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1.
J Photochem Photobiol B ; 200: 111645, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31671371

RESUMO

Antimicrobial peptide W3R6 was derived from chensinin-1b and showed potential as a novel antibiotics. However, W3R6 was susceptible to protease cleavage, which limited its therapeutic application. To improve the proteolytic resistance of W3R6, D-amino acids were incorporated into its sequence by specific amino acid substitution or whole sequence substitution according to the specificity of the cleavage site. In this study, partially substituted analog D-Arg-W3R6 and completely substituted D-enantiomer D-W3R6 were synthesized. The resistance of D-Arg-W3R6 and D-W3R6 to cleavage by the tested protease increased, particularly of D-W3R6. The antimicrobial activity of D-Arg-W3R6 was almost the same as that of the parent peptide W3R6, but the antimicrobial activity of D-W3R6 was slightly decreased. The hemolytic activity of both D-Arg-W3R6 and D-W3R6 was negligible. The CD spectrum of D-W3R6 exhibited symmetry with that of W3R6 in a membrane-mimetic environment. The membrane interaction between the D-amino acid substituted analogs and a real/mimic bacterial cell membrane was examined. The outer membrane depolarization, inner membrane permeability and dye leakage in three types of liposomes treated with D-Arg-W3R6 and D-W3R6 were not obviously different from W3R6, which could be due to the similar physical and chemical properties. In addition, these three peptides showed the binding ability with LPS micelles detected by ITC, and their ability to disrupt the LPS micelles was examined by DLS experiment and even neutralize the surface negative charge of E. coli cells. These results suggest that D-Arg-W3R6 is a promising antibiotic molecule.


Assuntos
Aminoácidos/química , Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/química , Anti-Infecciosos/síntese química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Parede Celular/química , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Lipossomos/química , Lipossomos/metabolismo , Peptídeos/química , Permeabilidade/efeitos dos fármacos , Estabilidade Proteica
2.
Life Sci ; 235: 116827, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31479680

RESUMO

OBJECTIVE: This study aims to evaluate the effective of azoles and MTX for patients with invasive candidiasis. METHODS: We used the disk diffusion assay and the checkerboard assay to evaluate the in vitro interactions between MTX and antifungals. In addition, we used the transmission electron microscopy to observe the ultrastructure of the effect of MTX and fluconazole on Candida albicans. RESULTS: The rates of synergy for the combination of MTX with fluconazole (FLC), itraconazole (ITC), and voriconazole (VRZ) were 91.3%, 65.2%, and 87% in checkerboard testing. No antagonism was found between methotrexate and azole antifungals in any of the strains. Furthermore, MTX treated C. albicans showed extensive cell wall vacuolations and the inhibition of blastospores growth, as observed using transmission electron microscopy. There was an apparent destruction of the cell membrane and cell wall resulting in the destruction of cytoplasm, a phenomenon observed when MTX was combined with azoles. CONCLUSION: This study provides evidence that the combination of azoles and MTX is effective for patients with invasive candidiasis, which on the other hand, will reduce the side effects of the drugs.


Assuntos
Candida albicans/efeitos dos fármacos , Sinergismo Farmacológico , Fluconazol/farmacologia , Itraconazol/farmacologia , Metotrexato/farmacologia , Voriconazol/farmacologia , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Gástrula/efeitos dos fármacos , Técnicas In Vitro , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão
3.
J Microbiol Biotechnol ; 29(8): 1221-1229, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31370112

RESUMO

Mycobacterium tuberculosis, a causative pathogen of tuberculosis (TB), still threatens human health worldwide. To find a novel drug to eradicate this pathogen, we tested taurine-5- bromosalicylaldehyde Schiff base (TBSSB) as an innovative anti-mycobacterial drug using Mycobacterium smegmatis as a surrogate model for M. tuberculosis. We investigated the antimicrobial activity of TBSSB against M. smegmatis by plotting growth curves, examined the effect of TBSSB on biofilm formation, observed morphological changes by scanning electron microscopy and transmission electron microscopy, and detected differentially expressed proteins using two-dimensional gel electrophoresis coupled with mass spectrometry. TBSSB inhibited mycobacterial growth and biofilm formation, altered cell ultrastructure and intracellular content, and inhibited cell division. Furthermore, M. smegmatis adapted itself to TBSSB inhibition by regulating the metabolic pathways and enzymatic activities of the identified proteins. NDMA-dependent methanol dehydrogenase, NAD(P)H nitroreductase, and amidohydrolase AmiB1 appear to be pivotal factors to regulate the M. smegmatis survival under TBSSB. Our dataset reinforced the idea that Schiff base-taurine compounds have the potential to be developed as novel anti-mycobacterial drugs.


