ABSTRACT
Introducción Los estudios genéticos han demostrado asociaciones de varios polimorfismos de un solo nucleótido (SNP) con diferentes tasas de progresión y variación en la susceptibilidad a la infección por VIH. Este estudio tuvo como objetivo estimar la frecuencia de los polimorfismos ccr5Δ32, IL-6-174G/C, IFN-γ+874T/A e IL-10-1082A/G en pacientes cubanos infectados por VIH y un grupo de parejas serodiscordantes para evaluar su influencia sobre el riesgo y la progresión de la enfermedad. Métodos Se realizó un estudio transversal en 120 sujetos atendidos en el Instituto de Medicina Tropical «Pedro Kour» (IPK) y el Hospital Hermanos Ameijeiras entre junio de 2018 y diciembre de 2019. La amplificación de los fragmentos de los genes ccr5, IL-6, IFN-γ e IL-10 se realizó mediante reacción en cadena de la polimerasa seguida por el análisis del polimorfismo de fragmentos de restricción utilizando la enzima NlaIII para la IL-6. El sistema de mutación refractario a la amplificación por PCR se utilizó en el caso de los genes IFN-γ e IL-10. Resultados Las distribuciones alélicas y genotípicas de los genes ccr5, IL-6, IFN-γ e IL-10 no difirieron significativamente entre los dos grupos. Los recuentos celulares y los valores de carga viral en plasma no difirieron significativamente entre los genotipos de los genes ccr5, IL-6, IFN-γ e IL-10. Solo el genotipo IL-6 GC se asoció con valores más altos de carga viral. La combinación de alelos de los cuatro SNP considerados mostró un aumento muy significativo del riesgo de infección por VIH para uno de ellos, pero con una frecuencia muy baja (<1%). Conclusión Este estudio contribuye a evaluar la frecuencia de estos polimorfismos y su influencia en los biomarcadores de la progresión de la infección por VIH en la población cubana con infección por el VIH. (AU)
Introduction Genetic studies have shown associations of several single nucleotide polymorphisms (SNP) with different rates of progression and variation in susceptibility to HIV infection. This study aimed to estimate the frequency of ccr5Δ32, IL-6-174G/C, IFN-γ+874T/A and IL-10-1082A/G polymorphisms in Cuban HIV-infected patients and a group of sero-discordant couples to assess their influence on risk and disease progression. Methods A cross-sectional study was carried out on 120 subjects registered at the Institute of Tropical Medicine «Pedro Kour» (IPK) and the Ameijeiras Hospital from June 2018 until December 2019. The amplification of fragments of the ccr5, IL-6, IFN-γ and IL-10 genes was performed by polymerase chain reaction followed by identification of polymorphisms using the restriction fragment length polymorphism analysis for IL-6 with the restriction enzymes Nla III. Amplification refractory mutation system was used for IFN-γ and IL-10 genes. Results The allelic and genotypic distributions of the genes ccr5, IL-6, IFN-γ and IL-10 did not differ significantly between the two groups. Cell counts and plasma viral load values did not differ significantly between genotypes of the ccr5, IL-6, IFN-γ and IL-10 genes. Only the IL-6 GC genotype was associated with higher viral load values. The combination of alleles of the four considered SNPs showed a highly significant increase in the risk of HIV infection for one of them, but with a very low frequency (<1%). Conclusion This study contributes to evaluating the frequency of these polymorphisms and their influence on biomarkers of the progression of HIV infection in the Cuban HIV-population. (AU)
Subject(s)
Humans , Young Adult , Adult , Middle Aged , Aged , Aged, 80 and over , HIV Infections , Polymorphism, Single Nucleotide , Acquired Immunodeficiency Syndrome , Genetic Predisposition to Disease , Cross-Sectional Studies , CubaABSTRACT
Introducción Los estudios genéticos han demostrado asociaciones de varios polimorfismos de un solo nucleótido (SNP) con diferentes tasas de progresión y variación en la susceptibilidad a la infección por VIH. Este estudio tuvo como objetivo estimar la frecuencia de los polimorfismos ccr5Δ32, IL-6-174G/C, IFN-γ+874T/A e IL-10-1082A/G en pacientes cubanos infectados por VIH y un grupo de parejas serodiscordantes para evaluar su influencia sobre el riesgo y la progresión de la enfermedad. Métodos Se realizó un estudio transversal en 120 sujetos atendidos en el Instituto de Medicina Tropical «Pedro Kour» (IPK) y el Hospital Hermanos Ameijeiras entre junio de 2018 y diciembre de 2019. La amplificación de los fragmentos de los genes ccr5, IL-6, IFN-γ e IL-10 se realizó mediante reacción en cadena de la polimerasa seguida por el