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We present the specific cooperative effect of a semisynthetic glycopeptide antibiotic teicoplanin and chiral ionic liquids containing the (1R,2S,5R)-(-)-menthol moiety on the chiral recognition of enantiomers of mandelic acid, vanilmandelic acid, and phenyllactic acid. Experiments were performed chromatographically on an Astec Chirobiotic T chiral stationary phase applying the mobile phase with the addition of the chiral ionic liquids. The stereoselective binding of enantiomers to teicoplanin in presence of new chiral ionic liquids were evaluated applying thermodynamic measurements and the docking simulations. Both the experimental and theoretical methods revealed that the chiral recognition of enantiomers in the presence of new chiral ionic liquids was enthalpy driven. The changes of the teicoplanin conformation occurring upon binding of the chiral ionic liquids are responsible for the differences in the standard changes in Gibbs energy (ΔG0 ) values obtained for complexes formed by the R and S enantiomers and teicoplanin. Docking simulations revealed the steric adjustment between the chiral ionic liquids cyclohexane ring (chair conformation) and the β-d-glucosamine ring of teicoplanin and additionally hydrophobic interactions between the decanoic aliphatic chain of teicoplanin and the alkyl group of the tested salts. The obtained terpene derivatives can be considered as "structural task-specific ionic liquids" responsible for enhancing the chiral resolution in synergistic systems with two chiral selectors.

Thermophilic Campylobacter species colonize the intestine of agricultural and domestic animals commensally but cause severe gastroenteritis in humans. In contrast to other enteropathogenic bacteria, Campylobacter has been considered to be non-glycolytic, a metabolic property originally used for their taxonomic classification. Contrary to this dogma, we demonstrate that several Campylobacter coli strains are able to utilize glucose as a growth substrate. Isotopologue profiling experiments with (13) C-labeled glucose suggested that these strains catabolize glucose via the pentose phosphate and Entner-Doudoroff (ED) pathways and use glucose efficiently for de novo synthesis of amino acids and cell surface carbohydrates. Whole genome sequencing of glycolytic C. coli isolates identified a genomic island located within a ribosomal RNA gene cluster that encodes for all ED pathway enzymes and a glucose permease. We could show in vitro that a non-glycolytic C. coli strain could acquire glycolytic activity through natural transformation with chromosomal DNA of C. coli and C. jejuni subsp. doylei strains possessing the ED pathway encoding plasticity region. These results reveal for the first time the ability of a Campylobacter species to catabolize glucose and provide new insights into how genetic macrodiversity through intra- and interspecies gene transfer expand the metabolic capacity of this food-borne pathogen.

The focus of the study was to develop a bio-analytical assay for a 1,2,4-triazole derivative from plasma and brain tissue homogenate samples. The goal was to compare analytical techniques that facilitate high accuracy with simplified sample processing. In this study, commonly used standard protein precipitation and solid-phase extraction methods utilizing C18 and cartridges of Hybrid technology were compared in terms of their ability for sample pretreatment and removal of biological matrices before high-performance liquid chromatography quantification. Fast classical reversed-phase chromatography on a C18 column paired with selective sample preparation using Hybrid solid-phase extraction technology resulted in the most precise bio-analytical determination of the hydrophobic 1,2,4-triazole derivative in both biological samples studied. The obtained recovery values were above 95% with the coefficient of variation lower than 5%.

In this study a new RP-HPLC with photo-diode array detection method for the determination of ibuprofen ((RS)-2-(4-isobutylphenyl)propionic acid) in human plasma samples was developed. Samples were prepared by SPE and analyzed by an isocratic elution mode over a C18 column using 80% methanol. A novel sample pretreatment method, based on the addition of ionic liquids possessing chaotropic ions to small human plasma sample (100 μL), was elaborated. 1-Butyl-3-methylimidazolium chloride and 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM BF4) were tested from the point of view of extraction yield. Quantification was based on calibration curve applying diclofenac as the internal standard. Owing to dilution of plasma sample by 2 mM aqueous solution of BMIM BF4 before SPE, appropriate sample purification and extraction yields higher than 95% with precision lower than 2% can be achieved. Linear coefficients of correlation (r2) were >0.99 in the range of 0.3–5 μg/mL ibuprofen concentration in plasma. The limit of quantification was 65 ng/mL and the detection limit for ibuprofen was 19.5 ng/mL.

