RESUMO
Graham Hoyle was an important neuroscientist, muscle biologist, and zoologist throughout much of the second half of the twentieth century. A native of England, Hoyle studied under Bernard Katz in London before earning his D.Sc. in neurophysiology from the University of Glasgow. He immigrated to the United States in the mid-1950s and worked with C.A.G. Wiersma at Caltech, with whom he shared a love for crustacean neuromuscular physiology. Hoyle accepted a position at the University of Oregon in 1961 and remained there as a professor until his death in 1985 at the age of 61. Hoyle was active scientifically at a time when the basics of muscle biology were still being discovered. He made many important contributions to the field of neuromuscular physiology, particularly in the realm of comparative physiology. Hoyle was passionate about the importance of a comparative approach in physiology and emphasized that "as a comparative physiologist, I value knowledge of the diverse forms not only for its own sake, but also because it embodies the general truth." Perhaps Hoyle's most lasting legacy is embodied in the many students and postdocs who trained with him early in their careers. Many of these young scientists went on to build prominent careers and trained numerous students of their own. In addition to offering an overview of Hoyle's career, this article revisits some of Hoyle's central contributions to muscle biology and assesses them in light of our current understanding of muscle structure and function.NEW & NOTEWORTHY Graham Hoyle was an important neuroscientist, muscle biologist, and zoologist throughout much of the second half of the twentieth century. He was trained by Bernard Katz at University College London and later worked with C.A.G. Wiersma at Caltech. As a professor at the University of Oregon, Hoyle helped found the Institute of Neuroscience and trained many prominent scientists in the fields of neuromuscular biology and neuroethology.
Assuntos
Fenômenos Fisiológicos do Sistema Nervoso , Neurofisiologia , Humanos , Estados Unidos , Pesquisadores , Pessoal de Saúde , MúsculosRESUMO
The frog sciatic nerve provides a robust physiological preparation students may conveniently use to investigate the properties of compound action potentials. Electrical stimulation with standard physiology teaching equipment elicits compound action potentials that are easily recorded by upper-level undergraduate students. The amplitude of compound action potentials increases with greater stimulation voltages, up until a maximum response is achieved. Plotting action potential size as a function of stimulating voltage produces a curve that illustrates the responsiveness of a nerve. In the present study, several local anesthetics (MS-222, procaine, lidocaine, benzocaine, and tetracaine) were used to reversibly suppress compound action potentials within a time frame consistent with the limitations of teaching labs. Highly responsive nerves generate steep response curves that reach asymptotes at relatively low stimulating voltages. Less active nerves require higher stimulating voltages and appear "right-shifted." Anesthetized response curves may also appear "flatter," exhibiting lower peak amplitude, when compared to fully active nerves. The magnitude of action potential suppression and time course of recovery depended upon the specific anesthetic applied. Nerves anesthetized with MS-222 were the fastest to recover, reaching their original responsiveness within 20 min. Tetracaine had the most dramatic effects, with nerves typically requiring more than a day to fully recover physiological responses. Carefully dissected nerves maintained their physiological responses for many days when stored in Ringer solution at 4°C, making this preparation particularly useful for undergraduate lab experiences. Quantitative analyses may be performed on the data collected, providing students with opportunities to design and implement their own experiments.NEW & NOTEWORTHY The frog sciatic nerve preparation represents a "classical" physiology lab for demonstrating principles of action potentials. Local anesthetics provide an inexpensive tool to manipulate the physiological activity of nerves and other excitable tissues. Isolated nerves retain their physiological activity for up to several days when kept in Ringer solution at 4°C. Quantitative data analysis from this robust nerve preparation should present students with many opportunities for designing their own experiments with anesthetics.