análisis del polimorfismo de fragmentos de restricción utilizando la enzima NlaIII para la IL-6. El sistema de mutación refractario a la amplificación por PCR se utilizó en el caso de los genes IFN-γ e IL-10. Resultados Las distribuciones alélicas y genotípicas de los genes ccr5, IL-6, IFN-γ e IL-10 no difirieron significativamente entre los dos grupos. Los recuentos celulares y los valores de carga viral en plasma no difirieron significativamente entre los genotipos de los genes ccr5, IL-6, IFN-γ e IL-10. Solo el genotipo IL-6 GC se asoció con valores más altos de carga viral. La combinación de alelos de los cuatro SNP considerados mostró un aumento muy significativo del riesgo de infección por VIH para uno de ellos, pero con una frecuencia muy baja (<1%). Conclusión Este estudio contribuye a evaluar la frecuencia de estos polimorfismos y su influencia en los biomarcadores de la progresión de la infección por VIH en la población cubana con infección por el VIH. (AU)
Introduction Genetic studies have shown associations of several single nucleotide polymorphisms (SNP) with different rates of progression and variation in susceptibility to HIV infection. This study aimed to estimate the frequency of ccr5Δ32, IL-6-174G/C, IFN-γ+874T/A and IL-10-1082A/G polymorphisms in Cuban HIV-infected patients and a group of sero-discordant couples to assess their influence on risk and disease progression. Methods A cross-sectional study was carried out on 120 subjects registered at the Institute of Tropical Medicine «Pedro Kour» (IPK) and the Ameijeiras Hospital from June 2018 until December 2019. The amplification of fragments of the ccr5, IL-6, IFN-γ and IL-10 genes was performed by polymerase chain reaction followed by identification of polymorphisms using the restriction fragment length polymorphism analysis for IL-6 with the restriction enzymes Nla III. Amplification refractory mutation system was used for IFN-γ and IL-10 genes. Results The allelic and genotypic distributions of the genes ccr5, IL-6, IFN-γ and IL-10 did not differ significantly between the two groups. Cell counts and plasma viral load values did not differ significantly between genotypes of the ccr5, IL-6, IFN-γ and IL-10 genes. Only the IL-6 GC genotype was associated with higher viral load values. The combination of alleles of the four considered SNPs showed a highly significant increase in the risk of HIV infection for one of them, but with a very low frequency (<1%). Conclusion This study contributes to evaluating the frequency of these polymorphisms and their influence on biomarkers of the progression of HIV infection in the Cuban HIV-population. (AU)
Subject(s)
Humans , Young Adult , Adult , Middle Aged , Aged , Aged, 80 and over , HIV Infections , Polymorphism, Single Nucleotide , Acquired Immunodeficiency Syndrome , Genetic Predisposition to Disease , Cross-Sectional Studies , CubaABSTRACT
INTRODUCTION: Genetic studies have shown associations of several single nucleotide polymorphisms (SNP) with different rates of progression and variation in susceptibility to HIV infection. This study aimed to estimate the frequency of ccr5Δ32, IL-6-174G/C, IFN-γ+874T/A and IL-10-1082A/G polymorphisms in Cuban HIV-infected patients and a group of sero-discordant couples to assess their influence on risk and disease progression. METHODS: A cross-sectional study was carried out on 120 subjects registered at the Institute of Tropical Medicine «Pedro Kour¼ (IPK) and the Ameijeiras Hospital from June 2018 until December 2019. The amplification of fragments of the ccr5, IL-6, IFN-γ and IL-10 genes was performed by polymerase chain reaction followed by identification of polymorphisms using the restriction fragment length polymorphism analysis for IL-6 with the restriction enzymes Nla III. Amplification Refractory Mutation System was used for IFN-γ and IL-10 genes. RESULTS: The allelic and genotypic distributions of the genes ccr5, IL-6, IFN-γ and IL-10 did not differ significantly between the two groups. Cell counts and plasma viral load values did not differ significantly between genotypes of the ccr5, IL-6, IFN-γ and IL-10 genes. Only the IL-6 GC genotype was associated with higher viral load values. The combination of alleles of the four considered SNPs showed a highly significant increase in the risk of HIV infection for one of them, but with a very low frequency (<1%). CONCLUSION: This study contributes to evaluating the frequency of these polymorphisms and their influence on biomarkers of the progression of HIV infection in the Cuban HIV-population.