This paper is devoted to application of ionic liquids as surfactants in LC of organic compounds, derivatives of 1,4-thiosemicarbazides. According to HPLC requirements the most advantageous conditions such as transparency for ultraviolet light, low CMC, additional inorganic salt additives, and appropriate organic solvent were established. The CMC was determined using conductivity measurements. Suitability of two different stationary phases: RP-C18 and cyanopropyl bonded phase was examined under micellar conditions. Chosen ionic liquid surfactant was compared to common traditional amphiphilic reagent - SDS. Elaborated on chromatographic micellar conditions were tested as a pilot technique for prediction of distribution coefficients of organic analytes in ionic liquid-based aqueous two-phase system.

New synthesized 1,4-disubstituted thiosemicarbazide derivatives were analyzed in the RP system, modified with the addition of salts; chaotropic (sodium hexafluorophosphate - Na PF(6)), cosmotropic (sodium phosphate - NaH(2)PO(4)), and neutral (NaCl) on Zorbax XDB C18 column. The effect of the eluent composition on the analytes retention (k), system efficiency (N), peak symmetry (A(s)), and LOD values were all examined and compared to unmodified organic-aqueous mobile phase system. It was established that eluent modified with chaotropic salts addition was also the most advantageous according to other peak parameters such as the theoretical plates numbers and asymmetry factors. The lower LOD values were achieved in comparison to unmodified organic-aqueous eluent system. Compatibility of lipophilicity parameters calculated by the use of computer software with experimental ones measured by RP-HPLC was also the best for chaotropic modified mobile phase. To explain the observed phenomena, molecular modeling was performed for chosen representative compound in different environment representing examined mobile phase composition.

Summary DivIVA is a well-conserved coiled-coil protein present in most Gram-positive bacteria and has been implicated in division site selection, peptidoglycan biosynthesis and sporulation. DivIVA proteins bind lipid membranes and characteristically accumulate at curved membrane areas, i.e. the cell poles and the division site, to which they recruit various interaction partners. We have studied the role of this morphogen in the human pathogen Listeria monocytogenes and our results suggest a novel mechanism by which DivIVA contributes to cell division. Contrary to expectation a ΔdivIVA mutant exhibited a pronounced chaining phenotype rather than a defect in cell division which we attributed to reduced extracellular levels of the autolytic enzymes p60 and MurA. We demonstrate that this is due to a malfunction in secretion of these autolysins and phenotypic comparison of the ΔdivIVA strain with a ΔsecA2 mutant suggests that DivIVA influences the activity of the SecA2 secretion route in L. monocytogenes. Also from the phenotypic analysis it was clear that divIVA affected swarming motility, biofilm formation, invasiveness and cell-to-cell spread in cell culture infection models. Thus, our experiments show that DivIVA is an important factor for various listerial traits that are essential for the pathogenicity of this organism.

Sodium hexafluorophosphate, perchlorate and tetrafluoroborate were applied as the ion-pair reagents in reversed-phase chromatography of several imidazolium-based ionic liquids. The optimization of the retention was performed by changing the kind of organic modifier (methanol, acetonitrile), concentration and the kind of the ion-pair reagents in the mobile phase and the column kind (Zorbax SB-C18, Zorbax SB-Phenyl, Zorbax SB-CN, Zorbax SB-NH2 and Supelcosil LC-F). The selectivity of the proposed chromatographic systems according to the cation kind was compared on the basis of the resolution of ionic liquid mixture. The perturbation method was applied to identify the anion kind. The formation of ion-associated complexes between promethazine as counter-cation and chaotropic anions controlling their retention was confirmed.

