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1.
Int J Sports Med ; 31(6): 367-71, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20419620

RESUMO

Exercise induced proteinuria is a common phenomenon in high performance sports. Based on the appearance of so called "effort urines" in routine doping analysis the purpose of this study was to investigate the influence of exercise induced proteinuria on IEF profiles and SDS-PAGE relative mobility values (rMVs) of endogenous human erythropoietin (EPO). Twenty healthy subjects performed cycle-ergometer exercise until exhaustion. VO (2)max, blood lactate, urinary proteins and urinary creatinine were analysed to evaluate the exercise performance and proteinuria. IEF and SDS-PAGE analyses were performed to test for differences in electrophoretic behaviour of the endogenous EPO before and after exercise. All subjects showed increased levels of protein/creatinine ratio after performance (8.8+/-5.2-26.1+/-14.4). IEF analysis demonstrated an elevation of the relative amount of basic band areas (13.9+/-11.3-36.4+/-12.6). Using SDS-PAGE analysis we observed a decrease in rMVs after exercise and no shift in direction of the recombinant human EPO (rhEPO) region (0.543+/-0.013-0.535+/-0.012). Following identification criteria of the World Anti Doping Agency (WADA) all samples were negative. The implementation of the SDS-PAGE method represents a good solution to distinguish between results influenced by so called effort urines and results of rhEPO abuse. Thus this method can be used to confirm adverse analytical findings.


Assuntos
Eletroforese em Gel de Poliacrilamida , Eritropoetina/sangue , Eritropoetina/urina , Teste de Esforço/métodos , Esforço Físico/fisiologia , Adulto , Dopagem Esportivo/prevenção & controle , Feminino , Alemanha , Humanos , Focalização Isoelétrica/métodos , Masculino
2.
J Cell Biochem ; 75(3): 382-92, 1999 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-10536362

RESUMO

Understanding the mechanisms that control the proliferation and commitment of human stem cells into cells of the osteogenic lineage for the preservation of skeletal structure is of basic importance in bone physiology. This study examines some aspects of the differentiation in vitro of human bone marrow fibroblastic cells cultured in the absence (basal media) or presence of 1nM dexamethasone and 50 micrograms/ml ascorbate for 6, 10, 14, and 21 days. Northern blot analysis and in situ hybridisation with digoxygenin-labelled riboprobes for Type I collagen, osteocalcin, bone morphogenetic proteins 2 (BMP-2), and 4 (BMP-4) and the estrogen receptor alpha (ERalpha), together with immunocytochemical analysis of ERalpha expression and histochemical staining of alkaline phosphatase was performed. In basal media, alkaline phosphatase activity and collagen expressions were detected at day 6, ERalpha from day 10 and osteocalcin from day 10. In the presence of dexamethasone and ascorbate, cell proliferation and alkaline phosphatase were markedly stimulated over 10 to 14 days with a dramatic increase in the temporal expression of Type I collagen, ERalpha, and osteocalcin mRNAs in these cultures. Northern blot analysis showed cells cultured in basal media, expressed the highest levels of the mRNA for each marker protein at day 14, whereas in the presence of ascorbate and dexamethasone, the highest levels for alkaline phosphatase, ERalpha, osteocalcin, BMP-2, and BMP-4 were observed at day 21. ERalpha, BMP-2, and BMP-4 expression were found to correlate temporally with induction of the osteoblast phenotype as determined by alkaline phosphatase, collagen, and osteocalcin expression. These results give additional information on the development of the osteoblast phenotype from early fibroblastic stem cells and on the biological factors involved in this process. These studies suggest a role for estrogen and BMP-2 and -4 in the differentiation of osteoprogenitor cells.


Assuntos
Proteínas Morfogenéticas Ósseas/genética , Células-Tronco Hematopoéticas/metabolismo , Osteoblastos/metabolismo , RNA Mensageiro/genética , Receptores de Estrogênio/genética , Fator de Crescimento Transformador beta , Idoso , Idoso de 80 Anos ou mais , Fosfatase Alcalina/metabolismo , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Diferenciação Celular , Células Cultivadas , Colágeno/genética , Receptor alfa de Estrogênio , Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Humanos , Hibridização In Situ , Osteoblastos/citologia , Osteocalcina/genética , RNA Mensageiro/metabolismo
3.
J Cell Biochem ; 74(3): 372-85, 1999 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-10412039

