, cloxacillin, dicloxacillin, and methicillin (Figure 1); it shows promise of application to other acid chlorides. The determination is more selective than the usual titration methods, which do not differentiate among acids with similar pK's. Relative standard deviations of the acid chloride determination are 1.0-2.5%. Residual carboxylic acid can be repetitively determined within a range of 0.6% absolute. | [
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PMID:4465 | Demonstration and some properties of cytosol-binding proteins for thyroxine and triiodothyronine in human liver. | Cytosol-binding proteins for L-thyroxine (T4) and triiodo-L-thyronine (T3) were studied in human liver specimens obtained at autopsy from 5 male and 2 female subjects. The liver cytosol containing 131I-T4 or T3, together with or without added stable hormones, was fractionated by Pevikon thin-layer electrophoresis at pH 8.6, 8.0, and7.4. It was demonstrated in all the specimens that besides a small amount of serum T4-binding globulin, there existed three T4-binding proteins, termed hT4-1, hT4-2 and hT4-3, with the electrophoretic mobilities of alpha2- and beta-globulins, and two T3-binding proteins, termed hT3-1 and hT3-2, with the mobilities of gamma-globulin. Binding of hormones by the cytosol proteins was pH-dependent, and a preliminary dialysis had no effect on the hormone binding. The major band of T4, hT4-2, bound more than half the tracer T4, and possessed the maximal binding capacity of 110 mug/100 ml of 33% cytosol at pH 7.4. However, it showed no apparent affinity for T3, because the bound T4 could not be displaced with a T3 load of 600 mug/100 ml. The major band of T3, hT3-2, bound more than 70% of the tracer T3, and appeared to have a large capacity for the hormone although secondary binding sites on the same molecule might be responsible for the large capacity. The binding sites appeared almost specific for T3, because only a small, insignificant displacement was noted with a T4 load of 600 mug/100 ml. The results provide evidence for distinct binding proteins for T4 and T3 in the human liver cytosol, though their physiological roles remain to be elucidated. | Demonstration and some properties of cytosol-binding proteins for thyroxine and triiodothyronine in human liver. Cytosol-binding proteins for L-thyroxine (T4) and triiodo-L-thyronine (T3) were studied in human liver specimens obtained at autopsy from 5 male and 2 female subjects. The liver cytosol containing 131I-T4 or T3, together with or without added stable hormones, was fractionated by Pevikon thin-layer electrophoresis at pH 8.6, 8.0, and7.4. It was demonstrated in all the specimens that besides a small amount of serum T4-binding globulin, there existed three T4-binding proteins, termed hT4-1, hT4-2 and hT4-3, with the electrophoretic mobilities of alpha2- and beta-globulins, and two T3-binding proteins, termed hT3-1 and hT3-2, with the mobilities of gamma-globulin. Binding of hormones by the cytosol proteins was pH-dependent, and a preliminary dialysis had no effect on the hormone binding. The major band of T4, hT4-2, bound more than half the tracer T4, and possessed the maximal binding capacity of 110 mug/100 ml of 33% cytosol at pH 7.4. However, it showed no apparent affinity for T3, because the bound T4 could not be displaced with a T3 load of 600 mug/100 ml. The major band of T3, hT3-2, bound more than 70% of the tracer T3, and appeared to have a large capacity for the hormone although secondary binding sites on the same molecule might be responsible for the large capacity. The binding sites appeared almost specific for T3, because only a small, insignificant displacement was noted with a T4 load of 600 mug/100 ml. The results provide evidence for distinct binding proteins for T4 and T3 in the human liver cytosol, though their physiological roles remain to be elucidated. | [
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PMID:4466 | Glucagon-sensitive adenylate cyclase in human renal medulla. | In cell-free preparations (washed 600 x g pellets) of human renal medulla, glucagon produced a dose-dependent stimulation of adenylate cyclase. The stimulation of renal medullary adenylate cyclase by saturating concentrations of glucagon was additive to the saturating doses of vasopressin. Furthermore, L-isoproterenol stimulated renal medullary adenylate cyclase in a dose-dependent manner, and this stimulation was blocked by DL-propranolol. Stimulation of the renal medullary adenylate cyclase by maximal doses of glucagon and L-isoproterenol was additive. DL-Propranolol did not inhibit stimulation of glucagon. Thus, the results indicate the existence of a specific adenylate cyclase that is responsive to glucagon--distinct from the isoproterenol-sensitive adenylate cyclase and the previously described vasopressin-sensitive adenylate cyclase in human renal medulla. We suggest that the renal tubular effect of glucagon may be mediated by glucagon-dependent cyclic-AMP production in renal tissue. | Glucagon-sensitive adenylate cyclase in human renal medulla. In cell-free preparations (washed 600 x g pellets) of human renal medulla, glucagon produced a dose-dependent stimulation of adenylate cyclase. The stimulation of renal medullary adenylate cyclase by saturating concentrations of glucagon was additive to the saturating doses of vasopressin. Furthermore, L-isoproterenol stimulated renal medullary adenylate cyclase in a dose-dependent manner, and this stimulation was blocked by DL-propranolol. Stimulation of the renal medullary adenylate cyclase by maximal doses of glucagon and L-isoproterenol was additive. DL-Propranolol did not inhibit stimulation of glucagon. Thus, the results indicate the existence of a specific adenylate cyclase that is responsive to glucagon--distinct from the isoproterenol-sensitive adenylate cyclase and the previously described vasopressin-sensitive adenylate cyclase in human renal medulla. We suggest that the renal tubular effect of glucagon may be mediated by glucagon-dependent cyclic-AMP production in renal tissue. | [