Assuntos
Aldeídos/farmacologia , Antibacterianos/farmacologia , Mycobacterium smegmatis/efeitos dos fármacos , Proteômica , Bases de Schiff/farmacologia , Taurina/análogos & derivados , Proteínas de Bactérias/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Parede Celular/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/ultraestrutura , Mycobacterium tuberculosis , Taurina/farmacologia
4.
Int J Nanomedicine ; 14: 4801-4816, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31308659

RESUMO

Background: Silver nanoparticles (AgNPs) inhibit the proliferation of various fungi; however, their mechanisms of action remain poorly understood. To better understand the inhibitory mechanisms, we focused on the early events elicited by 5 nm AgNPs in pathogenic Candida albicans and non-pathogenic Saccharomyces cerevisiae. Methods: The effect of 5 nm and 100 nm AgNPs on fungus cell proliferation was analyzed by growth kinetics monitoring and spot assay. We examined cell cycle progression, reactive oxygen species (ROS) production, and cell death using flow cytometry. Glucose uptake was assessed using tritium-labeled 2-deoxyglucose. Results: The growth of both C. albicans and S. cerevisiae was suppressed by treatment with 5 nm AgNPs but not with 100 nm AgNPs. In addition, 5 nm AgNPs induced cell cycle arrest and a reduction in glucose uptake in both fungi after 30 minutes of culture in a dose-dependent manner (P<0.05). However, in C. albicans only, an increase in ROS production was detected after exposure to 5 nm AgNPs. Concordantly, an ROS scavenger blocked the effect of 5 nm AgNPs on the cell cycle and glucose uptake in C. albicans only. Furthermore, the growth-inhibition effect of 5 nm AgNPs was not greater in S. cerevisiae mutant strains deficient in oxidative stress response genes than it was in wild type. Finally, 5 nm AgNPs together with a glycolysis inhibitor, 3-bromopyruvate, synergistically enhanced cell death in C. albicans (P<0.05) but not in S. cerevisiae. Conclusion: AgNPs exhibit antifungal activity in a manner that may or may not be ROS dependent, according to the fungal species. The combination of AgNPs with 3-bromopyruvate may be more useful against infection with C. albicans.


Assuntos
Candida albicans/citologia , Ciclo Celular/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Piruvatos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/citologia , Prata/farmacologia , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Depuradores de Radicais Livres/farmacologia , Fase G1/efeitos dos fármacos , Genes Fúngicos , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento
5.
Int J Nanomedicine ; 14: 4667-4679, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31308652

RESUMO

Purpose: The pathogenicity in Candida spp was attributed by several virulence factors such as production of tissue damaging extracellular enzymes, germ tube formation, hyphal morphogenesis and establishment of drug resistant biofilm. The objective of present study was to investigate the effects of silver nanoparticles (AgNPs) on growth, cell morphology and key virulence attributes of Candida species. Methods: AgNPs were synthesized by the using seed extract of Syzygium cumini (Sc), and were characterized by UV-Vis spectrophotometer, Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), and transmission electron microscopy (TEM). ScAgNPs were used to evaluate their antifungal and antibacterial activity as well as their potent inhibitory effects on germ tube and biofilm formation and extracellular enzymes viz. phospholipases, proteinases, lipases and hemolysin secreted by Candida spp. Results: The MICs values of ScAgNPs were ranged from 0.125-0.250 mg/ml, whereas the MBCs and MFCs were 0.250 and 0.500 mg/ml, respectively. ScAgNPs significantly inhibit the production of phospholipases by 82.2, 75.7, 78.7, 62.5, and 65.8%; proteinases by 82.0, 72.0, 77.5, 67.0, and 83.7%; lipase by 69.4, 58.8, 60.0, 42.9, and 65.0%; and hemolysin by 62.8, 69.7, 67.2, 73.1, and 70.2% in C. albicans, C. tropicalis, C. dubliniensis, C. parapsilosis and C. krusei, respectively, at 500 µg/ml. ScAgNPs inhibit germ tube formation in C. albicans up to 97.1% at 0.25 mg/ml. LIVE/DEAD staining results showed that ScAgNPs almost completely inhibit biofilm formation in C. albicans. TEM analysis shows that ScAgNPs not only anchored onto the cell surface but also penetrated and accumulated in the cytoplasm that causes severe damage to the cell wall and cytoplasmic membrane. Conclusion: To summarize, the biosynthesized ScAgNPs strongly suppressed the multiplication, germ tube and biofilm formation and most importantly secretion of hydrolytic enzymes (viz. phospholipases, proteinases, lipases and hemolysin) by Candia spp. The present research work open several avenues of further study, such as to explore the molecular mechanism of inhibition of germ tubes and biofilm formation and suppression of production of various hydrolytic enzymes by Candida spp.


