META060 inhibits multiple kinases in the NF-kappaB pathway and suppresses LPS--mediated inflammation in vitro and ex vivo.

OBJECTIVE: We investigated whether a novel candidate META060 targeted the inflammatory signal transduction without affecting constitutive COX-2 enzymatic activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We also investigated its bioavailability in humans and its anti-inflammatory effect ex vivo. METHODS: We measured prostaglandin E(2), nitric oxide, TNFalpha and IL-6 by ELISA, COX-2 protein by Western blot, NF-kappaB nuclear binding by electrophoretic mobility shift assays, and NF-kappaB activation by luciferase assay. Kinase inhibitions were measured by cell-free assays. Bioavailability was tested in 4 human subjects consuming 940 mg META060. LPS-activated TNFalpha and IL-6 were measured in peripheral blood mononuclear cells (PBMC) isolated from 1 subject up to 6 hours post administration. RESULTS: META060 dose-dependently inhibited prostaglandin E(2) and nitric oxide formation, COX-2 abundance, and NF-kappaB activation. In cell-free assays, META060 inhibited multiple kinases in the NF-kappaB signaling pathway, including BTK, PI3K, and GSK3. META060 was detected in the plasma of the subjects; isolated PBMC were resistant to LPS-stimulated TNFalpha and IL-6 production. CONCLUSION: Without inhibiting COX-2 enzyme, META060 reduces the inflammation by inhibiting multiple kinases involved in NF-kappaB pathway, and may have potential as a safe anti-inflammatory therapeutic.

Gastric mucosal cell model for estimating relative gastrointestinal toxicity of non-steroidal anti-inflammatory drugs.

The study objective was to characterize the AGS human gastric mucosal cell line as a model for estimating gastrointestinal toxicity of COX-inhibiting compounds. Rofecoxib, celecoxib, nimesulide, ibuprofen, indomethacin, aspirin, salicylic acid, naproxen and acetaminophen were tested for inhibition of COX-2-mediated prostaglandin E2 synthesis in A549 and AGS cells. The IC50 ratio AGS/A549 was calculated as an estimate of the therapeutic index (TI) for gastrointestinal toxicity. Calculated IC50 values of non-steroidal anti-inflammatory drugs (NSAIDs) in A549 cells were in excellent agreement with published values (r = 0.996; P < 0.005). Calcium ionophore induction of arachidonic acid release in AGS cells provided TI similar to those using platelets and A549 cells (r = 0.918; P < 0.01). The AGS/A549 model exhibited lower TI than the platelet/A549 model. Spearman ranking correlated clinical NSAID gastropathy with lower AGS TI values. The AGS cell line has excellent potential to serve as a model for assessing the gastrointestinal effects of COX-inhibiting compounds.

The biosynthetic origin of the pyridone ring of efrotomycin.

Nocardia lactamdurans has been shown to catabolyse uracil via the reductive pathway. The end product of this pathway, beta-alanine, is incorporated into the pyridone ring of efrotomycin. Support for this proposal includes: (1) reversal of thymine inhibition of efrotomycin biosynthesis by dihydrouracil and N-carbamoyl-beta-aline, two intermediates of the catabolic pathway; (2) incorporation of [5,6-3H]-uracil into efrotomycin with a relative molar specific activity of approximately 0.5, close to the theoretical maximum; and (3) 13C coupling at C4 and C5 of efrotomycin after feeding resting cells with [4,5-13C]-uracil. Our results do not rule out the possibility of an alternative source of beta-alanine or the coexistence of uracil catabolism via oxidative reactions.

Isolation of a thymidine kinase negative mutant of Nocardia lactamdurans.

By selection for resistance to fluorodeoxyuridine in a fluorouracil/fluorouridine resistant background, we have isolated a thymidine kinase negative mutant of Nocardia lactamdurans. This strain is characterized by the inability to incorporate exogenous [2-14C]-thymidine into DNA. The incorporation of radioactive thymine is similarly reduced even in the presence of deoxyadenosine. This phenotype is readily explained by the inability to detect the enzyme thymidine kinase in crude extracts.

Oxidative and defluorinative metabolism of fludalanine, 2-2H-3-fluoro-D-alanine.

The antibacterial agent fludalanine [2-2H-3-fluoro-D-alanine (DFA)] is a potent inhibitor of bacterial alanine racemase, an enzyme required for the generation of D-alanine, an essential component of the bacterial cell wall. Primary metabolism of DFA involves its oxidation to fluoropyruvate (FP); this organic fluoride is then rapidly reduced to fluorolactate (FL) which is the major organic metabolite in laboratory animals. Gas-liquid chromatographic chemical ionization mass spectrometric assays were developed for these two metabolites. FL is the predominant organofluoride metabolite of DFA in the circulation. FP was detected in the urine although recovery was very low. The rapid conversion of FP to FL precludes assay of the former in serum. Maximum serum FL concentrations in the rat appear about 1 hr after the dose of DFA and are relatively constant for several hours thereafter. The peak FL concentration is proportional to the dose of DFA; repeated daily dosing of DFA appears to cause neither saturation nor induction of metabolic pathways. Comparison of FL concentrations determined using the GC/MS assay with those based on an enzymic method specific for L-(+)-FL demonstrated that only the latter isomer is found in the plasma of monkeys dosed with DFA. In vivo exchange studies involving the alpha-proton of FL indicate that a small FP pool exists and is in equilibrium with FL. A crude pyruvate dehydrogenase complex isolated from beef heart mitochondria was shown to produce equimolar quantities of acetate, CO2, and fluoride from FP.