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PMID:4467 | Simultaneous comparison of delta 5-3beta-hydroxysteroid levels in the fetoplacental circulation of normal pregnancy in labor and not in labor. | Concentrations of pregnenolone (delta5P), dehydroepiandrosterone (DHEA), 16alpha-hydroxydehydroepiandrosterone (16alpha-OH DHEA), pregnenolone sulfate (delta5P-S), and dehydroepiandrosterone sulfate (DHEA-S) were measured simultaneously by radioimmunoassay in individual, paired umbilical artery (UA) and vein (UV) sera from 18 normal term pregnancies, 6 in labor, 12 not in labor. Mean UA and UV levels +/- SEM (ng/ml) were for delta5P: 30.39 +/- 1.69, 35.55 +/- 3.06; DHEA: 12.31 +/- 2.34, 3.66 +/- 0.38; 16alpha-OH DHEA: 7.48 +/- 0.63, 10.59 +/- 0.78; delta5P-S: 1,652 +/- 154, 1,486 +/- 130; DHEA-S: 2,122 +/- 134, +/- 134, 1,906 +/- 134. Umbilical artery delta5P-S, DHEA-S, and DHEA levels were significantly higher than UV levels, whereas the reverse was true for delta5P and 16alpha-OH DHEA. The inverse arterio-venous (A-V) gradient for 16alpha-OH DHEA was contrary to previous published reports using pooled samples. Comparison by linear regression of paired UA and UV steroid concentrations of delta5P, delta5P-S, DHEA, and DHEA-S revealed a significant correlation (P less than 0.01) for each steroid. Labor was associated with a significant increase in UA levels of DHEA-S and a smaller, but not quite significant, increase in UA levels of delta5P-S, while similar changes for unconjugated delta5-3beta-hydroxysteroids were not observed. Mean A-V gradients between the group of patients in labor and those not in labor were not significantly different. These data demonstrate that: 1) a significant difference between UA and UV concentrations exists for delta5P, DHEA, 16alpha-OH DHEA, delta5P-S, and DHEA-S; 2) there is a significant correlation between UA and UV concentrations for delta5P, DHEA, delta5P-S, and DHEA-S, implying that each fetoplacental unit maintains an equilibrium relative to these steroid concentrations in the umbilical circulation; 3) labor is associated with a significant increase in UA levels of DHEA-S and probably of delta5P-S. | Simultaneous comparison of delta 5-3beta-hydroxysteroid levels in the fetoplacental circulation of normal pregnancy in labor and not in labor. Concentrations of pregnenolone (delta5P), dehydroepiandrosterone (DHEA), 16alpha-hydroxydehydroepiandrosterone (16alpha-OH DHEA), pregnenolone sulfate (delta5P-S), and dehydroepiandrosterone sulfate (DHEA-S) were measured simultaneously by radioimmunoassay in individual, paired umbilical artery (UA) and vein (UV) sera from 18 normal term pregnancies, 6 in labor, 12 not in labor. Mean UA and UV levels +/- SEM (ng/ml) were for delta5P: 30.39 +/- 1.69, 35.55 +/- 3.06; DHEA: 12.31 +/- 2.34, 3.66 +/- 0.38; 16alpha-OH DHEA: 7.48 +/- 0.63, 10.59 +/- 0.78; delta5P-S: 1,652 +/- 154, 1,486 +/- 130; DHEA-S: 2,122 +/- 134, +/- 134, 1,906 +/- 134. Umbilical artery delta5P-S, DHEA-S, and DHEA levels were significantly higher than UV levels, whereas the reverse was true for delta5P and 16alpha-OH DHEA. The inverse arterio-venous (A-V) gradient for 16alpha-OH DHEA was contrary to previous published reports using pooled samples. Comparison by linear regression of paired UA and UV steroid concentrations of delta5P, delta5P-S, DHEA, and DHEA-S revealed a significant correlation (P less than 0.01) for each steroid. Labor was associated with a significant increase in UA levels of DHEA-S and a smaller, but not quite significant, increase in UA levels of delta5P-S, while similar changes for unconjugated delta5-3beta-hydroxysteroids were not observed. Mean A-V gradients between the group of patients in labor and those not in labor were not significantly different. These data demonstrate that: 1) a significant difference between UA and UV concentrations exists for delta5P, DHEA, 16alpha-OH DHEA, delta5P-S, and DHEA-S; 2) there is a significant correlation between UA and UV concentrations for delta5P, DHEA, delta5P-S, and DHEA-S, implying that each fetoplacental unit maintains an equilibrium relative to these steroid concentrations in the umbilical circulation; 3) labor is associated with a significant increase in UA levels of DHEA-S and probably of delta5P-S. | [
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PMID:4468 | Isolation of human eosinophil phospholipase D. | Phospholipase D preferentially contained in human eosinophil polymorphonuclear leukocytes as compared to other leukocytes was isolated by sequential asion and cation exchange chromatography and gel filtration. The purified eosinophil enzyme specifically liberated choline from I-alpha-phosphatidyl choline with a pH optimum of 4.5-6.0 and exhibited a pI of 5.8-6.2 on polyacrylamide-gel isoelectric focusing, which are properties shared by phospholipase D from plant sources; however, its apparent mol wt of 60,000 is approximately one-half that of the plant enzymes. Eosinophil and cabbage phospholipase D inactivated a partially purified rat platelet-activating factor (PAF) in a time- and dose-dependent reaction. The cleavage of this PAF activity was attributed to the inherent phospholipase D activity of the eosinophil enzyme since the two activities chromatographed together at each purification step, and there was apparent reciprocal inhibition of choline-generating activity by PAF and of PAF-inactivating activity by phosphatidyl choline. Thus, possible regulatory functions of the eosinophil in immediate hypersensitivity reactions include inactivation of a PAF by phospholipase D as well as degradation of slow-reacting substance of anaphylaxis by arylsulfatase B. | Isolation of human eosinophil phospholipase D. Phospholipase D preferentially contained in human eosinophil polymorphonuclear leukocytes as compared to other leukocytes was isolated by sequential asion and cation exchange chromatography and gel filtration. The purified eosinophil enzyme specifically liberated choline from I-alpha-phosphatidyl choline with a pH optimum of 4.5-6.0 and exhibited a pI of 5.8-6.2 on polyacrylamide-gel isoelectric focusing, which are properties shared by phospholipase D from plant sources; however, its apparent mol wt of 60,000 is approximately one-half that of the plant enzymes. Eosinophil and cabbage phospholipase D inactivated a partially purified rat platelet-activating factor (PAF) in a time- and dose-dependent reaction. The cleavage of this PAF activity was attributed to the inherent phospholipase D activity of the eosinophil enzyme since the two activities chromatographed together at each purification step, and there was apparent reciprocal inhibition of choline-generating activity by PAF and of PAF-inactivating activity by phosphatidyl choline. Thus, possible regulatory functions of the eosinophil in immediate hypersensitivity reactions include inactivation of a PAF by phospholipase D as well as degradation of slow-reacting substance of anaphylaxis by arylsulfatase B. | [