Assuntos
Antifúngicos/farmacologia , Candida/crescimento & desenvolvimento , Candida/patogenicidade , Nanopartículas Metálicas/química , Prata/farmacologia , Antifúngicos/química , Biofilmes/efeitos dos fármacos , Candida/citologia , Candida/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Proteínas Hemolisinas/metabolismo , Humanos , Hidrólise , Nanopartículas Metálicas/ultraestrutura , Testes de Sensibilidade Microbiana , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Syzygium/química , Virulência/efeitos dos fármacos , Fatores de Virulência
6.
Cell Physiol Biochem ; 53(2): 285-300, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31334617

RESUMO

BACKGROUND/AIMS: Although naturally-derived antifungals have been investigated for their ability to inactivate Candida albicans, which is a major cause of candidiasis, they have shown a less than 3 log reduction in C. albicans or required treatment times of longer than 3 h. Thus, the naturally-derived antifungals used in previous studies could not substantially eradicate C. albicans within a short period of time. METHODS: To improve the fungicidal effects of naturallyderived antifungals against C. albicans within short time periods, we developed composites showing antifungal synergism using caprylic acid (CA), carvacrol (CAR) and thymol (THM) for 1-10 min at 22/37°C. Using flow cytometry, we examined the mode of action for the synergism of these compounds on membrane integrity and efflux pump activity. RESULTS: Whereas the maximum reduction by individual treatments was 0.6 log CFU/ml, CA + CAR/THM (all 1.5 mM) eliminated all pathogens (> 6.8 log reduction) after 1 min at 37°C and after 10 min at 22°C. The flow cytometry results showed that exposure to CA damaged the membranes in 15.7-36.5% of cells and inhibited efflux pumps in 15.4-31.3% of cells. Treatments with CAR/THM slightly affected cell membranes (in 1.8-6.9% of cells) but damaged efflux pumps in 14.4-29.6% of cells. However, the combined treatments clearly disrupted membranes (> 83.1% of cells) and pumps (> 95.0% of cells). The mechanism of this synergism may involve membrane damage by CA, which facilitates the entry of antifungals into the cytoplasm, and the inhibition of efflux pumps by CA, CAR or THM, causing their accumulation within cells and, leading to cell death. CONCLUSION: Antifungal composites (CA + CAR/THM) showing synergism (i.e., an additional 6 log reduction) within minutes at room/body temperature can be used to treat candidiasis and improve the microbiological safety of facilities contaminated with fungi as a novel alternative to synthetic antifungals.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Caprilatos/farmacologia , Proteínas Fúngicas/metabolismo , Monoterpenos/farmacologia , Timol/farmacologia , Candida albicans/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Sinergismo Farmacológico , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Temperatura Ambiente
7.
Nat Commun ; 10(1): 2733, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227716

RESUMO

Cell wall antibiotics are crucial for combatting the emerging wave of resistant bacteria. Yet, our understanding of antibiotic action is limited, as many strains devoid of all resistance determinants display far higher antibiotic tolerance in vivo than suggested by the antibiotic-target binding affinity in vitro. To resolve this conflict, here we develop a comprehensive theory for the bacterial cell wall biosynthetic pathway and study its perturbation by antibiotics. We find that the closed-loop architecture of the lipid II cycle of wall biosynthesis features a highly asymmetric distribution of pathway intermediates, and show that antibiotic tolerance scales inversely with the abundance of the targeted pathway intermediate. We formalize this principle of minimal target exposure as intrinsic resistance mechanism and predict how cooperative drug-target interactions can mitigate resistance. The theory accurately predicts the in vivo efficacy for various cell wall antibiotics in different Gram-positive bacteria and contributes to a systems-level understanding of antibiotic action.


Assuntos
Vias Biossintéticas/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Bactérias Gram-Positivas/metabolismo , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Parede Celular/metabolismo , Bactérias Gram-Positivas/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/microbiologia , Modelos Biológicos , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurâmico/biossíntese
8.
Subcell Biochem ; 92: 1-5, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31214982

RESUMO

Astonishing progress has been made in recent years to understand the structural complexity and functions of the biosynthetic pathways of the bacterial and archaeal envelopes. This progress has prompted me to assemble the present book that provides a detailed overview and the state-of-art of the respective research field. Ideally, the book will provide students and advanced scientists an up to date picture of the different parts of the bacterial and archaeal cell envelope and enable them to understand their functional roles.


