id
stringlengths 14
28
| title
stringclasses 18
values | content
stringlengths 2
999
| contents
stringlengths 19
1.02k
|
|---|---|---|---|
Surgery_Schwartz_3302
|
Surgery_Schwartz
|
the resulting proteins can undergo selec-tive activation, inactivation, or compartmentalization (protein activity control).Because a large number of genes are regulated at the tran-scriptional level, regulation of gene transcripts (i.e., mRNA) often is referred to as gene regulation in a narrow definition. Each of the steps during transcription is properly regulated in eukaryotic cells. Because genes are differentially regulated from one another, one gene can be differentially regulated in differ-ent cell types or at different developmental stages. Therefore, gene regulation at the level of transcription is largely context dependent. However, there is a common scheme that applies to transcription at the molecular level (Fig. 15-6). Each gene promoter possesses unique sequences called TATA boxes that can be recognized and bound by a large complex containing RNA polymerase II, forming the basal transcription machinery. Usually located upstream of the TATA box (but sometimes lon-ger
|
Surgery_Schwartz. the resulting proteins can undergo selec-tive activation, inactivation, or compartmentalization (protein activity control).Because a large number of genes are regulated at the tran-scriptional level, regulation of gene transcripts (i.e., mRNA) often is referred to as gene regulation in a narrow definition. Each of the steps during transcription is properly regulated in eukaryotic cells. Because genes are differentially regulated from one another, one gene can be differentially regulated in differ-ent cell types or at different developmental stages. Therefore, gene regulation at the level of transcription is largely context dependent. However, there is a common scheme that applies to transcription at the molecular level (Fig. 15-6). Each gene promoter possesses unique sequences called TATA boxes that can be recognized and bound by a large complex containing RNA polymerase II, forming the basal transcription machinery. Usually located upstream of the TATA box (but sometimes lon-ger
|
Surgery_Schwartz_3303
|
Surgery_Schwartz
|
TATA boxes that can be recognized and bound by a large complex containing RNA polymerase II, forming the basal transcription machinery. Usually located upstream of the TATA box (but sometimes lon-ger distances) are a number of regulatory sequences referred to as enhancers that are recognized by regulatory proteins called transcription factors. These transcription factors specifically bind to the enhancers, often in response to environmental or developmental cues, and cooperate with each other and with basal transcription factors to initiate transcription. Regulatory sequences that negatively regulate the initiation of transcription also are present on the promoter DNA. The transcription factors that bind to these sites are called repressors, in contrast to the activators that activate transcription. The molecular interactions Figure 15-6. Transcriptional control by RNA polymerase. DNA is packaged into a chromatin structure. TATA = the common sequence on the promoter recognized by TBP
|
Surgery_Schwartz. TATA boxes that can be recognized and bound by a large complex containing RNA polymerase II, forming the basal transcription machinery. Usually located upstream of the TATA box (but sometimes lon-ger distances) are a number of regulatory sequences referred to as enhancers that are recognized by regulatory proteins called transcription factors. These transcription factors specifically bind to the enhancers, often in response to environmental or developmental cues, and cooperate with each other and with basal transcription factors to initiate transcription. Regulatory sequences that negatively regulate the initiation of transcription also are present on the promoter DNA. The transcription factors that bind to these sites are called repressors, in contrast to the activators that activate transcription. The molecular interactions Figure 15-6. Transcriptional control by RNA polymerase. DNA is packaged into a chromatin structure. TATA = the common sequence on the promoter recognized by TBP
|
Surgery_Schwartz_3304
|
Surgery_Schwartz
|
The molecular interactions Figure 15-6. Transcriptional control by RNA polymerase. DNA is packaged into a chromatin structure. TATA = the common sequence on the promoter recognized by TBP and polymerase II holoenzyme; TBP = TATA-binding protein and associated factors; TF = hypothetical transcription factor; TFBS = transcription factor binding site; ball-shaped structures = nucleosomes. Coactivator or corepressor is a factor linking the TF with the Pol II complex.TFCoactivator orCorepressorPol IIHoloenzymeTBPTBPTATATFBSbetween transcription factors and promoter DNA, as well as between the cooperative transcription factors, are highly regu-lated and context-dependent. Specifically, the recruitment of transcription factors to the promoter DNA occurs in response to physiologic signals. A number of structural motifs in these DNA-binding transcription factors facilitate this recognition and interaction. These include the helix-turn-helix, the homeodo-main motif, the zinc finger, the leucine
|
Surgery_Schwartz. The molecular interactions Figure 15-6. Transcriptional control by RNA polymerase. DNA is packaged into a chromatin structure. TATA = the common sequence on the promoter recognized by TBP and polymerase II holoenzyme; TBP = TATA-binding protein and associated factors; TF = hypothetical transcription factor; TFBS = transcription factor binding site; ball-shaped structures = nucleosomes. Coactivator or corepressor is a factor linking the TF with the Pol II complex.TFCoactivator orCorepressorPol IIHoloenzymeTBPTBPTATATFBSbetween transcription factors and promoter DNA, as well as between the cooperative transcription factors, are highly regu-lated and context-dependent. Specifically, the recruitment of transcription factors to the promoter DNA occurs in response to physiologic signals. A number of structural motifs in these DNA-binding transcription factors facilitate this recognition and interaction. These include the helix-turn-helix, the homeodo-main motif, the zinc finger, the leucine
|
Surgery_Schwartz_3305
|
Surgery_Schwartz
|
of structural motifs in these DNA-binding transcription factors facilitate this recognition and interaction. These include the helix-turn-helix, the homeodo-main motif, the zinc finger, the leucine zipper, and the helix-loop-helix motifs.Human GenomeGenome is a collective term for all genes present in one organ-ism. The human genome contains DNA sequences of 3 billion base pairs, carried by 23 pairs of chromosomes. The human genome has an estimated 25,000 to 30,000 genes, and overall it is 99.9% identical in all people.7,8 Approximately 3 million locations where single-base DNA differences exist have been identified and termed single nucleotide polymorphisms. Single nucleotide polymorphisms may be critical determinants of human variation in disease susceptibility and responses to envi-ronmental factors.The completion of the human genome sequence in 2003 represented another great milestone in modern science. The Human Genome Project created the field of genomics, which is the study of
|
Surgery_Schwartz. of structural motifs in these DNA-binding transcription factors facilitate this recognition and interaction. These include the helix-turn-helix, the homeodo-main motif, the zinc finger, the leucine zipper, and the helix-loop-helix motifs.Human GenomeGenome is a collective term for all genes present in one organ-ism. The human genome contains DNA sequences of 3 billion base pairs, carried by 23 pairs of chromosomes. The human genome has an estimated 25,000 to 30,000 genes, and overall it is 99.9% identical in all people.7,8 Approximately 3 million locations where single-base DNA differences exist have been identified and termed single nucleotide polymorphisms. Single nucleotide polymorphisms may be critical determinants of human variation in disease susceptibility and responses to envi-ronmental factors.The completion of the human genome sequence in 2003 represented another great milestone in modern science. The Human Genome Project created the field of genomics, which is the study of
|
Surgery_Schwartz_3306
|
Surgery_Schwartz
|
factors.The completion of the human genome sequence in 2003 represented another great milestone in modern science. The Human Genome Project created the field of genomics, which is the study of genetic material in detail (see Fig. 15-1). The medical field is building on the knowledge, resources, and technologies emanating from the human genome to further the understanding of the relationship of the genes and their muta-tions to human health and disease. This expansion of genomics into human health applications resulted in the field of genomic medicine.The emergence of genomics as a science will transform the practice of medicine and surgery in this century. This break-through has allowed scientists the opportunity to gain remarkable insights into the lives of humans. Ultimately, the goal is to use this information to develop new ways to treat, cure, or even prevent the thousands of diseases that afflict humankind. In the 21st century, work will begin to incorporate the information
|
Surgery_Schwartz. factors.The completion of the human genome sequence in 2003 represented another great milestone in modern science. The Human Genome Project created the field of genomics, which is the study of genetic material in detail (see Fig. 15-1). The medical field is building on the knowledge, resources, and technologies emanating from the human genome to further the understanding of the relationship of the genes and their muta-tions to human health and disease. This expansion of genomics into human health applications resulted in the field of genomic medicine.The emergence of genomics as a science will transform the practice of medicine and surgery in this century. This break-through has allowed scientists the opportunity to gain remarkable insights into the lives of humans. Ultimately, the goal is to use this information to develop new ways to treat, cure, or even prevent the thousands of diseases that afflict humankind. In the 21st century, work will begin to incorporate the information
|
Surgery_Schwartz_3307
|
Surgery_Schwartz
|
is to use this information to develop new ways to treat, cure, or even prevent the thousands of diseases that afflict humankind. In the 21st century, work will begin to incorporate the information embedded in the human genome sequence into surgical practices. By doing so, the genomic information can be used for diagnosing and predicting disease and disease suscep-tibility. Diagnostic tests can be designed to detect errant genes in patients suspected of having particular diseases or of being at risk for developing them. Furthermore, exploration into the function of each human gene is now possible, which will shed 3Brunicardi_Ch15_p0479-p0510.indd 48518/02/19 11:12 AM 486BASIC CONSIDERATIONSPART Ilight on how faulty genes play a role in disease causation. This knowledge also makes possible the development of a new gen-eration of therapeutics based on genes. Drug design is being revolutionized as researchers create new classes of medicines based on a reasoned approach to the use of
|
Surgery_Schwartz. is to use this information to develop new ways to treat, cure, or even prevent the thousands of diseases that afflict humankind. In the 21st century, work will begin to incorporate the information embedded in the human genome sequence into surgical practices. By doing so, the genomic information can be used for diagnosing and predicting disease and disease suscep-tibility. Diagnostic tests can be designed to detect errant genes in patients suspected of having particular diseases or of being at risk for developing them. Furthermore, exploration into the function of each human gene is now possible, which will shed 3Brunicardi_Ch15_p0479-p0510.indd 48518/02/19 11:12 AM 486BASIC CONSIDERATIONSPART Ilight on how faulty genes play a role in disease causation. This knowledge also makes possible the development of a new gen-eration of therapeutics based on genes. Drug design is being revolutionized as researchers create new classes of medicines based on a reasoned approach to the use of
|
Surgery_Schwartz_3308
|
Surgery_Schwartz
|
the development of a new gen-eration of therapeutics based on genes. Drug design is being revolutionized as researchers create new classes of medicines based on a reasoned approach to the use of information on gene sequence and protein structure function rather than the tradi-tional trial-and-error method. Drugs targeted to specific sites in the body promise to have fewer side effects than many of today’s medicines. Finally, other applications of genomics will involve the transfer of genes to replace defective versions or the use of gene therapy to enhance normal functions such as immunity.Proteomics refers to the study of the structure and expression of proteins as well as the interactions among pro-teins encoded by a human genome (see Fig. 15-1).9 A num-ber of Internet-based repositories for protein sequences exist, including Swiss-Prot (www.expasy.ch). These databases allow comparisons of newly identified proteins with previously char-acterized sequences to allow prediction of
|
Surgery_Schwartz. the development of a new gen-eration of therapeutics based on genes. Drug design is being revolutionized as researchers create new classes of medicines based on a reasoned approach to the use of information on gene sequence and protein structure function rather than the tradi-tional trial-and-error method. Drugs targeted to specific sites in the body promise to have fewer side effects than many of today’s medicines. Finally, other applications of genomics will involve the transfer of genes to replace defective versions or the use of gene therapy to enhance normal functions such as immunity.Proteomics refers to the study of the structure and expression of proteins as well as the interactions among pro-teins encoded by a human genome (see Fig. 15-1).9 A num-ber of Internet-based repositories for protein sequences exist, including Swiss-Prot (www.expasy.ch). These databases allow comparisons of newly identified proteins with previously char-acterized sequences to allow prediction of
|
Surgery_Schwartz_3309
|
Surgery_Schwartz
|
for protein sequences exist, including Swiss-Prot (www.expasy.ch). These databases allow comparisons of newly identified proteins with previously char-acterized sequences to allow prediction of similarities, identifi-cation of splice variants, and prediction of membrane topology and posttranslational modifications. Tools for proteomic profil-ing include two-dimensional gel electrophoresis, time-of-flight mass spectrometry, matrix-assisted laser desorption/ionization, and protein microarrays. Structural proteomics aims to describe the three-dimensional structure of proteins that is critical to understanding function. Functional genomics seeks to assign a biochemical, physiologic, cell biologic, and/or developmental function to each predicted gene. An ever-increasing arsenal of approaches, including transgenic animals, RNA interference (RNAi), and various systematic mutational strategies, will allow dissection of functions associated with newly discovered genes. Although the potential
|
Surgery_Schwartz. for protein sequences exist, including Swiss-Prot (www.expasy.ch). These databases allow comparisons of newly identified proteins with previously char-acterized sequences to allow prediction of similarities, identifi-cation of splice variants, and prediction of membrane topology and posttranslational modifications. Tools for proteomic profil-ing include two-dimensional gel electrophoresis, time-of-flight mass spectrometry, matrix-assisted laser desorption/ionization, and protein microarrays. Structural proteomics aims to describe the three-dimensional structure of proteins that is critical to understanding function. Functional genomics seeks to assign a biochemical, physiologic, cell biologic, and/or developmental function to each predicted gene. An ever-increasing arsenal of approaches, including transgenic animals, RNA interference (RNAi), and various systematic mutational strategies, will allow dissection of functions associated with newly discovered genes. Although the potential
|
Surgery_Schwartz_3310
|
Surgery_Schwartz
|
including transgenic animals, RNA interference (RNAi), and various systematic mutational strategies, will allow dissection of functions associated with newly discovered genes. Although the potential of this field of study is vast, it is in its early stages.It is anticipated that a genomic and proteomic approach to human disease will lead to a new understanding of pathogenesis that will aid in the development of effective strategies for early diagnosis and treatment.10 For example, identification of altered protein expression in organs, cells, subcellular structures, or protein complexes may lead to development of new biomark-ers for disease detection. Moreover, improved understanding of how protein structure determines function will allow rational identification of therapeutic targets, and thereby not only accel-erate drug development, but also lead to new strategies to evalu-ate therapeutic efficacy and potential toxicity.9Cell Cycle and ApoptosisEvery organism is composed of many
|
Surgery_Schwartz. including transgenic animals, RNA interference (RNAi), and various systematic mutational strategies, will allow dissection of functions associated with newly discovered genes. Although the potential of this field of study is vast, it is in its early stages.It is anticipated that a genomic and proteomic approach to human disease will lead to a new understanding of pathogenesis that will aid in the development of effective strategies for early diagnosis and treatment.10 For example, identification of altered protein expression in organs, cells, subcellular structures, or protein complexes may lead to development of new biomark-ers for disease detection. Moreover, improved understanding of how protein structure determines function will allow rational identification of therapeutic targets, and thereby not only accel-erate drug development, but also lead to new strategies to evalu-ate therapeutic efficacy and potential toxicity.9Cell Cycle and ApoptosisEvery organism is composed of many
|
Surgery_Schwartz_3311
|
Surgery_Schwartz
|
and thereby not only accel-erate drug development, but also lead to new strategies to evalu-ate therapeutic efficacy and potential toxicity.9Cell Cycle and ApoptosisEvery organism is composed of many different cell types at dif-ferent developmental stages. Some cell types continue to grow, while some cells stop growing after a developmental stage or resume growth after a break. For example, embryonic stem cells grow continuously, while nerve cells and striated muscle cells stop dividing after maturation. Cell cycle is the process for every cell including DNA replication and protein synthe-sis, DNA segregation in half, and package DNA and protein in two newly formed cells to enable passage of identical genetic information from one parental cell to two daughter cells. Thus, the cell cycle is the fundamental mechanism to maintain tissue homeostasis. A cell cycle comprises four periods: G1 (first gap phase before DNA synthesis), S (synthesis phase when DNA replication occurs), G2 (the gap
|
Surgery_Schwartz. and thereby not only accel-erate drug development, but also lead to new strategies to evalu-ate therapeutic efficacy and potential toxicity.9Cell Cycle and ApoptosisEvery organism is composed of many different cell types at dif-ferent developmental stages. Some cell types continue to grow, while some cells stop growing after a developmental stage or resume growth after a break. For example, embryonic stem cells grow continuously, while nerve cells and striated muscle cells stop dividing after maturation. Cell cycle is the process for every cell including DNA replication and protein synthe-sis, DNA segregation in half, and package DNA and protein in two newly formed cells to enable passage of identical genetic information from one parental cell to two daughter cells. Thus, the cell cycle is the fundamental mechanism to maintain tissue homeostasis. A cell cycle comprises four periods: G1 (first gap phase before DNA synthesis), S (synthesis phase when DNA replication occurs), G2 (the gap
|
Surgery_Schwartz_3312
|
Surgery_Schwartz
|
the fundamental mechanism to maintain tissue homeostasis. A cell cycle comprises four periods: G1 (first gap phase before DNA synthesis), S (synthesis phase when DNA replication occurs), G2 (the gap phase before mitosis), and M (mitosis, the phase when two daughter cells with identical DNA are generated) (Fig. 15-7). After a full cycle, the daughter Figure 15-7. The cell cycle and its control system. M is the mito-sis phase, when the nucleus and the cytoplasm divide; S is the phase when DNA is duplicated; G1 is the gap between M and S; G2 is the gap between S and M. A complex of cyclin and cyclin-dependent kinase (CDK) controls specific events of each phase. Without cyclin, CDK is inactive. Different cyclin/CDK complexes are shown around the cell cycle. A, B, D, and E stand for cyclin A, cyclin B, cyclin D, and cyclin E, respectively.B/CDK1A/CDK1A/CDK2E/CDK2D/CDK4D/CDK6G1G2SMMitosisDNA replicationcells enter G1 again, and when they receive appropriate signals, undergo another cycle,
|
Surgery_Schwartz. the fundamental mechanism to maintain tissue homeostasis. A cell cycle comprises four periods: G1 (first gap phase before DNA synthesis), S (synthesis phase when DNA replication occurs), G2 (the gap phase before mitosis), and M (mitosis, the phase when two daughter cells with identical DNA are generated) (Fig. 15-7). After a full cycle, the daughter Figure 15-7. The cell cycle and its control system. M is the mito-sis phase, when the nucleus and the cytoplasm divide; S is the phase when DNA is duplicated; G1 is the gap between M and S; G2 is the gap between S and M. A complex of cyclin and cyclin-dependent kinase (CDK) controls specific events of each phase. Without cyclin, CDK is inactive. Different cyclin/CDK complexes are shown around the cell cycle. A, B, D, and E stand for cyclin A, cyclin B, cyclin D, and cyclin E, respectively.B/CDK1A/CDK1A/CDK2E/CDK2D/CDK4D/CDK6G1G2SMMitosisDNA replicationcells enter G1 again, and when they receive appropriate signals, undergo another cycle,
|
Surgery_Schwartz_3313
|
Surgery_Schwartz
|
cyclin B, cyclin D, and cyclin E, respectively.B/CDK1A/CDK1A/CDK2E/CDK2D/CDK4D/CDK6G1G2SMMitosisDNA replicationcells enter G1 again, and when they receive appropriate signals, undergo another cycle, and so on. The machinery that drives cell cycle progression is made up of a group of enzymes called cyclin-dependent kinases (CDKs). Cyclin expression fluctuates during the cell cycle, and cyclins are essential for CDK activi-ties and form complexes with CDK. The cyclin A/CDK1 and cyclin B/CDK1 drive the progression for the M phase, while cyclin A/CDK2 is the primary S phase complex. Early G1 cyclin D/CDK4/6 or late G1 cyclin E/CDK2 controls the G1-S transi-tion. There also are negative regulators for CDK termed CDK inhibitors, which inhibit the assembly or activity of the cyclin-CDK complex. Expression of cyclins and CDK inhibitors often is regulated by developmental and environmental factors.The cell cycle is connected with signal transduction path-ways as well as gene expression.
|
Surgery_Schwartz. cyclin B, cyclin D, and cyclin E, respectively.B/CDK1A/CDK1A/CDK2E/CDK2D/CDK4D/CDK6G1G2SMMitosisDNA replicationcells enter G1 again, and when they receive appropriate signals, undergo another cycle, and so on. The machinery that drives cell cycle progression is made up of a group of enzymes called cyclin-dependent kinases (CDKs). Cyclin expression fluctuates during the cell cycle, and cyclins are essential for CDK activi-ties and form complexes with CDK. The cyclin A/CDK1 and cyclin B/CDK1 drive the progression for the M phase, while cyclin A/CDK2 is the primary S phase complex. Early G1 cyclin D/CDK4/6 or late G1 cyclin E/CDK2 controls the G1-S transi-tion. There also are negative regulators for CDK termed CDK inhibitors, which inhibit the assembly or activity of the cyclin-CDK complex. Expression of cyclins and CDK inhibitors often is regulated by developmental and environmental factors.The cell cycle is connected with signal transduction path-ways as well as gene expression.