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PMID:4469 | Role of expectancy set in the systematic desensitization of speech anxiety: an extension of prior research. | The influence of expectancy set with regard to therapy outcome on the effectiveness of systematic desensitization (SD) for reducing public speaking anxiety was investigated. The 7 Ss given a high expectancy set for favorable therapy outcome were informed about psychological research that indicates that SD is effective to reduce public speaking fears. SD was administered with the standard instructions to the 11 Ss given a neutral expectancy set. This expectancy manipulation did not require deception and perhaps could be used with actual SD therapy clients. As in previous research by Woy and Efran, the expectancy set manipulation significantly modified Ss' self-report of subjective perceptions of anxiety from pretreatment to posttreatment speeches, but did not affect overt behavioral or physiological indices of anxiety. Since subjective perceptions of anxiety responses are psychologically significant behaviors, these data suggest the importance of conveying a high expectation of improvement to SD and perhaps also to other types of therapy clients. SD sessions administered to small groups of clients on consecutive days, as in this study, appeared to be as effective to reduce speech anxiety as SD sessions administered to each client individually at 1-week intervals, as in the Woy and Efran study. | Role of expectancy set in the systematic desensitization of speech anxiety: an extension of prior research. The influence of expectancy set with regard to therapy outcome on the effectiveness of systematic desensitization (SD) for reducing public speaking anxiety was investigated. The 7 Ss given a high expectancy set for favorable therapy outcome were informed about psychological research that indicates that SD is effective to reduce public speaking fears. SD was administered with the standard instructions to the 11 Ss given a neutral expectancy set. This expectancy manipulation did not require deception and perhaps could be used with actual SD therapy clients. As in previous research by Woy and Efran, the expectancy set manipulation significantly modified Ss' self-report of subjective perceptions of anxiety from pretreatment to posttreatment speeches, but did not affect overt behavioral or physiological indices of anxiety. Since subjective perceptions of anxiety responses are psychologically significant behaviors, these data suggest the importance of conveying a high expectation of improvement to SD and perhaps also to other types of therapy clients. SD sessions administered to small groups of clients on consecutive days, as in this study, appeared to be as effective to reduce speech anxiety as SD sessions administered to each client individually at 1-week intervals, as in the Woy and Efran study. | [
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PMID:4470 | Graduated approach modeling in an aversive task. | Sixty female Ss were assigned randomly to six conditions that involved self-administration of electrical shock. These were: (1) no model-one trial S response; (2) no model-graduated trial S response; (3) one trial model-one trial S response; (4) one trial model-graduated S response; (5) graduated model-one trial response; and (6) graduated model-graduated response. Results indicated that graduated exposure of S to an aversive stimuli was more effective than a single such exposure and that type of exposure provided by a model had little effect. | Graduated approach modeling in an aversive task. Sixty female Ss were assigned randomly to six conditions that involved self-administration of electrical shock. These were: (1) no model-one trial S response; (2) no model-graduated trial S response; (3) one trial model-one trial S response; (4) one trial model-graduated S response; (5) graduated model-one trial response; and (6) graduated model-graduated response. Results indicated that graduated exposure of S to an aversive stimuli was more effective than a single such exposure and that type of exposure provided by a model had little effect. | [
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PMID:4471 | Cephradine excretion in humans with pH-altered urine. | In a random crossover study, ten healthy men were pretreated with ammonium chloride or sodium bicarbonate to alter urinary pH, and then were given a single 1-Gm dose of cephradine, either orally or intravenously. No significant changes were found in the terminal phase rate constant nor in the time or magnitude of peak concentration. Total recovery was greater than 90 per cent whenever degradation of cephradine did not occur. An unexplained but significant reduction in area under curve was present in data for prealkalinized subjects and for all subjects who took cephradine orally. Trapping of cephradine was not significant in the excretory pathway. | Cephradine excretion in humans with pH-altered urine. In a random crossover study, ten healthy men were pretreated with ammonium chloride or sodium bicarbonate to alter urinary pH, and then were given a single 1-Gm dose of cephradine, either orally or intravenously. No significant changes were found in the terminal phase rate constant nor in the time or magnitude of peak concentration. Total recovery was greater than 90 per cent whenever degradation of cephradine did not occur. An unexplained but significant reduction in area under curve was present in data for prealkalinized subjects and for all subjects who took cephradine orally. Trapping of cephradine was not significant in the excretory pathway. | [
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PMID:4472 | Partial agonist properties and toxicity of oral oxilorphan. | Alternative pharmacologic adjuncts are needed for the management of opiate abuse. Oxilorphan, a narcotic antagonist, was studied at 5 different dose levels (1, 2, 4, 6, and 8 mg) in 30 normal subjects to determine the relation of single oral doses and toxicity. The drug causes pupillary constriction and mild central nervous system side effects (nausea, dizziness) at all doses. Mean urine volume increased (P less than 0.05) during the 12 hours after 1 and 2 mg. Oxilorphan has partial agonist properties similar to dl-cyclazocine. | Partial agonist properties and toxicity of oral oxilorphan. Alternative pharmacologic adjuncts are needed for the management of opiate abuse. Oxilorphan, a narcotic antagonist, was studied at 5 different dose levels (1, 2, 4, 6, and 8 mg) in 30 normal subjects to determine the relation of single oral doses and toxicity. The drug causes pupillary constriction and mild central nervous system side effects (nausea, dizziness) at all doses. Mean urine volume increased (P less than 0.05) during the 12 hours after 1 and 2 mg. Oxilorphan has partial agonist properties similar to dl-cyclazocine. | [
