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2.
Biomed Res Int ; 2021: 3598000, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34761004

RESUMO

Amides derived from ferulic acid have a wide spectrum of pharmacological activities, including antitumor and antifungal activity. In the present study, a series of ten amides were obtained by coupling reactions using the reagents (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyBOP) and N,N'-dicyclohexylcarbodiimide (DCC). All the compounds were identified on the basis of their IR, 1H- and 13C-NMR, HRMS data, and with yields ranging from 43.17% to 91.37%. The compounds were subjected to cytotoxic tests by the alamar blue technique and antifungal screening by the broth microdilution method to determine the minimum inhibitory concentration (MIC). The amides 10 and 11 displayed the best result in both biological evaluations, and compound 10 was the most potent and selective in HL-60 cancer cells, with no cytotoxicity on healthy cells. This amide had antifungal activity in all strains and had the lowest MIC against Candida albicans and Candida tropicalis. The possible mechanism of antifungal action occurs via the fungal cell wall. Molecular modeling suggested that compounds 10 and 11 interact with the enzymes GWT1 and GSC1, which are essential for the development of C. albicans. The findings of the present study demonstrated that compounds 10 and 11 may be used as a platform in drug development in the future.


Assuntos
Ácidos Cumáricos/farmacologia , Dicicloexilcarbodi-Imida/química , Compostos Organofosforados/química , Triazóis/química , Amidas/química , Amidas/farmacologia , Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Ácidos Cumáricos/química , Dicicloexilcarbodi-Imida/farmacologia , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Óleos Voláteis/química , Compostos Organofosforados/farmacologia , Triazóis/farmacologia
3.
IUBMB Life ; 73(6): 883-892, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33773019

RESUMO

Escherichia coli is able to utilize the mixture of carbon sources and produce molecular hydrogen (H2 ) via formate hydrogen lyase (FHL) complexes. In current work role of transcriptional activator of formate regulon FhlA in generation of fermentation end products and proton motive force, N'N'-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase activity at 20 and 72 hr growth during utilization of mixture of glucose, glycerol, and formate were investigated. It was shown that in fhlA mutant specific growth rate was ~1.5 fold lower compared to wt, while addition of DCCD abolished the growth in fhlA but not in wt. Formate was not utilized in fhlA mutant but wt cells simultaneously utilized formate with glucose. Glycerol utilization started earlier (from 2 hr) in fhlA than in wt. The DCCD-sensitive ATPase activity in wt cells membrane vesicles increased ~2 fold at 72 hr and was decreased 70% in fhlA. Addition of formate in the assays increased proton ATPase activity in wt and mutant strain. FhlA absence mainly affected the ΔpH but not ΔΨ component of Δp in the cells grown at 72 hr but not in 24 hr. The Δp in wt cells decreased from 24 to 72 hr of growth ~40 mV while in fhlA mutant it was stable. Taken together, it is suggested that FhlA regulates the concentration of fermentation end products and via influencing FO F1 -ATPase activity contributes to the proton motive force generation.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Força Próton-Motriz/genética , ATPases Translocadoras de Prótons/genética , Transativadores/fisiologia , Acetatos/metabolismo , Carbono/metabolismo , Dicicloexilcarbodi-Imida/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Fermentação , Formiatos/metabolismo , Formiatos/farmacologia , Glucose/metabolismo , Glicerol/metabolismo , Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Oxirredução , Transativadores/genética
4.
Lett Appl Microbiol ; 72(6): 669-676, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32955753

RESUMO

Acanthopanax (A.) henryi (Oliv.) Harms contain many bioactive compounds commonly used in traditional Chinese medicine. The objective of the present study was to investigate the antibacterial activity of the single constituent, Eleutheroside K (ETSK) isolated from the leaves of A. henryi (Oliv.) Harms, against methicillin-resistant Staphylococcus (S.) aureus (MRSA). Broth microdilution assay was used to measure the minimal inhibitory concentration (MIC) and the MIC values of ETSK against eight clinical S. aureus strains were all 50 µg ml-1 . At sub-inhibitory concentrations, a synergistic effect between oxacillin (OXA) and ETSK was confirmed using checkerboard dilution assay and time-kill curve analysis. The bacteriostatic effect became more pronounced when ETSK was used in combination with detergent (Triton X-100) or ATPase inhibitor (N, N'-dicyclohexylcarbodiimide). According to western blot analysis, the down-regulated expression of Penicillin-binding protein 2a (PBP2a) further validated that the bacterial activity was inhibited when treated with ETSK in a dose-dependent manner. Results based on our study verified that ETSK significantly suppressed MRSA infections and emphasized the potential application of ETSK as a novel anti-MRSA natural drug.


