RESUMO
AIM: To investigate effects of hormone replacement therapy in postmenopausal women on factors associated with metabolic flexibility related to whole-body parameters including fat oxidation, resting energy expenditure, body composition and plasma concentrations of fatty acids, glucose, insulin, cortisol, and lipids, and for the mitochondrial level, including mitochondrial content, respiratory capacity, efficiency, and hydrogen peroxide emission. METHODS: 22 postmenopausal women were included. 11 were undergoing estradiol and progestin treatment (HT), and 11 were matched non-treated controls (CONT). Peak oxygen consumption, maximal fat oxidation, glycated hemoglobin, body composition, and resting energy expenditure were measured. Blood samples were collected at rest and during 45 min of ergometer exercise (65% VO2peak). Muscle biopsies were obtained at rest and immediately post-exercise. Mitochondrial respiratory capacity, efficiency, and hydrogen peroxide emission in permeabilized fibers and isolated mitochondria were measured, and citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HAD) activity were assessed. RESULTS: HT showed higher absolute mitochondrial respiratory capacity and post-exercise hydrogen peroxide emission in permeabilized fibers and higher CS and HAD activities. All respiration normalized to CS activity showed no significant group differences in permeabilized fibers or isolated mitochondria. There were no differences in resting energy expenditure, maximal, and resting fat oxidation or plasma markers. HT had significantly lower visceral and total fat mass compared to CONT. CONCLUSION: Use of hormone therapy is associated with higher mitochondrial content and respiratory capacity and a lower visceral and total fat mass. Resting energy expenditure and fat oxidation did not differ between HT and CONT.
Assuntos
Metabolismo Energético , Pós-Menopausa , Humanos , Feminino , Pós-Menopausa/metabolismo , Pessoa de Meia-Idade , Metabolismo Energético/efeitos dos fármacos , Idoso , Consumo de Oxigênio/efeitos dos fármacos , Terapia de Reposição Hormonal , Terapia de Reposição de Estrogênios , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Composição Corporal/efeitos dos fármacos , Estradiol/sangue , Estradiol/metabolismo , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Tecido Adiposo/metabolismo , Tecido Adiposo/efeitos dos fármacosRESUMO
Heterotrimeric guanine nucleotide binding proteins (G proteins) transduce extracellular signals received by transmembrane receptors to effector proteins. The multigene family of G protein alpha subunits, which interact with receptors and effectors, exhibit a high level of sequence diversity. In mammals, 15 G alpha subunit genes can be grouped by sequence and functional similarities into four classes. We have determined the murine chromosomal locations of all 15 G alpha subunit genes using an interspecific backcross derived from crosses of C57BL/6J and Mus spretus mice. These data, in combination with mapping studies in humans, have provided insight into the events responsible for generating the genetic diversity found in the mammalian alpha subunit genes and a framework for elucidating the role of the G alpha subunits in disease.
Assuntos
Evolução Biológica , Proteínas de Ligação ao GTP/genética , Família Multigênica , Animais , Sequência de Bases , Mapeamento Cromossômico , Cruzamentos Genéticos , DNA/genética , Sondas de DNA , Feminino , Ligação Genética , Humanos , Invertebrados/genética , Masculino , Camundongos , Dados de Sequência MolecularRESUMO
Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are dominantly inherited chondrodysplasias characterized by short stature and early-onset osteoarthrosis. The disease genes in families with PSACH and MED have been localized to an 800 kilobase interval on the short arm of chromosome 19. Recently the gene for cartilage oligomeric matrix protein (COMP) was localized to chromosome 19p13.1. In three patients with these diseases, we identified COMP mutations in a region of the gene that encodes a Ca++ binding motif. Our data demonstrate that PSACH and some forms of MED are allelic and suggest an essential role for Ca++ binding in COMP structure and function.