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PMID:4474 | Characteristics of the release of adenosine 3':5'-monophosphate from micropipets by microiontophoresis. | The transfer number for radio-labelled cyclic AMP released from microiontophoretic pipets into brain pieces was determined for a large number of samples by radioassay. Release of cyclic AMP was linearly related to both iontophoretic current intensity and time as predicted by Faraday's Law. The results revealed that cyclic AMP has a rather low transfer number. In addition, an unusually large amount of variation of release, both within and among pipets was found under a variety of times and currents. The cause of the variation is not known but could be due to the unusual structure of the cyclic AMP molecule and the fact that it must be iontophoresed as a negative ion. These characteristics of cyclic AMP release may contribute to the difficulty in obtaining positive responses from appropriate neuronal target cells in vivo. | Characteristics of the release of adenosine 3':5'-monophosphate from micropipets by microiontophoresis. The transfer number for radio-labelled cyclic AMP released from microiontophoretic pipets into brain pieces was determined for a large number of samples by radioassay. Release of cyclic AMP was linearly related to both iontophoretic current intensity and time as predicted by Faraday's Law. The results revealed that cyclic AMP has a rather low transfer number. In addition, an unusually large amount of variation of release, both within and among pipets was found under a variety of times and currents. The cause of the variation is not known but could be due to the unusual structure of the cyclic AMP molecule and the fact that it must be iontophoresed as a negative ion. These characteristics of cyclic AMP release may contribute to the difficulty in obtaining positive responses from appropriate neuronal target cells in vivo. | [
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PMID:4475 | Effects of egg factors on cyclic nucleotide metabolism in sea urchin sperm. | Cyclic AMP in Strongylocentrotus purpuratus sperm was elevated approximately 2-fold by theophylline or 1-methyl-3-isobutylxanthine. Factors released from sea urchin eggs (FRE) elevated sperm cyclic AMP by about 7-fold within 1 min, and the combination of FRE with theophylline increased sperm cyclic AMP up to 100-fold within 1 min. Cyclic GMP in sea urchin sperm was slightly elevated by theophylline, but was lowered by FRE. Cyclic GMP in sperm treated with FRE plus theophylline was not higher than in sperm treated with theophylline alone. The ability of FRE-containing sea water to increase sperm cyclic AMP in the presence of theophylline was altered only slightly if at all by boiling, but it was decreased by about 50% by dialysis and destroyed by ashing. Filtration of FRE on Sephadex G-50 columns yielded two peaks of cyclic AMP-elevating activity. One peak (peak I) was eluted at the column void volume, and the other (peak II) was retained by the column. The cyclic GMP-lowering activity was located in fractions approximately corresponding to peak I of cyclic AMP-elevating activity. Dialysis of FRE-containing sea water before its application to the G-50 column virtually eliminated peak II of the cyclic AMP-elevating activity. When the cyclic AMP-elevating activity in peak I was filtered on Bio Gel A-5m columns, it also migrated at or near the column void volume. Fractions corresponding to peak I contained material that inhibited both guanylate and adenylate cyclase activities in broken cell preparations of sperm and guanylate cyclase from rat lung. The inhibitory material was stable to boiling, non-dialyzable, and destroyed by ashing. Under a variety of conditions, FRE-containing sea water or cyclic AMP-elevating peaks I or II did not stimulate sperm adenylate cyclase activity in broken cell preparations. | Effects of egg factors on cyclic nucleotide metabolism in sea urchin sperm. Cyclic AMP in Strongylocentrotus purpuratus sperm was elevated approximately 2-fold by theophylline or 1-methyl-3-isobutylxanthine. Factors released from sea urchin eggs (FRE) elevated sperm cyclic AMP by about 7-fold within 1 min, and the combination of FRE with theophylline increased sperm cyclic AMP up to 100-fold within 1 min. Cyclic GMP in sea urchin sperm was slightly elevated by theophylline, but was lowered by FRE. Cyclic GMP in sperm treated with FRE plus theophylline was not higher than in sperm treated with theophylline alone. The ability of FRE-containing sea water to increase sperm cyclic AMP in the presence of theophylline was altered only slightly if at all by boiling, but it was decreased by about 50% by dialysis and destroyed by ashing. Filtration of FRE on Sephadex G-50 columns yielded two peaks of cyclic AMP-elevating activity. One peak (peak I) was eluted at the column void volume, and the other (peak II) was retained by the column. The cyclic GMP-lowering activity was located in fractions approximately corresponding to peak I of cyclic AMP-elevating activity. Dialysis of FRE-containing sea water before its application to the G-50 column virtually eliminated peak II of the cyclic AMP-elevating activity. When the cyclic AMP-elevating activity in peak I was filtered on Bio Gel A-5m columns, it also migrated at or near the column void volume. Fractions corresponding to peak I contained material that inhibited both guanylate and adenylate cyclase activities in broken cell preparations of sperm and guanylate cyclase from rat lung. The inhibitory material was stable to boiling, non-dialyzable, and destroyed by ashing. Under a variety of conditions, FRE-containing sea water or cyclic AMP-elevating peaks I or II did not stimulate sperm adenylate cyclase activity in broken cell preparations. | [
