RESUMO
Hypertension is the leading contributor to cardiovascular disease worldwide; the prevalence of hypertension is higher among black adults than other racial/ethnic groups. One of the cellular defense mechanisms against reactive oxygen species are the antioxidants, such as the enzyme superoxide dismutase (SOD). Therefore, this study aimed to analyze the influence of the SNP Val16Ala of the SOD2 gene on oxidative stress and hypertension in a community population of self-declared black individuals in southern Brazil. The 158 participants declared themselves black (black/brown) regarding their skin color, being 89 (56.3%) self-declared black and 69 (43.7%) brown. A real-time polymerase chain reaction determined the MnSOD Ala16Val polymorphism, and oxidative stress marker levels were significant, in addition to differences in the hypertensive group regarding the levels of carbonyl (p = .016), thiobarbituric acid reactive substances (p = .040), ischemia-modified albumin (p = .046), total antioxidant capacity (p = .011), and Nitric oxide metabolites (p = .029). The SOD Val/Val genotype was considered a risk factor regardless of the other variables for hypertension (p = .034). The Val16Ala polymorphism of the MnSOD gene presented an association with hypertension.
Assuntos
Hipertensão , Albumina Sérica , Adulto , Antioxidantes , Biomarcadores , Predisposição Genética para Doença , Genótipo , Humanos , Hipertensão/genética , Polimorfismo Genético , Albumina Sérica/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
Promiscuous activities have been related to the capacity to catalyze reactions different from those a protein has evolved to sustain. In this work, we rethought the serum albumin's promiscuous behavior using evolutionary and structural analysis. We found that the cross aldol condensation of acetone and p-formylbenzonitrile is a promiscuous reaction conserved in humans serum albumin and in closely related albumins from other mammals. Evolutionary analysis indicates that the residues involved in this promiscuous reaction are evolving under positive selection, an evolutionary pattern indicating a putative functional adaptation. Also, key residues are located in an evolutionary conserved cavity connected with the protein surface with an also conserved tunnel and mutations involving these residues are described in human diseases. Overall, our results suggest that albumin could have evolved to sustain a still unknown biological function among the many others it maintains. Our results could contribute to better characterize the serum albumin family and raise questions about the evolution of protein promiscuity and function.
Assuntos
Evolução Molecular , Albumina Sérica , Adaptação Fisiológica , Animais , Catálise , Humanos , Mamíferos , Albumina Sérica/genéticaRESUMO
The aim of this study was to analyze the effect of linker length on the expression and biological activity of recombinant protein onconase (ONC) in fusion with human serum albumin (HSA) in Pichia pastoris. Four flexible linkers with different lengths namely Linker L0, L1: (GGGGS)1, L2: (GGGGS)2, and L3:(GGGGS)3 were inserted into the fusion gene and referred to as HSA-n-ONC, where N = 0, 5, 10, or 15. The sequence of the fusion gene HSA-ONC was designed based on the GC content and codon bias in P. pastoris; the signal peptide of albumin was used as the secretion signal. Gene sequences coding for the fusion protein with different linkers were inserted into pPICZα-A to form recombinant plasmids pPICZα-A/HSA-n-ONC, which were then transformed into P. pastoris X-33 for protein expression. Ideal conditions for expression of the fusion proteins were optimized at a small scale, using shake flasks before proceeding to mass production in 10-L fermenters. The recombinant fusion proteins were purified by aqueous two-phase extraction coupled with DEAE anion exchange chromatography, and their cytotoxic effect on the tumor cell was evaluated by the sulforhodamine B assay. The results showed that the expressed amount of fusion proteins had no significant relationship with the length of different linkers and rHSA-0-ONC had no cytotoxic effect on the tumor cells. While rHSA-5-ONC and rHSA-10-ONC had a weak cytotoxic effect, rHSA-15-ONC could kill various tumor cells in vitro. In summary, the biological activity of the fusion protein gradually improved with increasing length of the linker.
