Enterococcal cytolysin: a novel two component peptide system that serves as a bacterial defense against eukaryotic and prokaryotic cells

The cytolysin is a novel, two-peptide lytic toxin produced by some strains of Enterococcus faecalis. It is toxic in animal models of enterococcal infection, and associated with acutely terminal outcome in human infection. The cytolysin exerts activity against a broad spectrum of cell types including a wide range of gram positive bacteria, eukaryotic cells such as human, bovine and horse erythrocytes, retinal cells, polymorphonuclear leukocytes, and human intestinal epithelial cells. The cytolysin likely originated as a bacteriocin involved with niche control in the complex microbial ecologies associated with eukaryotic hosts. However, additional anti-eukaryotic activities may have been selected for as enterococci adapted to eukaryotic cell predation in water or soil ecologies. Cytolytic activity requires two unique peptides that possess modifications characteristic of the lantibiotic bacteriocins, and these peptides are broadly similar in size to most cationic eukaryotic defensins. Expression of the cytolysin is tightly controlled by a novel mode of gene regulation in which the smaller peptide signals high-level expression of the cytolysin gene cluster. This complex regulation of cytolysin expression may have evolved to balance defense against eukaryotic predators with stealth.     

Solvent effect in cis-1,4 polymerization of 1,3-butadiene by a catalyst based on neodymium

In this work a laboratory polymerization scale process was studied for the production of polybutadiene with high content of cis-1,4 repeating units. A Ziegler-Natta catalytic system based on neodymium versatate (catalyst), diisobutylaluminium hydride (cocatalyst) and tert-butyl chloride (chlorinating agent) was used. The influence of solvent nature (pure grade) and possible contaminants (electron donors) in a recovered solvent from a butadiene-styrene anionic polymerization industrial plant on the stereoselectivity and catalytic activity, molecular weight and molecular weight distribution of the resultant polybutadienes was studied. The polymers were characterized by infrared spectroscopy and size exclusion chromatography. Polybutadienes with cis-1,4 units content in the range of 99-98% were produced. The polymers weight-average molecular weight, , varied from 2.23 × 10⁵ to 4.47 × 10⁵ and the molecular weight distribution, MWD, from 3.1 to 5.1.     

Lipase production by solid-state fermentation

The production of lipase by Penicillium simplicissimum in solid-state fermentation was studied using babassu cake as the basal medium. Tray-type and packed-bed bioreactors were employed. In the former, the influence of temperature; content of the medium, and medium supplementation with olive oil, sugarcane molasses, corn steep liquor, and yeast hydrolysate was studied. For all combinations of supplements, a temperature of 30°C, a moisture content of 70%, and a concentration of carbon source of 6.25% (m/m, dry basis) provided optimum conditions for lipase production. When used as single supplements olive oil and molasses also were able to provide high lipase activities (20 U/g). Using packed-bed bioreactors and molasses-supplemented medium, optimum conditions for enzyme production were air superficial velocities above 55 cm/min and temperatures below 28°C. The lower temperature optimum found for these reactors is probably related to radial heat gradient formation inside the packed bed. Maximum lipase activities obtained in these bioreactors (26.4 U/g) were 30% higher than in tray-type reactors.     

Salmonella in foods: new enrichment procedure for TECRA Salmonella Visual Immunoassay using a single rv(R10) only, TT only, or dual rv(R10) and TT selective enrichment broths (AOAC official method 998.09): collaborative study

A collaborative study was conducted to compare a new enrichment procedure for the TECRA Salmonella Visual Immunoassay (TSVIA) with the reference method given in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (7th Ed.). Three food types (milk powder, pepper, and soy flour) were analyzed in Australia and 3 food types (milk chocolate, dried egg, and raw turkey) were analyzed in the United States. Thirty-eight collaborators participated in the study. The TECRA method was evaluated using both Rappaport-Vassiliadis R10 (RV(R10)) and tetrathionate (TT) broths for selective enrichment. M broth cultures arising from each of the 2 selective enrichment broths were tested in the TSVIA using 2 individual wells, one for each selective broth, and a single well to test the pooled selective enrichment broths. The results for the pooled enrichment broths were reported elsewhere. This study presents the results for the use of single enrichment broths, i.e., RV(R10) only or TT only, with the TSVIA. No significant differences (p > 0.05) were observed for the pairwise comparison of the proportion of positive samples for either RV(R10) or TT used as a single enrichment broth for the TSVIA with that for the reference method.     