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PMID:4476 | Dephosphorylation of bovine casein by milk alkaline phosphatase. | The pH of optimum activity of alkaline phosphatase from cow's milk depended on the substrate, being 10-1 for rho-nitrophenylphosphate, 8-6 for phosphoserine, 8-0 for phosvitin and 6-8 for casein. Individual casein components were dephosphorylated more rapidly than mixtures of alphas- and beta-caseins or of alphas-, beta-and kappa-caseins and micellar casein. Mixtures of 2 components involving kappa-casein were more readily dephosphorylated than alphas- and beta-casein mixtures. At pH 6-8, lactose, whey proteins and phosphate ions had an inhibitory effect. beta-Lactoglobulin had an inhibitory effect only when the pH of the reaction was lower than the optimum pH value of the enzyme. Mg2+ and Zn2+ were not inhibitory. The optimum conditions for dephosphorylation of casein are described. | Dephosphorylation of bovine casein by milk alkaline phosphatase. The pH of optimum activity of alkaline phosphatase from cow's milk depended on the substrate, being 10-1 for rho-nitrophenylphosphate, 8-6 for phosphoserine, 8-0 for phosvitin and 6-8 for casein. Individual casein components were dephosphorylated more rapidly than mixtures of alphas- and beta-caseins or of alphas-, beta-and kappa-caseins and micellar casein. Mixtures of 2 components involving kappa-casein were more readily dephosphorylated than alphas- and beta-casein mixtures. At pH 6-8, lactose, whey proteins and phosphate ions had an inhibitory effect. beta-Lactoglobulin had an inhibitory effect only when the pH of the reaction was lower than the optimum pH value of the enzyme. Mg2+ and Zn2+ were not inhibitory. The optimum conditions for dephosphorylation of casein are described. | [
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PMID:4473 | Effect of flurazepam on common clinical laboratory tests. | Twenty-two clinical laboratory tests performed on blood samples from 16 normal subjects following one week of either flurazepam or placebo administered in a double-blind method showed no apparent chemical interference by flurazepam on any of the testing procedures. | Effect of flurazepam on common clinical laboratory tests. Twenty-two clinical laboratory tests performed on blood samples from 16 normal subjects following one week of either flurazepam or placebo administered in a double-blind method showed no apparent chemical interference by flurazepam on any of the testing procedures. | [
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PMID:4477 | A kinetic concepto of lipid transport in ruminants. | Summarization of the literature shows a strong correlation between dietary fatty acid intake and total lipid concentration in plasma in lactating cows whereas total milk fat secreted is related to neither of these. In the process of plasma triglyceride removal, chylomicra and very low density lipoproteins are converted to low density lipoproteins. Limited kinetic data indicate that the fractional removal rates for chulomicra and very low density lipoproteins are rapid in lactating cows whereas fractional removal of low density lipoproteins is slower, resulting in accumulation of the latter in plasma. Under such conditions, low density lipoprotein concentrations of plasma would not be expected to reflect quantitatively the transfer of plasma triglyceride fatty acids to milk fat. Quantitative analysis or triglyceride fatty acid turnover in density less than 1.006 lipoproteins should delineate the role of plasma lipid transport in milk fat synthesis. High fat diets protected from rumen biohydrogenation have proven to be a useful approach in studying ruminant fat metabolism and may be used more extensively to elucidate the role of cholesterol in plasma lipid transport and the metabolism of essential fatty acids in ruminants. | A kinetic concepto of lipid transport in ruminants. Summarization of the literature shows a strong correlation between dietary fatty acid intake and total lipid concentration in plasma in lactating cows whereas total milk fat secreted is related to neither of these. In the process of plasma triglyceride removal, chylomicra and very low density lipoproteins are converted to low density lipoproteins. Limited kinetic data indicate that the fractional removal rates for chulomicra and very low density lipoproteins are rapid in lactating cows whereas fractional removal of low density lipoproteins is slower, resulting in accumulation of the latter in plasma. Under such conditions, low density lipoprotein concentrations of plasma would not be expected to reflect quantitatively the transfer of plasma triglyceride fatty acids to milk fat. Quantitative analysis or triglyceride fatty acid turnover in density less than 1.006 lipoproteins should delineate the role of plasma lipid transport in milk fat synthesis. High fat diets protected from rumen biohydrogenation have proven to be a useful approach in studying ruminant fat metabolism and may be used more extensively to elucidate the role of cholesterol in plasma lipid transport and the metabolism of essential fatty acids in ruminants. | [
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PMID:4478 | Bovine pancreatic lipase.I.Isolation, homogeneity, and characterization. | Bovine pancreatic lipase was isolated in pure form by lyophilization of fresh bovine pancreas, extraction of the enzyme with sucrose solution, fractional precipitation with ammonium sulfate and acetone, followed by chromatography on Sephadex G-100. The specific activity of the purest lipase fraction was 1750 micromoles fatty acid, liberated in 30 min per milligram of protein, indicating a purification of approximately 473-fold, with an overall yield of about 42%. Homogeneity of the enzyme was confirmed by rechromatography on Sephadex G-100 as well as with the gel electrophoretic and ultracentrifugal techniques. The purified enzyme gave a typical protein ultraviolet absorption spectrum with maximum absorption at 276 nm and minimum at 252 nm. The purified enzyme exhibited a single pH optimum of 8.8 and an isoelectric point near pH 5.5. Its optimum temperature was 37 C, and its optimum substrate concentration was 10%. These properties resembled those of milk lipase. | Bovine pancreatic lipase.I.Isolation, homogeneity, and characterization. Bovine pancreatic lipase was isolated in pure form by lyophilization of fresh bovine pancreas, extraction of the enzyme with sucrose solution, fractional precipitation with ammonium sulfate and acetone, followed by chromatography on Sephadex G-100. The specific activity of the purest lipase fraction was 1750 micromoles fatty acid, liberated in 30 min per milligram of protein, indicating a purification of approximately 473-fold, with an overall yield of about 42%. Homogeneity of the enzyme was confirmed by rechromatography on Sephadex G-100 as well as with the gel electrophoretic and ultracentrifugal techniques. The purified enzyme gave a typical protein ultraviolet absorption spectrum with maximum absorption at 276 nm and minimum at 252 nm. The purified enzyme exhibited a single pH optimum of 8.8 and an isoelectric point near pH 5.5. Its optimum temperature was 37 C, and its optimum substrate concentration was 10%. These properties resembled those of milk lipase. | [