|
Surgery_Schwartz_3314
|
Surgery_Schwartz
|
Expression of cyclins and CDK inhibitors often is regulated by developmental and environmental factors.The cell cycle is connected with signal transduction path-ways as well as gene expression. Although the S and M phases rarely are subjected to changes imposed by extracellular sig-nals, the G1 and G2 phases are the primary periods when cells decide whether or not to move on to the next phase. During the G1 phase, cells receive greenor red-light signals, S phase entry or G1 arrest, respectively. Growing cells proliferate only when supplied with appropriate mitogenic growth factors. Cells become committed to entry of the cell cycle only toward the end of G1. Mitogenic signals stimulate the activity of early G1 CDKs (e.g., cyclin D/CDK4) that inhibit the activity of pRb protein and activate the transcription factor called E2F to induce the expression of batteries of genes essential for G1-S progression. Meanwhile, cells also receive antiproliferative signals such as those from tumor
|
Surgery_Schwartz. Expression of cyclins and CDK inhibitors often is regulated by developmental and environmental factors.The cell cycle is connected with signal transduction path-ways as well as gene expression. Although the S and M phases rarely are subjected to changes imposed by extracellular sig-nals, the G1 and G2 phases are the primary periods when cells decide whether or not to move on to the next phase. During the G1 phase, cells receive greenor red-light signals, S phase entry or G1 arrest, respectively. Growing cells proliferate only when supplied with appropriate mitogenic growth factors. Cells become committed to entry of the cell cycle only toward the end of G1. Mitogenic signals stimulate the activity of early G1 CDKs (e.g., cyclin D/CDK4) that inhibit the activity of pRb protein and activate the transcription factor called E2F to induce the expression of batteries of genes essential for G1-S progression. Meanwhile, cells also receive antiproliferative signals such as those from tumor
|
Surgery_Schwartz_3315
|
Surgery_Schwartz
|
the transcription factor called E2F to induce the expression of batteries of genes essential for G1-S progression. Meanwhile, cells also receive antiproliferative signals such as those from tumor suppressors. These antiproliferative signals also act in the G1 phase to stop cells’ progress into the S phase by inducing CKI production. For example, when DNA is dam-aged, cells will repair the damage before entering the S phase. Therefore, G1 contains one of the most important checkpoints for cell cycle progression. If the analogy is made that CDK is to a cell as an engine is to a car, then cyclins and CKI are the gas pedal and brake, respectively. Accelerated proliferation or Brunicardi_Ch15_p0479-p0510.indd 48618/02/19 11:12 AM 487MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15improper cell cycle progression with damaged DNA would be disastrous. Genetic gain-of-function mutations in oncogenes (that often promote expression or activity of the
|
Surgery_Schwartz. the transcription factor called E2F to induce the expression of batteries of genes essential for G1-S progression. Meanwhile, cells also receive antiproliferative signals such as those from tumor suppressors. These antiproliferative signals also act in the G1 phase to stop cells’ progress into the S phase by inducing CKI production. For example, when DNA is dam-aged, cells will repair the damage before entering the S phase. Therefore, G1 contains one of the most important checkpoints for cell cycle progression. If the analogy is made that CDK is to a cell as an engine is to a car, then cyclins and CKI are the gas pedal and brake, respectively. Accelerated proliferation or Brunicardi_Ch15_p0479-p0510.indd 48618/02/19 11:12 AM 487MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15improper cell cycle progression with damaged DNA would be disastrous. Genetic gain-of-function mutations in oncogenes (that often promote expression or activity of the
|
Surgery_Schwartz_3316
|
Surgery_Schwartz
|
AND PRECISION SURGERYCHAPTER 15improper cell cycle progression with damaged DNA would be disastrous. Genetic gain-of-function mutations in oncogenes (that often promote expression or activity of the cyclin/CDK complex) or loss-of-function mutations in tumor suppressor (that stimulate production of CKI) are causal factors for malig-nant transformation.In addition to cell cycle control, cells use genetically pro-grammed mechanisms to kill cells. This cellular process, called apoptosis or programmed cell death, is essential for the mainte-nance of tissue homeostasis (Fig. 15-8).Normal tissues undergo proper apoptosis to remove unwanted cells, those that have completed their jobs or have been damaged or improperly proliferated. Apoptosis can be activated by many physiologic stimuli such as death receptor signals (e.g., Fas or cytokine tumor necrosis factor), growth fac-tor deprivation, DNA damage, and stress signals. Two major pathways control the biochemical mechanisms governing
|
Surgery_Schwartz. AND PRECISION SURGERYCHAPTER 15improper cell cycle progression with damaged DNA would be disastrous. Genetic gain-of-function mutations in oncogenes (that often promote expression or activity of the cyclin/CDK complex) or loss-of-function mutations in tumor suppressor (that stimulate production of CKI) are causal factors for malig-nant transformation.In addition to cell cycle control, cells use genetically pro-grammed mechanisms to kill cells. This cellular process, called apoptosis or programmed cell death, is essential for the mainte-nance of tissue homeostasis (Fig. 15-8).Normal tissues undergo proper apoptosis to remove unwanted cells, those that have completed their jobs or have been damaged or improperly proliferated. Apoptosis can be activated by many physiologic stimuli such as death receptor signals (e.g., Fas or cytokine tumor necrosis factor), growth fac-tor deprivation, DNA damage, and stress signals. Two major pathways control the biochemical mechanisms governing
|
Surgery_Schwartz_3317
|
Surgery_Schwartz
|
as death receptor signals (e.g., Fas or cytokine tumor necrosis factor), growth fac-tor deprivation, DNA damage, and stress signals. Two major pathways control the biochemical mechanisms governing apop-tosis: the death receptor and mitochondrial. However, recent advances in apoptosis research suggest an interconnection of the two pathways. What is central to the apoptotic machinery is the activation of a cascade of proteinases called caspases. Similar to CDK in the cell cycle, activities and expression of caspases are well controlled by positive and negative regulators. The complex machinery of apoptosis must be tightly controlled. Perturbations of this process can cause neoplastic transforma-tion or other diseases.Signal Transduction PathwaysGene expression in a genome is controlled in a temporal and spatial manner, at least in part by signaling pathways.11 A sig-naling pathway generally begins at the cell surface and, after a signaling relay by a cascade of intracellular effectors,
|
Surgery_Schwartz. as death receptor signals (e.g., Fas or cytokine tumor necrosis factor), growth fac-tor deprivation, DNA damage, and stress signals. Two major pathways control the biochemical mechanisms governing apop-tosis: the death receptor and mitochondrial. However, recent advances in apoptosis research suggest an interconnection of the two pathways. What is central to the apoptotic machinery is the activation of a cascade of proteinases called caspases. Similar to CDK in the cell cycle, activities and expression of caspases are well controlled by positive and negative regulators. The complex machinery of apoptosis must be tightly controlled. Perturbations of this process can cause neoplastic transforma-tion or other diseases.Signal Transduction PathwaysGene expression in a genome is controlled in a temporal and spatial manner, at least in part by signaling pathways.11 A sig-naling pathway generally begins at the cell surface and, after a signaling relay by a cascade of intracellular effectors,
|
Surgery_Schwartz_3318
|
Surgery_Schwartz
|
temporal and spatial manner, at least in part by signaling pathways.11 A sig-naling pathway generally begins at the cell surface and, after a signaling relay by a cascade of intracellular effectors, ends up in the nucleus (Fig. 15-9). All cells have the ability to sense changes in their external environment. The bioactive substances to which cells can respond are many and include proteins, short peptides, amino acids, nucleotides/nucleosides, steroids, reti-noids, fatty acids, and dissolved gases. Some of these substances are lipophilic and thereby can cross the plasma membrane by NucleusDeath signal(e.g., TNF or Fas)DeathreceptorPlasmamembraneActivation ofcaspase cascadeCytochrome creleaseDeathreceptorsignalingpathwayMitochondrionNormal target cellApoptotic target cellFigure 15-8. A simplified view of the apop-tosis pathways. Extracellular death receptor pathways include the activation of Fas and tumor necrosis factor (TNF) receptors and consequent activation of the caspase path-way.
|
Surgery_Schwartz. temporal and spatial manner, at least in part by signaling pathways.11 A sig-naling pathway generally begins at the cell surface and, after a signaling relay by a cascade of intracellular effectors, ends up in the nucleus (Fig. 15-9). All cells have the ability to sense changes in their external environment. The bioactive substances to which cells can respond are many and include proteins, short peptides, amino acids, nucleotides/nucleosides, steroids, reti-noids, fatty acids, and dissolved gases. Some of these substances are lipophilic and thereby can cross the plasma membrane by NucleusDeath signal(e.g., TNF or Fas)DeathreceptorPlasmamembraneActivation ofcaspase cascadeCytochrome creleaseDeathreceptorsignalingpathwayMitochondrionNormal target cellApoptotic target cellFigure 15-8. A simplified view of the apop-tosis pathways. Extracellular death receptor pathways include the activation of Fas and tumor necrosis factor (TNF) receptors and consequent activation of the caspase path-way.
|
Surgery_Schwartz_3319
|
Surgery_Schwartz
|
view of the apop-tosis pathways. Extracellular death receptor pathways include the activation of Fas and tumor necrosis factor (TNF) receptors and consequent activation of the caspase path-way. Intracellular death pathway indicates the release of cytochrome c from mitochon-dria, which also triggers the activation of the caspase cascade. During apoptosis, cells undergo DNA fragmentation and nuclear and cell membrane breakdown and are eventually digested by other cells.Ligand(e.g., growth factor)Cell-surfacereceptorPlasmamembraneNucleusGeneexpressionLigand(e.g., hormone)Signaling cascadeIntracellularreceptorFigure 15-9. Cell-surface and intracellular receptor pathways. Extracellular signaling pathway: Most growth factors and other hydrophilic signaling molecules are unable to move across the plasma membrane and directly activate cell-surface receptors such as G-protein–coupled receptors and enzyme-linked receptors. The receptor serves as the receiver and in turn activates the downstream
|
Surgery_Schwartz. view of the apop-tosis pathways. Extracellular death receptor pathways include the activation of Fas and tumor necrosis factor (TNF) receptors and consequent activation of the caspase path-way. Intracellular death pathway indicates the release of cytochrome c from mitochon-dria, which also triggers the activation of the caspase cascade. During apoptosis, cells undergo DNA fragmentation and nuclear and cell membrane breakdown and are eventually digested by other cells.Ligand(e.g., growth factor)Cell-surfacereceptorPlasmamembraneNucleusGeneexpressionLigand(e.g., hormone)Signaling cascadeIntracellularreceptorFigure 15-9. Cell-surface and intracellular receptor pathways. Extracellular signaling pathway: Most growth factors and other hydrophilic signaling molecules are unable to move across the plasma membrane and directly activate cell-surface receptors such as G-protein–coupled receptors and enzyme-linked receptors. The receptor serves as the receiver and in turn activates the downstream
|
Surgery_Schwartz_3320
|
Surgery_Schwartz
|
plasma membrane and directly activate cell-surface receptors such as G-protein–coupled receptors and enzyme-linked receptors. The receptor serves as the receiver and in turn activates the downstream signals in the cell. Intracellular signaling pathway: Hormones or other diffusible molecules enter the cell and bind to the intracel-lular receptor in the cytoplasm or in the nucleus. Either extracel-lular or intracellular signals often reach the nucleus to control gene expression.Brunicardi_Ch15_p0479-p0510.indd 48718/02/19 11:12 AM 488BASIC CONSIDERATIONSPART Idiffusion to bind to a specific target protein within the cyto-plasm (intracellular receptor). Other substances bind directly with a transmembrane protein (cell-surface receptor). Binding of ligand to receptor initiates a series of biochemical reactions (signal transduction) typically involving protein-protein inter-actions and the transfer of high-energy phosphate groups, lead-ing to various cellular end responses.Control and
|
Surgery_Schwartz. plasma membrane and directly activate cell-surface receptors such as G-protein–coupled receptors and enzyme-linked receptors. The receptor serves as the receiver and in turn activates the downstream signals in the cell. Intracellular signaling pathway: Hormones or other diffusible molecules enter the cell and bind to the intracel-lular receptor in the cytoplasm or in the nucleus. Either extracel-lular or intracellular signals often reach the nucleus to control gene expression.Brunicardi_Ch15_p0479-p0510.indd 48718/02/19 11:12 AM 488BASIC CONSIDERATIONSPART Idiffusion to bind to a specific target protein within the cyto-plasm (intracellular receptor). Other substances bind directly with a transmembrane protein (cell-surface receptor). Binding of ligand to receptor initiates a series of biochemical reactions (signal transduction) typically involving protein-protein inter-actions and the transfer of high-energy phosphate groups, lead-ing to various cellular end responses.Control and
|
Surgery_Schwartz_3321
|
Surgery_Schwartz
|
biochemical reactions (signal transduction) typically involving protein-protein inter-actions and the transfer of high-energy phosphate groups, lead-ing to various cellular end responses.Control and specificity through simple protein-protein interactions—referred to as adhesive interactions—is a com-mon feature of signal transduction pathways in cells.12 Signaling also involves catalytic activities of signaling molecules, such as protein kinases/phosphatases, that modify the structures of key signaling proteins. Upon binding and/or modification by upstream signaling molecules, downstream effectors undergo a conformational (allosteric) change and, consequently, a change in function. The signal that originates at the cell surface and is relayed by the cytoplasmic proteins often ultimately reaches the transcriptional apparatus in the nucleus. It alters the DNA binding and activities of transcription factors that directly turn genes on or off in response to the stimuli. Abnormal
|
Surgery_Schwartz. biochemical reactions (signal transduction) typically involving protein-protein inter-actions and the transfer of high-energy phosphate groups, lead-ing to various cellular end responses.Control and specificity through simple protein-protein interactions—referred to as adhesive interactions—is a com-mon feature of signal transduction pathways in cells.12 Signaling also involves catalytic activities of signaling molecules, such as protein kinases/phosphatases, that modify the structures of key signaling proteins. Upon binding and/or modification by upstream signaling molecules, downstream effectors undergo a conformational (allosteric) change and, consequently, a change in function. The signal that originates at the cell surface and is relayed by the cytoplasmic proteins often ultimately reaches the transcriptional apparatus in the nucleus. It alters the DNA binding and activities of transcription factors that directly turn genes on or off in response to the stimuli. Abnormal
|
Surgery_Schwartz_3322
|
Surgery_Schwartz
|
reaches the transcriptional apparatus in the nucleus. It alters the DNA binding and activities of transcription factors that directly turn genes on or off in response to the stimuli. Abnormal alterations in signaling activities and capacities in otherwise normal cells can lead to diseases such as cancer.Advances in biology in the last two decades have dramati-cally expanded the view on how cells are wired with signal-ing pathways. In a given cell, many signaling pathways operate simultaneously and crosstalk with one another. A cell gener-ally may react to a hormonal signal in a variety of ways: (a) by changing its metabolite or protein, (b) by generating an electric current, or (c) by contracting. Cells continually are subject to multiple input signals that simultaneously and sequentially acti-vate multiple receptorand non–receptor-mediated signal trans-duction pathways, which form a signaling network. Although the regulators responsible for cell behavior are rapidly identified as a
|
Surgery_Schwartz. reaches the transcriptional apparatus in the nucleus. It alters the DNA binding and activities of transcription factors that directly turn genes on or off in response to the stimuli. Abnormal alterations in signaling activities and capacities in otherwise normal cells can lead to diseases such as cancer.Advances in biology in the last two decades have dramati-cally expanded the view on how cells are wired with signal-ing pathways. In a given cell, many signaling pathways operate simultaneously and crosstalk with one another. A cell gener-ally may react to a hormonal signal in a variety of ways: (a) by changing its metabolite or protein, (b) by generating an electric current, or (c) by contracting. Cells continually are subject to multiple input signals that simultaneously and sequentially acti-vate multiple receptorand non–receptor-mediated signal trans-duction pathways, which form a signaling network. Although the regulators responsible for cell behavior are rapidly identified as a
|
Surgery_Schwartz_3323
|
Surgery_Schwartz
|
acti-vate multiple receptorand non–receptor-mediated signal trans-duction pathways, which form a signaling network. Although the regulators responsible for cell behavior are rapidly identified as a result of genomic and proteomic techniques, the specific functions of the individual proteins, how they assemble, and the networks that control cellular behavior remain to be defined. An increased understanding of cell regulatory pathways—and how they are disrupted in disease—will likely reveal common themes based on protein interaction domains that direct associa-tions of proteins with other polypeptides, phospholipids, nucleic acids, and other regulatory molecules. Advances in the under-standing of signaling networks will require methods of inves-tigation that move beyond traditional “linear” approaches into medical informatics and computational biology. The bewilder-ing biocomplexity of such networks mandates multidisciplinary and transdisciplinary research collaboration. The vast amount
|
Surgery_Schwartz. acti-vate multiple receptorand non–receptor-mediated signal trans-duction pathways, which form a signaling network. Although the regulators responsible for cell behavior are rapidly identified as a result of genomic and proteomic techniques, the specific functions of the individual proteins, how they assemble, and the networks that control cellular behavior remain to be defined. An increased understanding of cell regulatory pathways—and how they are disrupted in disease—will likely reveal common themes based on protein interaction domains that direct associa-tions of proteins with other polypeptides, phospholipids, nucleic acids, and other regulatory molecules. Advances in the under-standing of signaling networks will require methods of inves-tigation that move beyond traditional “linear” approaches into medical informatics and computational biology. The bewilder-ing biocomplexity of such networks mandates multidisciplinary and transdisciplinary research collaboration. The vast amount
|
Surgery_Schwartz_3324
|
Surgery_Schwartz
|
approaches into medical informatics and computational biology. The bewilder-ing biocomplexity of such networks mandates multidisciplinary and transdisciplinary research collaboration. The vast amount of information that is rapidly emerging from genomic and pro-teomic data mining will require the development of new model-ing methodologies within the emerging disciplines of medical mathematics and physics.Signaling pathways often are grouped according to the properties of signaling receptors. Many hydrophobic signaling molecules are able to diffuse across plasma membranes and directly reach specific cytoplasmic targets. Steroid hormones, thyroid hormones, retinoids, and vitamin D are examples that exert their activity upon binding to structurally related recep-tor proteins that are members of the nuclear hormone receptor superfamily. Ligand binding induces a conformational change that enhances transcriptional activity of these receptors. Most extracellular signaling molecules interact
|
Surgery_Schwartz. approaches into medical informatics and computational biology. The bewilder-ing biocomplexity of such networks mandates multidisciplinary and transdisciplinary research collaboration. The vast amount of information that is rapidly emerging from genomic and pro-teomic data mining will require the development of new model-ing methodologies within the emerging disciplines of medical mathematics and physics.Signaling pathways often are grouped according to the properties of signaling receptors. Many hydrophobic signaling molecules are able to diffuse across plasma membranes and directly reach specific cytoplasmic targets. Steroid hormones, thyroid hormones, retinoids, and vitamin D are examples that exert their activity upon binding to structurally related recep-tor proteins that are members of the nuclear hormone receptor superfamily. Ligand binding induces a conformational change that enhances transcriptional activity of these receptors. Most extracellular signaling molecules interact
|
Surgery_Schwartz_3325
|
Surgery_Schwartz
|
of the nuclear hormone receptor superfamily. Ligand binding induces a conformational change that enhances transcriptional activity of these receptors. Most extracellular signaling molecules interact with transmembrane protein receptors that couple ligand binding to intracellular sig-nals, leading to biologic actions.There are three major classes of cell-surface receptors: transmitter-gated ion channels, seven-transmembrane G-protein–coupled receptors (GPCRs), and enzyme-linked receptors. The superfamily of GPCRs is one of the largest families of proteins, representing over 800 genes of the human genome. Members of this superfamily share a characteristic seven-transmembrane configuration. The ligands for these receptors are diverse and include hormones, chemokines, neurotransmitters, protein-ases, inflammatory mediators, and even sensory signals such as odorants and photons. Most GPCRs signal through het-erotrimeric G proteins, which are guanine-nucleotide regula-tory complexes. Thus,
|
