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1.
Proc Natl Acad Sci U S A ; 119(34): e2211991119, 2022 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-35960847
3.
Acta Crystallogr F Struct Biol Commun ; 78(Pt 7): 252-264, 2022 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-35787552

RESUMO

The unintended crystallization of proteins which generally originate from the expression host instead of the target recombinant proteins is periodically reported. Despite the massive technological advances in the field, assigning a structural model to the corresponding diffraction data is not a trivial task. Here, the structure of acyl-carrier protein synthase (AcpS) from Mycobacterium smegmatis (msAcpS), which crystallized inadvertently in an experimental setup to grow crystals of a Mycobacterium tuberculosis protein using M. smegmatis as an expression system, is reported. After numerous unsuccessful attempts to solve the structure of the target protein by the molecular-replacement method no convincing solutions were obtained, indicating that the diffraction data may correspond to a crystal of an artifactual protein, which was finally identified by the Sequence-Independent Molecular replacement Based on Available Databases (SIMBAD) server. The msAcpS structure was solved at 2.27 Šresolution and structural analysis showed an overall conserved fold. msAcpS formed a trimeric structure similar to those of other reported structures of AcpS from various organisms; however, the residues involved in trimer formation are not strictly conserved. An unrelated metal ion (Ni2+), which was possibly incorporated during protein purification, was observed in the proximity of His49 and His116. Structural and sequence differences were observed in the loop connecting the α3 and α4 helices that is responsible for the open and closed conformations of the enzyme. Moreover, the structural analysis of msAcpS augments the current understanding of this enzyme, which plays a crucial role in the functional activation of acyl-carrier proteins in the fatty-acid biosynthesis pathway.


Assuntos
Proteínas de Bactérias , Mycobacterium smegmatis/enzimologia , Transferases/química , Proteína de Transporte de Acila/química , Proteínas de Bactérias/química , Cristalização , Cristalografia por Raios X , Mycobacterium smegmatis/metabolismo
4.
J Chromatogr A ; 1677: 463318, 2022 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-35853422

RESUMO

Heparin is a linear sulfated polysaccharide with a complex structure. It is important to figure out the sequences at the terminals of the sugar chains, as it will help us understand the heparin structure deeper and control its quality properly. The tetrasaccharide linkage region (LR) could be a tag to help us find out heparin terminals after digestion by different combinations of heparinases. In this work, orthogonal chromatographic approaches including SAX, SEC-MS and 2D-LC-MS were applied to qualitatively and quantitatively analyze the heparinase released LR-terminals. The disaccharides next to LR are those ones with low or non-sulfation, UA-GlcNAc and UA-GlcNAc6S, and then they are extended with the highly sulfated disaccharides, IdoA2S-GlcNS and IdoA2S-GlcNS6S. It is suggested that the sulfo transferases did not work at the sugar residues next to LR terminal, especially the 2-O-sulfo and N-sulfo transferases, which could be affected by steric hindrance from LR, when heparin is biosynthesized. This conclusion will be theoretical fundamental to help us understand heparin's structure deeper. The methods provided in this work could be potential ways to control heparin's quality and monitor the production processes of heparin properly.


Assuntos
Dissacarídeos , Heparina , Dissacarídeos/química , Heparina/química , Heparina Liase , Oligossacarídeos/química , Transferases
5.
Phytomedicine ; 104: 154325, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35820303

RESUMO

BACKGROUND: Tetrandrine (TET), a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra S. Moore, is the only approved medicine in China for silicosis. However, TET-induced hepatotoxicity has raised safety concerns. The underlying toxic targets and mechanism induced by TET remain unclear; there are no targeted detoxification strategies developed for TET-induced hepatotoxicity. Ursolic acid (UA), a pentacyclic triterpene with liver protective effects, may have detoxification effects on TET-induced hepatotoxicity. PURPOSE: This study aims to explore toxic targets and mechanism of TET and present UA as a potential targeted therapy for alleviating TET-induced hepatotoxicity. METHODS: A TET-induced liver-injury model was established to evaluate TET toxicity and the potential UA detoxification effect. Alkenyl-modified TET and UA probes were designed to identify potential liver targets. Pharmacological and molecular biology methods were used to explore the underlying toxicity/detoxification mechanism. RESULTS: TET induced liver injury by covalently binding to the substrate-binding pocket (H-site) of glutathione S-transferases (GSTs) and inhibiting GST activity. The covalent binding led to toxic metabolite accumulation and caused redox imbalance and liver injury. UA protected the liver from TET-induced damage by competitively binding to the GST H-site. CONCLUSION: The mechanism of TET-induced hepatotoxicity is related to irreversible binding with the GST H-site and GST-activity inhibition. UA, a natural antidote, competed with TET on H-site binding and reversed the redox imbalance. This study revealed the hepatotoxic mechanism of TET and provided a targeted detoxifying agent, UA, to alleviate hepatotoxicity caused by GST inhibition.