Cefoxitin resistance to beta-lactamase: a major factor for susceptibility of bacteroides fragilis to the antibiotic.

Toluene-treated cell suspensions of Bacteroides fragilis were used to screen clinical isolates for the production of beta-lactamase. Approximately one-third of the isolates possessed considerable cephalosporinase activity. A significant correlation was found between beta-lactamase production and resistance to cephalosporin antibiotics. Several isolates were resistant to cefuroxime and cefamandole and produced enzymes capable of hydrolyzing these antibiotics. However, none of the 79 strains tested could hydrolyze the cephamycin derivative, cefoxitin. A large percentage (>90%) of the strains were susceptible to cefoxitin. Therefore, resistance to lactamase hydrolysis is a major factor for the effectiveness of cefoxitin against B. fragilis. Detailed studies of four isolates suggest that two different enzymes may be produced. Both are cephalosporinases but differ with regard to cellular distribution and substrate specificity. Cefoxitin is not a substrate for either enzyme, but it is an excellent competitive inhibitor (K(i) approximately 0.1 muM).

Discriminant analysis of antibiotic susceptibility as a means of bacterial identification.

This study shows that antibiotic susceptibility data can be used effectively in the presumptive identification of bacteria. Using 12 antibiotics and determining the zone sizes for each, 82% of the isolates considered were correctly identified without any other information. If the inability to distinguish between Escherichia coli and Shigella is disregarded, the percentage of correct identification is 92%. The method involves determining a set of discriminant functions and defining each taxon by a unique function. An unknown isolate is identified by evaluating each discriminant function and assigning the isolate to the taxon whose discriminant function has the largest value. A total of 468 isolates were examined. After eliminating the multiply resistant isolates, the remaining 369 isolates were used to determine the discriminant functions for the eight taxa considered.

Principal component analysis of infraspecific variation in bacteria.

In certain types of ecological investigations it may be desirable to investigate infraspecific variation in bacteria. Principal component analysis is demonstrated to be satisfactory for this purpose. Hypothetical bacterial populations were used to show that such analysis can be used to compare collections of bacterial isolates taken at different times or from different sources. Alternatively, given n isolates, whether they represent a single bacterial population can be determined. The method is applied to authentic collections of bacteria in three separate analyses. The results are compatible with current taxonomic tenets.

Biochemical and serological characterization of hydrogen sulfide-positive variants of Escherichia coli.

Over 200 H(2)S-positive, gram-negative rods have been characterized by standard biochemical and serological techniques. The results indicate that the isolates are H(2)S-positive variants of Escherichia coli. Comparison of the variants with biochemically typical E. coli suggests that they represent a rather limited subgroup within the species. The H(2)S-positive strains were more resistant to antibiotics than the typical strains; 54% of the H(2)S-positive isolates were resistant to three or more antibiotics compared with only 25% of the typical strains. Similar differences were also seen in the distribution of O and H antigens and in the results of certain biochemical tests.

A thermophilic, acidophilic mycoplasma isolated from a coal refuse pile.

A thermophilic, acidophilic procaryote lacking a cell wall has been isolated from a coal refuse pile which had undergone self-heating. Electron micrographs, chemical assays for hexosamine, and the inability of vancomycin to inhibit growth confirm the lack of a cell wall. The apparent ability of the organism to reproduce by budding and the low guanine plus cytosine content of its DNA indicate a relation to the mycoplasmas. The temperature optimum of the organism is 59 degrees C, and growth occurs over a range of 45 degrees to 62 degrees C. No growth occurs at 37 degrees C or at 65 degrees C. The optimum pH for growth is between 1 and 2, and growth occurs between pH 0.96 and 3.5 but does not occur at pH 0.35 and only poorly at pH 4.0. We propose to call this organism Thermoplasma acidophila. The existence of this organism extends considerably the range of habitats in which mycoplasma may occur.

Limits of microbial existence: temperature and pH.

A microscopic survey made to detect the presence of bacteria in hot springs of varying temperature and pH characteristics revealed that in neutral and alkaline hot springs bacteria are found at temperatures up to the boiling point of water (92 degrees to 100 degrees C, depending on the altitude). In hot springs of increasing acidity the upper temperature limit at which bacteria are found decreases; at pH 2 to 3 the upper temperature limit is 75 degrees to 80 degrees C. Bacteria have thus been able to evolve with the ability to grow at either high temperature or high acidity, but not at both high temperature and high acidity. These results suggest that there are physicochemical limitations of the environment beyond which life is impossible.