RESUMO
The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.
Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/fisiologia , Catálise , Domínio Catalítico , Dioxigenases/genética , Humanos , Ácidos Cetoglutáricos/metabolismo , Processamento de Proteína Pós-Traducional/genética , RNA Mensageiro/genética , Espalhamento a Baixo Ângulo , Relação Estrutura-Atividade , Difração de Raios X/métodosRESUMO
Protein kinase CK2 is a highly pleiotropic protein kinase capable of phosphorylating hundreds of protein substrates. It is involved in numerous cellular functions, including cell viability, apoptosis, cell proliferation and survival, angiogenesis, or ER-stress response. As CK2 activity is found perturbed in many pathological states, including cancers, it becomes an attractive target for the pharma. A large number of low-mass ATP-competitive inhibitors have already been developed, the majority of them halogenated. We tested the binding of six series of halogenated heterocyclic ligands derived from the commercially available 4,5-dihalo-benzene-1,2-diamines. These ligand series were selected to enable the separation of the scaffold effect from the hydrophobic interactions attributed directly to the presence of halogen atoms. In silico molecular docking was initially applied to test the capability of each ligand for binding at the ATP-binding site of CK2. HPLC-derived ligand hydrophobicity data are compared with the binding affinity assessed by low-volume differential scanning fluorimetry (nanoDSF). We identified three promising ligand scaffolds, two of which have not yet been described as CK2 inhibitors but may lead to potent CK2 kinase inhibitors. The inhibitory activity against CK2α and toxicity against four reference cell lines have been determined for eight compounds identified as the most promising in nanoDSF assay.
Assuntos
Caseína Quinase II/química , Halogenação , Compostos Heterocíclicos/síntese química , Fenilenodiaminas/química , Trifosfato de Adenosina/química , Domínio Catalítico , Cromatografia Líquida de Alta Pressão/métodos , Fluorometria/métodos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Simulação de Acoplamento MolecularRESUMO
INTRODUCTION: Allergen-specific immunotherapy (AIT) is the core treatment in allergic rhinitis and asthma. Although widely used, some patients do not benefit from treatment and there is no efficacy objective marker. AIM: To define the profile of gene transcripts during the build-up phase of AIT and their comparison to the control group and then search for a viable efficacy marker in relation to patient symptoms. MATERIAL AND METHODS: AIT was administered in 22 patients allergic to grass pollen. Analysis of 15 selected transcript expression was performed in whole blood samples taken before AIT (sample A) and after reaching the maintenance dose (sample B). The control group included 25 healthy volunteers (sample C). The primary endpoint was Relative Quantification. The gene expression analysis was followed by clinical evaluation with the use of Allergy Control Score (ACS). RESULTS: Comparison between samples A and B of gene expression showed a significant increase in IFNG expression (p = 0.03). In relation to the control group, pretreatment samples from patients showed higher levels of AFAP1L1 (p = 0.006), COMMD8 (p = 0.001), PIK3CD (p = 0.027) and TWIST2 (p = 0.0003) in univariate analysis. A generalized linear regression model was built according to the Bayesian Information Criterion based on the IFNG, FCER1A and PCDHB10 expression pattern for prediction of the AIT outcome. The model showed a correlation in predicted and observed changes in ACS. CONCLUSIONS: There is a significant change in the expression of IFNG during the build-up phase of AIT. The authors propose an in vitro model of AIT efficacy prediction for further validation.
RESUMO
BACKGROUND: Hymenoptera venom allergy (HVA) is of great concern because of the possibility of anaphylaxis, which may be fatal. Venom immunotherapy (VIT) is the only disease-modifying treatment in HVA and, although efficient, its mechanism remains partially unknown. Gene expression analysis may be helpful for establishing a proper model of tolerance induction during the build-up phase of VIT. The present study aimed to analyze how the start of VIT changes the expression of 15 selected genes. METHODS: Forty-five patients starting VIT with a wasp venom allergy were enrolled. The diagnosis was established based on anaphylaxis history (third or fourth grade on the Mueller scale) and positive soluble immunoglobulin E and/or skin tests. Two blood collections were performed in the patient group: before and after 3 months of VIT. One sample was taken in the control group. Gene expression analysis was performed using a reverse transcriptase-polymerase chain reaction with microfluidic cards and normalized to the 18S housekeeping gene. RESULTS: Commd8 was the only gene that changed expression significantly after the start of VIT (p = 0.012). Its expression decreased towards the levels observed in the healthy controls. Twelve out of 15 genes (commd8, cldn1, cngb3, fads1, hes6, hla-drb5, htr3b, prlr, slc16a4, snx33, socs3 and twist2) revealed a significantly different expression compared to the healthy controls. CONCLUSIONS: The present study shows that commd8 changes significantly its expression during initial phase of VIT. This gene might be a candidate for VIT biomarker in future studies.