Assuntos
Antineoplásicos , Benzilisoquinolinas , Doença Hepática Induzida por Substâncias e Drogas , Benzilisoquinolinas/farmacologia , Benzilisoquinolinas/uso terapêutico , Sítios de Ligação , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Humanos , Transferases/metabolismo , Triterpenos
6.
J Dairy Sci ; 105(8): 6639-6653, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35787321

RESUMO

This study was performed to determine the early-life (first month of age) supplementation of liquid feed with fennel seed powder (FSP) or oregano leaf powder (OLP) on growth performance, health, and blood biochemical attributes in preweaning dairy calves. Holstein female calves (n = 57; 1 d of age; 34.1 ± 0.97 kg of BW; mean ± SE) were assigned randomly to receive liquid feed (colostrum and milk) with no added herbal plants (CON) or supplemented with FSP (3 g/d) or OLP (30 g/d) during the first month of age. The calves received pooled colostrum (4.5 kg/d on the first 2 d of life; total solids = 25.0% ± 1.24; mean ± SD) and then pooled waste milk (6 kg/d from d 3 to 44, 5 kg/d from d 45 to 46, 4 kg/d from d 47 to 48, and 3 kg/d from d 49 to 50 of the trial; total solids = 12.54% ± 0.50) to ensure they receive same mixed liquid feed daily. The calves had unlimited access to the starter feed and fresh water and remained in the study until weaning on d 51 of age. The average mean temperature-humidity index was 70.1 units (ranging between 61.9 to 78.2) during the experiment, indicating a borderline degree of environmental heat-load. The amount of starter feed offered and refused was recorded daily. The calves were weighed immediately after birth and every 10 d thereafter, before the morning feeding. Jugular blood samples were taken immediately before and 24 h after colostrum feeding, at first month of age, and at weaning to quantify serum concentrations of glucose, urea N, cholesterol, triglycerides, total proteins, albumin, globulin, aspartate transferase, alanine transferase, total antioxidant status, and malondialdehyde. Health checks including rectal temperature, general appearance (on a 1-5 score system), fecal score (on a 1-5 score system), and bovine respiratory disease (BRD; scored using the University of Wisconsin Calf Health Chart) were performed daily, by a veterinarian who was unaware of the calf treatment allocations, for all calves over the study period. A repeated-measures ANOVA was used to compare growth performance and blood metabolites among treatment groups, and a logistic regression model using a binomial distribution (PROC GLIMMIX, SAS v. 9.4, SAS Institute Inc.) was used to assess the chance of elevated rectal temperature (≥39.4°C), general appearance (≥2), diarrhea (≥3), and BRD. A Poisson regression model (PROC GENMOD) was also used to test group differences in the experience of days with elevated rectal temperature and general appearance, and frequency and duration of diarrhea or BRD. Total nutrient intake (DM, CP, and ME, but not ether extract) originating from milk and starter feed was greater in OLP- (but not FSP-) supplemented calves compared with CON group, being partially associated with difference in milk refusal. Calves receiving FSP and OLP had greater average daily gain, BW gain, skeletal gain (withers height or heart girth, respectively), and feed efficiency compared with CON animals with no difference between FSP and OLP. Rectal temperature was lower in FSP- (but not OLP-) supplemented calves compared with CON animals. The CON animals had a greater chance of experiencing higher rectal temperature (≥39.4°C; odds ratio = 1.55 and confidence interval = 1.12-2.15 and odds ratio = 1.33 and confidence interval = 0.92-1.90, respectively, compared with FSP and OLP) and general appearance (≥2; odds ratio = 1.99 and confidence interval = 1.45-2.74 and odds ratio = 1.45 and confidence interval = 1.03-2.05), and diarrhea (odds ratio = 1.47 and confidence interval = 1.11-1.97 and odds ratio = 1.49 and confidence interval = 1.07-2.08) compared with those receiving FSP or OLP; with the chance of BRD being lower in FSP- (but not OLP-) supplemented versus CON animals (odds ratio = 1.59 and confidence interval = 1.13-2.23). As compared with OLP and CON groups, FSP treatment resulted in shorter days with elevated rectal temperature and general appearance. Supplementing FSP or OLP decreased the duration of diarrhea and BRD but not their frequency compared with CON. Duration of diarrhea was not different between FSP- or OLP-supplemented calves but calves supplemented with FSP had shorter days with BRD compared with OLP-supplemented calves. Of the blood constituents measured on d 30, concentration of aspartate transferase was higher in FSP- (but not CON) versus OLP-supplemented calves, indicating a transient liver tissue damage or dysfunction which was subsequently ameliorated. At weaning, blood concentration of triglycerides was higher in FSP and OLP groups compared with CON group. Supplementation of the liquid feed with FSP or OLP, especially FSP, had beneficial effects on calf growth performance and health. Further experiments are warranted for optimizing the dosage and duration of feeding FSP or OLP as feed additives for dairy calves.


