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1.
Biol Reprod ; 65(5): 1522-7, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11673270

RESUMO

The mouse lactate dehydrogenase c gene (mldhc) is transcribed only in cells of the germinal epithelium. Cloning and analysis of the mldhc promoter revealed that a 100-base pair fragment was able to drive testis-specific transcription in vitro and in transgenic mice. Several testis-specific genes are believed to be regulated at least in part through differential methylation of CpG dinucleotides. We investigated the possibility that transcriptional repression of the mldhc gene is mediated in somatic tissues by hypermethylation of CpG dinucleotides. The CpG dinucleotides within a fragment of the mldhc promoter containing a GC box and tandem activating transcription factor/cAMP-responsive element binding sites are hypermethylated in somatic tissues and hypomethylated in testis. Methylation of the activating transcription factor/cAMP-responsive elements altered the protein binding pattern observed in electrophoretic mobility shift assays using mouse liver but not testis nuclear extract. Furthermore, methylation of an extended mldhc promoter fragment driving lac Z silenced transcription from the promoter in a transient transfection assay. These data suggest that tissue-specific differential methylation plays a role in mldhc silencing in somatic tissues.


Assuntos
Ilhas de CpG/fisiologia , Metilação de DNA , Isoenzimas/genética , L-Lactato Desidrogenase/genética , Regiões Promotoras Genéticas , Testículo/metabolismo , Transcrição Gênica , Fatores Ativadores da Transcrição , Animais , Sítios de Ligação , Proteínas Sanguíneas/metabolismo , Núcleo Celular/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Fígado/ultraestrutura , Masculino , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Análise de Sequência de DNA/métodos , Sulfitos , Testículo/ultraestrutura , Fatores de Transcrição/metabolismo , Transfecção
2.
J Biol Chem ; 276(38): 35414-21, 2001 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-11447215

RESUMO

Developmental and testis-specific expression of the mouse lactate dehydrogenase C (mldhc) gene requires mechanisms for activation in germ cells and repression in somatic cells. Promoter activity restricted to the testis has been demonstrated using in vitro transcription assays with a 60-base pair promoter sequence upstream of the transcription initiation site. This promoter fragment has a TATA box and an overlapping 31-base pair palindromic sequence. Here we have explored the role of the palindrome as a silencer of the ldhc gene in somatic tissues. A gel retardation assay detected two sites within the palindrome that were important for protein binding. A member of the NF-I/CTF family was identified as the protein binding to one of the sites. In transiently transfected mouse L cells, a promoter fragment in which the NF-I site was mutated showed a 4-fold greater activity as compared with the wild-type sequence. Overexpression of the four NF-I proteins, NF-IA, -B, -C, or -X, in mouse L cells transiently transfected with an ldhc promoter-reporter construct resulted in a 20-50% decrease in activity of the wild-type promoter but had no effect when the NF-I binding element in the palindrome was mutated. These results indicate a role for the NF-I proteins in regulation of the mldhc gene.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Núcleo Celular/enzimologia , Regulação Enzimológica da Expressão Gênica , Isoenzimas/genética , L-Lactato Desidrogenase/genética , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , DNA , Células Germinativas/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição NFI , Ligação Proteica , Testículo/metabolismo
3.
Mol Hum Reprod ; 4(11): 1058-64, 1998 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9835358