Assuntos
Biomarcadores/metabolismo , Dessensibilização Imunológica/métodos , Himenópteros/imunologia , Hipersensibilidade Imediata/terapia , Mordeduras e Picadas de Insetos/terapia , Venenos de Vespas/uso terapêutico , Vespas/imunologia , Adolescente , Adulto , Idoso , Animais , Estudos de Casos e Controles , Dessaturase de Ácido Graxo Delta-5 , Feminino , Perfilação da Expressão Gênica , Humanos , Himenópteros/patogenicidade , Hipersensibilidade Imediata/etiologia , Hipersensibilidade Imediata/metabolismo , Imunoglobulina E/imunologia , Mordeduras e Picadas de Insetos/complicações , Mordeduras e Picadas de Insetos/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Testes Cutâneos , Adulto JovemRESUMO
A series of chlorine-substituted benzotriazole derivatives, representing all possible substitution patterns of halogen atoms attached to the benzotriazole benzene ring, were synthetized as potential inhibitors of human protein kinase CK2. Basic ADME parameters for the free solutes (hydrophobicity, electronic properties) together with their binding affinity to the catalytic subunit of protein kinase CK2 were determined with reverse-phase HPLC, spectrophotometric titration, and Thermal Shift Assay Method, respectively. The analysis of position-dependent thermodynamic contribution of a chlorine atom attached to the benzotriazole ring confirmed the previous observation for brominated benzotriazoles, in which substitution at positions 5 and 6 with bromine was found crucial for ligand binding. In all tested halogenated benzotriazoles the replacement of Br with Cl decreases the hydrophobicity, while the electronic properties remain virtually unaffected. Supramolecular architecture identified in the just resolved crystal structures of three of the four possible dichloro-benzotriazoles shows how substitution distant from the triazole ring affects the pattern of intermolecular interactions. Summarizing, the benzotriazole benzene ring substitution pattern has been identified as the main driver of ligand binding, predominating the non-specific hydrophobic effect.
Assuntos
Caseína Quinase II/metabolismo , Triazóis/química , Triazóis/metabolismo , Caseína Quinase II/química , Domínio Catalítico , Cristalografia por Raios X , Humanos , Hidrocarbonetos Halogenados/síntese química , Hidrocarbonetos Halogenados/química , Hidrocarbonetos Halogenados/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Eletricidade Estática , Relação Estrutura-Atividade , Triazóis/síntese químicaRESUMO
A series of halogenated derivatives of natural flavonoids: baicalein and chrysin were designed and investigated as possible ligands for the catalytic subunit of tumor-associated human kinase CK2. Thermal shift assay method, in silico modeling, and high-performance liquid chromatography-derived hydrophobicity together with IC50 values determined in biochemical assay were used to explain the ligand affinity to the catalytic subunit of human protein kinase CK2. Obtained results revealed that substitution of baicalein and chrysin with halogen atom increases their binding affinity to hCK2α, and for 8-chlorochrysin the observed effect is even stronger than for the reference CK2 inhibitor-4,5,6,7-tetrabromo-1H-benzotriazole. The cytotoxic activities of the baicalein and chrysin derivatives in the in vitro model have been evaluated for MV4-11 (human biphenotypic B myelomonocytic leukemia), A549 (human lung adenocarcinoma), LoVo (human colon cancer), and MCF-7 (human breast cancer) as well as on the nontumorigenic human breast epithelial MCF-10A cell lines. Among the baicalein derivatives, the strongest cytotoxic effect was observed for 8-bromobaicalein, which exhibited the highest activity against breast cancer cell line MCF-7 (IC50 10 ± 3 µM). In the chrysin series, the strongest cytotoxic effect was observed for unsubstituted chrysin, which exhibited the highest activity against leukemic cell line MV4-11 (IC50 10 ± 4 µM).
