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INTRODUCTION: The thyroid cancer has the ninth larger incidence of cancer in the world. Investigations related to the exposure to metals have become important due to the sensibility of the thyroid gland to them. Studies reveal that carcinogenic progressions are associated to the deficiency of the essential trace elements. In this context, the zinc is highlighted, essential for the metabolism of the thyroidal hormone and has a potential relation with the pathogenesis of the thyroid cancer. The objective of this systematic review and meta-analysis is to evaluate the low serum zinc as a risk factor for thyroid cancer in adults. METHODS AND ANALYSIS: PubMed/MEDLINE, Scopus, Embase and LILACS databases will be searched for observational studies investigating the low serum zinc as a risk factor for thyroid cancer in adults. No language or publication period restrictions will be imposed. The primary outcome will be that the low serum zinc is a risk factor for thyroid cancer. Three independent reviewers will select the studies and extract data from the original publications. The risk-of-bias will be assessed by using the Newcastle-Ottawa Quality Assessment Scale (NOS). Data synthesis will be performed using the R software (V.4.3.1) and to assess heterogeneity, we will compute the I2 statistic and the results will be based on either random-effects or fixed-effects models, depending on the heterogeneity. The Grading of Recommendations, Development, and Evaluation (GRADE) system will be used to evaluate the reliability and quality of evidence. PROSPERO REGISTRATION NUMBER: International Prospective Register of Systematic Reviews (PROSPERO) CRD42023463747.
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Metanálise como Assunto , Revisões Sistemáticas como Assunto , Neoplasias da Glândula Tireoide , Zinco , Humanos , Neoplasias da Glândula Tireoide/sangue , Neoplasias da Glândula Tireoide/epidemiologia , Zinco/sangue , Fatores de RiscoRESUMO
BACKGROUND & AIMS: Duchenne muscular dystrophy (DMD) involves muscle fragility, sarcolemma instability, and chronic inflammation. This study aims to identify the inflammatory profile of DMD patients and evaluate associations between clinical and nutritional variables. METHODS: We performed a cross-sectional study nested in a cohort to obtain sociodemographics, illness time, use of medications, and supplement data through interviews and the patient's medical records. Then, we assessed the relationships between illness time, cytokine levels, and nutritional status. RESULTS: Forty-four male participants, aged 4.3-24.2 years, were evaluated. Concerning nutritional status, 18 participants were eutrophic. The fat mass increased and the lean mass decreased from the beginning of the first signs of DMD. Cytokines levels in DMD patients, even under corticosteroids therapy, are higher than values described in the literature on healthy subjects. The regression models demonstrated that illness time and BMI/A z-scores are associated with higher values of interleukin-6. CONCLUSIONS: A persistent inflammatory profile was observed in the patients evaluated. The data suggest that maintaining adequate nutritional status and body composition is important for determining the inflammation presented by individuals with DMD.
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Composição Corporal , Inflamação , Distrofia Muscular de Duchenne , Estado Nutricional , Humanos , Distrofia Muscular de Duchenne/complicações , Masculino , Estudos Transversais , Adolescente , Criança , Adulto Jovem , Pré-Escolar , Citocinas/sangue , Índice de Massa Corporal , Interleucina-6/sangueRESUMO
Protein tyrosine phosphatases (PTPs) are key virulence factors in pathogenic bacteria, consequently, they have become important targets for new approaches against these pathogens, especially in the fight against antibiotic resistance. Among these targets of interest YopH (Yersinia outer protein H) from virulent species of Yersinia is an example. PTPs can be reversibly inhibited by nitric oxide (NO) since the oxidative modification of cysteine residues may influence the protein structure and catalytic activity. We therefore investigated the effects of NO on the structure and enzymatic activity of Yersinia enterocolitica YopH in vitro. Through phosphatase activity assays, we observe that in the presence of NO YopH activity was inhibited by 50%, and that this oxidative modification is partially reversible in the presence of DTT. Furthermore, YopH S-nitrosylation was clearly confirmed by a biotin switch assay, high resolution mass spectrometry (MS) and X-ray crystallography approaches. The crystal structure confirmed the S-nitrosylation of the catalytic cysteine residue, Cys403, while the MS data provide evidence that Cys221 and Cys234 might also be modified by NO. Interestingly, circular dichroism spectroscopy shows that the S-nitrosylation affects secondary structure of wild type YopH, though to a lesser extent on the catalytic cysteine to serine YopH mutant. The data obtained demonstrate that S-nitrosylation inhibits the catalytic activity of YopH, with effects beyond the catalytic cysteine. These findings are helpful for designing effective YopH inhibitors and potential therapeutic strategies to fight this pathogen or others that use similar mechanisms to interfere in the signal transduction pathways of their hosts.
