RESUMEN
CpG methylation is essential for mouse development as well as gene regulation and genome stability. Many features of mammalian DNA methylation are consistent with the action of a de novo methyltransferase that establishes methylation patterns during early development and the post-replicative maintenance of these patterns by a maintenance methyltransferase. The mouse methyltransferase Dnmt1 (encoded by Dnmt) shows a preference for hemimethylated substrates in vitro, making the enzyme a candidate for a maintenance methyltransferase. Dnmt1 also has de novo methylation activity in vitro, but the significance of this finding is unclear, because mouse embryonic stem (ES) cells contain a de novo methylating activity unrelated to Dnmt1 (ref. 10). Recently, the Dnmt3 family of methyltransferases has been identified and shown in vitro to catalyse de novo methylation. To analyse the function of these enzymes, we expressed Dnmt and Dnmt3a in transgenic Drosophila melanogaster. The absence of endogenous methylation in Drosophila facilitates detection of experimentally induced methylation changes. In this system, Dnmt3a functioned as a de novo methyltransferase, whereas Dnmt1 had no detectable de novo methylation activity. When co-expressed, Dnmt1 and Dnmt3a cooperated to establish and maintain methylation patterns. Genomic DNA methylation impaired the viability of transgenic flies, suggesting that cytosine methylation has functional consequences for Drosophila development.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Drosophila melanogaster/genética , Genoma , Animales , Animales Modificados Genéticamente , Islas de CpG/genética , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Metiltransferasa 3A , Drosophila melanogaster/embriología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Genes Letales/genética , Genes Letales/fisiología , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Ratones , Fenotipo , Pupa/genética , Pupa/crecimiento & desarrollo , Pupa/metabolismo , Transgenes/genética , Transgenes/fisiologíaRESUMEN
The ability of a viral vector to safely deliver and stably integrate large transgene units (transgenons), which not only include one or several therapeutic genes, but also requisite native transcriptional regulatory elements, would be of significant benefit for diseases presently refractory to available technologies. The herpes simplex virus type-1 (HSV-1) amplicon vector has the largest known payload capacity of approximately 130 kb, but its episomal maintenance within the transduced cell nucleus and induction of host cell silencing mechanisms limits the duration of the delivered therapeutic gene(s). Our laboratory developed an integration-competent version of the HSV-1 amplicon by adaptation of the Sleeping Beauty (SB) transposon system, which significantly extends transgene expression in vivo. The maximum size limit of the amplicon-vectored transposable element remains unknown, but previously published plasmid-centric studies have established that DNA segments longer than 6-kb are inefficiently transposed. Here, we compared the transposition efficiency of SB transposase in the context of both the HSV amplicon vector as well as the HSV amplicon plasmid harboring 7 and 12-kb transposable reporter transgene units. Our results indicate that the transposition efficiency of the 12-kb transposable unit via SB transposase was significantly reduced as compared with the 7-kb transposable unit when the plasmid version of the HSV amplicon was used. However, the packaged HSV amplicon vector form provided a more amenable platform from which the 12-kb transposable unit was mobilized at efficiency similar to that of the 7-kb transposable unit via the SB transposase. Overall, our results indicate that SB is competent in stably integrating transgenon units of at least 12 kb in size within the human genome upon delivery of the platform via HSV amplicons.
Asunto(s)
Elementos Transponibles de ADN/genética , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos , Herpesvirus Humano 1/genética , Transposasas/genética , Integración Viral , Células HeLa , Humanos , TransgenesRESUMEN
Histone variant macroH2A1 (macroH2A1) contains an NH(2)-terminal domain that is highly similar to core histone H2A and a larger COOH-terminal domain of unknown function. MacroH2A1 is expressed at similar levels in male and female embryonic stem (ES) cells and adult tissues, but a portion of total macroH2A1 protein localizes to the inactive X chromosomes (Xi) of differentiated female cells in concentrations called macrochromatin bodies. Here, we show that centrosomes of undifferentiated male and female ES cells harbor a substantial store of macroH2A1 as a nonchromatin-associated pool. Greater than 95% of centrosomes from undifferentiated ES cells contain macroH2A1. Cell fractionation experiments confirmed that macroH2A1 resides at a pericentrosomal location in close proximity to the known centrosomal proteins gamma-tubulin and Skp1. Retention of macroH2A1 at centrosomes was partially labile in the presence of nocodazole suggesting that intact microtubules are necessary for accumulation of macroH2A1 at centrosomes. Upon differentiation of female ES cells, Xist RNA expression became upregulated and monoallelic as judged by fluorescent in situ hybridization, but early Xist signals lacked associated macroH2A1. Xi acquired macroH2A1 soon thereafter as indicated by the colocalization of Xist RNA and macroH2A1. Accumulation of macroH2A1 on X chromosomes occurred with a corresponding loss of centrosomal macroH2A1. Our results define a sequence for the loading of macroH2A1 on the Xi and place this event in the context of differentiation and Xist expression. Furthermore, these results suggest a role for the centrosome in the X inactivation process.