Assuntos
Caseína Quinase II/antagonistas & inibidores , Flavanonas/química , Flavonoides/química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Caseína Quinase II/química , Caseína Quinase II/metabolismo , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Flavanonas/metabolismo , Flavanonas/farmacologia , Flavonoides/metabolismo , Flavonoides/farmacologia , Halogenação , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Inibidores de Proteínas Quinases/metabolismo , Relação Estrutura-AtividadeRESUMO
MicroRNAs are small non-coding molecules playing a significant regulatory role in several allergic diseases. However their role in tolerance induction remains unclear. The aim of this study was to determine the expression of selected microRNAs during the first three months of wasp venom immunotherapy (VIT). 5 adult patients with a history of severe systemic reactions after stinging by wasps and confirmed sensitization were included. Venous blood samples were collected before VIT, 24 hours after completing its initial phase and after 3 months of the maintenance therapy. A control group was comprised of 5 healthy individuals with no history of allergy. In the blood samples expression of 96 microRNAs was determined with the use of microfluidic cards. In a statistical analysis the expression was compared between the study groups as well as between the pre- and post-VIT samples. Significant differences were found between the patients with wasp venom allergy and the healthy controls in the expression of miR-601 and miR-1201 upregulated in allergic patients at every time point (p = 0.04; p = 0.015, respectively). During VIT profile of microRNA was changing with lower expression of 6 microRNAs (including miR-182, miR-342, miR-375) and higher of 11 microRNAs (including let-7d, miR-34b, miR-143). To conclude, VIT has led to some changes in the expression of microRNA associated with Th2-type inflammation and tolerance induction.
Assuntos
Mordeduras e Picadas/imunologia , Expressão Gênica/imunologia , Imunoterapia/métodos , MicroRNAs/genética , Venenos de Vespas/imunologia , Vespas/imunologia , Adulto , Animais , Dessensibilização Imunológica/métodos , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Venenos de Vespas/administração & dosagem , Adulto JovemRESUMO
PURPOSE: Central serous chorioretinopathy (CSCR) is a complex ocular entity that, in its chronic form, can lead to serious visual impairment and morphological damage to the retina. The aim of the current retrospective study was to evaluate the damage present after long-standing but resolved central serous chorioretinopathy and refer it to healthy individuals. Correlations between measurable factors-for example, duration of the disease, baseline retinal morphological parameters, or patient age and/or their degree of impairment-were also assessed. MATERIALS AND METHODS: The study group consisted of thirty-two eyes (13 female and 19 male, mean age 49.6 years SD +/- 10.5) with chronic central serous chorioretinopathy (mean duration 18.9 months SD +/- 15.4) in which complete resolution of subretinal fluid was achieved after subthreshold micropulse laser treatment. Inclusion criterion was a lack of subretinal fluid within the whole area of the central retina scanned by the spectral domain optical coherence tomography. The group was extracted out of 51 cases of chronic CSCR that were treated with that method. They were analyzed according to final best-corrected visual acuity and retinal morphological parameters as measured by spectral optical coherence tomography with angiography option (OCTA). Results were compared with the outcomes of a control group, which consisted of 40 eyes of healthy individuals with full distance visual acuity (0.0 logMAR, 1.0 Snellen) never treated with subthreshold micropulse laser. Statistical analysis included regarding correlation between final visual acuity and final central retinal thickness and retinal and functional parameters prior to treatment. RESULTS: Final best-corrected visual acuity after chronic central serous chorioretinopathy was 0.23 logMAR (0.6 Snellen) and central retinal thickness was 39.32 µm smaller than in controls. No correlation was found between final visual acuity and retinal thickness and duration of the disease, patient age, and baseline morphological retinal parameters. OCTA scans revealed impaired choriocapillaries flow signal even following resolution of the disease. CONCLUSION: Chronic central serous chorioretinopathy is a potentially damaging clinical entity that results in serious visual impairment, retinal thinning, and choroidal flow defects. Further research is needed to determine precisely the timepoint of this damage.