Assuntos
Anestésicos Locais , Tetracaína , Humanos , Anestésicos Locais/farmacologia , Tetracaína/farmacologia , Potenciais de Ação/fisiologia , Solução de Ringer/farmacologia , Nervo IsquiáticoRESUMO
Ghost crabs are the fastest and most aerobically fit of the land crabs. The exceptional locomotory capacity of these invertebrate athletes seemingly depends upon effective coupling between the cardiovascular system and skeletal muscles, but how these systems are integrated has not been well defined. In the present study, we investigated the relationship between aerobic muscle fibers within the skeletal muscles used to power running and the blood vessels supplying these muscles. We used histochemical staining techniques to identify aerobic versus glycolytic fibers and to characterize membrane invaginations within the aerobic fibers. We also determined how the diameters of these two fiber types scale as a function of body size, across two orders of magnitude. Vascular casts were made of the blood vessels perfusing these muscles, and special attention was given to small, capillary-like vessels supplying the fibers. Finally, we injected fluorescent microspheres into the hearts of living crabs and tracked their deposition into different muscle regions to quantify relative hemolymph flow to metabolic fiber types. Collectively, these analyses demonstrate that ghost crab muscles are endowed with an extensive arterial hemolymph supply. Moreover, the hemolymph flow to aerobic fibers is significantly greater than to glycolytic fibers within the same muscles. Aerobic fibers are increasingly subdivided by membrane invaginations as crabs increase in size, keeping the diffusive distances relatively constant. These findings support a functional coupling between a well-developed circulatory system and metabolically active muscle fibers in these invertebrates.
Assuntos
Braquiúros , Animais , Artérias , Hemolinfa , Humanos , Fibras Musculares Esqueléticas , Músculo EsqueléticoRESUMO
The crayfish stretch receptor organ (SRO) preparation represents a robust experimental model for undergraduate laboratory experiences. For example, this preparation may be included as part of a course-based undergraduate research experience (CURE), where students work independently to plan and carry out their own experiments. In the current paper, we provide an example of how local anesthetics may be used to manipulate the SRO preparation and to perform quantitative analyses of SRO action potential firing rates. Local anesthetics provide interesting tools for manipulating physiological responses within the nervous system. A variety of inexpensive anesthetics are available for student use and each of these is expected to inhibit neurophysiological responses. While specific anesthetics exhibit subtle differences in chemical organization, they are generally understood to block voltage gated sodium channels. In the current study, we investigated the effects of two local anesthetics, MS-222 and procaine, on the action potential firing rate from the crayfish SRO. Using quantitative analyses of SRO action potential generation, we determined that each anesthetic has unique inhibitory effects on action potential firing rate that may be explained by their neuropharmacological properties. This manipulation may thus be utilized as an interesting experimental tool in undergraduate teaching laboratories. Local anesthetics applied to crayfish SRO preparations can thus be used to deepen student understanding of local anesthetics, exercise quantitative analyses, and provide experimental tools for independent experimental design.
RESUMO
Skeletal muscle fibers are classified according to the myosin heavy chain (MHC) isoforms and other myofibrillar proteins expressed within these cells. In addition to 'pure' fibers expressing single MHC isoforms, many fibers are 'hybrids' that co-express two or more different isoforms of MHC or other myofibrillar proteins. Although hybrid fibers have been recognized by muscle biologists for more than three decades, uncertainty persists about their prevalence in normal muscles, their role in fiber-type transitions, and what they might tell us about fiber-type regulation at the cellular and molecular levels. This Review summarizes current knowledge on the relative abundance of hybrid fibers in a variety of muscles from different species. Data from more than 150 muscles from 39 species demonstrate that hybrid fibers are common, frequently representing 25% or more of the fibers in normal muscles. Hybrid fibers appear to have two main roles: (1) they function as intermediates during the fiber-type transitions associated with skeletal muscle development, adaptation to exercise and aging; and (2) they provide a functional continuum of fiber phenotypes, as they possess physiological properties that are intermediate to those of pure fiber types. One aspect of hybrid fibers that is not widely recognized is that fiber-type asymmetries - such as dramatic differences in the MHC composition along the length of single fibers - appear to be a common aspect of many fibers. The final section of this Review examines the possible role of differential activities of nuclei in different myonuclear domains in establishing fiber-type asymmetries.