Assuntos
Antibacterianos/farmacologia , Archaea/citologia , Archaea/efeitos dos fármacos , Bactérias/citologia , Bactérias/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos
9.
Enzyme Microb Technol ; 128: 40-48, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31186109

RESUMO

The overuse and misuse of antibiotics in treating bacterial infections cause the rapid emergence of drug-resistant bacteria, suggesting that the development of alternative strategies to control antibiotic-resistant bacteria is urgently needed. Endolysins are bacteriophage-encoded enzymes that can degrade peptidoglycan in bacterial cell walls, and they have great potential as alternative antimicrobial agents. However, exogenous application of recombinant endolysin is limited to Gram-positive bacteria because endolysins cannot penetrate the outer membrane of Gram-negative bacteria. Here, a liposome-mediated endolysin encapsulation system was developed, and its ability to penetrate the outer membrane of Gram-negative bacteria was tested. The phage-derived endolysin BSP16Lys was isolated, characterized, and used for encapsulation into a cationic liposome comprised of dipalmitoylphosphatidylcholine (DPPC), cholesterol, and hexadecylamine. The BSP16Lys-encapsulated liposome had a high zeta potential value (over 30 mV) with an average diameter of 303 nm. The encapsulation efficiency of BSP16Lys into the liposome was 35.27%. Salmonella Typhimuriumand Escherichia coli cells treated with BSP16Lys-encapsulated liposomes showed 2.2-log CFU/mL and 1.6-log CFU/mL reductions in the viable cell numbers, respectively, without treatment of a membrane permeabilizer. These results showed potential for liposome-mediated delivery of endolysin for exogenous application against Gram-negative bacteria.


Assuntos
Antibacterianos/metabolismo , Endopeptidases/metabolismo , Enzimas Imobilizadas/metabolismo , Escherichia coli/efeitos dos fármacos , Lipossomos/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Salmonella typhimurium/efeitos dos fármacos , Antibacterianos/síntese química , Parede Celular/efeitos dos fármacos , Contagem de Colônia Microbiana , Lipossomos/síntese química , Peptidoglicano/metabolismo
10.
Photochem Photobiol Sci ; 18(7): 1700-1708, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31214675

RESUMO

The ever growing world-population poses challenges concerning the need for more food free of pesticide residues. The most common means to control plant pathogens is through the application of pesticides, which raises concerns over safety for humans and the environment. Recently, Photodynamic Inactivation (PDI) of microorganisms using natural photosensitizers has shown itself to be a powerful tool to combat bacteria and fungi. This study investigates the efficacy of PDI against the Gram(+) bacterial plant pathogen Rhodococcus fascians and Gram(-) Xanthomonas axonopodis and Erwinia amylovora using two chlorin e6 derivatives as photosensitizers: anionic sodium magnesium chlorophyllin (Chl, approved as food additive E140) in combination with cell wall permeabilizing agents (Na2EDTA or Polyaspartic acid sodium salt (PA)) and B17-0024, a mixture of chlorin e6 derivatives with cationic moieties at physiological pH. Both photosensitizers show excellent efficacy against R. fascians, whereby B17-0024 is phototoxic at a one order of magnitude lower concentration than Chl (10 µM B17-0024: relative inactivation (r.i.) >7.5 × 106, 100 µM Chl: r.i. 2.2 × 106, illumination with 26.6 J cm-2, 395 nm). The phototreatment of Gram(-) bacteria with Chl requires the obligatory use of cell wall permeabilizing agents like Na2EDTA (X. axonopodis) or PA (E. amylovora) to induce significant killing (more than 7 log units at 100 µM). On the other hand, B17-0024 proves to be a highly effective photosensitizer inducing bacterial inactivation at very low concentrations (10 µM for R. fascians and X. axonopodis, 100 µM for E. amylovora) without additives. In summary, PDI using both the natural photosensitizer Chl in combination with cell wall permeabilizing agents is effective and environmentally friendly. As an alternative, B17-0024 is highly photoactive against all model strains tested - even without cell wall permeabilizing agents. The photodynamic approach based on chlorin e6 derivatives should add to the growers' toolbox as a preferred alternative for the control of phytopathogens.


Assuntos
Produtos Agrícolas/microbiologia , Erwinia amylovora/efeitos da radiação , Luz , Rhodococcus/efeitos da radiação , Xanthomonas axonopodis/efeitos da radiação , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Erwinia amylovora/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/química , Porfirinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Rhodococcus/efeitos dos fármacos , Xanthomonas axonopodis/efeitos dos fármacos
11.
J Plant Physiol ; 239: 10-17, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31177026

