RESUMO
BACKGROUND AND AIM: Circulating amino acids are modified by sex, body mass index (BMI) and insulin resistance (IR). However, whether the presence of genetic variants in branched-chain amino acid (BCAA) catabolic enzymes modifies circulating amino acids is still unknown. Thus, we determined the frequency of two genetic variants, one in the branched-chain aminotransferase 2 (BCAT2) gene (rs11548193), and one in the branched-chain ketoacid dehydrogenase (BCKDH) gene (rs45500792), and elucidated their impact on circulating amino acid levels together with clinical, anthropometric and biochemical parameters. METHODS AND RESULTS: We performed a cross-sectional comparative study in which we recruited 1612 young adults (749 women and 863 men) aged 19.7 ± 2.1 years and with a BMI of 24.9 ± 4.7 kg/m2. Participants underwent clinical evaluation and provided blood samples for DNA extraction and biochemical analysis. The single nucleotide polymorphisms (SNPs) were determined by allelic discrimination using real-time polymerase chain reaction (PCR). The frequencies of the less common alleles were 15.2 % for BCAT2 and 9.83 % for BCKDH. The subjects with either the BCAT2 or BCKDH SNPs displayed no differences in the evaluated parameters compared with subjects homozygotes for the most common allele at each SNP. However, subjects with both SNPs had higher body weight, BMI, blood pressure, glucose, and circulating levels of aspartate, isoleucine, methionine, and proline than the subjects homozygotes for the most common allele (P < 0.05, One-way ANOVA). CONCLUSION: Our findings suggest that the joint presence of both the BCAT2 rs11548193 and BCKDH rs45500792 SNPs induces metabolic alterations that are not observed in subjects without either SNP.
Assuntos
3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/genética , Aminoácidos/sangue , Antígenos de Histocompatibilidade Menor/genética , Polimorfismo de Nucleotídeo Único , Proteínas da Gravidez/genética , Transaminases/genética , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/metabolismo , Adolescente , Fatores Etários , Biomarcadores/sangue , Glicemia/análise , Pressão Sanguínea , Índice de Massa Corporal , Estudos Transversais , Feminino , Frequência do Gene , Estudos de Associação Genética , Homozigoto , Humanos , Masculino , México , Antígenos de Histocompatibilidade Menor/metabolismo , Fenótipo , Proteínas da Gravidez/metabolismo , Transaminases/metabolismo , Adulto JovemRESUMO
Experimental models of maternal diabetes lead to the intrauterine programming of Gestational Diabetes Mellitus (GDM) in the offspring, together with an intrauterine proinflammatory environment, feto-placental metabolic alterations and fetal overgrowth. The aim of this work was to evaluate the effect of the mitochondrial antioxidant Idebenone given to F0 mild pregestational diabetic rats on the development of GDM in their F1 offspring and the intergenerational programming of a pro-oxidant/proinflammatory environment that affects the placentas of F2 fetuses. Control and mild pregestational diabetic female rats (F0) were mated with control males, and Idebenone or vehicle was administered to diabetic rats from day 1 of gestation to term. The F1 female offspring were mated with control males and maternal and fetal plasma samples were obtained for metabolic determinations at term. The F2 fetuses and placentas were weighed, and placental protein levels and peroxynitrite-induced damage (immunohistochemistry), mRNA levels (PCR), nitric oxide production (Griess reaction), and number of apoptotic cells (TUNEL) were evaluated. The F1 offspring of F0 diabetic rats (treated or not with Idebenone) developed GDM. The placentas of GDM rats showed a decrease in the mRNA levels of manganese superoxide dismutase and an increase in the production of nitric oxide, peroxynitrite-induced damage, and connective tissue growth factor levels, alterations that were prevented by the maternal Idebenone treatment in F0 rats. In conclusion, the maternal treatment with Idebenone in pregestational diabetic F0 rats ameliorates the pro-oxidant/proinflammatory environment that affects the placentas of F2 fetuses, although it does not prevent F1 rats from developing GDM.