Assuntos
Proteínas de Anfíbios/genética , Antineoplásicos/farmacologia , Clonagem Molecular/métodos , Pichia/genética , Proteínas Recombinantes de Fusão/genética , Ribonucleases/genética , Proteínas de Anfíbios/biossíntese , Proteínas de Anfíbios/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica , Humanos , Concentração Inibidora 50 , Extração Líquido-Líquido , Pichia/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Engenharia de Proteínas , Sinais Direcionadores de Proteínas , Rana pipiens/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/farmacologia , Rodaminas/química , Ribonucleases/biossíntese , Ribonucleases/farmacologia , Albumina Sérica/biossíntese , Albumina Sérica/genética , Relação Estrutura-Atividade , Transformação GenéticaRESUMO
Advanced glycation end products (AGE) are elevated in diabetes mellitus (DM) and predict the development of atherosclerosis. AGE-albumin induces oxidative stress, which is linked to a reduction in ABCA-1 and cholesterol efflux. We characterized the glycation level of human serum albumin (HSA) isolated from poorly controlled DM2 (n = 11) patients compared with that of control (C, n = 12) individuals and determined the mechanism by which DM2-HSA can interfere in macrophage lipid accumulation. The HSA glycation level was analyzed by MALDI/MS. Macrophages were treated for 18 h with C- or DM2-HSA to measure the (14) C-cholesterol efflux, the intracellular lipid accumulation and the cellular ABCA-1 protein content. Agilent arrays (44000 probes) were used to analyze gene expression, and the differentially expressed genes were validated by real-time RT-PCR. An increased mean mass was observed in DM2-HSA compared with C-HSA, reflecting the condensation of at least 5 units of glucose. The cholesterol efflux mediated by apo AI, HDL3 , and HDL2 was impaired in DM2-HSA-treated cells, which was related to greater intracellular lipid accumulation. DM2-HSA decreased Abcg1 mRNA expression by 26%. Abca1 mRNA was unchanged, although the final ABCA-1 protein content decreased. Compared with C-HAS-treated cells, NADPH oxidase 4 mRNA expression increased in cells after DM2-HSA treatment. Stearoyl-Coenzyme A desaturase 1, janus kinase 2, and low density lipoprotein receptor mRNAs were reduced by DM2-HSA. The level of glycation that occurs in vivo in DM2-HSA-treated cells selectively alters macrophage gene expression, impairing cholesterol efflux and eliciting intracellular lipid accumulation, which contribute to atherogenesis, in individuals with DM2.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Colesterol/metabolismo , Diabetes Mellitus Tipo 2/genética , Macrófagos/metabolismo , Albumina Sérica/metabolismo , Adulto , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Transporte Biológico/genética , Transporte Biológico/fisiologia , Colesterol/genética , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Expressão Gênica/fisiologia , Produtos Finais de Glicação Avançada , Humanos , Masculino , Camundongos , Estresse Oxidativo/genética , Albumina Sérica/genética , Albumina Sérica GlicadaRESUMO
Chronic obstructive pulmonary disease (COPD) is a growing cause of morbidity and mortality throughout the world. The purpose of our study was to uncover biomarkers and explore its pathogenic mechanisms at the molecular level. The gene expression profiles of COPD samples and normal controls were downloaded from Gene Expression Omnibus. Matlab was used for data preprocessing and SAM4.0 was applied to determine the differentially expressed genes (DEGs). Furthermore, a protein-protein interaction (PPI) network was constructed by mapping the DEGs into PPI data, and functional analysis of the network was conducted with BiNGO. A total of 348 DEGs and 765 interactive genes were identified. The hub genes were mainly involved in metabolic processes and ribosome biogenesis. Several genes related to COPD in the PPI network were found, including CAMK1D, ALB, KIT, and DDX3Y. In conclusion, CAMK1D, ALB, KIT, and DDX3Y were chosen as candidate genes, which have the potential to be biomarkers or candidate target molecules to apply in clinical diagnosis and treatment of COPD.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/genética , Doença Pulmonar Obstrutiva Crônica/genética , Transdução de Sinais/genética , Biomarcadores/metabolismo , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Predisposição Genética para Doença/genética , Humanos , Antígenos de Histocompatibilidade Menor , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Albumina Sérica/genética , Albumina Sérica/metabolismo , Albumina Sérica HumanaRESUMO