Assuntos
Coriorretinopatia Serosa Central/fisiopatologia , Retina/patologia , Transtornos da Visão/etiologia , Acuidade Visual/fisiologia , Coriorretinopatia Serosa Central/complicações , Coriorretinopatia Serosa Central/diagnóstico , Corioide/patologia , Doença Crônica , Feminino , Angiofluoresceinografia , Seguimentos , Fundo de Olho , Humanos , Masculino , Pessoa de Meia-Idade , Vasos Retinianos/diagnóstico por imagem , Estudos Retrospectivos , Tomografia de Coerência Óptica , Transtornos da Visão/diagnóstico , Transtornos da Visão/fisiopatologiaRESUMO
1,N6-α-hydroxypropanoadenine (HPA) is an exocyclic DNA adduct of acrolein - an environmental pollutant and endocellular oxidative stress product. Escherichia coli AlkB dioxygenase belongs to the superfamily of α-ketoglutarate (αKG)- and iron-dependent dioxygenases which remove alkyl lesions from bases via an oxidative mechanism, thereby restoring native DNA structure. Here, we provide in vivo and in vitro evidence that HPA is mutagenic and is effectively repaired by AlkB dioxygenase. HPA generated in plasmid DNA caused A â C and A â T transversions and, less frequently, A â G transitions. The lesion was efficiently repaired by purified AlkB protein; the optimal pH, Fe(II), and αKG concentrations for this reaction were determined. In vitro kinetic data show that the protonated form of HPA is preferentially repaired by AlkB, albeit the reaction is stereoselective. Moreover, the number of reaction cycles carried out by an AlkB molecule remains limited. Molecular modeling of the T(HPA)T/AlkB complex demonstrated that the R stereoisomer in the equatorial conformation of the HPA hydroxyl group is strongly preferred, while the S stereoisomer seems to be susceptible to AlkB-directed oxidative hydroxylation only when HPA adopts the syn conformation around the glycosidic bond. In addition to the biochemical activity assays, substrate binding to the protein was monitored by differential scanning fluorimetry allowing identification of the active protein form, with cofactor and cosubstrate bound, and monitoring of substrate binding. In contrast FTO, a human AlkB homolog, failed to bind an ssDNA trimer carrying HPA.
Assuntos
Adenina/análogos & derivados , Enzimas AlkB/metabolismo , Carcinógenos Ambientais/metabolismo , Adutos de DNA/metabolismo , Reparo do DNA , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Mutagênicos/metabolismo , Adenina/química , Adenina/metabolismo , Adenina/toxicidade , Enzimas AlkB/química , Enzimas AlkB/genética , Sítios de Ligação , Biocatálise , Carcinógenos Ambientais/química , Carcinógenos Ambientais/toxicidade , Adutos de DNA/química , Adutos de DNA/toxicidade , DNA Bacteriano/química , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/metabolismo , Estabilidade Enzimática , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Hidroxilação , Conformação Molecular , Simulação de Dinâmica Molecular , Mutagênese/efeitos dos fármacos , Mutagênicos/química , Mutagênicos/toxicidade , Oxirredução , Conformação Proteica , Teoria Quântica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Especificidade por SubstratoRESUMO
Gestational diabetes mellitus (GDM) is a glucose intolerance that occurs during pregnancy. Several studies suggest that inflammation contributes to pregnancy-induced insulin resistance and the development of glucose intolerance. The aim of this study is to examine the association between the CCL2, CCL5, IL4 and IL15 gene polymorphisms and the development of GDM. This study included 411 pregnant women who underwent a 75 g oral glucose tolerance test at 24-28 weeks of gestation. Participants were categorised into 2 groups according to results of the oral glucose tolerance test (OGTT). The GDM group included 204 pregnant women who were diagnosed with GDM. The normal glucose tolerance group included 207 pregnant women with normal values in the OGTT. To discriminate the CCL2 rs1024611 and rs4586, CCL5 rs2107538, IL4 rs2243250, IL15 rs2857261 and rs2254514 alleles, TaqMan® Pre-Designed SNP Genotyping Assays were used. GDM was significantly associated with genotypes and alleles of the CCL2 rs1024611 and rs4586 polymorphisms, while there was no statistically significant association between the CCL5 rs2107538, IL4 rs2243250, IL15 rs2857261, and rs2254514 gene polymorphisms and GDM. In a multivariate regression analysis, age and BMI before pregnancy were independent significant predictors of a higher risk of GDM, while a lower number of G alleles CCL2 rs1024611 was protective against GDM. Moreover, women with the GG CCL2 rs1024611 and CC rs4586 genotype tended to have lower body mass and BMI increases during pregnancy, as well as lower newborn body mass. The results of our study suggest an association between CCL2 gene polymorphisms and GDM.