RESUMO

Auxin is one of the crucial plant hormones which stimulates and controls cell and plant growth. The effects of auxin IBA (indole-3-butyric acid) during 10-days on maize plants growth in controlled conditions (hydroponic, 16-h photoperiod, 70% humidity, 25/20 °C temperature), depended on its concentration in the substrate. A high concentration (10-7 M) of IBA inhibited root growth, evoked the development of apoplasmic barriers (Casparian bands and suberin lamellae) closer to the root apex, and elevated the amount of lignin in roots. A low concentration (10-11 M) of IBA stimulated root growth but affected neither the development of apoplasmic barriers, nor the amount of lignin. Auxin in a 10-8 M concentration influenced the root growth to a minimal extent compare to the control, and it was the non-effective concentration. Plant cell walls as cell structures ensure cell enlargement and plant growth, and have to react to auxin stimulus by modification of their components. We found the most significant changes in the composition of the PF III fraction (lignocellulosic complex) of the cell wall. The presence of auxin in the substrate affected all three components of this fraction - Klason lignin and both the by acid (2 M TFA) non-hydrolysable and the hydrolysable parts of this complex. The ratio of the non-hydrolysable part to the Klason lignin increased from 1.3 to 3.3 with increasing auxin concentrations in the substrate. This may be related to the deposition of polysaccharides and lignin in the cell wall, which help maintain the specific tensile stress of, and turgor pressure on, the cell walls.


Assuntos
Indóis/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Zea mays/efeitos dos fármacos , Zea mays/fisiologia , Parede Celular/efeitos dos fármacos , Parede Celular/fisiologia , Relação Dose-Resposta a Droga , Ácidos Indolacéticos/administração & dosagem , Ácidos Indolacéticos/farmacologia , Indóis/administração & dosagem , Lipídeos/química , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/fisiologia , Xilema/efeitos dos fármacos , Xilema/fisiologia
12.
Carbohydr Polym ; 217: 126-134, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31079668

RESUMO

Longan pulp is an excellent source of polysaccharides and other nutrients that have many health benefits. However, longans is susceptible to pulp breakdown after harvest and loses its nutrition values. To solve this problem, this study aimed to study the effects of a novel chitosan, Kadozan, on pulp breakdown index, contents of pectin, cellulose and hemicelluloses, and activities of enzymes in longan pulp relating to disassembly of polysaccharides (XET, PE, PG, ß-Gal, and cellulase). The data illustrated that, compared to the control longans, chitosan-treated longans contained higher amounts of CWM, CSP, ISP, cellulose and hemicelluloses, but exhibited lower pulp breakdown index, lower activities of cell wall-disassembling enzymes, and contained lower WSP amount. These results suggested that Kadozan with a dilution of 1:500 (VKadozan: VKadozan + Water) could significantly decrease activities of disassembling-enzymes and depolymerization of polysaccharides in cell wall, and subsequently alleviate pulp breakdown and prolong storage-life of postharvest longans.


Assuntos
Parede Celular/efeitos dos fármacos , Quitosana/farmacologia , Inibidores Enzimáticos/farmacologia , Frutas/metabolismo , Polissacarídeos/metabolismo , Sapindaceae/metabolismo , Parede Celular/metabolismo , Celulose/metabolismo , Conservação de Alimentos/métodos , Qualidade dos Alimentos , Glicosídeo Hidrolases/antagonistas & inibidores , Hidrólise/efeitos dos fármacos , Pectinas/metabolismo
13.
J Med Microbiol ; 68(6): 848-859, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31136294

RESUMO

PURPOSE: The purpose of the present study was to determine the relatedness of Staphylococcus aureus strains successively isolated over a 7-day period from a single bacteraemic patient undergoing antibiotic treatment with vancomycin. METHODS: The S. aureus strains had been isolated and sequenced previously. Antibiotic susceptibility testing, population analysis profiling, and lysostaphin sensitivity and phagocytic killing assays were used to characterize these clonal isolates. RESULTS: The seven isolates (MEH1-MEH7) were determined to belong to a common multilocus sequence type (MLST) and spa type. Within the third and fifth day of vancomycin treatment, mutations were observed in the vraS and rpsU genes, respectively. Population analysis profiles revealed that the initial isolate (MEH1) was vancomycin-susceptible S. aureus (VSSA), while those isolated on day 7 were mostly heteroresistant vancomycin-intermediate S. aureus (hVISA). Supporting these findings, MEH7 was also observed to be slower in growth, to have an increase in cell wall width and to have reduced sensitivity to lysostaphin, all characteristics of VISA and hVISA strains. In addition, MEH7, although phagocytosed at numbers comparable to the initial isolate, MEH1, survived in higher numbers in RAW 264.7 macrophages. Macrophages infected with MEH7 also released more TNF-α and IFN-1ß. CONCLUSION: We report an increasing resistance to vancomycin coupled with daptomycin that occurred within approximately 3 days of receiving vancomycin and steadily increased until the infection was cleared with an alternative antibiotic therapy. This study reiterates the need for rapid, efficient and accurate detection of hVISA and VISA infections, especially in high-bacterial load, metastatic infections like bacteraemia.