Assuntos
Antioxidantes/farmacologia , Ubiquinona/análogos & derivados , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Gestacional/metabolismo , Feminino , Macrossomia Fetal/metabolismo , Macrossomia Fetal/fisiopatologia , Feto/metabolismo , Masculino , Óxido Nítrico/metabolismo , Placenta/metabolismo , Gravidez , Proteínas da Gravidez , Ratos , Ratos Wistar , Ubiquinona/farmacologiaRESUMO
Previously, we reported that the presence of multiple day 7 (D7) bovine embryos in the uterus induces systemic immune responses in circulating polymorphonuclear neutrophils (PMNs), but with unknown mechanism. Thus, this study aimed to investigate the direct impact of D7 bovine embryo on PMNs' immune responses in vitro and whether these PMNs can amplify and transfer embryo signals further to another PMN population. PMNs were directly stimulated by embryo culture media (ECM) or interferon tau (IFNT) (10 ng/ml) followed by evaluating mRNA expression by real-time PCR and phenotypic analysis by flow cytometry. To test whether PMNs can transfer embryo signals to a new PMN population, PMNs triggered by ECM or IFNT, were thoroughly washed and diluted to remove any media components, and again were incubated in fresh culture media for 3 h, from which culture supernatants were collected and used as PMN conditioned media (CM) to stimulate a new PMN population. Similar to ECM, IFNT directly stimulated expressions of IFNs (IFNA, IFNG), interferon-stimulated genes (ISGs; OAS1, ISG15, MX1), STAT1, TGFB and IL8, and downregulated TNFA in PMNs. Flow cytometrical analyses demonstrated that IFNT stimulated expressions of pregnancy-related phenotypic markers, CD16 and arginase-1 (ARG1), in PMNs. Most importantly, PMN CM induced ISGs and STAT1 mRNA in fresh PMNs. Since IFNT directly upregulated IFNA expression in PMNs, the impact of IFNA on PMNs' immune responses was further tested. Stimulation of PMNs with IFNA, especially at a low level (1 pg/ml), induced IFNT-like immune responses comparable to those induced by PMN CM. Together, these findings indicated that D7 bovine embryos induce direct anti-inflammatory responses with upregulation of ISGs expressions in PMNs mainly via IFNT. Additionally, PMNs can amplify and transfer embryo signals to a new PMN population in a cell-to-cell communication mechanism possibly mediated in part by IFNA. Such a novel immunological crosstalk might contribute to embryo tolerance and pregnancy establishment in cattle.
Assuntos
Embrião de Mamíferos/imunologia , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica , Interferon Tipo I/imunologia , Neutrófilos/imunologia , Proteínas da Gravidez/imunologia , Gravidez/genética , Gravidez/imunologia , Animais , Arginase/genética , Bovinos , Meios de Cultivo Condicionados/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade Inata , Técnicas In Vitro , Interferon Tipo I/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fenótipo , Proteínas da Gravidez/farmacologia , Receptores de IgG/genéticaRESUMO
The prion protein (PrP) misfolding to its infectious form is critical to the development of prion diseases, whereby various ligands are suggested to participate, such as copper and nucleic acids (NA). The PrP globular domain was shown to undergo NA-driven liquid-liquid phase separation (LLPS); this latter may precede pathological aggregation. Since Cu(II) is a physiological ligand of PrP, we argue whether it modulates phase separation altogether with nucleic acids. Using recombinant PrP, we investigate the effects of Cu(II) (at 6 M equivalents) and a previously described PrP-binding GC-rich DNA (equimolarly to protein) on PrP conformation, oligomerization, and phase transitions using a range of biophysical techniques. Raman spectroscopy data reveals the formation of the ternary complex. Microscopy suggests that phase separation is mainly driven by DNA, whereas Cu(II) has no influence. Our results show that DNA can be an adjuvant, leading to the structural conversion of PrP, even in the presence of an endogenous ligand, copper. These results provide new insights into the role of Cu(II) and NA on the phase separation, structural conversion, and aggregation of PrP, which are critical events leading to neurodegeneration.
Assuntos
Cobre/química , Oligonucleotídeos/química , Proteínas da Gravidez/química , Agregados Proteicos , Animais , Cátions Bivalentes , Clonagem Molecular , Cobre/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Camundongos , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The increasing detection of infections of Trypanosoma cruzi, the etiological agent of Chagas disease, in non-endemic regions beyond Latin America has risen to be a major public health issue. With an impact in the millions of people, current treatments rely on antiquated drugs that produce severe side effects and are considered nearly ineffective for the chronic phase. The minimal progress in the development of new drugs highlights the need for advances in basic research on crucial biochemical pathways in T. cruzi to identify new targets. Here, we report on the T. cruzi presenilin-like transmembrane aspartyl enzyme, a protease of the aspartic class in a unique phylogenetic subgroup with T. vivax separate from protozoans. Computational analyses suggest it contains nine transmembrane domains and an active site with the characteristic PALP motif of the A22 family. Multiple linear B-cell epitopes were identified by SPOT-synthesis analysis with Chagasic patient sera. Two were chosen to generate rabbit antisera, whose signal was primarily localized to the flagellar pocket, intracellular vesicles, and endoplasmic reticulum in parasites by whole-cell immunofluorescence. The results suggest that the parasitic presenilin-like enzyme could have a role in the secretory pathway and serve as a target for the generation of new therapeutics specific to the T. cruzi.