Assuntos
Acondroplasia/genética , Proteínas da Matriz Extracelular , Glicoproteínas/genética , Mutação , Osteocondrodisplasias/genética , Acondroplasia/diagnóstico por imagem , Acondroplasia/metabolismo , Alelos , Sequência de Aminoácidos , Sequência de Bases , Cálcio/metabolismo , Calmodulina/genética , Cartilagem , Proteína de Matriz Oligomérica de Cartilagem , Mapeamento Cromossômico , Cromossomos Humanos Par 19 , DNA Satélite/genética , Fator de Crescimento Epidérmico/genética , Feminino , Genes Dominantes , Ligação Genética , Glicoproteínas/metabolismo , Humanos , Masculino , Proteínas Matrilinas , Dados de Sequência Molecular , Osteocondrodisplasias/diagnóstico por imagem , Osteocondrodisplasias/metabolismo , Linhagem , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Radiografia , Sequências Repetitivas de Ácido NucleicoRESUMO
A metric physical map of human chromosome 19 has been generated. The foundation of the map is sets of overlapping cosmids (contigs) generated by automated fingerprinting spanning over 95% of the euchromatin, about 50 megabases (Mb). Distances between selected cosmid clones were estimated using fluorescence in situ hybridization in sperm pronuclei, providing both order and distance between contigs. An average inter-marker separation of 230 kb has been obtained across the non-centromeric portion of the chromosome. Various types of larger insert clones were used to span gaps between contigs. Currently, the map consists of 51 'islands' containing multiple clone types, whose size, order and relative distance are known. Over 450 genes, genetic markers, sequence tagged sites (STSs), anonymous cDNAs, and other markers have been localized. In addition, EcoRI restriction maps have been generated for > 41 Mb (approximately 83%) of the chromosome.
Assuntos
Mapeamento Cromossômico/métodos , Cromossomos Humanos Par 19 , Sequência de Bases , Cosmídeos/genética , Impressões Digitais de DNA , Desoxirribonuclease EcoRI , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Masculino , Dados de Sequência Molecular , Mapeamento por Restrição , EspermatozoidesRESUMO
A powerful convergence of genetics, neuroimaging and epidemiological research has identified the biological pathways mediating individual differences in complex behavioral processes and the related risk for disease. Orthologous genetic variation in non-human primates (NHPs) represents a unique opportunity to characterize the detailed molecular and cellular mechanisms that bias behaviorally and clinically relevant brain function. We report that a rhesus macaque orthologue of a common polymorphism of the serotonin transporter gene (rh5-HTTLPR) has strikingly similar effects on behavior and brain morphology to those in humans. Specifically, the rh5-HTTLPR (S)hort allele broadly affects cognitive choice behavior and brain morphology without observably affecting the 5-hydroxytryptamine (5-HT) transporter or 5-HT(1A) concentrations in vivo. Collectively, our findings indicate that 5-HTTLPR-associated behavioral effects reflect genotype-dependent biases in cortical development rather than static differences in serotonergic signaling mechanisms. Moreover, these data highlight the vast potential of NHP models in advancing our understanding of human genetic variation affecting behavior and neuropsychiatric disease liability.
Assuntos
Comportamento de Escolha/fisiologia , Cognição/fisiologia , Polimorfismo Genético/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Serotonina/metabolismo , Transmissão Sináptica/genética , Animais , Aprendizagem da Esquiva/fisiologia , Comportamento Animal/fisiologia , Benzilaminas/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Mapeamento Encefálico , Isótopos de Carbono/metabolismo , Genótipo , Macaca mulatta , Imageamento por Ressonância Magnética/métodos , Masculino , Testes Neuropsicológicos , Piperazinas/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Piridinas/metabolismo , Receptor 5-HT1A de Serotonina/genética , Serotonina/genética , Fatores de Tempo , Trítio/metabolismoRESUMO
To illuminate the function and evolutionary history of both genomes, we sequenced mouse DNA related to human chromosome 19. Comparative sequence alignments yielded confirmatory evidence for hypothetical genes and identified exons, regulatory elements, and candidate genes that were missed by other predictive methods. Chromosome-wide comparisons revealed a difference between single-copy HSA19 genes, which are overwhelmingly conserved in mouse, and genes residing in tandem familial clusters, which differ extensively in number, coding capacity, and organization between the two species. Finally, we sequenced breakpoints of all 15 evolutionary rearrangements, providing a view of the forces that drive chromosome evolution in mammals.