RESUMO

Characterization and classification of human zona pellucida glycoproteins is essential to understand the functions of these components during fertilization. To achieve this, antibodies were raised in rabbits against recombinant non-human primate [Bonnet Monkey (Macaca radiata)] zona pellucida proteins, bmZP1, bmZP2 and bmZP3 expressed in Escherichia coli. Antibodies against the three recombinant zona proteins reacted with human zonae as revealed by indirect immunofluorescence. Such antibodies were used as specific probes to further characterize human zona pellucida glycoproteins in Western blot of heat solubilized human zonae pellucidae (hSIZP) resolved by one dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Under non-reduced conditions human (h) hZP1, hZP2 and hZP3 resolved as 60, 100 and 53 kDa bands respectively. Under reduced conditions, dominant reactivity of hZP1, hZP2 and hZP3 was localized to 63, 65 and 58 kDa and faint reactivity to 53, 96 and 138 kDa bands respectively. In two-dimensional SDS-PAGE, hZP1 was shown to comprise two chains at 63-58 and 55-45 kDa, each consisting of multiple isomers. hZP2 was less acidic when compared with hZP1 and hZP3 and comprised a major component of 65 kDa and a minor component of approximately 96 kDa. The 65 kDa component displayed a higher degree of charged isomers in comparison with the 96 kDa component. hZP3 comprised a broad band in the range 68-58 kDa. These studies show conclusively that the hZP1 heavy train overlaps with hZP3 and that in previous studies, hZP2 was likely to have been misinterpreted as being hZP1. Our studies failed to distinguish two distinct species of hZP3, unlike previous reports. These studies will further help in our understanding of the nature of human zona pellucida glycoproteins.


Assuntos
Proteínas do Ovo/análise , Glicoproteínas de Membrana/análise , Receptores de Superfície Celular , Zona Pelúcida/metabolismo , Animais , Anticorpos/imunologia , Western Blotting , Proteínas do Ovo/imunologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Masculino , Glicoproteínas de Membrana/imunologia , Coelhos , Proteínas Recombinantes de Fusão/imunologia , Glicoproteínas da Zona Pelúcida
4.
Mol Reprod Dev ; 50(2): 229-39, 1998 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9590540

RESUMO

Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for an immunocontraceptive vaccine. The efficacy of such a vaccine has to be evaluated in nonhuman primates, thus necessitating the characterization of their ZP glycoproteins. A bonnet monkey (Macaca radiata) ovarian cDNA lambda gt11 library was screened for ZP2 (bZP2) using full-length human ZP2 cDNA as a probe. Two identical full-length clones with an open reading frame of 2235 nt encoding a polypeptide of 745 aa residues were isolated. The deduced aa sequence of bZP2 revealed high sequence identity (94.2%) with human ZP2. The bZP2 cDNA (115-1914 nt, 1.8 kb), excluding sequences coding for N-terminal signal sequence and C-terminal transmembranelike domain, was PCR amplified and Sac1-Sal1 restricted fragment cloned in frame downstream of the T5 promoter under the lac operator control in a pQE-30 vector. Recombinant bZP2 (r-bZP2) was expressed as a polyhistidine fusion protein in Escherichia coli strain M15 [pREP4]. Immunoblot with rabbit polyclonal antibodies against bZP2 synthetic peptide (corresponding to aa residues 429-444; K434 replaced by R and 1436 by V) revealed a major band of 68 kDa. Immunization of male rabbits with the r-bZP2 protein purified on Ni-NTA resin under denaturing conditions generated antibodies reactive with r-bZP2 in ELISA as well as with native protein as revealed by positive fluorescence of ZP of bonnet monkey ovary. The availability of r-bZP2 and its aa sequence will help in the development and evaluation of a contraceptive vaccine based on ZP2.


Assuntos
Proteínas do Ovo/genética , Escherichia coli/metabolismo , Vetores Genéticos , Glicoproteínas de Membrana/genética , Receptores de Superfície Celular , Zona Pelúcida/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , DNA Complementar , Proteínas do Ovo/imunologia , Proteínas do Ovo/metabolismo , Feminino , Expressão Gênica , Humanos , Macaca radiata , Masculino , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Glicoproteínas da Zona Pelúcida
5.
Hum Reprod Update ; 3(4): 311-24, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9459277