Assuntos
Antibacterianos/farmacologia , Macrófagos/fisiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Resistência a Vancomicina/genética , Vancomicina/farmacologia , Idoso , Bacteriemia/microbiologia , Parede Celular/efeitos dos fármacos , Daptomicina/farmacologia , Humanos , Lisostafina/farmacologia , Masculino , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Mutação , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/virologia
14.
Biomed Res Int ; 2019: 1871239, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119154

RESUMO

Pathogenic species of mycobacteria are known to use the host cholesterol during lung infection as an alternative source of carbon and energy. Mycobacteria culture in minimal medium (MM) has been used as an in vitro experimental model to study the consumption of exogenous cholesterol. Once in MM, different species of mycobacteria start to consume the cholesterol and initiate transcriptional and metabolic adaptations, upregulating the enzymes of the methylcitrate cycle (MCC) and accumulating a variety of primary metabolites that are known to be important substrates for cell wall biosynthesis. We hypothesized that stressful pressure of cultures in MM is able to induce critical adaptation for the bacteria which win the infection. To identify important modifications in the biosynthesis of the cell wall, we cultured the fast-growing and nonpathogenic Mycobacterium smegmatis in MM supplemented with or without glycerol and/or cholesterol. Different from the culture in complete medium Middlebrook 7H9 broth, the bacteria when cultured in MM decreased growth and changed in the accumulation of cell wall molecules. However, the supplementation of MM with glycerol and/or cholesterol recovered the accumulation of phosphatidylinositol mannosides (PIMs) and other phospholipids but maintained growth deceleration. The biosynthesis of lipomannan (LM) and of lipoarabinomannan (LAM) was significantly modulated after culture in MM, independently of glycerol and/or cholesterol supplementation, where LM size was decreased (LM13-25KDa) and LAM increased (LAM37-100KDa), when compared these molecules after bacteria culture in complete medium (LM17-25KDa and LAM37-50KDa). These changes modified the cell surface hydrophobicity and susceptibility against H2O2. The infection of J774 macrophages with M. smegmatis, after culture in MM, induced the formation of granuloma-like structures, while supplementation with cholesterol induced the highest rate of formation of these structures. Taken together, our results identify critical changes in mycobacterial cell wall molecules after culture in MM that induces cholesterol accumulation, helping the mycobacteria to increase their capacity to form granuloma-like structures.


Assuntos
Parede Celular/metabolismo , Microambiente Celular/efeitos dos fármacos , Colesterol/farmacologia , Mycobacterium smegmatis/metabolismo , Membrana Celular/metabolismo , Parede Celular/efeitos dos fármacos , Meios de Cultura/química , Meios de Cultura/farmacologia , Granuloma/metabolismo , Granuloma/patologia , Peróxido de Hidrogênio/farmacologia , Lipopolissacarídeos/biossíntese , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/patogenicidade
15.
Plant Physiol Biochem ; 141: 105-113, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31136933

RESUMO

The boron (B) is an essential nutrient and plays an important role in the stability of the primary cell wall (CW). Due to the narrow window between B deficiency and toxicity, mismanagement practices lead to B toxicity that inhibit root growth and overall crop productivity. However, the exact cause of root growth inhibition remains unclear. The present study examined the potential causes and targets of B toxicity by studying intercellular mechanism. The trifoliate seedlings were cultured under excess B conditions. The results indicated that plant growth was inhibited by excess B, nevertheless, the effects were prominent on roots and leaves. B toxicity exacerbated oxidative stress and root cell death. The analysis of CW functional groups, CW microstructure and B forms lead to the conclusion that alterations in CW, and accumulation of free-B and carbohydrates might cause inhibition of growth and visible symptoms of B toxicity.


Assuntos
Boro/farmacologia , Parede Celular/efeitos dos fármacos , Estresse Oxidativo , Poncirus/efeitos dos fármacos , Poncirus/metabolismo , Anemia Hipocrômica , Antioxidantes/química , Sítios de Ligação , Membrana Celular/metabolismo , Folhas de Planta/efeitos dos fármacos , Raízes de Plantas/metabolismo , Análise de Componente Principal , Plântula/metabolismo , Solo/química , Espectroscopia de Infravermelho com Transformada de Fourier , Xantina Oxidase/química
16.
J Plant Physiol ; 238: 63-71, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31146183