Summary Various non-spore forming bacteria, including Escherichia coli, enter a dormant-like state, the viable but non-culturable (VBNC) state, characterized by the presence of viable cells but the inability to grow on routine laboratory media. Upon resuscitation, these VBNC cells recover both culturability and pathogenicity. In 2011, a large outbreak involving more than 3000 cases of bloody diarrhoea and haemolytic uremic syndrome was caused by an E. coli O104:H4 strain expressing genes characteristic of both enterohaemorrhagic (EHEC) and enteroaggregative E. coli (EAEC). The ability of the outbreak strain to enter the VBNC state may have complicated its detection in the suspected sources. In this paper, we investigated the ability of the outbreak strain to enter and subsequently recover from the VBNC state. We found that in a nutrient-poor micro-environment, various stresses such as toxic concentrations of copper ions or certain types of tap water are able to render the bacteria unculturable within a few days. Without copper ion stress, the majority of cells remained culturable for at least 40 days. Incubation with the stressors at 23°C compared with 4°C hastened this observed loss of culturability. The integrity of a considerable fraction of copper ion- and tap water 1-stressed bacteria was demonstrated by live/dead staining and microscopy. Relieving stress by copper-ion chelation facilitated resuscitation of these bacteria while preserving their fitness, major virulence gene markers (stx2, aggR, aggA genes) and specific phenotypes (ESBL resistance, autoaggregation typical for EAEC strains).

Highly hydrophilic compounds belonging to biogenic amines were analysed in the reversed-phase system, modified with the addition of ionic liquids: 1-ethyl-3-methyl-imidazolium hexafluorophosphate (EMIM PF(6)) and chaotropic salt NaPF(6) on Discovery HS C18 column at acidic conditions. The effect of the additives concentration and the presence of organic solvent on the analytes' chromatographic parameters such as retention factor, tailing factor and theoretical plates number were all determined. On the basis of k versus ionic liquid concentration in aqueous-organic mobile phase with constant amount of phosphate buffer, retention mechanism was studied. It was established that the presence of organic solvent with low dielectric constant and ionic liquid with both chaotropic ions allows achieving typical Langmuir shape of this relationship. Investigated mobile phase additives are comparable according to efficiency and selectivity towards biogenic amines analysis. However, the sensitivity was found to be better for the eluent system modified with chaotropic salt.

Synchroma grande (Synchromophyceae, Heterokontophyta) is a marine amoeboid alga, which was isolated from a benthic habitat. This species has sessile cell stages (amoeboid cells with lorica and cysts) and non-sessile cell stages (migrating and floating amoebae) during its life cycle. The different cell types and their transitions within the life cycle are described, as are their putative functions. Cell proliferation was observed only in cells attached to the substrate but not in free-floating or migrating cells. We also characterised the phagotrophy of the meroplasmodium in comparison to other amoeboid algae and the formation of the lorica. The functional adaptations of S. grande during its life cycle were compared to the cell stages of other amoeboid algae of the red and green chloroplast lineages. S. grande was found to be highly adapted to the benthic habitat. One sexual and two asexual reproductive strategies (haplo-diploid life cycle) support the ability of this species to achieve rapid diversification and high adaptivity in its natural habitat.

The enantioseparation of carboxylic acids, including amino acid dansyl-derivatives, 2-arylpropionic acids and 2-aryloxypropionic acids, was tested by CEC on a porous silica sol-gel monolithic column that was prepared by polycondensation of tetramethoxysilane in acidic conditions and post-gelation heat treatment (120 degrees C for 3 h) in the presence of urea, and successively, by anchoring to the silica (+)-1-allyl-(5R,8S,10R)-terguride as the chiral selector. The bimodal structure of the sorbent showed through pores with a median value of 1.39 mu m and a mesopore size distribution ranging between 6 and 12 nm (average pore size of 9.9 nm). To attain optimum separation conditions, the influence of the pH and the concentration of the buffer solution in the mobile phase on resolution were investigated. The monolithic column showed: (i) for the compounds studied resolution values two or three times higher in comparison with previously developed separation systems where the same chiral selector was used. For example, on the monolithic column Dns-serine enantiomers were much better separated (8 min with a selectivity factor of 1.34) than by HPLC (20 min, alpha = 1.17); (ii) high chemical and mechanical stability as demonstrated by the use of such column for hundreds of analysis along about 1 year without significant variations of the resolution and the retention parameters.