Assuntos
Quimiocina CCL2/genética , Quimiocina CCL5/genética , Diabetes Gestacional/genética , Interleucina-15/genética , Interleucina-4/genética , Polimorfismo de Nucleotídeo Único , Peso ao Nascer/genética , Índice de Massa Corporal , Estudos de Casos e Controles , Diabetes Gestacional/sangue , Feminino , Genótipo , Teste de Tolerância a Glucose , Humanos , Recém-Nascido , Obesidade/genética , Gravidez , Complicações na Gravidez/genética , Aumento de Peso/genéticaRESUMO
BACKGROUND: Chronic central serous chorioretinopathy is a serious therapeutic problem as it may lead to significant visual impairment. The aim of this retrospective study is to evaluate functional and morphological effects, as well as factors influencing visual outcome in patients treated by subthreshold micropulse laser. METHODS: Fifty-one eyes with chronic central serous chorioretinopathy lasting longer than 4 months (18 months on average) underwent up to two sessions of subtreshold micropulse laser treatment. Change in best corrected visual acuity, central retinal thickness, central retinal volume, average central retinal thickness and maximum subretinal fluid height were measured. Relation between the effects of the treatment and the initial retinal morphological and functional parameters was then analyzed. RESULTS: The total resorption of subretinal fluid was achieved in 36 cases (70.6%). There was, however, only a minor improvement in best corrected visual acuity of approximately one line on the Snellen chart. No correlation was observed between the effects of the treatment and the duration of the symptoms, retinal morphology and initial visual acuity. Younger patients responded better to the therapy. CONCLUSION: Subthreshold micropulse laser treatment in chronic serous chorioretinopathy provides good morphological results, however significant improvement of visual acuity is not to be expected.
Assuntos
Coriorretinopatia Serosa Central/cirurgia , Fotocoagulação a Laser/métodos , Acuidade Visual/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Coriorretinopatia Serosa Central/diagnóstico , Coriorretinopatia Serosa Central/fisiopatologia , Doença Crônica , Feminino , Angiofluoresceinografia , Seguimentos , Fundo de Olho , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de Tempo , Tomografia de Coerência Óptica , Resultado do TratamentoRESUMO
PURPOSE: Gestational diabetes mellitus (GDM) is the glucose intolerance occurring during pregnancy. The prevalence of GDM is increased in obese women. Leptin and adiponectin are adipokines that play an important role in the regulation of insulin secretion and glucose and lipid metabolism. The aim of this study was to examine the association between adiponectin and leptin gene polymorphisms and the development of GDM. METHODS: This case-control study included 204 pregnant women with GDM and 207 pregnant women with normal glucose tolerance (NGT). The diagnosis of GDM was based on a 75-g oral glucose tolerance test (OGTT) at 24-28 weeks' gestation. To discriminate the ADIPOQ rs266729, rs1501299 and LEP rs2167270 alleles, TaqMan® Pre-Designed SNP Genotyping Assays were used. RESULTS: There was a statistically significant association between the ADIPOQ rs266729 gene polymorphism and GDM. Among women with GDM, a higher prevalence of the G allele was observed (GG and CG genotypes). Multivariate logistic regression analysis, taking into account age, BMI before pregnancy, past pregnancies and the ADIPOQ rs266729 gene polymorphism, revealed that the presence of a G allele is an independent risk factor for GDM. Moreover, there was the association between the LEP rs2167270 polymorphism and the requirement for daily insulin, which was significantly higher in women with the A allele (AA and GA genotypes). CONCLUSIONS: The results of our study suggest an association between adiponectin gene rs266729 as well as leptin gene rs2167270 polymorphisms and GDM.