Subject(s)
Acquired Immunodeficiency Syndrome , HIV Infections , Humans , HIV Infections/genetics , Acquired Immunodeficiency Syndrome/genetics , Interleukin-6/genetics , Interleukin-10/genetics , Cross-Sectional Studies , Gene Frequency , Genetic Predisposition to Disease , Polymorphism, Single Nucleotide , Receptors, CCR5/geneticsABSTRACT
Although antibiotics against Mycobacterium tuberculosis have decreased the incidence of tuberculosis infections significantly, the emergence of drug-resistant strains of this deadly pathogen renders current treatments ineffective. Therefore, it is imperative to identify biochemical pathways in M. tuberculosis that can serve as targets for new anti-mycobacterial drugs. We recently cloned, expressed, and purified MT CYP51, a soluble protein from M. tuberculosis that is similar in sequence to CYP51 (lanosterol-14alpha-demethylase) isozymes, pharmacological targets for several anti-mycotic compounds. Its striking amino acid sequence similarity to that of mammalian and fungal CYP51s led to the hypothesis that MT CYP51 plays an important role in mycobacterial biology that can be targeted for drug action. In this manuscript, we established through spectral analysis that several azole antifungals bind MT CYP51 with high affinity. The effects of several azole compounds on the growth of M. bovis and M. smegmatis, two mycobacterial species that closely resemble M. tuberculosis were examined. We established a correlation between the affinity of azole compounds to MT CYP51 and their ability to impair the growth of M. bovis and M. smegmatis. These results suggest that the metabolic functions of MT CYP51 may be comparable to those of CYP51 in yeast and fungi and may lead to the development of a new generation of anti-mycobacterial agents.
Subject(s)
Antitubercular Agents/pharmacology , Azoles/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Mycobacterium tuberculosis/drug effects , Oxidoreductases/metabolism , Amino Acid Sequence , Antifungal Agents/pharmacology , Base Sequence , Cell Division/drug effects , Microbial Sensitivity Tests , Molecular Sequence Data , Mycobacterium bovis/cytology , Mycobacterium bovis/drug effects , Mycobacterium smegmatis/cytology , Mycobacterium smegmatis/drug effects , Mycobacterium tuberculosis/enzymology , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spectrum Analysis , Sterol 14-DemethylaseABSTRACT
Brown adipose tissue (BAT) hyperplasia is a fundamental physiological response to cold; it involves an acute phase of mitotic cell growth followed by a prolonged differentiation phase. Peroxisome proliferator-activated receptors (PPARs) are key regulators of fatty acid metabolism and adipocyte differentiation and may therefore mediate important metabolic changes during non-shivering thermogenesis. In the present study we have investigated PPAR mRNA expression in relation to peroxisome proliferation in rat BAT during cold acclimatization. By immunoelectron microscopy we show that the number of peroxisomes per cytoplasmic volume and acyl-CoA oxidase immunolabeling density remained constant (thus increasing in parallel with tissue mass and cell number) during the initial proliferative phase and the acute thermogenic response but increased after 14 days of cold exposure, correlating with terminal differentiation of BAT. A pronounced decrease in BAT PPARalpha and PPARgamma mRNA levels was found within hours of exposure to cold, which was reversed after 14 days, suggesting a role for either or both of these subtypes in the proliferation and induction of peroxisomes and peroxisomal beta-oxidation enzymes. In contrast, PPARdelta mRNA levels increased progressively during cold exposure. Transactivation assays in HIB 1B and HEK-293 cells demonstrated an adrenergic stimulation of peroxisome proliferator response element reporter activity via PPAR, establishing a role for these nuclear receptors in hormonal regulation of gene transcription in BAT.
