Currently, it is clear that the luxS gene has an impact on the process of biofilm formation in Campylobacter jejuni. However, even within the species, naturally occurring strains of Campylobacter lacking the luxS gene exist, which can form biofilms. In order to better understand the genetic determinants and the role of quorum sensing through the LuxS/AI-2 pathway in biofilm formation, a set of mutant/complemented strains of C. jejuni 81-176 were prepared. Additionally, the impact of the mutagenic strategy used against the luxS gene was investigated. Biofilm formation was affected by both the presence and absence of the luxS gene, and by the mutagenic strategy used. Analysis by CLSM showed that all mutant strains formed significantly less biofilm mass when compared to the wild-type. Interestingly, the deletion mutant (∆luxS) showed a larger decrease in biofilm mass than the substitution (∙luxS) and insertional inactivated ([Formula: see text]luxS) mutants, even though all the mutant strains lost the ability to produce autoinducer-2 molecules. Moreover, the biofilm of the ∆luxS mutant lacked the characteristic microcolonies observed in all other strains. The complementation of all mutant strains resulted in restored ability to produce AI-2, to form a complex biofilm, and to develop microcolonies at the level of the wild-type.
Persulfidation contributes to a group of redox post-translational modifications (PTMs), which arise exclusively on the sulfhydryl group of cysteine as a result of hydrogen sulfide (H2S) action. Redox-active molecules, including H2S, contribute to sperm development; therefore, redox PTMs represent an extremely important signalling pathway in sperm life. In this path, persulfidation prevents protein damage caused by irreversible cysteine hyperoxidation and thus maintains this signalling pathway. In our study, we detected both H2S and its production by all H2S-releasing enzymes (cystathionine γ-lyase (CTH), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (MPST)) in male reproduction, including spermatozoa. We provided evidence that sperm H2S leads to persulfidation of proteins, such as glyceraldehyde-3-phosphate dehydrogenase, tubulin, and anchor protein A-kinase. Overall, this study suggests that persulfidation, as a part of the redox signalling pathway, is tightly regulated by enzymatic H2S production and is required for sperm viability.
- MeSH
- cystathionin-gama-lyasa metabolismus MeSH
- cystein metabolismus MeSH
- lidé MeSH
- rozmnožování MeSH
- sperma metabolismus MeSH
- sulfan * metabolismus MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
A bacteriocin termed plantaricin MX with a broad antimicrobial spectrum was produced by Lactobacillus plantarum NMD-17, which was isolated from Inner Mongolia traditional koumiss of china. Among 300 strains of lactic acid bacteria (LAB) belonging to the genera Lactococcus, Lactobacillus, Streptococcus, Leuconostoc, and Enterococcus, five strains including Lactobacillus reuteri NMD-86, Lactobacillus helveticus NMD-137, Lactococcus lactis NMD-152, Enterococcus faecalis NMD-178, and Enterococcus faecium NMD-219 were revealed to significantly induce the bacteriocin synthesis and greatly increase the cell numbers of Lactobacillus plantarum NMD-17 and activity of AI-2 signaling molecule. Bacteriocin synthesis was not increased by cell-free supernatants and autoclaved cultures of inducing strains, demonstrating that intact cells of inducing strains were essential to the induction of bacteriocin synthesis. The existence of bacteriocin structural plnEF genes and the plnD and luxS genes involved in quorum sensing was confirmed by PCR, and the presence of plnB gene encoding histidine protein kinase was determined by single oligonucleotide nested PCR (Son-PCR). Quantitative real-time PCR demonstrated that plnB, plnD, luxS, plnE, and plnF genes of L. plantarum NMD-17 were upregulated significantly (P < 0.01) in co-cultivation with L. reuteri NMD-86. The results showed that the bacteriocin synthesis of L. plantarum NMD-17 in co-cultivation might have a close relationship with LuxS-mediated quorum sensing system.