Surgery_Schwartz. of the nuclear hormone receptor superfamily. Ligand binding induces a conformational change that enhances transcriptional activity of these receptors. Most extracellular signaling molecules interact with transmembrane protein receptors that couple ligand binding to intracellular sig-nals, leading to biologic actions.There are three major classes of cell-surface receptors: transmitter-gated ion channels, seven-transmembrane G-protein–coupled receptors (GPCRs), and enzyme-linked receptors. The superfamily of GPCRs is one of the largest families of proteins, representing over 800 genes of the human genome. Members of this superfamily share a characteristic seven-transmembrane configuration. The ligands for these receptors are diverse and include hormones, chemokines, neurotransmitters, protein-ases, inflammatory mediators, and even sensory signals such as odorants and photons. Most GPCRs signal through het-erotrimeric G proteins, which are guanine-nucleotide regula-tory complexes. Thus,
|
Surgery_Schwartz_3326
|
Surgery_Schwartz
|
inflammatory mediators, and even sensory signals such as odorants and photons. Most GPCRs signal through het-erotrimeric G proteins, which are guanine-nucleotide regula-tory complexes. Thus, the receptor serves as the receiver, the G protein serves as the transducer, and the enzyme serves as the effector arm. Enzyme-linked receptors possess an extracellular ligand-recognition domain and a cytosolic domain that either has intrinsic enzymatic activity or directly links with an enzyme. Structurally, these receptors usually have only one transmembrane-spanning domain. Of at least five forms of enzyme-linked recep-tors classified by the nature of the enzyme activity to which they are coupled, the growth factor receptors such as tyrosine kinase receptor or serine/threonine kinase receptors mediate diverse cellular events including cell growth, differentiation, metabolism, and survival/apoptosis. Dysregulation (particularly mutations) of these receptors is thought to underlie conditions of
|
Surgery_Schwartz. inflammatory mediators, and even sensory signals such as odorants and photons. Most GPCRs signal through het-erotrimeric G proteins, which are guanine-nucleotide regula-tory complexes. Thus, the receptor serves as the receiver, the G protein serves as the transducer, and the enzyme serves as the effector arm. Enzyme-linked receptors possess an extracellular ligand-recognition domain and a cytosolic domain that either has intrinsic enzymatic activity or directly links with an enzyme. Structurally, these receptors usually have only one transmembrane-spanning domain. Of at least five forms of enzyme-linked recep-tors classified by the nature of the enzyme activity to which they are coupled, the growth factor receptors such as tyrosine kinase receptor or serine/threonine kinase receptors mediate diverse cellular events including cell growth, differentiation, metabolism, and survival/apoptosis. Dysregulation (particularly mutations) of these receptors is thought to underlie conditions of
|
Surgery_Schwartz_3327
|
Surgery_Schwartz
|
diverse cellular events including cell growth, differentiation, metabolism, and survival/apoptosis. Dysregulation (particularly mutations) of these receptors is thought to underlie conditions of abnormal cellular proliferation in the context of cancer. The following sections will further review two examples of growth factor signaling pathways and their connection with human diseases.Insulin Pathway and Diabetes.13 The discovery of insulin in the early 1920s is one of the most dramatic events in the treatment of human disease. Insulin is a peptide hormone that is secreted by the β-cell of the pancreas. Insulin is required for the growth and metabolism of most mammalian cells, which contain cell-surface insulin receptors (InsR). Insulin binding to InsR activates the kinase activity of InsR. InsR then adds phosphoryl groups, a process referred to as phosphorylation, and subsequently activates its immediate intracellular effector, called insulin receptor substrate (IRS). IRS plays a
|
Surgery_Schwartz. diverse cellular events including cell growth, differentiation, metabolism, and survival/apoptosis. Dysregulation (particularly mutations) of these receptors is thought to underlie conditions of abnormal cellular proliferation in the context of cancer. The following sections will further review two examples of growth factor signaling pathways and their connection with human diseases.Insulin Pathway and Diabetes.13 The discovery of insulin in the early 1920s is one of the most dramatic events in the treatment of human disease. Insulin is a peptide hormone that is secreted by the β-cell of the pancreas. Insulin is required for the growth and metabolism of most mammalian cells, which contain cell-surface insulin receptors (InsR). Insulin binding to InsR activates the kinase activity of InsR. InsR then adds phosphoryl groups, a process referred to as phosphorylation, and subsequently activates its immediate intracellular effector, called insulin receptor substrate (IRS). IRS plays a
|
Surgery_Schwartz_3328
|
Surgery_Schwartz
|
InsR. InsR then adds phosphoryl groups, a process referred to as phosphorylation, and subsequently activates its immediate intracellular effector, called insulin receptor substrate (IRS). IRS plays a central role in coordinating the signaling of insulin by activating distinct sig-naling pathways, the PI3K-Akt pathway and MAPK pathway, both of which possess multiple protein kinases that can control transcription, protein synthesis, and glycolysis (Fig. 15-10).The primary physiologic role of insulin is in glucose homeostasis, which is accomplished through the stimulation of glucose uptake into insulin-sensitive tissues such as fat and skeletal muscle. Defects in insulin synthesis/secretion and/or responsiveness are major causal factors in diabetes, one of the leading causes of death and disability in the United States, affecting an estimated 16 million Americans. Type 2 diabetes accounts for about 90% of all cases of diabetes. Clustering of type 2 diabetes in certain families and ethnic
|
Surgery_Schwartz. InsR. InsR then adds phosphoryl groups, a process referred to as phosphorylation, and subsequently activates its immediate intracellular effector, called insulin receptor substrate (IRS). IRS plays a central role in coordinating the signaling of insulin by activating distinct sig-naling pathways, the PI3K-Akt pathway and MAPK pathway, both of which possess multiple protein kinases that can control transcription, protein synthesis, and glycolysis (Fig. 15-10).The primary physiologic role of insulin is in glucose homeostasis, which is accomplished through the stimulation of glucose uptake into insulin-sensitive tissues such as fat and skeletal muscle. Defects in insulin synthesis/secretion and/or responsiveness are major causal factors in diabetes, one of the leading causes of death and disability in the United States, affecting an estimated 16 million Americans. Type 2 diabetes accounts for about 90% of all cases of diabetes. Clustering of type 2 diabetes in certain families and ethnic
|
Surgery_Schwartz_3329
|
Surgery_Schwartz
|
in the United States, affecting an estimated 16 million Americans. Type 2 diabetes accounts for about 90% of all cases of diabetes. Clustering of type 2 diabetes in certain families and ethnic populations points to a strong genetic background for the disease. More than 90% of affected individuals have insulin resistance, which develops when the body is no longer able to respond correctly to insu-lin circulating in the blood. Although relatively little is known about the biochemical basis of this metabolic disorder, it is clear that the insulin-signaling pathways malfunction in this disease. It is also known that genetic mutations in the InsR or IRS cause type 2 diabetes, although which one is not certain. The majority of type 2 diabetes cases may result from defects in downstream-signaling components in the insulin-signaling pathway. Brunicardi_Ch15_p0479-p0510.indd 48818/02/19 11:12 AM 489MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER
|
Surgery_Schwartz. in the United States, affecting an estimated 16 million Americans. Type 2 diabetes accounts for about 90% of all cases of diabetes. Clustering of type 2 diabetes in certain families and ethnic populations points to a strong genetic background for the disease. More than 90% of affected individuals have insulin resistance, which develops when the body is no longer able to respond correctly to insu-lin circulating in the blood. Although relatively little is known about the biochemical basis of this metabolic disorder, it is clear that the insulin-signaling pathways malfunction in this disease. It is also known that genetic mutations in the InsR or IRS cause type 2 diabetes, although which one is not certain. The majority of type 2 diabetes cases may result from defects in downstream-signaling components in the insulin-signaling pathway. Brunicardi_Ch15_p0479-p0510.indd 48818/02/19 11:12 AM 489MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER
|
Surgery_Schwartz_3330
|
Surgery_Schwartz
|
components in the insulin-signaling pathway. Brunicardi_Ch15_p0479-p0510.indd 48818/02/19 11:12 AM 489MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15PlasmamembraneNucleusInsulinreceptor(InsR)GeneexpressionMAPKcascadeLipid & glucosemetabolismCellsurvivalIRSInsulinAdaptorPI3KFigure 15-10. Insulin-signaling pathway. Insulin is a peptide growth factor that binds to and activates the heterotetrameric recep-tor complex (InsR). InsR possesses protein tyrosine kinase activity and is able to phosphorylate the downstream insulin receptor sub-strate (IRS). Phosphorylated IRS serves as a scaffold and controls the activation of multiple downstream pathways for gene expres-sion, cell survival, and glucose metabolism. Inactivation of the insulin pathway can lead to type 2 diabetes.Type 2 diabetes also is associated with declining β-cell func-tion, resulting in reduced insulin secretion; these pathways are under intense study. A full understanding of the basis of
|
Surgery_Schwartz. components in the insulin-signaling pathway. Brunicardi_Ch15_p0479-p0510.indd 48818/02/19 11:12 AM 489MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15PlasmamembraneNucleusInsulinreceptor(InsR)GeneexpressionMAPKcascadeLipid & glucosemetabolismCellsurvivalIRSInsulinAdaptorPI3KFigure 15-10. Insulin-signaling pathway. Insulin is a peptide growth factor that binds to and activates the heterotetrameric recep-tor complex (InsR). InsR possesses protein tyrosine kinase activity and is able to phosphorylate the downstream insulin receptor sub-strate (IRS). Phosphorylated IRS serves as a scaffold and controls the activation of multiple downstream pathways for gene expres-sion, cell survival, and glucose metabolism. Inactivation of the insulin pathway can lead to type 2 diabetes.Type 2 diabetes also is associated with declining β-cell func-tion, resulting in reduced insulin secretion; these pathways are under intense study. A full understanding of the basis of
|
Surgery_Schwartz_3331
|
Surgery_Schwartz
|
2 diabetes.Type 2 diabetes also is associated with declining β-cell func-tion, resulting in reduced insulin secretion; these pathways are under intense study. A full understanding of the basis of insulin resistance is crucial for the development of new therapies for type 2 diabetes. Furthermore, apart from type 2 diabetes, insulin resistance is a central feature of several other common human disorders, including atherosclerosis and coronary artery disease, hypertension, and obesity.Transforming Growth Factor-a (TGF-a) Pathway and Cancers.14 Growth factor signaling controls cell growth, differ-entiation, and apoptosis. Although insulin and many mitogenic growth factors promote cell proliferation, some growth factors and hormones inhibit cell proliferation. TGF-β is one of them. The balance between mitogens and TGF-β plays an important role in controlling the proper pace of cell cycle progression. The growth inhibition function of TGF-β signaling in epithelial cells plays a major role
|
Surgery_Schwartz. 2 diabetes.Type 2 diabetes also is associated with declining β-cell func-tion, resulting in reduced insulin secretion; these pathways are under intense study. A full understanding of the basis of insulin resistance is crucial for the development of new therapies for type 2 diabetes. Furthermore, apart from type 2 diabetes, insulin resistance is a central feature of several other common human disorders, including atherosclerosis and coronary artery disease, hypertension, and obesity.Transforming Growth Factor-a (TGF-a) Pathway and Cancers.14 Growth factor signaling controls cell growth, differ-entiation, and apoptosis. Although insulin and many mitogenic growth factors promote cell proliferation, some growth factors and hormones inhibit cell proliferation. TGF-β is one of them. The balance between mitogens and TGF-β plays an important role in controlling the proper pace of cell cycle progression. The growth inhibition function of TGF-β signaling in epithelial cells plays a major role
|
Surgery_Schwartz_3332
|
Surgery_Schwartz
|
between mitogens and TGF-β plays an important role in controlling the proper pace of cell cycle progression. The growth inhibition function of TGF-β signaling in epithelial cells plays a major role in maintaining tissue homeostasis.The TGF-β superfamily comprises a large number of struc-turally related growth and differentiation factors that act through a receptor complex at the cell surface (Fig. 15-11). The com-plex consists of transmembrane serine/threonine kinases. The receptor signals through activation of heterotrimeric complexes of intracellular effectors called SMADs (which are contracted from homologous Caenorhabditis elegans Sma and Drosophila Mad, two evolutionarily conserved genes for TGF-β signaling). Upon phosphorylation by the receptors, SMAD complexes translocate into the nucleus, where they bind to gene promoters and cooperate with specific transcription factors to regulate the expression of genes that control cell proliferation and differen-tiation. For example,
|
Surgery_Schwartz. between mitogens and TGF-β plays an important role in controlling the proper pace of cell cycle progression. The growth inhibition function of TGF-β signaling in epithelial cells plays a major role in maintaining tissue homeostasis.The TGF-β superfamily comprises a large number of struc-turally related growth and differentiation factors that act through a receptor complex at the cell surface (Fig. 15-11). The com-plex consists of transmembrane serine/threonine kinases. The receptor signals through activation of heterotrimeric complexes of intracellular effectors called SMADs (which are contracted from homologous Caenorhabditis elegans Sma and Drosophila Mad, two evolutionarily conserved genes for TGF-β signaling). Upon phosphorylation by the receptors, SMAD complexes translocate into the nucleus, where they bind to gene promoters and cooperate with specific transcription factors to regulate the expression of genes that control cell proliferation and differen-tiation. For example,
|
Surgery_Schwartz_3333
|
Surgery_Schwartz
|
the nucleus, where they bind to gene promoters and cooperate with specific transcription factors to regulate the expression of genes that control cell proliferation and differen-tiation. For example, TGF-β strongly induces the transcription PlasmamembraneTGF b receptorGeneexpressionNucleusSMADTGF bAnti-proliferationFigure 15-11. TGF-β signaling pathway. The TGF-β family has at least 29 members encoded in the human genome. They are also peptide growth factors. Each member binds to a heterotetrameric complex consisting of a distinct set of type I and type II recep-tors. TGF-β receptors are protein serine/threonine kinases and can phosphorylate the downstream substrates called SMAD proteins. Phosphorylated SMADs are directly transported into the nucleus, where they bind to the DNA and regulate gene expression that is responsible for inhibition of cell proliferation. Inactivation of the TGF-β pathway through genetic mutations in the TGF-β receptors or SMADs is frequent in human cancer,
|
Surgery_Schwartz. the nucleus, where they bind to gene promoters and cooperate with specific transcription factors to regulate the expression of genes that control cell proliferation and differen-tiation. For example, TGF-β strongly induces the transcription PlasmamembraneTGF b receptorGeneexpressionNucleusSMADTGF bAnti-proliferationFigure 15-11. TGF-β signaling pathway. The TGF-β family has at least 29 members encoded in the human genome. They are also peptide growth factors. Each member binds to a heterotetrameric complex consisting of a distinct set of type I and type II recep-tors. TGF-β receptors are protein serine/threonine kinases and can phosphorylate the downstream substrates called SMAD proteins. Phosphorylated SMADs are directly transported into the nucleus, where they bind to the DNA and regulate gene expression that is responsible for inhibition of cell proliferation. Inactivation of the TGF-β pathway through genetic mutations in the TGF-β receptors or SMADs is frequent in human cancer,
|
Surgery_Schwartz_3334
|
Surgery_Schwartz
|
gene expression that is responsible for inhibition of cell proliferation. Inactivation of the TGF-β pathway through genetic mutations in the TGF-β receptors or SMADs is frequent in human cancer, leading to the uncontrolled proliferation of cancer cells.of a gene called p15INK4B (a type of CKI) and, at the same time, reduces the expression of many oncogenes such as c-Myc. The outcome of the altered gene expression leads to the inhibition of cell cycle progression. Meanwhile, the strength and dura-tion of TGF-β signaling is fine-tuned by a variety of positive or negative modulators, including protein phosphatases. Therefore, controlled activation of TGF-β signaling is an intrinsic mecha-nism for cells to ensure controlled proliferation.Resistance to TGF-β’s anticancer action is one hall-mark of human cancer cells. TGF-β receptors and SMADs are identified as tumor suppressors. The TGF-β signaling circuit can be disrupted in a variety of ways and in different types of human tumors. Some
|
Surgery_Schwartz. gene expression that is responsible for inhibition of cell proliferation. Inactivation of the TGF-β pathway through genetic mutations in the TGF-β receptors or SMADs is frequent in human cancer, leading to the uncontrolled proliferation of cancer cells.of a gene called p15INK4B (a type of CKI) and, at the same time, reduces the expression of many oncogenes such as c-Myc. The outcome of the altered gene expression leads to the inhibition of cell cycle progression. Meanwhile, the strength and dura-tion of TGF-β signaling is fine-tuned by a variety of positive or negative modulators, including protein phosphatases. Therefore, controlled activation of TGF-β signaling is an intrinsic mecha-nism for cells to ensure controlled proliferation.Resistance to TGF-β’s anticancer action is one hall-mark of human cancer cells. TGF-β receptors and SMADs are identified as tumor suppressors. The TGF-β signaling circuit can be disrupted in a variety of ways and in different types of human tumors. Some
|
Surgery_Schwartz_3335
|
Surgery_Schwartz
|
of human cancer cells. TGF-β receptors and SMADs are identified as tumor suppressors. The TGF-β signaling circuit can be disrupted in a variety of ways and in different types of human tumors. Some lose TGF-β responsiveness through downregulation or mutations of their TGF-β receptors. The cytoplasmic SMAD4 protein, which transduces signals from ligand-activated TGF-β receptors to downstream targets, may be eliminated through mutation of its encoding gene. The locus encoding cell cycle inhibitor p15INK4B may be deleted. Alterna-tively, the immediate downstream target of its actions, cyclin-dependent kinase 4 (CDK4), may become unresponsive to the inhibitory actions of p15INK4B because of mutations that block p15INK4B binding. The resulting cyclin D/CDK4 complexes con-stitutively inactivate tumor suppressor pRb by hyperphosphory-lation. Finally, functional pRb, the end target of this pathway, may be lost through mutation of its gene. For example, in pan-creatic and colorectal cancers,
|
Surgery_Schwartz. of human cancer cells. TGF-β receptors and SMADs are identified as tumor suppressors. The TGF-β signaling circuit can be disrupted in a variety of ways and in different types of human tumors. Some lose TGF-β responsiveness through downregulation or mutations of their TGF-β receptors. The cytoplasmic SMAD4 protein, which transduces signals from ligand-activated TGF-β receptors to downstream targets, may be eliminated through mutation of its encoding gene. The locus encoding cell cycle inhibitor p15INK4B may be deleted. Alterna-tively, the immediate downstream target of its actions, cyclin-dependent kinase 4 (CDK4), may become unresponsive to the inhibitory actions of p15INK4B because of mutations that block p15INK4B binding. The resulting cyclin D/CDK4 complexes con-stitutively inactivate tumor suppressor pRb by hyperphosphory-lation. Finally, functional pRb, the end target of this pathway, may be lost through mutation of its gene. For example, in pan-creatic and colorectal cancers,
|
Surgery_Schwartz_3336
|
Surgery_Schwartz
|
tumor suppressor pRb by hyperphosphory-lation. Finally, functional pRb, the end target of this pathway, may be lost through mutation of its gene. For example, in pan-creatic and colorectal cancers, 100% of cells derived from these cancers carry genetic defects in the TGF-β signaling pathway. Therefore, the antiproliferative pathway converging onto pRb Brunicardi_Ch15_p0479-p0510.indd 48918/02/19 11:12 AM 490BASIC CONSIDERATIONSPART Iand the cell division cycle is, in one way or another, disrupted in a majority of human cancer cells. Besides cancer, dysregu-lation of TGF-β signaling also has been associated with other human diseases such as Marfan’s syndrome and thoracic aortic aneurysm.Gene Therapy and Molecular Drugs in CancerModern advances in the use of molecular biology to manipulate genomes have greatly contributed to the understanding of the molecular basis for how cells live, die, or differentiate. Given the fact that human diseases arise from improper changes in the
|
Surgery_Schwartz. tumor suppressor pRb by hyperphosphory-lation. Finally, functional pRb, the end target of this pathway, may be lost through mutation of its gene. For example, in pan-creatic and colorectal cancers, 100% of cells derived from these cancers carry genetic defects in the TGF-β signaling pathway. Therefore, the antiproliferative pathway converging onto pRb Brunicardi_Ch15_p0479-p0510.indd 48918/02/19 11:12 AM 490BASIC CONSIDERATIONSPART Iand the cell division cycle is, in one way or another, disrupted in a majority of human cancer cells. Besides cancer, dysregu-lation of TGF-β signaling also has been associated with other human diseases such as Marfan’s syndrome and thoracic aortic aneurysm.Gene Therapy and Molecular Drugs in CancerModern advances in the use of molecular biology to manipulate genomes have greatly contributed to the understanding of the molecular basis for how cells live, die, or differentiate. Given the fact that human diseases arise from improper changes in the
|
Surgery_Schwartz_3337
|
Surgery_Schwartz
|
genomes have greatly contributed to the understanding of the molecular basis for how cells live, die, or differentiate. Given the fact that human diseases arise from improper changes in the genome, the continuous understanding of how the genome func-tions will make it possible to tailor medicine on an individual basis. Although significant hurdles remain, the course toward therapeutic application of molecular biology already has been mapped out by many proof-of-principle studies in the literature. In this section, cancer is used as an example to elaborate some therapeutic applications of molecular biology. Modern molecu-lar medicine includes gene therapy and molecular drugs that target genes or gene products within human cells.Cancer is a complex disease, involving uncontrolled growth and spread of tumor cells (Fig. 15-12). Cancer development depends on the acquisition and selection of specific character-istics that set the tumor cell apart from normal somatic cells. Cancer cells have