RESUMO

The specific effects of interferon alpha (IFNalpha), on the differentiation pathways of human osteogenic cells are not known. The aim of this study was to investigate possible effects of IFNalpha on osteogenic development by investigating cell differentiation, colony formation (colony forming unit-fibroblastic, CFU-F), cell proliferation, and gene expression, in particular bone morphogenetic protein (BMP) expression, of human bone marrow osteoprogenitor cells. Human bone marrow fibroblasts were cultured with or without the addition of IFNalpha (5-1,000 IU/ml) in the presence and absence of dexamethasone (10 nM) and ascorbate (100 microM), which are agents known to affect osteogenic differentiation. IFNalpha produced a significant dose-dependent inhibition of cell proliferation and alkaline phosphatase specific activity at concentrations as low as 50 IU/ml. IFNalpha (50-1,000 IU/ml) inhibited the stimulation of alkaline phosphatase specific activity induced by ascorbate and dexamethasone. Examination of CFU-F showed dose- and time-dependent inhibitions of colony formation and reductions in both colony size and alkaline phosphatase-positive CFU-F colonies particularly at earlier times. Reactivity with an antibody specific for osteoprogenitors (HOP-26), was reduced in IFNalpha-treated cultures. Northern blot analysis showed a significant dose-dependent up-regulation of BMP-2 mRNA, estrogen receptor alpha mRNA and osteocalcin mRNA expression in ascorbate/dexamethasone cultures. In contrast, IFNalpha significantly inhibited BMP-2 mRNA expression in the absence of ascorbate and dexamethasone. In conclusion, IFNalpha inhibits human osteoprogenitor cell proliferation, CFU- F formation, HOP-26 expression, and alkaline phosphatase specific activity and modulates BMP-2 gene expression. These results suggest a role for IFNalpha in local bone turnover through the specific and direct modulation of osteoprogenitor proliferation and differentiation.


Assuntos
Medula Óssea/efeitos dos fármacos , Interferon-alfa/farmacologia , Células-Tronco/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Biomarcadores , Contagem de Células/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Humanos , Osteocalcina/metabolismo , Fatores de Tempo
4.
Hum Mol Genet ; 5(5): 581-90, 1996 May.
Artigo em Inglês | MEDLINE | ID: mdl-8733123

RESUMO

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant, neuromuscular disorder characterized by progressive weakness of muscles in the face, shoulder and upper arm. Deletion of integral copies of a 3.3 kb repeated unit from the subtelomeric region on chromosome 4q35 has been shown to be associated with FSHD. These repeated units which are apparently not transcribed, map very close to the 4q telomere and belong to a 3.3 kb repeat family dispersed over heterochromatic regions of the genome. Hence, position effect variegation (PEV), inducing allele-specific transcriptional repression of a gene located more centromeric, has been postulated as the underlying genetic mechanism of FSHD. This hypothesis has directed the search for the FSHD gene to the region centromeric to the repeated units. A CpG island was identified and found to be associated with the 5' untranslated region of a novel human gene, FRG1 (FSHD Region Gene 1). This evolutionary conserved gene is located about 100 kb proximal to the repeated units and belongs to a multigene family with FRG1 related sequences on multiple chromosomes. The mature chromosome 4 FRG1 transcript is 1042 bp in length and contains nine exons which encode a putative protein of 258 amino acid residues. Transcription of FRG1 was detected in several human tissues including placenta, lymphocytes, brain and muscle. To investigate a possible PEV mechanism, allele-specific FRG1 steady-state transcript levels were determined using RNA-based single-strand conformation polymorphism (SSCP) analysis. A polymorphic fragment contained within the first exon of FRG1 was amplified from reverse transcribed RNA from lymphocytes and muscle biopsies of patients and controls. No evidence for PEV mediated repression of allelic transcription was obtained in these tissues. However, detection of PEV in FSHD patients may require analysis of more specific cell types at particular developmental stages.


Assuntos
Cromossomos Humanos Par 4 , Distrofias Musculares/genética , Proteínas/genética , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Mapeamento Cromossômico , DNA Complementar , Regulação da Expressão Gênica , Haplorrinos , Humanos , Hibridização in Situ Fluorescente , Proteínas dos Microfilamentos , Dados de Sequência Molecular , Família Multigênica , Proteínas Nucleares , Linhagem , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Proteínas de Ligação a RNA , Ratos , Mapeamento por Restrição , Ovinos
5.
Muscle Nerve Suppl ; 2: S19-26, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-7739621