RESUMO

This study aimed to investigate the firmness retention by ethylene treatment in olive fruit, as observed earlier. Ethylene concentrations up to 1000 µL L-1 were applied to dark green 'Konservolia' olives harvested shortly before the green maturation and exposed to 20 °C for up to 9 d. Surprisingly, the results indicated a tendency to fruit firmness increases in concentration-dependent manner in a non-climacteric fruit. The highest concentration increased the firmness within 12 h by approximately 1.35-fold, but transiently for approximately up to 5 d; all ethylene inhibitors tested, either of synthesis (ethoxyvinyl glycine or AVG), or perception (1 -methyl-cyclopropene or 1-MCP, and silver nitrate) prevented the firmness increase. Texture was evaluated by firmness and changes in lignin, cellulose (CL), total pectins (TPC), water soluble pectins (WSP) and total non-cellulosic sugars (total sugars) concentrations, and in pectin esterification degree (DE) in the alcohol insoluble residue (AIR) of 'Konservolia' fruit pericarp during 1.5-d, 5-d and 10-d treatments with 1000 µL L-1 ethylene at 20 °C. Pectins in AIR were also extracted sequentially with cyclohexane-trans-1,2-diaminetetra-acetate (CDTA), Na2CO3, 1 M and 4 M KOH. The results showed that on day 1.5, the increased firmness was consistent with increased CL (crystalline formation, as observed by microscopy), total sugars and DE levels, but reduced WSP, whereas softening reversed the changes and lowered TPC and CDTA-soluble pectins in all fruit on day 10. However, on day 5 ethylene-treated olives exhibited a transitional phase during softening, characterized by retention of high TPC concentration and energy demand, as indicated by elevated respiration rates. The inhibitor 1-MCP, applied before ethylene, did inhibit the responses to ethylene treatment. Ethylene firming effect and the respective cell wall changes in olives are demonstrated for first time. The experiments could be used for research on perception and transcription responses to ethylene in olive, a non-climacteric fruit. In practice, high ethylene concentrations could also be beneficial for firmness increase and/or short storage of dark green olives.


Assuntos
Parede Celular/metabolismo , Ciclopropanos/farmacologia , Etilenos/metabolismo , Frutas/metabolismo , Olea/metabolismo , Parede Celular/efeitos dos fármacos , Celulose/metabolismo , Produção Agrícola/métodos , Relação Dose-Resposta a Droga , Etilenos/antagonistas & inibidores , Etilenos/farmacologia , Qualidade dos Alimentos , Frutas/efeitos dos fármacos , Frutas/crescimento & desenvolvimento , Lignina/metabolismo , Olea/efeitos dos fármacos , Olea/crescimento & desenvolvimento , Pectinas/metabolismo
17.
Lett Appl Microbiol ; 69(1): 41-49, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31044446

RESUMO

Plumbagin (5-hydroxy-2-methyl-1,4-napthoquinone) is a bicyclic naphthoquinone, found in three major plant families viz. Plumbaginaceae, Ebenceae and Droseraceae. The phytochemical is reported to exhibit various pharmacological properties. In this study, plumbagin isolated from Plumbago zeylanica L. was investigated for its in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA). Against 100 MRSA isolates that included multi-drug-resistant phenotypes, plumbagin showed consistent activity with a narrow minimum inhibitory concentration (MIC) range of 4-8 µg ml-1 . The time-kill study revealed 99% kill of a reference MRSA strain, 8 h after exposure to plumbagin. In the combination MIC study using the reference MRSA strain, plumbagin showed synergistic effect with ciprofloxacin and piperacillin while additive or indifference effect with other commonly used antibiotics. The transmission electron micrograph of the reference MRSA strain treated with plumbagin confirmed cell wall and cytoplasmic changes. Our results demonstrated potent anti-MRSA activity of plumbagin which was not impacted by multi-drug resistance. This is a first ever study that evaluated in vitro anti-MRSA activity of plumbagin employing large number of MRSA isolates. The findings of this study support the need for the further investigation on this phytochemical agent for therapeutic application. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed phytochemical plumbagin's potent and consistent in vitro antibacterial activity against clinically problematic methicillin-resistant Staphylococcus aureus (MRSA) including multi-drug-resistant (MDR) phenotypes. The study results support further research to assess the clinical scope of plumbagin.


Assuntos
Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Naftoquinonas/farmacologia , Extratos Vegetais/farmacologia , Plumbaginaceae/química , Parede Celular/efeitos dos fármacos , Ciprofloxacino/farmacologia , Citoplasma/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Compostos Fitoquímicos/farmacologia , Piperacilina/farmacologia
18.
BMC Plant Biol ; 19(1): 152, 2019 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-31010418