Assuntos
Antibacterianos/farmacologia , Eleutherococcus/metabolismo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oxacilina/farmacologia , Extratos Vegetais/farmacologia , Dicicloexilcarbodi-Imida/farmacologia , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Eleutherococcus/química , Resistência a Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Octoxinol/farmacologia , Proteínas de Ligação às Penicilinas/biossíntese , Folhas de Planta/química
5.
Biochemistry (Mosc) ; 85(8): 930-937, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33045953

RESUMO

The effects of N,N'-dicyclohexylcarbodiimide (DCCD), non-specific inhibitor of various transport systems functioning in biological membranes, on Na+-transporting P-type ATPase of the green halotolerant microalga Dunaliella maritima were studied in the experiments with vesicular plasma membranes isolated from the alga cells. The effects of DCCD on electrogenic/ion transport function of the enzyme and its ATP hydrolase activity were investigated. Electrogenic/ion transport function of the enzyme was recorded as a Na+-dependent generation of electric potential on the vesicle membranes with the help of the potential-sensitive probe oxonol VI. It was found that unlike many other ion-transporting ATPases, the Na+-ATPase of D. maritima is insensitive to DCCD. This agent did not inhibit either ATP hydrolysis catalyzed by this enzyme or its transport activity. At the same time DCCD affected the ability of the vesicle membranes to maintain electric potential generated by the D. maritima Na+-ATPase. The observed effects can be explained based on the assumption that DCCD interacts with the Na+/H+ antiporter in the plasma membrane of D. maritima.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Clorofíceas/enzimologia , Dicicloexilcarbodi-Imida/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Microalgas/enzimologia , Transdução de Sinais/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Hidrólise/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , ATPases do Tipo-P/metabolismo , Prótons
6.
Commun Biol ; 3(1): 431, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770029

RESUMO

rnf genes are widespread in bacteria and biochemical and genetic data are in line with the hypothesis that they encode a membrane-bound enzyme that oxidizes reduced ferredoxin and reduces NAD and vice versa, coupled to ion transport across the cytoplasmic membrane. The Rnf complex is of critical importance in many bacteria for energy conservation but also for reverse electron transport to drive ferredoxin reduction. However, the enzyme has never been purified and thus, ion transport could not be demonstrated yet. Here, we have purified the Rnf complex from the anaerobic, fermenting thermophilic bacterium Thermotoga maritima and show that is a primary Na+ pump. These studies provide the proof that the Rnf complex is indeed an ion (Na+) translocating, respiratory enzyme. Together with a Na+-F1FO ATP synthase it builds a simple, two-limb respiratory chain in T. maritima. The physiological role of electron transport phosphorylation in a fermenting bacterium is discussed.


Assuntos
Proteínas de Bactérias/metabolismo , Fermentação , Sódio/metabolismo , Thermotoga maritima/enzimologia , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/isolamento & purificação , Dicicloexilcarbodi-Imida/farmacologia , Fermentação/efeitos dos fármacos , Ferredoxinas/metabolismo , Glucose/metabolismo , Hidrólise , Transporte de Íons/efeitos dos fármacos , Ionóforos/farmacologia , Lipossomos , Modelos Biológicos , Oxirredutases/metabolismo , Subunidades Proteicas/isolamento & purificação , ATPases Translocadoras de Prótons/isolamento & purificação , ATPases Translocadoras de Prótons/metabolismo , Thermotoga maritima/efeitos dos fármacos
7.
IUBMB Life ; 72(5): 915-921, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31856407