|
Surgery_Schwartz. genomes have greatly contributed to the understanding of the molecular basis for how cells live, die, or differentiate. Given the fact that human diseases arise from improper changes in the genome, the continuous understanding of how the genome func-tions will make it possible to tailor medicine on an individual basis. Although significant hurdles remain, the course toward therapeutic application of molecular biology already has been mapped out by many proof-of-principle studies in the literature. In this section, cancer is used as an example to elaborate some therapeutic applications of molecular biology. Modern molecu-lar medicine includes gene therapy and molecular drugs that target genes or gene products within human cells.Cancer is a complex disease, involving uncontrolled growth and spread of tumor cells (Fig. 15-12). Cancer development depends on the acquisition and selection of specific character-istics that set the tumor cell apart from normal somatic cells. Cancer cells have
|
Surgery_Schwartz_3338
|
Surgery_Schwartz
|
spread of tumor cells (Fig. 15-12). Cancer development depends on the acquisition and selection of specific character-istics that set the tumor cell apart from normal somatic cells. Cancer cells have defects in regulatory circuits that govern nor-mal cell proliferation and homeostasis. Many lines of evidence indicate that tumorigenesis in humans is a multistep process and that these steps reflect genetic alterations that drive the progres-sive transformation of normal human cells into highly malignant derivatives. The genomes of tumor cells are invariably altered at multiple sites, having suffered disruption through lesions as sub-tle as point mutations and as obvious as changes in chromosome complement. A succession of genetic changes, each conferring one or another type of growth advantage, leads to the progressive conversion of normal human cells into cancer cells.Cancer research in the past 20 years has generated a rich and complex body of knowledge, revealing cancer to be a
|
Surgery_Schwartz. spread of tumor cells (Fig. 15-12). Cancer development depends on the acquisition and selection of specific character-istics that set the tumor cell apart from normal somatic cells. Cancer cells have defects in regulatory circuits that govern nor-mal cell proliferation and homeostasis. Many lines of evidence indicate that tumorigenesis in humans is a multistep process and that these steps reflect genetic alterations that drive the progres-sive transformation of normal human cells into highly malignant derivatives. The genomes of tumor cells are invariably altered at multiple sites, having suffered disruption through lesions as sub-tle as point mutations and as obvious as changes in chromosome complement. A succession of genetic changes, each conferring one or another type of growth advantage, leads to the progressive conversion of normal human cells into cancer cells.Cancer research in the past 20 years has generated a rich and complex body of knowledge, revealing cancer to be a
|
Surgery_Schwartz_3339
|
Surgery_Schwartz
|
leads to the progressive conversion of normal human cells into cancer cells.Cancer research in the past 20 years has generated a rich and complex body of knowledge, revealing cancer to be a dis-ease involving dynamic changes in the genome. The causes of cancer include genetic predisposition, environmental influences, infectious agents, and aging. These transform normal cells into cancerous ones by derailing a wide spectrum of regulatory pathways including signal transduction pathways, cell cycle machinery, or apoptotic pathways.15,16 The early notion that cancer was caused by mutations in genes critical for the control of cell growth implied that genome stability is important for preventing oncogenesis. There are two classes of cancer genes in which alteration has been identified in human and animal cancer cells: oncogenes, with dominant gain-of-function muta-tions, and tumor suppressor genes, with recessive loss-of-function mutations. In normal cells, oncogenes promote cell growth by
|
Surgery_Schwartz. leads to the progressive conversion of normal human cells into cancer cells.Cancer research in the past 20 years has generated a rich and complex body of knowledge, revealing cancer to be a dis-ease involving dynamic changes in the genome. The causes of cancer include genetic predisposition, environmental influences, infectious agents, and aging. These transform normal cells into cancerous ones by derailing a wide spectrum of regulatory pathways including signal transduction pathways, cell cycle machinery, or apoptotic pathways.15,16 The early notion that cancer was caused by mutations in genes critical for the control of cell growth implied that genome stability is important for preventing oncogenesis. There are two classes of cancer genes in which alteration has been identified in human and animal cancer cells: oncogenes, with dominant gain-of-function muta-tions, and tumor suppressor genes, with recessive loss-of-function mutations. In normal cells, oncogenes promote cell growth by
|
Surgery_Schwartz_3340
|
Surgery_Schwartz
|
and animal cancer cells: oncogenes, with dominant gain-of-function muta-tions, and tumor suppressor genes, with recessive loss-of-function mutations. In normal cells, oncogenes promote cell growth by activating cell cycle progression, whereas tumor suppres-sors counteract oncogenes’ functions. Therefore, the balance between oncogenes and tumor suppressors maintains a well-controlled state of cell growth.During the development of most types of human cancer, cancer cells can break away from primary tumor masses, invade adjacent tissues, and hence travel to distant sites where they form new colonies. This spreading process of tumor cells, called metastasis, is the cause of 90% of human cancer deaths. Meta-static cancer cells that enter the bloodstream can reach virtu-ally all tissues of the body. Bones are one of the most common places for these cells to settle and start growing again. Bone Figure 15-12. Tumor clonal evolution and metastasis. A tumor develops from mutant cells with
|
Surgery_Schwartz. and animal cancer cells: oncogenes, with dominant gain-of-function muta-tions, and tumor suppressor genes, with recessive loss-of-function mutations. In normal cells, oncogenes promote cell growth by activating cell cycle progression, whereas tumor suppres-sors counteract oncogenes’ functions. Therefore, the balance between oncogenes and tumor suppressors maintains a well-controlled state of cell growth.During the development of most types of human cancer, cancer cells can break away from primary tumor masses, invade adjacent tissues, and hence travel to distant sites where they form new colonies. This spreading process of tumor cells, called metastasis, is the cause of 90% of human cancer deaths. Meta-static cancer cells that enter the bloodstream can reach virtu-ally all tissues of the body. Bones are one of the most common places for these cells to settle and start growing again. Bone Figure 15-12. Tumor clonal evolution and metastasis. A tumor develops from mutant cells with
|
Surgery_Schwartz_3341
|
Surgery_Schwartz
|
the body. Bones are one of the most common places for these cells to settle and start growing again. Bone Figure 15-12. Tumor clonal evolution and metastasis. A tumor develops from mutant cells with multiple genetic mutations. Through repeated alterations in the genome, mutant epithelial cells are able to develop into a cluster of cells (called a tumor clone) that proliferates in an uncontrollable fashion. Further changes in the tumor cells can transform the tumor cells into a population of cells that can enter the blood vessels and repopulate in a new location.Tumor cells escape fromblood vessel and proliferateto form metastatic tumorsBlood vesselTumor cells break looseand enter bloodstreamUncontrolled cellproliferationCell proliferationCell proliferationCell with multiplemutationsCell with two mutationsMutant epithelial cellNormal epithelial cellmetastasis is one of the most frequent causes of pain in people with cancer. It also can cause bones to break and create other symptoms and
|
Surgery_Schwartz. the body. Bones are one of the most common places for these cells to settle and start growing again. Bone Figure 15-12. Tumor clonal evolution and metastasis. A tumor develops from mutant cells with multiple genetic mutations. Through repeated alterations in the genome, mutant epithelial cells are able to develop into a cluster of cells (called a tumor clone) that proliferates in an uncontrollable fashion. Further changes in the tumor cells can transform the tumor cells into a population of cells that can enter the blood vessels and repopulate in a new location.Tumor cells escape fromblood vessel and proliferateto form metastatic tumorsBlood vesselTumor cells break looseand enter bloodstreamUncontrolled cellproliferationCell proliferationCell proliferationCell with multiplemutationsCell with two mutationsMutant epithelial cellNormal epithelial cellmetastasis is one of the most frequent causes of pain in people with cancer. It also can cause bones to break and create other symptoms and
|
Surgery_Schwartz_3342
|
Surgery_Schwartz
|
two mutationsMutant epithelial cellNormal epithelial cellmetastasis is one of the most frequent causes of pain in people with cancer. It also can cause bones to break and create other symptoms and problems for patients.The progression in the knowledge of cancer biology has been accelerating in recent years. All of the scientific knowledge Brunicardi_Ch15_p0479-p0510.indd 49018/02/19 11:12 AM 491MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15acquired through hard work and discovery has made it possible for cancer treatment and prevention. As a result of explosive new discoveries, some modern treatments were developed. The success of these therapies, together with traditional treatments such as surgical procedures, is further underscored by the fact that in 2002 the cancer rate was reduced in the United States. Current approaches to the treatment of cancer involve killing cancer cells with toxic chemicals, radiation, or surgery. Alterna-tively,
|
Surgery_Schwartz. two mutationsMutant epithelial cellNormal epithelial cellmetastasis is one of the most frequent causes of pain in people with cancer. It also can cause bones to break and create other symptoms and problems for patients.The progression in the knowledge of cancer biology has been accelerating in recent years. All of the scientific knowledge Brunicardi_Ch15_p0479-p0510.indd 49018/02/19 11:12 AM 491MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15acquired through hard work and discovery has made it possible for cancer treatment and prevention. As a result of explosive new discoveries, some modern treatments were developed. The success of these therapies, together with traditional treatments such as surgical procedures, is further underscored by the fact that in 2002 the cancer rate was reduced in the United States. Current approaches to the treatment of cancer involve killing cancer cells with toxic chemicals, radiation, or surgery. Alterna-tively,
|
Surgery_Schwartz_3343
|
Surgery_Schwartz
|
that in 2002 the cancer rate was reduced in the United States. Current approaches to the treatment of cancer involve killing cancer cells with toxic chemicals, radiation, or surgery. Alterna-tively, several new biologicand gene-based therapies are aimed at enhancing the body’s natural defenses against invading can-cers. Understanding the biology of cancer cells has led to the development of designer therapies for cancer prevention and treatment. Gene therapy, immune system modulation, geneti-cally engineered antibodies, and molecularly designed chemical drugs are all promising fronts in the war against cancer.Immunotherapy. The growth of the body is controlled by many natural signals through complex signaling pathways. Some of these natural agents have been used in cancer treatment and have been proven effective for fighting several cancers through the clinical trial process. These naturally occurring biologic agents, such as interferons, interleukins, and other cytokines, can now be
|
Surgery_Schwartz. that in 2002 the cancer rate was reduced in the United States. Current approaches to the treatment of cancer involve killing cancer cells with toxic chemicals, radiation, or surgery. Alterna-tively, several new biologicand gene-based therapies are aimed at enhancing the body’s natural defenses against invading can-cers. Understanding the biology of cancer cells has led to the development of designer therapies for cancer prevention and treatment. Gene therapy, immune system modulation, geneti-cally engineered antibodies, and molecularly designed chemical drugs are all promising fronts in the war against cancer.Immunotherapy. The growth of the body is controlled by many natural signals through complex signaling pathways. Some of these natural agents have been used in cancer treatment and have been proven effective for fighting several cancers through the clinical trial process. These naturally occurring biologic agents, such as interferons, interleukins, and other cytokines, can now be
|
Surgery_Schwartz_3344
|
Surgery_Schwartz
|
been proven effective for fighting several cancers through the clinical trial process. These naturally occurring biologic agents, such as interferons, interleukins, and other cytokines, can now be produced in the laboratory. These agents, as well as the synthetic agents that mimic the natural signals, are given to patients to influence the natural immune response agents either by directly altering the cancer cell growth or by acting indirectly to help healthy cells control the cancer. One of the most exciting applications of immunotherapy has come from the identification of certain tumor targets called antigens and the aiming of an antibody at these targets. This was first used as a means of local-izing tumors in the body for diagnosis and was more recently used to attack cancer cells. Trastuzumab (Herceptin) is an exam-ple of such a drug.17 Trastuzumab is a monoclonal antibody that neutralizes the mitogenic activity of cell-surface growth fac-tor receptor HER-2, which is
|
Surgery_Schwartz. been proven effective for fighting several cancers through the clinical trial process. These naturally occurring biologic agents, such as interferons, interleukins, and other cytokines, can now be produced in the laboratory. These agents, as well as the synthetic agents that mimic the natural signals, are given to patients to influence the natural immune response agents either by directly altering the cancer cell growth or by acting indirectly to help healthy cells control the cancer. One of the most exciting applications of immunotherapy has come from the identification of certain tumor targets called antigens and the aiming of an antibody at these targets. This was first used as a means of local-izing tumors in the body for diagnosis and was more recently used to attack cancer cells. Trastuzumab (Herceptin) is an exam-ple of such a drug.17 Trastuzumab is a monoclonal antibody that neutralizes the mitogenic activity of cell-surface growth fac-tor receptor HER-2, which is
|
Surgery_Schwartz_3345
|
Surgery_Schwartz
|
cells. Trastuzumab (Herceptin) is an exam-ple of such a drug.17 Trastuzumab is a monoclonal antibody that neutralizes the mitogenic activity of cell-surface growth fac-tor receptor HER-2, which is overexpressed in approximately 25% of breast cancers. HER-2–overexpressing tumors tend to grow faster and generally are more likely to recur than tumors that do not overproduce HER-2. Trastuzumab is designed to attack cancer cells that overexpress HER-2 by slowing or pre-venting the growth of these cells, resulting in increased survival of HER-2–positive breast cancer patients. Another significant example is the administration of interleukin-2 (IL-2) to patients with metastatic melanoma or kidney cancer, which has been shown to mediate the durable regression of metastatic cancer. IL-2, a cytokine produced by human helper T lymphocytes, has a wide range of immune regulatory effects, including the expansion of lymphocytes following activation by a specific antigen. Although IL-2 has no direct
|
Surgery_Schwartz. cells. Trastuzumab (Herceptin) is an exam-ple of such a drug.17 Trastuzumab is a monoclonal antibody that neutralizes the mitogenic activity of cell-surface growth fac-tor receptor HER-2, which is overexpressed in approximately 25% of breast cancers. HER-2–overexpressing tumors tend to grow faster and generally are more likely to recur than tumors that do not overproduce HER-2. Trastuzumab is designed to attack cancer cells that overexpress HER-2 by slowing or pre-venting the growth of these cells, resulting in increased survival of HER-2–positive breast cancer patients. Another significant example is the administration of interleukin-2 (IL-2) to patients with metastatic melanoma or kidney cancer, which has been shown to mediate the durable regression of metastatic cancer. IL-2, a cytokine produced by human helper T lymphocytes, has a wide range of immune regulatory effects, including the expansion of lymphocytes following activation by a specific antigen. Although IL-2 has no direct
|
Surgery_Schwartz_3346
|
Surgery_Schwartz
|
produced by human helper T lymphocytes, has a wide range of immune regulatory effects, including the expansion of lymphocytes following activation by a specific antigen. Although IL-2 has no direct impact on cancer cells, the impact of IL-2 on cancers in vivo derives from its ability to expand lymphocytes with antitumor activity. The expanded lymphocyte pool enables recognition of the antigen on cancer cells. Thus, the molecular identification of cancer antigens has opened new possibilities for the development of effective immunotherapies for patients with cancer. Clinical studies using immunization with peptides derived from cancer antigens have shown that high levels of lymphocytes with antitumor activity can be produced in cancer-bearing patients. Highly avid antitu-mor lymphocytes can be isolated from immunized patients and grown in vitro for use in cell-transfer therapies.Chemotherapy. The primary function of anticancer chemicals is to block different steps involved in cell
|
Surgery_Schwartz. produced by human helper T lymphocytes, has a wide range of immune regulatory effects, including the expansion of lymphocytes following activation by a specific antigen. Although IL-2 has no direct impact on cancer cells, the impact of IL-2 on cancers in vivo derives from its ability to expand lymphocytes with antitumor activity. The expanded lymphocyte pool enables recognition of the antigen on cancer cells. Thus, the molecular identification of cancer antigens has opened new possibilities for the development of effective immunotherapies for patients with cancer. Clinical studies using immunization with peptides derived from cancer antigens have shown that high levels of lymphocytes with antitumor activity can be produced in cancer-bearing patients. Highly avid antitu-mor lymphocytes can be isolated from immunized patients and grown in vitro for use in cell-transfer therapies.Chemotherapy. The primary function of anticancer chemicals is to block different steps involved in cell
|
Surgery_Schwartz_3347
|
Surgery_Schwartz
|
can be isolated from immunized patients and grown in vitro for use in cell-transfer therapies.Chemotherapy. The primary function of anticancer chemicals is to block different steps involved in cell growth and replica-tion. These chemicals often block a critical chemical reaction in a signal transduction pathway or during DNA replication or gene expression. For example, STI571, also known as Gleevec, is one of the first molecularly targeted drugs based on the changes that cancer causes in cells.18 STI571 offers promise for the treatment of chronic myeloid leukemia (CML) and may soon surpass interferon-γ as the standard treatment for the disease. In CML, STI571 is targeted at the Bcr-Abl kinase, an activated oncogene product in CML (Fig. 15-13). Bcr-Abl is an overly activated protein kinase resulting from a specific genetic abnor-mality generated by chromosomal translocation that is found in the cells of patients with CML. STI571-mediated inhibi-tion of Bcr-Abl kinase activity not only
|
Surgery_Schwartz. can be isolated from immunized patients and grown in vitro for use in cell-transfer therapies.Chemotherapy. The primary function of anticancer chemicals is to block different steps involved in cell growth and replica-tion. These chemicals often block a critical chemical reaction in a signal transduction pathway or during DNA replication or gene expression. For example, STI571, also known as Gleevec, is one of the first molecularly targeted drugs based on the changes that cancer causes in cells.18 STI571 offers promise for the treatment of chronic myeloid leukemia (CML) and may soon surpass interferon-γ as the standard treatment for the disease. In CML, STI571 is targeted at the Bcr-Abl kinase, an activated oncogene product in CML (Fig. 15-13). Bcr-Abl is an overly activated protein kinase resulting from a specific genetic abnor-mality generated by chromosomal translocation that is found in the cells of patients with CML. STI571-mediated inhibi-tion of Bcr-Abl kinase activity not only
|
Surgery_Schwartz_3348
|
Surgery_Schwartz
|
resulting from a specific genetic abnor-mality generated by chromosomal translocation that is found in the cells of patients with CML. STI571-mediated inhibi-tion of Bcr-Abl kinase activity not only prevents cell growth of Bcr-Abl–transformed leukemic cells, but also induces apoptosis. Clinically, the drug quickly corrects the blood cell abnormali-ties caused by the leukemia in a majority of patients, achiev-ing a complete disappearance of the leukemic blood cells and the return of normal blood cells. Additionally, the drug appears to have some effect on other cancers including certain brain tumors and gastrointestinal (GI) stromal tumors, a very rare type of stomach cancer.Gene Therapy. Gene therapy is an experimental treatment that involves genetically altering a patient’s own tumor cells or lym-phocytes (cells of the immune system, some of which can attack cancer cells). For years, the concept of gene therapy has held promise as a new, potentially potent weapon to attack cancer.
|
Surgery_Schwartz. resulting from a specific genetic abnor-mality generated by chromosomal translocation that is found in the cells of patients with CML. STI571-mediated inhibi-tion of Bcr-Abl kinase activity not only prevents cell growth of Bcr-Abl–transformed leukemic cells, but also induces apoptosis. Clinically, the drug quickly corrects the blood cell abnormali-ties caused by the leukemia in a majority of patients, achiev-ing a complete disappearance of the leukemic blood cells and the return of normal blood cells. Additionally, the drug appears to have some effect on other cancers including certain brain tumors and gastrointestinal (GI) stromal tumors, a very rare type of stomach cancer.Gene Therapy. Gene therapy is an experimental treatment that involves genetically altering a patient’s own tumor cells or lym-phocytes (cells of the immune system, some of which can attack cancer cells). For years, the concept of gene therapy has held promise as a new, potentially potent weapon to attack cancer.