- MeSH
- bakteriální proteiny * genetika MeSH
- bakteriociny * genetika MeSH
- kumys * mikrobiologie MeSH
- Lactobacillales * fyziologie MeSH
- Lactobacillus plantarum * genetika MeSH
- lyasy štěpící vazby C-S * genetika MeSH
- mikrobiální interakce * fyziologie MeSH
- quorum sensing genetika MeSH
- Publikační typ
- časopisecké články MeSH
Garlic is a well-known example of natural self-defence system consisting of an inactive substrate (alliin) and enzyme (alliinase) which, when combined, produce highly antimicrobial allicin. Increase of alliinase stability and its activity are of paramount importance in various applications relying on its use for in-situ synthesis of allicin or its analogues, e.g., pulmonary drug delivery, treatment of superficial injuries, or urease inhibitors in fertilizers. Here, we discuss the effect of temperature, pH, buffers, salts, and additives, i.e. antioxidants, chelating agents, reducing agents and cosolvents, on the stability and the activity of alliinase extracted from garlic. The effects of the storage temperature and relative humidity on the stability of lyophilized alliinase was demonstrated. A combination of the short half-life, high reactivity and non-specificity to particular proteins are reasons most bacteria cannot deal with allicin's mode of action and develop effective defence mechanism, which could be the key to sustainable drug design addressing serious problems with escalating emergence of multidrug-resistant (MDR) bacterial strains.
- MeSH
- antibakteriální látky farmakologie MeSH
- Bacteria účinky léků ultrastruktura MeSH
- biokatalýza účinky léků MeSH
- časové faktory MeSH
- česnek enzymologie MeSH
- chemické jevy * MeSH
- disulfidy chemie metabolismus MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- kyseliny sulfinové chemie metabolismus MeSH
- lyasy štěpící vazby C-S metabolismus MeSH
- lyofilizace MeSH
- mikrobiální testy citlivosti MeSH
- mikrobiální viabilita účinky léků MeSH
- pufry MeSH
- stabilita enzymů účinky léků MeSH
- stereoizomerie MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The aim of this study was to evaluate the mutual relationship among perivascular adipose tissue (PVAT) and endogenous and exogenous H2S in vasoactive responses of isolated arteries from adult normotensive (Wistar) rats and hypertriglyceridemic (HTG) rats, which are a nonobese model of metabolic syndrome. In HTG rats, mild hypertension was associated with glucose intolerance, dyslipidemia, increased amount of retroperitoneal fat, increased arterial contractility, and endothelial dysfunction associated with arterial wall injury, which was accompanied by decreased nitric oxide (NO)-synthase activity, increased expression of H2S producing enzyme, and an altered oxidative state. In HTG, endogenous H2S participated in the inhibition of endothelium-dependent vasorelaxation regardless of PVAT presence; on the other hand, aortas with preserved PVAT revealed a stronger anticontractile effect mediated at least partially by H2S. Although we observed a higher vasorelaxation induced by exogenous H2S donor in HTG rats than in Wistar rats, intact PVAT subtilized this effect. We demonstrate that, in HTG rats, endogenous H2S could manifest a dual effect depending on the type of triggered signaling pathway. H2S within the arterial wall contributes to endothelial dysfunction. On the other hand, PVAT of HTG is endowed with compensatory vasoactive mechanisms, which include stronger anti-contractile action of H2S. Nevertheless, the possible negative impact of PVAT during hypertriglyceridemia on the activity of exogenous H2S donors needs to be taken into consideration.
- MeSH
- aorta thoracica patofyziologie MeSH
- cévní endotel patofyziologie MeSH
- cystathionin-gama-lyasa metabolismus MeSH
- hypertriglyceridemie metabolismus MeSH
- metabolický syndrom metabolismus patofyziologie MeSH
- modely nemocí na zvířatech MeSH
- noradrenalin farmakologie MeSH
- oxidace-redukce MeSH
- potkani Wistar MeSH
- signální transdukce * MeSH
- superoxiddismutasa metabolismus MeSH
- superoxidy metabolismus MeSH
- synthasa oxidu dusnatého, typ III metabolismus MeSH
- tuková tkáň metabolismus MeSH
- vazodilatace fyziologie MeSH
- vazokonstrikce účinky léků MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The microaerophilic pathogen Campylobacter jejuni is a leading bacterial cause of human gastroenteritis in developed countries. Even though it has a reputation as a fastidious organism, C. jejuni is widespread and can be easily isolated from various animals, food, and environmental sources. It is suggested that an ability to form biofilms is probably necessary for the survival of C. jejuni under harsh environmental conditions. The first step required for successful biofilm formation is adhesion to a suitable surface. Therefore, in this work, the degree of adhesion was evaluated, followed by characterization and quantification of biofilms using confocal laser scanning microscopy (CLSM). A total of 15 isolates of C. jejuni were used in the experiments (12 isolates from surface and waste waters, 1 human clinical, 1 food and 1 ACTT BAA-2151 collection strain, all samples originated from the Czech Republic). Regardless of the sample origin, all C. jejuni isolates were able to adhere to the polystyrene surface within 30 min, with the number of attached cells increasing with the time of incubation. The resulting data showed that all isolates were able to form complex voluminous biofilms after 24 h of cultivation. The average amount of biovolume ranged from 3.59 × 106 μm3 to 17.50 × 106 μm3 in isolates obtained from different sources of water, 16.79 × 106 μm3 in the food isolate and 10.92 × 106 μm3 in the collection strain. However, the highest amount of biomass was produced by the human clinical isolate (25.48 × 106 μm3). Similar to the quantity, the architecture of the biofilms also differed, from a rugged flat monolayer of cells to large clustered structures. Further, all isolates were tested for the presence of the luxS gene, as the luxS/AI-2 (autoinducer-2) quorum sensing pathway has been previously connected with enhanced biofilm formation. Two isolates originated from surface waters did not possess the luxS gene. These isolates formed thinner and sparser biofilms lacking the presence of significant clusters. However, the ability to adhere to the surface was preserved. The sequencing of the luxS-containing fragments shown a high similarity of the luxS gene among the isolates.
