RESUMO
The Schistosoma mansoni SmKI-1 protein is composed of two domains: a Kunitz-type serine protease inhibitor motif (KD) and a C-terminus domain with no similarity outside the genera. Our previous work has demonstrated that KD plays an essential role in neutrophil elastase (NE) binding blockage, in neutrophil influx and as a potential anti-inflammatory molecule. In order to enhance NE blocking capacity, we analyzed the KD sequence from a structure-function point of view and designed specific point mutations in order to enhance NE affinity. We substituted the P1 site residue at the reactive site for a leucine (termed RL-KD), given its central role for KD's inhibition to NE. We have also substituted a glutamic acid that strongly interacts with the P1 residue for an alanine, to help KD to be buried on NE S1 site (termed EA-KD). KD and the mutant proteins were evaluated in silico by molecular docking to human NE, expressed in Escherichia coli and tested towards its NE inhibitory activity. Both mutated proteins presented enhanced NE inhibitory activity in vitro and RL-KD presented the best performance. We further tested RL-KD in vivo in an experimental model of monosodium urate (MSU)-induced acute arthritis. RL-KD showed reduced numbers of total cells and neutrophils in the mouse knee cavity when compared to KD. Nevertheless, both RL-KD and KD reduced mice hypernociception in a similar fashion. In summary, our results demonstrated that both mutated proteins showed enhanced NE inhibitory activity in vitro. However, RL-KD had a prominent effect in diminishing inflammatory parameters in vivo.
Assuntos
Leucina/efeitos dos fármacos , Leucina/genética , Mutação Puntual , Proteínas Secretadas Inibidoras de Proteinases/química , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Animais , Artrite , Leucina/química , Leucina/metabolismo , Elastase de Leucócito/efeitos dos fármacos , Camundongos , Simulação de Acoplamento Molecular , Neutrófilos , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Proteínas Recombinantes , Relação Estrutura-Atividade , Especificidade por Substrato , Receptor 4 Toll-Like/genética , TranscriptomaRESUMO
The AndesOrthohantavirus (ANDV), which causes the hantavirus cardiopulmonary syndrome, enters cells via integrins, and a change from leucine to proline at residue 33 in the PSI domain (L33P), impairs ANDV recognition. We assessed the association between this human polymorphism and ANDV infection. We defined susceptible and protective genotypes as "TT" (coding leucine) and "CC" (coding proline), respectively. TT was present at a rate of 89.2% (66/74) among the first cohort of ANDV cases and at 60% (63/105) among exposed close-household contacts, who remained uninfected (p < 0.05). The protective genotype (CC) was absent in all 85 ANDV cases, in both cohorts, and was present at 11.4% of the exposed close-household contacts who remained uninfected. Logistic regression modeling for risk of infection had an OR of 6.2â»12.6 (p < 0.05) in the presence of TT and well-known ANDV risk activities. Moreover, an OR of 7.3 was obtained when the TT condition was analyzed for two groups exposed to the same environmental risk. Host genetic background was found to have an important role in ANDV infection susceptibility, in the studied population.
Assuntos
Predisposição Genética para Doença , Infecções por Hantavirus/genética , Integrina alfaVbeta3/genética , Orthohantavírus , Polimorfismo de Nucleotídeo Único , Adulto , Características da Família , Feminino , Genótipo , Humanos , Leucina/genética , Masculino , Prolina/genética , Estudos Prospectivos , Medição de Risco , Fatores de RiscoRESUMO
The Na+/iodide (I-) symporter (NIS), a glycoprotein expressed at the basolateral plasma membrane of thyroid follicular cells, mediates I- accumulation for thyroid hormonogenesis and radioiodide therapy for differentiated thyroid carcinoma. However, differentiated thyroid tumors often exhibit lower I- transport than normal thyroid tissue (or even undetectable I- transport). Paradoxically, the majority of differentiated thyroid cancers show intracellular NIS expression, suggesting abnormal targeting to the plasma membrane. Therefore, a thorough understanding of the mechanisms that regulate NIS plasma membrane transport would have multiple implications for radioiodide therapy. In this study, we show that the intracellularly facing carboxy-terminus of NIS is required for the transport of the protein to the plasma membrane. Moreover, the carboxy-terminus contains dominant basolateral information. Using internal deletions and site-directed mutagenesis at the carboxy-terminus, we identified a highly conserved monoleucine-based sorting motif that determines NIS basolateral expression. Furthermore, in clathrin adaptor protein (AP)-1B-deficient cells, NIS sorting to the basolateral plasma membrane is compromised, causing the protein to also be expressed at the apical plasma membrane. Computer simulations suggest that the AP-1B subunit σ1 recognizes the monoleucine-based sorting motif in NIS carboxy-terminus. Although the mechanisms by which NIS is intracellularly retained in thyroid cancer remain elusive, our findings may open up avenues for identifying molecular targets that can be used to treat radioiodide-refractory thyroid tumors that express NIS intracellularly.