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Proteínas da Membrana Bacteriana Externa/química , Cisteína/química , Óxido Nítrico/química , Proteínas Tirosina Fosfatases/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Biotina/metabolismo , Catálise , Cristalografia por Raios X/métodos , Cisteína/metabolismo , Humanos , Espectrometria de Massas/métodos , Estrutura Molecular , Óxido Nítrico/metabolismo , Oxirredução , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais , Yersinia enterocolitica/metabolismoRESUMO
This study was conducted to evaluate the phenolic composition, toxicity, and antimicrobial activity of Licania rigida Benth, an underexploited wild Licania species. L. rigida leaf fractions (ethyl alcohol and ethyl acetate) were analyzed for their phenolic compound and flavonoid total, and high-performance liquid chromatography/ultraviolet spectra chromatographic profiles. Regarding the extract biological effects, toxicity was measured by acute oral toxicity in Wistar rats, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method, and apoptosis indicators with DAPI in VERO cells, whereas well-agar diffusion and broth microdilution assays were applied to evaluate the antimicrobial ability. The phytochemical analysis resulted in significant amounts of phenolic compounds and total flavonoids in the extract and fraction, with flavonol-3-O-glycosylates as the main constituent. Regarding the extract and fraction antimicrobial activity, the results showed a significant effect against gram-positive bacteria and fungi, among which Staphylococcus epidermidis and Candida krusei displayed more susceptibility. No toxicity effects were observed in animals. Concerning the cytotoxicity assay, only the highest dose tested exhibited a minimal toxic effect on the analyzed cell lines. These results are relevant considering the increase of multiresistant microorganisms to conventional treatments applied. Therefore, investigating the pharmacological properties of the genus Licania is promising in the search for new sources of antimicrobial compounds.
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Anti-Infecciosos , Chrysobalanaceae , Animais , Antibacterianos , Anti-Infecciosos/toxicidade , Antioxidantes , Chlorocebus aethiops , Testes de Sensibilidade Microbiana , Extratos Vegetais/toxicidade , Ratos , Ratos Wistar , Células VeroRESUMO
The genetics underlying non-syndromic familial non-medullary thyroid carcinoma (FNMTC) is still poorly understood. To identify susceptibility genes for FNMTC, we performed whole-exome sequencing (WES) in a Brazilian family affected by papillary thyroid carcinoma (PTC) in three consecutive generations. WES was performed in four affected and two unaffected family members. Manual inspection in over 100 previously reported susceptibility genes for FNMTC showed that no variants in known genes co-segregated with disease phenotype in this family. Novel candidate genes were investigated using PhenoDB and filtered using Genome Aggregation (gnomAD) and Online Archive of Brazilian Mutations (ABraOM) population databases. The missense variant p.Ile657Met in the NID1 gene was the only variant that co-segregated with the disease, while absent in unaffected family members and controls. The allele frequency for this variant was <0.0001 in the gnomAD and ABbraOM databases. In silico analysis predicted the variant to be deleterious or likely damaging to the protein function. Somatic mutations in NID1 gene were found in nearly 500 cases of different cancer subtypes in the intOGen platform. Immunohistochemistry analysis showed NID1 expression in PTC cells, while it was absent in normal thyroid tissue. Our findings were corroborated using data from the TCGA cohort. Moreover, higher expression of NID1 was associated with higher likelihood of relapse after treatment and N1b disease in PTCs from the TCGA cohort. Although replication studies are needed to better understand the role of this variant in the FNMTC susceptibility, the NID1 variant (c.1971T>G) identified in this study fulfills several criteria that suggest it as a new FNMTC predisposing gene.