Asunto(s)
Centrosoma/fisiología , Histonas/metabolismo , Células Madre/fisiología , Cromosoma X/fisiología , Animales , Diferenciación Celular , Células Cultivadas , Centrosoma/efectos de los fármacos , Centrosoma/ultraestructura , Citocalasina B/farmacología , Femenino , Histonas/análisis , Histonas/genética , Hibridación in Situ , Masculino , Ratones , Nocodazol/farmacología , Células Madre/citología , Tubulina (Proteína)/análisisRESUMEN
Two protein isoforms of histone macroH2A1 (mH2A1) are found in mammalian cells. One isoform, mH2A1.2 is highly concentrated on the heterochromatinized inactive X chromosome (Xi) of female cells. mH2A1.2 protein is also present in male cells, but fails to form dense concentrations. Another protein isoform, mH2A1.1, differs from mH2A1.2 by a single short segment of amino acids. In this study, we cloned and characterized the genomic locus of the mouse mH2A1 gene and mapped it to chromosome 13. Two alternatively spliced transcripts derived from the mH2A1 locus are responsible for the generation of the two mH2A1 protein isoforms with mH2A1.2 mRNA being the most abundant spliced form in all tissues examined. The absolute amount of mH2A1 mRNA is similar in male and female cells for most tissues with the exception of testes where it is par-ticularly abundant. Both spliced forms are present in all adult tissues analyzed as well as in female embryonic stem cells. In contrast, male embryonic stem cells expressed mH2A1.1 at low levels if at all. The relatively abundant expression of mH2A1 in both sexes suggests that mH2A1 has functions in addition to a possible involvement in X chromosome inactivation.
Asunto(s)
Empalme Alternativo/genética , Histonas/genética , ARN Mensajero/genética , Envejecimiento , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular , Línea Celular , Mapeo Cromosómico , Clonación Molecular , Compensación de Dosificación (Genética) , Exones/genética , Etiquetas de Secuencia Expresada , Femenino , Histonas/química , Histonas/fisiología , Masculino , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Isoformas de Proteínas/genética , ARN Mensajero/análisis , Células Madre/citología , Células Madre/metabolismo , Testículo/metabolismoRESUMEN
Bloom syndrome (BS) is characterized by genomic instability and cancer susceptibility caused by defects in BLM, a DNA helicase of the RecQ-family (J. German and N. A. Ellis, The Genetic Basis of Human Cancer, pp. 301-316, 1998). RecQ helicases and topoisomerase III proteins interact physically and functionally in yeast (S. Gangloff et al., Mol. Cell. Biol., 14: 8391-8398, 1994) and in Escherichia coli can function together to enable passage of double-stranded DNA (F. G. Harmon et al., Mol. Cell, 3: 611-620, 1999). We demonstrate in somatic and meiotic human cells an association between BLM and topoisomerase IIIalpha. These proteins colocalize in promyelocytic leukemia protein nuclear bodies, and this localization is disrupted in BS cells. Thus, mechanisms by which RecQ helicases and topoisomerase III proteins cooperate to maintain genomic stability in model organisms likely apply to humans.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , ADN Helicasas/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , Meiosis , Adenosina Trifosfatasas/genética , ADN Helicasas/genética , ADN-Topoisomerasas de Tipo I/genética , Regulación de la Expresión Génica , Humanos , RecQ Helicasas , Células Tumorales CultivadasRESUMEN