A utilização do soro fetal bovino (SFB), embora bastante disseminada na produção in vitro (PIV) de embriões bovinos, apresenta limitações por ser um meio indefinido e por causar efeitos que prejudicam a qualidade desses embriões. Por esse motivo, nos últimos anos, grande parte das pesquisas relacionadas à PIV está voltada para a substituição do SFB por outros compostos nos meios de cultura. No presente estudo, foram utilizados como compostos protéicos a albumina sérica bovina livre de ácidos graxos (BSA-FAF) e um produto comercial denominado fluido embriônico (FE) de maneira isolada ou em diferentes combinações e concentrações, com objetivo de substituir ou diminuir a concentração do SFB durante a maturação in vitro (MIV). [...] Ademais, o G3 também apresentou diminuição na taxa de maturação nuclear quando comparado ao G4. Quanto à maturação citoplasmática, nos grupos G2, G7, G6 e G3, houve redução (p<0,05) das taxas para 43,9por cento, 43,2 por cento, 43,1 por cento e 36,5 por cento, respectivamente, quando comparadas ao meio controle (G1), que permitiu a obtenção de valores médios de 62,4 por cento. Por outro lado, nos grupos G8, G4 e G5, a taxa de maturação citoplasmática não foi afetada com a redução do SFB, onde 59,3 por cento, 51,3 por cento e 50,8 por cento dos oócitos apresentaram os GC dispostos na periferia, respectivamente. Os resultados obtidos pelo teste de contrastes ortogonais complementam os obtidos na avaliação da maturação nuclear e migração de grânulos corticais, mostrando a necessidade do SFB durante a MIV, mesmo que em baixas concentrações, e a possibilidade de diminuir a sua concentração associando-o a BSA-FAF e/ou FE. Dessa forma, conclui-se que é possível reduzir a concentração de SFB no meio de MIV para até 3,5% sem prejuízo significativo aos índices de maturação nuclear e citoplasmática.
The use of fetal calf serum (FCS), although widely employed during in vitro production (IVP) of bovine embryos, has limitations. FCS is an undefined media and may have harmful effects on the quality of embryos. For this reason, in recent years, research efforts aimed at improving IVP of bovine embryos, have focused at the replacement of FCS by alternative compounds in culture media. In this study, fatty acid free bovine serum albumin (BSA-FAF) and embryonic fluid (EF) were used separately or in combination, in different concentrations, to replace or reduce the concentration of FCS during in vitro maturation (IVM). [...] Moreover, G3 also showed inferior nuclear maturation rate when compared to G4. Regarding cytoplasmic maturation, the rates were reduced to 43.9 percent, 43.2 percent, 43.1 percent and 36.5 percent in G2, G7, G6 and G3 groups, respectively, compared to the control group (G1; 62.4 percent). On the other hand, in the groups G8, G4 and G5, maturation rates were not affected by reduction of FCS, where 59.3 percent, 51.3 percent and 50.8 percent of the oocytes displayed CG arranged peripherally, respectively. The results obtained by the orthogonal contrast test are in accordance with the ones from the evaluation of the nuclear maturation and cortical granules migration. These data show the need of FCS on the MIV, even in low concentrations, and the possibility of decrease its concentration by associating it with BSA-FAF and/or EF. Therefore, we concluded that it is possible to reduce the concentration of FCS in IVM medium to a concentration of 3.5 percent without affecting nuclear and cytoplasmic maturation rates.