An assay of L-tyrosine (Tyr) hydroxylating activity operating in lincomycin biosynthesis is described. The assay development consisted of HPLC procedure development, assessing the effect of reaction mixture components on non-enzymatic Dopa and Tyr oxidation, and sample stability evaluation. The HPLC procedure with isocratic elution and fluorescence detection was developed and validated. The method showed a wide linear range of Dopa determination of 0.125-25 micromol/L with lower limit of quantification (LLOQ) of 0.125 micromol/L, RSD of 7.2% and accuracy of 101.7%. The studied linear range of Tyr was 15.625 mmol/L to 500 mmol/L with LLOQ of 15.625 mmol/L, RSD of 1.1%, and accuracy of 98.1%. Recoveries for Dopa and Tyr were 100.66 +/- 0.89% and 94.76 +/- 0.94%, respectively. The inter- and intra-day accuracies and precisions were all within 10%. Samples of the reaction mixture were stable for at least 24 h at room temperature (RT) and 28 days at -20 degrees C. The method was tested for the enzyme activity monitoring in purified as well as crude preparations and enabled micro preparation of the enzyme product during confirmation of its identity. The influence of pH and ascorbic acid content in reaction mixture was studied with respect to non-enzymatic Tyr oxidation.

Three urea derivatives of ergoline-based chiral selectors (CSs), differing in the size of the urea side chain, i.e. dimethyl- (CSI), diethyl- (CSII), and diisopropylurea (CSIII), were used to study the effect of steric hindrance on the enantioseparation of dansyl amino acids (Dns-AAs), pesticides, and mandelic acid under condition of capillary electrophoresis (CE) in linear polyacrylamide coated capillaries. A mixture of organic modifiers (MeOH/THF, 4:1 v/v) in a BGE consisting of 100 mM beta-alanine-acetate was used to increase the solubility of CSs up to 25 mM. The capillary was filled with CS (high UV absorption), and the inlet and outlet vials contained buffer solutions only. The best enantioseparation of Dns-AAs was achieved on CSI. Increased steric hindrance of the chiral binding site led to reduction of both enantioselectivity and resolution. The opposite pattern was observed for the separation of mandelic acid enantiomers, where the best enantioseparation and resolution was obtained with CSIII. Most of the pesticides studied reached maximum selectivity on the diethylurea ergoline derivative (CSII). Enantioseparation of fenoxaprop was found to be independent of steric hindrance.

The D/L ratio of aspartic acid enantiomers in proteins of low turnover is generally accepted as a reliable procedure for age determination. In our study, twelve samples of eyetooth dentin were analyzed for age determination. The pure insoluble collagen isolated from eyetooth dentin was obtained by an EDTA demineralization process. Free amino acids obtained after collagen hydrolysis were converted into o-phthaldialdehyde-N-acetyl-L-cysteine (OPA-NAC) derivatives for HPLC analysis under modified conditions and into trifluoroacetic acid isopropyl esters for GC analysis, respectively. The modified HPLC procedure used phosphate buffer and acidified sample matrix prior to injection which resulted in suppression of peak tailing of both diastereomers, thus allowing achievement of both good selectivity and good resolution. To ensure the high accuracy of the developed method the other parameters, i.e. specificity, precision, linearity, LOD, and LOQ, were also determined. Nine collagen samples covering the age range of 18 to 84 years were used for the determination of coefficient of racemization (KR) and calculation of parameters for age estimation. The regression equations for the data set analyzed were as follows: KR= 0.0005 x age + 0.0262 (R2 = 0.9639) for HPLC, and KR= 0.0006 x age + 0.0319 (R2 = 0.9374) for GC, respectively.

The cytokine macrophage migration inhibitory factor (MIF) is inducibly secreted by immune cells and certain other cell types to critically participate in the regulation of the host immune response. However, MIF does not contain a N-terminal signal sequence and the mechanism of MIF secretion is unknown. Here we show in a model of endotoxin-stimulated THP-1 monocytes that MIF does not enter the endoplasmatic reticulum and that MIF secretion is not inhibited by monensin or brefeldin A, demonstrating that MIF secretion occurs via a non-classical export route. Glyburide and probenicide but not other typical inhibitors of non-classical protein export strongly block MIF secretion, indicating that the export pathway of MIF involves an ABCA1 transporter.

