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1.
Molecules ; 26(5)2021 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-33800069

RESUMO

New drugs with novel antibacterial targets for Gram-negative bacterial pathogens are desperately needed. The protein LpxC is a vital enzyme for the biosynthesis of lipid A, an outer membrane component of Gram-negative bacterial pathogens. The ACHN-975 molecule has high enzymatic inhibitory capacity against the infectious diseases, which are caused by multidrug-resistant bacteria, but clinical research was halted because of its inflammatory response in previous studies. In this work, the structure of the recombinant UDP-3-O-(R-3-hydroxymyristol)-N-acetylglucosamine deacetylase from Aquifex aeolicus in complex with ACHN-975 was determined to a resolution at 1.21 Å. According to the solved complex structure, ACHN-975 was docked into the AaLpxC's active site, which occupied the site of AaLpxC substrate. Hydroxamate group of ACHN-975 forms five-valenced coordination with resides His74, His226, Asp230, and the long chain part of ACHN-975 containing the rigid alkynyl groups docked in further to interact with the hydrophobic area of AaLpxC. We employed isothermal titration calorimetry for the measurement of affinity between AaLpxC mutants and ACHN-975, and the results manifest the key residues (His74, Thr179, Tyr212, His226, Asp230 and His253) for interaction. The determined AaLpxC crystal structure in complex with ACHN-975 is expected to serve as a guidance and basis for the design and optimization of molecular structures of ACHN-975 analogues to develop novel drug candidates against Gram-negative bacteria.


Assuntos
Amidoidrolases/química , Amidoidrolases/metabolismo , Antibacterianos/química , Bactérias Gram-Negativas/efeitos dos fármacos , Amidoidrolases/genética , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Aquifex/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Calorimetria , Cristalografia por Raios X , Conformação Proteica , Termodinâmica
2.
Molecules ; 26(7)2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-33916405

RESUMO

The study aimed to investigate the antibacterial activity of Mustard (Brassica juncea) and Moringa (Moringa oleifera) leaf extracts and coagulant protein for their potential application in water treatment. Bacterial cell aggregation and growth kinetics studies were employed for thirteen bacterial strains with different concentrations of leaf extracts and coagulant protein. Moringa oleifera leaf extract (MOS) and coagulant protein showed cell aggregation against ten bacterial strains, whereas leaf extract alone showed growth inhibition of five bacterial strains for up to 6 h and five bacterial strains for up to 3 h. Brassica juncea leaf extract (BJS) showed growth inhibition for up to 6 h, and three bacterial strains showed inhibition for up to 3 h. The highest inhibition concentration with 2.5 mg/mL was 19 mm, and furthermore, the minimum inhibitory concentration (MIC) (0.5 mg/mL) and MBC (1.5 mg/mL) were determined to have a higher antibacterial effect for <3 KDa peptides. Based on LCMS analysis, napin was identified in both MOS and BJS; furthermore, the mode of action of napin peptide was determined on lipoprotein X complex (LpxC) and four-chained structured binding protein of bacterial type II topoisomerase (4PLB). The docking analysis has exhibited moderate to potent inhibition with a range of dock score -912.9 Kcal/mol. Thus, it possesses antibacterial-coagulant potential bioactive peptides present in the Moringa oleifera purified protein (MOP) and Brassica juncea purified protein (BJP) that could act as an effective antimicrobial agent to replace currently available antibiotics. The result implies that MOP and Brassica juncea purified coagulant (BJP) proteins may perform a wide degree of antibacterial functions against different pathogens.


Assuntos
Albuminas 2S de Plantas/química , Antibacterianos/química , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Moringa oleifera/química , Mostardeira/química , Albuminas 2S de Plantas/isolamento & purificação , Albuminas 2S de Plantas/farmacologia , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/química , Amidoidrolases/genética , Amidoidrolases/metabolismo , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Sítios de Ligação , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/enzimologia , Bactérias Gram-Positivas/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Extratos Vegetais/química , Folhas de Planta/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas
3.
BMC Genomics ; 22(1): 287, 2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879046