Second‐Generation Inhibitors for the Metalloprotease Neprilysin Based on Bicyclic Heteroaromatic Scaffolds: Synthesis,Biological Activity,and X‐Ray Crystal‐Structure Analysis

A new class of nonpeptidic inhibitors of the Zn^II‐dependent metalloprotease neprilysin with IC₅₀ values in the nanomolar activity range (0.034–0.30 μM ) were developed based on structure‐based de novo design (Figs. 1 and 2). The inhibitors feature benzimidazole and imidazo[4,5‐c]pyridine moieties as central scaffolds to undergo H‐bonding to Asn542 and Arg717 and to engage in favorable π‐π stacking interactions with the imidazole ring of His711. The platform is decorated with a thiol vector to coordinate to the Zn^II ion and an aryl residue to occupy the hydrophobic S1′ pocket, but lack a substituent for binding in the S2′ pocket, which remains closed by the side chains of Phe106 and Arg110 when not occupied. The enantioselective syntheses of the active compounds (+)‐ 1 , (+)‐ 2 , (+)‐ 25 , and (+)‐ 26 were accomplished using Evans auxiliaries (Schemes 2, 4, and 5). The inhibitors (+)‐ 2 and (+)‐ 26 with an imidazo[4,5‐c]pyridine core are ca. 8 times more active than those with a benzimidazole core ((+)‐ 1 and (+)‐ 25 ) (Table 1). The predicted binding mode was established by X‐ray analysis of the complex of neprilysin with (+)‐ 2 at 2.25‐Å resolution (Fig. 4 and Table 2). The ligand coordinates with its sulfanyl residue to the Zn^II ion, and the benzyl residue occupies the S1′ pocket. The 1H‐imidazole moiety of the central scaffold forms the required H‐bonds to the side chains of Asn542 and Arg717. The heterobicyclic platform additionally undergoes π‐π stacking with the side chain of His711 as well as edge‐to‐face‐type interactions with the side chain of Trp693. According to the X‐ray analysis, the substantial advantage in biological activity of the imidazo‐pyridine inhibitors over the benzimidazole ligands arises from favorable interactions of the pyridine N‐atom in the former with the side chain of Arg102. Unexpectedly, replacement of the phenyl group pointing into the deep S1′ pocket by a biphenyl group does not enhance the binding affinity for this class of inhibitors.     

Solid State NMR and TG/MS Study on the Transformation of Methyl Groups During Pyrolysis of Preceramic Precursors to SiOC Glasses

The sol-gel method was used to prepare two different starting gels containing SiCH₃-groups for the preparation of SiOC ceramics. To understand the role of Si—H bonds in the incorporation of carbon into the SiOC network, gels prepared from a 1:2 mixture of triethoxysilane and methyldiethoxysilane (T^HD^H2) and solely methyltriethoxysilane (T^Me) were investigated. Thermogravimetric analysis coupled with mass spectroscopy (TG-MS) in inert atmosphere was performed to attain an insight into the decomposition reactions involved during gel-glass transformation. Samples calcined at different temperatures up to 1000°C were characterized by ²⁹Si and ¹³C magic angle spinning nuclear magnetic resonance (MAS-NMR) spectroscopy. The presence of SiH groups in the starting gel allows an efficient conversion of Si—CH₃ groups into CSi₄ sites at lower temperatures. As a result, despite a much lower amount of carbon in the starting T^HD^H2 gel (C/Si = 0.33) compared to the T^Me gel (C/Si = 1), the amount of carbon inserted into the SiOC network of both glasses is equivalent, but the T^Me sample contains the 10 fold amount of free carbon.     