Assuntos
Ácido Aspártico Proteases/metabolismo , Membrana Celular/metabolismo , Proteínas da Gravidez/metabolismo , Presenilinas/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Ácido Aspártico Proteases/análise , Ácido Aspártico Proteases/genética , Membrana Celular/química , Membrana Celular/genética , Humanos , Filogenia , Proteínas da Gravidez/análise , Proteínas da Gravidez/genética , Presenilinas/análise , Presenilinas/genética , Proteínas de Protozoários/análise , Proteínas de Protozoários/genética , Coelhos , Análise de Sequência de Proteína , Trypanosoma cruzi/química , Trypanosoma cruzi/genéticaRESUMO
Interferon tau (IFNT) is the cytokine responsible for the maternal recognition of pregnancy in ruminants and plays a role modulating embryo-maternal communication in the oviduct inducing a local response from immune cells. We aimed to investigate IFNT production, reactive oxygen species, and oxidative stress under the influence of heat stress (HS) during different stages of bovine in vitro embryo production. HS was established when the temperature was gradually raised from 38.5°C to 40.5°C in laboratory incubator, sustained for 6 hr, and decreased back to 38.5°C. To address the HS effects on IFNT production, reactive oxygen species, and oxidative stress, ovaries from a slaughterhouse were used according to treatments: control group (38.5°C); oocytes matured under HS; oocytes fertilized under HS; zygotes cultured in the first day under HS; and cells submitted to HS at oocyte maturation, fertilization, and the first day of zygote culture. The HS negatively affected cleavage and blastocyst rates, in all HS groups. On Day 7, all HS-treated embryos showed decrease IFNT gene and protein expressions, whereas reactive oxygen species were increased in comparison to the control. In conclusion, the compromised early embryo development due to higher temperatures during in vitro oocyte maturation, fertilization, and/or zygote stage have diminished IFNT expression and increased reactive oxygen species in bovine.
Assuntos
Bovinos/embriologia , Desenvolvimento Embrionário/fisiologia , Resposta ao Choque Térmico/fisiologia , Oócitos/fisiologia , Estresse Oxidativo/fisiologia , Zigoto/fisiologia , Animais , Células Cultivadas , Embrião de Mamíferos , Feminino , Fertilização in vitro/veterinária , Transtornos de Estresse por Calor/embriologia , Transtornos de Estresse por Calor/metabolismo , Transtornos de Estresse por Calor/fisiopatologia , Temperatura Alta , Técnicas de Maturação in Vitro de Oócitos , Interferon Tipo I/metabolismo , Oócitos/citologia , Oogênese/fisiologia , Proteínas da Gravidez/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Zigoto/citologiaRESUMO
INTRODUCTION AND OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is multistage with heterogeneous outcomes. We studied the influence of insulin resistance (IR) on the hepatic transcriptome of early NAFLD stages, to understand disease development. MATERIALS AND METHODS: In this cross-sectional study, possible clinicopathological risk factors were compared between mild-NAFL (Nâ¯=â¯72) and non-alcoholic steatohepatitis (NASH; Nâ¯=â¯51) patients. Liver tissue-transcriptome difference was studied between a subset of 25 mild-NAFL and 20 NASH biopsies and validated on another subset of 12 mild-NAFL and 13 NASH biopsies, using RT-PCR. The relationship between IR driven gene expression changes with fibrosis in NASH was investigated. RESULTS: Significantly higher weight (pâ¯=â¯0.005) and elevated levels of HbA1c (pâ¯=â¯0.009), FBG (pâ¯=â¯0.03) and HOMA-IR (pâ¯=â¯0.009) were found in NASH patients. Five differentially expressed genes (DEGs, fold changeâ¯>â¯1.5) were identified in NASH-FABP4, FABP5L2, CD24, PRAP1, and SPP1. The DEGs were positively associated with disease severity and HOMA-IR, and were found to be efficient classifiers of mild-NAFL and NASH. Additional 1218 genes identified related to IR (IrCGs), which can classify NASH-with-fibrosis patients separately from mild-NAFL and NASH patients. IrCGs can promote intra-hepatic fat accumulation, dysregulation of the lipid metabolism, lipotoxicity, and activation of cell survival pathways including activation of cell proliferation and differentiation pathways. CONCLUSIONS: Hepatic expression of genes associated with insulin resistance may drive NAFLD development and progression.