Assuntos
Cromossomos Humanos Par 19/genética , Sequência Conservada/genética , Evolução Molecular , Animais , Quebra Cromossômica/genética , Mapeamento de Sequências Contíguas , DNA Satélite/genética , Éxons/genética , Etiquetas de Sequências Expressas , Dosagem de Genes , Ordem dos Genes/genética , Ligação Genética/genética , Genoma , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética , Camundongos , Família Multigênica/genética , Fases de Leitura Aberta/genética , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Elementos Nucleotídeos Curtos e Dispersos/genética , Sequências Repetidas Terminais/genéticaRESUMO
Labeled probes of unique-sequence human X chromosomal deoxyribonucleic acid, prepared by two different procedures, were used to measure the amount of human X chromosomal deoxyribonucleic acid in 12 mouse cell lines expressing human hypoxanthine phosphoribosyltransferase after chromosome-mediated gene transfer. The amount of X chromosomal deoxyribonucleic acid detected by this procedure ranged from undetectable levels in the three stable transformants and some unstable transformants examined to about 20% of the human X chromosome in two unstable transformants. Reassociation kinetics of the X chromosomal probe with deoxyribonucleic acid from the two unstable transformants containing 15 to 20% of the human X chromosome indicate that a single copy of these sequences is present. In one of these lines, the X chromosomal sequences exist as multiple fragments which were not concordantly segregated when the cells were selected for loss of hprt.
Assuntos
Transfecção , Cromossomo X , Animais , DNA/genética , Humanos , Células Híbridas/enzimologia , Hipoxantina Fosforribosiltransferase/genética , Camundongos , FenótipoRESUMO
A basic ribosomal phosphoprotein of 30,000 molecular weight was rapidly dephosphorylated in cultured Drosophila melanogaster cells heat shocked at 37 degrees C. The protein was associated with the 40S ribosomal subunit and had an electrophoretic mobility similar to that of purified rat liver protein S6 on basic two-dimensional polyacrylamide gels as well as a similar partial proteolysis peptide map. In logarithmically growing cultures, this D. melanogaster S6 protein appeared to have a single phosphorylated species consisting of 30 to 40% of the total cellular S6. Thus, the nearly complete dephosphorylation of this protein observed in heat shock involves a large fraction of the cellular S6. The significance of this dephosphorylation in the expression of the heat shock response was investigated by examining the phosphorylation status of S6 in recovery from heat shock and in response to chemical inducers of the heat shock response. During recovery from a 30-min heat shock, the recovery of normal protein synthesis was almost complete in 2 to 4 hr, whereas there was no significant rephosphorylation of S6 for 8 h. Two chemical inducers of the heat shock response, canavanine and sodium arsenite, induced the synthesis of heat shock proteins in D. melanogaster cells. Sodium arsenite also caused an inhibition of normal protein synthesis similar to that observed in heat shock. Neither agent, however, caused significant dephosphorylation of S6. These results suggest that the dephosphorylation of S6, although invariably observed in heat-shocked cells, may in some cases be dissociated from both the induction of heat shock protein synthesis and the turnoff of normal protein synthesis which occur in a heat shock response.
Assuntos
Arsenitos , Drosophila melanogaster/metabolismo , Temperatura Alta , Proteínas Ribossômicas/metabolismo , Compostos de Sódio , Animais , Arsênio/farmacologia , Células Cultivadas , Células HeLa/metabolismo , Proteínas de Choque Térmico/biossíntese , Humanos , Peso Molecular , Fosfoproteínas/metabolismo , Fosforilação , Conformação Proteica , Proteína S6 RibossômicaRESUMO
Transfer of genetic information can be effected by incubation of cultured eucaryotic cells with isolated metaphase chromosomes. In most cases, a resulting transformed cell contains only a fragment of a donor chromosome. The amount of transferred donor DNA has been quantified in 11 independent mouse A9 transformants by nucleic acid hybridization analysis. Each transformant had been selected for hprt (hypoxanthine phosphoribosyltransferase; EC 2.4.2.8) transfer and contained part of the human X chromosome. A labeled probe of transcribed human X-chromosomal DNA was prepared by hybridization of nick-translated unique-sequence human DNA with whole cellular RNA from a human-mouse hybrid cell line, A9/HRBC2-A, containing a single human chromosome., X. The amount of human X-chromosomal DNA in the transformants was quantitated by comparing the hybridization of this probe with transformant and A9/HRBC2-A DNAs. Two unstable transformants which had a microscopically detectable donor chromosome fragment contained 15% of the human X-chromosomal single-copy DNA. Four other unstable transformants contained 4 to 7% of human X-chromosomal DNA sequences. The transferred DNA was below the level of detection in three other unstable and in all three stable transformants. We conclude that the initial transfer event can introduce a substantial amount of genetic information but only smaller amounts of DNA are stably incorporated by integration.