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There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two "druggable" pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.
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Metiltransferases/química , RNA Helicases/química , SARS-CoV-2/química , Proteínas não Estruturais Virais/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Apoenzimas/química , Apoenzimas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Modelos Moleculares , Fosfatos/química , Fosfatos/metabolismo , Conformação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , RNA Viral/química , RNA Viral/metabolismo , SARS-CoV-2/enzimologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismoRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate-ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.
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Domínio Catalítico/fisiologia , Ligação Proteica/fisiologia , Proteínas não Estruturais Virais/metabolismo , Domínio Catalítico/genética , Cristalografia por Raios X , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Proteínas não Estruturais Virais/genética , Tratamento Farmacológico da COVID-19RESUMO
COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.
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Betacoronavirus/química , Cisteína Endopeptidases/química , Fragmentos de Peptídeos/química , Proteínas não Estruturais Virais/química , Betacoronavirus/enzimologia , Sítios de Ligação , Domínio Catalítico , Proteases 3C de Coronavírus , Cristalografia por Raios X , Cisteína Endopeptidases/metabolismo , Desenho de Fármacos , Espectrometria de Massas , Modelos Moleculares , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , SARS-CoV-2 , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Eletricidade Estática , Proteínas não Estruturais Virais/metabolismoRESUMO
Nucleoside diphosphate kinases (NDPKs) are crucial to keep the high triphosphate nucleotide levels in the biological process. The enzymatic mechanism has been extensively described; however, the structural characteristics and kinetic parameters have never been fully determined. In Schistosoma mansoni, NDPK (SmNDPK) is directly involved in the pyrimidine and purine salvage pathways, being essential for nucleotide metabolism. The SmNDPK enzymatic activity is the highest of the known purine metabolisms when compared to the mammalian NDPKs, suggesting the importance of this enzyme in the worm metabolism. Here, we report the recombinant expression of SmNDPK that resulted in 1.7 and 1.9 Å apo-form structure in different space-groups, as well as the 2.1 Å SmNDPK.ADP complex. The binding and kinetic assays reveal the ATP-dependence for enzyme activation. Moreover, in situ hybridization showed that SmNDPK transcripts are found in reproductive organs and in the esophagus gland of adult worms, which can be intrinsically related with the oviposition and digestive processes. These results will help us fully understand the crucial participation of this enzyme in Schistosoma mansoni and its importance for the pathology of the disease.
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Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Núcleosídeo-Difosfato Quinase/química , Núcleosídeo-Difosfato Quinase/metabolismo , Schistosoma mansoni/enzimologia , Esquistossomose mansoni/parasitologia , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Esôfago/química , Esôfago/enzimologia , Feminino , Trato Gastrointestinal/química , Trato Gastrointestinal/enzimologia , Proteínas de Helminto/genética , Humanos , Cinética , Masculino , Modelos Moleculares , Núcleosídeo-Difosfato Quinase/genética , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Alinhamento de SequênciaRESUMO
Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, SmPNP2. Thermofluorescence screening of different ligands suggested cytidine and cytosine are potential ligands. The binding of cytosine and cytidine were confirmed by isothermal titration calorimetry, with a KD of 27 µM for cytosine, and a KM of 76.3 µM for cytidine. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystal structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capacity of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site thus permitting an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site enhances our understanding of the correlation between structure and nucleotide selectivity, thus suggesting new ways to analyse PNP activity.