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PMID:4479 | Distribution and removal of cadmium from milk. | Distribution of added cadmium in milk systems and the feasibility of removing cadmium were investigated. In milk containing 1 ppm cadmium, 96% of the added cadmium chloride was dispersed in the skim milk fraction, and 3% was associated with the cream fraction. Cadmium was not bound strongly to any protein fraction. The association of cadmium with acid casein, whey proteins, and the fat globule membrane was 18,6, and .5% of total added cadmium. With a 5-min exposure thiosuccinylated amino-ethyl cellulose, thionitrocarboxyphenylated aminoethyl cellulose, and reduced human hair removed 72, 70, and 30%, respectively, of added cadmium in skim milk previously equilibrated for 2 h at 37 C. Increasing equilibration time beyond 24 h had no effect on removal efficiency whereas increasing pH decreased cadmium removal markedly. | Distribution and removal of cadmium from milk. Distribution of added cadmium in milk systems and the feasibility of removing cadmium were investigated. In milk containing 1 ppm cadmium, 96% of the added cadmium chloride was dispersed in the skim milk fraction, and 3% was associated with the cream fraction. Cadmium was not bound strongly to any protein fraction. The association of cadmium with acid casein, whey proteins, and the fat globule membrane was 18,6, and .5% of total added cadmium. With a 5-min exposure thiosuccinylated amino-ethyl cellulose, thionitrocarboxyphenylated aminoethyl cellulose, and reduced human hair removed 72, 70, and 30%, respectively, of added cadmium in skim milk previously equilibrated for 2 h at 37 C. Increasing equilibration time beyond 24 h had no effect on removal efficiency whereas increasing pH decreased cadmium removal markedly. | [
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PMID:4480 | Influence of viable yogurt microflora on digestion of lactose by the rat. | Laboratory rats were fed experimental diets including yogurt, pasteurized yogurt, and simulated yogurt with sucrose or lactose for 7 days followed by a single experimental meal of yogurt, pasteurized yogurt, or simulated yogurt. Assays of blood galactose demonstrated that animals fed natural yogurt containing the viable culture microflora were able to absorb galactose more efficiently. Intestinal lactase activity of yogurt-fed animals was greater than in animals fed other experimental diets including pasteurized yogurt. Gastrointestinal survival of culture organisms was demonstrated in vivo up to 3 h after feeding, and thus, the viable cells resulted in more efficient hydrolysis which favored lactose digestion in natural yogurt. | Influence of viable yogurt microflora on digestion of lactose by the rat. Laboratory rats were fed experimental diets including yogurt, pasteurized yogurt, and simulated yogurt with sucrose or lactose for 7 days followed by a single experimental meal of yogurt, pasteurized yogurt, or simulated yogurt. Assays of blood galactose demonstrated that animals fed natural yogurt containing the viable culture microflora were able to absorb galactose more efficiently. Intestinal lactase activity of yogurt-fed animals was greater than in animals fed other experimental diets including pasteurized yogurt. Gastrointestinal survival of culture organisms was demonstrated in vivo up to 3 h after feeding, and thus, the viable cells resulted in more efficient hydrolysis which favored lactose digestion in natural yogurt. | [
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PMID:4481 | Nonimmunologic aspects of caries resistance. | A variety of components provide salivary secretions with an array of potentially effective means of combating cariogenic challenges. These defense factors range from a laissez-faire mechanical cleansing to exquisitely controlled production of highly specific antibodies. In between the two extremes are antibacterial systems whose operating characteristics are only beginning to be understood. These systems are well worth our attention. They may be the key to our understanding of variations in individual susceptibility, and could provide valuable leads for development of anticaries agents. | Nonimmunologic aspects of caries resistance. A variety of components provide salivary secretions with an array of potentially effective means of combating cariogenic challenges. These defense factors range from a laissez-faire mechanical cleansing to exquisitely controlled production of highly specific antibodies. In between the two extremes are antibacterial systems whose operating characteristics are only beginning to be understood. These systems are well worth our attention. They may be the key to our understanding of variations in individual susceptibility, and could provide valuable leads for development of anticaries agents. | [