A vital step in the development of heart failure is the transition from compensatory cardiac hypertrophy to decompensated dilated cardiomyopathy (DCM) during cardiac remodeling under mechanical or pathological stress. However, the molecular mechanisms underlying the development of DCM and heart failure remain incompletely understood. In the present study, we investigate whether Gab1, a scaffolding adaptor protein, protects against hemodynamic stress-induced DCM and heat failure. We first observed that the protein levels of Gab1 were markedly reduced in hearts from human patients with DCM and from mice with experimental viral myocarditis in which DCM developed. Next, we generated cardiac-specific Gab1 knockout mice (Gab1-cKO) and found that Gab-cKO mice developed DCM in hemodynamic stress-dependent and age-dependent manners. Under transverse aorta constriction (TAC), Gab1-cKO mice rapidly developed decompensated DCM and heart failure, whereas Gab1 wild-type littermates exhibited adaptive left ventricular hypertrophy without changes in cardiac function. Mechanistically, we showed that Gab1-cKO mouse hearts displayed severe mitochondrial damages and increased cardiomyocyte apoptosis. Loss of cardiac Gab1 in mice impaired Gab1 downstream MAPK signaling pathways in the heart under TAC. Gene profiles further revealed that ablation of Gab1 in heart disrupts the balance of anti- and pro-apoptotic genes in cardiomyocytes. These results demonstrate that cardiomyocyte Gab1 is a critical regulator of the compensatory cardiac response to aging and hemodynamic stress. These findings may provide new mechanistic insights and potential therapeutic target for DCM and heart failure.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis , Cardiomiopatía Dilatada/metabolismo , Hipertrofia Ventricular Derecha/metabolismo , Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Envejecimiento/genética , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Cardiomiopatía Dilatada/fisiopatología , Modelos Animales de Enfermedad , Humanos , Hipertrofia Ventricular Derecha/genética , Hipertrofia Ventricular Derecha/patología , Hipertrofia Ventricular Derecha/fisiopatología , Ratones , Ratones Noqueados , Mitocondrias Cardíacas/genética , Mitocondrias Cardíacas/patología , Miocitos Cardíacos/patología , Fosfoproteínas/genética , Estrés Fisiológico/genéticaRESUMEN
PURPOSE: The present study was undertaken to compare cardiorespiratory, metabolic, and perceptual responses to upper body exercise on an arm ergometer (AE) and a modified leg ergometer (LE). METHODS: Seventeen male and seven female subjects completed two experimental trials. During each trial, the subjects performed two successive 8-min steady-state arm crank exercises on either an AE or an LE. The crank frequency was kept constant at 50 rev x min(-1) during all exercise bouts. The two power outputs selected were 50 and 75 W for male subjects and 25 and 50 W for female subjects. To achieve these power outputs, the brake resistance was set at 1, 2, and 3 kg at a power output of 25, 50, and 75 W, respectively, for the AE and 0.5, 1, and 1.5 kg at a power output of 25, 50, and 75 W, respectively, for the LE. Oxygen uptake (VO2), heart rate (HR), respiratory exchange ratio (RER), expired ventilation (VE), gross efficiency (GE), and ratings of perceived exertion (RPE) were measured every minute during the last 2 min of each exercise bout. RESULTS: In male subjects, VO2, HR, RER, VE, and RPE were higher (P < 0.05), whereas GE was lower (P < 0.05) during arm crank exercise on an AE than an LE at power outputs of 50 and 70 W. In female subjects, similar differences in these variables between the two ergometers were also observed when exercise was performed at 50 W. However, VO2, RER, VE, and GE did not differ between the two ergometers when exercise was performed at 25 W. CONCLUSIONS: Upper body exercise elicits greater cardiorespiratory, metabolic, and perceptual responses on an AE than an LE at the same power output when power output is computed according to the manufacturer's instructions.
