Characterization of a bacteriocin-producing strain of Enterococcus faecalis from cow’s milk used in the production of Moroccan traditional dairy foods

A bacteriocin-producing lactic acid bacterium (strain 2.5) isolated from cow’s milk used in cheese production from Northern Morocco was selected for its strong anti-listerial activity. The producer strain was identified as Enterococcus faecalis by molecular methods. Strain 2.5 carried the genetic determinants for the two-peptide enterococcal bacteriocin enterocin 1071, and the active bacteriocin was purified to homogeneity by reversed-phase chromatography from culture broths of the producer strain. Strain 2.5 carried two plasmids (of ∼7 and 40 kb). Characterization of strain 2.5 at biosafety level indicated that this strain is non-haemolytic, and lacks the genetic determinants for most of the virulence factors described in enterococci (cylB, cylM, gelE, ace and agg) although it carried the genetic determinants cylA, efaAfs as well as determinants for the sex pheromone peptides cpd, cob, and ccf. Strain 2.5 was resistant to tetracycline, rifampicin, and ciprofloxacin, but it was sensitive to penicillin, ampicillin, vancomycin, and teicoplanin. Results from the present study support the potential role of strain 2.5 as an anti-listerial agent to be tested in traditional fermented foods.     

Impact of Cu(II) ions on the structure and antimicrobial properties of sisomicin,an aminoglycoside antibiotic

The formation of copper(II) complexes of an aminoglycoside antibiotic – sisomicin – was studied by potentiometry and spectroscopic techniques (UV–Vis, CD, NMR and EPR). At physiological pH, Cu(II) is bound to both amino functions and hydroxyl oxygen of the 2-deoxystreptamine moiety. When pH increases slightly, another amino group located at the aminosugar ring becomes engaged in the coordination process. Microbiological studies with the use of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa showed that copper(II) does not interfere with the bactericidal action of sisomicin.     

Bio-oxidation of tripropylene glycol under aerobic conditions

Aerobic biodegradation of tripropylene glycol (PG3) was investigated under the conditions of the OECD screening test 301E and the Continuous Flow Activated Sludge Simulation test (CFAS). A modified two-chamber facility with a denitrification stage was used for the CFAS test. Primary PG3 biodegradation was measured by the HPLC with fluorimetric detection and analyte derivatisation. Metabolites were identified by LC-MS with electrospray ionisation and GC-MS with electron impact ionisation, as well as semiquantitatively determined by the LC-MS technique. PG3 was found to be inherently biodegradable and it exhibits a strong poisonous effect on activated sludge after exceeding the threshold concentration (10 mg l⁻¹). Metabolite accumulation onto the activated sludge is probably responsible for this poisonous effect. Probable biotransformation products of tripropylene glycol under the aerobic conditions include metabolites with a single terminal aldehyde or a ketone group and metabolites with two terminal aldehyde or ketone groups. Their concentration rises at the end of the OECD screening test.     

Radiation of the Tnt1 retrotransposon superfamily in three Solanaceae genera

Background  

Tnt1 was the first active plant retrotransposon identified in tobacco after nitrate reductase gene disruption. The Tnt1 superfamily comprises elements from Nicotiana (Tnt1 and Tto1) and Lycopersicon (Retrolyc1 and Tlc1) species. The study presented here was conducted to characterise Tnt1-related sequences in 20 wild species of Solanum and five cultivars of Solanum tuberosum.     

The effects of insulin and β-adrenergic stimulation on glucose transport,glut 4 and PKB activation in the myocardium of lean and obese non-insulin dependent diabetes mellitus rats

Glucose uptake, glut 4 translocation and activation of protein kinase B were measured in Langendorff perfused hearts from (i) Wistar control, (ii) lean, neonatal Streptozotocin induced (Stz) and (iii) Zucker (fa/fa) obese diabetic rats of 10–12 weeks old. Hearts were subjected to stimulation with insulin, isoproterenol (-adrenergic agonist) or a combination of insulin and isoproterenol, during the perfusion protocol. Basal myocardial glucose uptake was impaired in both diabetic models, but could be stimulated significantly by insulin. In the Zucker rats, the time-course of insulin action was delayed. Insulin and -stimulation of glucose uptake were not additive. Evaluation of sarcolemmal membranes from these hearts showed that the affinity of glut 4 was significantly lower in the Zucker but not in the Stz hearts while a reduced affinity found with a combination of insulin and -stimulation in control hearts, was absent in both diabetic models. Total membrane lysates were analyzed for glut 4 expression while an intracellular component was generated to quantify translocation on stimulation as well as activity of protein kinase B (PKB). At this age, the neonatal Streptozotocin induced diabetic animals presented with more faulty regulation concerning adrenergic stimulated effects on elements of this signal transduction pathway while the Zucker fa/fa animals showed larger deviations in insulin stimulated effects. The overall response of the Zucker myocardium was poorer than that of the Stz group. No significant modulation of -adrenergic signaling on insulin stimulated glucose uptake was found. The PI-3-kinase inhibitor wortmannin, could abolish glucose uptake as well as PKB activation elicited by both insulin and isoproterenol.     