|
Surgery_Schwartz_3349
|
Surgery_Schwartz
|
or lym-phocytes (cells of the immune system, some of which can attack cancer cells). For years, the concept of gene therapy has held promise as a new, potentially potent weapon to attack cancer. Although a rapid progression in the understanding of the molec-ular and clinical aspects of gene therapy has been witnessed in the past decade, gene therapy treatment has not yet been shown to be superior to standard treatments in humans.Inactive(In the absence of ATP)Overly activeUncontrolledcell proliferationBlockedcell proliferationBlocked activityATPSubstrateBcr-AblkinasePO4TyrSTI571SubstrateBcr-AblkinaseTyrSubstrateBcr-AblkinaseTyrFigure 15-13. Mechanism of STI571 as a molecular drug. Bcr-Abl is an overly activated oncogene product resulting from a specific genetic abnormality generated by chromo-somal translocation that is found in cells of patients with chronic myeloid leukemia. Bcr-Abl is an activated protein kinase and thus requires adenosine triphosphate (ATP) to phosphorylate
|
Surgery_Schwartz. or lym-phocytes (cells of the immune system, some of which can attack cancer cells). For years, the concept of gene therapy has held promise as a new, potentially potent weapon to attack cancer. Although a rapid progression in the understanding of the molec-ular and clinical aspects of gene therapy has been witnessed in the past decade, gene therapy treatment has not yet been shown to be superior to standard treatments in humans.Inactive(In the absence of ATP)Overly activeUncontrolledcell proliferationBlockedcell proliferationBlocked activityATPSubstrateBcr-AblkinasePO4TyrSTI571SubstrateBcr-AblkinaseTyrSubstrateBcr-AblkinaseTyrFigure 15-13. Mechanism of STI571 as a molecular drug. Bcr-Abl is an overly activated oncogene product resulting from a specific genetic abnormality generated by chromo-somal translocation that is found in cells of patients with chronic myeloid leukemia. Bcr-Abl is an activated protein kinase and thus requires adenosine triphosphate (ATP) to phosphorylate
|
Surgery_Schwartz_3350
|
Surgery_Schwartz
|
by chromo-somal translocation that is found in cells of patients with chronic myeloid leukemia. Bcr-Abl is an activated protein kinase and thus requires adenosine triphosphate (ATP) to phosphorylate substrates, which in turn promote cell proliferation. STI571 is a small molecule that competes with the ATP-bind-ing site and thus blocks the transfer of phos-phoryl group to substrate. PO4 = phosphate; Tyr = tyrosine.Brunicardi_Ch15_p0479-p0510.indd 49118/02/19 11:12 AM 492BASIC CONSIDERATIONSPART ISeveral problems must be resolved to transform it into a clinically relevant form of therapy. The major issues that limit its translation to the clinic are improving the selectivity of tumor targeting, improving the delivery to the tumor, and the enhance-ment of the transduction rate of the cells of interest. In most gene therapy trials for malignant diseases, tumors can be accessed and directly injected (in situ gene therapy). In situ gene therapy also offers a better distribution of the
|
Surgery_Schwartz. by chromo-somal translocation that is found in cells of patients with chronic myeloid leukemia. Bcr-Abl is an activated protein kinase and thus requires adenosine triphosphate (ATP) to phosphorylate substrates, which in turn promote cell proliferation. STI571 is a small molecule that competes with the ATP-bind-ing site and thus blocks the transfer of phos-phoryl group to substrate. PO4 = phosphate; Tyr = tyrosine.Brunicardi_Ch15_p0479-p0510.indd 49118/02/19 11:12 AM 492BASIC CONSIDERATIONSPART ISeveral problems must be resolved to transform it into a clinically relevant form of therapy. The major issues that limit its translation to the clinic are improving the selectivity of tumor targeting, improving the delivery to the tumor, and the enhance-ment of the transduction rate of the cells of interest. In most gene therapy trials for malignant diseases, tumors can be accessed and directly injected (in situ gene therapy). In situ gene therapy also offers a better distribution of the
|
Surgery_Schwartz_3351
|
Surgery_Schwartz
|
of interest. In most gene therapy trials for malignant diseases, tumors can be accessed and directly injected (in situ gene therapy). In situ gene therapy also offers a better distribution of the vector virus throughout the tumor. Finally, a combination of gene therapy strategies will be more effective than the use of a single gene therapy system. An important aspect of effective gene therapy involves the choice of appropriate genes for manipulation. Genes that promote the production of messenger chemicals or other immune-active sub-stances can be transferred into the patient’s cells. These include genes that inhibit cell cycle progression, induce apoptosis, enhance host immunity against cancer cells, block the ability of cancer cells to metastasize, and cause tumor cells to undergo suicide. Recent development of RNAi technology, which uses a loss-of-function approach to block gene functions, ensures a new wave of hopes for gene therapy. Nonetheless, gene therapy is still experimental
|
Surgery_Schwartz. of interest. In most gene therapy trials for malignant diseases, tumors can be accessed and directly injected (in situ gene therapy). In situ gene therapy also offers a better distribution of the vector virus throughout the tumor. Finally, a combination of gene therapy strategies will be more effective than the use of a single gene therapy system. An important aspect of effective gene therapy involves the choice of appropriate genes for manipulation. Genes that promote the production of messenger chemicals or other immune-active sub-stances can be transferred into the patient’s cells. These include genes that inhibit cell cycle progression, induce apoptosis, enhance host immunity against cancer cells, block the ability of cancer cells to metastasize, and cause tumor cells to undergo suicide. Recent development of RNAi technology, which uses a loss-of-function approach to block gene functions, ensures a new wave of hopes for gene therapy. Nonetheless, gene therapy is still experimental
|
Surgery_Schwartz_3352
|
Surgery_Schwartz
|
Recent development of RNAi technology, which uses a loss-of-function approach to block gene functions, ensures a new wave of hopes for gene therapy. Nonetheless, gene therapy is still experimental and is being studied in clinical trials for many different types of cancer. The mapping of genes respon-sible for human cancer is likely to provide new targets for gene therapy in the future. The preliminary results of gene therapy for cancer are encouraging, and as advancements are made in the understanding of the molecular biology of human cancer, the future of this rapidly developing field holds great potential for treating cancer.It is noteworthy that the use of multiple therapeutic meth-ods has proven more powerful than a single method. The use of chemotherapy after surgery to destroy the few remaining cancerous cells in the body is called adjuvant therapy. Adju-vant therapy was first tested and found to be effective in breast cancer. It was later adopted for use in other cancers. A
|
Surgery_Schwartz. Recent development of RNAi technology, which uses a loss-of-function approach to block gene functions, ensures a new wave of hopes for gene therapy. Nonetheless, gene therapy is still experimental and is being studied in clinical trials for many different types of cancer. The mapping of genes respon-sible for human cancer is likely to provide new targets for gene therapy in the future. The preliminary results of gene therapy for cancer are encouraging, and as advancements are made in the understanding of the molecular biology of human cancer, the future of this rapidly developing field holds great potential for treating cancer.It is noteworthy that the use of multiple therapeutic meth-ods has proven more powerful than a single method. The use of chemotherapy after surgery to destroy the few remaining cancerous cells in the body is called adjuvant therapy. Adju-vant therapy was first tested and found to be effective in breast cancer. It was later adopted for use in other cancers. A
|
Surgery_Schwartz_3353
|
Surgery_Schwartz
|
few remaining cancerous cells in the body is called adjuvant therapy. Adju-vant therapy was first tested and found to be effective in breast cancer. It was later adopted for use in other cancers. A major discovery in chemotherapy is the advantage of multiple che-motherapeutic agents (known as combination or cocktail chemotherapy) over single agents. Some types of fast-growing leukemias and lymphomas (tumors involving the cells of the bone marrow and lymph nodes) responded extremely well to combination chemotherapy, and clinical trials led to gradual improvement of the drug combinations used. Many of these tumors can be cured today by combination chemotherapy. As cancer cells carry multiple genetic defects, the use of combina-tion chemotherapy, immunotherapy, and gene therapies may be more effective in treating cancers.Stem Cell ResearchStem cell biology represents a cutting-edge scientific research field with potential clinical applications.19 It may have an enor-mous impact on human
|
Surgery_Schwartz. few remaining cancerous cells in the body is called adjuvant therapy. Adju-vant therapy was first tested and found to be effective in breast cancer. It was later adopted for use in other cancers. A major discovery in chemotherapy is the advantage of multiple che-motherapeutic agents (known as combination or cocktail chemotherapy) over single agents. Some types of fast-growing leukemias and lymphomas (tumors involving the cells of the bone marrow and lymph nodes) responded extremely well to combination chemotherapy, and clinical trials led to gradual improvement of the drug combinations used. Many of these tumors can be cured today by combination chemotherapy. As cancer cells carry multiple genetic defects, the use of combina-tion chemotherapy, immunotherapy, and gene therapies may be more effective in treating cancers.Stem Cell ResearchStem cell biology represents a cutting-edge scientific research field with potential clinical applications.19 It may have an enor-mous impact on human
|
Surgery_Schwartz_3354
|
Surgery_Schwartz
|
effective in treating cancers.Stem Cell ResearchStem cell biology represents a cutting-edge scientific research field with potential clinical applications.19 It may have an enor-mous impact on human health by offering hope for curing human diseases such as diabetes mellitus, Parkinson’s disease, neuro-logic degeneration, and congenital heart disease. Stem cells are endowed with two remarkable properties (Fig. 15-14). First, stem cells can proliferate in an undifferentiated but pluripotent state and, as a result, can self-renew. Second, they have the abil-ity to differentiate into many specialized cell types. There are two groups of stem cells: embryonic stem (ES) cells and adult stem cells.Human ES cells (hESCs) are derived from early preim-plantation embryos called blastocysts (5 days postfertilization) and are capable of generating all differentiated germ layers in the body by chimera assays or 2-D/3-D differentiation in a Self-renewalStem
|
Surgery_Schwartz. effective in treating cancers.Stem Cell ResearchStem cell biology represents a cutting-edge scientific research field with potential clinical applications.19 It may have an enor-mous impact on human health by offering hope for curing human diseases such as diabetes mellitus, Parkinson’s disease, neuro-logic degeneration, and congenital heart disease. Stem cells are endowed with two remarkable properties (Fig. 15-14). First, stem cells can proliferate in an undifferentiated but pluripotent state and, as a result, can self-renew. Second, they have the abil-ity to differentiate into many specialized cell types. There are two groups of stem cells: embryonic stem (ES) cells and adult stem cells.Human ES cells (hESCs) are derived from early preim-plantation embryos called blastocysts (5 days postfertilization) and are capable of generating all differentiated germ layers in the body by chimera assays or 2-D/3-D differentiation in a Self-renewalStem
|
Surgery_Schwartz_3355
|
Surgery_Schwartz
|
embryos called blastocysts (5 days postfertilization) and are capable of generating all differentiated germ layers in the body by chimera assays or 2-D/3-D differentiation in a Self-renewalStem cellDifferentiationTerminallydifferentiatedcellFigure 15-14. Stem cells. A stem cell is capable of self-renewal (unlimited cell cycle) and differentiation (becoming nondividing cells with specialized functions). Differentiating stem cells often undergo additional cell divisions before they become fully mature cells that carry out specific tissue functions.dish—ectoderm, mesoderm, and endoderm—and therefore are considered pluripotent. There are two pluripotent states associ-ated with hESCs, one of which is the classic culture with bFGF (basic fibroblast growth factor) and knock out replacer (KSR), termed as “primed” pluripotent state. More recently, “naive” hESC culture methods have been introduced based on mouse studies, by supplementing 2i inhibitors (MEK1 and GSK3β inhibitors) into the medium
|
Surgery_Schwartz. embryos called blastocysts (5 days postfertilization) and are capable of generating all differentiated germ layers in the body by chimera assays or 2-D/3-D differentiation in a Self-renewalStem cellDifferentiationTerminallydifferentiatedcellFigure 15-14. Stem cells. A stem cell is capable of self-renewal (unlimited cell cycle) and differentiation (becoming nondividing cells with specialized functions). Differentiating stem cells often undergo additional cell divisions before they become fully mature cells that carry out specific tissue functions.dish—ectoderm, mesoderm, and endoderm—and therefore are considered pluripotent. There are two pluripotent states associ-ated with hESCs, one of which is the classic culture with bFGF (basic fibroblast growth factor) and knock out replacer (KSR), termed as “primed” pluripotent state. More recently, “naive” hESC culture methods have been introduced based on mouse studies, by supplementing 2i inhibitors (MEK1 and GSK3β inhibitors) into the medium
|
Surgery_Schwartz_3356
|
Surgery_Schwartz
|
as “primed” pluripotent state. More recently, “naive” hESC culture methods have been introduced based on mouse studies, by supplementing 2i inhibitors (MEK1 and GSK3β inhibitors) into the medium in addition to bFGF.Adult stem cells are present in and can be isolated from adult tissues. They often are tissue specific and only can gen-erate the cell types comprising a particular tissue in the body; therefore, they are considered multipotent. However, in some cases, they can transdifferentiate into cell types found in other tissues, called transdifferentiation. For example, hematopoietic stem cells are adult stem cells. They reside in bone marrow and are capable of generating all cell types of the blood and immune system. Another example is mesenchymal stem cells (MSCs), which is initially identified in the bone marrow (BM) to sup-port hematopoietic stem cell homeostasis. In addition to BM, MSCs are also present in adipose tissue, umbilical cord, pla-centa, amniotic fluid, dental pulp,
|
Surgery_Schwartz. as “primed” pluripotent state. More recently, “naive” hESC culture methods have been introduced based on mouse studies, by supplementing 2i inhibitors (MEK1 and GSK3β inhibitors) into the medium in addition to bFGF.Adult stem cells are present in and can be isolated from adult tissues. They often are tissue specific and only can gen-erate the cell types comprising a particular tissue in the body; therefore, they are considered multipotent. However, in some cases, they can transdifferentiate into cell types found in other tissues, called transdifferentiation. For example, hematopoietic stem cells are adult stem cells. They reside in bone marrow and are capable of generating all cell types of the blood and immune system. Another example is mesenchymal stem cells (MSCs), which is initially identified in the bone marrow (BM) to sup-port hematopoietic stem cell homeostasis. In addition to BM, MSCs are also present in adipose tissue, umbilical cord, pla-centa, amniotic fluid, dental pulp,
|
Surgery_Schwartz_3357
|
Surgery_Schwartz
|
identified in the bone marrow (BM) to sup-port hematopoietic stem cell homeostasis. In addition to BM, MSCs are also present in adipose tissue, umbilical cord, pla-centa, amniotic fluid, dental pulp, skeletal muscle, tendons, and synovial, etc., and are reported to obtain the ability to differen-tiate into osteogenic, chondrogenic, and adipogenic lineages in vitro. Due to their characteristics of easy acquisition (from adi-pose tissue, for example), strong ex vivo proliferation, immune-modulatory function, and ability to migrate to damaged tissue, MSCs have been utilized in regenerative medicine.Stem cells can be grown in culture and be induced to dif-ferentiate into a particular cell type, either in vitro or in vivo. With the recent and continually increasing improvement in culturing stem cells, scientists are beginning to understand the molecular mechanisms of stem cell self-renewal and differentia-tion in response to environmental cues. It is believed that dis-covery of the signals
|
Surgery_Schwartz. identified in the bone marrow (BM) to sup-port hematopoietic stem cell homeostasis. In addition to BM, MSCs are also present in adipose tissue, umbilical cord, pla-centa, amniotic fluid, dental pulp, skeletal muscle, tendons, and synovial, etc., and are reported to obtain the ability to differen-tiate into osteogenic, chondrogenic, and adipogenic lineages in vitro. Due to their characteristics of easy acquisition (from adi-pose tissue, for example), strong ex vivo proliferation, immune-modulatory function, and ability to migrate to damaged tissue, MSCs have been utilized in regenerative medicine.Stem cells can be grown in culture and be induced to dif-ferentiate into a particular cell type, either in vitro or in vivo. With the recent and continually increasing improvement in culturing stem cells, scientists are beginning to understand the molecular mechanisms of stem cell self-renewal and differentia-tion in response to environmental cues. It is believed that dis-covery of the signals
|
Surgery_Schwartz_3358
|
Surgery_Schwartz
|
cells, scientists are beginning to understand the molecular mechanisms of stem cell self-renewal and differentia-tion in response to environmental cues. It is believed that dis-covery of the signals that control self-renewal vs differentiation will be extremely important for the therapeutic use of stem cells in treating disease. It is possible that success in the study of the changes in signal transduction pathways in stem cells will lead to the development of therapies to replace diseased or damaged cells in the body using stem cell derivatives. Recently, stem cell research has been transformed by the discovery from the Shinya Yamanaka group and the James Thomsen group, who have found that a simple genetic manipulation can reprogram adult differentiated cells back into pluripotent stem cells.20,21 This exciting discovery not only bypasses the ethical issues of using early embryos to generate ES cells, but also ensures a Brunicardi_Ch15_p0479-p0510.indd 49218/02/19 11:12 AM
|
Surgery_Schwartz. cells, scientists are beginning to understand the molecular mechanisms of stem cell self-renewal and differentia-tion in response to environmental cues. It is believed that dis-covery of the signals that control self-renewal vs differentiation will be extremely important for the therapeutic use of stem cells in treating disease. It is possible that success in the study of the changes in signal transduction pathways in stem cells will lead to the development of therapies to replace diseased or damaged cells in the body using stem cell derivatives. Recently, stem cell research has been transformed by the discovery from the Shinya Yamanaka group and the James Thomsen group, who have found that a simple genetic manipulation can reprogram adult differentiated cells back into pluripotent stem cells.20,21 This exciting discovery not only bypasses the ethical issues of using early embryos to generate ES cells, but also ensures a Brunicardi_Ch15_p0479-p0510.indd 49218/02/19 11:12 AM
|
Surgery_Schwartz_3359
|
Surgery_Schwartz
|
stem cells.20,21 This exciting discovery not only bypasses the ethical issues of using early embryos to generate ES cells, but also ensures a Brunicardi_Ch15_p0479-p0510.indd 49218/02/19 11:12 AM 493MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15potentially limitless source of patient-specific stem cells for tis-sue engineering and regenerative medicine.The Atomic Theory of Disease22As early as the 5th century b.c., the ancient Greek Democritus first proposed that matter is composed of indivisible particles called “atoms.” In the 17th and 18th centuries, Isaac Newton described the expansion of gases as rushed atoms into empty space. The existence of atoms was doubted until the discovery of subatomic particles in the 20th century, which demonstrated that the atom was actually divisible into protons, neutrons, and electrons.Over 100 years after this discovery, direct impacts from utilizing subatomic particles were revealed. This began with the
|
Surgery_Schwartz. stem cells.20,21 This exciting discovery not only bypasses the ethical issues of using early embryos to generate ES cells, but also ensures a Brunicardi_Ch15_p0479-p0510.indd 49218/02/19 11:12 AM 493MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15potentially limitless source of patient-specific stem cells for tis-sue engineering and regenerative medicine.The Atomic Theory of Disease22As early as the 5th century b.c., the ancient Greek Democritus first proposed that matter is composed of indivisible particles called “atoms.” In the 17th and 18th centuries, Isaac Newton described the expansion of gases as rushed atoms into empty space. The existence of atoms was doubted until the discovery of subatomic particles in the 20th century, which demonstrated that the atom was actually divisible into protons, neutrons, and electrons.Over 100 years after this discovery, direct impacts from utilizing subatomic particles were revealed. This began with the
|
Surgery_Schwartz_3360
|
Surgery_Schwartz
|
that the atom was actually divisible into protons, neutrons, and electrons.Over 100 years after this discovery, direct impacts from utilizing subatomic particles were revealed. This began with the discovery of the X-ray. The most advanced and well-applied atomic technologies include accurate imaging such as X-ray computed tomography (CT) scan, magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), and positron emission tomography (PET). Additional applica-tions include radiation oncology, which utilizes ionizing parti-cles to treat malignant diseases by inducing double-strand DNA breaks resulting in programed cell death.The staggering advances in anatomy, physiology, and molecular biology over the past centuries have led us to our current state in which the atom is now the anatomy of the 21st century. As 99% of the body is composed of six elements (oxygen, carbon, hydrogen, nitrogen, calcium, and phosphorus), the next great advance in medicine will be
|
Surgery_Schwartz. that the atom was actually divisible into protons, neutrons, and electrons.Over 100 years after this discovery, direct impacts from utilizing subatomic particles were revealed. This began with the discovery of the X-ray. The most advanced and well-applied atomic technologies include accurate imaging such as X-ray computed tomography (CT) scan, magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), and positron emission tomography (PET). Additional applica-tions include radiation oncology, which utilizes ionizing parti-cles to treat malignant diseases by inducing double-strand DNA breaks resulting in programed cell death.The staggering advances in anatomy, physiology, and molecular biology over the past centuries have led us to our current state in which the atom is now the anatomy of the 21st century. As 99% of the body is composed of six elements (oxygen, carbon, hydrogen, nitrogen, calcium, and phosphorus), the next great advance in medicine will be
|
Surgery_Schwartz_3361
|
Surgery_Schwartz
|
atom is now the anatomy of the 21st century. As 99% of the body is composed of six elements (oxygen, carbon, hydrogen, nitrogen, calcium, and phosphorus), the next great advance in medicine will be bridging the sub-atomic, molecular, and genomic levels by forming an atomic theory of disease, which states that alterations in the composi-tion of subatomic particles are the root cause of disease. The atomic theory of disease would include genetic alterations at the atomic/subatomic level that are akin to single nucleotide polymorphisms (SNPs), in which alleles for a gene differ on the exact nucleotide in a single location, which can change the ulti-mate protein structure. This can lead to subtle changes in func-tion or dramatic results that cause pathology. We hypothesize that on a subatomic level, there could potentially be polymor-phisms as well, in which there are subtle changes in the sea of subatomic particles. Isotopes, discovered 100 years ago, would fall into this category of
|
Surgery_Schwartz. atom is now the anatomy of the 21st century. As 99% of the body is composed of six elements (oxygen, carbon, hydrogen, nitrogen, calcium, and phosphorus), the next great advance in medicine will be bridging the sub-atomic, molecular, and genomic levels by forming an atomic theory of disease, which states that alterations in the composi-tion of subatomic particles are the root cause of disease. The atomic theory of disease would include genetic alterations at the atomic/subatomic level that are akin to single nucleotide polymorphisms (SNPs), in which alleles for a gene differ on the exact nucleotide in a single location, which can change the ulti-mate protein structure. This can lead to subtle changes in func-tion or dramatic results that cause pathology. We hypothesize that on a subatomic level, there could potentially be polymor-phisms as well, in which there are subtle changes in the sea of subatomic particles. Isotopes, discovered 100 years ago, would fall into this category of
|
Surgery_Schwartz_3362
|
Surgery_Schwartz
|