A method of separation of dansyl amino acid (Dns-AA) enantiomers by capillary electrophoresis (CE) using terguride-based chiral selectors (CSs) was developed using linear polyacrylamide coated capillaries. Due to the high UV absorption of all studied CSs, only the capillary was filled with CS, while the inlet and outlet vials contained buffer solution only. Conditions were found for baseline separation of all Dns-AA under study. L-Dns-AAs were eluted prior to D-Dns-AAs on all studied CSs. Use of the optical antipode of allyl-terguride resulted in reversal of elution order of all Dns-AAs (the D-form was eluted prior to the L-form). The best enantioselectivity was achieved for hydroxyl group-containing AAs, i. e. serine and threonine. This finding is in good agreement with the proposed mechanism of chiral recognition of these AAs determined by X-ray studies. Seven CSs with different N(1)-side chain units, five of them designed for the preparation of silica based HPLC chiral stationary phases, were tested for their ability to resolve enantiomers of Dns-AAs. Different polarity and hydrophobicity of N(1)-side chains of the CSs resulted in different CS mobilities, thus influencing the effective separation time in the capillary. 1-(5-Aminopentyl)-terguride showed the best enantioseparation and resolution for the Dns-AAs studied.

Flobufen (F) is the original nonsteroidal antiinflammatory drug (NSAID) containing two enantiomers. The aim of this investigation was to elucidate the biotransformation pathway of F at chiral level in phase I of biotransformation. Stereoselectivity and stereospecificity of the respective enzymes were studied in male rats in vitro (microsomal and cytosolic fractions, hepatocytes suspension) and in vivo. The rac-F, (+)-R-F and (−)-S-F were used as substrates. Amounts of F enantiomers, 4-dihydroflobufen diastereoisomers (DHF) and other metabolites (M-17203, UM) were determined with a chiral HPLC method in two chromatographic runs on R,R-ULMO and allyl-terguride bonded columns. Stereoselective biotransformation of the two enantiomers of F was observed at all tested levels and significant bidirectional chiral inversion of enantiomers of F was observed in hepatocytes. Mean enantiomeric ratios of F concentrations (S-/R-), after rac-F incubations, ranging from 1.09 in cytosolic fraction to 18.23 in hepatocytes. Stereospecificity of the respective F reductases was also observed. (2R;4S)-DHF and (2S;4S)-DHF are the principal metabolites of F in microsomes and hepatocytes. Neither DHF diastereoisomers nor M-17203 were found in cytosolic fraction. Only the nonchiral metabolite, M-17203, was found in all urine and feces samples after oral administration of F. Chirality 13:754–759, 2001. © 2001 Wiley-Liss, Inc.

The content of ergot alkaloids (ergovaline and chanoclavine), and their production in October 1996 and during the period May–September 1997, were investigated in seventeen ecotypes of perennial ryegrass (Lolium perenne L.) and in nineteen ecotypes of meadow fescue (Festuca pratensis Huds.), naturally infected with Neotyphodium spp. The ecotypes were collected in the north-eastern part of the Czech Republic. In 1996 the content of ergovaline in the ecotypes of perennial ryegrass ranged from 0·00 to 2·73 μg g–1 dry matter (DM) (one cut), and in 1997 from 0·00 to 4·65 μg g–1 DM (five cuts). In meadow fescue the content of ergovaline varied from 0·00 to 0·61 μg g–1 DM (one cut) in 1996, and in 1997 from 0·00 to 2·31 μg g–1 DM (five cuts). The content of chanoclavine (investigated in 1997 in four cuts only) in perennial ryegrass ranged between 0·00 and 3·39 μg g–1 DM, and in meadow fescue between 0·00 and 2·26 μg g–1 DM. Most ecotypes of L. perenne reacted to the high temperature and heavy rainfall in June and July of 1997 with an enhanced production of ergovaline, whereas the content of chanoclavine was not changed. Such reaction to stress conditions was not observed in the ecotypes of F. pratensis. Large differences in the production of both ergot alkaloids between different ecotypes of both plant species were observed.