RESUMO

Escherichia coli is able to ferment not only single but also mixtures of carbon sources. The formate metabolism and effect of formate on various enzymes have been extensively studied during sole glucose but not mixed carbon sources utilization. It was revealed that in membrane vesicles (MV) of wild type cells grown at pH 7.5 during fermentation of the mixture of glucose (2 g/L), glycerol (10 g/L), and formate (0.68 g/L), in the assays, the addition of formate (10 mM) increased the N,N'-dicyclohexylcarbodiimide (DCCD)-inhibited ATPase activity on ~30% but no effect of potassium ions (100 mM) had been detected. In selC (coding formate dehydrogenases) and fdhF (coding formate dehydrogenase H) single mutants, formate increased DCCD-inhibited ATPase activity on ~40 and ~70%, respectively. At pH 5.5, in wild type cells MV, formate decreased the DCCD-inhibited ATPase activity ~60% but unexpectedly in the presence of potassium ions, it was stimulated ~5.8 fold. The accessible SH or thiol groups number in fdhF mutant was less by 28% compared with wild type. In formate assays, the available SH groups number was less ~10% in wild type but not in fdhF mutant. Taken together, the data suggest that proton ATPase activity depends on externally added formate in the presence of potassium ions at low pH. This effect might be regulated by the changes in the number of redox-active thiol groups via formate dehydrogenase H, which might be directly related to proton ATPase FO subunit.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Formiato Desidrogenases/genética , Formiatos/farmacologia , Hidrogenase/genética , Complexos Multienzimáticos/genética , Potássio/farmacologia , ATPases Translocadoras de Prótons/genética , Carbono/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Dicicloexilcarbodi-Imida/farmacologia , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/metabolismo , Fermentação , Formiato Desidrogenases/metabolismo , Formiatos/metabolismo , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , Glucose/farmacologia , Glicerol/metabolismo , Glicerol/farmacologia , Concentração de Íons de Hidrogênio , Hidrogenase/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Complexos Multienzimáticos/metabolismo , Mutação , Potássio/metabolismo , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/metabolismo
8.
Sci Rep ; 9(1): 4279, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862913

RESUMO

During fermentation Escherichia coli excrete succinate mainly via Dcu family carriers. Current work reveals the total and N,N'-dicyclohexylcarbodiimide (DCCD) inhibited ATPase activity at pH 7.5 and 5.5 in E. coli wild type and dcu mutants upon glycerol fermentation. The overall ATPase activity was highest at pH 7.5 in dcuABCD mutant. In wild type cells 50% of the activity came from the FOF1-ATPase but in dcuD mutant it reached ~80%. K+ (100 mM) stimulate total but not DCCD inhibited ATPase activity 40% and 20% in wild type and dcuD mutant, respectively. 90% of overall ATPase activity was inhibited by DCCD at pH 5.5 only in dcuABC mutant. At pH 7.5 the H+ fluxes in E. coli wild type, dcuD and dcuABCD mutants was similar but in dcuABC triple mutant the H+ flux decreased 1.4 fold reaching 1.15 mM/min when glycerol was supplemented. In succinate assays the H+ flux was higher in the strains where DcuD is absent. No significant differences were determined in wild type and mutants specific growth rate except dcuD strain. Taken together it is suggested that during glycerol fermentation DcuD has impact on H+ fluxes, FOF1-ATPase activity and depends on potassium ions.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Glicerol/metabolismo , Adenosina Trifosfatases/genética , Transportadores de Ácidos Dicarboxílicos/genética , Transportadores de Ácidos Dicarboxílicos/metabolismo , Dicicloexilcarbodi-Imida/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fermentação/efeitos dos fármacos , Fermentação/genética , Fermentação/fisiologia , Concentração de Íons de Hidrogênio
9.
Mater Sci Eng C Mater Biol Appl ; 97: 356-366, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30678921

RESUMO

Synthesis of noble metal nanoparticles (NPs) on modified graphene oxide with biocompatible polymers has attracted significant due to their unique properties and various applications. In this research, covalent-modified graphene oxide (MGO) with diacid terminated poly (ethylene glycol) (PEG) was used as a substrate and stabilizing of Au (ш). The reduction of MGO-Au (ш) complex with hydrazine monohydrate under reflux condition obtained biocompatible reduced MGO (rMGO)-Au NPs. Diacid terminated PEG obtained from the reaction of PEG with succinic anhydride in the presence of N,N- dicyclohexylcarbodiimide (DCC) and 4-methylamino pyridine (DMAP) was attached to GO sheets to prevent from the aggregation of rMGO sheets and Au NPs. The resulting aqueous suspension was characterized through UV-vis, FT-IR, Raman, XRD, DLS-zeta potential, SEM, EDX and TEM. Furthermore, nanocomposite showed good catalytic behavior in Betti reaction- synthesis of 1-(α-aminoalkyl)-2-naphthols. The favorable properties of colloidal nanocomposite were attributed to the stable and well distribution Au NPs on rMGO.