Asunto(s)
Ergometría/instrumentación , Ejercicio Físico/fisiología , Consumo de Oxígeno/fisiología , Adolescente , Adulto , Brazo/fisiología , Femenino , Frecuencia Cardíaca , Humanos , Masculino , Percepción , RespiraciónRESUMEN
Level and incline backward treadmill walking techniques are used in the rehabilitation of certain lower extremity injuries (eg., anterior cruciate ligament reconstruction). Of interest to clinicians is the maintenance of cardiorespiratory fitness resulting from these activities. The purpose of the present study was to determine the cardiorespiratory and metabolic stress of backward walking compared with forward walking. The metabolic cost of backward incline walking above a 1% grade has previously not been reported. Seventeen volunteers (11 males and six females, age = 25 +/- 2 years) underwent a forward maximal running test and four random-ordered 6-minute submaximal walking bouts at 93.8 m/min (3.5 mph). The bouts consisted of forward walking at 0% and 5% elevation and backward walking at 0% and 5% elevation. Measurements taken for each exercise session were oxygen uptake, expired ventilation, heart rate, and rating of perceived exertion. Statistical analysis of these dependent variables indicates that: 1) at a given elevation, backward walking elicited greater cardiorespiratory, metabolic, and perceptual responses than forward walking and 2) backward walking at 5% elevation could provide a sufficient stimulus to maintain cardiorespiratory fitness.
Asunto(s)
Fenómenos Fisiológicos Cardiovasculares , Metabolismo Energético/fisiología , Fenómenos Fisiológicos Respiratorios , Caminata/fisiología , Adulto , Análisis de Varianza , Prueba de Esfuerzo , Femenino , Humanos , Masculino , Consumo de Oxígeno , Valores de ReferenciaRESUMEN
STUDY DESIGN: A within-subject experimental design. OBJECTIVES: To compare the magnitude of metabolic and cardiorespiratory changes produced during box lifting and lowering among combinations of lift technique (leg lift and leg-torso lift) and lift weight (10.8 and 15.4 kg). BACKGROUND: Continuous box lifting and lowering can be used as an exercise in a low-back rehabilitation program. Awareness of the possible cardiovascular stress of this activity is important to the clinician because some patients may have existing cardiovascular pathologies or possess unknown risk factors for cardiovascular disease. METHODS AND MEASURES: A group of 17 nonimpaired men 26 +/- 8 years of age (mean +/- SD) performed the 4 experimental trials on different days in a counterbalanced order determined by a Latin Square design. Lifting and lowering was performed for 6 continuous minutes at a rate of 12 cycles per minute. Physiologic variables were oxygen uptake, minute ventilation, heart rate, systolic blood pressure, diastolic blood pressure, metabolic equivalent, and rate-pressure product. RESULTS: There were stepwise increases in the values for oxygen uptake, minute ventilation, heart rate, metabolic equivalent, and rate-pressure product from the leg-torso lift to the leg lift and from 10.8 to 15.4 kg of weight within each lift technique (with the exception that minute ventilation and heart rate did not differ between the leg-torso lift at 15.4 kg and the leg lift at 10.8 kg). For the 4 lifts, values (mean +/- SD) varied from 20.3 +/- 5.4 to 28.8 +/- 5.8 mL x kg x min(-1) for oxygen uptake, 42.2 +/- 11.1 to 66.4 +/- 15.2 L x min(-2) for minute ventilation,129 +/- 20.6 to 156 +/- 16.5 beats x min(-1) for heart rate, 5.8 +/- 1.6 to 8.2 +/- 1.6 for metabolic equivalent, and 197 +/- 49.4 to 245 +/- 41.2 for rate-pressure product (x10(-2)). CONCLUSION: The leg lift with the 15.4-kg weight produced the greatest physiologic stress. Because of the magnitude of the increase in the variables measured for all 4 types of lifts, clinicians should closely monitor patients' response to this type of exercise.