Enantioseparation of 2-arylpropionic acids (Flobufen) and dansyl-derivatives of amino acids can be achieved in 10–15 min by electrochromatography using a capillary coated with poly-terguride. It was found that the analytes, in the range of the buffer pH between 2.5–4.0, were driven by anodic electroosmotic flow originated by the positively charged moieties of the ergolinic skeleton, and, only partially, by their anodic electrophoretic mobility. The enantiomers eluted with the same sequence, e.g. l- or (S)- isomer more retained than d- or (R)-, which was observed in HPLC separations on terguride-silica based CSPs, thus indicating that racemates were resolved by a similar chiral recognition mechanism. The retention of the solutes was affected by the concentration and composition of the effluent. An other mobile phase variable, strongly affecting the selectivity, was the pH of the buffer. © 1997 John Wiley & Sons, Ltd.

Designed and synthesized 4-alkyl-1,2,4-triazole-3-thione derivatives showed significant anticonvulsant activity, determined in the maximal electroshock-induced seizure (MES) test. The chemical structure of all new compounds was confirmed by spectral methods ((1)H NMR, (13)C NMR, IR, MS). A sensitive and selective method was elaborated for the determination of the anticonvulsant compounds levels in mice brain tissue, based on HPLC with diode array detector (DAD). Chromatographic tests showed that lack of anticonvulsant effect of two derivatives (15, 16) with long alkyl chains at N-4 position of the 1,2,4-triazole ring was due to the inability to cross the blood-brain barrier (BBB).

Electrocoagulation is an evolving technology that has been effectively applied for wastewater treatment but its applications in biotechnology and nanotechnology are very limited. This method was applied for the preparation of nanoparticles from soluble exopolysaccharide (EPS) produced by Claviceps viridis in a submerged batch culture. A cathode/anode pair electrode (Al or Fe) system was used for determination of the separation rates of electrocoagulation and the yields of EPS nanoparticles production. The separation rates of 0.170 ± 0.003 mg EPS/sec (Fe electrodes) and 0.250 ± 0.003 mg EPS/sec (Al electrodes) were calculated for voltage gradient 1 V/1 cm of electrodes distance and were constant during experiments. The specific yield of EPS nanoparticles production based on the consumed electric power was dependent on the material of the electrodes and its value was determined as 0.71 ± 0.01 mg EPS/W for Fe electrodes and 0.91 ± 0.01 mg EPS/W for Al electrodes, respectively. © 2003 Wiley Periodicals, Inc.

A new polyene macrolide, closely related to the pentaene macrolide antibiotic roflamycoin, is isolated from both fermentation broth and biomass of Streptomyces durmitorensis wild-type strain MS405.

Subunits II and III of the photosystem I reaction centre are encoded by the nuclear genes PsaD and PsaF, respectively. In spinach, the expression of both genes is highly synchronized with regard to time, space and in response to stimulators such as light. Nevertheless, promoter sequences as well as the design and location of regulatory elements are strikingly different. Promoter and leader of PsaF, when fused to the GUS reporter gene, direct a positive light response in the cotyledons of transgenic tobacco seedlings. In contrast, the equivalent PsaD regions confer a negative-light regulation to the GUS gene. If a 6-kb fragment that contains 1802 bp of the promoter, the transcription unit as well as additional 2.5 kb downstream of the PsaD gene is introduced into tobacco, the transcript level from the PsaD transgene is positively light-regulated in tobacco cotyledons. Thus, regulatory elements of the spinach PsaD and PsaF promoters are arranged in a very different way and essential cis-determinants for the positive light response of the PsaD gene can be located within the coding region and/or even further downstream.

A series of nine naturally occurring and/or semi-synthetic elymoclavine mono- and oligoglycosides containing various sugar units was investigated by fast atom bombardment, high-resolution measurements and linked scans. The fragmentations of the [M + H]+ and [M + Na]+ ions are described and their different contributions to the structure elucidation are discussed.

A new covalently-bonded, silica-based stationary phase, using as the chiral selector the 1-(3-aminopropyl) derivative of (+)-(5R,8S,10R)-terguride, has been developed to resolve optically active isomers by HPLC. Good resolution of structurally related racemic ergot alkaloids were obtained using water-methanol mixtures as the eluent. Analysis of the influence of the type and concentration of the organic modifier, and the pH of the buffer in the mobile phase allowed the enantioseparation of these compounds to be optimized. Determination of the optical purity of a lisuride-containig drug is reported. © 1994 Wiley-Liss, Inc.