RESUMO

BACKGROUND: Alanine:glyoxylate aminotransferase 2 (AGXT2; EC 2.6.1.44) is the only enzyme that degrades the R-form of 3-aminoisobutyrate, an intermediate metabolite of thymine. AGXT2, as well as diaminoarginine dimethylaminohydrolase 1 (DDAH1; EC 3.5.3.18), works as an enzyme that degrades asymmetric dimethylarginine (ADMA), which competitively inhibits the nitric oxide synthase family. Thus, these two enzyme activities may change vascular vulnerability for a lifetime via the nitric oxide (NO) system. We investigated the association between vascular conditions and diseases such as hypertension and diabetes mellitus and polymorphisms of these two genes in 750 older Japanese subjects (mean age ± standard deviation, 77.0 ± 7.6 years) recruited using the complete enumeration survey method in the Nakayama study. Demographic and biochemical data, such as blood pressure (BP) and casual blood sugar (CBS), were obtained. Four functional single nucleotide polymorphisms (SNPs; rs37370, rs37369, rs180749, and rs16899974) of AGXT2 and one functional insertion/deletion polymorphism in the promotor region with four SNPs (rs307894, rs669173, rs997251, and rs13373844) of DDAH1 were investigated. Plasma ADMA was also analyzed in 163 subjects. RESULTS: The results of multiple regression analysis showed that a loss of the functional haplotype of AGXT2, CAAA, was significantly positively correlated with BP (systolic BP, p = 0.034; diastolic BP, p = 0.025) and CBS (p = 0.021). No correlation was observed between DDAH1 and either BP or CBS. ADMA concentrations were significantly elevated in subjects with two CAAA haplotypes compared with subjects without the CAAA haplotype (p = 0.033). CONCLUSIONS: Missense variants of AGXT2, but not DDAH1, may be related to vulnerability to vascular diseases such as hypertension and DM via the NO system.


Assuntos
Glicemia , Polimorfismo de Nucleotídeo Único , Amidoidrolases/genética , Arginina , Pressão Sanguínea/genética , Humanos , Japão , Inquéritos e Questionários
4.
Artigo em Inglês | MEDLINE | ID: mdl-33722890

RESUMO

Pyrazinamide (PZA) is a widely used antitubercular chemotherapeutic. Typically, PZA resistance (PZA-R) emerges in Mycobacterium tuberculosis strains with existing resistance to isoniazid and rifampin (i.e., multidrug resistance [MDR]) and is conferred by loss-of-function pncA mutations that inhibit conversion to its active form, pyrazinoic acid (POA). PZA-R departing from this canonical scenario is poorly understood. Here, we genotyped pncA and purported alternative PZA-R genes (panD, rpsA, and clpC1) with long-read sequencing of 19 phenotypically PZA-monoresistant isolates collected in Sweden and compared their phylogenetic and genomic characteristics to a large set of MDR PZA-R (MDRPZA-R) isolates. We report the first association of ClpC1 mutations with PZA-R in clinical isolates, in the ClpC1 promoter (clpC1p -138) and the N terminus of ClpC1 (ClpC1Val63Ala). Mutations have emerged in both these regions under POA selection in vitro, and the N-terminal region of ClpC1 has been implicated further, through its POA-dependent efficacy in PanD proteolysis. ClpC1Val63Ala mutants spanned 4 Indo-Oceanic sublineages. Indo-Oceanic isolates invariably harbored ClpC1Val63Ala and were starkly overrepresented (odds ratio [OR] = 22.2, P < 0.00001) among PZA-monoresistant isolates (11/19) compared to MDRPZA-R isolates (5/80). The genetic basis of Indo-Oceanic isolates' overrepresentation in PZA-monoresistant tuberculosis (TB) remains undetermined, but substantial circumstantial evidence suggests that ClpC1Val63Ala confers low-level PZA resistance. Our findings highlight ClpC1 as potentially clinically relevant for PZA-R and reinforce the importance of genetic background in the trajectory of resistance development.


Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Amidoidrolases/genética , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Farmacorresistência Bacteriana/genética , Humanos , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/genética , Filogenia , Pirazinamida/farmacologia , Suécia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico
5.
Pestic Biochem Physiol ; 173: 104770, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33771270

RESUMO

Chitin deacetylases (CDAs, EC 3.5.1.41) catalyze the N-deacetylation of chitin to produce chitosan, which is essential for insect survival. Hence, CDAs are promising targets for the development of novel insecticidal drugs. In this study, the putative Group I chitin deacetylase genes HpCDA1, HpCDA2-1 and HpCDA2-2 were identified from Holotrichia parallela. Conserved domain database search identified a chitin-binding peritrophin-A domain (ChBD), a low-density lipoprotein receptor class A domain (LDLa), and a putative CDA-like catalytic domain. RT-qPCR analysis showed that the Group I HpCDAs were expressed in various tissues and predominant in the integument. The developmental expression patterns from the first-instar to third-instar larvae showed that HpCDAs were highly expressed on the first day and gradually declined after molting. The functional characteristics of the Group I CDAs in cuticle organization were examined using RNA interference (RNAi) and transmission electron microscopy (TEM) methods. Administration of double-stranded HpCDA (dsHpCDA) through larval injection could suppress the expression levels of HpCDA1 and HpCDA2, thus resulting in abnormal or lethal phenotypes. TEM analysis revealed that RNAi of either HpCDA1 or HpCDA2 remarkably affected the cuticle integrity, as evidenced by cuticle disorganization and chitin laminae disruption, suggesting the crucial role of CDAs in chitin modification. These experimental results demonstrate the important contribution of putative key genes involved in chitin metabolism, and provide a foundation for developing new strategies to control H. parallela.