Assuntos
Adiponectina/genética , Diabetes Gestacional/genética , Estudos de Associação Genética , Leptina/genética , Adulto , Alelos , Diabetes Gestacional/patologia , Feminino , Predisposição Genética para Doença , Genótipo , Teste de Tolerância a Glucose , Humanos , Insulina/genética , Polimorfismo de Nucleotídeo Único , GravidezRESUMO
BACKGROUND: Allergen-specific immunotherapy is the most effective method of treatment in allergy to wasp venom. However, its mechanism of action is still not fully understood. The aim of this study is to describe changes in microRNA (miRNA) expression in patients undergoing the buildup phase of venom immunotherapy. METHODS: The study group comprised 7 adult patients with a history of severe systemic reactions after stinging by a wasp. In all patients, sensitization to wasp venom had been confirmed by skin tests and serum IgE. The buildup phase of wasp venom immunotherapy (VIT) was conducted according to an ultrarush protocol. In blood samples collected before and 24 h after completing the VIT buildup phase, 740 miRNAs were assessed. RESULTS: Of the 740 miRNAs, 440 were detected in the study group, and in 5 expression was significantly changed after the buildup phase of VIT: miR-370, miR-539, miR-502-3p, miR-299, and miR-29c. Another 62 miRNAs changed 2-fold in some patients (nonsignificant), including increases in miR-143 (stimulating FOXp3 expression) and let-7d (reducing expression of IL-13, IL-6, and TLR4), and decreases in proinflammatory miR-301, miR-146b, miR-106, and miR-485. CONCLUSIONS: Several changes in miRNA expression have been found as a result of the buildup phase of wasp VIT, with lower expression of some miRNAs involved in allergic inflammation and higher expression of those possibly involved in tolerance induction. However, the role of the most significant changes is uncertain.
Assuntos
Dessensibilização Imunológica , Regulação da Expressão Gênica , Mordeduras e Picadas de Insetos/genética , Mordeduras e Picadas de Insetos/terapia , MicroRNAs/genética , Venenos de Vespas/administração & dosagem , Vespas , Adulto , Idoso , Animais , Feminino , Perfilação da Expressão Gênica , Humanos , Tolerância Imunológica , Masculino , Pessoa de Meia-Idade , Projetos PilotoRESUMO
Efficient repair by Escherichia coli AlkB dioxygenase of exocyclic DNA adducts 3,N(4)-ethenocytosine, 1,N(6)-ethenoadenine, 3,N(4)-α-hydroxyethanocytosine, and reported here for the first time 3,N(4)-α-hydroxypropanocytosine requires higher Fe(II) concentration than the reference 3-methylcytosine. The pH optimum for the repair follows the order of pK(a) values for protonation of the adduct, suggesting that positively charged substrates favorably interact with the negatively charged carboxylic group of Asp-135 side chain in the enzyme active center. This interaction is supported by molecular modeling, indicating that 1,N(6)-ethenoadenine and 3,N(4)-ethenocytosine are bound to AlkB more favorably in their protonated cationic forms. An analysis of the pattern of intermolecular interactions that stabilize the location of the ligand points to a role of Asp-135 in recognition of the adduct in its protonated form. Moreover, ab initio calculations also underline the role of substrate protonation in lowering the free energy barrier of the transition state of epoxidation of the etheno adducts studied. The observed time courses of repair of mixtures of stereoisomers of 3,N(4)-α-hydroxyethanocytosine or 3,N(4)-α-hydroxypropanocytosine are unequivocally two-exponential curves, indicating that the respective isomers are repaired by AlkB with different efficiencies. Molecular modeling of these adducts bound by AlkB allowed evaluation of the participation of their possible conformational states in the enzymatic reaction.