Assuntos
Animais , Albumina Sérica/genética , Bovinos/embriologia , Soroalbumina Bovina/genética , Técnicas de Maturação in Vitro de Oócitos/veterinária , Fertilização in vitro/veterinária , Técnicas de Cultura/veterináriaRESUMO
A utilização do soro fetal bovino (SFB), embora bastante disseminada na produção in vitro (PIV) de embriões bovinos, apresenta limitações por ser um meio indefinido e por causar efeitos que prejudicam a qualidade desses embriões. Por esse motivo, nos últimos anos, grande parte das pesquisas relacionadas à PIV está voltada para a substituição do SFB por outros compostos nos meios de cultura. No presente estudo, foram utilizados como compostos protéicos a albumina sérica bovina livre de ácidos graxos (BSA-FAF) e um produto comercial denominado fluido embriônico (FE) de maneira isolada ou em diferentes combinações e concentrações, com objetivo de substituir ou diminuir a concentração do SFB durante a maturação in vitro (MIV). [...] Ademais, o G3 também apresentou diminuição na taxa de maturação nuclear quando comparado ao G4. Quanto à maturação citoplasmática, nos grupos G2, G7, G6 e G3, houve redução (p<0,05) das taxas para 43,9por cento, 43,2 por cento, 43,1 por cento e 36,5 por cento, respectivamente, quando comparadas ao meio controle (G1), que permitiu a obtenção de valores médios de 62,4 por cento. Por outro lado, nos grupos G8, G4 e G5, a taxa de maturação citoplasmática não foi afetada com a redução do SFB, onde 59,3 por cento, 51,3 por cento e 50,8 por cento dos oócitos apresentaram os GC dispostos na periferia, respectivamente. Os resultados obtidos pelo teste de contrastes ortogonais complementam os obtidos na avaliação da maturação nuclear e migração de grânulos corticais, mostrando a necessidade do SFB durante a MIV, mesmo que em baixas concentrações, e a possibilidade de diminuir a sua concentração associando-o a BSA-FAF e/ou FE. Dessa forma, conclui-se que é possível reduzir a concentração de SFB no meio de MIV para até 3,5% sem prejuízo significativo aos índices de maturação nuclear e citoplasmática.(AU)
The use of fetal calf serum (FCS), although widely employed during in vitro production (IVP) of bovine embryos, has limitations. FCS is an undefined media and may have harmful effects on the quality of embryos. For this reason, in recent years, research efforts aimed at improving IVP of bovine embryos, have focused at the replacement of FCS by alternative compounds in culture media. In this study, fatty acid free bovine serum albumin (BSA-FAF) and embryonic fluid (EF) were used separately or in combination, in different concentrations, to replace or reduce the concentration of FCS during in vitro maturation (IVM). [...] Moreover, G3 also showed inferior nuclear maturation rate when compared to G4. Regarding cytoplasmic maturation, the rates were reduced to 43.9 percent, 43.2 percent, 43.1 percent and 36.5 percent in G2, G7, G6 and G3 groups, respectively, compared to the control group (G1; 62.4 percent). On the other hand, in the groups G8, G4 and G5, maturation rates were not affected by reduction of FCS, where 59.3 percent, 51.3 percent and 50.8 percent of the oocytes displayed CG arranged peripherally, respectively. The results obtained by the orthogonal contrast test are in accordance with the ones from the evaluation of the nuclear maturation and cortical granules migration. These data show the need of FCS on the MIV, even in low concentrations, and the possibility of decrease its concentration by associating it with BSA-FAF and/or EF. Therefore, we concluded that it is possible to reduce the concentration of FCS in IVM medium to a concentration of 3.5 percent without affecting nuclear and cytoplasmic maturation rates.(AU)
Assuntos
Animais , Bovinos/embriologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Soroalbumina Bovina/genética , Albumina Sérica/genética , Técnicas de Cultura/veterinária , Fertilização in vitro/veterináriaRESUMO
The clinical course of patients with chronic hepatitis B (CH-B) was greatly changed by the introduction of nucleoside analogues. We often encounter patients where the serum level of albumin recovers quickly following the treatment. In this study, we focused carefully on the changes in serum albumin level noted during nucleoside analogue therapy, in an effort to clarify the mechanism behind the restoration of albumin production. We observed changes in serum albumin levels during nucleoside analogue therapy in 12 patients with CH-B and studied the mechanism behind the restoration of albumin production following the therapy. The serum level of albumin was significantly increased very soon after the treatment was started. Prior to treatment with nucleoside analogues, the albumin signal for mRNA was only slightly seen in the peri-portal area, whereas 12 months after the treatment, the liver tissue presented an obvious signal of albumin mRNA. Serum levels of hepatocyte growth factor (HGF) were significantly decreased 12 months after the treatment. In this study, we demonstrated that nucleoside analogues decrease HGF through the suppression of hepatocyte damage, leading to the restoration of albumin production in patients with CH-B.