Assuntos
Besouros , Amidoidrolases/genética , Amidoidrolases/metabolismo , Animais , Quitina/metabolismo , Besouros/genética , Besouros/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Interferência de RNA
6.
Int J Mol Sci ; 22(3)2021 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-33530477

RESUMO

In migraine pain, cannabis has a promising analgesic action, which, however, is associated with side psychotropic effects. To overcome these adverse effects of exogenous cannabinoids, we propose migraine pain relief via activation of the endogenous cannabinoid system (ECS) by inhibiting enzymes degrading endocannabinoids. To provide a functional platform for such purpose in the peripheral and central parts of the rat nociceptive system relevant to migraine, we measured by activity-based protein profiling (ABPP) the activity of the main endocannabinoid-hydrolases, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). We found that in trigeminal ganglia, the MAGL activity was nine-fold higher than that of FAAH. MAGL activity exceeded FAAH activity also in DRG, spinal cord and brainstem. However, activities of MAGL and FAAH were comparably high in the cerebellum and cerebral cortex implicated in migraine aura. MAGL and FAAH activities were identified and blocked by the selective and potent inhibitors JJKK-048/KML29 and JZP327A, respectively. The high MAGL activity in trigeminal ganglia implicated in the generation of nociceptive signals suggests this part of ECS as a priority target for blocking peripheral mechanisms of migraine pain. In the CNS, both MAGL and FAAH represent potential targets for attenuation of migraine-related enhanced cortical excitability and pain transmission.


Assuntos
Amidoidrolases/metabolismo , Endocanabinoides/metabolismo , Transtornos de Enxaqueca/etiologia , Transtornos de Enxaqueca/metabolismo , Monoacilglicerol Lipases/metabolismo , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/genética , Animais , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiopatologia , Modelos Animais de Doenças , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Hidrólise , Masculino , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/genética , Nervos Periféricos/metabolismo , Nervos Periféricos/fisiopatologia , Ratos
7.
Int J Cardiol ; 331: 189-198, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33535073

RESUMO

BACKGROUND: Endothelial dysfunction is a key event in the development of vascular diseases, including atherosclerosis. Endothelial progenitor cells (EPCs) play an important role in vascular repair. Decreased dimethylarginine dimethylaminohydrolase (DDAH) activity is observed in several pathological conditions, and it is associated with an increased risk of vascular disease. We hypothesized that bone marrow-derived EPCs and combination therapy with DDAH2-EPCs could reduce plaque size and ameliorate endothelial dysfunction in an atherosclerosis rabbit model. METHOD: Four groups of rabbits (n = 8 per group) were subjected to a hyperlipidemic diet for a month. After establishing the atherosclerosis model, rabbits received 4 × 106 EPC, EPCs expressing DDAH2, through femoral vein injection, or saline (the control group with basic food and the untreated group). One month after transplantation, plaque thickness, endothelial function, oxidative stress, and inflammatory mRNAs, DDAH, and eNOS function were assessed. RESULTS: DDAH2-EPCs transplantation (p < 0.05) and EPCs transplantation (p < 0.05) were both associated with a reduction in plaque size compared to the control saline injection. The antiproliferative and antiatherogenic effects of EPCs were further enhanced by the overexpression of DDAH2 (p < 0.05, DDAH2-EPCs vs. EPCs). Furthermore, DDAH2-EPCs transplantation significantly increased endothelium integrity compared to the EPCs transplantation. CONCLUSION: Transplantation of EPCs overexpressing DDAH2 may enhance the repair of injured endothelium by reducing inflammation and restoring endothelial function. Therefore, pCMV6-mediated DDAH2 gene-transfected EPCs are a potentially valuable tool for the treatment of atherosclerosis.


