RESUMO
Depression is a serious and high-priority public health problem. In Chilean population, prevalence ranges from 5 to 27,3 percent. Therapy is based mainly in the use of selective serotonin reuptake inhibitors (SSRIs). Combination of thyroid hormone, sodium liothyronine, associated to traditional antidepressants to improve or accelerate therapeutic response is currently accepted. The use of this combination is based on hypothalamus-hypophysis-thyroid axis (HHT) alterations and on the peripheral conversion to active hormone, the triiodothyronine (T3), by type 2 and 3deiodinases (D2 and D3). Subtle changes in enzyme activity could have a strong impact in T3 brain availability. In major depression as high as a 25 percent of altered responses of HHT axis to the TRH stimulus may be observed. Certain polymorphisms of the D2 gene could be associated to enzyme activity changes. Isotopic studies are able to assess brain flow in diverse conditions, like global or specific regional perfusion variations in patients with mild hypothyroidism, pre and post T4 or SSRIs therapy in depressive patients.
Assuntos
Humanos , Adulto , Depressão/tratamento farmacológico , Doenças da Glândula Tireoide/psicologia , Antidepressivos/uso terapêuticoRESUMO
Several dominant, late-onset neurodegenerative diseases (e.g. Huntington's disease) are caused by expansion of polyglutamine (polyQ) repeats within specific proteins. The diverse, yet overlapping, pathology of these diseases could be due to novel deleterious functions unique to each protein or to a common pathophysiology mediated by the long polyQ chains themselves. By engineering Drosophila to express different polyQ peptides, we find that expanded polyQ chains alone are intrinsically cytotoxic and cause neuronal degeneration and early adult death. We further find that this intrinsic toxicity is dependent on cell type and polyQ length and that the inclusion of other amino acids modifies and reduces toxicity. This is the first in vivo evidence that polyQs, when removed from their disease gene context, cause neurotoxicity. These studies provide a basis for understanding the diverse clinical presentations in terms of the intrinsic cytotoxic effect of polyQ peptides being modulated by protein context. Parallel experiments in which cytotoxic polyQ expansions were engineered into Dishevelled, a Drosophila protein containing a naturally occurring polyQ tract, strongly suggest that the effect of a toxic polyQ peptide can be neutralized by protein context. This animal model provides a simple and effective means of screening for therapeutics that relieves the polyQ-induced lethality, independent of any particular disease gene. By quantifying the degree of lethality in several transgenic lines, we have identified a number of genetically modified strains that are suitable for eventual testing of compounds or genes that ameliorate the pathology of polyQ peptides.
Assuntos
Drosophila melanogaster/genética , Degeneração Neural/genética , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Animais Geneticamente Modificados , Sequência de Bases , Proteínas Desgrenhadas , Proteínas de Drosophila , Olho/patologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Dados de Sequência Molecular , Mortalidade , Fenótipo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Sequências Repetitivas de Aminoácidos , Frações SubcelularesRESUMO
The dominant kanr marker gene plays an important role in gene disruption experiments in budding yeast, as this marker can be used in a variety of yeast strains lacking the conventional yeast markers. We have developed a loxP-kanMX-loxP gene disruption cassette, which combines the advantages of the heterologous kanr marker with those from the Cre-lox P recombination system. This disruption cassette integrates with high efficiency via homologous integration at the correct genomic locus (routinely 70%). Upon expression of the Cre recombinase the kanMX module is excised by an efficient recombination between the loxP sites, leaving behind a single loxP site at the chromosomal locus. This system allows repeated use of the kanr marker gene and will be of great advantage for the functional analysis of gene families.