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PMID:4482 | Adherence of serotype e Streptococcus mutans and the inhibitory effect of Lancefield group E and S mutans type e antiserum. | S mutans strain MT703 from an active carious lesion in the tooth of a child had type e specificity and showed a cross-reaction with the Lancefield group E cell wall streptococcal polysaccharide antigen. Heat-killed cells MT703 adhered to a glass surface in the presence of CGT MT703 and sucrose. Pretreatment of the cells with anti-MT703 whole cell serums inhibited adherecne. The removal of glycerol teichoic acid antibody and group E antibody from the MT703 serum did not result in a loss of inhibitory activity. Antiserum with or without adsorption significantly inhibited glucan synthesis by CGT from sucrose. Antibodies specific for the polyglycerol phosphate of teichoic acid did not inhibit adherence. Anti-group E serum and serums specific for other types of S mutans, did not show adherence inhibitory activity except for an occasional type c specific antiserum. Antibody specific for the type e antigen produced significant inhibition of the binding of CGT to the MT703 cell wall, and adherence of these cells did not occur. Antibody to CGT inhibited glucan synthesis. Treatment of the cells with dextranase, dextran antibody, or trypsin caused a significant reduction in adherence. The results suggest that the type antigen and dextran on the surface of the S mutans type e cell are functional in adherence, and that these polymers are associated with cell wall protein. | Adherence of serotype e Streptococcus mutans and the inhibitory effect of Lancefield group E and S mutans type e antiserum. S mutans strain MT703 from an active carious lesion in the tooth of a child had type e specificity and showed a cross-reaction with the Lancefield group E cell wall streptococcal polysaccharide antigen. Heat-killed cells MT703 adhered to a glass surface in the presence of CGT MT703 and sucrose. Pretreatment of the cells with anti-MT703 whole cell serums inhibited adherecne. The removal of glycerol teichoic acid antibody and group E antibody from the MT703 serum did not result in a loss of inhibitory activity. Antiserum with or without adsorption significantly inhibited glucan synthesis by CGT from sucrose. Antibodies specific for the polyglycerol phosphate of teichoic acid did not inhibit adherence. Anti-group E serum and serums specific for other types of S mutans, did not show adherence inhibitory activity except for an occasional type c specific antiserum. Antibody specific for the type e antigen produced significant inhibition of the binding of CGT to the MT703 cell wall, and adherence of these cells did not occur. Antibody to CGT inhibited glucan synthesis. Treatment of the cells with dextranase, dextran antibody, or trypsin caused a significant reduction in adherence. The results suggest that the type antigen and dextran on the surface of the S mutans type e cell are functional in adherence, and that these polymers are associated with cell wall protein. | [
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PMID:4483 | The effect of beta adrenergic blockade on bronchial sensitivity to acetyl-beta-methacholine in normal and allergic rhinitis subjects. | The effect of propranolol inhalation on sensitivity to methacholine inhalation was studied in normal and allergic rhinitis subjects to determine whether beta adrenergic blockade alters sensitivity to mediators in nonasthmatic atopic individuals. A partial beta adrenergic blockade is suggested as being instrumental in asthma. Hay fever patients studied showed similar effects and also developed asthma for the first time. | The effect of beta adrenergic blockade on bronchial sensitivity to acetyl-beta-methacholine in normal and allergic rhinitis subjects. The effect of propranolol inhalation on sensitivity to methacholine inhalation was studied in normal and allergic rhinitis subjects to determine whether beta adrenergic blockade alters sensitivity to mediators in nonasthmatic atopic individuals. A partial beta adrenergic blockade is suggested as being instrumental in asthma. Hay fever patients studied showed similar effects and also developed asthma for the first time. | [
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PMID:4484 | Effect of beta adrenergic stimulation and blockade on cutaneous reactivity to histamine. | It has been previously demonstrated that iontophoresis of beta adrenergic agents will alter the size of immediate hypersensitivity skin tests. It was unclear whether this alteration was due to an effect on the dermal mast cell (inhibition of histamine release) or on the cutaneous vasculature (inhibition of capillary permeability). For this reason isoproterenol, propranolol, diphenhydramine as a positive control, and saline as a negative control were iontophoresed onto the forearm of 10 atopic and 10 nonatopic adult subjects. In order to bypass histamine release from mast cells the patients were then challenged directly with histamine by the "prick" technique. The size of the resultant wheals was noted. The data obtained allowed the following conclusions: (1) The atopic group responded to histamine with greater wheal size than the nonatopic group. (2) Iontophoresis of diphyenhydramine effectively reduced the magnitude of the histamine wheal in both groups. (3) Isoproterenol decreased the wheal size in both groups. (4) Propranolol increased the wheal size in only the nonatopic group. (5) The successful modulation of the histamine-induced wheal and flare indicated that these drugs, regardless of their effect on the dermal mast cell, exert a measurable effect on the target organ (vasculature). | Effect of beta adrenergic stimulation and blockade on cutaneous reactivity to histamine. It has been previously demonstrated that iontophoresis of beta adrenergic agents will alter the size of immediate hypersensitivity skin tests. It was unclear whether this alteration was due to an effect on the dermal mast cell (inhibition of histamine release) or on the cutaneous vasculature (inhibition of capillary permeability). For this reason isoproterenol, propranolol, diphenhydramine as a positive control, and saline as a negative control were iontophoresed onto the forearm of 10 atopic and 10 nonatopic adult subjects. In order to bypass histamine release from mast cells the patients were then challenged directly with histamine by the "prick" technique. The size of the resultant wheals was noted. The data obtained allowed the following conclusions: (1) The atopic group responded to histamine with greater wheal size than the nonatopic group. (2) Iontophoresis of diphyenhydramine effectively reduced the magnitude of the histamine wheal in both groups. (3) Isoproterenol decreased the wheal size in both groups. (4) Propranolol increased the wheal size in only the nonatopic group. (5) The successful modulation of the histamine-induced wheal and flare indicated that these drugs, regardless of their effect on the dermal mast cell, exert a measurable effect on the target organ (vasculature). | [