Assuntos
Aterosclerose , Células Progenitoras Endoteliais , Amidoidrolases/genética , Animais , Arginina , Aterosclerose/genética , Aterosclerose/terapia , Coelhos
8.
Med Sci Monit ; 27: e928813, 2021 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-33619241

RESUMO

BACKGROUND Aminoacylase 1 (ACY-1) is a cytosolic enzyme that catalyzes amino acid deacylation and has been reported to participate in various human diseases. However, the role and mechanism of ACY-1 in neuroblastoma (NB) are not completely understood. The aim of this study was to elucidate the role of ACY-1 in NB. MATERIAL AND METHODS Overexpression and knockdown of ACY-1 in human NB cells were performed, and the transfection efficiency was assessed through fluorescence microscopy, real-time PCR, and western blotting. The effect of ACY-1 on tumorigenesis and metastasis was determined by cell counting, colony formation, wound healing, flow cytometry, and transwell invasion assays in vitro, and the signaling pathway was examined using western blotting. RESULTS ACY-1 overexpression inhibited proliferation and induced apoptosis in human NB cells. ACY-1 inhibited the colony formation ability, migration, and invasion of SH-SY5Y cell lines. Moreover, the ERK1/2 and TGF-ß1 signaling pathways were more active when ACY-1 was overexpressed in NB cells. However, the knockdown of ACY-1 in SH-SY5Y cell lines showed the opposite effects. CONCLUSIONS ACY-1 regulates the proliferation, migration, and invasion of human NB cells through the ERK1/2 and TGF-ß1 signaling pathways, implying that ACY-1 may serve as a therapeutic target for patients with NB.


Assuntos
Amidoidrolases/metabolismo , Neuroblastoma/metabolismo , Amidoidrolases/genética , Apoptose/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Transfecção , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/fisiologia
9.
Nat Commun ; 12(1): 545, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33483499

RESUMO

In Rhizobiales bacteria, such as Sinorhizobium meliloti, cell elongation takes place only at new cell poles, generated by cell division. Here, we show that the role of the FtsN-like protein RgsS in S. meliloti extends beyond cell division. RgsS contains a conserved SPOR domain known to bind amidase-processed peptidoglycan. This part of RgsS and peptidoglycan amidase AmiC are crucial for reliable selection of the new cell pole as cell elongation zone. Absence of these components increases mobility of RgsS molecules, as well as abnormal RgsS accumulation and positioning of the growth zone at the old cell pole in about one third of the cells. These cells with inverted growth polarity are able to complete the cell cycle but show partially impaired chromosome segregation. We propose that amidase-processed peptidoglycan provides a landmark for RgsS to generate cell polarity in unipolarly growing Rhizobiales.


Assuntos
Amidoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Sinorhizobium meliloti/metabolismo , Amidoidrolases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Western Blotting , Divisão Celular/genética , Proteínas de Membrana/genética , Microscopia de Fluorescência , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/crescimento & desenvolvimento
10.
Biomed Pharmacother ; 134: 111149, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33385683

RESUMO

E. coli is associated with high rates of infection and resistance to drugs not only in China but also the rest of the world. In addition, the number of E. coli biofilm infections continue to increase with time. Notably, biofilms are attractive targets for the prevention of infections caused by multidrug-resistant bacteria. Moreover, the pgaABCD-encoded Poly-ß-1,6-N-acetyl-d-glucosamine (PNAG) plays an important role in biofilm formation. Therefore, this study aimed to explore the specific effect of the (R)-(+)-pulegone (PU) on growth and biofilm formation in multi-drug resistant E. coli. The molecular mechanisms involved were also examined. The results showed that PU had significant antibacterial and antibiofilm formation activity against E. coli K1, with MIC and MBC values of 23.68 and 47.35 mg/mL, respectively. On the other hand, the maximum inhibition rate for biofilm formation in the bacterium was 52.36 % at 94.70 mg/mL of PU. qRT-PCR data showed that PU significantly down-regulated expression of the pgaABCD genes (P < 0.05). PU was also broadly effective against biofilm formation in MG1655 and MG1655/ΔpgaABCD, exhibiting the maximum inhibition rates were 98.23 % and 93.35 %, respectively. In addition, PU destroyed pre-formed mature biofilm in both MG1655 and MG1655/ΔpgaABCD about 95.03 % and 92.4 %, respectively. The study therefore verified that pgaA was a potential and key target for PU in E. coli although it was not the only one. Overall, the findings indicated that PU is a potential and novel inhibitor of drug resistance, This therefore gives insights on new ways of preventing and treating biofilm-associated infections in the food industry as well as in clinical practice.