Assuntos
Integrases , Mutagênese Insercional/métodos , Saccharomyces cerevisiae/genética , Proteínas Virais , Sequência de Bases , DNA Nucleotidiltransferases , Genes Fúngicos , Marcadores Genéticos , Resistência a Canamicina , Modelos Genéticos , Dados de Sequência MolecularRESUMO
Hyperekplexia is a rare, autosomal dominant neurological disorder characterized by hypertonia, especially in infancy, and by an exaggerated startle response. This disorder is caused by mutations in the alpha 1 subunit of the inhibitory glycine receptor (GLRA1). We previously reported two GLRA1 point mutations detected in 4 unrelated hyperekplexia families; both mutations were at nucleotide 1192 and resulted in the replacement of Arg271 by a glutamine (R271Q) in one case and a leucine (R271L) in the other. Here, 5 additional hyperekplexia families are shown to have the most common G-to-A transition mutation at nucleotide 1192. Haplotype analysis using polymorphisms within and close to the GLRA1 locus suggests that this mutation has arisen at least twice (and possibly four times). In 2 additional families, a third mutation is also presented that changes a tyrosine at amino acid 279 to a cysteine (Y279C). Five patients with atypical clinical features and equivocal or absent family history of hyperekplexia and 1 patient with a classical presentation but not family history are presented in whom a mutation in the GLRA1 gene was not detected. Thus, only clinically typical hyperekplexia appears to be consistently associated with GLRA1 mutations, and these affect a specific extracellular domain of the protein.
Assuntos
Análise Mutacional de DNA , Rigidez Muscular/genética , Reflexo de Sobressalto/genética , Sequência de Bases , Éxons , Haplótipos , Humanos , Lactente , Dados de Sequência Molecular , Polimorfismo GenéticoRESUMO
Female BALB/c mice were immunized intranasally with the mouse pneumonitis biovar of Chlamydia trachomatis and subsequently challenged in the ovarian bursa (C. trachomatis immunized, C. trachomatis challenged). Two groups of mice served as controls. One group was sham immunized intranasally with mock-infected HeLa 229 cell extracts and was challenged in the ovarian bursa with C. trachomatis MoPn (sham immunized, C. trachomatis challenged). The second control group was sham immunized and not challenged (sham immunized, nonchallenged). Before challenge, the C. trachomatis-immunized, C. trachomatis-challenged animals mounted a significant humoral response as shown by high immunoglobulin G (IgG), IgM, and IgA levels and high levels of neutralizing antibodies in serum and moderate IgG and IgA titers in vaginal secretions. Reactivity by Western blot (immunoblot) to the lipopolysaccharide, 30-, 40- (major outer membrane protein), and 60-kDa cysteine-rich proteins and 75- and 100-kDa chlamydial components could be demonstrated. However, reactivity to the 60-kDa heat shock protein was only observed 22 days after challenge. In addition, this group of animals mounted a significant immune response to chlamydial antigens, as shown by a lymphocyte proliferation assay, compared with the sham-immunized nonchallenged mice. After intrabursal challenge, there was no C. trachomatis shedding from the vagina in the C. trachomatis-immunized, C. trachomatis-challenged animals, while 63% of the sham-immunized, C. trachomatis-challenged mice had a positive C. trachomatis culture. In addition, histological sections from the genital tract showed, at 2 weeks postchallenge, a marked acute inflammatory reaction in the sham-immunized, C. trachomatis-challenged animals while in the C. trachomatis-immunized, C. trachomatis-challenged mice there was minimal inflammatory reaction. When the animals were mated, only 12% of the mice from the sham-immunized, C. trachomatis-challenged mice were fertile. In contrast, 94 and 80% of the sham-immunized, nonchallenged and C. trachomatis-immunized, C. trachomatis-challenged mice, respectively, were fertile.