MIND-BEST: Web server for drugs and target discovery; design, synthesis, and assay of MAO-B inhibitors and theoretical-experimental study of G3PDH protein from Trichomonas gallinae

Many drugs with very different affinity to a large number of receptors are described. Thus, in this work, we selected drug-target pairs (DTPs/nDTPs) of drugs with high affinity/nonaffinity for different targets. Quantitative structure-activity relationship (QSAR) models become a very useful tool in this context because they substantially reduce time and resource-consuming experiments. Unfortunately, most QSAR models predict activity against only one protein target and/or they have not been implemented on a public Web server yet, freely available online to the scientific community. To solve this problem, we developed a multitarget QSAR (mt-QSAR) classifier combining the MARCH-INSIDE software for the calculation of the structural parameters of drug and target with the linear discriminant analysis (LDA) method in order to seek the best model. The accuracy of the best LDA model was 94.4% (3,859/4,086 cases) for training and 94.9% (1,909/2,012 cases) for the external validation series. In addition, we implemented the model into the Web portal Bio-AIMS as an online server entitled MARCH-INSIDE Nested Drug-Bank Exploration & Screening Tool (MIND-BEST), located at http://miaja.tic.udc.es/Bio-AIMS/MIND-BEST.php . This online tool is based on PHP/HTML/Python and MARCH-INSIDE routines. Finally, we illustrated two practical uses of this server with two different experiments. In experiment 1, we report for the first time a MIND-BEST prediction, synthesis, characterization, and MAO-A and MAO-B pharmacological assay of eight rasagiline derivatives, promising for anti-Parkinson drug design. In experiment 2, we report sampling, parasite culture, sample preparation, 2-DE, MALDI-TOF and -TOF/TOF MS, MASCOT search, 3D structure modeling with LOMETS, and MIND-BEST prediction for different peptides as new protein of the found in the proteome of the bird parasite Trichomonas gallinae, which is promising for antiparasite drug targets discovery.     

Rho-mediated Contractility Exposes a Cryptic Site in Fibronectin and Induces Fibronectin Matrix Assembly

Many factors influence the assembly of fibronectin into an insoluble fibrillar extracellular matrix. Previous work demonstrated that one component in serum that promotes the assembly of fibronectin is lysophosphatidic acid (Zhang, Q., W.J. Checovich, D.M. Peters, R.M. Albrecht, and D.F. Mosher. 1994. J. Cell Biol. 127:1447–1459). Here we show that C3 transferase, an inhibitor of the low molecular weight GTP-binding protein Rho, blocks the binding of fibronectin and the 70-kD NH₂-terminal fibronectin fragment to cells and blocks the assembly of fibronectin into matrix induced by serum or lysophosphatidic acid. Microinjection of recombinant, constitutively active Rho into quiescent Swiss 3T3 cells promotes fibronectin matrix assembly by the injected cells. Investigating the mechanism by which Rho promotes fibronectin polymerization, we have used C3 to determine whether integrin activation is involved. Under conditions where C3 decreases fibronectin assembly we have only detected small changes in the state of integrin activation. However, several inhibitors of cellular contractility, that differ in their mode of action, inhibit cell binding of fibronectin and the 70-kD NH₂-terminal fibronectin fragment, decrease fibronectin incorporation into the deoxycholate insoluble matrix, and prevent fibronectin's assembly into fibrils on the cell surface. Because Rho stimulates contractility, these results suggest that Rho-mediated contractility promotes assembly of fibronectin into a fibrillar matrix. One mechanism by which contractility could enhance fibronectin assembly is by tension exposing cryptic self-assembly sites within fibronectin that is being stretched. Exploring this possibility, we have found a monoclonal antibody, L8, that stains fibronectin matrices differentially depending on the state of cell contractility. L8 was previously shown to inhibit fibronectin matrix assembly (Chernousov, M.A., A.I. Faerman, M.G. Frid, O.Y. Printseva, and V.E. Koteliansky. 1987. FEBS (Fed. Eur. Biochem. Soc.) Lett. 217:124–128). When it is used to stain normal cultures that are developing tension, it reveals a matrix indistinguishable from that revealed by polyclonal anti-fibronectin antibodies. However, the staining of fibronectin matrices by L8 is reduced relative to the polyclonal antibody when the contractility of cells is inhibited by C3. We have investigated the consequences of mechanically stretching fibronectin in the absence of cells. Applying a 30–35% stretch to immobilized fibronectin induced binding of soluble fibronectin, 70-kD fibronectin fragment, and L8 monoclonal antibody. Together, these results provide evidence that self-assembly sites within fibronectin are exposed by tension.     