Assuntos
Desenvolvimento Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Isoformas de Proteínas/metabolismo , AnimaisRESUMO
Frogs are routinely used in physiology teaching laboratories to demonstrate important physiological processes. There have been recent directives that promote the use of the anesthetic MS-222 (tricaine methanesulfonate), rather than lowering body temperature with a cold water bath to prepare reptiles and amphibians for physiological experiments or euthanasia. Indeed, the most recent edition of the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals proclaims that chilling in water is not an appropriate method and advocates for the usage of MS-222 or other anesthetics. However, prominent researchers have responded to this position by highlighting evidence that cooling ectothermic vertebrates is, in fact, an effective and appropriate method. Furthermore, MS-222 is a known voltage-gated Na+ channel blocker, and this anesthetic's impact on the physiology of excitable tissues suggests that its use might be incompatible with experiments on nerve and muscle tissues. In the present study, I examined the effects of MS-222 at a concentration of 1.5 g/l on nerve, skeletal muscle, and cardiac muscle physiology of frogs. I found that immersion of frogs in this anesthetic blocked basic nerve and muscle physiology, making the frogs unsuitable for laboratory experiments. Applying MS-222 directly to the sciatic nerve dramatically blocked normal excitation-contraction coupling in skeletal muscle preparations, and direct application to the heart caused the organs to stop contracting. Based on these results, I conclude that MS-222 at the concentration studied may be incompatible with physiological preparations that rely on electrically excitable tissues for their normal function. Physiology educators who must use MS-222 with frogs should empirically determine an appropriate dosage and recovery time before using the anesthetic in the teaching laboratory.
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Aminobenzoatos/farmacologia , Anestésicos/farmacologia , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Fisiologia/educação , Nervo Isquiático/fisiologia , Animais , Humanos , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Rana pipiens , Nervo Isquiático/efeitos dos fármacosRESUMO
The role of hybrid fibers as intermediates in fiber type transformations is not completely understood. In some cases hybrids are clearly transitional fibers changing from one type to another, but in others they represent phenotypically stable fibers in normal muscles. In the current study, our goal was to understand the fate of hybrid fibers in fiber type transitions that take place during muscle maturation. Previous studies have reported high proportions of hybrid fibers during postnatal development, but few have followed the fate of these fibers past the time of weaning. We quantified proportions of hybrid fibers in three different mouse skeletal muscles from newly weaned to 6-month-old mice. Hybrid fibers were very prevalent in the brachioradialis (BR) and tibialis anterior (TA) muscles from newly weaned mice, where they constituted 50 and 40% of the fibers, respectively. These hybrids declined steadily to about 15-30% over the next several months. In the soleus muscle the proportion of hybrids did not change, but constituted approximately 20% of fibers. The reduction in IIX/IIB hybrids resulted from different processes in the BR and the TA. In the BR, the reduction was coincident with an increase in type IIX fibers. In the TA, the number of IIX/IIB hybrids was inversely correlated with the proportion of IIB fibers. These patterns reveal that the role of hybrid fibers as intermediates in muscle development is complex. Some hybrid fibers in maturing muscles represent transitional fiber types, while others are phenotypically stable. Moreover, the fate of transitional fibers may be distinct among similar fiber types within different muscles.
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Desenvolvimento Muscular/fisiologia , Fibras Musculares Esqueléticas/citologia , Cadeias Pesadas de Miosina/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismoRESUMO
Most undergraduate physiology laboratories are very limited in how they treat renal physiology. It is common to find teaching laboratories equipped with the capability for high-resolution digital recordings of physiological functions (muscle twitches, ECG, action potentials, respiratory responses, etc.), but most urinary laboratories still rely on a "dipstick" approach of urinalysis. Although this technique can provide some basic insights into the functioning of the kidneys, it overlooks the dynamic processes of filtration, reabsorption, and secretion. In the present article, we provide a straightforward approach of using renal clearance measurements to estimate glomerular filtration rate, fractional water reabsorption, glucose clearance, and other physiologically relevant parameters. The estimated values from our measurements in laboratory are in close agreement with those anticipated based on textbook parameters. For example, we found glomerular filtration rate to average 124 ± 45 ml/min, serum creatinine to be 1.23 ± 0.4 mg/dl, and fractional water reabsorption to be â¼96.8%. Furthermore, analyses for the class data revealed significant correlations between parameters like fractional water reabsorption and urine concentration, providing opportunities to discuss urine concentrating mechanisms and other physiological processes. The procedures outlined here are general enough that most undergraduate physiology laboratory courses should be able to implement them without difficulty.