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PMID:4573 | Further observations on the potentiation of the antibacterial effect of methenamine by acetohydroxamic acid. | The use of methenamine in the treatment of urinary tract infections due to Proteus species is limited by urine alkalinity. Acetohydroxamic acid, an inhibitor of urease, maintains acidity despite growth of Proteus in urine. Easily achievable concentrations of acetohydroxamic acid in vitro systems that simulated the dynamics of the urinary tract potentiated the antibacterial effect of methenamine against Proteus species. The combined use of a urease inhibitor and methenamine may be effective in the treatment of urinary infection caused by these organisms. | Further observations on the potentiation of the antibacterial effect of methenamine by acetohydroxamic acid. The use of methenamine in the treatment of urinary tract infections due to Proteus species is limited by urine alkalinity. Acetohydroxamic acid, an inhibitor of urease, maintains acidity despite growth of Proteus in urine. Easily achievable concentrations of acetohydroxamic acid in vitro systems that simulated the dynamics of the urinary tract potentiated the antibacterial effect of methenamine against Proteus species. The combined use of a urease inhibitor and methenamine may be effective in the treatment of urinary infection caused by these organisms. | [
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PMID:4575 | Hematopoietic thymocyte precursors. I. Assay and kinetics of the appearance of progeny. | A quantitative assay for the hematopoietic precursor of thymocytes has been developed. Using this assay the kinetics of appearance of the progeny of transfused bone marrow and spleen cells in the thymus of irradiated (760 R) mice has been studied. Precursor cells are seven to eightfold more common in bone marrow than in spleen and are absent from peripheral lymph nodes. They decline in number as the animals age. When hematopoietic cells are injected immediately after lethal irradiation only a small number of cells actually enter the gland. Their progeny are not detectable in the thymus for 8-12 days. The time of their detection depends both upon the size of the residual endogenous thymocyte population and the number of progenitor cells injected. Evidence has been presented that excludes thymic injury as the basis for the delay in the appearance of donor type cells and indicates that neither the production of a "homing" signal in the irradiated animal nor the development of precursor cells are limiting factors in the rate of thymic repopulation. These studies indicate that only an exceedingly small number (less than 100) of prothymocytes are required to repopulate the thymus of an irradiated mouse. This restricted number of progenitors must produce the entire repertory of T-cell immunologic responsiveness seen in the first weeks after repopulation. | Hematopoietic thymocyte precursors. I. Assay and kinetics of the appearance of progeny. A quantitative assay for the hematopoietic precursor of thymocytes has been developed. Using this assay the kinetics of appearance of the progeny of transfused bone marrow and spleen cells in the thymus of irradiated (760 R) mice has been studied. Precursor cells are seven to eightfold more common in bone marrow than in spleen and are absent from peripheral lymph nodes. They decline in number as the animals age. When hematopoietic cells are injected immediately after lethal irradiation only a small number of cells actually enter the gland. Their progeny are not detectable in the thymus for 8-12 days. The time of their detection depends both upon the size of the residual endogenous thymocyte population and the number of progenitor cells injected. Evidence has been presented that excludes thymic injury as the basis for the delay in the appearance of donor type cells and indicates that neither the production of a "homing" signal in the irradiated animal nor the development of precursor cells are limiting factors in the rate of thymic repopulation. These studies indicate that only an exceedingly small number (less than 100) of prothymocytes are required to repopulate the thymus of an irradiated mouse. This restricted number of progenitors must produce the entire repertory of T-cell immunologic responsiveness seen in the first weeks after repopulation. | [
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PMID:4576 | Frog lysozyme. I. Its identification, occurrence as isozymes, and quantitative distribution in tissues of the leopard frog, Rana pipiens. | In the course of examining the etiology of the Lucké renal adenocarcinoma of the frog, Rana pipiens, it was found that organs of the normal adult contain bacteriolytic enzymes. These enzymes all satisfied the six criteria for the identification of lysozymes and at least eight forms were separable by polyacrylamide gel electrophoresis. Their qualitative and quantitative distribution was organ-specific. All eight isozymes were found in normal kidney, while liver and spleen contained seven forms; skin, six; ovarian egg, five; and serum, two. In quantitative assays using a radial diffusion test, spleen had the greatest lysozyme concentration, followed in descending order by kidney, liver, skin, and ovary. Serum contained very low amounts. In terms of enzyme activity per animal, ovary was the highest ranking organ. As such a large number of lysozyme isozymes has not been reported in any other organism, their origins and functions are considered in the context of their presence in an ectotherm. | Frog lysozyme. I. Its identification, occurrence as isozymes, and quantitative distribution in tissues of the leopard frog, Rana pipiens. In the course of examining the etiology of the Lucké renal adenocarcinoma of the frog, Rana pipiens, it was found that organs of the normal adult contain bacteriolytic enzymes. These enzymes all satisfied the six criteria for the identification of lysozymes and at least eight forms were separable by polyacrylamide gel electrophoresis. Their qualitative and quantitative distribution was organ-specific. All eight isozymes were found in normal kidney, while liver and spleen contained seven forms; skin, six; ovarian egg, five; and serum, two. In quantitative assays using a radial diffusion test, spleen had the greatest lysozyme concentration, followed in descending order by kidney, liver, skin, and ovary. Serum contained very low amounts. In terms of enzyme activity per animal, ovary was the highest ranking organ. As such a large number of lysozyme isozymes has not been reported in any other organism, their origins and functions are considered in the context of their presence in an ectotherm. | [
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