Asunto(s)
Hemodinámica/fisiología , Músculo Esquelético/fisiología , Mecánica Respiratoria/fisiología , Levantamiento de Peso/fisiología , Adulto , Pruebas Respiratorias , Prueba de Esfuerzo , Humanos , Masculino , Consumo de Oxígeno , Valores de ReferenciaRESUMEN
Y-box proteins bind DNA and RNA and are characterized by a cold shock domain and a carboxyl-terminus containing clusters of aromatic and basic residues that alternate with clusters of acidic residues. Y-box proteins 1 and 3 in mouse testis were cloned here by 3' rapid amplification of cDNA ends (RACE) using a degenerate primer. Northern blots and reverse transcription-polymerase chain reaction (RT-PCR) established that the levels of Y-box protein 1 and 3 mRNAs are regulated individually: (i) Y-box protein 1 mRNA is strongly expressed in kidney, whereas Y-box protein 3 mRNA is strongly expressed in heart and muscle; (ii) Y-box protein 1 and 3 mRNAs are weakly expressed in early prepubertal testis and strongly expressed in pachytene spermatocytes, round spermatids, and elongated spermatids; and (iii) prepubertal testes and meiotic and haploid spermatogenic cells express two alternatively spliced Y-box protein 3 mRNAs encoding isoforms with different carboxyl termini, whereas somatic tissues primarily express one form. Sucrose gradients reveal that approximately 27% of both Y-box protein 3 mRNAs are translationally active in adult testis. In conclusion, spermatogenic cells in mice express five isoforms of Y-box proteins including Y-box protein 1, and two isoforms each of Y-box proteins 2 and 3. This multiplicity is intriguing because Y-box proteins are thought to activate transcription and repress translation in spermatogenic cells.
Asunto(s)
Empalme Alternativo , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , ARN Mensajero , Espermatogénesis/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Centrifugación por Gradiente de Densidad , ADN Complementario , Perfilación de la Expresión Génica/métodos , Masculino , Ratones , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , SacarosaRESUMEN
The mouse Pabp2 retroposon encodes an isoform of poly(A) binding protein that is expressed in meiotic and early haploid spermatogenic cells. In the present study, we have determined the transcription start site of the Pabp2 gene to clarify the source of its promoter, a prerequisite for expression of retroposons and preservation of their function by natural selection. The 5' end of the mouse Pabp2 retroposon exhibits extensive similarity to the entire 5' UTR of the human PABP1 mRNA, but there is no similarity upstream of the transcription start site of the human PABP1 mRNA, indicating that the Pabp2 gene lacks 5' flanking sequences of the parental PABP1 gene. Oligonucleotide-directed RNase H cleavage and 5' rapid amplification of cDNA ends both indicate that the transcription start site of the mouse Pabp2 gene is located approximately 330 bases downstream of the capsite of the PABP1 mRNA, indicating that the Pabp2 promoter is derived from the PABP1 5' UTR.
Asunto(s)
Regiones no Traducidas 5' , Proteínas de Unión al ADN/genética , Regiones Promotoras Genéticas , Proteínas de Unión al ARN/genética , Retroelementos , Animales , Secuencia de Bases , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Proteína II de Unión a Poli(A) , Proteínas de Unión a Poli(A) , Proteínas de Unión al ARN/química , Ribonucleasa H , Testículo/química , Transcripción GenéticaRESUMEN
Male germ cells in mice develop normally at 32 degrees C and spermatogenesis is severely inhibited by higher temperatures, including abdominal temperature, 37 degrees C. To examine the effects of heat stress on protein synthesis in various testicular cell types, seminiferous tubules were cultured at 32 degrees or 37 degrees C for 70 min or 42.5 degrees or 44 degrees C for 10 min followed by incubation for 60 min at 32 degrees C. Cultures were labeled with [35S]methionine, and the proteins that are soluble in 4% trichloroacetic acid were analyzed by acid-urea polyacrylamide gel electrophoresis. This culture system preserves the cytoarchitecture of the seminiferous epithelium and avoids breaking late haploid cells (elongated spermatids) during tissue dissociation. Incorporation of [35S]methionine into histone H1t, the testis-specific subtype of histone H1, in pachytene primary spermatocytes (meiotic cells) was reduced by about 33-50% following incubation at 37 degrees and 42.5 degrees C and by >/=90% after incubation at 44 degrees C. In contrast, exposure to 37 degrees, 42.5 degrees, and 44 degrees C had minimal effects on incorporation into transition proteins 1 and 2 in elongated spermatids. To determine whether heat stress inhibits translational initiation, the distribution of several mRNAs in cytoplasmic extracts of cultured tubules was analyzed by sucrose gradients and Northern blots. Exposure to 37 degrees and 44 degrees C produces incremental reductions in the size of polysomes translating H1t mRNA in pachytene spermatocytes and the sulfated glycoprotein 2 mRNA in Sertoli cells, the somatic cell type in the germinal epithelium. Neither 37 degrees nor 44 degrees C reduces the size or proportion of polysomal protamine 2 mRNA in elongated spermatids. These results demonstrate that the initiation of translation in pachytene spermatocytes and Sertoli cells is inhibited by exposure to abdominal temperature and that elongated spermatids are much more resistant to thermal stress.
