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Pharmacodynamic Evaluation of the Neutralization of Endotoxin by PMX622 in Mice. Philip Lake, 2004.Polymyxin B (PMB) binds to and neutralizes endotoxin, but its systemic clinical utility is limited by neuro- and nephrotoxicity . PMX622 is a covalent conjugate of PMB and Dextran-70 designed to retain the ability of PMB to neutralize endotoxin and to retain the favorable colloidal, pharmacokinetic, and metabolic properties of Dextran-70 . PMX622 has demonstrated efficacy in a number of animal models and effectively neutralized endotoxin in phase I clinical trials . Here, we systematically evaluated the pharmacodynamic properties of PMX622 in a murine model of endotoxin-induced lethality in galactosamine-sensitized mice . PMX622 completely and dose dependently inhibited lethality in this model . A stoichiometric relationship was found between the endotoxin challenge dose and the dose of PMX622 needed for protection . PMX622 neutralized endotoxin from four different genera of gram-negative bacteria but not Neisseria meningitidis . PMX622 was significantly less toxic than PMB in the mouse, suggesting that PMX622 has a better margin of safety than PMB . The timing of PMX622 administration relative to endotoxin was crucial . PMX622 was active for several hours prior to the endotoxin challenge; however, PMX622 did not protect mice if administered  15 min after endotoxin challenge . This suggests that PMX622 would best be clinically used prophylactically rather than therapeutically . These studies will be crucial in designing and interpreting human clinical trials assessing PMX622 efficacy .
Ability of Thermophilic Lactic Acid Bacteria To Produce Aroma Compounds from Amino Acids. Sandra Helinck, 2004.Although a large number of key odorants of Swiss-type cheese result from amino acid catabolism, the amino acid catabolic pathways in the bacteria present in these cheeses are not well known . In this study, we compared the in vitro abilities of Lactobacillus delbrueckii subsp . lactis, Lactobacillus helveticus, and Streptococcus thermophilus to produce aroma compounds from three amino acids, leucine, phenylalanine, and methionine, under mid-pH conditions of cheese ripening (pH 5.5), and we investigated the catabolic pathways used by these bacteria . In the three lactic acid bacterial species, amino acid catabolism was initiated by a transamination step, which requires the presence of an  -keto acid such as
-ketoglutarate (-KG) as the amino group acceptor, and produced
-keto acids . Only S . thermophilus exhibited glutamate dehydrogenase activity, which produces
-KG from glutamate, and consequently only S . thermophilus was capable of catabolizing amino acids in the reaction medium without
-KG addition . In the presence of
-KG, lactobacilli produced much more varied aroma compounds such as acids, aldehydes, and alcohols than S . thermophilus, which mainly produced
-keto acids and a small amount of hydroxy acids and acids . L . helveticus mainly produced acids from phenylalanine and leucine, while L . delbrueckii subsp . lactis produced larger amounts of alcohols and/or aldehydes . Formation of aldehydes, alcohols, and acids from
-keto acids by L . delbrueckii subsp . lactis mainly results from the action of an
-keto acid decarboxylase, which produces aldehydes that are then oxidized or reduced to acids or alcohols . In contrast, the enzyme involved in the
-keto acid conversion to acids in L . helveticus and S . thermophilus is an
-keto acid dehydrogenase that produces acyl coenzymes A .
Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae. Ping Luo, 2003.Natural transformation in Streptococcus pneumoniae is regulated by a quorum-sensing system that acts through accumulation and sensing of a peptide pheromone (competence-stimulating peptide [CSP]) to control many competence-specific genes acting in DNA uptake, processing, and integration . The period of competence induced by CSP lasts only 15 min (quarter-height peak width) . The recently identified regulator ComX is required for the CSP-dependent expression of many competence-specific genes that share an unusual consensus sequence (TACGAATA) at their promoter regions . To test the hypothesis that this regulator acts as a transient alternative sigma factor, ComX was purified from an Escherichia coli overexpression strain and core RNA polymerase was purified from a comX-deficient S . pneumoniae strain . The reconstituted ComX-polymerase holoenzyme produced transcripts for the competence-specific genes ssbB, cinA, cglA, celA, and dalA and was inhibited by anti-ComX antibody, but not by anti-  70 antibody . Western blotting using antibodies specific for ComX,
70, and poly-His revealed a transient presence of ComX for a period of 15 to 20 min after CSP treatment, while RNA polymerase remained at a constant level and
A remained between 60 and 125% of its normal level . ComX reached a molar ratio to RNA polymerase of at least 1.5 . We conclude that ComX is unstable and acts as a competence-specific sigma factor .
An Exocellular Protein from the Oil-Degrading Microbe Acinetobacter venetianus RAG-1 Enhances the Emulsifying Activity of the Polymeric Bioemulsifier Emulsan. Horacio Bach, 2003.The oil-degrading microorganism Acinetobacter venetianus RAG-1 produces an extracellular polyanionic, heteropolysaccharide bioemulsifier termed emulsan . Emulsan forms and stabilizes oil-water emulsions with a variety of hydrophobic substrates . Removal of the protein fraction yields a product, apoemulsan, which exhibits much lower emulsifying activity on hydrophobic substrates such as n-hexadecane . One of the key proteins associated with the emulsan complex is a cell surface esterase . The esterase (molecular mass, 34.5 kDa) was cloned and overexpressed in Escherichia coli BL21(DE3) behind the phage T7 promoter with the His tag system . After overexpression, about 80 to 90% of the protein was found in inclusion bodies . The overexpressed esterase was recovered from the inclusion bodies by solubilization with deoxycholate and, after slow dialysis, was purified by metal chelation affinity chromatography . Mixtures containing apoemulsan and either the catalytically active soluble form of the recombinant esterase isolated from cell extracts or the solubilized inactive form of the enzyme recovered from the inclusion bodies formed stable oil-water emulsions with very hydrophobic substrates such as hexadecane under conditions in which emulsan itself was ineffective . Similarly, a series of esterase-defective mutants were generated by site-directed mutagenesis, cloned, and overexpressed in E . coli. Mutant proteins defective in catalytic activity as well as others apparently affected in protein conformation were also active in enhancing the apoemulsan-mediated emulsifying activity . Other proteins, including a His-tagged overexpressed esterase from the related organism Acinetobacter calcoaceticus BD4, showed no enhancement . Genetic and Biochemical Characterization of the Phosphoenolpyruvate:Glucose/Mannose Phosphotransferase System of Streptococcus thermophilus. Armelle Cochu, 2003.
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