Assuntos
Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Biofilmes/efeitos dos fármacos , Monoterpenos Cicloexânicos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli K12/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Amidoidrolases/genética , Biofilmes/crescimento & desenvolvimento , Escherichia coli K12/genética , Escherichia coli K12/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Testes de Sensibilidade Microbiana
11.
Cell Host Microbe ; 29(1): 1-3, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33444551

RESUMO

As vectors of microbial diseases in vertebrates, ticks are excellent at regulating bacterial proliferation in and around them. In a recent issue of Cell, Hayes et al. (2020) reveal acarid toxins of bacterial origin that help eliminate microbes that are pathogenic to black-legged ticks but commensal to their vertebrate hosts.


Assuntos
Ixodes , Amidoidrolases/genética , Animais , Fatores Imunológicos , Simbiose , Vertebrados
12.
J Pharmacol Exp Ther ; 376(1): 1-11, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33087396

RESUMO

The endocannabinoid, anandamide (AEA), stimulates cannabinoid receptors (CBRs) and is enriched in the kidney, especially the renal medulla. AEA infused into the renal outer medulla of mice stimulates urine flow rate and salt excretion. Here we show that these effects are blocked by the CBR type 1 (CB1) inverse agonist, rimonabant. Immunohistochemical analysis demonstrated the presence of CB1 in thick ascending limb (TAL) tubules. Western immunoblotting demonstrated the presence of CB1 (52 kDa) in the cortex and outer medulla of mouse kidney. The effect of direct [CP55940 (CP) or AEA] or indirect [fatty acyl amide hydrolase (FAAH) inhibitor, PF3845 (PF)] cannabinoidimetics on Na+ transport in isolated mouse TAL tubules was studied using the Na+-sensitive dye, SBFI-AM. Switching from 0 Na+ solution to control Ringer's solution (CR) rapidly increased TAL cell [Na+]i Addition of CP to CR produced a further elevation, similar in magnitude to that of ouabain, a Na+-K+-ATPase inhibitor. This [Na+]i-elevating effect of CP was time-dependent, required the presence of Na+ in the bathing solution, and was insensitive to Na+-K+-2Cl- cotransporter inhibition. Addition of PF to CR elevated [Na+]i in FAAH wild-type but not FAAH knockout (KO) TALs, whereas the additions of CP and AEA to PF-treated FAAH KO TALs increased [Na+]i An interaction between cannabinoidimetics and ouabain (Ou) was observed. Ou produced less increase in [Na+]i after cannabinoidimetic treatment, whereas cannabinoidimetics had less effect after Ou treatment. It is concluded that cannabinoidimetics, including CP and AEA, inhibit Na+ transport in TALs by inhibiting Na+ exit via Na+-K+-ATPase. SIGNIFICANCE STATEMENT: Cannabinoids including endocannabinoids induce renal urine and salt excretion and are proposed to play a physiological role in the regulation of blood pressure. Our data suggest that the mechanism of the cannabinoids involves inhibition of the sodium pump, Na+-K+-ATPase, in thick ascending limb cells and, likely, other proximal and distal tubular segments of the kidney nephron.


Assuntos
Antagonistas de Receptores de Canabinoides/farmacologia , Cicloexanóis/farmacologia , Diurese , Alça do Néfron/metabolismo , Natriurese , Rimonabanto/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/genética , Animais , Ácidos Araquidônicos/farmacologia , Agonistas de Receptores de Canabinoides/farmacologia , Endocanabinoides/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ouabaína/farmacologia , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Piridinas/farmacologia , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores
13.
Protein Expr Purif ; 177: 105751, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32931916

RESUMO

A novel amidase (TAM) was identified and cloned from the genome of Thauera sinica K11. The recombinant protein was purified to homogeneity by one-step affinity chromatography for up to 26.4-fold with a yield of 38.1%. Gel filtration chromatography and SDS-PAGE revealed that the enzyme was a tetramer with a subunit of approximately 37.5 kDa. The amidase exhibited the maximum acyl transfer activity at 45 °C and pH 7.0, and it was highly stable over a wide pH range of 6.0-11.0. Inhibition of enzyme activity was observed in the presence of metal ions, thiol reagents and organic solvents. TAM showed a broad substrate spectrum toward aliphatic, aromatic and heterocyclic amides. For linear aliphatic monoamides, the acyl transfer activity of TAM was decreased with the extension of the carbon chain length, and thus the highest activity of 228.2 U/mg was obtained when formamide was used as substrate. This distinct selectivity of amidase to linear aliphatic monoamides expanded the findings of signature amidases to substrate specificity.