Assuntos
Cromossomos Sexuais , Transformação Genética , Cromossomo X , Animais , Sequência de Bases , DNA/análise , Feminino , Células HeLa , Humanos , Células Híbridas , Células L , Camundongos , Hibridização de Ácido Nucleico , RNA/análise , Transcrição GênicaRESUMO
PurposeTo evaluate Damato Multifixation Campimetry Online (DMCO), a free-of-charge internet-based visual field test. DMCO exists in three versions: DMCO BASIC, DMCO STANDARD, and DMCO ADVANCED. The main focus was (i) to investigate the sensitivity and the specificity of the existing DMCO versions in the detection of glaucomatous visual field loss and (ii) to define and evaluate algorithms for the interpretation of DMCO results.MethodsThe study design was an evaluation of a diagnostic test and included 97 individuals performing DMCO and white-on-white perimetry. Interpretation algorithms were devised to define abnormality, and these were evaluated using the Glaucoma Staging System as gold standard. Receiver operating characteristic (ROC) curves and area under the ROC (AUC) were calculated.ResultsAUCs from 15 algorithms ranged from 0.79 to 0.90. The most promising algorithm combined results from two successive DMCO STANDARD tests. The sensitivity was highly dependent on the severity of glaucoma. Hence, for eyes with mild, moderate, advanced, and severe glaucoma, the DMCO test demonstrated a sensitivity of 11.8, 71.4, 100, and 100%, respectively. The specificity was as high as 98.1%. Median duration per eye to complete the DMCO STANDARD test was 86 s for the control group and 125 s in participants with glaucoma.ConclusionsDMCO shows promise as a free-of-charge online tool to identify glaucomatous visual field defects in a preselected population. Ongoing studies are evaluating the use of DMCO in a nonselected population.
Assuntos
Diagnóstico por Computador , Glaucoma/diagnóstico , Transtornos da Visão/diagnóstico , Testes de Campo Visual/métodos , Campos Visuais , Algoritmos , Área Sob a Curva , Humanos , Sistemas On-Line , Curva ROC , Sensibilidade e EspecificidadeRESUMO
PDE4C is one of four mammalian genes that encode multiple PDE4 cyclic AMP-specific phosphodiesterase isoforms that are inhibited by rolipram. Fluorescent in situ hybridisation localised PDE4C to the p13.1 region of human chromosome 19. Overlapping cosmid clones spanning the human PDE4C gene were identified and characterised. Analysis of this locus indicated that the PDE4C gene spans at least 38 kb, consists of at least 18 exons, and contains the marker D19S212 within an intron. Comparison of published human PDE4C cDNA sequences with those of the genomic DNA identified four alternatively spliced exons and the possibility that the PDE4C locus contains at least three alternative promoters. PDE4C-containing cosmids also contained the genes for the growth regulatory transcription factor, JUND, and the mini guanine nucleotide regulatory protein, RAB3A. The RAB3A gene was shown to consist of 5 exons spanning 7.9 kb, while the JUND gene was found to contain no introns. Analysis of cosmids containing PDE4C, JUND, and RAB3A showed that 27 kb separate JUND and PDE4C, while only 3.7 kb separate PDE4C and RAB3A. The three genes share the same orientation of transcription and are arranged in the order cen- 5'- JUND-PDE4C-RAB3A-3'-tel.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/genética , Cromossomos Humanos Par 19 , AMP Cíclico/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Sequência de Bases , Mapeamento Cromossômico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , DNA Complementar , Humanos , Dados de Sequência Molecular , Especificidade por Substrato , Proteína rab3A de Ligação ao GTP/genéticaRESUMO
Three rearrangements in ALL disrupt E2A and create E2A fusion proteins: the t(1;19)(q23;p13) and E2A-PBX1, t(17;19)(q22;p13) and E2A-HLF and a cryptic inv(19)(p13;q13) and E2A-FB1. While E2A is fused to PBX1 in most ALLs with a t(1;19), 5-10% of cases have translocations that appear identical, but do not affect E2A or PBX1. Because more intensive therapy improves the outcome of patients with E2A-PBX1positive (1;19) translocations, it is critical to identify this subset of patients so that appropriate therapy can be administered. In addition, there are balanced and unbalanced variants of the t(1;19) and controversy exists regarding the clinical significance of this distinction. We have developed a two-color fluorescence in situ hybridization assay that accurately detects E2A translocations in metaphase and interphase cells, distinguishes between balanced and unbalanced variants and identifies patients with a t(1;19) who lack E2A-PBX1 fusion. We found that clonal microheterogeneity is common in patients with E2A translocations and most patients have mixtures of cells with balanced and unbalanced translocations, suggesting that this distinction represents two ends of a continuum rather than distinct biological entities. These reagents should have widespread clinical utility and be useful for translational and basic research studies involving E2A translocations and this region of chromosome 19p13.