RESUMO

Facioscapulohumeral muscular dystrophy (FSHD) is caused by deletions of 3.3-kb tandemly repeated units contained within a large polymorphic EcoRI fragment close to the telomere of chromosome 4q. Since the rearrangements were assumed to interfere with the structure or function of the putative FSHD gene, the gene search was focused on cosmids containing these repeat units and, in addition, cosmids spanning 75 kb of upstream sequences. cDNA selection hybridization was applied to four overlapping cosmid clones, yielding a total of 150 putative cDNA clones. These clones showed a random distribution across the cosmid contig, except for three regions which contained a much larger number of clones. Nine cDNA clones hybridized to a 2.2-kb EcoRI fragment, located 22 kb centromeric to the 3.3-kb repeated units. This 2.2-kb fragment showed evolutionary conservation, and analysis of the sequence by "GRAIL" predicted the presence of several exons. Transcripts homologous to this fragment could be identified but none of them originated from the 4q35 locus. Strikingly, most clones revealed 4-10 homologous loci, and no single copy clones could be isolated. These findings are in line with earlier observations by fluorescent in situ hybridization (FISH) showing hybridization of individual cosmid clones to multiple chromosomes. The presence of homologous regions on other chromosomes seriously complicates the cloning of the FSHD gene.


Assuntos
Cromossomos Humanos Par 4 , DNA Complementar/genética , Genes , Distrofias Musculares/genética , Northern Blotting , Desoxirribonuclease EcoRI , Face , Humanos , Úmero , Escápula
6.
Muscle Nerve Suppl ; (2): S19-26, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-23573582

RESUMO

Facioscapulohumeral muscular dystrophy (FSHD) is caused by deletions of 3.3-kb tandemly repeated units contained within a large polymorphic EcoRI fragment close to the telomere of chromosome 4q. Since the rearrangements were assumed to interfere with the structure or function of the putative FSHD gene, the gene search was focused on cosmids containing these repeat units and, in addition, cosmids spanning 75 kb of upstream sequences. cDNA selection hybridization was applied to four overlapping cosmid clones, yielding a total of 150 putative cDNA clones. These clones showed a random distribution across the cosmid contig, except for three regions which contained a much larger number of clones. Nine cDNA clones hybridized to a 2.2-kb EcoRI fragment, located 22 kb centromeric to the 3.3-kb repeated units. This 2.2-kb fragment showed evolutionary conservation, and analysis of the sequence by "GRAIL" predicted the presence of several exons. Transcripts homologous to this fragment could be identified but none of them originated from the 4q35 locus. Strikingly, most clones revealed 4-10 homologous loci, and no single copy clones could be isolated. These findings are in line with earlier observations by fluorescent in situ hybridization (FISH) showing hybridization of individual cosmid clones to multiple chromosomes. The presence of homologous regions on other chromosomes seriously complicates the cloning of the FSHD gene.


Assuntos
DNA Complementar , Hibridização in Situ Fluorescente , Mapeamento Cromossômico , Cromossomos Humanos Par 4 , Humanos , Distrofias Musculares/genética , Distrofia Muscular Facioescapuloumeral
7.
J Immunol ; 152(7): 3476-82, 1994 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-7511649

RESUMO

CD4-CD8- gamma delta T cells of ruminants uniquely express a 220-kDa surface Ag recognized by several mAbs clustered as WC1. We recently reported the isolation of a cDNA clone encoding a WC1 Ag. Southern blotting suggested that the bovine genome contains multiple sequences highly related to the isolated WC1 cDNA. Here, we demonstrate that some of the clustered WC1 mAbs stain predominantly nonoverlapping subsets of bovine CD4-CD8- gamma delta T cells. By the isolation of two additional cDNA clones encoding molecules highly related to the original WC1 Ag, we provide a molecular basis for this phenomenon. Cells transfected with cDNAs encoding individual WC1 Ags are differentially recognized by various WC1 mAbs. Thus, expression of members of the WC1 gene family divides bovine CD4-CD8- gamma delta T cells into phenotypical subsets. Field inversion gel electrophoresis revealed that all WC1 genes map to a single, large (> 1 Mbp) Notl fragment. Although the function of WC1 remains unknown, it likely involves interaction with ligands that originate from a similarly complex genetic system.


Assuntos
Antígenos de Superfície/genética , Glicoproteínas de Membrana/metabolismo , Subpopulações de Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Superfície/metabolismo , Sequência de Bases , Antígenos CD4/análise , Antígenos CD8/análise , Bovinos , Mapeamento Cromossômico , Epitopos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T gama-delta/análise , Mapeamento por Restrição , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico
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