Acid-base versus structural properties of an aminoglycoside antibiotic--sisomicin: NMR and potentiometric approach

Aminoglycoside antibiotics constitute a class of the drugs of high interest, whose therapeutic action is based upon the electrostatic interaction with the variety of RNA molecules. The positive charge of these drugs molecules, located at their amino functions, has a prevailing influence on this process. The potentiometry and (1)H NMR spectroscopy are applied hereby to achieve the characteristics of the acid-base properties of particular protonating groups. We found that the pK values of deprotonation processes cover a wide values range 6-9.8. The correlation spectra of sisomicin, both COSY and TOCSY, allowed attributing unambiguously individual signals to the corresponding protons. These spectra involve a lot of the cross-peaks originating from the B and C rings protons, while the analogous signals originating from A rings protons are less numerous. Molecular modeling provided that the methylated amino group of A ring is located too far from the protonated functions of the remaining rings to affect their pK values. The phenomena observed herein are discussed in line of strength of the analogous processes observed for other aminoglycosides. As the result, four types of amino groups consisted within these antibiotics are distinguished.     

Sphingomyelin-rich domains are sites of lysenin oligomerization: Implications for raft studies

Lysenin is a self-assembling, pore-forming toxin which specifically recognizes sphingomyelin. Mutation of tryptophan 20 abolishes lysenin oligomerization and cytolytic activity. We studied the interaction of lysenin WT and W20A with sphingomyelin in membranes of various lipid compositions which, according to atomic force microscopy studies, generated either homo- or heterogeneous sphingomyelin distribution. Liposomes composed of SM/DOPC, SM/DOPC/cholesterol and SM/DPPC/cholesterol could bind the highest amounts of GST-lysenin WT, as shown by surface plasmon resonance analysis. These lipid compositions enhanced the release of carboxyfluorescein from liposomes induced by lysenin WT, pointing to the importance of heterogeneous sphingomyelin distribution for lysenin WT binding and oligomerization. Lysenin W20A bound more weakly to sphingomyelin-containing liposomes than did lysenin WT. The same amounts of lysenin W20A bound to sphingomyelin mixed with either DOPC or DPPC, indicating that the binding was not affected by sphingomyelin distribution in the membranes. The mutant lysenin had a limited ability to penetrate hydrophobic region of the membrane as indicated by measurements of surface pressure changes. When applied to detect sphingomyelin on the cell surface, lysenin W20A formed large conglomerates on the membrane, different from small and regular clusters of lysenin WT. Only lysenin WT recognized sphingomyelin pool affected by formation of raft-based signaling platforms. During fractionation of Triton X-100 cell lysates, SDS-resistant oligomers of lysenin WT associated with membrane fragments insoluble in Triton X-100 while monomers of lysenin W20A partitioned to Triton X-100-soluble membrane fractions. Altogether, the data suggest that oligomerization of lysenin WT is a prerequisite for its docking in raft-related domains.     

The newly synthesized plant growth regulator <Emphasis Type="Italic">S</Emphasis>-methylmethionine salicylate may provide protection against high salinity in wheat

High salinity is one of the major environmental factors limiting the productivity of crop species worldwide. Improving the stress tolerance of cultivated plants and thus increasing crop yields in an environmentally friendly way is a crucial task in agriculture. In the present work the ability of a new derivative, S-methylmethionine-salicylate (MMS), to improve the salt tolerance of wheat plants was tested parallel with its related compounds salicylic acid and S-methylmethionine. The results show that while these compounds are harmful at relatively high concentration (0.5 mM), they may provide protection against high salinity at lower (0.1 mM) concentration. This was confirmed by gas exchange, chlorophyll content and chlorophyll-a fluorescence induction measurements. While osmotic adjustment probably plays a critical role in the improved salt tolerance, neither Na or K transport from the roots to the shoots nor proline synthesis are the main factors in the tolerance induced by the compounds tested. MMS, S-methylmethionine and Na-salicylate had different effects on flavonol biosynthesis. It was also shown that salt treatment had a substantial influence on the SA metabolism in wheat roots and leaves. Present results suggest that the investigated compounds can be used to improve salt tolerance in plants.