Assuntos
Doenças dos Bovinos , Foeniculum , Origanum , Ração Animal/análise , Animais , Ácido Aspártico , Peso Corporal , Bovinos , Dieta/veterinária , Suplementos Nutricionais , Feminino , Humanos , Leite , Pós , Sementes , Transferases , Triglicerídeos , Desmame
7.
BMC Cancer ; 22(1): 685, 2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35729618

RESUMO

BACKGROUND: Glutathione-S transferases (GSTs) comprise a series of critical enzymes involved in detoxification of endogenous or xenobiotic compounds. Among several GSTs, Glutathione S-transferases mu (GSTM) has been implicated in a number of cancer types. However, the prognostic value and potential functions of the GSTM family genes have not been investigated in lung adenocarcinoma (LUAD). METHODS: We examined the expression of GSTM5 in LUAD and identified associations among GSTM5 expression, clinicopathological features, survival data from the Cancer Genome Atlas (TCGA). The correlation between GSTM5 DNA methylation and its expression was analyzed using the MEXPRESS tool and UCSC Xena browser. The methylation status of GSTM5 in the promoter region in lung cancer cells was measured by methylation-specific PCR (MSP). After 5-aza-2'-deoxycytidine treatment of lung cancer cells, expression of GSTM5, cell proliferation and migration were assessed by RT-PCR, CCK-8 and transwell assays, respectively. RESULTS: The results showed that GSTM5 was abnormally down-regulated in LUAD patients' tissues, and patients with low GSTM5 expression level had significantly shorter OS. Cox regression analyses revealed that GSTM5 was associated with overall survival (OS) of LUAD patients, which expression was an independent prognostic indicator in terms of OS (hazard ratio: 0.848; 95% CI: 0.762-0.945; P = 0.003). In addition, we found the promoter region of GSTM5 was hypermethylated in the tumor tissue compared with adjacent normal tissues, and the average methylation level of GSTM5 were moderately correlated with its expression. Moreover, methylation-specific PCR also showed that the GSTM5 gene promoter was hypermethylated in lung cancer cells, and treatment with 5-Aza-CdR can restore the gene expression and inhibit cell proliferation and migration. Finally, Gene Set Enrichment Analysis (GSEA) revealed that low GSTM5 expression was significantly related to DNA repair pathways. CONCLUSION: Our data demonstrate that low GSTM5 expression and its high DNA methylation status may act as a novel putative molecular target gene for LUAD.


Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Adenocarcinoma de Pulmão/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , DNA/metabolismo , Metilação de DNA , Decitabina , Regulação Neoplásica da Expressão Gênica , Glutationa/metabolismo , Glutationa Transferase , Humanos , Neoplasias Pulmonares/patologia , Prognóstico , Transferases/genética , Transferases/metabolismo
8.
Int J Mol Sci ; 23(11)2022 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-35682614

RESUMO

Chemical probes can be used to understand the complex biological nature of diseases. Due to the diversity of cancer types and dynamic regulatory pathways involved in the disease, there is a need to identify signaling pathways and associated proteins or enzymes that are traceable or detectable in tests for cancer diagnosis and treatment. Currently, fluorogenic chemical probes are widely used to detect cancer-associated proteins and their binding partners. These probes are also applicable in photodynamic therapy to determine drug efficacy and monitor regulating factors. In this review, we discuss the synthesis of chemical probes for different cancer types from 2016 to the present time and their application in monitoring the activity of transferases, hydrolases, deacetylases, oxidoreductases, and immune cells. Moreover, we elaborate on their potential roles in photodynamic therapy.