Assuntos
Materiais Biocompatíveis/química , Grafite/química , Nanocompostos/química , Aminopiridinas/química , Catálise , Coloides/química , Dicicloexilcarbodi-Imida/química , Nanopartículas Metálicas/química , Microscopia Eletrônica de Varredura , Naftóis/síntese química , Naftóis/química , Óxidos/química , Polietilenoglicóis/química , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Anidridos Succínicos/química , Difração de Raios X
10.
Lett Appl Microbiol ; 68(1): 31-37, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30269343

RESUMO

Lactobacillus acidophilus is one of the widespread probiotic bacteria that can overcome acid and bile barrier of stomach and intestine, respectively, and then have beneficial effects on the host improving its intestinal microbial balance. The cell membrane FO F1 -ATPase is an important factor in the response and tolerance to low pH through the action of controlling the H+ concentration between the cell cytoplasm and external medium. In this study, the effects of extremely high-frequency EMI at the frequencies of 51·8 GHz and 53 GHz and cetfazidime ( µmol l-1 ) on survival of L. acidophilus VKM B-1660 in the gastrointestinal model in vitro and on ATPase activity of their membrane vesicles were investigated. Irradiated L. acidophilus survived in media with acid pH; the irradiation stimulated N,N'-dicyclohexylcarbodiimide-sensitive FO F1 -ATPase activity under acidic conditions, but enhanced the inhibitory effects of ceftazidime. Probably irradiated L. acidophilus is overcoming the acid barrier even in the presence of ceftazidime due to the FO F1 -ATPase. The obtained results can allow the use of L. acidophilus in food industry, veterinary and medicine. SIGNIFICANCE AND IMPACT OF THE STUDY: The probiotic property of lactobacilli is defined with survival in different conditions of human digestive tract even in the presence of antibiotics and subjected to electromagnetic irradiation (EMI) at the extremely high frequency. Despite the fact that EMI and antibiotic ceftazidime affected Lactobacillus acidophilus; the viable number of bacterial cells was decreased in in vitro gastrointestinal model, but they could to grow in fresh growth medium. The changes in the FO F1 -ATPase activity were obtained at acidic pH. Thus, these bacteria can overcome acid barrier due to the FO F1 -ATPase: the irradiation stimulates the FO F1 -ATPase activity in the acidic conditions, but enhances the effects of ceftazidime. The results are important for identifying the mechanisms of lactobacilli survival for physical and chemical factors and valuable for use.


Assuntos
Antibacterianos/farmacologia , Ceftazidima/farmacologia , Radiação Eletromagnética , Lactobacillus acidophilus/crescimento & desenvolvimento , ATPases Translocadoras de Prótons/metabolismo , Bile/metabolismo , Membrana Celular/efeitos dos fármacos , Dicicloexilcarbodi-Imida , Trato Gastrointestinal/microbiologia , Humanos , Concentração de Íons de Hidrogênio , Lactobacillus acidophilus/efeitos dos fármacos , Lactobacillus acidophilus/efeitos da radiação , Probióticos
11.
IUBMB Life ; 70(10): 1040-1047, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30161297

RESUMO

During fermentation Escherichia coli produces di-hydrogen (H2 ) via reversible membrane-bound [Ni-Fe]-hydrogenases (Hyd). This study describes the total and N,N'-dicyclohexylcarbodiimide (DCCD) inhibited ATPase activity and H2 production at various pHs in E. coli wild type and mutants encoding Hyd enzymes and formate dehydrogenases (FDH) on fermentation of glucose, glycerol, and formate. The highest total ATPase activity was detected at pH 7.5 in hyaB hybC selC (lacking large subunits of Hyd-1 and Hyd-2 and FDH, respectively) triple mutant. This ATPase activity was mainly due to the proton-translocating ATPase but in FDH mutant the DCCD inhibition was less compared to wild type. Potassium ions stimulated total ATPase activity at pH 5.5 ~50% and ~35% in wild type and hypF (lacking all Hyd enzymes) mutant, respectively. Moreover, K+ also stimulated DCCD inhibited ATPase activity ~1.7-fold-2-fold in strains where FDH was absent only at pH 5.5. DCCD inhibited H2 production only at pH 5.5 in all assays. Taken together it is suggested that at low pH, FDH, and Hyd enzymes are linked with the FO F1 -ATPase for regulating and maintaining the cytoplasmatic pH and thus proton motive force generation. FDH and Hyd enzymes have impact on the FO F1 -ATPase activity depending on external pH and potassium ions. © 2018 IUBMB Life, 70(10):1040-1047, 2018.