Assuntos
Amidas/metabolismo , Amidoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Clonagem Molecular/métodos , Subunidades Proteicas/metabolismo , Thauera/enzimologia , Amidoidrolases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Genoma Bacteriano , Concentração de Íons de Hidrogênio , Cinética , Filogenia , Multimerização Proteica , Subunidades Proteicas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Temperatura , Thauera/classificação , Thauera/genética
14.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(11): 1241-1243, 2020 Nov 10.
Artigo em Chinês | MEDLINE | ID: mdl-33179229

RESUMO

OBJECTIVE: To analyze the molecular etiology of a Chinese child affected with dihydropyrimidinase deficiency. METHODS: Genomic DNA was extracted from peripheral blood samples of the family members. Pathogenic variant was determined by whole exome sequencing and verified by Sanger sequencing. RESULTS: The child was found to harbor homozygous c.905G>A (p.Arg302Gln) variants in exon 5 of the DPYS gene, for which her parents were both heterozygous carriers. CONCLUSION: The homozygous c.905G>A (p.Arg302Gln) variants of the DPYS gene probably underlies the dihydropyrimidinase deficiency in the child. Above result has enabled genetic counseling and prenatal diagnosis for this family.


Assuntos
Amidoidrolases/genética , Erros Inatos do Metabolismo/genética , Grupo com Ancestrais do Continente Asiático/genética , Criança , Éxons , Feminino , Humanos , Mutação , Linhagem
15.
PLoS One ; 15(7): e0235643, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735615

RESUMO

BACKGROUND: Pyrazinamide is an important drug against the latent stage of tuberculosis and is used in both first- and second-line treatment regimens. Pyrazinamide-susceptibility test usually takes a week to have a diagnosis to guide initial therapy, implying a delay in receiving appropriate therapy. The continued increase in multi-drug resistant tuberculosis and the prevalence of pyrazinamide resistance in several countries makes the development of assays for prompt identification of resistance necessary. The main cause of pyrazinamide resistance is the impairment of pyrazinamidase function attributed to mutations in the promoter and/or pncA coding gene. However, not all pncA mutations necessarily affect the pyrazinamidase function. OBJECTIVE: To develop a methodology to predict pyrazinamidase function from detected mutations in the pncA gene. METHODS: We measured the catalytic constant (kcat), KM, enzymatic efficiency, and enzymatic activity of 35 recombinant mutated pyrazinamidase and the wild type (Protein Data Bank ID = 3pl1). From all the 3D modeled structures, we extracted several predictors based on three categories: structural stability (estimated by normal mode analysis and molecular dynamics), physicochemical, and geometrical characteristics. We used a stepwise Akaike's information criterion forward multiple log-linear regression to model each kinetic parameter with each category of predictors. We also developed weighted models combining the three categories of predictive models for each kinetic parameter. We tested the robustness of the predictive ability of each model by 6-fold cross-validation against random models. RESULTS: The stability, physicochemical, and geometrical descriptors explained most of the variability (R2) of the kinetic parameters. Our models are best suited to predict kcat, efficiency, and activity based on the root-mean-square error of prediction of the 6-fold cross-validation. CONCLUSIONS: This study shows a quick approach to predict the pyrazinamidase function only from the pncA sequence when point mutations are present. This can be an important tool to detect pyrazinamide resistance.


Assuntos
Amidoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/enzimologia , Amidoidrolases/química , Amidoidrolases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clonagem Molecular , Cinética , Modelos Lineares , Simulação de Dinâmica Molecular , Mutagênese , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
16.
Am J Physiol Heart Circ Physiol ; 319(3): H582-H603, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32762558

RESUMO

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by null mutations in dystrophin and characterized by muscle degeneration. Cardiomyopathy is common and often prevalent at similar frequency in female DMD carriers irrespective of whether they manifest skeletal muscle disease. Impaired muscle nitric oxide (NO) production in DMD disrupts muscle blood flow regulation and exaggerates postexercise fatigue. We show that circulating levels of endogenous methylated arginines including asymmetric dimethylarginine (ADMA), which act as NO synthase inhibitors, are elevated by acute necrotic muscle damage and in chronically necrotic dystrophin-deficient mice. We therefore hypothesized that excessive ADMA impairs muscle NO production and diminishes exercise tolerance in DMD. We used transgenic expression of dimethylarginine dimethylaminohydrolase 1 (DDAH), which degrades methylated arginines, to investigate their contribution to exercise-induced fatigue in DMD. Although infusion of exogenous ADMA was sufficient to impair exercise performance in wild-type mice, transgenic DDAH expression did not rescue exercise-induced fatigue in dystrophin-deficient male mdx mice. Surprisingly, DDAH transgene expression did attenuate exercise-induced fatigue in dystrophin-heterozygous female mdx carrier mice. Improved exercise tolerance was associated with reduced heart weight and improved cardiac ß-adrenergic responsiveness in DDAH-transgenic mdx carriers. We conclude that DDAH overexpression increases exercise tolerance in female DMD carriers, possibly by limiting cardiac pathology and preserving the heart's responses to changes in physiological demand. Methylated arginine metabolism may be a new target to improve exercise tolerance and cardiac function in DMD carriers or act as an adjuvant to promote NO signaling alongside therapies that partially restore dystrophin expression in patients with DMD.NEW & NOTEWORTHY Duchenne muscular dystrophy (DMD) carriers are at risk for cardiomyopathy. The nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is released from damaged muscle in DMD and impairs exercise performance. Transgenic expression of dimethylarginine dimethylaminohydrolase to degrade ADMA prevents cardiac hypertrophy, improves cardiac function, and improves exercise tolerance in DMD carrier mice. These findings highlight the relevance of ADMA to muscular dystrophy and have important implications for therapies targeting nitric oxide in patients with DMD and DMD carriers.