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Nucleosídeos/metabolismo , Purina-Núcleosídeo Fosforilase/química , Purina-Núcleosídeo Fosforilase/metabolismo , Schistosoma mansoni/enzimologia , Schistosoma mansoni/genética , Adenosina/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Citidina/metabolismo , Citosina/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Inosina/metabolismo , Cinética , Modelos Moleculares , Mutação , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Purina-Núcleosídeo Fosforilase/genética , Schistosoma mansoni/química , Especificidade por SubstratoRESUMO
BACKGROUND: Suspicion of mandibular invasion directly influences perioperative strategy, requiring marginal or segmental mandibulectomy, or reconstruction in some cases. This has a considerable impact on outcome and quality of life. The aim of this study was to evaluate the accuracy of magnetic resonance and computed tomography in the prediction of mandibular invasion in patients with oral cavity cancer. METHOD: A systematic review was conducted, including diagnostic studies comparing magnetic resonance imaging with computed tomography in the prediction of bone invasion. Sensitivity, specificity, positive and negative likelihood values and summary receiver operating characteristic (sROC) curves were calculated. RESULTS: The electronic and manual search identified 346 articles. Of these, 11 studies were included in the systematic review for a total of 477 patients. The sensitivity, specificity, and positive and negative likelihood values for MRI were 78%, 86%, 5.29 and 0.23, respectively. For CT, they were 76%, 89%, 6.00 and 0.28, respectively. The sROC curves for MRI and CT were 82.3% and 82.5%, respectively. CONCLUSION: No superiority was observed between the diagnostic methods regarding mandibular invasion detection.
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Imageamento por Ressonância Magnética/métodos , Mandíbula/diagnóstico por imagem , Mandíbula/patologia , Neoplasias Bucais/patologia , Invasividade Neoplásica/diagnóstico por imagem , Tomografia Computadorizada por Raios X/métodos , HumanosRESUMO
Type 1 diabetes mellitus (T1DM) and estrogen deficiency are associated with several alterations in bone turnover. Zinc (Zn) is required for growth, development, and overall health. Zinc has been used in complementary therapy against bone loss in several diseases. We hypothesized that Zn supplementation represents a potential therapy against severe bone loss induced by the combined effect of estrogen deficiency and T1DM. We evaluated the protective effect of Zn against bone alterations in a chronic model of these disorders. Female Wistar rats were ramdomized into 3 groups (5 rats each): control, OVX/T1DM (ovariectomized rats with streptozotocin-induced T1DM), and OVX/T1DM+Zn (OVX/T1DM plus daily Zn supplementation). Serum biochemical, bone histomorphometric, and molecular analyses were performed. Histomorphometric parameters were similar between the control and OVX/T1DM+Zn groups, suggesting that Zn prevents bone architecture alterations. In contrast, the OVX/T1DM group showed significantly lower trabecular width and bone area as well as greater trabecular separation than the control. The OVX/T1DM and OVX/T1DM+Zn groups had significantly higher serum alkaline phosphatase activity than the control. The supplemented group had higher levels of serum-ionized calcium and phosphorus than the nonsupplemented group. The RANKL/OPG ratio was similar between the control and OVX/T1DM+Zn groups, whereas it was higher in the OVX/T1DM group. In conclusion, Zn supplementation prevents bone alteration in chronic OVX/T1DM rats, as demonstrated by the reduced RANKL/OPG ratio and preservation of bone architecture. The findings may represent a novel therapeutic approach to preventing OVX/T1DM-induced bone alterations.