Assuntos
Cromossomos Humanos Par 17 , Cromossomos Humanos Par 19 , Cromossomos Humanos Par 1 , Leucemia/genética , Fatores de Transcrição/genética , Translocação Genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Humanos , Hibridização in Situ Fluorescente , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição/análise , Células Tumorais CultivadasRESUMO
The amino acid hypusine is formed post-translationally in a single cellular protein, the eukaryotic translation initiation factor 5A, by two enzymes, namely deoxyhypusine synthase and deoxyhypusine hydroxylase. Hypusine is found in all eukaryotes and in some archaebacteria, but not in eubacteria. The deoxyhypusine synthase cDNA was cloned and mapped by fluorescence in situ hybridization on chromosome 19p13.11-p13.12. Rare cDNAs containing internal deletions were also found. We localized the deoxyhypusine synthase gene on a high resolution cosmid/BAC contig map of chromosome 19 to a region in 19p13.2-distal 19p13.1 between MANB and JUNB. Analysis of the genomic exon/intron structure of the gene coding region showed that it consists of nine exons and spans a length of 6.6kb. From observation of the genomic structure, it seems likely that the internally deleted forms of mature RNA are the result of alternative splicing, rather than of artifacts.
Assuntos
Cromossomos Humanos Par 19/genética , Genes/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Processamento Alternativo/genética , Mapeamento Cromossômico , DNA Complementar/química , DNA Complementar/genética , Éxons/genética , Humanos , Íntrons/genética , Isoenzimas/química , Isoenzimas/genética , Dados de Sequência MolecularRESUMO
The P/Q-type Ca(2+) channel alpha(1A) subunit gene (CACNA1A) was cloned on the short arm of chromosome 19 between the markers D19S221 and D19S179 and found to be responsible for Episodic Ataxia type 2, Familial Hemiplegic Migraine and Spinocerebellar Ataxia type 6. This region was physically mapped by 11 cosmid contigs spanning about 1. 4Mb, corresponding to less than 70% of the whole region. The cosmid contig used to characterize the CACNA1A gene accounted only for the coding region of the gene lacking, therefore, the promoter and possible regulation regions. The present study improves the physical map around and within the CACNA1A by giving a complete cosmid or BAC contig coverage of the D19S221-D19S179 interval. A number of new STSs, whether polymorphic or not, were characterized and physically mapped within this region. Four ESTs were also assigned to cosmids belonging to specific contigs.
Assuntos
Canais de Cálcio/genética , Cromossomos Humanos Par 19 , Mapeamento de Sequências Contíguas , Southern Blotting , Clonagem Molecular , Cosmídeos , Etiquetas de Sequências Expressas , Biblioteca Gênica , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Modelos Genéticos , Sitios de Sequências RotuladasRESUMO
We report here on a system for automated preparation of high-density colony filters of arrayed libraries using the high density replicating system (HDR) for the Beckman Biomek 1000 robotic workstation. This system, consisting of a 96-pin tool, a sterilization station and controlling software, transfers samples from microplates onto target membranes in arrays up to 36 times the density of a 96-well microplate. The transfer operation can be completely automated with the addition of the Biomek Side Loader System, which consists of a robotic arm capable of transferring plates and filters between the Biomek working tablet and a storage area. Using the complete system, we are able to plate 32 replica filters (8 x 12 cm), each containing the clones from 16 different microplates (i.e., 1536 clones per filter), in a 16-h overnight run without any operator intervention. We describe conditions used for transfer of bacterial yeast colonies and fixation of DNA to the membranes, and we illustrate hybridization results obtained with cosmid and YAC filters.