- MeSH
- bakteriální proteiny MeSH
- biofilmy MeSH
- Campylobacter jejuni * genetika MeSH
- lidé MeSH
- lyasy štěpící vazby C-S MeSH
- quorum sensing MeSH
- voda MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
The role of hydrogen sulfide (H2S) is addressed in Xenopuslaevis oocytes. Three enzymes involved in H2S metabolism, cystathionine β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase, were detected in prophase I and metaphase II-arrested oocytes and drove an acceleration of oocyte meiosis resumption when inhibited. Moreover, meiosis resumption is associated with a significant decrease in endogenous H2S. On another hand, a dose-dependent inhibition was obtained using the H2S donor, NaHS (1 and 5 mM). NaHS impaired translation. NaHS did not induce the dissociation of the components of the M-phase promoting factor (MPF), cyclin B and Cdk1, nor directly impacted the MPF activity. However, the M-phase entry induced by microinjection of metaphase II MPF-containing cytoplasm was diminished, suggesting upstream components of the MPF auto-amplification loop were sensitive to H2S. Superoxide dismutase and catalase hindered the effects of NaHS, and this sensitivity was partially dependent on the production of reactive oxygen species (ROS). In contrast to other species, no apoptosis was promoted. These results suggest a contribution of H2S signaling in the timing of amphibian oocytes meiosis resumption.
- MeSH
- apoptóza účinky léků MeSH
- cyklin B metabolismus MeSH
- cystathionin-beta-synthasa antagonisté a inhibitory metabolismus MeSH
- cystathionin-gama-lyasa antagonisté a inhibitory metabolismus MeSH
- cytoplazma metabolismus MeSH
- faktor podporující zrání metabolismus MeSH
- fosfatasy cdc25 metabolismus MeSH
- katalasa metabolismus MeSH
- meióza účinky léků MeSH
- metafáze účinky léků MeSH
- oocyty chemie enzymologie metabolismus MeSH
- profáze meiózy I účinky léků MeSH
- proteinkinasy metabolismus MeSH
- proteiny buněčného cyklu metabolismus MeSH
- proteiny Xenopus metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- signální transdukce účinky léků MeSH
- sulfan metabolismus MeSH
- sulfidy metabolismus farmakologie MeSH
- sulfurtransferasy antagonisté a inhibitory metabolismus MeSH
- superoxiddismutasa metabolismus MeSH
- viabilita buněk účinky léků MeSH
- Xenopus laevis MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The luxS gene is responsible for the synthesis of AI-2 (autoinducer-2), a signaling molecule that participates in quorum sensing regulation in a large number of bacteria. In this work, we investigated which phenotypes are regulated by luxS gene in Serratia proteamaculans 94, psychrotrophic strain isolated from spoiled refrigerated meat. AI-2 was identified in S. proteamaculans 94, and the luxS gene involved in its synthesis was cloned and sequenced. A mutant with the inactivated luxS gene was constructed. Inactivation of the luxS gene was shown to lead to the absence of AI-2 synthesis, chitinolytic activity, swimming motility, suppression of the growth of fungal plant pathogens Rhizoctonia solani and Helminthosporium sativum by volatile compounds emitted by S. proteamaculans 94 strain, and to a decrease of extracellular proteolytic activity. The knockout of the luxS gene did not affect synthesis of N-acyl-homoserine lactones, lipolytic, and hemolytic activities of S. proteamaculans 94.