Assuntos
Hidrolases , Neoplasias , Corantes Fluorescentes/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Oxirredutases/metabolismo , Proteínas , Transferases
9.
J Pediatr Gastroenterol Nutr ; 74(4): 463-470, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-35703948

RESUMO

OBJECTIVES: Anti-neutrophil cytoplasmic antibody (ANCA) directed against proteinase 3 (PR3) is a marker for granulomatosis with polyangiitis, but is also found in patients with inflammatory bowel disease (IBD), mainly ulcerative colitis (UC). The aim of our study was to investigate ANCA and PR3-ANCA in paediatric IBD. METHODS: We tested 326 paediatric IBD patients and 164 controls for anti-Saccharomyces cerevisiae antibodies (ASCA), ANCA (indirect immunofluorescence, IIF) and PR3-ANCA (chemiluminescence immunoassay). We applied the Paris classification for paediatric IBD and documented liver manifestations such as primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH). RESULTS: We found PR3-ANCA in 49/121 (40%) of UC, 20/187 (11%) of Crohn disease (CD) and 2/18 (11%) of IBD-unclassified (IBD-U) patients but in none of the controls. 54% UC and 12% CD patients were positive for ANCA (IIF). PR3-ANCA positive UC patients were characterised by more extensive disease (P = .070). Fourteen of 21 (67%) of UC patients with backwash ileitis were anti-PR3 ANCA-positive (P = .011). We diagnosed PSC or PSC/AIH in 19 UC and 3 IBD-U patients. Fifteen of 22 (68%) patients with PSC or PSC/AIH were anti-PR3-ANCA positive in contrast to 36 of 117 (32%) patients without PSC (P = .001). PR3-ANCA positive patients showed higher levels of gamma-glutamyl transferase, alanine transaminase and aspartate transferase (P < 0.001, 0.001, 0.006, respectively). Forty-seven percent of CD and 6% of UC patients were ASCA-IgA positive. PR3-ANCA-positive and -negative patients showed no significant differences concerning age at diagnosis, disease activity, need for drugs, and number of hospitalisations. CONCLUSIONS: Our study provides data for PR3-ANCA as a potential serological marker for paediatric UC and PSC.


Assuntos
Colangite Esclerosante , Colite Ulcerativa , Doença de Crohn , Doenças Inflamatórias Intestinais , Anticorpos Anticitoplasma de Neutrófilos , Biomarcadores , Criança , Colangite Esclerosante/diagnóstico , Colite Ulcerativa/diagnóstico , Doença de Crohn/diagnóstico , Humanos , Mieloblastina , Transferases
10.
Shock ; 57(6): 218-227, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35759303

RESUMO

INTRODUCTION: Survivors of sepsis exhibit persistent immunosuppression. Epigenetic events may be responsible for some of these immunosuppressive changes. During sepsis circulating exosomes contain large quantities of DNA methyltransferase (DNMT) mRNAs. We hypothesized that exosomes directly transfer DNMT mRNAs to recipient monocytes with resultant methylation events and immunosuppression. METHODS: Exosomes containing DNMT mRNA were generated by stimulating monocytes with LPS. Confocal microscopy was used to determine uptake kinetics in the presence of pharmacologic inhibition. Expression and packaging of specific DNMT mRNA was controlled using DNMT siRNAs. Whole genome and gene specific methylation was assessed using bisulfite sequencing. Ingenuity pathway analysis was performed to determine the biological function of significance of differentially methylated regions. RESULTS: Exosomes effectively transferred DNMT mRNA to recipient monocytes. Pharmacologic inhibition of exosome uptake prevented this increase in DNMT mRNA expression. Recipient monocytes exhibited hypermethylation changes and gene suppression. siRNAs decreased the packaging of DNMT mRNAs and prevented TNFα gene suppression, restoring immunocompetence. CONCLUSION: These data support a role for exosome-mediated transfer of DNMT mRNA with resultant methylation and gene silencing. Pharmacologic uptake inhibition or targeted siRNA mediated DNMT gene silencing prevented DNMT mRNA transfer and maintained the cell's ability to express TNFα in response to LPS. This highlights the potential therapeutic value of targeting these exosome-mediated epigenetic events to maintain the host immune response during sepsis.