Assuntos
Antivirais/uso terapêutico , Guanina/análogos & derivados , Hepatite B Crônica/sangue , Lamivudina/uso terapêutico , Albumina Sérica/metabolismo , Adulto , Idoso , Antivirais/farmacologia , Feminino , Expressão Gênica , Guanina/farmacologia , Guanina/uso terapêutico , Hepatite B Crônica/tratamento farmacológico , Fator de Crescimento de Hepatócito/sangue , Humanos , Lamivudina/farmacologia , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Albumina Sérica/genética , Fator de Crescimento Transformador beta1/sangue , Adulto JovemRESUMO
BACKGROUND/AIM: The aim of this work was to establish an association between the single-nucleotide polymorphisms (SNPs) of TGFB1 (rs1800471), AT (rs3789679), MMP-1 (rs17886084), MMP-3 (rs35068180), and PAI-1 (rs1799889) and the histological grading of necroinflammation, staging of hepatic fibrosis, and liver function in Mexican patients with advanced liver fibrosis due to chronic hepatitis C virus infection. METHODS: AT, MMP-1, MMP-3, and PAI-1 gene polymorphisms were analyzed by polymerase chain reaction in real time, whereas TGFB1 polymorphism was detected by polymerase chain reaction-based restriction fragment length polymorphism in 38 patients with established advanced liver fibrosis and 50 subjects from the general population. Grading of necroinflammation and staging of liver fibrosis were assessed by liver biopsy and graded according to modified histological activity index Ishak score. RESULTS: Regarding TGFB1 SNP, significant differences were found between G/G and G/C genotypes of patients with hepatic necroinflammation (P = 0.05) and hepatic fibrosis (P = 0.002). There were also significant differences among genotypes of patients with the AT SNP in hepatic necroinflammation (P = 0.01). The albumin-globulin ratio between genotypes of patients with the MMP-3 SNP gene showed significant differences (P = 0.02). CONCLUSION: Our findings demonstrate that a specific combination of genotypes associated with biochemical values and a histological high score determine more severe liver disease. The presence of the G/G genotype of TGFB1 SNP in patients was significantly associated with severity of liver necroinflammation and fibrosis. Patients with the G/G genotype of AT SNP were associated with severe necroinflammation. The albumin-globulin ratio was increased in patients with the 6A allele of MMP-3 SNP. These results might contribute to diagnosis and further establishment of liver disease treatment.
Assuntos
Estudos de Associação Genética , Hepatite C Crônica/genética , Cirrose Hepática/genética , Metaloproteinase 3 da Matriz/genética , Polimorfismo de Nucleotídeo Único/genética , Fator de Crescimento Transformador beta1/genética , Idoso , Alelos , Feminino , Estudos de Associação Genética/métodos , Genótipo , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/etnologia , Humanos , Cirrose Hepática/diagnóstico , Cirrose Hepática/etnologia , Testes de Função Hepática/métodos , Masculino , México/etnologia , Pessoa de Meia-Idade , Necrose/diagnóstico , Necrose/etnologia , Necrose/genética , Albumina Sérica/genética , Soroglobulinas/genéticaRESUMO
BACKGROUND: We evaluated the effects of albumin isolated from control individuals and from patients with poorly controlled type 1 diabetes mellitus on macrophage gene expression and on reverse cholesterol transport. METHODS: Serum albumin was purified from control subjects (n = 12) and from patients with poorly controlled type 1 diabetes mellitus (n = 13). (14)C-cholesterol-labelled J774 macrophages treated with albumin were employed to measure cholesterol efflux mediated by apo A-I, HDL(3) or HDL(2), the intracellular lipid accumulation and the cellular ABCA-1 protein content. Agilent arrays (44000 probes) were used to analyse gene expression. Several differentially expressed genes were validated by real-time reverse transcription-PCR using TaqMan Two Step RT-PCR. RESULTS: Levels of glycation-modified and (carboxymethyl)lysine-modified albumin were higher in diabetic patients than in control subjects. Apo A-I-mediated and HDL(2)-mediated cellular cholesterol efflux were impaired in macrophages treated with albumin from diabetic patients in comparison with control albumin-treated cells, which was attributed to the reduction in ABCA-1 protein content. Even in the presence of cholesterol acceptors, a higher level of intracellular lipid was observed in macrophages exposed to albumin from diabetic individuals in comparison with the control. The reduction in ABCA-1 content was associated with enhanced expression of stearoyl CoA desaturase 1 and decreased expression of janus kinase 2, which were induced by albumin from patients with type 1 diabetes mellitus. CONCLUSIONS: (Carboxymethyl)lysine-modified albumin isolated from poorly controlled type 1 diabetic patients impairs ABCA-1-mediated reverse cholesterol transport and elicits intracellular lipid accumulation, possibly contributing to atherosclerosis.