Assuntos
Escherichia coli/enzimologia , Formiato Desidrogenases/genética , ATPases Translocadoras de Prótons/genética , Carbono/metabolismo , Dicicloexilcarbodi-Imida/farmacologia , Fermentação/efeitos dos fármacos , Formiato Desidrogenases/metabolismo , Formiatos/metabolismo , Glucose/metabolismo , Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Hidrogenase/genética , Proteínas Ferro-Enxofre/genética
12.
J Gen Appl Microbiol ; 64(4): 174-179, 2018 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-29669961

RESUMO

Although Streptococcus anginosus constitutes a proportion of the normal flora of the gastrointestinal and genital tracts, and the oral cavity, it has been reported that S. anginosus infection could be closely associated with abscesses at various body sites, infective endocarditis, and upper gastrointestinal cancers. The colonization in an acidic environment due to the aciduricity of S. anginosus could be the etiology of the systemic infection of the bacteria. To elucidate the aciduricity and acid tolerance mechanisms of the microbe, we examined the viability and growth of S. anginosus under acidic conditions. The viabilities of S. anginosus NCTC 10713 and Streptococcus mutans ATCC 25175 at pH 4.0 showed as being markedly higher than those of Streptococcus sanguinis ATCC 10556, Streptococcus gordonii ATCC 10558, and Streptococcus mitis ATCC 49456; however, the viability was partially inhibited by dicyclohexylcarbodiimide, an H+-ATPase inhibitor, suggesting that H+-ATPase could play a role in the viability of S. anginosus under acidic conditions. In addition, S. anginosus NCTC 10713 could grow at pH 5.0 and showed a marked arginine deiminase (ADI) activity, unlike its ΔarcA mutant, deficient in the gene encoding ADI, and other streptococcal species, which indicated that ADI could also be associated with aciduricity. These results suggest that S. anginosus has significant aciduric properties, which can be attributed to these enzyme activities.


Assuntos
Ácidos/metabolismo , Hidrolases/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Streptococcus anginosus/efeitos dos fármacos , Streptococcus anginosus/fisiologia , Ácidos/farmacologia , Arginina/metabolismo , Meios de Cultura , Dicicloexilcarbodi-Imida/farmacologia , Deleção de Genes , Concentração de Íons de Hidrogênio , Hidrolases/genética , Viabilidade Microbiana/efeitos dos fármacos , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/genética , Streptococcus anginosus/enzimologia , Streptococcus anginosus/genética
13.
Cell Biochem Biophys ; 76(1-2): 209-217, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29039057

RESUMO

Microorganisms are part of the natural environments and reflect the effects of different physical factors of surrounding environment, such as gamma (γ) radiation. This work was devoted to the study of the influence of low doses of γ radiation with the intensity of 2.56 µW (m2 s)-1 (absorbed doses were 3.8 mGy for the radiation of 15 min and 7.2 mGy-for 30 min) on Escherichia coli M-17 and Pseudomonas aeruginosa GRP3 wild type cells. The changes of bacterial, growth, survival, morphology, and membrane activity had been studied after γ irradiation. Verified microbiological (specific growth rate, lag phase duration, colony-forming units (CFU) number, and light microscopy digital image analysis), biochemical (ATPase activity of bacterial membrane vesicles), and biophysical (H+ fluxes throughout cytoplasmic membrane of bacteria) methods were used for assessment of radiation implications on bacteria. It was shown that growth specific rate, lag phase duration and CFU number of these bacteria were lowered after irradiation, and average cell surface area was decreased too. Moreover ion fluxes of bacteria were changed: for P. aeruginosa they were decreased and for E. coli-increased. The N,N'-dicyclohexylcarbodiimide (DCCD) sensitive fluxes were also changed which were indicative for the membrane-associated F0F1-ATPase enzyme. ATPase activity of irradiated membrane vesicles was decreased for P. aeruginosa and stimulated for E. coli. Furthermore, DCCD sensitive ATPase activity was also changed. The results obtained suggest that these bacteria especially, P. aeruginosa are sensitive to γ radiation and might be used for developing new monitoring methods for estimating environmental changes after γ irradiation.