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Educação de Graduação em Medicina , Testes de Função Renal , Laboratórios , Fisiologia/educação , Humanos , LouisianaRESUMO
Gene looping, defined as the interaction of the promoter and the terminator regions of a gene during transcription, requires transcription factor IIB (TFIIB). We have earlier demonstrated association of TFIIB with the distal ends of a gene in an activator-dependent manner (El Kaderi, B., Medler, S., Raghunayakula, S., and Ansari, A. (2009) J. Biol. Chem. 284, 25015-25025). The presence of TFIIB at the 3' end of a gene required its interaction with cleavage factor 1 (CF1) 3' end processing complex subunit Rna15. Here, employing affinity chromatography and glycerol gradient centrifugation, we show that TFIIB associates with poly(A) polymerase and the entire CF1 complex in yeast cells. The factors required for general transcription such as TATA-binding protein, RNA polymerase II, and TFIIH are not a component of the TFIIB complex. This holo-TFIIB complex was resistant to MNase digestion. The complex was observed only in the looping-competent strains, but not in the looping-defective sua7-1 strain. The requirement of Rna15 in gene looping has been demonstrated earlier. Here we provide evidence that poly(A) polymerase (Pap1) as well as CF1 subunits Rna14 and Pcf11 are also required for loop formation of MET16 and INO1 genes. Accordingly, cross-linking of TFIIB to the 3' end of genes was abolished in the mutants of Pap1, Rna14, and Pcf11. We further show that in sua7-1 cells, where holo-TFIIB complex is not formed, the kinetics of activated transcription is altered. These results suggest that a complex of TFIIB, CF1 subunits, and Pap1 exists in yeast cells. Furthermore, TFIIB interaction with the CF1 complex and Pap1 is crucial for gene looping and transcriptional regulation.
Assuntos
Complexos Multiproteicos/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIB/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Regiões 3' não Traduzidas/fisiologia , Cromatografia de Afinidade , DNA Fúngico/genética , DNA Fúngico/metabolismo , Genes Fúngicos/fisiologia , Complexos Multiproteicos/genética , Mutação , Mio-Inositol-1-Fosfato Sintase/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Associadas a Pancreatite , Polinucleotídeo Adenililtransferase/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fator de Transcrição TFIIB/genética , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Gene looping juxtaposes the promoter and terminator regions of RNA polymerase II-transcribed genes in yeast and mammalian cells. Here we report an activator-dependent interaction of transcription initiation and termination factors during gene looping in budding yeast. Chromatin analysis revealed that MET16, INO1, and GAL1p-BUD3 are in a stable looped configuration during activated transcription. Looping was nearly abolished in the absence of transcription activators Met28, Ino2, and Gal4 of MET16, INO1, and GAL1p-BUD3 genes, respectively. The activator-independent increase in transcription was not accompanied by loop formation, thereby suggesting an essential role for activators in gene looping. The activators did not facilitate loop formation directly because they did not exhibit an interaction with the 3' end of the genes. Instead, activators physically interacted with the general transcription factor TFIIB when the genes were activated and in a looped configuration. TFIIB cross-linked to both the promoter and the terminator regions during the transcriptionally activated state of a gene. The presence of TFIIB on the terminator was dependent on the Rna15 component of CF1 3' end processing complex. Coimmunoprecipitation revealed a physical interaction of Rna15 with TFIIB. We propose that the activators facilitate gene looping through their interaction with TFIIB during transcriptional activation of genes.
Assuntos
Transcrição Gênica , Cromatina/química , Imunoprecipitação da Cromatina , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Modelos Biológicos , Modelos Genéticos , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fator de Transcrição TFIIB/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Although skeletal muscle fiber types are often defined as belonging to discrete categories, many muscles possess fibers with intermediate phenotypes. These hybrid fiber types can be identified by their expression of two or more myosin heavy chain (MHC) isoforms within the same single fiber. In mouse muscles, the most common hybrid fibers are those coexpressing the IIX and IIB MHC isoforms. In the present study, we focused on these IIX/IIB fibers from normal mouse muscles to determine the relative proportions of MHC isoforms at both the protein and mRNA levels and to examine the longitudinal distribution of isoforms within single fibers. We found that IIX/IIB hybrids represent â¼25 and 50% of the fibers in the mouse tibialis anterior and brachioradialis, respectively. The relative proportion of the IIX and IIB isoforms in these fibers spans a continuum, from predominantly IIB-like hybrids to IIX-like hybrids. Quantitative assessment of mRNA levels using real-time PCR from single fibers indicated that IIB expression dominated over IIX expression in most fibers and that a general correlation existed between mRNA isoform levels and MHC protein content. However, the match between mRNA levels and protein content was not precise. Finally, we measured MHC isoform proportions in adjacent fiber segments and discovered that â¼30% of hybrids possessed significant differences in isoform content along their length. In some instances, the muscle fiber type as defined by MHC content changed completely along the length of a fiber. This pattern of asymmetrical MHC isoform content along the length of single fibers suggests that the multiple myonuclei of a muscle fiber may express distinct myofibrillar isoforms in an uncoordinated fashion.