The effects of gamma irradiation on the microbiological, physical and sensory qualities of diced tomatoes

Diced Roma tomatoes were treated with gamma irradiation and evaluated for changes in microbial, physical, chemical and sensory properties. Dosages for Trial 1 were 0.0, 0.39, 0.56 and 1.82 kGy and in Trial 2, 0.0, 0.50, 1.24 and 3.70 kGy. Irradiation at 3.70 kGy resulted in no aerobic populations through day 12 and significantly fewer colonies through day 15 whereas yeast and mold populations experienced a 2 log reduction through day 12. Color, titratable acidity, and °Brix were not significantly affected by irradiation. Tissue firmness decreased with increasing dose but not with storage time. Treatment with 3.7 kGy decreased firmness by 50% and 20% with 0.5 kGy, however, the reduced firmness induced by 0.50 kGy was undetected by a 9 member trained sensory panel. A significant (p0.05) inverse correlation between changes in texture and water-soluble pectin (WSP) was determined while total pectin remained relatively constant and oxalate soluble pectin content decreased slightly with irradiation dose. The significant inverse correlation between the loss of firmness and WSP indicates that the changes in WSP play an important role in the tissue softening of tomatoes, This study indicates that irradiation at 0.5 kGy can reduce microbial counts substantially to improve microbial shelf life without adverse effects on sensory qualities.     

Reactions of the Dirhenium(II) Complexes Re(2)X(4)(&mgr;-dppm)(2) (X = Cl, Br; dppm = Ph(2)PCH(2)PPh(2)) with Isocyanides. 11.(1) A Triply Bonded Dirhenium Complex Containing a Labile Acetonitrile Ligand. Synthesis, Structural Characterization, and Reactivity of [(XylNC)(CH(3)CN)ClRe(&mgr;-dppm)(2)ReCl(2)(CO)]O(3)SCF(3)

The reaction of the open bioctahedral form of Re(2)Cl(4)(&mgr;-dppm)(2)(CO)(CNXyl) (1), where XylNC = 2,6-dimethylphenyl isocyanide, with TlO(3)SCF(3) in the presence of acetonitrile proceeds with retention of stereochemistry at the dirhenium unit to afford the complex [Re(2)Cl(3)(&mgr;-dppm)(2)(CO)(CNXyl)(NCCH(3))]O(3)SCF(3) (3). The single-crystal X-ray structure determination of 3 shows that a Re&tbd1;Re bond is retained (the Re-Re distance is 2.378(3) ?) and that it is the chloride ligand trans to the XylNC ligand of 1 which is labilized. Complex 1 reacts with TlO(3)SCF(3) in a noncoordinating solvent to produce the unsymmetrical complex [Re(2)Cl(3)(&mgr;-dppm)(2)(CO)(CNXyl)]O(3)SCF(3) (2), through loss of this same chloride ligand of 1 and CO transfer from the adjacent Re center. The acetonitrile ligand of 3 is very labile and is readily displaced by XylNC and t-BuNC, with retention of stereochemistry, to produce complexes of stoichiometry [Re(2)Cl(3)(&mgr;-dppm)(2)(CO)(CNXyl)(CNR)]O(3)SCF(3) (R = Xyl, 4a; R = t-Bu, 4b). In a noncoordinating solvent, the nitrile ligand of 3 is lost and 2 is formed following CO transfer; this conversion is reversed upon the reaction of 2 with acetonitrile. When 3 is treated with CO, the acetonitrile ligand is again displaced, but in this instance the reaction is accompanied by a structure change to produce an edge-sharing bioctahedral complex of the type [Re(2)(&mgr;-CO)(&mgr;-Cl)(&mgr;-dppm)(2)Cl(2)(CO)(CNXyl)]O(3)SCF(3) (5).     