Assuntos
Escherichia coli/efeitos da radiação , Raios gama , Pseudomonas aeruginosa/efeitos da radiação , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/metabolismo , Dicicloexilcarbodi-Imida/farmacologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Microscopia , ATPases Translocadoras de Prótons/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo
14.
Biochim Biophys Acta Biomembr ; 1860(2): 586-599, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29179995

RESUMO

Cardiolipin (CL) is an anionic phospholipid at the inner mitochondrial membrane (IMM) that facilitates the formation of transient non-bilayer (non-lamellar) structures to maintain mitochondrial integrity. CL modulates mitochondrial functions including ATP synthesis. However, the biophysical mechanisms by which CL generates non-lamellar structures and the extent to which these structures contribute to ATP synthesis remain unknown. We hypothesized that CL and ATP synthase facilitate the formation of non-bilayer structures at the IMM to stimulate ATP synthesis. By using 1H NMR and 31P NMR techniques, we observed that increasing the temperature (8°C to 37°C), lowering the pH (3.0), or incubating intact mitochondria with CTII - an IMM-targeted toxin that increases the formation of immobilized non-bilayer structures - elevated the formation of non-bilayer structures to stimulate ATP synthesis. The F0 sector of the ATP synthase complex can facilitate the formation of non-bilayer structures as incubating model membranes enriched with IMM-specific phospholipids with exogenous DCCD-binding protein of the F0 sector (DCCD-BPF) elevated the formation of immobilized non-bilayer structures to a similar manner as CTII. Native PAGE assays revealed that CL, but not other anionic phospholipids, specifically binds to DCCD-BPF to promote the formation of stable lipid-protein complexes. Mechanistically, molecular docking studies identified two lipid binding sites for CL in DCCD-BPF. We propose a new model of ATP synthase regulation in which CL mediates the formation of non-bilayer structures that serve to cluster protons and ATP synthase complexes as a mechanism to enhance proton translocation to the F0 sector, and thereby increase ATP synthesis.


Assuntos
Cardiolipinas/metabolismo , Bicamadas Lipídicas/metabolismo , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bovinos , Dicicloexilcarbodi-Imida/metabolismo , Espectroscopia de Ressonância Magnética , Mitocôndrias Cardíacas/metabolismo , Modelos Biológicos , Simulação de Acoplamento Molecular , Ligação Proteica , Prótons , Lipossomas Unilamelares/metabolismo
15.
Proc Natl Acad Sci U S A ; 114(47): E10083-E10091, 2017 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-29114048

RESUMO

EmrE is a small multidrug resistance transporter found in Escherichia coli that confers resistance to toxic polyaromatic cations due to its proton-coupled antiport of these substrates. Here we show that EmrE breaks the rules generally deemed essential for coupled antiport. NMR spectra reveal that EmrE can simultaneously bind and cotransport proton and drug. The functional consequence of this finding is an exceptionally promiscuous transporter: not only can EmrE export diverse drug substrates, it can couple antiport of a drug to either one or two protons, performing both electrogenic and electroneutral transport of a single substrate. We present a free-exchange model for EmrE antiport that is consistent with these results and recapitulates ∆pH-driven concentrative drug uptake. Kinetic modeling suggests that free exchange by EmrE sacrifices coupling efficiency but boosts initial transport speed and drug release rate, which may facilitate efficient multidrug efflux.