Asunto(s)
Respuesta al Choque Térmico , Chaperonas Moleculares , Biosíntesis de Proteínas , Células de Sertoli/metabolismo , Espermátides/metabolismo , Espermatocitos/metabolismo , Animales , Centrifugación por Gradiente de Densidad , Proteínas Cromosómicas no Histona/biosíntesis , Clusterina , Técnicas de Cultivo , Glicoproteínas/genética , Histonas/biosíntesis , Histonas/genética , Masculino , Metionina/metabolismo , Ratones , Protaminas/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Túbulos SeminíferosRESUMEN
Sucrose gradients have been widely used to study the translational activity of mRNA species in meiotic and haploid spermatogenic cells in mammals. Unfortunately, the results of these studies have been very inconsistent. The purpose of the present study was to obtain accurate and reproducible measurements of the translational activity of a large number of testicular mRNA in sucrose gradients. Extracts of adult testes and cultured seminiferous tubules were sedimented on sucrose gradients, and the distribution of 18 mRNA species was quantified by phosphoimaging. The proportions of various mRNA species sedimenting with polysomes in meiotic and haploid cells (approximately 6-74%) is less than typical of efficiently translated mRNAs (85-90%), demonstrating that the initiation of translation of virtually all mRNA species is at least partially inhibited and that the extent of inhibition is mRNA-specific. Most mRNA species in meiotic and early haploid spermatogenic cells are translated on polysomes in which the ribosome spacing is somewhat wider than in somatic cells, 100-150 verses 80-100 bases. However, the ribosome spacing on protamine mRNAs is unusually close (40-50 bases), and the spacing on poly(A) binding protein mRNA is unusually wide (212-272 bases), thus suggesting that the rate of translational initiation, termination and/or elongation is regulated on translationally active forms of certain mRNA.
Asunto(s)
Biosíntesis de Proteínas , ARN Mensajero/análisis , ARN Mensajero/genética , Testículo/fisiología , Animales , Centrifugación por Gradiente de Densidad/métodos , Masculino , Ratones , Ratones Endogámicos , Ribosomas/genética , SacarosaRESUMEN
The present investigation was undertaken to examine whether maximal oxygen uptake (VO2max) and anaerobic threshold (AT) measured during incremental treadmill exercise would be affected by the exercise protocol in trained and untrained individuals. Fifteen untrained men, 10 untrained women, and 12 trained individuals participated in this study. The Astrand, Bruce, and Costill/Fox protocols were selected for comparison. Each subject was tested using all three protocols and the three tests were conducted in a randomized counterbalanced order. During each test, oxygen uptake was measured every 30 s and the test was terminated according to the standard criteria. The VO2max was determined by averaging the two consecutive highest measurements, whereas AT was determined using ventilatory parameters following the V-slope technique. The Astrand, Bruce, and Costill/Fox protocols produced test durations of 9.8 (SEM 0.5), 12.4 (SEM 0.4), and 4.9 (SEM 0.3) min, respectively, in the untrained men, 9.0 (SEM 0.8), 11.0 (SEM 0.6), and 5.3 (SEM 0.6) min, respectively, in the untrained women, and 14.5 (SEM 0.5), 17.0 (SEM 0.5) and 10.4 (SEM 0.4) min, respectively, in the trained men. In the untrained men and women, no differences in VO2max were observed among the three different protocols, but AT was lower when using the Bruce compared to the Astrand protocol. In the trained men, VO2max and AT were lower when using the Bruce protocol than either the Astrand or Costill/Fox protocols. In conclusion, VO2max measured during treadmill exercise is not affected by the protocol of the test and using a running protocol of short duration (i.e. about 5 min) could be a time-efficient way of assessing VO2max in healthy untrained subjects. In trained subjects, however, a protocol consisting of running with small increments in gradient is effective in eliciting a higher VO2max. The lower AT associated with the Bruce protocol seen in both untrained and trained groups suggests this aerobic parameter is protocol dependent and this protocol dependency is not affected by training status.