Assuntos
Proteínas de Escherichia coli/fisiologia , Oxigenases de Função Mista/fisiologia , Domínio Catalítico , DNA/química , Adutos de DNA/química , Reparo do DNA , Relação Dose-Resposta a Droga , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Concentração de Íons de Hidrogênio , Ferro/química , Cinética , Ligantes , Peroxidação de Lipídeos , Oxigenases de Função Mista/química , Modelos Químicos , Conformação Molecular , Estresse Oxidativo , Ligação Proteica , Prótons , EstereoisomerismoRESUMO
Degraded and low template DNA is often analyzed in forensic genetics laboratories. Reliable analysis of degraded and low template DNA is of great importance, because its results impact the quality and reliability of expert testimonies. Recently, a number of whole-genome amplification (WGA) methods have been proposed as preamplification tools improving quantity and quality of DNA. We chose, investigated, and compared 7 WGA methods to evaluate their ability to "recover" degraded and nondegraded DNA. These methods include degenerate oligonucleotide primed polymerase chain reaction, primer extension preamplification (PEP) polymerase chain reaction, GenomePlex WGA (Sigma), multiple displacement amplification, GenomiPhi Amplification Kit (Amersham Biosciences), restriction and circularization aided rolling circle amplification, and blunt-end ligation-mediated WGA. Recently, we have described the comparison of these methods' efficiency and reliability using SGMPlus kit. However, Y-chromosome profiling is also often used in analysis of both nondegraded and degraded DNA. This includes criminal cases and investigation of kinship in male linage. Here we demonstrate the impact of WGA methods on Y-chromosome loci (Yfiler) reactivation.The best results for nondegraded DNA were obtained with GenomiPhi kit and PEP method. In the case of degraded DNA (200 base pairs), the most complete profiles were obtained with GenomePlex kit and PEP method. None of the analyzed methods allowed full reactivation of degraded (200 base pairs) DNA in terms of Y-chromosome loci profiling.
Assuntos
Cromossomos Humanos Y , Genoma Humano , Técnicas de Amplificação de Ácido Nucleico/métodos , DNA/isolamento & purificação , Degradação Necrótica do DNA , Impressões Digitais de DNA/métodos , Loci Gênicos , Humanos , Masculino , Músculo Esquelético/químicaRESUMO
Degraded DNA is often analyzed in forensic genetics laboratories. Reliable analysis of degraded DNA is of great importance, since its results impact the quality and reliability of expert testimonies. Recently, a number of whole genome amplification (WGA) methods have been proposed as preamplification tools. They work on the premise of being able to generate microgram quantities of DNA from as little as the quantity of DNA from a single cell. We chose, investigated, and compared seven WGA methods to evaluate their ability to "recover" degraded and nondegraded DNA: degenerate oligonucleotide-primed PCR, primer extension preamplification PCR, GenomePlex™ WGA commercial kit (Sigma), multiple displacement amplification, GenomiPhi™ Amplification kit (Amersham Biosciences), restriction and circularization-aided rolling circle amplification, and blunt-end ligation-mediated WGA. The efficiency and reliability of those methods were analyzed and compared using SGMPlus, YFiler, mtDNA, and Y-chromosome SNP typing. The best results for nondegraded DNA were obtained with GenomiPhi and PEP methods. In the case of degraded DNA (200 bp), the best results were obtained with GenomePlex which successfully amplified also severely degraded DNA (100 bp), thus enabling correct typing of mtDNA and Y-SNP loci. WGA may be very useful in analysis of low copy number DNA or degraded DNA in forensic genetics, especially after introduction of some improvements (sample pooling and replicate DNA typing).
Assuntos
Degradação Necrótica do DNA , Genoma Humano , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Alelos , Cromossomos Humanos Y , Impressões Digitais de DNA , Primers do DNA , DNA Mitocondrial/genética , Genética Forense , Humanos , Masculino , Repetições de Microssatélites , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , Análise de Sequência de DNARESUMO
There is a necessity for deceased identification as a result of many accidents and sometimes bones are the only accessible source of DNA. So far, a universal method that allows for extraction of DNA from materials at different stages of degradation does not exist. The aims of this study were: the comparison of three methods of DNA extraction from bones with different degree of degradation and an evaluation of the usefulness of these methods in forensic genetics. The efficiency of DNA extraction, the degree of extract contamination by polymerase chain reaction (PCR) inhibitors and the possibility of determining the STR loci profile were especially being compared. Nuclear DNA from bones at different states of degradation was isolated using three methods: classical, organic phenol-chloroform extraction, DNA extraction from crystal aggregates and extraction by total demineralisation. Total demineralisation is the best method for most cases of DNA extraction from bones, although it does not provide pure DNA. DNA extraction from aggregates removes inhibitors much better and is also a good method of choice when identity determination of exhumed remains is necessary. In the case of not buried bones (remains found outside) total demineralisation or phenol-chloroform protocols are more efficient for successful DNA extraction.