Assuntos
Perfilação da Expressão Gênica , Resistência à Insulina/genética , Cirrose Hepática/genética , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Transcriptoma , Adulto , Glicemia/metabolismo , Antígeno CD24/genética , Antígeno CD24/metabolismo , Estudos Transversais , Progressão da Doença , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Hemoglobinas Glicadas/metabolismo , Humanos , Insulina/sangue , Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Osteopontina/genética , Osteopontina/metabolismo , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Índice de Gravidade de DoençaRESUMO
We aimed to evaluate the accuracy of Interferon-tau stimulated genes (ISG) abundance in peripheral blood polymorphonuclear cells (PMNs) on D20 after fixed-time artificial insemination (FTAI; D0) as a pregnancy diagnosis method against CL evaluation by Doppler ultrasonography and progesterone (P4) concentrations on D20, as well as Pregnancy Associated Glycoproteins (PAG) concentrations on D25. Additionally, we evaluated the potential of ISG abundance in PMNs as pregnancy loss predictors. Nelore heifers (n = 103) and cows (n = 144) underwent estrous synchronization and were artificially inseminated on D0. Pregnancy was diagnosed by B-mode ultrasonography on D30 and D70, and after the final diagnosis, females were classified in four groups: Pregnant; Non-pregnant; Functional CL on D20 but non-pregnant (CL-NP) and Pregnancy loss between D30 and D70 (PL). After determining cutoff values, the Sensitivity (SE), Specificity (SP), Positive Predictive Value (PPV), Negative Predictive Value (NPV) and Accuracy (ACC) were determined for each method. All methods were classified as significant (P < 0.05) predictors of pregnancy. Both ISG expression and PAG concentrations were greater (P < 0.05) in pregnant females than in non-pregnant and CL-NP females but did not differ (P > 0.05) from the PL group. ISG15 expression was greater (P < 0.05) in heifers than in cows, but this difference was not found in OAS1 expression and PAG concentrations. All the methods evaluated were proven to be adequate predictors of pregnancy, but greater accuracies were obtained through PAG concentrations and Doppler-US, due to the decreased number of false positive and false negative results.
Assuntos
Bovinos , Interferon Tipo I/farmacologia , Neutrófilos/efeitos dos fármacos , Proteínas da Gravidez/farmacologia , Testes de Gravidez/veterinária , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Neutrófilos/metabolismo , Paridade , Gravidez , Proteínas da Gravidez/sangue , Testes de Gravidez/métodos , Progesterona/sangue , Sensibilidade e Especificidade , Ultrassonografia DopplerRESUMO
Preimplantation Factor (PIF) is a novel fifteen amino acid linear peptide (MVRIKPGSANKPSDD), which has different biological functions in mammalian species e.g. its role in neuron restoration, pregnancy and related disorders, and also in autoimmune diseases. Since all clinical studies have shown that PIF has both local and systemic effects, it can be considered as an integrated therapy for the treatment of inflammation conditions, along with the prevention of advanced disease. The synthetic PIF (sPIF) analog is a good representative of native PIF action, and it regulates peripheral immune cells to achieve endurance without immune suppression - an effective agent in nonpregnant autoimmune models. This study provides information, from evidence-based studies so far about PIF's different functional aspects.
Assuntos
Proteínas da Gravidez , Animais , Doenças Autoimunes , Feminino , Humanos , Sistema Imunitário/imunologia , Camundongos , Peptídeos , Gravidez , Proteínas da Gravidez/imunologia , Proteínas da Gravidez/fisiologia , Técnicas de Reprodução AssistidaRESUMO
INTRODUCTION AND OBJECTIVES: Liver regeneration plays a valuable significance for hepatectomies, and is mainly attributed to hepatocyte proliferation. MicroRNA-125a-3p was reported to be highly associated with liver regeneration process. We studied the underlying mechanism of the functional role of miR-125a-3p in liver regeneration. MATERIALS AND METHODS: The miR-125a-3p mimics and inhibitor vector were constructed and transfected into primary human liver HL-7702 cells, the transfected cell viability was detected using cell counting kit-8 (CCK-8). Cell cycle distribution was analyzed by flow cytometry. With Targetscan and OUGene prediction, the potential targets of miR-125 were verified by real-time quantitative PCR (qPCR) and luciferase reporter assays in turn. The overexpression vector of proline-rich acidic protein 1 (PRAP1) was constructed and co-transfected with miR-125a-3p mimics into HL-7702 cells, detecting the changes of proliferative capacity and cell cycle distribution. Western blot and qPCR performed to analyze gene expressions. RESULTS: Overexpressed miR-125a-3p notably increased the hepatocyte viability at 48h, and decreased the number of G1 phase cells (p<0.05). However, miR-125a-3p inhibition suppressed the development of hepatocytes. PRAP1 was the target of miR-125a-3p. After co-transfection with PRAP1 vector, hepatocyte viability was decrease and the G1 phase cell number was increased (p<0.05). More importantly, overexpressed PRAP1 notably decreased the mRNA and protein levels of cyclin D1, cyclin-dependent kinase 2 (CDK2) and cell division cycle 25A (CDC25A). CONCLUSION: The elevated miR-125a-3p positively correlated with hepatocyte viability and cell cycle progression due to the modulation of PRAP1, and miR-125a-3p may contribute to improving liver regeneration.