Assuntos
Arginina/análogos & derivados , Cardiomiopatias/metabolismo , Circulação Coronária , Tolerância ao Exercício , Heterozigoto , Distrofia Muscular de Duchenne/metabolismo , Miocárdio/metabolismo , Músculo Quadríceps/metabolismo , Amidoidrolases/genética , Amidoidrolases/metabolismo , Animais , Arginina/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/fisiopatologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatologia , Miocárdio/patologia , Necrose , Músculo Quadríceps/patologia , Músculo Quadríceps/fisiopatologia , Função Ventricular Esquerda
17.
Cancer Sci ; 111(9): 3359-3366, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32619063

RESUMO

Cancer treatment with a fluoropyrimidine (FP) is often accompanied by severe toxicity that may be dependent on the activity of catalytic enzymes encoded by the DPYD, DPYS, and UPB1 genes. Genotype-guided dose individualization of FP therapy has been proposed in western countries, but our knowledge of the relevant genetic variants in East Asian populations is presently limited. To investigate the association between these genetic variations and FP-related high toxicity in a Japanese population, we obtained blood samples from 301 patients who received this chemotherapy and sequenced the coding exons and flanking intron regions of their DPYD, DPYS, and UPB1 genes. In total, 24 single nucleotide variants (15 in DPYD, 7 in DPYS and 2 in UPB1) were identified including 3 novel variants in DPYD and 1 novel variant in DPYS. We did not find a significant association between FP-related high toxicity and each of these individual variants, although a certain trend toward significance was observed for p.Arg181Trp and p.Gln334Arg in DPYS (P = .0813 and .087). When we focused on 7 DPYD rare variants (p.Ser199Asn, p.IIe245Phe, p.Thr305Lys, p.Glu386Ter, p.Ser556Arg, p.Ala571Asp, p.Trp621Cys) which have an allele frequency of less than 0.01% in the Japanese population and are predicted to be loss-of-function mutations by in silico analysis, the group of patients who were heterozygous carriers of at least one these rare variants showed a strong association with FP-related high toxicity (P = .003). Although the availability of screening of these rare loss-of-function variants is still unknown, our data provide useful information that may help to alleviate FP-related toxicity in Japanese patients with cancer.


Assuntos
Amidoidrolases/genética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/etiologia , Predisposição Genética para Doença , Variação Genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/tratamento farmacológico , Neoplasias/patologia
18.
BMC Med Genet ; 21(1): 138, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32600357

RESUMO

BACKGROUND: Dihidropyrimidinase (DHP) deficiency is an inherited inborn error of pyrimidine metabolism with a variable clinical presentation and even asymptomatic subjects. Dihydropyrimidinase is encoded by the DPYS gene, thus pathogenic mutations in this gene can cause DHP deficiency. To date, several variations in the DPYS gene have been reported but only 23 of them have been confirmed to be pathogenic. Therefore, the biochemical, clinical and genetic aspects of this disease are still unclear. CASE PRESENTATION: Here, we report a 22-year-old woman with DHP deficiency. To identify the genetic cause of DHP deficiency in this patient, Whole Exome Sequencing (WES) was performed, which revealed a novel homozygote stop gain mutation (NM_001385: Exon 9, c.1501 A > T, p.K501X) in the DPYS gene. Sanger sequencing was carried out on proband and other family members in order to confirm the identified mutation. According to the homozygote genotype of the patient and heterozygote genotype of her parents, the autosomal recessive pattern of inheritance was confirmed. In addition, bioinformatics analysis of the identified variant using Mutation Taster and T-Coffee Multiple Sequence Alignment showed the pathogenicity of mutation. Moreover, mRNA expression level of DPYS gene in the proband's liver biopsy showed about 6-fold reduction compared to control, which strongly suggested the pathogenicity of the identified mutation. CONCLUSIONS: This study identified a novel pathogenic stop gain mutation in DPYS gene in a DHP deficient patient. Our findings can improve the knowledge about the genetic basis of the disease and also provide information for accurate genetic counseling for the families at risk of these types of disorders.