Assuntos
Membrana Celular/metabolismo , Simportadores/química , Simportadores/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Transporte Biológico , Membrana Celular/genética , Humanos , Iodetos/metabolismo , Leucina/genética , Leucina/metabolismo , Transporte Proteico , Ratos , Alinhamento de Sequência , Simportadores/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismoRESUMO
We report a new transthyretin (ATTR) gene c.272C>G mutation and variant protein, p.Leu32Val, in a kindred of Bolivian origin with a rapid progressive peripheral neuropathy and cardiomyopathy. Three individuals from a kindred with peripheral nerve and cardiac amyloidosis were examined. Analysis of the TTR gene was performed by Sanger direct sequencing. Neuropathologic examination was obtained on the index patient with mass spectrometry study of the ATTR deposition. Direct DNA sequence analysis of exons 2, 3, and 4 of the TTR gene demonstrated a c.272 C>G mutation in exon 2 (p.L32V). Sural nerve biopsy revealed massive amyloid deposition in the perineurium, endoneurium and vasa nervorum. Mass spectrometric analyses of ATTR immunoprecipitated from nerve biopsy showed the presence of both wild-type and variant proteins. The observed mass results for the wild-type and variant proteins were consistent with the predicted values calculated from the genetic analysis data. The ATTR L32V is associated with a severe course. This has implications for treatment of affected individuals and counseling of family members.
Assuntos
Neuropatias Amiloides Familiares/genética , Saúde da Família , Leucina/genética , Mutação/genética , Pré-Albumina/genética , Valina/genética , Neuropatias Amiloides Familiares/fisiopatologia , Bolívia , Análise Mutacional de DNA , Feminino , Humanos , Pessoa de Meia-Idade , Condução Nervosa/genética , Pré-Albumina/metabolismoRESUMO
The most common type of endocrine disease is type 2 diabetes mellitus (T2DM); genetic factors contribute to the development to T2DM. In this study, we investigated the role of the Leu53Leu, Arg213Gly, and Ala40Thr polymorphisms in extracellular superoxide dismutase (EC-SOD) gene in the development of T2DM in a Chinese population. DNA was extracted from peripheral blood samples obtained from 256 T2DM patients and 324 control subjects recruited from our hospital between January 2013 and March 2015. DNA was genotyped by polymerase chain reaction-restriction fragment length polymorphism. The obtained data was then statistically analyzed. The chi-square test revealed a statistically significant difference in the genotype frequencies of EC-SOD Ala40Thr (χ2 = 13.26, P = 0.001) between the patients and controls. Unconditional regression analysis indicated that the GA and AA genotypes of EC-SOD Ala40Thr were associated with an increased risk of T2DM compared to the GG genotype {adjusted odds ratio (OR) [95% confidence interval (CI)] = 1.46 (1.01-2.11) and 2.67 (1.48-4.85), respectively}. In the dominant model, the GA+AA genotype of EC-SOD Ala40Thr was correlated with a higher risk of T2DM, in comparison with the GG genotype (OR = 1.64, 95%CI = 1.16-2.33). In the recessive model, AA of EC-SOD Ala40Thr showed a 2.19-fold higher risk of developing T2DM than the GG+GA genotype. In conclusion, people with the Ala40Thr polymorphism in EC-SOD are at a higher risk of developing T2DM; therefore, this may be utilized as a biomarker for early screening of T2DM in a Chinese population.