Subject(s)
Adaptation, Physiological , Adipose Tissue, Brown/physiology , Cold Temperature , Receptors, Cytoplasmic and Nuclear/physiology , Transcription Factors/physiology , Acyl-CoA Oxidase , Adaptation, Physiological/genetics , Adipose Tissue, Brown/enzymology , Adipose Tissue, Brown/ultrastructure , Amino Acid Sequence , Animals , Base Sequence , CCAAT-Enhancer-Binding Proteins , Carbon-Carbon Double Bond Isomerases/genetics , Cloning, Molecular , DNA, Complementary , DNA-Binding Proteins/genetics , Dodecenoyl-CoA Isomerase , Female , Immunohistochemistry , Lipoprotein Lipase/genetics , Male , Microbodies/metabolism , Microscopy, Immunoelectron , Molecular Sequence Data , Nuclear Proteins/genetics , Oxidoreductases/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Cytoplasmic and Nuclear/genetics , Transcription Factors/genetics , TransfectionABSTRACT
The negative glucocorticoid regulation of CRH gene expression is a critical control element in the hypothalamic-pituitary-adrenal axis. In this study, the molecular mechanisms mediating the glucocorticoid repression of cAMP-induced CRH-reporter expression in AtT-20 cells have been examined. In these cells, dexamethasone decreases forskolin-induced expression of CRH-reporter activity in a dose-dependent manner. This repression is mediated by the glucocorticoid receptor (GR) and does not require ongoing protein synthesis. Several binding sites for the GR DNA-binding domain were identified within the CRH 5'-flanking and 5'-untranslated regions utilizing in vitro DNase I protection assays. These sites were independently mutated and/or deleted. Functional studies in transfected cells suggest that none of the protected DNA sequences mediate the glucocorticoid regulation and that the regulatory element(s) mediating negative glucocorticoid regulation is contained within the CRH DNA sequences from -248 to +4 bp relative to the major transcription initiation site. To further localize the DNA sequence(s) responsive to glucocorticoids, DNA fragments containing various amounts of human CRH 5'flanking sequences were inserted 5' to the SV40 promoter. An 18-bp DNA fragment containing the CRH cAMP-responsive element is sufficient to confer both positive cAMP regulation and glucocorticoid repression of cAMP-stimulated expression to the SV40 promoter. These results suggest that glucocorticoid repression of forskolin-activated CRH-reporter expression in AtT-20 cells occurs via interference with the cAMP-mediated activation of gene expression, possibly via direct or indirect interactions between the GR and the cAMP-responsive element-binding proteins.
Subject(s)
Corticotropin-Releasing Hormone/genetics , Cyclic AMP/physiology , Dexamethasone/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Pituitary Neoplasms/pathology , Recombinant Fusion Proteins/biosynthesis , Second Messenger Systems/physiology , Animals , Binding Sites , Chloramphenicol O-Acetyltransferase/biosynthesis , Chloramphenicol O-Acetyltransferase/genetics , Colforsin/pharmacology , Corticotropin-Releasing Hormone/biosynthesis , Depression, Chemical , Genes, Reporter , Humans , Mice , Regulatory Sequences, Nucleic Acid , Transcription, Genetic/drug effects , TransfectionABSTRACT
Membrane depolarization is a critical element of neuronal signaling. In this study, the biochemical and molecular mechanisms involved in transcriptional regulation of the corticotropin-releasing hormone (CRH) gene by depolarization were investigated. In PC-12 cells, potassium-induced membrane depolarization increased expression of a CRH-reporter construct in a cAMP-dependent manner. This synergistic activation was mediated via calcium influx, predominantly via L-type calcium channels, and calmodulin. RNase protection assays demonstrated increased levels of CRH-reporter transcripts in stably transfected cells after treatment with cAMP and potassium, with the induced transcripts initiating at the major transcription initiation site of the human CRH gene. At the genomic level, the CRH cAMP-responsive element conferred both positive cAMP and synergistic cAMP/depolarization regulation to a heterologous promoter. Additionally, DNase I protection assays demonstrated similar nuclear protein/DNA binding profiles across the cAMP-responsive element after treatment of PC-12 cells with potassium or potassium/cAMP. These results support a model in which the protein(s) binding to the cAMP-responsive element integrates signals initiated by multiple pathways (cAMP and calcium) and transmits that integrated signal to the basal transcription machinery, resulting in increased levels of gene expression.