Assuntos
Fibras Musculares Esqueléticas/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Análise de Variância , Animais , Feminino , Modelos Lineares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cadeias Pesadas de Miosina/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Hybrid muscle fibres co-expressing two or more myosin heavy chain (MHC) isoforms represent a significant proportion of fibres in many muscles, but the prevalence and precise composition of these fibres varies significantly among muscles and animal species. In the present study, we used a forced running protocol for 6 weeks to determine the effects of running exercise on the relative proportion of hybrid muscle fibre types in mouse muscles. In the course of this experiment, we also determined the relative proportions of these fibres in several different skeletal muscles, since data about hybrid fibres in the mouse are sparse. We found that the proportions of hybrid fibres in mouse muscles varied significantly among specific muscles (2-25%), but these proportions were unaffected by 6 weeks of forced running exercise. In contrast, weight-bearing muscles significantly increased in mass in response to running. These data suggest that hybrid muscle fibres are relatively refractory to the effects of moderate exercise and represent a stable phenotype in normal mouse muscles. The precise nature and physiological function of these fibres remain incompletely understood, but it is clear that hybrid fibres represent a common phenotype in many muscles.
Assuntos
Fibras Musculares Esqueléticas/fisiologia , Condicionamento Físico Animal , Corrida/fisiologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/classificação , Músculo Esquelético/fisiologia , Condicionamento Físico Animal/métodos , Fatores de TempoRESUMO
A cDNA encoding a myostatin (Mstn)-like gene from an astacuran crustacean, Homarus americanus, was cloned and characterized. Mstn inhibits skeletal muscle growth in vertebrates and may play a role in crustacean muscle as a suppressor of protein synthesis. Sequence analysis and three-dimensional modeling of the Ha-Mstn protein predicted a high degree of conservation with vertebrate and other invertebrate myostatins. Qualitative polymerase chain reaction (PCR) demonstrated ubiquitous expression of transcript in all tissues, including skeletal muscles. Quantitative PCR analysis was used to determine the effects of natural molting and eyestalk ablation (ESA) on Ha-Mstn expression in the cutter claw (CT) and crusher claw (CR) closer muscles and deep abdominal (DA) muscle. In intermolt lobsters, the Ha-Mstn mRNA level in the DA muscle was significantly lower than the mRNA levels in the CT and CR muscles. Spontaneous molting decreased Ha-Mstn mRNA during premolt, with the CR muscle, which is composed of slow-twitch (S1) fibers, responding preferentially (82% decrease) to the atrophic signal compared to fast fibers in CT (51% decrease) and DA (69% decrease) muscles. However, acute increases in circulating ecdysteroids caused by ESA had no effect on Ha-Mstn mRNA levels in the three muscles. These data indicate that the transcription of Ha-Mstn is differentially regulated during the natural molt cycle and it is an important regulator of protein turnover in molt-induced claw muscle atrophy.
Assuntos
Regulação da Expressão Gênica , Muda/genética , Músculo Esquelético/metabolismo , Miostatina/genética , Nephropidae/genética , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Miostatina/química , Miostatina/metabolismo , Fases de Leitura Aberta/genética , Fator 2 de Elongação de Peptídeos/genética , Fator 2 de Elongação de Peptídeos/metabolismo , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de SequênciaRESUMO
The transcription cycle of RNAPII is comprised of three consecutive steps; initiation, elongation and termination. It has been assumed that the initiation and termination steps occur in spatial isolation, essentially as independent events. A growing body of evidence, however, has challenged this dogma. First, factors involved in initiation and termination exhibit both a genetic and a physical interaction during transcription. Second, the initiation and termination factors have been found to occupy both ends of a transcribing gene. Third, physical interaction of initiation and termination factors occupying distal ends of a gene sometime results in the entire terminator region of a genes looping back and contact its cognate promoter, thereby forming a looped gene architecture during transcription. A logical interpretation of these findings is that the initiation and termination steps of transcription do not occur in isolation. There is extensive communication of factors occupying promoter and terminator ends of a gene during transcription cycle. This review entails a discussion of the promoter-terminator crosstalk and its implication in the context of transcription.