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Densidade Óssea/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Suplementos Nutricionais , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Zinco/administração & dosagem , Fosfatase Alcalina/sangue , Animais , Glicemia/metabolismo , Osso e Ossos/efeitos dos fármacos , Cálcio/sangue , Diabetes Mellitus Tipo 1/tratamento farmacológico , Feminino , Osteoprotegerina/genética , Ovariectomia , Fósforo/sangue , Ligante RANK/genética , Ratos , Ratos WistarRESUMO
The steady expansion in the capacity of modern beamlines for high-throughput data collection, enabled by increasing X-ray brightness, capacity of robotics and detector speeds, has pushed the bottleneck upstream towards sample preparation. Even in ligand-binding studies using crystal soaking, the experiment best able to exploit beamline capacity, a primary limitation is the need for gentle and nontrivial soaking regimens such as stepwise concentration increases, even for robust and well characterized crystals. Here, the use of acoustic droplet ejection for the soaking of protein crystals with small molecules is described, and it is shown that it is both gentle on crystals and allows very high throughput, with 1000 unique soaks easily performed in under 10â min. In addition to having very low compound consumption (tens of nanolitres per sample), the positional precision of acoustic droplet ejection enables the targeted placement of the compound/solvent away from crystals and towards drop edges, allowing gradual diffusion of solvent across the drop. This ensures both an improvement in the reproducibility of X-ray diffraction and increased solvent tolerance of the crystals, thus enabling higher effective compound-soaking concentrations. The technique is detailed here with examples from the protein target JMJD2D, a histone lysine demethylase with roles in cancer and the focus of active structure-based drug-design efforts.
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Acústica/instrumentação , Cristalização/instrumentação , Proteínas/química , Cristalização/economia , Cristalização/métodos , Cristalografia por Raios X , Desenho de Equipamento , Fatores de TempoRESUMO
Schistosoma mansoni is the parasite responsible for schistosomiasis, a disease that affects about 218 million people worldwide. Currently, both direct treatment and disease control initiatives rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, have stimulated efforts to develop new drugs for the treatment of schistosomiasis. Schistosomes do not have the de novo purine biosynthetic pathway, and instead depend entirely on the purine salvage pathway to supply its need for purines. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenylosuccinate lyase (SmADSL) is an enzyme in this pathway, which cleaves adenylosuccinate (ADS) into adenosine 5'-monophosphate (AMP) and fumarate. SmADSL kinetic characterization was performed by isothermal titration calorimetry (ITC) using both ADS and SAICAR as substrates. Structures of SmADSL in Apo form and in complex with AMP were elucidated by x-ray crystallography revealing a highly conserved tetrameric structure required for their function since the active sites are formed from residues of three different subunits. The active sites are also highly conserved between species and it is difficult to identify a potent species-specific inhibitor for the development of new therapeutic agents. In contrast, several mutagenesis studies have demonstrated the importance of dimeric interface residues in the stability of the quaternary structure of the enzyme. The lower conservation of these residues between SmADSL and human ADSL could be used to lead the development of anti-schistosomiasis drugs based on disruption of subunit interfaces. These structures and kinetics data add another layer of information to Schistosoma mansoni purine salvage pathway.
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Adenilossuccinato Liase/química , Adenilossuccinato Liase/metabolismo , Schistosoma mansoni/enzimologia , Monofosfato de Adenosina/metabolismo , Adenilossuccinato Liase/genética , Animais , Domínio Catalítico , Sequência Conservada , Cristalografia por Raios X , Fumaratos/metabolismo , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Estabilidade ProteicaRESUMO
The parasite Schistosoma mansoni possesses all pathways for pyrimidine biosynthesis, in which dihydrofolate reductase (DHFR), thymidylate cycle participants, is essential for nucleotide metabolism to obtain energy and structural nucleic acids. Thus, DHFRs have been widely suggested as therapeutic targets for the treatment of infectious diseases. In this study, we expressed recombinant SmDHFR in a heterologous manner to obtain structural, biochemical and kinetic information. X-ray diffraction of recombinant SmDHFR at 1.95Å resolution showed that the structure exhibited the canonical DHFR fold. Isothermal titration calorimetry was used to determine the kinetic constants for NADP+ and dihydrofolate. Moreover, inhibition assays were performed using the commercial folate analogs methotrexate and aminopterin; these analogs are recognized as folate competitors and are used as chemotherapeutic agents in cancer and autoimmune diseases. This study provides information that may prove useful for the future discovery of novel drugs and for understanding these metabolic steps from this pathway of S. mansoni, thus aiding in our understanding of the function of these essential pathways for parasite metabolism.