Assuntos
Povo Asiático/genética , Diabetes Mellitus Tipo 2/genética , Polimorfismo de Nucleotídeo Único , Superóxido Dismutase/genética , Adulto , Alanina/genética , Arginina/genética , Feminino , Predisposição Genética para Doença , Genótipo , Glutamina/genética , Humanos , Leucina/genética , Masculino , Pessoa de Meia-Idade , Análise de Regressão , Treonina/genéticaRESUMO
Although the Val34Leu polymorphism in blood coagulation factor XIII-A (FXIII-A) has been implicated in the pathogenesis of intracerebral hemorrhage (ICH), the results of research conducted thus far have been inconclusive. In this meta-analysis, we have assessed the association between the FXIII-A Val34Leu polymorphism and ICH risk. Published reports pertaining to this association were retrieved from the PubMed database, and the data from these studies were pooled and statistically analyzed with Stata 12.0. Summary odds ratios (OR) and 95% confidence intervals (95%CI) were calculated according to a fixed-effect or a random-effect model (as appropriate). The initial search identified 520 articles, only seven of which (retrospective studies) met the inclusion criteria and were included in this meta-analysis. These studies comprised 727 ICH patients and 1968 controls. The results of a combined analysis showed no significant association between the FXIII-A Val34Leu polymorphism and ICH risk in the overall population (Leu/Leu vs Val/Val: OR = 1.41, 95%CI = 0.82-2.43; Val/Leu vs Val/Val: OR = 1.08, 95%CI = 0.89-1.30; dominant model: OR = 1.14, 95%CI = 0.95-1.36; recessive model: OR = 0.72, 95%CI = 0.43-1.22). The results of this meta-analysis suggest that the FXIII-A Val34Leu polymorphism is not associated with ICH risk in a Caucasian population. Further large and well-designed studies must be conducted to confirm this preliminary conclusion.
Assuntos
Hemorragia Cerebral/genética , Fator XIIIa/genética , Substituição de Aminoácidos , Predisposição Genética para Doença , Humanos , Leucina/genética , Polimorfismo de Nucleotídeo Único , Valina/genéticaRESUMO
INTRODUCTION: obesity affects more than a third of Mexican population. Oxidative stress participates actively in the etiology of this phenomenon. Glutathione peroxidase-1 (GPX-1) plays a protective role against oxidative stress. The SNP Pro200Leu (rs10504050) has been reported to affect the activity of the enzyme. OBJECTIVE: to determine the frequency of rs10504050 polymorphism in women with obesity and normal weight control, asses the concentration of peripheral TBARS and evaluate the consumption of pro and antioxidants. METHODS: 104 women with obesity and 70 healthy controls (CG) were included in the study. Anthropometric, biochemical, clinical and dietary features were evaluated. GPx-1 rs10504050 was determined by PCR/RFLP method. TBARS was assayed spectrophotometrically in plasma. The subjects were stratified and compared by obesity grades and by subgroups of prediabetes and diabetes condition. Statistical analysis included ANOVA of Kruskal Wallis, Xi squared and Pearson correlation. RESULTS: for rs10504050 polymorphism there were differences (Xi2 = 6; p = 0.01) between frequency (0.61) of obese carriers (Pro/Leu plus Leu/Leu) and CG carriers (0.42), and between (Xi2 = 8; p = 0.004) morbid (IMC > 40) obesity (0.74) and CG carriers. The obese group (OB) showed a prevalence of 66% of prediabetes plus diabetes. There were no differences in frequencies of rs10504050 in OB with pre or diabetes versus CG, or versus obese participants without diabetes. TBARS concentration was greater in all the degrees of OB versus CG. CONCLUSION: GPx-1 Pro200Leu polymorphism was associated with obesity especially with morbid obesity, but not with obese participants with prediabetes or diabetes. Oxidative stress is present in all grades of obesity significantly.