Assuntos
Amidoidrolases/genética , Códon sem Sentido/genética , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Mutação/genética , Amidoidrolases/química , Sequência de Aminoácidos , Sequência de Bases , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Masculino , Linhagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adulto Jovem
19.
Exp Anim ; 69(4): 388-394, 2020 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-32507787

RESUMO

We recently demonstrated that aspartoacylase (Aspa) and hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (Hcn1) genes were causative of essential tremor (ET) in rats. This finding was obtained using Aspaem34Kyo/Hcn1A354V double-mutant rats, but they were bred on a heterogeneous genetic background of two strains, F344 and WTC. Here, we developed an Aspaem34Kyo/Hcn1em1Kyo double-knockout rat strain with a homogenous F344 genetic background and studied the ability of glutamate receptor antagonists to suppress ET. The F344-Aspa/Hcn1 double-knockout rats exhibited spontaneous, intense body tremor equivalent to that in the double-mutant rats. N-acetyl-aspartate (NAA), a substrate of ASPA, showed accumulation in all brain regions and in the spinal cord. However, N-acetyl-aspartyl-glutamate (NAAG), which is derived from NAA and interacts with glutamatergic receptors, was decreased in the medulla oblongata of the double-knockout rats. The tremor was suppressed by 3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid, an N-methyl-D-aspartate (NMDA) receptor antagonist, in F344-Aspa/Hcn1 double-knockout rats. The non-NMDA glutamate receptor antagonist NBQX weakly inhibited the tremor, while the metabotropic glutamate receptor antagonist LY341495 showed no effect. In addition, both NR2B subunit-specific (Ro 25-6981) and NR2C/NR2D subunit-specific (cis-piperidine dicarboxylic acid) NMDA receptor antagonists suppressed the tremor. These data indicated that the pathogenesis of tremor in Aspa/Hcn1 double-knockout rats involved ionotropic glutamate receptors, particularly NMDA receptors.


Assuntos
Amidoidrolases/genética , Tremor Essencial/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais de Potássio/genética , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/fisiologia , Amidoidrolases/metabolismo , Animais , Encéfalo/metabolismo , Tremor Essencial/tratamento farmacológico , Técnicas de Inativação de Genes , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Terapia de Alvo Molecular , Fenóis/farmacologia , Fenóis/uso terapêutico , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Canais de Potássio/metabolismo , Quinoxalinas/farmacologia , Quinoxalinas/uso terapêutico , Ratos Endogâmicos F344 , Ratos Mutantes , Medula Espinal/metabolismo
20.
PLoS One ; 15(5): e0232543, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32413052

RESUMO

INTRODUCTION: Susceptibility testing for pyrazinamide (PZA), a cornerstone anti-TB drug is not commonly done in Uganda because it is expensive and characterized with technical difficulties thus resistance to this drug is less studied. Resistance is commonly associated with mutations in the pncA gene and its promoter region. However, these mutations vary geographically and those conferring phenotypic resistance are unknown in Uganda. This study determined the prevalence of PZA resistance and its association with pncA mutations. MATERIALS AND METHODS: Using a cross-sectional design, archived isolates collected during the Uganda national drug resistance survey between 2008-2011 were sub-cultured. PZA resistance was tested by BACTEC Mycobacterial Growth Indicator Tube (MGIT) 960 system. Sequence reads were downloaded from the NCBI Library and bioinformatics pipelines were used to screen for PZA resistance-conferring mutations. RESULTS: The prevalence of phenotypic PZA resistance was found to be 21%. The sensitivity and specificity of pncA sequencing were 24% (95% CI, 9.36-45.13%) and 100% (73.54% - 100.0%) respectively. We identified four mutations associated with PZA phenotypic resistance in Uganda; K96R, T142R, R154G and V180F. CONCLUSION: There is a high prevalence of phenotypic PZA resistance among TB patients in Uganda. The low sensitivity of pncA gene sequencing confirms the already documented discordances suggesting other mechanisms of PZA resistance in Mycobacterium tuberculosis.


Assuntos
Amidoidrolases/genética , Antituberculosos/uso terapêutico , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Pulmonar/tratamento farmacológico , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/genética , Prevalência , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Pulmonar/microbiologia , Uganda/epidemiologia
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