Assuntos
Osso e Ossos/química , DNA/isolamento & purificação , Antropologia Forense/métodos , Genética Forense/métodos , Técnica de Descalcificação , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase Multiplex , Reação em Cadeia da Polimerase , Fatores de TempoRESUMO
Peroxysome proliferator-activated receptor gamma coactivator-1-alpha (PGC-1α; encoded by the gene PPARGC1A in humans) is a crucial component in training-induced muscle adaptation because it is a co-activator of transcriptional factors that control gene expression in coordinated response to exercise. It has been suggested that a Gly482Ser substitution in PPARGC1A has functional relevance in the context of human disorders and athletic performance. To test this hypothesis, we examined the genotype distribution of PPARGC1A Gly482Ser in a group of Polish athletes and confirmed the results obtained in a replication study of Russian athletes. We found that the 482Ser allele was under-represented in the cohort of Polish and Russian athletes examined compared with unfit controls (P < 0.0001). A statistically significant low frequency of the 482Ser allele was observed among the endurance,strength-endurance, and sprint-strength groups of Polish athletes (P = 0.019, P = 0.022, and P < 0.0001, respectively). The replication study revealed that the 482Ser allele was also less prevalent in Russian endurance and strength-endurance athletes (P = 0.029 and P < 0.0001, respectively). Our results suggest that the PPARGC1A Gly482Ser polymorphism is associated with elite endurance athletic status. These findings support the hypothesis that the PPARGC1A 482Ser allele may impair aerobic capacity: thus, the Gly482 allele may be considered a beneficial factor for endurance performance.
Assuntos
Atletas , Proteínas de Choque Térmico/genética , Resistência Física/genética , Fatores de Transcrição/genética , Adulto , Desempenho Atlético , Estudos de Coortes , Feminino , Humanos , Masculino , Força Muscular/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Polônia , Polimorfismo Genético , Federação Russa , População Branca/genética , Adulto JovemRESUMO
The aims of this study were to determine the distribution of the AMPD1 genotype among groups of high-level Polish power-oriented athletes, and to investigate potential associations between genetic polymorphism in exon 2 of the AMPD1 gene and power-oriented athlete status. Altogether, 158 male Polish power-oriented athletes were genotyped by PCR-RFLP. The genetic control group comprised 160 unrelated male volunteers. We observed significant differences in genotype distribution when all 158 athletes (89.25% CC, 10.75% CT, 0.00% TT; P = 0.0025) were compared with controls (75.00% CC, 23.75% CT, 1.25% TT). A significant deficiency of the T allele was noted in all subgroups (short-distance runners: 5.21%, P = 0.032; short-distance swimmers: 5.56%, P = 0.031; weightlifters: 5.36%, P = 0.024) compared with controls (13.13%), while this trend was even stronger when the frequency of the T allele was compared between controls and all 158 athletes (5.38%, P = 0.0007). Our results indicate a lower frequency of the AMPD1 exon 2 T34 allele in elite Polish power-oriented athletes. Our data suggest that the C allele may help athletes to attain elite status in power-oriented sports.
Assuntos
AMP Desaminase/genética , Atletas , Polimorfismo Genético , Adulto , Éxons , Frequência do Gene , Humanos , Masculino , Polônia , Corrida , Natação , Levantamento de Peso , População Branca/genética , Adulto JovemRESUMO
The transcription factor hypoxia-inducible factor 1 (HIF1) is one of the key regulators of cellular metabolism. The aim of this study was to analyze the possible importance of the HIF1A Pro582Ser polymorphisms in rowing. One hundred twenty-seven male Polish rowers (both former and current competitors) were recruited for this study. Genotyping was carried out by polymerase chain reaction. Significance was assessed by Chi-square (χ(2)) analysis. The results obtained revealed that frequency of the HIF1A Pro/Ser genotype (32.28 vs. 18.91%; p = 0.006) and Ser allele (16.93 vs. 10.00%; p = 0.01) were significantly higher in the rowers compared with those in controls. The results obtained confirm the significance of the HIF1A gene as a useful genetic marker in rowing. This kind of information would presumably be applicable in a program to search for the most predisposed individuals and also in the planning of training programs.