Introducción: la obesidad afecta a una tercera parte de la población mexicana. El estrés oxidativo (EO) participa activamente en la etiología del fenómeno. La glutatión peroxidasa-1 (GPx-1) juega un papel protector contra el EO. El SNP Pro200Leu (rs10504050) afecta a la actividad de la enzima. Objetivo: determinar la frecuencia del polimorfismo rs10504050 en mujeres con obesidad (OB) y normopeso (CG), determinar la concentración de TBARS en sangre periférica y evaluar el consumo de pro y antioxidantes. Métodos: en el estudio se incluyeron 104 mujeres con obesidad y 70 controles. El polimorfismo rs10504050 se determinó por el método PCR/RFLP. La concentración de TBARS se cuantificó mediante espectrofotometría en plasma sanguíneo. Las participantes se estratificaron y compararon por grados de obesidad y subgrupos de prediabetes y diabetes. Se emplearon las pruebas estadísticas ANOVA de Kruskal Wallis, Xi cuadrada y correlación de Pearson. Resultados: el polimorfismo rs10504050 mostró diferencias estadísticas (Xi2 = 6; p = 0,01) entre la frecuencia del grupo OB (0,61) por arrastre (Pro/Leu+Leu/Leu) y el CG (0,42), así como (Xi2 = 8; p = 0,004) entre personas con obesidad mórbida (0,74) comparadas con el CG. No hubo diferencia significativa entre las frecuencias del rs10504050 en OB con pre o diabetes, comparado con el CG, ni con personas con obesidad sin diabetes. Las concentraciones de TBARS fueron mayores en todos los grados de OB comparados con el CG. Conclusión: el polimorfismo rs10504050 se asoció con obesidad, especialmente mórbida, pero no se asoció con diabetes o prediabetes. El estrés oxidativo está presente de manera significativa en todos los grados de obesidad.
Assuntos
Diabetes Mellitus/genética , Glutationa Peroxidase/genética , Obesidade Mórbida/genética , Polimorfismo Genético/genética , Estado Pré-Diabético/genética , Adulto , Diabetes Mellitus/epidemiologia , Feminino , Frequência do Gene , Heterozigoto , Humanos , Leucina/genética , México/epidemiologia , Pessoa de Meia-Idade , Obesidade Mórbida/epidemiologia , Estado Pré-Diabético/epidemiologia , Substâncias Reativas com Ácido Tiobarbitúrico/análise , Glutationa Peroxidase GPX1RESUMO
The mandible condylar process cartilage (CP) of Wistar rats is a secondary cartilage and acts as a mandibular growth site. This phenomenon depends on adequate proteins intake and hormone actions, including insulin. Objectives The present study evaluated the morphological aspects and the expression of the insulin receptor (IR) in the cartilage of the condylar process (CP) of rats subjected to protein undernourishment. Material and Methods The nourished group received a 20% casein diet, while the undernourished group (U) received a 5% casein diet. The re-nourished groups, R and RR, were used to assess the effects of re-nutrition during puberty and adulthood, respectively. CPs were processed and stained with picro-sirius red, safranin-O and azocarmine. Scanning electron microscopy and immunohistochemistry were also performed. Results The area of the CP cartilage and the number of cells in the chondroblastic layer decreased in the U group, as did the thickness of the CP layer in the joint and hypertrophic layer. Renourishment during the pubertal stage, but not during the adult phase, restored these parameters. The cell number was restored when re-nutrition occurred in the pubertal stage, but not in the adult phase. The extracellular matrix also decreased in the U group, but was restored by re-nutrition during the pubertal stage and further increased in the adult phase. IR expression was observed in all CPs, being higher in the chondroblastic and hypertrophic cartilage layers. The lowest expression was found in the U and RR groups. Conclusions Protein malnutrition altered the cellularity, the area, and the fibrous cartilage complex, as well as the expression of the IRs. .