Assuntos
Infecções por Chlamydia/complicações , Chlamydia trachomatis/imunologia , Infertilidade Feminina/prevenção & controle , Salpingite/complicações , Administração Intranasal , Animais , Anticorpos Antibacterianos/análise , Linfócitos B/imunologia , Western Blotting , Infecções por Chlamydia/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Salpingite/patologia , VacinaçãoRESUMO
Achondroplasia (ACH) is the most common genetic form of dwarfism. This disorder is inherited as an autosomal dominant trait, although the majority of cases are sporadic. A gene for ACH was recently localized to 4p16.3 by linkage analyses. The ACH candidate region includes the gene encoding fibroblast growth factor receptor 3 (FGFR3), which was originally considered as a candidate for the Huntington's disease gene. DNA studies revealed point mutations in the FGFR3 gene in ACH heterozygotes and homozygotes. The mutation on 15 of the 16 ACH-affected chromosomes was the same, a G-->A transition, at nucleotide 1138 of the cDNA. The mutation on the only ACH-affected chromosome 4 without the G-->A transition at nucleotide 1138 had a G-->C transversion at this same position. Both mutations result in the substitution of an arginine residue for a glycine at position 380 of the mature protein, which is in the transmembrane domain of FGFR3.
Assuntos
Acondroplasia/genética , Mutação Puntual/genética , Proteínas Tirosina Quinases , Receptores de Fatores de Crescimento de Fibroblastos/genética , Alelos , Sequência de Aminoácidos , Linfócitos B , Sequência de Bases , Células Cultivadas , Criança , Análise Mutacional de DNA , DNA Complementar/biossíntese , Feminino , Fibroblastos , Heterozigoto , Homozigoto , Humanos , Masculino , Dados de Sequência Molecular , Mutação Puntual/fisiologia , Polimorfismo de Fragmento de Restrição , Receptor Tipo 3 de Fator de Crescimento de FibroblastosRESUMO
A Swiss Webster white mouse model of salpingitis was used to characterize the immune response following an intrauterine infection with the Chlamydia trachomatis mouse pneumonitis biovar. Western blot (immunoblot) analyses of the serum samples showed that the immunodominant bands corresponded to molecular masses of 72, 60, 42, and 28 kDa and to the lipopolysaccharide. Antibodies to the 60-kDa heat shock protein and to the 60-kDa cysteine-rich protein were detected at 2 and 3 weeks postinfection, respectively. Neutralization was observed in an in vitro assay with serum samples as early as the 3rd day postinfection and remained high for the 7 weeks of observation. The mice were mated in the 7th week following infection. Of the infected experimental mice, 71.4% were found to be either unilaterally or bilaterally infertile, whereas only 27.4% of the noninfected control mice were found to be infertile.
Assuntos
Infecções por Chlamydia/imunologia , Chlamydia trachomatis , Infertilidade Feminina/etiologia , Pneumonia/etiologia , Doenças Uterinas/imunologia , Animais , Anticorpos Antibacterianos/sangue , Proteínas da Membrana Bacteriana Externa/imunologia , Reações Cruzadas , Modelos Animais de Doenças , Feminino , Proteínas de Choque Térmico/imunologia , Masculino , Camundongos , Salpingite/imunologia , Vagina/microbiologiaRESUMO
Three monoclonal antibodies (MAbs), E4, L1-4, and L1-24, to the major outer membrane protein (MOMP) of Chlamydia trachomatis were identified that neutralized in vitro the infectivity of members of the B- and C-related complex as well as the mouse pneumonitis strain. MAbs L1-4, L1-24, and E4 gave a strong signal in an indirect immunofluorescence assay and/or Western immunoblot with all serovars of the lymphogranuloma venereum and trachoma biovars and a weak signal with the mouse biovar. In addition, C. psittaci and C. pneumoniae were also weakly recognized by MAbs L1-4 and L1-24. As determined by the technique of pneumoniae were also weakly recognized by MAbs L1-4 and L1-24. As determined by by the technique of overlapping peptides, all three MAbs showed reactivity to variable domain (VD) IV of MOMP. While all three MAbs had different recognition patterns, all strongly bound to the peptides TLNPTI and LNPTIA within the species-conserved region of VD IV. MAb E4 also recognized the peptide SATAIF in the subspecies region of VD IV. Peptides corresponding to VD IV of MOMP were synthesized and used in competitive inhibition experiments to determine the functional location of the epitope recognized by these three MAbs. Both the serological and neutralizing activities of MAb E4 were inhibited by the peptides ATAIFDTTTLNPTIAG and FDTTTLNPTIAG; however, none of the peptides made to the VD IV region blocked the neutralizing activity of MAbs L1-4 and L1-24. Therefore, the neutralizable domain of the epitope recognized by MAb E4 is contiguous and may be an important candidate for inclusion in a subunit vaccine.