Assuntos
DNA (Citosina-5-)-Metiltransferases , Sepse , DNA , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Humanos , Lipopolissacarídeos , Monócitos/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Interferente Pequeno , Sepse/genética , Transferases/genética , Fator de Necrose Tumoral alfa/genética
11.
Commun Biol ; 5(1): 602, 2022 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-35760847

RESUMO

Single-cell RNA-sequencing (scRNA-seq) is valuable for analyzing cellular heterogeneity. Cell composition accuracy is critical for analyzing cell-cell interaction networks from scRNA-seq data. However, droplet- and plate-based scRNA-seq techniques have cell sampling bias that could affect the cell composition of scRNA-seq datasets. Here we developed terminator-assisted solid-phase cDNA amplification and sequencing (TAS-Seq) for scRNA-seq based on a terminator, terminal transferase, and nanowell/bead-based scRNA-seq platform. TAS-Seq showed high tolerance to variations in the terminal transferase reaction, which complicate the handling of existing terminal transferase-based scRNA-seq methods. In murine and human lung samples, TAS-Seq yielded scRNA-seq data that were highly correlated with flow-cytometric data, showing higher gene-detection sensitivity and more robust detection of important cell-cell interactions and expression of growth factors/interleukins in cell subsets than 10X Chromium v2 and Smart-seq2. Expanding TAS-Seq application will improve understanding and atlas construction of lung biology at the single-cell level.


Assuntos
Perfilação da Expressão Gênica , Análise de Célula Única , Animais , DNA Complementar/genética , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transferases
12.
Elife ; 112022 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-35731045

RESUMO

Background: Type 2 diabetes (T2D) accounts for ~90% of all cases of diabetes, resulting in an estimated 6.7 million deaths in 2021, according to the International Diabetes Federation. Early detection of patients with high risk of developing T2D can reduce the incidence of the disease through a change in lifestyle, diet, or medication. Since populations of lower socio-demographic status are more susceptible to T2D and might have limited resources or access to sophisticated computational resources, there is a need for accurate yet accessible prediction models. Methods: In this study, we analyzed data from 44,709 nondiabetic UK Biobank participants aged 40-69, predicting the risk of T2D onset within a selected time frame (mean of 7.3 years with an SD of 2.3 years). We started with 798 features that we identified as potential predictors for T2D onset. We first analyzed the data using gradient boosting decision trees, survival analysis, and logistic regression methods. We devised one nonlaboratory model accessible to the general population and one more precise yet simple model that utilizes laboratory tests. We simplified both models to an accessible scorecard form, tested the models on normoglycemic and prediabetes subcohorts, and compared the results to the results of the general cohort. We established the nonlaboratory model using the following covariates: sex, age, weight, height, waist size, hip circumference, waist-to-hip ratio, and body mass index. For the laboratory model, we used age and sex together with four common blood tests: high-density lipoprotein (HDL), gamma-glutamyl transferase, glycated hemoglobin, and triglycerides. As an external validation dataset, we used the electronic medical record database of Clalit Health Services. Results: The nonlaboratory scorecard model achieved an area under the receiver operating curve (auROC) of 0.81 (95% confidence interval [CI] 0.77-0.84) and an odds ratio (OR) between the upper and fifth prevalence deciles of 17.2 (95% CI 5-66). Using this model, we classified three risk groups, a group with 1% (0.8-1%), 5% (3-6%), and the third group with a 9% (7-12%) risk of developing T2D. We further analyzed the contribution of the laboratory-based model and devised a blood test model based on age, sex, and the four common blood tests noted above. In this scorecard model, we included age, sex, glycated hemoglobin (HbA1c%), gamma glutamyl-transferase, triglycerides, and HDL cholesterol. Using this model, we achieved an auROC of 0.87 (95% CI 0.85-0.90) and a deciles' OR of ×48 (95% CI 12-109). Using this model, we classified the cohort into four risk groups with the following risks: 0.5% (0.4-7%); 3% (2-4%); 10% (8-12%); and a high-risk group of 23% (10-37%) of developing T2D. When applying the blood tests model using the external validation cohort (Clalit), we achieved an auROC of 0.75 (95% CI 0.74-0.75). We analyzed several additional comprehensive models, which included genotyping data and other environmental factors. We found that these models did not provide cost-efficient benefits over the four blood test model. The commonly used German Diabetes Risk Score (GDRS) and Finnish Diabetes Risk Score (FINDRISC) models, trained using our data, achieved an auROC of 0.73 (0.69-0.76) and 0.66 (0.62-0.70), respectively, inferior to the results achieved by the four blood test model and by the anthropometry models. Conclusions: The four blood test and anthropometric models outperformed the commonly used nonlaboratory models, the FINDRISC and the GDRS. We suggest that our models be used as tools for decision-makers to assess populations at elevated T2D risk and thus improve medical strategies. These models might also provide a personal catalyst for changing lifestyle, diet, or medication modifications to lower the risk of T2D onset. Funding: The funders had no role in study design, data collection, interpretation, or the decision to submit the work for publication.