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Schistosoma mansoni/enzimologia , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Animais , Antagonistas do Ácido Fólico/farmacologia , Humanos , Cinética , Metotrexato/farmacologia , Proteínas Recombinantes , Difração de Raios XRESUMO
A current metagenomics focus is to interpret and transform collected genomic data into biological information. By combining structural, functional and genomic data we have assessed a novel bacterial protein selected from a carbohydrate-related activity screen in a microbial metagenomic library from Capra hircus (domestic goat) gut. This uncharacterized protein was predicted as a bacterial cell wall-modifying enzyme (CWME) and shown to contain four domains: an N-terminal, a cysteine protease, a peptidoglycan-binding and an SH3 bacterial domain. We successfully cloned, expressed and purified this putative cysteine protease (PCP), which presented autoproteolytic activity and inhibition by protease inhibitors. We observed cell wall hydrolytic activity and ampicillin binding capacity, a characteristic of most bacterial CWME. Fluorimetric binding analysis yielded a Kb of 1.8 × 105 M-1 for ampicillin. Small-angle X-ray scattering (SAXS) showed a maximum particle dimension of 95 Å with a real-space Rg of 28.35 Å. The elongated molecular envelope corroborates the dynamic light scattering (DLS) estimated size. Furthermore, homology modeling and SAXS allowed the construction of a model that explains the stability and secondary structural changes observed by circular dichroism (CD). In short, we report a novel cell wall-modifying autoproteolytic PCP with insight into its biochemical, biophysical and structural features.
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Ampicilina/metabolismo , Bactérias/enzimologia , Clonagem Molecular/métodos , Cisteína Proteases/química , Cisteína Proteases/metabolismo , Cabras/microbiologia , Animais , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/enzimologia , Parede Celular/genética , Cisteína Proteases/genética , Hidrólise , Metagenoma , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Schistosoma mansoni do not have de novo purine pathways and rely on purine salvage for their purine supply. It has been demonstrated that, unlike humans, the S. mansoni is able to produce adenine directly from adenosine, although the enzyme responsible for this activity was unknown. In the present work we show that S. mansoni 5´-deoxy-5´-methylthioadenosine phosphorylase (MTAP, E.C. 2.4.2.28) is capable of use adenosine as a substrate to the production of adenine. Through kinetics assays, we show that the Schistosoma mansoni MTAP (SmMTAP), unlike the mammalian MTAP, uses adenosine substrate with the same efficiency as MTA phosphorolysis, which suggests that this enzyme is part of the purine pathway salvage in S. mansoni and could be a promising target for anti-schistosoma therapies. Here, we present 13 SmMTAP structures from the wild type (WT), including three single and one double mutant, and generate a solid structural framework for structure description. These crystal structures of SmMTAP reveal that the active site contains three substitutions within and near the active site when compared to it mammalian counterpart, thus opening up the possibility of developing specific inhibitors to the parasite MTAP. The structural and kinetic data for 5 substrates reveal the structural basis for this interaction, providing substract for inteligent design of new compounds for block this enzyme activity.