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Chlamydia trachomatis/imunologia , Sequência de Aminoácidos , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Sequência de Bases , Ligação Competitiva , Clonagem Molecular , Epitopos , Dados de Sequência Molecular , Oligonucleotídeos/química , Peptídeos/imunologia , Reação em Cadeia da Polimerase , Alinhamento de SequênciaRESUMO
The gene encoding the major outer membrane protein of the Chlamydia trachomatis mouse pneumonitis biovar was sequenced and the amino acid sequence deduced. The primary structure of this protein is similar to that of the lymphogranuloma venereum and trachoma biovars in that it consists of four variable domains interspersed with five constant domains. This protein may be an ideal candidate for a vaccine in chlamydia-infected mouse experimental models.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Chlamydia trachomatis/genética , Porinas , Sequência de Aminoácidos , Animais , Sequência de Bases , Chlamydia trachomatis/metabolismo , DNA Bacteriano , Genes Bacterianos , Pneumopatias/microbiologia , Camundongos , Dados de Sequência Molecular , Especificidade da EspécieRESUMO
DNA encoding the major outer membrane protein of Chlamydia trachomatis serovar L3 was sequenced following amplification by the polymerase chain reaction. A comparison with the deduced amino acid (aa) sequence of the C. trachomatis serovar L2 showed that the L3 had three extra aa and 55 aa substitutions.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Chlamydia trachomatis/genética , Genes Bacterianos , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
DNA from Chlamydia trachomatis serovars L3, C, and E corresponding to the open reading frames of the 60-kDa protein and of a putative 15-kDa protein was sequenced. The open reading frames coding for the 60-kDa protein had 1,641 bp in the three serovars. Compared with the L3 serovar, there were 9 and 11 amino acid changes in the C and E serovars, respectively. The open reading frames corresponding to the putative 15-kDa protein had 450, 456, and 453 bp for the L3, C, and E serovars, respectively. When compared with the L3 serovar, the C and E serovars had 14 and 16 amino acid differences, respectively.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Chlamydia trachomatis/genética , Linfogranuloma Venéreo/microbiologia , Tracoma/microbiologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/genética , Variação Genética , Dados de Sequência Molecular , Fases de Leitura AbertaRESUMO
The testosterone dose-dependency of several mating and nonmating behaviors was examined in the male rat, chemically castrated with a GnRH antagonist analog. Graded doses of testosterone enanthate (TE) were given to male rats to reinstate behaviors abolished by GnRH antagonist treatment. GnRH antagonist treatment alone markedly lowered serum LH, FSH and T concentrations and ventral prostate and testis weights. Open field behaviors were not significantly affected by GnRH antagonist treatment or castration. Scent-marking behavior was markedly suppressed by both castration and GnRH antagonist and restored by the lowest dose of TE (0.05 mg). All measures of male sexual behavior were impaired by GnRH antagonist treatment and castration and restored by the lowest dose of TE (0.05 mg). The doses of TE required to restore normal ventral prostate weights and testis weights were higher than those required to maintain scent marking and mating behaviors. No direct behavioral effects of the GnRH antagonist, other than those that can be explained by GnRH antagonist-induced suppression of testosterone were observed. The finding that sexual and nonsexual behaviors in the male rat have different testosterone requirements from those maintaining spermatogenesis and fertility may have significant implications for contraception.