Assuntos
Diabetes Mellitus Tipo 2 , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/epidemiologia , Hemoglobina A Glicada , Humanos , Modelos Logísticos , Fatores de Risco , Transferases , Triglicerídeos
13.
Bioorg Med Chem ; 68: 116875, 2022 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-35716588

RESUMO

Nicotinamide adenine dinucleotide (NAD+) is an important biomolecule with essential roles at the intersection of energy metabolism, epigenetic regulation and cell signalling. Synthetic analogues of NAD+ are therefore of great interest as chemical tools for medicinal chemistry, chemical biology and drug discovery. Herein, we report the chemical synthesis and full analytical characterisation of three stereoisomers of 2″-amino NAD+, and their biochemical evaluation against two classes of NAD+-consuming enzymes: the human sirtuins 1-3, and the bacterial toxin TccC3. To rationalise the observed activities, molecular docking experiments were carried out with SIRT1 and SIRT2, which identified the correct orientation of the pyrophosphate linkage as a major determinant for activity in this series. These results, together with results from stability tests and a conformational analysis, allow, for the first time, a side-by-side comparison of the chemical and biochemical features, and analytical properties, of different 2″-amino NAD+ stereoisomers. Our findings provide insight into the recognition of co-substrate analogues by sirtuins, and will greatly facilitate the application of these important NAD+ analogues as chemical tool compounds for mechanistic studies with these as well as other NAD+-dependent enyzmes.


Assuntos
Sirtuínas , Difosfato de Adenosina , Epigênese Genética , Humanos , Simulação de Acoplamento Molecular , NAD/metabolismo , Sirtuína 2/metabolismo , Sirtuínas/metabolismo , Estereoisomerismo , Transferases/metabolismo
14.
Cell Rep ; 39(7): 110834, 2022 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-35584675

RESUMO

The evolution of zinc (Zn) as a protein cofactor altered the functional landscape of biology, but dependency on Zn also created an Achilles' heel, necessitating adaptive mechanisms to ensure Zn availability to proteins. A debated strategy is whether metallochaperones exist to prioritize essential Zn-dependent proteins. Here, we present evidence for a conserved family of putative metal transferases in human and fungi, which interact with Zn-dependent methionine aminopeptidase type I (MetAP1/Map1p/Fma1). Deletion of the putative metal transferase in Saccharomyces cerevisiae (ZNG1; formerly YNR029c) leads to defective Map1p function and a Zn-deficiency growth defect. In vitro, Zng1p can transfer Zn2+ or Co2+ to apo-Map1p, but unlike characterized copper chaperones, transfer is dependent on GTP hydrolysis. Proteomics reveal mis-regulation of the Zap1p transcription factor regulon because of loss of ZNG1 and Map1p activity, suggesting that Zng1p is required to avoid a compounding effect of Map1p dysfunction on survival during Zn limitation.


Assuntos
Proteínas de Saccharomyces cerevisiae , Transferases , Zinco , Aminopeptidases/genética , Aminopeptidases/metabolismo , Guanosina Trifosfato , Humanos , Metais/metabolismo , Metionina , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Transferases/fisiologia , Zinco/metabolismo
15.
Am J Med Genet A ; 188(8): 2339-2350, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35499143

RESUMO

Pontocerebellar hypoplasia (PCH) type 12 is a rare, perinatal lethal neurodegenerative genetic disorder caused by biallelic mutations in the COASY gene. Herein, we describe the clinical and neuroradiological profile of nine affected fetuses/neonates from five families identified with a common COASY: c.1486-3C>G biallelic variant. Four of the five families were identified after data reanalysis of unresolved, severe PCH like phenotype and the fifth family through collaboration. The common antenatal phenotype was cerebellar hypoplasia. Microcephaly, arthrogryposis, and intrauterine growth restriction were the shared postnatal findings. The neurological manifestations included seizures, poor sucking, and spasticity. Novel findings of corpus callosum agenesis, simplified gyral pattern, normal sized pons, optic neuropathy, and a small thorax are reported in this series. The allele frequency of the COASY: c.1486-3C>G variant was 0.62% in the available Asian Indian database. We describe this as a possible common Indian origin variant. To the best of our knowledge, this is the largest PCH12 series reported.