Assuntos
RNA Polimerase II/metabolismo , Transcrição Gênica , Cromatina/metabolismo , Eucariotos/metabolismo , Proteínas Fúngicas/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Saccharomycetales/metabolismo , Regiões Terminadoras Genéticas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Cyclically contracting muscles provide power for a variety of processes including locomotion, pumping blood, respiration, and sound production. In the current study, we apply a computational model derived from force-velocity relationships to explore how sustained power output is systematically affected by shortening velocity, operational frequency, and strain amplitude. Our results demonstrate that patterns of frequency dependent power output are based on a precise balance between a muscle's intrinsic shortening velocity and strain amplitude. We discuss the implications of this constraint for skeletal muscle design, and then explore implications for physiological processes based on cyclical muscle contraction. One such process is animal locomotion, where musculoskeletal systems make use of resonant properties to reduce the amount of metabolic energy used for running, swimming, or flying. We propose that skeletal muscle phenotype is tuned to this operational frequency, since each muscle has a limited range of frequencies at which power can be produced efficiently. This principle also has important implications for our understanding muscle plasticity, because skeletal muscles are capable of altering their active contractile properties in response to a number of different stimuli. We discuss the possibility that muscles are dynamically tuned to match the resonant properties of the entire musculoskeletal system.
Assuntos
Modelos Biológicos , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Animais , Simulação por Computador , Transferência de Energia/fisiologia , Teste de Esforço , Humanos , Reprodutibilidade dos TestesRESUMO
Skeletal muscles comprise hundreds of individual muscle fibers, with each possessing specialized contractile properties. Skeletal muscles are recognized as being highly plastic, meaning that the physiological properties of single muscle fibers can change with appropriate use. During fiber type transitions, one myosin heavy chain isoform is exchanged for another and over time the fundamental nature of the fiber adapts to become a different fiber type. Within the rat triceps surae complex, the soleus muscle starts out as a muscle comprised of a mixture type IIA and type I fibers. As neonatal rats grow and mature, the soleus undergoes a near complete transition into a muscle with close to 100% type I fibers at maturity. We used immunohistochemistry and single fiber SDS-PAGE to track the transformation of type IIA into type I fibers. We found that transitioning fibers progressively incorporate new myofibrils containing type I myosin into existing type IIA fibers. During this exchange, distinct type I-containing myofibrils are segregated among IIA myofibrils. The individual myofibrils within existing muscle fibers thus appear to represent the functional unit that is exchanged during fiber type transitions that occur as part of normal muscle development.
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Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Ratos/crescimento & desenvolvimento , Adenosina Trifosfatases/análise , Adenosina Trifosfatases/metabolismo , Animais , Fibras Musculares de Contração Rápida/ultraestrutura , Fibras Musculares de Contração Lenta/ultraestrutura , Músculo Esquelético/ultraestrutura , Cadeias Pesadas de Miosina/análise , Cadeias Pesadas de Miosina/metabolismo , Ratos Sprague-DawleyRESUMO
This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here is a powerful experimental approach for analyzing the transcription termination defect under physiological conditions. Like the traditional TRO assay, it relies on the presence of a transcriptionally active polymerase beyond the 3' end of the gene as an indicator of a transcription termination defect1. It overcomes two major problems encountered with the traditional TRO assay. First, it can detect if the polymerase reading through the termination signal is the one that initiated transcription from the promoter-proximal region, or if it is simply representing a pervasively transcribing polymerase that initiated non-specifically from somewhere in the body or the 3' end of the gene. Secondly, it can distinguish if the transcriptionally active polymerase signal beyond the terminator region is truly the readthrough sense mRNA transcribing polymerase or a terminator-initiated non-coding anti-sense RNA signal. Briefly, the protocol involves permeabilizing the exponentially growing yeast cells, allowing the transcripts that initiated in vivo to elongate in the presence of the BrUTP nucleotide, purifying BrUTP-labelled RNA by the affinity approach, reverse transcribing the purified nascent RNA and amplifying the cDNA using strand-specific primers flanking the promoter and the terminator regions of the gene2.