Assuntos
Biblioteca Gênica , Técnicas Genéticas/instrumentação , Biotecnologia , Mapeamento Cromossômico/instrumentação , Cromossomos Fúngicos , Cosmídeos , Estudos de Avaliação como Assunto , Hibridização de Ácido Nucleico/instrumentação , RobóticaRESUMO
We report on a 9-month-old girl with an interstitial duplication of 19p, developmental delay, and multiple anomalies including bifrontal prominence, obtuse frontonasal angle, short columella, additional midline philtral pillar, midline ridge on the tongue, vertical midline ridge at the mental symphysis, and a complex congenital heart defect including severe branch pulmonary artery stenosis, secundum atrial septal defect (ASD), and several ventricular septal defects (VSDs). Use of fluorescent in situ hybridization (FISH) with chromosome 19-specific probes showed a direct duplication of bands 19p13.13 and 19p13.2.
Assuntos
Anormalidades Múltiplas/genética , Aberrações Cromossômicas/genética , Cromossomos Humanos Par 19 , Deficiências do Desenvolvimento/genética , Face/anormalidades , Cardiopatias Congênitas/genética , Família Multigênica , Transtornos Cromossômicos , Mapeamento Cromossômico , Feminino , Humanos , Hibridização in Situ Fluorescente , LactenteRESUMO
The relative rates of transcription of the two genes for type I collagen have been measured in a runoff transcription assay using nuclei isolated from cultured human fibroblasts. Control experiments indicated that the ratio of pro alpha 1(I)/pro alpha 2(I) transcripts detected with a given nuclear preparation did not vary over a range of transcription times, nuclei concentrations, and amounts of filter-bound cDNA used in the assay. However, a significant difference in the ratio was observed when nuclei isolated from cells grown under different conditions were used. Nuclei from sub-confluent cultures generally transcribed the two genes in a ratio of 2:1 or lower, while nuclei from post-confluent cultures transcribed the genes in a much higher ratio of about 4:1. Analysis of the amount of pro alpha 1(I) and pro alpha 2(I) RNA transcribed suggests that it is the transcription of the pro alpha 1(I) gene, and not the pro alpha 2(I) gene, that varies with culture conditions. The steady state ratios of pro alpha 1(I)/pro alpha 2(I) RNA remained near 2:1 under all conditions. Thus, some post-transcriptional mechanism apparently results in maintenance of the steady-state pro alpha 1(I)/pro alpha 2(I) RNA ratios at approximately 2:1.
Assuntos
Colágeno/genética , Fibroblastos/metabolismo , RNA Mensageiro/metabolismo , Transcrição Gênica , Linhagem Celular , Colágeno/metabolismo , Humanos , Hibridização de Ácido NucleicoRESUMO
Hypoxanthine phosphoribosyltransferase (IMP:pryophosphate phosphoribosyltransferase, EC 2.4.2.8) from human erythrocytes has been purified 13 000-fold to apparent homogeneity. The native enzyme has a sedimentation coefficient of 5.9 S, determined by analytical ultracentrifugation, and a molecular weight of 81 000-83 000, determined by sedimentation equilibrium centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates a subunit molecular weight of 26 000, suggesting that the enzyme is a trimer. Isoelectric focusing resolves three peaks of enzyme activity at pH 5.6, 5.7 and 5.9. The amino acid composition of hypoxanthine phosphoribosyltrasferase is 17 Lys, 5 His, 12 Arg, 0 Trp, 31 Asx, 12 Thr, 14 Ser, 16 Glx, 14 Pro, 19 Gly, 12 Ala, 5 Cys, 18 Val, 5 Met, 11 Ile, 20 Leu, 10 Tyr, and 9 Phe. The enzyme appears to have a blocked N terminus.