Complexation of the triply-bonded dirhenium(II) complex Re(2)Cl(4)(mu-dppm)(2) (dppm = Ph(2)PCH(2)PPh(2)) by up to three acetylene molecules

The triply bonded dirhenium(II) synthons Re(2)X(4)(mu-dppm)(2) (X = Cl, Br; dppm = Ph(2)PCH(2)PPh(2)) react with acetylene at room temperature in CH(2)Cl(2) and acetone to afford the bis(acetylene) complexes Re(2)X(4)(mu-dppm)(2)(mu:eta(2),eta(2)-HCCH)(eta(2)-HCCH) (X = Cl (3), Br(4)). Compound 3 has been derivatized by reaction with RNC ligands in the presence of TlPF(6) to give unsymmetrical complexes of the type [Re(2)Cl(3)(mu-dppm)(2)(mu:eta(2),eta(2)-HCCH)(eta(2)-HCCH)(CNR)]PF(6) (R = Xyl (5), Mes (6), t-Bu (7)), in which the RCN ligand has displaced the chloride ligand cis to the eta(2)-HCCH ligand. The reaction of 3 with an additional 1 equiv of acetylene in the presence of TlPF(6) gives the symmetrical all-cis isomer of [Re(2)Cl(3)(mu-dppm)(2)(mu:eta(2),eta(2)-HCCH)(eta(2)-HCCH)(2)]PF(6) (8). The two terminal eta(2)-HCCH ligands in 8 are very labile and can be displaced by CO and XylNC to give the complexes [Re(2)Cl(3)(mu-dppm)(2)(mu:eta(2),eta(2)-HCCH)(L)(2)]Y (L = CO when Y = PF(6) (9); L = CO when Y = (PF(6))(0.5)/(H(2)PO(4))(0.5) (10); L = XylNC when Y = PF(6) (11)). These substitution reactions proceed with retention of the all-cis stereochemistry. Single-crystal X-ray structure determinations have been carried out on complexes 3, 5, 8, 10, and 11. In no instance have we found that the acetylene ligands undergo reductive coupling reactions.     

Quantification of the neurotransmitters melatonin and N-acetyl-serotonin in human serum by supercritical fluid chromatography coupled with tandem mass spectrometry

The aim of this study was developing a supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS) method and an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method, for the analysis of N-acetyl-serotonin (NAS) and melatonin (Mel) in human serum, and to compare the performance of these methods. Deuterated isotopologues of the neurotransmitters were synthesized and evaluated for suitability as internal standards in sample preparation. Liquid-liquid extraction was selected as sample preparation procedure. With chloroform, the best extraction solvent tested, an extraction yield of 48 ± 2% for N-acetyl-serotonin and 101 ± 10% for melatonin was achieved. SFC separation was accomplished within 3 min on a BEH stationary phase, employing isocratic elution with 90% carbon dioxide and 0.1% formic acid as well as 0.05% ammonium formate in methanol. For the 4 min UHPLC gradient separation with 0.1% formic acid in water and methanol, respectively, a Kinetex XB-C18 was used as stationary phase. Both chromatographic techniques were optimized regarding mobile phase composition, additives to the mobile phase and column temperature. Multiple reaction monitoring (MRM) analysis was used for quantification of the metabolites. Both methods were validated regarding retention time stability, LOD, LOQ, repeatability and reproducibility of quantification, process efficiency, extraction recovery and matrix effects. LOD and LOQ were 0.017 and 0.05 pg μL⁻¹ for NAS and 0.006 and 0.018 pg μL⁻¹ for Mel in SFC-MS/MS compared to 0.028 and 0.1 pg μL⁻¹ for NAS and 0.006 and 0.017 pg μL⁻¹ for Mel in UHPLC-MS/MS.