Assuntos
Doenças Cerebelares , Microcefalia , Transferases , Doenças Cerebelares/genética , Feminino , Humanos , Microcefalia/genética , Mutação , Fenótipo , Gravidez , Transferases/genética
16.
J Investig Med High Impact Case Rep ; 10: 23247096221097523, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35593442

RESUMO

Pure red cell aplasia (PRCA) is a rare disorder mainly affecting the erythroid precursor cells. It presents with severe isolated reticulocytopenia with relatively normal counts in the myeloid and megakaryocytic lineages. It has been attributed to numerous congenital and acquired causes. DNA Methyl Transferase 3 Alpha (DNMT3A) mutation has been typically associated with myeloid and lymphoid malignancies. There is a scarcity of data regarding the association of DNMT3A mutation with PRCA. We report a case of a 73-year-old man who initially presented with anemia and reticulocytopenia. After a thorough evaluation and eventual bone marrow biopsy, he was diagnosed with PRCA. Further genetic testing identified a DNMT3A mutation. We are reporting this rare case to highlight the fact that DNMT3A mutation can also present as isolated PRCA in and of itself without the co-occurrence of leukemia, lymphoma, or myelodysplastic syndrome (MDS).


Assuntos
Síndromes Mielodisplásicas , Aplasia Pura de Série Vermelha , Idoso , DNA , Humanos , Masculino , Mutação , Síndromes Mielodisplásicas/complicações , Síndromes Mielodisplásicas/genética , Aplasia Pura de Série Vermelha/complicações , Aplasia Pura de Série Vermelha/diagnóstico , Aplasia Pura de Série Vermelha/genética , Transferases
17.
Nat Commun ; 13(1): 2776, 2022 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-35589706

RESUMO

Toxic dipeptide-repeat (DPR) proteins are produced from expanded G4C2 repeats in the C9ORF72 gene, the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of ≥20 repeats inhibit the ribosome's peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryogenic electron microscopy (cryo-EM) reveals that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center (PTC). Consistent with these findings, the macrolide erythromycin, which binds in the tunnel, competes with poly-PR and restores peptidyl-transferase activity. Our results demonstrate that strong and specific binding of poly-PR and poly-GR in the ribosomal tunnel blocks translation, revealing the structural basis of their toxicity in C9ORF72-ALS/FTD.


Assuntos
Esclerose Amiotrófica Lateral , Demência Frontotemporal , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Microscopia Crioeletrônica , Dipeptídeos/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Humanos , Proteínas/genética , Proteínas/metabolismo , Ribossomos/metabolismo , Transferases
18.
Sci Rep ; 12(1): 7221, 2022 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-35508530

RESUMO

The development of drug resistance by Mycobacterium tuberculosis and other pathogenic bacteria emphasizes the need for new antibiotics. Unlike animals, most bacteria synthesize isoprenoid precursors through the MEP pathway. 1-Deoxy-D-xylulose 5-phosphate synthase (DXPS) catalyzes the first reaction of the MEP pathway and is an attractive target for the development of new antibiotics. We report here the successful use of a loop truncation to crystallize and solve the first DXPS structures of a pathogen, namely M. tuberculosis (MtDXPS). The main difference found to other DXPS structures is in the active site where a highly coordinated water was found, showing a new mechanism for the enamine-intermediate stabilization. Unlike other DXPS structures, a "fork-like" motif could be identified in the enamine structure, using a different residue for the interaction with the cofactor, potentially leading to a decrease in the stability of the intermediate. In addition, electron density suggesting a phosphate group could be found close to the active site, provides new evidence for the D-GAP binding site. These results provide the opportunity to improve or develop new inhibitors specific for MtDXPS through structure-based drug design.