Asunto(s)
Umbral Anaerobio/fisiología , Ejercicio Físico/fisiología , Adulto , Prueba de Esfuerzo , Femenino , Humanos , Masculino , Consumo de Oxígeno/fisiología , Carrera/fisiologíaRESUMEN
The outer membranes of mitochondria of mammalian sperm are encased in a keratinous structure known as the mitochondrial capsule. The experiments in the present study were designed to resolve a controversy surrounding the intracellular localization, developmental expression, and selenium-content of a cysteine-rich 17-20 kD protein that has been reported to constitute the major structural protein in the mitochondrial capsule of mammals. An antibody to a synthetic oligopeptide based on the predicted sequence of mouse cysteinerich protein recognizes a 24 kD protein in epididymal sperm tails of mice. The 24 kD protein does not appear to be a selenoprotein because: (1) it is not labeled with 75Se-selenite in seminiferous tubule culture; (2) cleavage with cyanogen bromide and translation of T7 RNA polymerase transcripts in vitro indicate that the translation start site is located downstream of potential UGA selenocysteine codons in the mouse cysteine-rich mRNA; (3) the reading frame encoding the cysteine-rich protein in rat lacks inphase UGA selenocysteine codons. Light and electron microscopy immunocytochemistry detects the cysteine-rich protein first during step 11 of spermiogenesis in the mouse demonstrating that the cysteine-rich protein mRNA is under temporal translational control. Electron microscope immunocytochemistry reveals that the cysteine-rich protein is evenly distributed in the cytoplasm in spermatids in steps 11 through early step 16 in mouse, and that it is associated with the outer mitochondrial membranes of spermatids in late step 16 and epididymal spermatozoa.
Asunto(s)
Expresión Génica , Proteínas/genética , Espermatozoides/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Codón Iniciador , ADN Complementario , Técnicas para Inmunoenzimas , Marcaje Isotópico , Masculino , Ratones , Microscopía Inmunoelectrónica , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Proteínas/metabolismo , Conejos , Ratas , Selenio/metabolismo , Selenoproteínas , Túbulos Seminíferos/citología , Túbulos Seminíferos/metabolismoRESUMEN
The gene encoding the testis-specific isoform of mouse poly(A) binding protein (Pabp2) has been isolated and sequenced. Unexpectedly, comparison of the sequence of genomic and cDNAs demonstrated that the Pabp2 gene lacks introns, whereas all other functional Pabp genes in plants, amphibians, and mammals contain introns. Thus, the mouse Pabp2 gene is a retroposon, created by synthesizing a reverse transcriptase copy of a processed mRNA and inserting the copy into the genome. The Pabp2 retroposon is unusual because it is functional: previous work demonstrates that its promoter drives the accumulation of Pabp2 mRNA in meiotic and early haploid spermatogenic cells, and the Pabp2 mRNA encodes a protein whose size and RNA-binding specificities are characteristic of PABP in plants, yeast, and mammals (Kleene et al. 1994). Two novel factors can be implicated in the retention of function of the Pabp2 retroposon. First, the promoter of the Pabp2 gene is not derived from its intron-containing progenitor, Pabp1. Second, mRNAs encoding somatic PABP isoform, PABP1, are present at high levels in meiotic and haploid spermatogenic cells. Both features contrast with the phosphoglycerate kinase 2 retroposon, which is believed to compensate for the depletion of the somatic isoform due to X-chromosome inactivation in meiotic spermatogenic cells. We also document that more functional retroposons are expressed in meiotic and haploid spermatogenic cells than in any other tissue and speculate that transcriptional derepression in spermatogenic cells favors the creation of expressed retroposons.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Retroelementos/genética , Espermatogénesis , Testículo/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Proteína II de Unión a Poli(A) , Isoformas de Proteínas , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Espermatogénesis/genéticaRESUMEN