Assuntos
Técnicas Genéticas , Saccharomycetales/genética , Transcrição Gênica , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ligação ao GTP/genética , Mutação , Regiões Promotoras Genéticas , RNA Antissenso , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae/genética , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/química , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
The organization of skeletal muscles in decapod crustaceans is significantly altered during molting and development. Prior to molting, the claw muscles atrophy dramatically, facilitating their removal from the base of the claw. During development, lobster claw muscles exhibit fiber switching over several molt cycles. Such processes may be influenced by the secretion of steroid molting hormones, known collectively as ecdysteroids. To assay the effects of these hormones, we used eyestalk ablation to trigger an elevation of circulating ecdysteroids and then quantified myofibrillar mRNA levels with real-time PCR and myofibrillar protein levels by SDS-PAGE. Levels of myosin heavy chain (MHC) and actin proteins and the mRNA encoding them were largely unaffected by eyestalk ablation, but in muscles from intact animals, myofibrillar gene expression was modestly elevated in premolt and postmolt animals. In contrast, polyubiquitin mRNA was significantly elevated (about 2-fold) in claw muscles from eyestalk-ablated animals with elevated circulating ecdysteroids. Moreover, patterns of MHC and actin gene expression are significantly different among slow and fast claw muscles. Consistent with these patterns, the three muscle types differed in the relative amounts of myosin heavy chain and actin proteins. All three muscles also co-expressed fast and slow myosin isoforms, even in fibers that are generally regarded as exclusively fast or slow. These results are consistent with other recent data demonstrating co-expression of myosin isoforms in lobster muscles.
Assuntos
Actinas/metabolismo , Ecdisteroides/metabolismo , Regulação da Expressão Gênica/fisiologia , Hormônios de Invertebrado/deficiência , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Nephropidae/metabolismo , Actinas/genética , Análise de Variância , Animais , California , Ecdisteroides/sangue , Ecdisteroides/farmacologia , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônios de Invertebrado/metabolismo , Miofibrilas/metabolismo , Cadeias Pesadas de Miosina/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
TFIIH is a general transcription factor with kinase and helicase activities. The kinase activity resides in the Kin28 subunit of TFIIH. The role of Kin28 kinase in the early steps of transcription is well established. Here we report a novel role of Kin28 in the termination of transcription. We show that RNAPII reads through a termination signal upon kinase inhibition. Furthermore, the recruitment of termination factors towards the 3' end of a gene was compromised in the kinase mutant, thus confirming the termination defect. A concomitant decrease in crosslinking of termination factors near the 5' end of genes was also observed in the kinase-defective mutant. Simultaneous presence of termination factors towards both the ends of a gene is indicative of gene looping; while the loss of termination factor occupancy from the distal ends suggest the abolition of a looped gene conformation. Accordingly, CCC analysis revealed that the looped architecture of genes was severely compromised in the Kin28 kinase mutant. In a looping defective sua7-1 mutant, even the enzymatically active Kin28 kinase could not rescue the termination defect. These results strongly suggest a crucial role of Kin28 kinase-dependent gene looping in the termination of transcription in budding yeast.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Genes Fúngicos , Conformação de Ácido Nucleico , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Terminação da Transcrição Genética , Regulação Fúngica da Expressão Gênica , Complexos Multiproteicos/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Regiões Terminadoras GenéticasRESUMO
Skeletal muscles are highly plastic tissues capable dramatic remodeling in response to use, disuse, disease, and other factors. Growing evidence suggests that adipose tissues exert significant effects on the basic fiber-type composition of skeletal muscles. In the current study, we investigated the long-term effects of a high-fat diet and subsequent obesity on the muscle fiber types in C57 BLK/6J mice. Litters of mice were randomly assigned to either a high-fat diet or a control group at the time of weaning, and were maintained on this diet for approximately 1 year. Single fibers were harvested from the soleus and plantaris muscles, and fiber types were determined using SDS-PAGE. The high-fat diet mice were significantly heavier than the control mice (39.17 ± 2.7 g vs. 56.87 ± 3.4 g; P < 0.0003), but muscle masses were not different. In male mice, the high-fat diet was associated with a significantly lower proportion of slow, type I fibers in the soleus muscle (40.4 ± 3.5% vs. 29.33 ± 2.6%; P < 0.0165). Moreover, the proportion of type I fibers in the soleus of male mice was inversely proportional to the relative fatness of the male mice (P < 0.003; r (2) = 0.65), but no association was observed in female mice. In male mice, the decline in type I fibers was correlated with an increase in type I/IIA hybrid fibers, suggesting that the type I fibers were transformed primarily into these hybrids. The reported trends indicate that type I fibers are most susceptible to the effects of obesity, and that these fiber-type changes can be sex specific.