- MeSH
- bakteriální proteiny genetika MeSH
- biofilmy růst a vývoj MeSH
- fenotyp MeSH
- homoserin analogy a deriváty metabolismus MeSH
- laktony metabolismus MeSH
- lyasy štěpící vazby C-S genetika MeSH
- maso mikrobiologie MeSH
- mikrobiální interakce MeSH
- quorum sensing genetika MeSH
- regulace genové exprese u bakterií MeSH
- Serratia genetika metabolismus MeSH
- těkavé organické sloučeniny analýza MeSH
- umlčování genů * MeSH
- Publikační typ
- časopisecké články MeSH
Methionine deprivation induces growth arrest and death of cancer cells. To eliminate l-methionine we produced, purified, and characterized the recombinant pyridoxal 5'-phosphate (PLP)-dependent l-methionine γ-lyase (MGL)- BL929 from the cheese-ripening Brevibacterium aurantiacum Transformation of an Escherichia coli strain with the gene BL929 from B. aurantiacum optimized for E. coli expression led to production of the MGL-BL929. Elimination of l-methionine and cytotoxicity in vitro were assessed, and methylation-sensitive epigenetics was explored for changes resulting from exposure of cancer cells to the enzyme. A bioreactor was built by encapsulation of the protein in human erythrocytes to achieve sustained elimination of l-methionine in extracellular fluids. Catalysis was limited to α,γ-elimination of l-methionine and l-homocysteine. The enzyme had no activity on other sulfur-containing amino acids. Enzyme activity decreased in presence of serum albumin or plasma resulting from reduction of PLP availability. Elimination of l-methionine induced cytotoxicity on a vast panel of human cancer cell lines and spared normal cells. Exposure of colorectal carcinoma cells to the MGL-BL929 reduced methyl-CpG levels of hypermethylated gene promoters including that of CDKN2A, whose mRNA expression was increased, together with a decrease in global histone H3 dimethyl lysine 9. The MGL-erythrocyte bioreactor durably preserves enzyme activity in vitro and strongly eliminates l-methionine from medium.
- MeSH
- bioreaktory MeSH
- Brevibacterium enzymologie MeSH
- dospělí MeSH
- erytrocyty účinky léků metabolismus MeSH
- lidé MeSH
- lyasy štěpící vazby C-S farmakologie MeSH
- methionin metabolismus MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- rekombinantní proteiny farmakologie MeSH
- tobolky MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Reactive dicarbonyls stimulate production of advanced glycation endproducts, increase oxidative stress and inflammation and contribute to the development of vascular complications. We measured concentrations of dicarbonyls - methylglyoxal (MG), glyoxal (GL) and 3-deoxyglucosone (3-DG) - in the heart and kidney of a model of metabolic syndrome - hereditary hypertriglyceridemic rats (HHTg) and explored its modulation by metformin. Adult HHTg rats were fed a standard diet with or without metformin (300 mg/kg b.w.) and dicarbonyl levels and metabolic parameters were measured. HHTg rats had markedly elevated serum levels of triacylglycerols (p<0.001), FFA (p<0.01) and hepatic triacylglycerols (p<0.001) along with increased concentrations of reactive dicarbonyls in myocardium (MG: p<0.001; GL: p<0.01; 3-DG: p<0.01) and kidney cortex (MG: p<0.01). Metformin treatment significantly reduced reactive dicarbonyls in the myocardium (MG: p<0.05, GL: p<0.05, 3-DG: p<0.01) along with increase of myocardial concentrations of reduced glutathione (p<0.01) and glyoxalase 1 mRNA expression (p<0.05). Metformin did not have any significant effect on dicarbonyls, glutathione or on glyoxalase 1 expression in kidney cortex. Chronically elevated hypertriglyceridemia was associated with increased levels of dicarbonyls in heart and kidney. Beneficial effects of metformin on reactive dicarbonyls and glyoxalase in the heart could contribute to its cardioprotective effects.
- MeSH
- deoxyglukosa analogy a deriváty metabolismus MeSH
- dieta MeSH
- fyziologický stres MeSH
- glutathion metabolismus MeSH
- glyoxal metabolismus MeSH
- hypertriglyceridemie farmakoterapie genetika patofyziologie MeSH
- hypoglykemika terapeutické užití MeSH
- krysa rodu rattus MeSH
- laktoylglutathionlyasa metabolismus MeSH
- metformin terapeutické užití MeSH
- myokard metabolismus MeSH
- potkani Wistar MeSH
- pyruvaldehyd metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