Assuntos
Mycobacterium tuberculosis , Animais , Antibacterianos/farmacologia , Sítios de Ligação , Mycobacterium tuberculosis/metabolismo , Pentosefosfatos , Transferases/metabolismo
19.
World J Gastroenterol ; 28(16): 1671-1680, 2022 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-35581967

RESUMO

BACKGROUND: Coronavirus disease 2019 (COVID-19) has a spectrum of clinical syndromes with serious involvement of the lung and frequent effection of the liver and hemostatic system. Blood biomarkers are affordable, rapid, objective, and useful in the evaluation and prognostication of COVID-19 patients. AIM: To investigate the association between aspartate transferase-to-platelet ratio index (APRI) and in-hospital mortality to develop a COVID-19 mortality prediction model. METHODS: A multicenter cohort study with a retrospective design was conducted. Medical records of all consecutive adult patients admitted to Al-Azhar University Hospital (Assiut, Egypt) and Chest Hospital (Assiut, Egypt) with confirmed COVID-19 from July 1, 2020 to October 1, 2020, were retrieved and analyzed. The patient cohort was classified into the following two categories based on the APRI: (1) COVID-19 presenting with APRI ≤ 0.5; and (2) COVID-19 presenting with APRI (> 0.5 and ≤ 1.5). The association between APRI and all-cause in-hospital mortality was analyzed, and the new model was developed through logistic regression analyses. RESULTS: Of the 353 patients who satisfied the inclusion criteria, 10% were admitted to the intensive care unit (n = 36) and 7% died during the hospital stay (n = 25). The median age was 40 years and 50.7% were male. On admission, 49% had aspartate transferase-dominant liver injury. On admission, APRI (> 0.5 and ≤ 1.5) was independently associated with all-cause in-hospital mortality in unadjusted regression analysis and after adjustment for age and sex; after stepwise adjustment for several clinically relevant confounders, APRI was still significantly associated with all-cause in-hospital mortality. On admission, APRI (> 0.5 and ≤ 1.5) increased the odds of mortality by five-times (P < 0.006). From these results, we developed a new predictive model, the APRI-plus, which includes the four predictors of age, aspartate transferase, platelets, and serum ferritin. Performance for mortality was very good, with an area under the receiver operating curve of 0.90. CONCLUSION: APRI-plus is an accurate and simplified prediction model for mortality among patients with COVID-19 and is associated with in-hospital mortality, independent of other relevant predictors.


Assuntos
COVID-19 , Adulto , Aspartato Aminotransferases , Ácido Aspártico , Biomarcadores , Plaquetas , Estudos de Coortes , Feminino , Humanos , Cirrose Hepática , Masculino , Contagem de Plaquetas , Estudos Retrospectivos , Fatores de Risco , Transferases
20.
Molecules ; 27(10)2022 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-35630573

RESUMO

The house fly Musca domestica L. is one of the medical and veterinary pests that can develop resistance to different insecticides. Mixing insecticides is a new strategy for accelerating pest control; furthermore, it can overcome insect resistance to insecticides. This study aims to evaluate three insecticides, chlorfenapyr, abamectin, and lambda-cyhalothrin, individually and their binary mixtures against 2nd instar larvae of M. domestica laboratory strain. Chlorfenapyr exhibited the most toxic effect on larvae, followed by abamectin then the lambda-cyhalothrin. The half-lethal concentrations (LC50) values were 3.65, 30.6, and 94.89 ppm, respectively. These results revealed that the high potentiation effect was the mixture of abamectin/chlorfenapyr in all the mixing ratios. In contrast, the tested combination of lambda-cyhalothrin/abamectin showed an antagonism effect at all mixing ratios against house fly larvae. The total protein, esterases, glutathione-S-transferase (GST), and cytochrome P-450 activity were also measured in the current investigation in the larvae treated with chlorfenapyr. Our results indicate that GST may play a role in detoxifying chlorfenapyr in M. domestica larvae. The highest activity of glutathione-S-transferase was achieved in treated larvae with chlorfenapyr, and an increase in cytochrome P-450 activity in the larvae was observed post-treatment with Abamectin/chlorfenapyr.


Assuntos
Moscas Domésticas , Inseticidas , Animais , Sistema Enzimático do Citocromo P-450 , Glutationa , Resistência a Inseticidas , Inseticidas/farmacologia , Ivermectina/análogos & derivados , Larva , Nitrilas , Piretrinas , Transferases
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