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Adenosina/metabolismo , Modelos Moleculares , Purina-Núcleosídeo Fosforilase/química , Purina-Núcleosídeo Fosforilase/metabolismo , Schistosoma mansoni/enzimologia , Animais , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Humanos , Cinética , Redes e Vias Metabólicas , Mutação , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/isolamento & purificação , Purinas/metabolismo , Schistosoma mansoni/genética , Alinhamento de SequênciaRESUMO
RESUMO Objetivos: identificar as características audiológicas de pacientes com diabetes mellitus tipo 2. Métodos: estudo transversal, realizado em unidade de atenção secundária para diabetes em Fortaleza, de abril a julho de 2010. Amostra composta por 152 pacientes diabéticos tipo 2, independente do sexo, entre 36 e 60 anos. Realizou-se audiometria tonal liminar (ATL), emissões otoacústicas evocadas por estímulo transiente (EOAT) e emissões otoacústicas evocadas-produto de distorção (EOAPD). Resultados: associação da idade e do tempo de diagnóstico com a presença de perda auditiva, e do tempo de diagnóstico com a ausência de EOAPD. Observou-se perda auditiva sensorioneural em 63,2% dos pacientes, das quais 71,9% eram bilaterais e 75% simétricas. Aproximadamente, 50% das perdas auditivas apresentaram configuração plana. As EOAT estavam ausentes em 75% dos pacientes e as EOAPD em 78,9%. Houve ausência de emissões otoacústicas na presença de ATL normal, em média, em 32% dos pacientes à direita e 48% à esquerda. Já a presença de emissões otoacústicas na ocorrência de perda auditiva foi observada em, aproximadamente, 30% dos pacientes para EOAT e 14% para EOAPD à direita; e 25% para EOAT e 11% para EOAPD à esquerda. Conclusão: predomínio de perda auditiva sensorioneural bilateral simétrica com configuração plana, e ausência de EOAT e EOAPD. A análise da associação dos resultados da ATL e das emissões otoacústicas sugere prejuízo das células ciliadas externas da cóclea ou possível neuropatia auditiva. Tais achados justificariam o monitoramento da audição destes pacientes, bem como a realização de testes específicos para avaliação do sistema auditivo central.
ABSTRACT Purpose: to identify the audiological characteristics of patients with type 2 diabetes mellitus. Methods: Cross-sectional study in secondary care unit for diabetes in Fortaleza, April-July 2010. The sample comprised 152 patients with type 2 diabetes, regardless of sex, between 36 and 60 years of age. Held pure tone audiometry (PTA), transient-evoked otoacoustic emissions (TEOAE) and distortion product otoacoustic emissions (DPOAE). Results: association of age and time of diagnosis with hearing loss, and the time of diagnosis with the absence of DPOAE. It was observed sensorineural hearing loss in 63.2% of patients, which 71.9% were bilateral and 75% symmetric. Approximately 50% of hearing loss had flat configuration. TOAEs were absent in 75% of the patients and DPOAE in 78.9%. There were no otoacoustic emissions in the presence of normal PTA, on average, 32% of the patients on the right and 48% on the left. The presence of otoacoustic emissions on the occurrence of hearing loss was observed in approximately 30% of patients for TEOAE and 14% for DPOAE on the right; and 25% for TEOAE and 11% for DPOAE on the left. Conclusions: Prevalence of symmetrical bilateral sensorineural hearing loss with a flat configuration, and absence of TEOAE and DPOAE. The association analysis of the results of PTA and OAE suggests loss of outer hair cells of the cochlea or possible auditory neuropathy. These findings justify the hearing monitoring of these patients, as well as conducting specific tests to evaluate the central auditory system.
RESUMO
The sequences of all seven polypeptide chains from the giant haemoglobin of the free-living earthworm Glossoscolex paulistus (HbGp) are reported together with the three-dimensional structure of the 3.6 MDa complex which they form. The refinement of the full particle, which has been solved at 3.2 Å resolution, the highest resolution reported to date for a hexagonal bilayer haemoglobin composed of 12 protomers, is reported. This has allowed a more detailed description of the contacts between subunits which are essential for particle stability. Interpretation of features in the electron-density maps suggests the presence of metal-binding sites (probably Zn(2+) and Ca(2+)) and glycosylation sites, some of which have not been reported previously. The former appear to be important for the integrity of the particle. The crystal structure of the isolated d chain (d-HbGp) at 2.1 Å resolution shows different interchain contacts between d monomers compared with those observed in the full particle. Instead of forming trimers, as seen in the complex, the isolated d chains associate to form dimers across a crystallographic twofold axis. These observations eliminate the possibility that trimers form spontaneously in solution as intermediates during the formation of the dodecameric globin cap and contribute to understanding of the possible ways in which the particle self-assembles.