Assuntos
Comportamento Animal/efeitos dos fármacos , Hormônios Liberadores de Hormônios Hipofisários/antagonistas & inibidores , Comportamento Sexual Animal/efeitos dos fármacos , Testosterona/análogos & derivados , Animais , Relação Dose-Resposta a Droga , Hormônio Foliculoestimulante/sangue , Hormônio Luteinizante/sangue , Masculino , Orquiectomia , Tamanho do Órgão/efeitos dos fármacos , Próstata/anatomia & histologia , Ratos , Ratos Endogâmicos , Testículo/anatomia & histologia , Testosterona/farmacologiaRESUMO
Antagonist analogues of gonadotropin-releasing hormone (GnRH-A) alone inhibit spermatogenesis in experimental animals, but concomitant decline in serum testosterone leads to abolition of mating behavior. We assessed if the antifertility effects of GnRH-A could be dissociated from its effects on mating behavior by combining it with a small dose of androgen. Seven groups of six adult male Wistar rats were treated for 70 days as follows: I) controls, II) GnRH-A alone (250 micrograms/day), III) GnRH-A + 0.05 mg testosterone enanthate (TE), IV) GnRH-A + 0.15 mg TE, V) GnRH-A + 0.50 mg TE, VI) GnRH-A + 1.50 mg TE, and VII) GnRH-A alone (recovery group). Testes, prostate, and seminal vesicle weights were markedly reduced by GnRH-A treatment alone. Doses of TE required to maintain prostate and seminal vesicle weights were between 0.15 and 0.50 mg. Testis weights were not restored to normal even by the highest dose of TE. Intratesticular sperm counts were markedly decreased by GnRH-A treatment and restored only at the highest dose of TE (1.50 mg). Five out of six animals in group II, six out of six animals in group III, and one out of six in group IV were azoospermic. When mated with normal females, all animals in groups I and VI were fertile, all animals in groups II, III, and IV were infertile, whereas only four out of six animals in group V were fertile. All measures of mating behavior were impaired by GnRH-A treatment and restored by the smallest dose of TE (0.05 mg).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Fertilidade/efeitos dos fármacos , Hormônios Liberadores de Hormônios Hipofisários/antagonistas & inibidores , Comportamento Sexual Animal/efeitos dos fármacos , Animais , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Contagem de Espermatozoides , Testículo/anatomia & histologia , Testosterona/análogos & derivados , Testosterona/sangue , Testosterona/farmacologiaRESUMO
Both testosterone (T) and gonadotropin-releasing hormone (GnRH)-antagonist (GnRH-A) when given alone lower serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in intact and castrated rats. However, when graded doses of testosterone enanthate (T.E.) were given to GnRH-A-treated intact male rats, a paradoxical dose-dependent increase in serum FSH occurred; whereas serum LH remained suppressed. This surprising finding led us to ask whether the paradoxical increase in serum FSH in GnRH-A-suppressed animals was a direct stimulatory effect of T on the hypothalamic-pituitary axis or the result of a T effect on a testicular regulator of FSH. To test these hypotheses, we treated adult male castrated rats with GnRH-A and graded doses of T.E. In both intact and castrated rats, serum LH remained undetectable in GnRH-A-treated rats with or without T.E. However, addition of T.E. to GnRH-A led to a dose-dependent increase in serum FSH in castrated animals as well, thus pointing against mediation by a selective testicular regulator of FSH. These data provide evidence that pituitary LH and FSH responses may be differentially regulated under certain conditions. When the action of GnRH is blocked (such as in GnRH-A-treated animals), T directly and selectively increases pituitary FSH secretion.