Assuntos
Antiporters/química , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Oniocompostos/metabolismo , Compostos Organofosforados/metabolismo , Prótons , Xenobióticos/metabolismo , Antiporters/genética , Antiporters/metabolismo , Sítios de Ligação , Transporte Biológico , Dicicloexilcarbodi-Imida/toxicidade , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Oniocompostos/química , Oniocompostos/farmacologia , Compostos Organofosforados/química , Compostos Organofosforados/farmacologia , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteolipídeos/química , Proteolipídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica , Xenobióticos/química , Xenobióticos/farmacologia
16.
J Biol Chem ; 292(52): 21320-21329, 2017 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-29089385

RESUMO

Twin-arginine translocation (Tat) systems transport folded proteins across cellular membranes with the concerted action of mostly three membrane proteins: TatA, TatB, and TatC. Hetero-oligomers of TatB and TatC form circular substrate-receptor complexes with a central binding cavity for twin-arginine-containing signal peptides. After binding of the substrate, energy from an electro-chemical proton gradient is transduced into the recruitment of TatA oligomers and into the actual translocation event. We previously reported that Tat-dependent protein translocation into membrane vesicles of Escherichia coli is blocked by the compound N,N'-dicyclohexylcarbodiimide (DCCD, DCC). We have now identified a highly conserved glutamate residue in the transmembrane region of E. coli TatC, which when modified by DCCD interferes with the deep insertion of a Tat signal peptide into the TatBC receptor complex. Our findings are consistent with a hydrophobic binding cavity formed by TatB and TatC inside the lipid bilayer. Moreover, we found that DCCD mediates discrete intramolecular cross-links of E. coli TatC involving both its N- and C-tails. These results confirm the close proximity of two distant sequence sections of TatC proposed to concertedly function as the primary docking site for twin-arginine signal peptides.


Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Arginina/metabolismo , Membrana Celular/metabolismo , Cristalografia por Raios X/métodos , Dicicloexilcarbodi-Imida/farmacologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana Transportadoras/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Sinais Direcionadores de Proteínas/fisiologia , Especificidade por Substrato
17.
Biochimie ; 140: 73-81, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28652018

RESUMO

The mitochondrial F-ATPase can be activated either by the classical cofactor Mg2+ or, with lower efficiency, by Ca2+. The latter may play a role when calcium concentration rises in mitochondria, a condition associated with cascade events leading to cell death. Common and distinctive features of these differently activated mitochondrial ATPases were pointed out in swine heart mitochondria. When Ca2+ replaces the natural cofactor Mg2+, the enzyme responsiveness to the transmembrane electrochemical gradient and to the classical F-ATPase inhibitors DCCD and oligomycin as well as the oligomycin sensitivity loss by thiol oxidation, are maintained. Consistently, the two mitochondrial ATPases apparently share the F1FO complex basic structure and mechanism. Peculiar cation-dependent properties, which may affect the F1 catalytic mechanism and/or the FO proton binding site features, may be linked to a different physiological role of the mitochondrial Ca-activated F-ATPase with respect to the Mg-activated F-ATPase.


Assuntos
Cálcio/farmacocinética , Magnésio/farmacologia , Mitocôndrias Cardíacas/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Animais , Cálcio/metabolismo , Dicicloexilcarbodi-Imida/farmacologia , Magnésio/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Oligomicinas/farmacologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , Suínos
18.
Microbiology (Reading) ; 162(7): 1243-1252, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27166225

RESUMO

Escherichia coli has an l-alanine export system that protects the cells from toxic accumulation of intracellular l-alanine in the presence of l-alanyl-l-alanine (l-Ala-l-Ala). When a DadA-deficient strain was incubated with 6.0 mM l-Ala-l-Ala, we detected l-alanine and d-alanine using high-performance liquid chromatography (HPLC) analysis at a level of 7.0 mM and 3.0 mM, respectively, after 48 h incubation. Treatment of the culture supernatant with d-amino acid oxidase resulted in the disappearance of a signal corresponding to d-alanine. Additionally, the culture supernatant enabled a d-alanine auxotroph to grow without d-alanine supplementation, confirming that the signal detected by HPLC was authentic d-alanine. Upon introduction of an expression vector harbouring the alanine racemase genes, alr or dadX, the extracellular level of d-alanine increased to 11.5 mM and 8.5 mM, respectively, under similar conditions, suggesting that increased metabolic flow from l-alanine to d-alanine enhanced d-alanine secretion. When high-density DadA-deficient cells preloaded with l-Ala-l-Ala were treated with 20 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP), secretion of both l-alanine and d-alanine was enhanced ~twofold compared with that in cells without CCCP treatment. In contrast, the ATPase inhibitor dicyclohexylcarbodiimide did not exert such an effect on the l-alanine and d-alanine secretion. Furthermore, inverted membrane vesicles prepared from DadA-deficient cells lacking the l-alanine exporter AlaE accumulated [3H]D-alanine in an energy-dependent manner. This energy-dependent accumulation of [3H]D-alanine was strongly inhibited by CCCP. These results indicate that E. coli has a transport system(s) that exports d-alanine and that this function is most likely modulated by proton electrochemical potential.