level, there could potentially be polymor-phisms as well, in which there are subtle changes in the sea of subatomic particles. Isotopes, discovered 100 years ago, would fall into this category of subatomic polymorphism, as they dif-fer in the number of neutrons present in the atom. Differences in other particles may not change the mass of the atom, but may alter some of the characteristics of the atom. This is where the basic human variations originate because an atomic polymerism would result in particular genetic change. Remarkably, somatic point mutations of KRAS and P53 are caused by a single proton shift in cytosine, which is known as tautomerization of cyto-sine. In turn, the tautomerized cytosine binds with adenine, and not guanine, thus leading to point mutations in KRAS and P53, which are well known driver mutations for many cancers. This is another example of alterations in subatomic particles that directly cause disease.A known example of a change in the subatomic milieu of
|
Surgery_Schwartz. level, there could potentially be polymor-phisms as well, in which there are subtle changes in the sea of subatomic particles. Isotopes, discovered 100 years ago, would fall into this category of subatomic polymorphism, as they dif-fer in the number of neutrons present in the atom. Differences in other particles may not change the mass of the atom, but may alter some of the characteristics of the atom. This is where the basic human variations originate because an atomic polymerism would result in particular genetic change. Remarkably, somatic point mutations of KRAS and P53 are caused by a single proton shift in cytosine, which is known as tautomerization of cyto-sine. In turn, the tautomerized cytosine binds with adenine, and not guanine, thus leading to point mutations in KRAS and P53, which are well known driver mutations for many cancers. This is another example of alterations in subatomic particles that directly cause disease.A known example of a change in the subatomic milieu of
|
Surgery_Schwartz_3363
|
Surgery_Schwartz
|
are well known driver mutations for many cancers. This is another example of alterations in subatomic particles that directly cause disease.A known example of a change in the subatomic milieu of an element leading to a disease process is that of methemo-globinemia, a disorder characterized by an overabundance of methemoglobin. Methemoglobin contains an oxidized form of iron (carrying an extra electron), as opposed to the reduced form in normal hemoglobin. This results in a shift in the oxygen-hemoglobin dissociation curve to the left, causing hypoxia. Met-hemoglobinemia can be congenital, due to a defect in an enzyme that normally reduces methemoglobin back to hemoglobin, or acquired, caused by breakdown products of drugs that can oxi-dize hemoglobin. Although there is less than 1% of methemo-globin normally present in human tissues, affecting local blood flow and inflammation through its effects on nitric oxide and heme, large quantities can lead to respiratory failure and
|
Surgery_Schwartz. are well known driver mutations for many cancers. This is another example of alterations in subatomic particles that directly cause disease.A known example of a change in the subatomic milieu of an element leading to a disease process is that of methemo-globinemia, a disorder characterized by an overabundance of methemoglobin. Methemoglobin contains an oxidized form of iron (carrying an extra electron), as opposed to the reduced form in normal hemoglobin. This results in a shift in the oxygen-hemoglobin dissociation curve to the left, causing hypoxia. Met-hemoglobinemia can be congenital, due to a defect in an enzyme that normally reduces methemoglobin back to hemoglobin, or acquired, caused by breakdown products of drugs that can oxi-dize hemoglobin. Although there is less than 1% of methemo-globin normally present in human tissues, affecting local blood flow and inflammation through its effects on nitric oxide and heme, large quantities can lead to respiratory failure and
|
Surgery_Schwartz_3364
|
Surgery_Schwartz
|
1% of methemo-globin normally present in human tissues, affecting local blood flow and inflammation through its effects on nitric oxide and heme, large quantities can lead to respiratory failure and death.Another example would be exposure to external energy such as radiation that leads to instability of nuclear genome. In Chernobyl and Fukushima, radiolabeled food was metabolized and incorporated into body cells and decay to emit gamma radi-ation, causing DNA damage. This radiation damage occurs pri-marily at a subatomic level from a radiobiologic point of view with a direct or indirect ionization of atoms. The clinical results depend on the tissue characteristics and the equilibrium between the damage applied to normal and diseased tissues.TECHNOLOGIES OF MOLECULAR AND CELL BIOLOGYDNA CloningSince the advent of recombinant DNA technology three decades ago, hundreds of thousands of genes have been identi-fied. Recombinant DNA technology is the technology that uses advanced enzymatic
|
Surgery_Schwartz. 1% of methemo-globin normally present in human tissues, affecting local blood flow and inflammation through its effects on nitric oxide and heme, large quantities can lead to respiratory failure and death.Another example would be exposure to external energy such as radiation that leads to instability of nuclear genome. In Chernobyl and Fukushima, radiolabeled food was metabolized and incorporated into body cells and decay to emit gamma radi-ation, causing DNA damage. This radiation damage occurs pri-marily at a subatomic level from a radiobiologic point of view with a direct or indirect ionization of atoms. The clinical results depend on the tissue characteristics and the equilibrium between the damage applied to normal and diseased tissues.TECHNOLOGIES OF MOLECULAR AND CELL BIOLOGYDNA CloningSince the advent of recombinant DNA technology three decades ago, hundreds of thousands of genes have been identi-fied. Recombinant DNA technology is the technology that uses advanced enzymatic
|
Surgery_Schwartz_3365
|
Surgery_Schwartz
|
the advent of recombinant DNA technology three decades ago, hundreds of thousands of genes have been identi-fied. Recombinant DNA technology is the technology that uses advanced enzymatic and microbiologic techniques to manipu-late DNA.23 Pure pieces of any DNA can be inserted into bac-teriophage DNA or other carrier DNA such as plasmids to produce recombinant DNA in bacteria. In this way, DNA can be reconstructed, amplified, and used to manipulate the functions of individual cells or even organisms. This technology, often referred to as DNA cloning, is the basis of all other DNA analy-sis methods. It is only with the awesome power of recombinant DNA technology that the completion of the Human Genome Project was possible. It also has led to the identification of the entire gene complements of organisms such as viruses, bacteria, worms, flies, and plants.Molecular cloning refers to the process of cloning a DNA fragment of interest into a DNA vector that ultimately is deliv-ered into
|
Surgery_Schwartz. the advent of recombinant DNA technology three decades ago, hundreds of thousands of genes have been identi-fied. Recombinant DNA technology is the technology that uses advanced enzymatic and microbiologic techniques to manipu-late DNA.23 Pure pieces of any DNA can be inserted into bac-teriophage DNA or other carrier DNA such as plasmids to produce recombinant DNA in bacteria. In this way, DNA can be reconstructed, amplified, and used to manipulate the functions of individual cells or even organisms. This technology, often referred to as DNA cloning, is the basis of all other DNA analy-sis methods. It is only with the awesome power of recombinant DNA technology that the completion of the Human Genome Project was possible. It also has led to the identification of the entire gene complements of organisms such as viruses, bacteria, worms, flies, and plants.Molecular cloning refers to the process of cloning a DNA fragment of interest into a DNA vector that ultimately is deliv-ered into
|
Surgery_Schwartz_3366
|
Surgery_Schwartz
|
of organisms such as viruses, bacteria, worms, flies, and plants.Molecular cloning refers to the process of cloning a DNA fragment of interest into a DNA vector that ultimately is deliv-ered into bacterial or mammalian cells or tissues24,25 (Fig. 15-15). This represents a very basic technique that is widely used in almost all areas of biomedical research. DNA vectors often are called plasmids, which are extrachromosomal molecules of DNA that vary in size and can replicate and be transmitted from bacterial cell to cell. Plasmids can be propagated either in the cytoplasm or after insertion, as part of the bacterial chromosome in Escherichia coli. The process of molecular cloning involves several steps of manipulation of DNA. First, the vector plasmid DNA is cleaved with a restriction enzyme to create compatible ends with the foreign DNA fragment to be cloned. The vec-tor and the DNA fragment are then joined in vitro by a DNA ligase. Alternatively, DNA cloning can be simply done through
|
Surgery_Schwartz. of organisms such as viruses, bacteria, worms, flies, and plants.Molecular cloning refers to the process of cloning a DNA fragment of interest into a DNA vector that ultimately is deliv-ered into bacterial or mammalian cells or tissues24,25 (Fig. 15-15). This represents a very basic technique that is widely used in almost all areas of biomedical research. DNA vectors often are called plasmids, which are extrachromosomal molecules of DNA that vary in size and can replicate and be transmitted from bacterial cell to cell. Plasmids can be propagated either in the cytoplasm or after insertion, as part of the bacterial chromosome in Escherichia coli. The process of molecular cloning involves several steps of manipulation of DNA. First, the vector plasmid DNA is cleaved with a restriction enzyme to create compatible ends with the foreign DNA fragment to be cloned. The vec-tor and the DNA fragment are then joined in vitro by a DNA ligase. Alternatively, DNA cloning can be simply done through
|
Surgery_Schwartz_3367
|
Surgery_Schwartz
|
to create compatible ends with the foreign DNA fragment to be cloned. The vec-tor and the DNA fragment are then joined in vitro by a DNA ligase. Alternatively, DNA cloning can be simply done through the so-called Gateway Technology that allows for the rapid and efficient transfer of DNA fragments between different cloning vectors while maintaining reading frame and orientation, with-out the use of restriction endonucleases and DNA ligase. The technology, which is based on the site-specific recombination system of bacteriophage l, is simple, fast, robust, and automat-able and thus compatible for high-throughput DNA cloning.Finally, the ligation product or the Gateway reaction prod-uct is introduced into competent host bacteria; this procedure is called transformation, which can be done by either calcium/heat shock or electroporation. Precautions must be taken in every step of cloning to generate the desired DNA construct. Brunicardi_Ch15_p0479-p0510.indd 49318/02/19 11:12 AM
|
Surgery_Schwartz. to create compatible ends with the foreign DNA fragment to be cloned. The vec-tor and the DNA fragment are then joined in vitro by a DNA ligase. Alternatively, DNA cloning can be simply done through the so-called Gateway Technology that allows for the rapid and efficient transfer of DNA fragments between different cloning vectors while maintaining reading frame and orientation, with-out the use of restriction endonucleases and DNA ligase. The technology, which is based on the site-specific recombination system of bacteriophage l, is simple, fast, robust, and automat-able and thus compatible for high-throughput DNA cloning.Finally, the ligation product or the Gateway reaction prod-uct is introduced into competent host bacteria; this procedure is called transformation, which can be done by either calcium/heat shock or electroporation. Precautions must be taken in every step of cloning to generate the desired DNA construct. Brunicardi_Ch15_p0479-p0510.indd 49318/02/19 11:12 AM
|
Surgery_Schwartz_3368
|
Surgery_Schwartz
|
by either calcium/heat shock or electroporation. Precautions must be taken in every step of cloning to generate the desired DNA construct. Brunicardi_Ch15_p0479-p0510.indd 49318/02/19 11:12 AM 494BASIC CONSIDERATIONSPART IFigure 15-15. Generation of recombinant DNA. The vector is a circular DNA molecule that is capable of replicating in Escherichia coli cells. Insert DNA (often your favorite gene) is ligated to the vector after ends of both DNA are properly treated with restriction enzymes. Ligated DNA (i.e., the recombinant plasmid DNA) is then transformed into E. coli cells, where it replicates to produce recombinant progenies. E. coli cells carrying the recombinant plasmid can be propagated to yield large quantities of plasmid DNA.InsertDNA of interestDigest withrestriction enzymeVectorLigationRecombinantplasmidIntroduceinto E. coliE. coli containingrecombinant plasmidPropagationE. coli containingrecombinant plasmidThe vector must be correctly prepared to maximize the creation
|
Surgery_Schwartz. by either calcium/heat shock or electroporation. Precautions must be taken in every step of cloning to generate the desired DNA construct. Brunicardi_Ch15_p0479-p0510.indd 49318/02/19 11:12 AM 494BASIC CONSIDERATIONSPART IFigure 15-15. Generation of recombinant DNA. The vector is a circular DNA molecule that is capable of replicating in Escherichia coli cells. Insert DNA (often your favorite gene) is ligated to the vector after ends of both DNA are properly treated with restriction enzymes. Ligated DNA (i.e., the recombinant plasmid DNA) is then transformed into E. coli cells, where it replicates to produce recombinant progenies. E. coli cells carrying the recombinant plasmid can be propagated to yield large quantities of plasmid DNA.InsertDNA of interestDigest withrestriction enzymeVectorLigationRecombinantplasmidIntroduceinto E. coliE. coli containingrecombinant plasmidPropagationE. coli containingrecombinant plasmidThe vector must be correctly prepared to maximize the creation
|
Surgery_Schwartz_3369
|
Surgery_Schwartz
|
E. coliE. coli containingrecombinant plasmidPropagationE. coli containingrecombinant plasmidThe vector must be correctly prepared to maximize the creation of recombinants; for example, it must be enzymatically treated to prevent self-ligation. Host bacteria must be made sufficiently competent to permit the entry of recombinant plasmids into cells. The selection of desired recombinant plasmid-bearing E. coli normally is achieved by the property of drug resistance conferred by the plasmid vectors. The plasmids encoding mark-ers provide specific resistance to (i.e., the ability to grow in the presence of) antibiotics such as ampicillin, kanamycin, and tetracycline. The foreign component in the plasmid vector can be a mammalian expression cassette, which can direct expres-sion of foreign genes in mammalian cells. The resulting plasmid vector can be amplified in E. coli to prepare large quantities of DNA for its subsequent applications such as transfection, gene therapy, transgenics, and
|
Surgery_Schwartz. E. coliE. coli containingrecombinant plasmidPropagationE. coli containingrecombinant plasmidThe vector must be correctly prepared to maximize the creation of recombinants; for example, it must be enzymatically treated to prevent self-ligation. Host bacteria must be made sufficiently competent to permit the entry of recombinant plasmids into cells. The selection of desired recombinant plasmid-bearing E. coli normally is achieved by the property of drug resistance conferred by the plasmid vectors. The plasmids encoding mark-ers provide specific resistance to (i.e., the ability to grow in the presence of) antibiotics such as ampicillin, kanamycin, and tetracycline. The foreign component in the plasmid vector can be a mammalian expression cassette, which can direct expres-sion of foreign genes in mammalian cells. The resulting plasmid vector can be amplified in E. coli to prepare large quantities of DNA for its subsequent applications such as transfection, gene therapy, transgenics, and
|
Surgery_Schwartz_3370
|
Surgery_Schwartz
|
in mammalian cells. The resulting plasmid vector can be amplified in E. coli to prepare large quantities of DNA for its subsequent applications such as transfection, gene therapy, transgenics, and knockout mice.Detection of Nucleic Acids and ProteinsSouthern Blot Hybridization. Southern blotting refers to the technique of transferring DNA fragments from an electropho-resis gel to a membrane support and the subsequent analysis of the fragments by hybridization with a radioactively or chemi-luminescently labeled probe (Fig. 15-16).26 Southern blotting is named after E. M. Southern, who in 1975 first described the technique of DNA analysis. It enables reliable and efficient analysis of size-fractionated DNA fragments in an immobilized membrane support. Southern blotting is composed of several steps. It normally begins with the digestion of the DNA samples with appropriate restriction enzymes, which will discriminate wild-type and mutant DNA by size and the separation of DNA samples in an
|
Surgery_Schwartz. in mammalian cells. The resulting plasmid vector can be amplified in E. coli to prepare large quantities of DNA for its subsequent applications such as transfection, gene therapy, transgenics, and knockout mice.Detection of Nucleic Acids and ProteinsSouthern Blot Hybridization. Southern blotting refers to the technique of transferring DNA fragments from an electropho-resis gel to a membrane support and the subsequent analysis of the fragments by hybridization with a radioactively or chemi-luminescently labeled probe (Fig. 15-16).26 Southern blotting is named after E. M. Southern, who in 1975 first described the technique of DNA analysis. It enables reliable and efficient analysis of size-fractionated DNA fragments in an immobilized membrane support. Southern blotting is composed of several steps. It normally begins with the digestion of the DNA samples with appropriate restriction enzymes, which will discriminate wild-type and mutant DNA by size and the separation of DNA samples in an
|
Surgery_Schwartz_3371
|
Surgery_Schwartz
|
steps. It normally begins with the digestion of the DNA samples with appropriate restriction enzymes, which will discriminate wild-type and mutant DNA by size and the separation of DNA samples in an agarose gel by electrophoresis with appropriate DNA size markers, called the DNA ladder. The DNA gel is stained with a dye, usually ethidium bromide, and photographed with a ruler laid alongside the gel so that band positions can later be identified on the membrane. The DNA gel then is treated so the DNA fragments are denatured (i.e., strand separation). The DNA then is transferred onto a nitrocellulose membrane by capillary diffusion or under electricity. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a radioactively or chemilumi-nescently labeled probe to be identified.The development of Southern transfer and the associated hybridization techniques made it possible for the first time to obtain information about the
|
Surgery_Schwartz. steps. It normally begins with the digestion of the DNA samples with appropriate restriction enzymes, which will discriminate wild-type and mutant DNA by size and the separation of DNA samples in an agarose gel by electrophoresis with appropriate DNA size markers, called the DNA ladder. The DNA gel is stained with a dye, usually ethidium bromide, and photographed with a ruler laid alongside the gel so that band positions can later be identified on the membrane. The DNA gel then is treated so the DNA fragments are denatured (i.e., strand separation). The DNA then is transferred onto a nitrocellulose membrane by capillary diffusion or under electricity. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a radioactively or chemilumi-nescently labeled probe to be identified.The development of Southern transfer and the associated hybridization techniques made it possible for the first time to obtain information about the
|
Surgery_Schwartz_3372
|
Surgery_Schwartz
|
chemilumi-nescently labeled probe to be identified.The development of Southern transfer and the associated hybridization techniques made it possible for the first time to obtain information about the physical organization of single and multicopy sequences in complex genomes. The later applica-tion of Southern blotting hybridization to the study of restriction fragment length polymorphisms opened up new possibilities DNA is digested withrestriction enzymes.DNA fragments are denaturedand separated by gelelectrophoresis.DNA fragments are transferredto a membrane filter.The filter is hybridized with a radioactive DNA probe.DNA fragment that is hybridized to the radioactive DNA is detected by autoradiography.Radioactive probeFigure 15-16. Southern blotting. Restriction enzymatic fragments of DNA are separated by agarose gel electrophoresis, transferred to a membrane filter, and then hybridized to a radioactive probe.such as genetic fingerprinting and prenatal diagnosis of genetic
|
Surgery_Schwartz. chemilumi-nescently labeled probe to be identified.The development of Southern transfer and the associated hybridization techniques made it possible for the first time to obtain information about the physical organization of single and multicopy sequences in complex genomes. The later applica-tion of Southern blotting hybridization to the study of restriction fragment length polymorphisms opened up new possibilities DNA is digested withrestriction enzymes.DNA fragments are denaturedand separated by gelelectrophoresis.DNA fragments are transferredto a membrane filter.The filter is hybridized with a radioactive DNA probe.DNA fragment that is hybridized to the radioactive DNA is detected by autoradiography.Radioactive probeFigure 15-16. Southern blotting. Restriction enzymatic fragments of DNA are separated by agarose gel electrophoresis, transferred to a membrane filter, and then hybridized to a radioactive probe.such as genetic fingerprinting and prenatal diagnosis of genetic
|
Surgery_Schwartz_3373
|
Surgery_Schwartz
|
of DNA are separated by agarose gel electrophoresis, transferred to a membrane filter, and then hybridized to a radioactive probe.such as genetic fingerprinting and prenatal diagnosis of genetic diseases.Northern Blot Hybridization. Northern blotting refers to the technique of size fractionation of RNA in a gel and the transfer-ring of an RNA sample to a solid support (membrane) in such a manner that the relative positions of the RNA molecules are maintained. The resulting membrane then is hybridized with a labeled probe complementary to the mRNA of interest. Signals generated from detection of the membrane can be used to deter-mine the size and abundance of the target RNA. In principle, Brunicardi_Ch15_p0479-p0510.indd 49418/02/19 11:12 AM 495MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15Northern blot hybridization is similar to Southern blot hybrid-ization (and hence its name), with the exception that RNA, not DNA, is on the membrane. Although
|
Surgery_Schwartz. of DNA are separated by agarose gel electrophoresis, transferred to a membrane filter, and then hybridized to a radioactive probe.such as genetic fingerprinting and prenatal diagnosis of genetic diseases.Northern Blot Hybridization. Northern blotting refers to the technique of size fractionation of RNA in a gel and the transfer-ring of an RNA sample to a solid support (membrane) in such a manner that the relative positions of the RNA molecules are maintained. The resulting membrane then is hybridized with a labeled probe complementary to the mRNA of interest. Signals generated from detection of the membrane can be used to deter-mine the size and abundance of the target RNA. In principle, Brunicardi_Ch15_p0479-p0510.indd 49418/02/19 11:12 AM 495MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15Northern blot hybridization is similar to Southern blot hybrid-ization (and hence its name), with the exception that RNA, not DNA, is on the membrane. Although
|
Surgery_Schwartz_3374
|
Surgery_Schwartz
|
DISEASE, AND PRECISION SURGERYCHAPTER 15Northern blot hybridization is similar to Southern blot hybrid-ization (and hence its name), with the exception that RNA, not DNA, is on the membrane. Although reverse-transcriptase PCR has been used in many applications (described in the next sec-tion, “Polymerase Chain Reaction”), Northern analysis is the only method that provides information regarding mRNA size and has remained a standard method for detection and quantita-tion of mRNA. The process of Northern hybridization involves several steps, as does Southern hybridization, including elec-trophoresis of RNA samples in an agarose-formaldehyde gel, transfer to a membrane support, and hybridization to a radio-actively labeled DNA probe. Data from hybridization allow Etc.Double-strandedDNAHeat toseparatestrandsHybridization of primers+DNA polymerase+dATP+dGTP+dCTP+dTTP5'5'DNAfromsynthesisprimersStep 1Step 2Step 3First cycleSeparate DNA strandsand add primerDNAsynthesisRegion ofdouble-stranded
|
Surgery_Schwartz. DISEASE, AND PRECISION SURGERYCHAPTER 15Northern blot hybridization is similar to Southern blot hybrid-ization (and hence its name), with the exception that RNA, not DNA, is on the membrane. Although reverse-transcriptase PCR has been used in many applications (described in the next sec-tion, “Polymerase Chain Reaction”), Northern analysis is the only method that provides information regarding mRNA size and has remained a standard method for detection and quantita-tion of mRNA. The process of Northern hybridization involves several steps, as does Southern hybridization, including elec-trophoresis of RNA samples in an agarose-formaldehyde gel, transfer to a membrane support, and hybridization to a radio-actively labeled DNA probe. Data from hybridization allow Etc.Double-strandedDNAHeat toseparatestrandsHybridization of primers+DNA polymerase+dATP+dGTP+dCTP+dTTP5'5'DNAfromsynthesisprimersStep 1Step 2Step 3First cycleSeparate DNA strandsand add primerDNAsynthesisRegion ofdouble-stranded
|
Surgery_Schwartz_3375
|
Surgery_Schwartz
|
of primers+DNA polymerase+dATP+dGTP+dCTP+dTTP5'5'DNAfromsynthesisprimersStep 1Step 2Step 3First cycleSeparate DNA strandsand add primerDNAsynthesisRegion ofdouble-stranded DNA to beamplifiedDNA oligonucleotideprimersSeparate DNA strandsand annual primerDNAsynthesisSeparate DNA strandsand annual primerDNAsynthesisFirst cycleProducing two double-strandedDNA moleculesSecond cycleProducing four double-strandedDNA moleculesThird cycleProducing eight double-strandedDNA moleculesABFigure 15-17. Amplification of DNA using the polymerase chain reaction (PCR) technique. Knowledge of the DNA sequence to be ampli-fied is used to design two synthetic DNA oligonucleotides, each complementary to the sequence on one strand of the DNA double helix at opposite ends of the region to be amplified. These oligonucleotides serve as primers for in vitro DNA synthesis, which is performed by a DNA polymerase, and they determine the segment of the DNA that is amplified. A. PCR starts with a double-stranded DNA,