Assuntos
Hormônio Foliculoestimulante/sangue , Hormônio Luteinizante/sangue , Hormônios Liberadores de Hormônios Hipofisários/antagonistas & inibidores , Testosterona/farmacologia , Animais , Castração , Hormônio Foliculoestimulante/metabolismo , Hormônio Luteinizante/metabolismo , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Testículo/anatomia & histologia , Testículo/efeitos dos fármacos , Testículo/fisiologiaRESUMO
We and others have observed that the response of serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) to chronic gonadotropin-releasing hormone-agonist (GnRH-A) treatment is substantially different in normal compared to hypogonadal males. These data suggested that products of the testes determine the gonadotropin response to GnRH-A. The present studies were designed to determine whether this effect is mediated by products of the interstitial (steroids) or the tubular compartment. To create experimental states with selective impairment of interstitial, tubular, or both compartments, 100 male sexually mature Wistar rats were divided into five groups: I, intact; II, castrated; III, castrated with 20-mm testosterone (T) implants; IV, bilaterally cryptorchid; and V, ketoconazole-treated animals. Cryptorchid animals have been shown to have impairment of tubular function while ketoconazole inhibits T biosynthesis. Each of the 5 groups was divided into 2 subgroups to receive daily injections of either saline or 1 microgram of a potent GnRH agonist, D-leu6 des-Gly10 GnRH N-ethylamide, for 4 wk. Unlike the intact animals, which showed an elevation of basal serum LH concentration after 4 wk of GnRH-A treatment, the castrated animals showed significant suppression below baseline. Animals with preferential impairment of tubular function (cryptorchid and castrated + T) also showed significant suppression of LH after GnRH-A treatment. However, the ketoconazole-treated animals (with inhibition of T biosynthesis and intact tubular function), behaved similarly to intact animals and demonstrated an elevation of LH after GnRH-A treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Hormônio Liberador de Hormônio do Crescimento/farmacologia , Hormônio Luteinizante/metabolismo , Testículo/fisiologia , Animais , Criptorquidismo/fisiopatologia , Hormônio Foliculoestimulante/metabolismo , Hormônio Liberador de Hormônio do Crescimento/análogos & derivados , Hipogonadismo/fisiopatologia , Cetoconazol/farmacologia , Masculino , Orquiectomia , Ratos , Ratos Endogâmicos , Testículo/efeitos dos fármacos , Testosterona/farmacologia , Testosterona/fisiologiaRESUMO
Ketoconazole, an antifungal agent, has been shown to lower serum testosterone (T) in man. Measurements of circulating precursors of T suggest that ketoconazole may inhibit 17,20-desmolase activity in the testis. To further elucidate its mechanism of action in vivo, we studied its effects on the pituitary-gonadal axis in the male rate. Two groups of normal male Sprague-Dawley rats were treated with either oil or 25 mg ketoconazole in oil by im injection every 8 h for 21 days. Serum ketoconazole concentrations in the rat 2 h after the 25-mg dose were similar to those after oral administration of a much lower (1/33rd) dose to man. Ketoconazole treatment led to 50% suppression of serum T and prostate and seminal vesicle weights. Testis weights were not significantly affected. Intratesticular T concentrations showed a 50% decrease below the control level. Testicular 17 alpha-hydroxylase, 17,20-desmolase, and 17 beta-hydroxysteroid dehydrogenase activities in the ketoconazole-treated animals were significantly decreased in proportion to the decreases in serum and intratesticular T concentrations. Elevations of serum LH and FSH concentrations in the ketoconazole-treated rats were not proportionate to the decline in serum T concentration. Therefore, to exclude an additional inhibitory effect of ketoconazole at the pituitary level, we treated two groups of castrated male Sprague-Dawley rats with the same dose of ketoconazole or oil for 3 days. Serum LH and FSH concentrations were not significantly different in the two groups. In separate experiments, combined treatment of intact rats with GnRH agonist and ketoconazole for 21 days led to lower mean serum T concentrations and accessory organ weights than those achieved with either agent alone. We conclude that ketoconazole inhibits T synthesis, primarily by inhibiting the activity of multiple enzymes in the T biosynthetic pathway and has no direct effect at the pituitary level; ketoconazole metabolism in the rat is considerably different from that in man; and ketoconazole enhances the inhibitory effects of GnRH agonist.