The purpose of this investigation was to examine the validity of regulating exercise intensity using ratings of perceived exertion (RPEs) during arm crank and leg cycle exercise at 50 and 70% peak oxygen consumption (VO2peak). Ten men and seven women [26 (1) years old; mean (SE)] participated in this study. Each subject completed a maximal estimation trial and two submaximal exercise bouts (production trials) on both an arm and leg ergometer. During each maximal estimation trial, subjects were asked to give a RPE for each stage of the exercise. RPEs, heart rates (HR), and power outputs (PO) equivalent to 50 and 70% VO2peak for each exercise mode were then estimated from plots of RPE versus oxygen consumption (VO2), HR versus VO2, and PO versus VO2, respectively. During the submaximal trials, subjects were instructed to select workloads on an arm and leg ergometer that produced the previously estimated RPEs. Comparisons were made for VO2, HR, and PO between the estimation and production trials for each mode at each exercise intensity. HR did not differ between the trials at either 50 or 70% VO2peak during arm and leg ergometry. In addition, VO2 and PO did not differ between the trials at either 50 or 70% VO2peak during arm ergometry and at 50% VO2peak during leg ergometry. However, these two parameters were lower (P < 0.05) during the production trial [1.88 (0.15) l x min(-1) and 89.1 (10.1) W, respectively] as compared to the estimation trial [2.08(0.14) l x min(-1) and 102.4 (6.5)W, respectively] during leg ergometry at 70% VO2peak. In conclusion, using RPEs to regulate exercise intensity is physiologically valid during arm ergometry at both 50 and 70% VO2peak and during leg ergometry at 50% VO2peak. However, this prescriptive approach remains questionable during leg cycle exercise at 70% VO2peak.
Asunto(s)
Brazo/fisiología , Ejercicio Físico/fisiología , Pierna/fisiología , Adulto , Prueba de Esfuerzo , Femenino , Frecuencia Cardíaca/fisiología , Humanos , Masculino , Consumo de Oxígeno/fisiologíaRESUMEN
BACKGROUND: Serum elevations of interleukin-6 (IL-6) correlate with multiple organ dysfunction syndrome and mortality in critically injured trauma patients. Data from rodent models of controlled hemorrhage suggest that recombinant IL-6 (rIL-6) infusion protects tissue at risk for ischemia-reperfusion injury. Exogenous rIL-6 administered during shock appears to abrogate inflammation, providing a protective rather than a deleterious influence. In an examination of this paradox, the current study aimed to determine whether rIL-6 decreases inflammation in a clinically relevant large animal model of uncontrolled hemorrhagic shock, (UHS), and to investigate the mechanism of protection. METHODS: Swine were randomized to four groups (8 animals in each): (1) sacrifice, (2) sham (splenectomy followed by hemodilution and cooling to 33 degrees C), (3) rIL-6 infusion (sham plus UHS using grade 5 liver injury with packing and resuscitation plus blinded infusion of rIL-6 [10 mcg/kg]), and (4) placebo (UHS plus blinded vehicle). After 4 hours, blood was sampled, estimated blood loss determined, animals sacrificed, and lung harvested for RNA isolation. Quantitative reverse transcriptase-polymerase chain reaction was used to assess granulocyte colony-stimulating factor (G-CSF), IL-6, and tumor necrosis factor-alpha (TNFalpha) messenger ribonucleic acid (mRNA) levels. Serum levels of IL-6 and TNFalpha were measured by enzyme-linked immunoassay (ELISA). RESULTS: As compared with placebo, IL-6 infusion in UHS did not increase estimated blood loss or white blood cell counts, nor decrease hematocrit or platelet levels. As compared with the sham condition, lung G-CSF mRNA production in UHS plus placebo increased eightfold (*p < 0.05). In contrast, rIL-6 infusion plus UHS blunted G-CSF mRNA levels, which were not significantly higher than sham levels (p = 0.1). Infusion of rIL-6 did not significantly affect endogenous production of either lung IL-6 or mRNA. As determined by ELISA, rIL-6 infusion did not increase final serum levels of IL-6 or TNFalpha over those of sham and placebo conditions. CONCLUSIONS: Exogenous rIL-6 blunts lung mRNA levels of the proinflammatory cytokine G-CSF. The administration of rIL-6 does not increase the local expression of IL-6 nor TNFalpha mRNA in the lung. Additionally, rIL-6 infusion does not appear to cause systemic toxicity.