RESUMO

The zona pellucida (ZP) surrounding a mammalian oocyte mediates the initial recognition and binding of spermatozoon to oocyte in a relatively species-specific manner and plays an important role in the subsequent activation events during the fertilization process. The ZP comprises three biochemically and immunologically distinct glycoproteins termed ZP1, ZP2 and ZP3. The critical role of ZP glycoproteins in reproduction together with their tissue-specific nature have led to their being considered as potential candidate antigens for immunocontraception. Immunization of females with ZP glycoproteins leads to a block of fertility in several animal models. However, it is invariably associated with either a transient or an irreversible alteration in the cyclicity, hormonal profile and follicular development in the ovary. To overcome these problems, attempts are being made to delineate relevant 'B' cell epitopes on ZP proteins so as to design immunocontraceptive vaccines based on synthetic peptides devoid of oophoritogenic 'T' cell epitopes. Monoclonal antibodies capable of inhibiting the gamete interaction are being employed to delineate such regions. Additionally, DNA-recombinant technology has made it feasible to obtain, in reasonably large quantities, the ZP glycoproteins from human and non-human primates. Availability of sequence information of these zona proteins and the availability of recombinant antigens (devoid of other ovarian-associated proteins) will further help in understanding more precisely their functions during fertilization and make it feasible to undertake immunization studies to determine their prospects as immunogens for fertility regulation.


Assuntos
Anticoncepção Imunológica/métodos , Proteínas do Ovo/imunologia , Glicoproteínas de Membrana/imunologia , Receptores de Superfície Celular , Vacinas , Sequência de Aminoácidos , Animais , Antígenos/química , Antígenos/imunologia , Proteínas do Ovo/química , Feminino , Humanos , Macaca radiata , Masculino , Glicoproteínas de Membrana/química , Dados de Sequência Molecular , Interações Espermatozoide-Óvulo , Espermatozoides/química , Espermatozoides/imunologia , Zona Pelúcida/química , Zona Pelúcida/imunologia , Glicoproteínas da Zona Pelúcida
6.
J Toxicol Clin Toxicol ; 34(2): 183-9, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8618252

RESUMO

OBJECTIVE: The present study focuses on serum testosterone concentrations in patients with skeletal fluorosis, in order to assess the hormonal status in fluoride toxicity. METHODS: Serum testosterones were compared for patients afflicted with skeletal fluorosis (n = 30) and healthy males consuming water containing less than 1 ppm fluoride (Control 1, n = 26) and a second category of controls (Control 2, n = 16): individuals living in the same house as the patients and consuming same water as patients but not exhibiting clinical manifestations of skeletal fluorosis. RESULTS: Circulating serum testosterones in skeletal fluorosis patients were significantly lower than those of Control 1 at p < 0.01. Testosterone concentrations of Control 2 were also lower than those of Control 1 at p < 0.05 but were higher than those of the patient group. CONCLUSIONS: Decreased testosterone concentrations in skeletal fluorosis patients and in males drinking the same water as the patients but with no clinical manifestations of the disease compared with those of normal, healthy males living in areas nonendemic for fluorosis suggest that fluoride toxicity may cause adverse effects in the reproductive system of males living in fluorosis endemic areas.


Assuntos
Doenças Ósseas/sangue , Intoxicação por Flúor/sangue , Testosterona/sangue , Adolescente , Adulto , Fertilidade/efeitos dos fármacos , Intoxicação por Flúor/urina , Fluoretos/análise , Fluoretos/sangue , Fluoretos/urina , Humanos , Masculino , Pessoa de Meia-Idade , Água/química
7.
Clin Biochem ; 27(6): 463-8, 1994 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-7697892

RESUMO

Circulating levels of haptoglobin and C-reactive protein were studied in patients of skeletal fluorosis and compared with two types of controls. The first type of control included normal healthy individuals consuming water containing permissible levels of fluoride (up to 1 mg/L). The second type of control included individuals consuming water contaminated with fluoride (1.2-14.5 mg/L) but not exhibiting clinical manifestations of skeletal fluorosis. A significant increase in the levels of haptoglobin (p < 0.01) and C-reactive protein (p < 0.01) as well as a raised erythrocyte sedimentation rate were seen in patients of skeletal fluorosis as compared to both types of controls. The present study suggests the possibility of a subclinical inflammatory reaction occurring in patients with skeletal fluorosis.


Assuntos
Doenças Ósseas/prevenção & controle , Proteína C-Reativa/análise , Intoxicação por Flúor/prevenção & controle , Haptoglobinas/análise , Sedimentação Sanguínea , Doenças Ósseas/sangue , Feminino , Intoxicação por Flúor/sangue , Humanos , Masculino , Abastecimento de Água
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