|
Surgery_Schwartz. of primers+DNA polymerase+dATP+dGTP+dCTP+dTTP5'5'DNAfromsynthesisprimersStep 1Step 2Step 3First cycleSeparate DNA strandsand add primerDNAsynthesisRegion ofdouble-stranded DNA to beamplifiedDNA oligonucleotideprimersSeparate DNA strandsand annual primerDNAsynthesisSeparate DNA strandsand annual primerDNAsynthesisFirst cycleProducing two double-strandedDNA moleculesSecond cycleProducing four double-strandedDNA moleculesThird cycleProducing eight double-strandedDNA moleculesABFigure 15-17. Amplification of DNA using the polymerase chain reaction (PCR) technique. Knowledge of the DNA sequence to be ampli-fied is used to design two synthetic DNA oligonucleotides, each complementary to the sequence on one strand of the DNA double helix at opposite ends of the region to be amplified. These oligonucleotides serve as primers for in vitro DNA synthesis, which is performed by a DNA polymerase, and they determine the segment of the DNA that is amplified. A. PCR starts with a double-stranded DNA,
|
Surgery_Schwartz_3376
|
Surgery_Schwartz
|
serve as primers for in vitro DNA synthesis, which is performed by a DNA polymerase, and they determine the segment of the DNA that is amplified. A. PCR starts with a double-stranded DNA, and each cycle of the reaction begins with a brief heat treatment to separate the two strands (Step 1). After strand separation, cooling of the DNA in the presence of a large excess of the two primer DNA oligonucleotides allows these primers to hybridize to complementary sequences in the two DNA strands (Step 2). This mixture is then incubated with DNA polymerase and the four deoxyribonucleoside triphosphates so that DNA is synthesized, starting from the two primers (Step 3). The entire cycle is then begun again by a heat treatment to separate the newly synthesized DNA strands. B. As the procedure is performed over and over again, the newly synthesized fragments serve as templates in their turn, and, within a few cycles, the predominant DNA is identical to the sequence bracketed by and including the
|
Surgery_Schwartz. serve as primers for in vitro DNA synthesis, which is performed by a DNA polymerase, and they determine the segment of the DNA that is amplified. A. PCR starts with a double-stranded DNA, and each cycle of the reaction begins with a brief heat treatment to separate the two strands (Step 1). After strand separation, cooling of the DNA in the presence of a large excess of the two primer DNA oligonucleotides allows these primers to hybridize to complementary sequences in the two DNA strands (Step 2). This mixture is then incubated with DNA polymerase and the four deoxyribonucleoside triphosphates so that DNA is synthesized, starting from the two primers (Step 3). The entire cycle is then begun again by a heat treatment to separate the newly synthesized DNA strands. B. As the procedure is performed over and over again, the newly synthesized fragments serve as templates in their turn, and, within a few cycles, the predominant DNA is identical to the sequence bracketed by and including the
|
Surgery_Schwartz_3377
|
Surgery_Schwartz
|
over and over again, the newly synthesized fragments serve as templates in their turn, and, within a few cycles, the predominant DNA is identical to the sequence bracketed by and including the two primers in the original template. Of the DNA put into the original reaction, only the sequence bracketed by the two primers is amplified because there are no primers attached anywhere else. In the example illustrated in B, three cycles of reaction produce 16 DNA chains, eight of which (boxed in brown) are the same length as and correspond exactly to one or the other strand of the original bracketed sequence shown at the far left; the other strands contain extra DNA downstream of the original sequence, which is replicated in the first few cycles. After three more cycles, 240 of the 256 DNA chains cor-respond exactly to the original bracketed sequence, and after several more cycles, essentially all of the DNA strands have this unique length. quantification of steady-state mRNA levels and, at
|
Surgery_Schwartz. over and over again, the newly synthesized fragments serve as templates in their turn, and, within a few cycles, the predominant DNA is identical to the sequence bracketed by and including the two primers in the original template. Of the DNA put into the original reaction, only the sequence bracketed by the two primers is amplified because there are no primers attached anywhere else. In the example illustrated in B, three cycles of reaction produce 16 DNA chains, eight of which (boxed in brown) are the same length as and correspond exactly to one or the other strand of the original bracketed sequence shown at the far left; the other strands contain extra DNA downstream of the original sequence, which is replicated in the first few cycles. After three more cycles, 240 of the 256 DNA chains cor-respond exactly to the original bracketed sequence, and after several more cycles, essentially all of the DNA strands have this unique length. quantification of steady-state mRNA levels and, at
|
Surgery_Schwartz_3378
|
Surgery_Schwartz
|
cor-respond exactly to the original bracketed sequence, and after several more cycles, essentially all of the DNA strands have this unique length. quantification of steady-state mRNA levels and, at the same time, provide information related to the presence, size, and integrity of discrete mRNA species. Thus, Northern blot analy-sis, also termed RNA gel blot analysis, commonly is used in molecular biology studies relating to gene expression.Polymerase Chain Reaction. PCR is an in vitro method for the polymerase-directed amplification of specific DNA sequences using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the tar-get DNA (Fig. 15-17).27 One cycle of PCR reaction involves template denaturation, primer annealing, and the extension of Brunicardi_Ch15_p0479-p0510.indd 49518/02/19 11:12 AM 496BASIC CONSIDERATIONSPART Ithe annealed primers by DNA polymerase. Because the primer extension products synthesized in one cycle can serve
|
Surgery_Schwartz. cor-respond exactly to the original bracketed sequence, and after several more cycles, essentially all of the DNA strands have this unique length. quantification of steady-state mRNA levels and, at the same time, provide information related to the presence, size, and integrity of discrete mRNA species. Thus, Northern blot analy-sis, also termed RNA gel blot analysis, commonly is used in molecular biology studies relating to gene expression.Polymerase Chain Reaction. PCR is an in vitro method for the polymerase-directed amplification of specific DNA sequences using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the tar-get DNA (Fig. 15-17).27 One cycle of PCR reaction involves template denaturation, primer annealing, and the extension of Brunicardi_Ch15_p0479-p0510.indd 49518/02/19 11:12 AM 496BASIC CONSIDERATIONSPART Ithe annealed primers by DNA polymerase. Because the primer extension products synthesized in one cycle can serve
|
Surgery_Schwartz_3379
|
Surgery_Schwartz
|
Brunicardi_Ch15_p0479-p0510.indd 49518/02/19 11:12 AM 496BASIC CONSIDERATIONSPART Ithe annealed primers by DNA polymerase. Because the primer extension products synthesized in one cycle can serve as a template in the next, the number of target DNA copies nearly doubles at each cycle. Thus, a repeated series of cycles result in the exponential accumulation of a specific fragment in which the termini are sharply defined by the 5′ ends of the primers. The introduction of the thermostable DNA polymerase (e.g., Taq polymerase) transforms the PCR into a simple and robust reaction. The reaction components (e.g., template, primers, Taq polymerase, 2′-deoxynucleoside 5′-triphosphates, and buffer) could all be assembled and the amplification reaction carried out by simply cycling the temperatures within the reaction tube. The specificity and yield in amplifying a particular DNA frag-ment by PCR reaction are affected by the proper setting of the reaction parameters (e.g., enzyme, primer, and
|
Surgery_Schwartz. Brunicardi_Ch15_p0479-p0510.indd 49518/02/19 11:12 AM 496BASIC CONSIDERATIONSPART Ithe annealed primers by DNA polymerase. Because the primer extension products synthesized in one cycle can serve as a template in the next, the number of target DNA copies nearly doubles at each cycle. Thus, a repeated series of cycles result in the exponential accumulation of a specific fragment in which the termini are sharply defined by the 5′ ends of the primers. The introduction of the thermostable DNA polymerase (e.g., Taq polymerase) transforms the PCR into a simple and robust reaction. The reaction components (e.g., template, primers, Taq polymerase, 2′-deoxynucleoside 5′-triphosphates, and buffer) could all be assembled and the amplification reaction carried out by simply cycling the temperatures within the reaction tube. The specificity and yield in amplifying a particular DNA frag-ment by PCR reaction are affected by the proper setting of the reaction parameters (e.g., enzyme, primer, and
|
Surgery_Schwartz_3380
|
Surgery_Schwartz
|
within the reaction tube. The specificity and yield in amplifying a particular DNA frag-ment by PCR reaction are affected by the proper setting of the reaction parameters (e.g., enzyme, primer, and Mg2+ concen-tration, as well as the temperature cycling profile). Modifying various PCR parameters to optimize the specificity of amplifi-cation yields more homogenous products, even in rare template reactions.The emergence of the PCR technique has dramatically altered the approach to both fundamental and applied bio-logic problems. The capability of amplifying a specific DNA fragment from a gene or the whole genome greatly advances the study of the gene and its function. It is simple, yet robust, speedy, and most of all, flexible. As a recombinant DNA tool, it underlies almost all of molecular biology. This revolution-ary technique enabled the modern methods for the isolation of genes, construction of a DNA vector, introduction of alterations into DNA, and quantitation of gene expression,
|
Surgery_Schwartz. within the reaction tube. The specificity and yield in amplifying a particular DNA frag-ment by PCR reaction are affected by the proper setting of the reaction parameters (e.g., enzyme, primer, and Mg2+ concen-tration, as well as the temperature cycling profile). Modifying various PCR parameters to optimize the specificity of amplifi-cation yields more homogenous products, even in rare template reactions.The emergence of the PCR technique has dramatically altered the approach to both fundamental and applied bio-logic problems. The capability of amplifying a specific DNA fragment from a gene or the whole genome greatly advances the study of the gene and its function. It is simple, yet robust, speedy, and most of all, flexible. As a recombinant DNA tool, it underlies almost all of molecular biology. This revolution-ary technique enabled the modern methods for the isolation of genes, construction of a DNA vector, introduction of alterations into DNA, and quantitation of gene expression,
|
Surgery_Schwartz_3381
|
Surgery_Schwartz
|
biology. This revolution-ary technique enabled the modern methods for the isolation of genes, construction of a DNA vector, introduction of alterations into DNA, and quantitation of gene expression, making it a fun-damental cornerstone of genetic and molecular analysis.Immunoblotting and Immunoprecipitation. Analyses of proteins are primarily carried out by antibody-directed immu-nologic techniques. For example, Western blotting, also called immunoblotting, is performed to detect protein levels in a popu-lation of cells or tissues, whereas immunoprecipitation is used to concentrate proteins from a larger pool. Using specific antibod-ies, microscopic analysis called immunofluorescence and immu-nohistochemistry is possible for the subcellular localization and expression of proteins in cells or tissues, respectively.Immunoblotting refers to the process of identifying a pro-tein from a mixture of proteins (Fig. 15-18). It consists of five steps: (a) sample preparation; (b) electrophoresis
|
Surgery_Schwartz. biology. This revolution-ary technique enabled the modern methods for the isolation of genes, construction of a DNA vector, introduction of alterations into DNA, and quantitation of gene expression, making it a fun-damental cornerstone of genetic and molecular analysis.Immunoblotting and Immunoprecipitation. Analyses of proteins are primarily carried out by antibody-directed immu-nologic techniques. For example, Western blotting, also called immunoblotting, is performed to detect protein levels in a popu-lation of cells or tissues, whereas immunoprecipitation is used to concentrate proteins from a larger pool. Using specific antibod-ies, microscopic analysis called immunofluorescence and immu-nohistochemistry is possible for the subcellular localization and expression of proteins in cells or tissues, respectively.Immunoblotting refers to the process of identifying a pro-tein from a mixture of proteins (Fig. 15-18). It consists of five steps: (a) sample preparation; (b) electrophoresis
|
Surgery_Schwartz_3382
|
Surgery_Schwartz
|
or tissues, respectively.Immunoblotting refers to the process of identifying a pro-tein from a mixture of proteins (Fig. 15-18). It consists of five steps: (a) sample preparation; (b) electrophoresis (separation of a protein mixture by sodium dodecyl sulfate-polyacrylamide gel electrophoresis); (c) transfer (the electrophoretic transfer of proteins from gel onto membrane support [e.g., nitrocel-lulose, nylon, or polyvinylidene difluoride]); (d) staining (the subsequent immunodetection of target proteins with specific antibody); and (e) development (colorimetric, chemilumines-cent, and recently fluorescent visualization of the antibody-recognized protein). Thus, immunoblotting combines the resolution of gel electrophoresis with the specificity of immu-nochemical detection. Immunoblotting is a powerful tool used to determine a number of important characteristics of proteins. For example, immunoblotting analysis will determine the pres-ence and the quantity of a protein in a given
|
Surgery_Schwartz. or tissues, respectively.Immunoblotting refers to the process of identifying a pro-tein from a mixture of proteins (Fig. 15-18). It consists of five steps: (a) sample preparation; (b) electrophoresis (separation of a protein mixture by sodium dodecyl sulfate-polyacrylamide gel electrophoresis); (c) transfer (the electrophoretic transfer of proteins from gel onto membrane support [e.g., nitrocel-lulose, nylon, or polyvinylidene difluoride]); (d) staining (the subsequent immunodetection of target proteins with specific antibody); and (e) development (colorimetric, chemilumines-cent, and recently fluorescent visualization of the antibody-recognized protein). Thus, immunoblotting combines the resolution of gel electrophoresis with the specificity of immu-nochemical detection. Immunoblotting is a powerful tool used to determine a number of important characteristics of proteins. For example, immunoblotting analysis will determine the pres-ence and the quantity of a protein in a given
|
Surgery_Schwartz_3383
|
Surgery_Schwartz
|
is a powerful tool used to determine a number of important characteristics of proteins. For example, immunoblotting analysis will determine the pres-ence and the quantity of a protein in a given cellular condition and its relative molecular weight. Immunoblotting also can be used to determine whether posttranslational modification such as phosphorylation has occurred on a protein. Importantly, through immunoblotting analysis, a comparison of the protein levels and modification states in normal vs diseased tissues is possible.Cell tissue lysates• Sample preparation• Gel electrophoresis Separation of proteins• Western transfer Transfer of proteins to membrane• Immunostaining Block membrane 1°/2° antibody staining• Development Colorimetric/chemiluminescence detection123456123456Figure 15-18. Immunoblotting. Proteins are prepared from cells or tissues, separated according to size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to a membrane filter.
|
Surgery_Schwartz. is a powerful tool used to determine a number of important characteristics of proteins. For example, immunoblotting analysis will determine the pres-ence and the quantity of a protein in a given cellular condition and its relative molecular weight. Immunoblotting also can be used to determine whether posttranslational modification such as phosphorylation has occurred on a protein. Importantly, through immunoblotting analysis, a comparison of the protein levels and modification states in normal vs diseased tissues is possible.Cell tissue lysates• Sample preparation• Gel electrophoresis Separation of proteins• Western transfer Transfer of proteins to membrane• Immunostaining Block membrane 1°/2° antibody staining• Development Colorimetric/chemiluminescence detection123456123456Figure 15-18. Immunoblotting. Proteins are prepared from cells or tissues, separated according to size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to a membrane filter.
|
Surgery_Schwartz_3384
|
Surgery_Schwartz
|
15-18. Immunoblotting. Proteins are prepared from cells or tissues, separated according to size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to a membrane filter. Detection of a protein of interest can be done by sequential incubation with a primary antibody directed against the protein, and then with an enzyme-conjugated secondary antibody that rec-ognizes the primary antibody. Visualization of the protein is carried out by using colorimetric or luminescent substrates for the conju-gated enzyme.Immunoprecipitation, another widely used immunochemi-cal technique, is a method that uses antibody to enrich a pro-tein of interest and any other proteins that are associated with it (Fig. 15-19). The principle of the technique lies in the property of a strong and specific affinity between antibodies and their anti-gens to locate and pull down target proteins in solution. Once the antibody-antigen (target protein) complexes are formed in the solution, they are
|
Surgery_Schwartz. 15-18. Immunoblotting. Proteins are prepared from cells or tissues, separated according to size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to a membrane filter. Detection of a protein of interest can be done by sequential incubation with a primary antibody directed against the protein, and then with an enzyme-conjugated secondary antibody that rec-ognizes the primary antibody. Visualization of the protein is carried out by using colorimetric or luminescent substrates for the conju-gated enzyme.Immunoprecipitation, another widely used immunochemi-cal technique, is a method that uses antibody to enrich a pro-tein of interest and any other proteins that are associated with it (Fig. 15-19). The principle of the technique lies in the property of a strong and specific affinity between antibodies and their anti-gens to locate and pull down target proteins in solution. Once the antibody-antigen (target protein) complexes are formed in the solution, they are
|
Surgery_Schwartz_3385
|
Surgery_Schwartz
|
specific affinity between antibodies and their anti-gens to locate and pull down target proteins in solution. Once the antibody-antigen (target protein) complexes are formed in the solution, they are collected and purified using small agarose beads with covalently attached protein A or protein G. Both protein A and protein G specifically interact with the antibodies, Brunicardi_Ch15_p0479-p0510.indd 49618/02/19 11:12 AM 497MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15Enriched YFP & YBPsWashSDS-PAGEAnti-YFP beadsAnti-YFP conjugated to beadsYour favorite protein (YFP)YFP-binding proteins (YBPs)Junk proteinsFigure 15-19. Immunoprecipitation. Proteins prepared from cells or tissues can be enriched using an antibody directed against them. The anti-body is first conjugated to agarose beads and then incubated with protein mixture. Due to the spe-cific high-affinity interaction between antibody and its antigen (the protein), the antigen-antibody complex
|
Surgery_Schwartz. specific affinity between antibodies and their anti-gens to locate and pull down target proteins in solution. Once the antibody-antigen (target protein) complexes are formed in the solution, they are collected and purified using small agarose beads with covalently attached protein A or protein G. Both protein A and protein G specifically interact with the antibodies, Brunicardi_Ch15_p0479-p0510.indd 49618/02/19 11:12 AM 497MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15Enriched YFP & YBPsWashSDS-PAGEAnti-YFP beadsAnti-YFP conjugated to beadsYour favorite protein (YFP)YFP-binding proteins (YBPs)Junk proteinsFigure 15-19. Immunoprecipitation. Proteins prepared from cells or tissues can be enriched using an antibody directed against them. The anti-body is first conjugated to agarose beads and then incubated with protein mixture. Due to the spe-cific high-affinity interaction between antibody and its antigen (the protein), the antigen-antibody complex
|
Surgery_Schwartz_3386
|
Surgery_Schwartz
|
first conjugated to agarose beads and then incubated with protein mixture. Due to the spe-cific high-affinity interaction between antibody and its antigen (the protein), the antigen-antibody complex can be collected on beads by centrifuga-tion. The immunoprecipitated protein can then be analyzed by immunoblotting. Alternatively, if pro-teins are radiolabeled in cells or tissues, detection of immunoprecipitated proteins can be achieved by simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography.thus forming a large immobilized complex of antibody-antigen bound to beads. The purified protein can then be analyzed by a number of biochemical methods. When immunoprecipitation is combined with immunoblotting, it can be used for the sensitive detection of proteins in low concentrations, which would other-wise be difficult to detect. Moreover, combined immunoprecipi-tation and immunoblotting analysis is very efficient in analyzing the protein-protein
|
Surgery_Schwartz. first conjugated to agarose beads and then incubated with protein mixture. Due to the spe-cific high-affinity interaction between antibody and its antigen (the protein), the antigen-antibody complex can be collected on beads by centrifuga-tion. The immunoprecipitated protein can then be analyzed by immunoblotting. Alternatively, if pro-teins are radiolabeled in cells or tissues, detection of immunoprecipitated proteins can be achieved by simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography.thus forming a large immobilized complex of antibody-antigen bound to beads. The purified protein can then be analyzed by a number of biochemical methods. When immunoprecipitation is combined with immunoblotting, it can be used for the sensitive detection of proteins in low concentrations, which would other-wise be difficult to detect. Moreover, combined immunoprecipi-tation and immunoblotting analysis is very efficient in analyzing the protein-protein
|
Surgery_Schwartz_3387
|
Surgery_Schwartz
|
proteins in low concentrations, which would other-wise be difficult to detect. Moreover, combined immunoprecipi-tation and immunoblotting analysis is very efficient in analyzing the protein-protein interactions or determining the posttransla-tional modifications of proteins. In addition, immunoprecipi-tated proteins can be used as preparative steps for assays such as intrinsic or associated enzymatic activities. The success of immunoprecipitation is influenced by two major factors: (a) the abundance of the protein in the original preparation and (b) the specificity and affinity of the antibody for this protein.Recently, immunoprecipitation is even used to enrich modi-fied DNA (for example, 5-methylcytosine) for bisulfite sequenc-ing. Besides proteins of interest, specific antibodies can also be raised against specially modified DNA. Like the protein immuno-precipitation, modified DNA can be pulled down, taking advan-tage of the specificity and affinity of antibody to antigen.DNA
|
Surgery_Schwartz. proteins in low concentrations, which would other-wise be difficult to detect. Moreover, combined immunoprecipi-tation and immunoblotting analysis is very efficient in analyzing the protein-protein interactions or determining the posttransla-tional modifications of proteins. In addition, immunoprecipi-tated proteins can be used as preparative steps for assays such as intrinsic or associated enzymatic activities. The success of immunoprecipitation is influenced by two major factors: (a) the abundance of the protein in the original preparation and (b) the specificity and affinity of the antibody for this protein.Recently, immunoprecipitation is even used to enrich modi-fied DNA (for example, 5-methylcytosine) for bisulfite sequenc-ing. Besides proteins of interest, specific antibodies can also be raised against specially modified DNA. Like the protein immuno-precipitation, modified DNA can be pulled down, taking advan-tage of the specificity and affinity of antibody to antigen.DNA
|
Surgery_Schwartz_3388
|
Surgery_Schwartz
|
can also be raised against specially modified DNA. Like the protein immuno-precipitation, modified DNA can be pulled down, taking advan-tage of the specificity and affinity of antibody to antigen.DNA Microarray. Now that the human genome sequence is completed, the primary focus of biologists is rapidly shifting toward gaining an understanding of how genes function. One of the interesting findings about the human genome is that there are only approximately 25,000 to 30,000 protein-encoding genes. However, it is known that genes and their products function in a complicated and yet orchestrated fashion and that the surprisingly small number of genes from the genome sequence is sufficient to make a human being. Nonetheless, with the tens of thousands of genes present in the genome, traditional methods in molecular biology, which generally work on a one-gene-in-one-experiment basis, cannot generate the whole picture of genome function. In the past several years, a new technology called DNA
|
Surgery_Schwartz. can also be raised against specially modified DNA. Like the protein immuno-precipitation, modified DNA can be pulled down, taking advan-tage of the specificity and affinity of antibody to antigen.DNA Microarray. Now that the human genome sequence is completed, the primary focus of biologists is rapidly shifting toward gaining an understanding of how genes function. One of the interesting findings about the human genome is that there are only approximately 25,000 to 30,000 protein-encoding genes. However, it is known that genes and their products function in a complicated and yet orchestrated fashion and that the surprisingly small number of genes from the genome sequence is sufficient to make a human being. Nonetheless, with the tens of thousands of genes present in the genome, traditional methods in molecular biology, which generally work on a one-gene-in-one-experiment basis, cannot generate the whole picture of genome function. In the past several years, a new technology called DNA