Assuntos
Animais , Camundongos , Anti-Inflamatórios não Esteroides/metabolismo , Ciclo-Oxigenase 1/metabolismo , /metabolismo , Inibidores de Ciclo-Oxigenase/metabolismo , Piroxicam/análogos & derivados , Tiazinas/metabolismo , Tiazóis/metabolismo , Substituição de Aminoácidos , Anti-Inflamatórios não Esteroides/química , Arginina/química , Arginina/genética , Arginina/metabolismo , Sítios de Ligação , Domínio Catalítico , Ciclo-Oxigenase 1/química , Ciclo-Oxigenase 1/genética , /química , /genética , Inibidores de Ciclo-Oxigenase/química , Ligação de Hidrogênio , Leucina/química , Leucina/genética , Leucina/metabolismo , Mutação , Piroxicam/química , Piroxicam/metabolismo , Estrutura Secundária de Proteína , Serina/química , Serina/genética , Serina/metabolismo , Tiazinas/química , Tiazóis/química , Tirosina/química , Tirosina/genética , Tirosina/metabolismo , ÁguaRESUMO
Leucine (Leu) participates in the activity of cationic amino acid (aa) transporters. Also, branched-chain aa [Leu, isoleucine (Ile), and valine (Val)] share intestinal transporters for absorption. We conducted an experiment with 16 young pigs (body weight of about 16 kg) to determine whether Leu and Ile affect expression of aa transporters b(0,+) and CAT-1 in the jejunum and expression of myosin in muscle, as well as serum concentration of essential aa, and growth performance in pigs. Dietary treatments were: wheat-based diets fortified with Lys, Thr, and Met; basal diet plus 0.50% Leu; basal diet plus 0.50% Ile, and basal diet plus 0.50% Leu and 0.50% Ile. After 28 days, the pigs were sacrificed to collect blood, jejunum, and semitendinosus and longissimus muscle samples. The effects of single and combined addition of Leu and Ile were analyzed. Leu alone or combined with Ile significantly decreased daily weight gain and reduced feed conversion. Leu and Ile, alone or in combination, significantly decreased expression of b(0,+) and significantly increased CAT-1. Ile alone or combined with Leu significantly decreased myosin expression in semitendinosus and significantly decreased it in longissimus muscle. Leu alone significantly decreased Lys, Ile and Thr serum concentrations; Ile significantly decreased Thr serum concentration; combined Leu and Ile significantly decreased Thr and significantly increased Val serum concentration. We conclude that dietary levels of Leu and Ile affect growth performance, expression of aa transporters and myosin, and aa serum concentrations in pigs.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos/genética , Expressão Gênica/genética , Isoleucina/metabolismo , Leucina/metabolismo , Miosinas/genética , Suínos/fisiologia , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Aminoácidos/sangue , Aminoácidos/genética , Aminoácidos/metabolismo , Ração Animal , Animais , Transportador 1 de Aminoácidos Catiônicos/genética , Transportador 1 de Aminoácidos Catiônicos/metabolismo , Dieta , Suplementos Nutricionais , Isoleucina/genética , Jejuno/metabolismo , Leucina/genética , Músculos/metabolismo , Miosinas/metabolismo , Suínos/genética , Suínos/crescimento & desenvolvimento , Suínos/metabolismo , Valina/genética , Valina/metabolismo , Aumento de PesoRESUMO
Invariant chain (Ii) is a transmembrane protein that associates with MHC class II molecules in the endoplasmic reticulum. The cytoplasmic tail of Ii contains two leucine residues able to direct Ii to the endocytic pathway. We obtained the pig Ii gene by RT-PCR. Mutated Ii was prepared via site directed mutagenesis by the PCR Megaprimer method to study the effect of the two leucines on the localization of pig Ii. These mutated fragments were ligated to the vector pmCherry-C1. The recombinant plasmids were transiently transfected into COS-7 cells with Lipofectamine(TM) 2000. Fluorescence of fusion proteins (mCherry-Ii) was observed with a fluorescent microscope. Amino acid sequence alignment showed that pig Ii has domains similar to those seen in other mammalian Ii, including the cytoplasmic, transmembrane, class II-associated Ii-derived peptide, and trimerization domains. Based on observations with the fluorescent microscope, we found that two leucine-based motifs are required for pig Ii intracellular localization, and that both motifs independently mediate this function in Ii.