RESUMO

BACKGROUND: During sexual reproduction, pollen grains land on the stigma, rehydrate and produce pollen tubes that grow through the female transmitting-tract tissue allowing the delivery of the two sperm cells to the ovule and the production of healthy seeds. Because pollen tubes are single cells that expand by tip-polarized growth, they represent a good model to study the growth dynamics, cell wall deposition and intracellular machineries. Aiming to understand this complex machinery, we used a low throughput chemical screen approach in order to isolate new tip-growth disruptors. The effect of a chemical inhibitor of monogalactosyldiacylglycerol synthases, galvestine-1, was also investigated. The present work further characterizes their effects on the tip-growth and intracellular dynamics of pollen tubes. RESULTS: Two small compounds among 258 were isolated based on their abilities to perturb pollen tube growth. They were found to disrupt in vitro pollen tube growth of tobacco, tomato and Arabidopsis thaliana. We show that these 3 compounds induced abnormal phenotypes (bulging and/or enlarged pollen tubes) and reduced pollen tube length in a dose dependent manner. Pollen germination was significantly reduced after treatment with the two compounds isolated from the screen. They also affected cell wall material deposition in pollen tubes. The compounds decreased anion superoxide accumulation, disorganized actin filaments and RIC4 dynamics suggesting that they may affect vesicular trafficking at the pollen tube tip. CONCLUSION: These molecules may alter directly or indirectly ROP1 activity, a key regulator of pollen tube growth and vesicular trafficking and therefore represent good tools to further study cellular dynamics during polarized-cell growth.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Tubo Polínico/crescimento & desenvolvimento , Bibliotecas de Moléculas Pequenas/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Arabidopsis/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Germinação/efeitos dos fármacos , Conformação Molecular , Tubo Polínico/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Superóxidos/metabolismo
19.
Chemosphere ; 228: 219-231, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31029968

RESUMO

Integration of chemical-genetic interaction data with biological functions provides a mechanistic understanding of how toxic compounds affect cells. Mono-(2-ethylhexyl)-phthalate (MEHP) is an active metabolite of di-(2-ethylhexyl)-phthalate (DEHP), a commonly used plasticizer. MEHP adversely affects human health causing hepatotoxicity and reproductive toxicity. How MEHP affects cellular physiology is not fully understood. We utilized a genome-wide competitive fitness-based assay called 'chemogenomic profiling' to determine the genetic interaction map of MEHP in Saccharomyces cerevisiae. Gene Ontology enrichment analysis of 218 genes that provide resistance to MEHP indicated that MEHP affects seven cellular processes namely: (1) cellular amino acid biosynthetic process, (2) sterol biosynthetic process, (3) cellular transport, (4) transcriptional and translational regulation, (5) protein glycosylation, (6) cytokinesis and cell morphogenesis and (7) ionic homeostasis. We show that MEHP protects yeast cells from membrane perturbing agents such as amphotericin B, dihydrosphingosine and phytosphingosine. Moreover, we also demonstrate that MEHP compromises the integrity of the yeast plasma membrane and cell wall. Our work provides a basis for further investigation of MEHP toxicity in humans.


Assuntos
Ácidos Ftálicos/toxicidade , Plastificantes/toxicidade , Vias Biossintéticas/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Dietilexilftalato/metabolismo , Humanos , Ácidos Ftálicos/farmacologia , Plastificantes/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo
20.
ACS Appl Mater Interfaces ; 11(17): 15332-15343, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30950609

RESUMO

In this study, we use Escherichia coli as a model to investigate the antimicrobial mechanism of a film made of a copolymer based on monomethylether poly(ethylene glycol), methyl methacrylate, and 2-dimethyl(aminoethyl) methacrylate, whose surface is active towards Gram-negative and Gram-positive bacteria. The polymer contains not quaternized amino groups that can generate a charged surface by protonation when in contact with water. For this purpose, we adopted a dual strategy based on the analysis of cell damage caused by contact with the polymer surface and on the evaluation of the cell response to the surface toxic action. The lithic effect on the protoplasts of E. coli showed that the polymer surface can affect the structure of cytoplasmic membranes, while assays of calcein leakage from large unilamellar vesicles at different phospholipid compositions indicated that action on membranes does not need a functionally active cell. On the other hand, the significant increase in sensitivity to actinomycin D demonstrates that the polymer interferes also with the structure of the outer membrane, modifying its permeability. The study on gene expression, based on the analysis of the transcripts in a temporal window where the contact with the polymer is not lethal and the damage is reversible, showed that some key genes of the synthesis and maintenance of the outer membrane structure ( fabR, fadR, fabA, waaA, waaC, kdsA, pldA, and pagP), as well as regulators of cellular response to oxidative stress ( soxS), are more expressed when bacteria are exposed to the polymer surface. All together these results identified the outer membrane as the main cellular target of the antimicrobial surface and indicated a specific cellular response to damage, providing more information on the antimicrobial mechanism. In this perspective, data reported here could play a pivotal role in a microbial growth control strategy based not only on the structural improvements of the materials but also on the possibility of intervening on the cellular pathways involved in the contrast reaction to these and other polymers with similar mechanisms.


Assuntos
Antibacterianos/metabolismo , Materiais Revestidos Biocompatíveis/química , Polímeros/química , Aciltransferases/genética , Aciltransferases/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Materiais Revestidos Biocompatíveis/farmacologia , Dactinomicina/química , Dactinomicina/metabolismo , Dactinomicina/farmacologia , Condutividade Elétrica , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Polietilenoglicóis/química , Polímeros/farmacologia , Polimetil Metacrilato/química , Propriedades de Superfície , Transativadores/genética , Transativadores/metabolismo , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo
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