|
Surgery_Schwartz_3389
|
Surgery_Schwartz
|
methods in molecular biology, which generally work on a one-gene-in-one-experiment basis, cannot generate the whole picture of genome function. In the past several years, a new technology called DNA microarray has attracted tremendous interest among biologists as well as cli-nicians. This technology promises to monitor the whole genome on a single chip so researchers can have a better picture of the interactions among thousands of genes simultaneously.DNA microarray, also called gene chip, DNA chip, and gene array, refers to large sets of probes of known sequences orderly arranged on a small chip, enabling many hybridization reactions to be carried out in parallel in a small device (Fig. 15-20).28 Like Southern and Northern hybridization, the underlying principle of this technology is the remarkable abil-ity of nucleic acids to form a duplex between two strands with complementary base sequences. DNA microarray provides a medium for matching known and unknown DNA samples based on
|
Surgery_Schwartz. methods in molecular biology, which generally work on a one-gene-in-one-experiment basis, cannot generate the whole picture of genome function. In the past several years, a new technology called DNA microarray has attracted tremendous interest among biologists as well as cli-nicians. This technology promises to monitor the whole genome on a single chip so researchers can have a better picture of the interactions among thousands of genes simultaneously.DNA microarray, also called gene chip, DNA chip, and gene array, refers to large sets of probes of known sequences orderly arranged on a small chip, enabling many hybridization reactions to be carried out in parallel in a small device (Fig. 15-20).28 Like Southern and Northern hybridization, the underlying principle of this technology is the remarkable abil-ity of nucleic acids to form a duplex between two strands with complementary base sequences. DNA microarray provides a medium for matching known and unknown DNA samples based on
|
Surgery_Schwartz_3390
|
Surgery_Schwartz
|
the remarkable abil-ity of nucleic acids to form a duplex between two strands with complementary base sequences. DNA microarray provides a medium for matching known and unknown DNA samples based on base-pairing rules and automating the process of identify-ing the unknowns. Microarrays require specialized robotics and imaging equipment that spot the samples on a glass or nylon substrate, carry out the hybridization, and analyze the data gener-ated. DNA microarrays containing different sets of genes from a variety of organisms are now commercially available, allowing biologists to simply purchase the chips and perform hybridiza-tion and data collection. The massive scale of microarray experi-ments requires the aid of computers. They are used during the capturing of the image of the hybridized target, the conversion of the image into usable measures of the extent of hybridization, and the interpretation of the extent of hybridization into a mean-ingful measure of the amount of the
|
Surgery_Schwartz. the remarkable abil-ity of nucleic acids to form a duplex between two strands with complementary base sequences. DNA microarray provides a medium for matching known and unknown DNA samples based on base-pairing rules and automating the process of identify-ing the unknowns. Microarrays require specialized robotics and imaging equipment that spot the samples on a glass or nylon substrate, carry out the hybridization, and analyze the data gener-ated. DNA microarrays containing different sets of genes from a variety of organisms are now commercially available, allowing biologists to simply purchase the chips and perform hybridiza-tion and data collection. The massive scale of microarray experi-ments requires the aid of computers. They are used during the capturing of the image of the hybridized target, the conversion of the image into usable measures of the extent of hybridization, and the interpretation of the extent of hybridization into a mean-ingful measure of the amount of the
|
Surgery_Schwartz_3391
|
Surgery_Schwartz
|
target, the conversion of the image into usable measures of the extent of hybridization, and the interpretation of the extent of hybridization into a mean-ingful measure of the amount of the complementary sequence in the target. Some data-analysis packages are available commer-cially or can be found in the core facility of certain institutions.DNA microarray technology has produced many signifi-cant results in quite different areas of application. There are two major application forms for the technology: identifica-tion of sequence (gene/gene mutation) in multiple regions of a genome and determination of expression level (abundance) of large numbers of genes simultaneously. For example, analysis of genomic DNA detects amplifications and deletions found in human tumors. Differential gene expression analysis also has uncovered networks of genes differentially present in can-cers that cannot be distinguished by conventional means. Sig-nificantly, recent advancements in next-generation
|
Surgery_Schwartz. target, the conversion of the image into usable measures of the extent of hybridization, and the interpretation of the extent of hybridization into a mean-ingful measure of the amount of the complementary sequence in the target. Some data-analysis packages are available commer-cially or can be found in the core facility of certain institutions.DNA microarray technology has produced many signifi-cant results in quite different areas of application. There are two major application forms for the technology: identifica-tion of sequence (gene/gene mutation) in multiple regions of a genome and determination of expression level (abundance) of large numbers of genes simultaneously. For example, analysis of genomic DNA detects amplifications and deletions found in human tumors. Differential gene expression analysis also has uncovered networks of genes differentially present in can-cers that cannot be distinguished by conventional means. Sig-nificantly, recent advancements in next-generation
|
Surgery_Schwartz_3392
|
Surgery_Schwartz
|
expression analysis also has uncovered networks of genes differentially present in can-cers that cannot be distinguished by conventional means. Sig-nificantly, recent advancements in next-generation sequencing (e.g., Solexa and 454 technology) have demonstrated the preci-sion and speed to analyze gene expression in any genome.Next-Generation Sequencing.29,30 First-generation sequenc-ing, also termed Sanger’s sequencing, requires a single-stranded DNA template, a specific DNA primer, a DNA polymerase, nor-mal deoxynucleoside triphosphates (dNTPs), and modified di-deoxynucleotidetriphosphates (ddNTPs). In the process of DNA sequencing, DNA polymerase adds random dNTP or ddNTP Brunicardi_Ch15_p0479-p0510.indd 49718/02/19 11:12 AM 498BASIC CONSIDERATIONSPART ICell #1Cell #2mRNAcDNADNA microarrayDNA microarray datacDNAmRNAFigure 15-20. DNA microarrays. DNA microarrays, also referred to as gene chips, have arrayed oligonucleotides or com-plementary DNAs (cDNAs) corresponding to tens or
|
Surgery_Schwartz. expression analysis also has uncovered networks of genes differentially present in can-cers that cannot be distinguished by conventional means. Sig-nificantly, recent advancements in next-generation sequencing (e.g., Solexa and 454 technology) have demonstrated the preci-sion and speed to analyze gene expression in any genome.Next-Generation Sequencing.29,30 First-generation sequenc-ing, also termed Sanger’s sequencing, requires a single-stranded DNA template, a specific DNA primer, a DNA polymerase, nor-mal deoxynucleoside triphosphates (dNTPs), and modified di-deoxynucleotidetriphosphates (ddNTPs). In the process of DNA sequencing, DNA polymerase adds random dNTP or ddNTP Brunicardi_Ch15_p0479-p0510.indd 49718/02/19 11:12 AM 498BASIC CONSIDERATIONSPART ICell #1Cell #2mRNAcDNADNA microarrayDNA microarray datacDNAmRNAFigure 15-20. DNA microarrays. DNA microarrays, also referred to as gene chips, have arrayed oligonucleotides or com-plementary DNAs (cDNAs) corresponding to tens or
|
Surgery_Schwartz_3393
|
Surgery_Schwartz
|
microarray datacDNAmRNAFigure 15-20. DNA microarrays. DNA microarrays, also referred to as gene chips, have arrayed oligonucleotides or com-plementary DNAs (cDNAs) corresponding to tens or hundreds of distinct genes. DNA microarray is used to comparatively analyze gene expression in different cells or tissues. Messenger RNAs (mRNAs) extracted from different sources are converted into cDNAs, which are then labeled with different fluorescent dyes. The two fluorescent cDNA probes are mixed and hybridized to the same DNA microarrays. The ratio of red to green fluorescence at each spot on the chip represents the relative expression of levels of that gene between two different cells. In the example shown in the figure, cDNA from cell #1 is labeled with red fluorescence and that from cell #2 is labeled with green fluorescence. On the micro-array, red spots demonstrate that the gene in the cell sample #1 is expressed at a higher level than the corresponding gene in cell sample #2. The green
|
Surgery_Schwartz. microarray datacDNAmRNAFigure 15-20. DNA microarrays. DNA microarrays, also referred to as gene chips, have arrayed oligonucleotides or com-plementary DNAs (cDNAs) corresponding to tens or hundreds of distinct genes. DNA microarray is used to comparatively analyze gene expression in different cells or tissues. Messenger RNAs (mRNAs) extracted from different sources are converted into cDNAs, which are then labeled with different fluorescent dyes. The two fluorescent cDNA probes are mixed and hybridized to the same DNA microarrays. The ratio of red to green fluorescence at each spot on the chip represents the relative expression of levels of that gene between two different cells. In the example shown in the figure, cDNA from cell #1 is labeled with red fluorescence and that from cell #2 is labeled with green fluorescence. On the micro-array, red spots demonstrate that the gene in the cell sample #1 is expressed at a higher level than the corresponding gene in cell sample #2. The green
|
Surgery_Schwartz_3394
|
Surgery_Schwartz
|
labeled with green fluorescence. On the micro-array, red spots demonstrate that the gene in the cell sample #1 is expressed at a higher level than the corresponding gene in cell sample #2. The green spots indicate that the gene in the cell sam-ple #2 is expressed at a higher level than the corresponding gene in the cell sample #1. Yellow spots represent equal expression of the gene in both cell samples.after the primer. If ddNTP is incorporated at the end of the chain, it terminizes the reaction and results in DNA fragments of different sizes. The ddNTPs could be radioactively or fluo-rescently labeled for auto-machine detection. Usually, Sanger’s sequencing is able to read sequence below 1 kb with the quality deteriorating after 700 bp. The accuracy and success rate largely depends on the DNA polymerase used.Recombinant DNA technology greatly impacts the com-pletion of the Human Genome Project due to the invention of shotgun sequencing, which includes breaking the genome DNA into
|
Surgery_Schwartz. labeled with green fluorescence. On the micro-array, red spots demonstrate that the gene in the cell sample #1 is expressed at a higher level than the corresponding gene in cell sample #2. The green spots indicate that the gene in the cell sam-ple #2 is expressed at a higher level than the corresponding gene in the cell sample #1. Yellow spots represent equal expression of the gene in both cell samples.after the primer. If ddNTP is incorporated at the end of the chain, it terminizes the reaction and results in DNA fragments of different sizes. The ddNTPs could be radioactively or fluo-rescently labeled for auto-machine detection. Usually, Sanger’s sequencing is able to read sequence below 1 kb with the quality deteriorating after 700 bp. The accuracy and success rate largely depends on the DNA polymerase used.Recombinant DNA technology greatly impacts the com-pletion of the Human Genome Project due to the invention of shotgun sequencing, which includes breaking the genome DNA into
|
Surgery_Schwartz_3395
|
Surgery_Schwartz
|
the DNA polymerase used.Recombinant DNA technology greatly impacts the com-pletion of the Human Genome Project due to the invention of shotgun sequencing, which includes breaking the genome DNA into small pieces and randomly cloning those pieces into DNA vectors that are easily sequenced. Based on the overlapping sequence of each clone, computer analysis can be programmed to map and align the DNA sequence that will ultimately cover the whole human genome.Based on shotgun sequencing, as the sequencing technol-ogies advance, next-generation sequencing (NGS), also called second-generation sequencing, has become one of the most powerful tools to analyze DNA mutation, identify epigenetic modification, and profile gene expression or ncRNA expression.31 The next-generation sequencing process usually includes library construction, sequencing, and data analysis. There are three major NGS platforms: Roche 454, Life Technologies Ion Tor-rent, and Illumina Solexa. Take the Illumina
|
Surgery_Schwartz. the DNA polymerase used.Recombinant DNA technology greatly impacts the com-pletion of the Human Genome Project due to the invention of shotgun sequencing, which includes breaking the genome DNA into small pieces and randomly cloning those pieces into DNA vectors that are easily sequenced. Based on the overlapping sequence of each clone, computer analysis can be programmed to map and align the DNA sequence that will ultimately cover the whole human genome.Based on shotgun sequencing, as the sequencing technol-ogies advance, next-generation sequencing (NGS), also called second-generation sequencing, has become one of the most powerful tools to analyze DNA mutation, identify epigenetic modification, and profile gene expression or ncRNA expression.31 The next-generation sequencing process usually includes library construction, sequencing, and data analysis. There are three major NGS platforms: Roche 454, Life Technologies Ion Tor-rent, and Illumina Solexa. Take the Illumina
|
Surgery_Schwartz_3396
|
Surgery_Schwartz
|
process usually includes library construction, sequencing, and data analysis. There are three major NGS platforms: Roche 454, Life Technologies Ion Tor-rent, and Illumina Solexa. Take the Illumina next-generation sequencing as an example: DNA are shared or digested into small pieces and then used to generate a DNA library with adapt-ers on both ends of each DNA piece. Then, the DNA library is diluted and loaded on a chamber of a slide, called a lane, for cluster amplification. Cycled fluorescent deoxyribonucleotide triphosphates (dNTPs) are then added to the chamber to enable DNA polymerization, resulting in different fluorescent emis-sion representing different dNTP reading on different clusters, into a microscope. The fluorescent signal is transformed into sequencing data that will be aligned and mapped to a standard genome database. The advantages of next-generation sequenc-ing include the following: no necessity of DNA cloning; fast and cost-effective; and a huge amount of data to
|
Surgery_Schwartz. process usually includes library construction, sequencing, and data analysis. There are three major NGS platforms: Roche 454, Life Technologies Ion Tor-rent, and Illumina Solexa. Take the Illumina next-generation sequencing as an example: DNA are shared or digested into small pieces and then used to generate a DNA library with adapt-ers on both ends of each DNA piece. Then, the DNA library is diluted and loaded on a chamber of a slide, called a lane, for cluster amplification. Cycled fluorescent deoxyribonucleotide triphosphates (dNTPs) are then added to the chamber to enable DNA polymerization, resulting in different fluorescent emis-sion representing different dNTP reading on different clusters, into a microscope. The fluorescent signal is transformed into sequencing data that will be aligned and mapped to a standard genome database. The advantages of next-generation sequenc-ing include the following: no necessity of DNA cloning; fast and cost-effective; and a huge amount of data to
|
Surgery_Schwartz_3397
|
Surgery_Schwartz
|
and mapped to a standard genome database. The advantages of next-generation sequenc-ing include the following: no necessity of DNA cloning; fast and cost-effective; and a huge amount of data to give good depth and accuracy of the sequence.Based on the applications, the most common next-generation sequencing technologies for whole-genome sequencing are whole-genome DNA sequencing, whole-genome bisulfite sequencing (BS-seq), RNA sequencing (RNA-seq), and chro-matin immunoprecipitation (ChIP) sequencing (ChIP-seq). Whole-genome DNA sequencing is purely to sequence the DNA sequence of a genome without any preprocessing of the DNA, reflecting any deletions, replications, and mutations within the genomic DNA. Given that the genomic information for human is enormous, to achieve great depth and accuracy the genome needs to be sequenced multiple times to reach statistic significance and pass quality control. Therefore, whole-genome DNA sequencing is still considered to be costly for diagnosis
|
Surgery_Schwartz. and mapped to a standard genome database. The advantages of next-generation sequenc-ing include the following: no necessity of DNA cloning; fast and cost-effective; and a huge amount of data to give good depth and accuracy of the sequence.Based on the applications, the most common next-generation sequencing technologies for whole-genome sequencing are whole-genome DNA sequencing, whole-genome bisulfite sequencing (BS-seq), RNA sequencing (RNA-seq), and chro-matin immunoprecipitation (ChIP) sequencing (ChIP-seq). Whole-genome DNA sequencing is purely to sequence the DNA sequence of a genome without any preprocessing of the DNA, reflecting any deletions, replications, and mutations within the genomic DNA. Given that the genomic information for human is enormous, to achieve great depth and accuracy the genome needs to be sequenced multiple times to reach statistic significance and pass quality control. Therefore, whole-genome DNA sequencing is still considered to be costly for diagnosis
|
Surgery_Schwartz_3398
|
Surgery_Schwartz
|
the genome needs to be sequenced multiple times to reach statistic significance and pass quality control. Therefore, whole-genome DNA sequencing is still considered to be costly for diagnosis as well as for research purposes. Under clinical settings, one of the most applied DNA sequencing technologies is whole-exome sequencing, i.e., using techniques to capture and analyze exons in all coding genes, given that most known diseases are due to mutations in spliced-in regions of coding genes. Whole-exome sequencing is mostly used for detecting single nucleotide vari-ants and is less reliable in detecting insertion-deletion variants or small copy number variants.BS-seq is commonly used to identify DNA methyla-tion on the genome (5-methylcytosine [5mC]). The process always involves a bisulfite treatment of DNA before library Brunicardi_Ch15_p0479-p0510.indd 49818/02/19 11:12 AM 499MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15construction, during which
|
Surgery_Schwartz. the genome needs to be sequenced multiple times to reach statistic significance and pass quality control. Therefore, whole-genome DNA sequencing is still considered to be costly for diagnosis as well as for research purposes. Under clinical settings, one of the most applied DNA sequencing technologies is whole-exome sequencing, i.e., using techniques to capture and analyze exons in all coding genes, given that most known diseases are due to mutations in spliced-in regions of coding genes. Whole-exome sequencing is mostly used for detecting single nucleotide vari-ants and is less reliable in detecting insertion-deletion variants or small copy number variants.BS-seq is commonly used to identify DNA methyla-tion on the genome (5-methylcytosine [5mC]). The process always involves a bisulfite treatment of DNA before library Brunicardi_Ch15_p0479-p0510.indd 49818/02/19 11:12 AM 499MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15construction, during which
|
Surgery_Schwartz_3399
|
Surgery_Schwartz
|
treatment of DNA before library Brunicardi_Ch15_p0479-p0510.indd 49818/02/19 11:12 AM 499MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15construction, during which the unmethylated cytosine will be transformed to a uracil, resulting in reading as a thymine in data output, whereas 5mC is protected and remains as cytosine in data output. Thus, 5mC and cytosine are distinguished this way. To perform whole-genome BS-seq, the library construc-tion starts with a decent amount of DNA followed by a complete conversion of unmethylated cytosine. Because it is also counted as whole-genome sequencing, a certain depth of data needs to be achieved for accurate and convincing results. To develop an affordable genome-wide DNA methylation analysis, the reduced representation bisulfite sequencing (RRBS) approach has been applied to enrich CpG-dense regions of the genome by digesting genomic DNA using a methylation-insensitive restric-tion enzyme, usually MspI. This
|
Surgery_Schwartz. treatment of DNA before library Brunicardi_Ch15_p0479-p0510.indd 49818/02/19 11:12 AM 499MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15construction, during which the unmethylated cytosine will be transformed to a uracil, resulting in reading as a thymine in data output, whereas 5mC is protected and remains as cytosine in data output. Thus, 5mC and cytosine are distinguished this way. To perform whole-genome BS-seq, the library construc-tion starts with a decent amount of DNA followed by a complete conversion of unmethylated cytosine. Because it is also counted as whole-genome sequencing, a certain depth of data needs to be achieved for accurate and convincing results. To develop an affordable genome-wide DNA methylation analysis, the reduced representation bisulfite sequencing (RRBS) approach has been applied to enrich CpG-dense regions of the genome by digesting genomic DNA using a methylation-insensitive restric-tion enzyme, usually MspI. This
|
Surgery_Schwartz_3400
|
Surgery_Schwartz
|
bisulfite sequencing (RRBS) approach has been applied to enrich CpG-dense regions of the genome by digesting genomic DNA using a methylation-insensitive restric-tion enzyme, usually MspI. This method covers a majority of promoters as well as some repetitive regions in the genome.RNA-seq is usually performed to analyze transcription for the same purpose as performing a microarray. However, RNA-seq is more accurate and provides more information such as splicing variants than traditional microarray. Usually, cDNA that is reversely transcribed from extracted RNA is used to generate libraries. Depending on the needs, mRNA and ncRNA can be enriched in different protocols for RNA extraction. Currently, techniques have been improved to perform RNA profiling on a single cell level. Single-cell RNA sequencing (scRNA-seq) allows expression analysis of individual cells in a population. One use is to examine heterogeneity in tumor cells. Although low copy number genes are usually poorly detected,
|
Surgery_Schwartz. bisulfite sequencing (RRBS) approach has been applied to enrich CpG-dense regions of the genome by digesting genomic DNA using a methylation-insensitive restric-tion enzyme, usually MspI. This method covers a majority of promoters as well as some repetitive regions in the genome.RNA-seq is usually performed to analyze transcription for the same purpose as performing a microarray. However, RNA-seq is more accurate and provides more information such as splicing variants than traditional microarray. Usually, cDNA that is reversely transcribed from extracted RNA is used to generate libraries. Depending on the needs, mRNA and ncRNA can be enriched in different protocols for RNA extraction. Currently, techniques have been improved to perform RNA profiling on a single cell level. Single-cell RNA sequencing (scRNA-seq) allows expression analysis of individual cells in a population. One use is to examine heterogeneity in tumor cells. Although low copy number genes are usually poorly detected,
|
Surgery_Schwartz_3401
|
Surgery_Schwartz
|
sequencing (scRNA-seq) allows expression analysis of individual cells in a population. One use is to examine heterogeneity in tumor cells. Although low copy number genes are usually poorly detected, scRNA-seq on a large number of cells can reveal rare cell subpopula-tions and uncommon RNA expression patterns among different subpopulations.ChIP-seq is always used to map the location of a DNA-binding protein in the genome. Prior to library construction, ChIP is performed to enrich DNA bound by the protein of interest (POI). First, POI and DNA are cross-linked before sonication. Then, a specific antibody is used to pull down POI and attached DNA fragments. After the protein and DNA are reverse cross-linked, DNA is purified to make the ChIP-seq library. If using an antibody against particular transcription fac-tor (TF), DNA sequences bound by this TF are pulled down CellisolationPrimaryculturePropagationTissuesampleProduction of recombinant proteinsAnalysis of gene
|
Surgery_Schwartz. sequencing (scRNA-seq) allows expression analysis of individual cells in a population. One use is to examine heterogeneity in tumor cells. Although low copy number genes are usually poorly detected, scRNA-seq on a large number of cells can reveal rare cell subpopula-tions and uncommon RNA expression patterns among different subpopulations.ChIP-seq is always used to map the location of a DNA-binding protein in the genome. Prior to library construction, ChIP is performed to enrich DNA bound by the protein of interest (POI). First, POI and DNA are cross-linked before sonication. Then, a specific antibody is used to pull down POI and attached DNA fragments. After the protein and DNA are reverse cross-linked, DNA is purified to make the ChIP-seq library. If using an antibody against particular transcription fac-tor (TF), DNA sequences bound by this TF are pulled down CellisolationPrimaryculturePropagationTissuesampleProduction of recombinant proteinsAnalysis of gene
|
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.