Assuntos
Alanina/metabolismo , Transporte Biológico/fisiologia , Dipeptídeos/metabolismo , Escherichia coli/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Alanina/química , Alanina Racemase/genética , Transporte Biológico/genética , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Cromatografia Líquida de Alta Pressão , D-Aminoácido Oxidase/farmacologia , Dicicloexilcarbodi-Imida/farmacologia , Escherichia coli/genética
19.
Biomed Res Int ; 2016: 9805675, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27213156

RESUMO

Avermectin-grafted-N,O-carboxymethyl chitosan (NOCC) derivative was obtained by esterification reaction using dicyclohexylcarbodiimide (DCC) as dehydrating agent and 4-methylaminopyridine as catalyst. The structures of the conjugate were confirmed by FT-IR, (1)H NMR, and XRD. Insecticidal activities against armyworms, carmine spider mites, black bean aphids, and brown plant hoppers were investigated at concentrations ranging from 0.16 to 1000 mg/L. At the concentration of 1000 mg/L and 500 mg/L, the lethal rate was 100%. Good insecticidal activity at 4 mg/L was still shown, especially against the black bean aphids and brown plant hoppers. Moreover, the photostability of the conjugate was evaluated and showed an apparent improvement. At 300 mins, the residual rate of the conjugate was 11.22%, much higher than 0.2% of the avermectin technical material. The conjugate we developed showed potential for further study and application in crop protection.


Assuntos
Afídeos/efeitos dos fármacos , Quitosana/farmacologia , Inseticidas/farmacologia , Ivermectina/análogos & derivados , Aminopiridinas/química , Animais , Quitosana/química , Dicicloexilcarbodi-Imida/química , Dicicloexilcarbodi-Imida/farmacocinética , Esterificação , Inseticidas/química , Ivermectina/química , Ivermectina/farmacologia , Espectroscopia de Ressonância Magnética , Espectroscopia de Infravermelho com Transformada de Fourier
20.
J Asian Nat Prod Res ; 18(5): 462-74, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26765144

RESUMO

Altholactone exhibited the anti-fungal activity with a high MIC value of 128 µg ml(-1) against Cryptococcus neoformans and Saccharomyces cerevisiae. Fifteen ester derivatives of altholactone 1-15 were modified by esterification and their structures were confirmed by spectroscopic methods. Most of the ester derivatives exhibited stronger anti-fungal activities than that of the precursor altholactone. 3-Bromo- and 2,4-dichlorobenzoates (7 and 15) exhibited the lowest minimal inhibitory concentration (MIC) values against C. neoformans at 16 µg ml(-1), while the 4-bromo-, 4-iodo-, and 1-bromo-3-chlorobenzoates (11-13) displayed potent activity against S. cerevisiae with MIC values of 1 µg ml(-1). In conclusion, this analysis indicates that the anti-fungal activity of altholactone is enhanced by addition of halogenated benzoyl group to the 3-OH group.


Assuntos
Antifúngicos/síntese química , Antifúngicos/farmacologia , Benzoatos/síntese química , Benzoatos/farmacologia , Furanos/síntese química , Furanos/farmacologia , Hidrocarbonetos Halogenados/síntese química , Hidrocarbonetos Halogenados/farmacologia , Pironas/síntese química , Pironas/farmacologia , 4-Aminopiridina/análogos & derivados , 4-Aminopiridina/química , Antifúngicos/química , Benzoatos/química , Cryptococcus neoformans/efeitos dos fármacos , Dicicloexilcarbodi-Imida/química , Furanos/química , Hidrocarbonetos Halogenados/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Pironas/química , Saccharomyces cerevisiae/efeitos dos fármacos , Estereoisomerismo
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