Identification of the rpmF-plsX-fabH genes of Rhodobacter capsulatus.

The rpmF-plsX-fabH gene cluster of Rhodobacter capsulatus homologous to that of Escherichia coli was identified. rpmF encodes ribosomal protein L32, plsX plays an undefined role in membrane lipid synthesis, and fabH encodes beta-ketoacyl-acyl carrier protein synthase III. The R. capsulatus plsX gene complemented a defect in an E. coli strain with the plsX50 mutation. Overproduction of the fabH gene product of R. capsulatus in E. coli resulted in dramatically increased beta-ketoacyl-acyl carrier protein synthase III activity. These results indicate that plsX and fabH apparently function the same in R. capsulatus as in E. coli.

Respiratory rate measurement in adults--how reliable is it?

Measurement of respiratory rate (RR) is essential in the evaluation of respiratory disorders. However, the variability in RR measurement in adults has never been adequately assessed. Respiratory rate was measured twice in 245 patients; the two measurements were performed by the same observer in 137 patients, by different observers in 58 patients and simultaneously by different observers in 50 patients. The mean (SD) difference between the first and second measurements was 0.03 (3); 95% limits of agreement-4.86-4.94 breaths min(-1), -5.7-5.7 breaths min(-1), and -4.2 to 4.4 breaths min(-1) for the same observer, different observer and simultaneous observer groups, respectively. The difference in RR measurements did not vary with RR. In conclusions on average, there is very good agreement between observers in RR measurement. Inter-observer variability may account for a difference of up to 6 breaths min(-1). This is relevant when applying clinical prediction rules based on threshold RR values.

Interleukin-1beta induced activation of nuclear factor-kappab can be inhibited by novel pharmacological agents in osteoarthritis.

OBJECTIVES: To investigate the importance of activation of the transcription factor, nuclear factor-kappaB (NF-kappaB) by interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) in the pathogenesis of osteoarthritis (OA) and assess its suitability as a target for therapy by determining its role in the induction of the cytokine IL-6 and the degenerative enzymes, matrix metalloproteinase (MMP)-1 and MMP-3 in vitro. METHODS: Three distinct cellular models, derived from primary OA tissue, were employed, namely, fibroblast-like synoviocytes (OA-SF); co-cultures containing phenotypic macrophage-like and fibroblast-like cells (OA-COCUL); and primary OA synovial tissue explants (OA-EXP). These were treated with specific inhibitors of IL-1beta, TNF-alpha and NF-kappaB to assess their differential role in the production of pathologically relevant mediators, specifically IL-6, MMP-1, MMP-3 and the tissue inhibitor of metalloproteinases-1 (TIMP-1), which were quantified by enzyme-linked immunosorbent assay. RESULTS: Inhibition of NF-kappaB by a novel agent, RO100 at a dose of 0.1 microM, exerted significant (P < 0.05) repression of IL-6, MMP-1 and MMP-3 production in OA-SF. Notably, neither TIMP-1 production nor cell viability was significantly affected at the dose tested. These data were reproduced in OA-EXP, which might be considered as having greater physiological relevance. Interestingly, comparable efficacy was noted using IL-1beta and TNF-alpha neutralizing antibodies in OA-COCUL. CONCLUSIONS: We have demonstrated that a novel pharmacological inhibitor of NF-kappaB, RO100 inhibits pathological mediators of OA progression with equivalent efficacy as established IL-1beta and TNF-alpha neutralizing strategies. Our findings highlight a potential for developing NF-kappaB targeted therapeutics for positively regulating disease activity and improving clinical outcome in OA.

Effect of cold shock on lipid A biosynthesis in Escherichia coli. Induction At 12 degrees C of an acyltransferase specific for palmitoleoyl-acyl carrier protein.

Palmitoleate is not present in lipid A isolated from Escherichia coli grown at 30 degrees C or higher, but it comprises approximately 11% of the fatty acyl chains of lipid A in cells grown at 12 degrees C. The appearance of palmitoleate at 12 degrees C is accompanied by a decline in laurate from approximately 18% to approximately 5.5%. We now report that wild-type E. coli shifted from 30 degrees C to 12 degrees C acquire a novel palmitoleoyl-acyl carrier protein (ACP)-dependent acyltransferase that acts on the key lipid A precursor Kdo2-lipid IVA. The palmitoleoyl transferase is induced more than 30-fold upon cold shock, as judged by assaying extracts of cells shifted to 12 degrees C. The induced activity is maximal after 2 h of cold shock, and then gradually declines but does not disappear. Strains harboring an insertion mutation in the lpxL(htrB) gene, which encodes the enzyme that normally transfers laurate from lauroyl-ACP to Kdo2-lipid IVA (Clementz, T., Bednarski, J. J., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 12095-12102) are not defective in the cold-induced palmitoleoyl transferase. Recently, a gene displaying 54% identity and 73% similarity at the protein level to lpxL was found in the genome of E. coli. This lpxL homologue, designated lpxP, encodes the cold shock-induced palmitoleoyl transferase. Extracts of cells containing lpxP on the multicopy plasmid pSK57 exhibit a 10-fold increase in the specific activity of the cold-induced palmitoleoyl transferase compared with cells lacking the plasmid. The elevated specific activity of the palmitoleoyl transferase under conditions of cold shock is attributed to greatly increased levels of lpxP mRNA. The replacement of laurate with palmitoleate in lipid A may reflect the desirability of maintaining the optimal outer membrane fluidity at 12 degrees C.

Inhibition of lipopolysaccharide biosynthesis and cell growth following inactivation of the kdtA gene in Escherichia coli.

The enzyme 3-deoxy-D-manno-octulosonic acid (Kdo) transferase is encoded by the kdtA gene in Escherichia coli. The enzyme is a single polypeptide that catalyzes the transfer of two Kdo residues to a tetraacyldisaccharide-1,4'-bisphosphate precursor of lipid A, designated lipid IVA (Belunis, C.J., and Raetz, C.R.H. (1992) J. Biol. Chem. 267, 9988-9997). To determine if Kdo transfer to lipid IVA is required for growth, we constructed a strain of E. coli with a chromosomal kdtA::kan insertion mutation. In mutants carrying the kdtA::kan allele on the chromosome, cell growth and Kdo transferase activity were dependent upon a copy of the intact kdtA gene on a plasmid. When the kdtA-bearing plasmid was itself temperature sensitive for replication, the growth of these strains was inhibited after several hours at 44 degrees C, and Kdo transferase activity in extracts became undetectable. Concomitantly, the cells accumulated massive amounts of lipid IVA, the precursor of (Kdo)2-lipid IVA. The kdtA::kan mutation could also be complemented by hybrid plasmids bearing the gseA gene of Chlamydia trachomatis. gseA specifies a distinct Kdo transferase that adds three Kdo moieties to lipid IVA. Lipopolysaccharide from E. coli kdtA::kan constructs complemented by gseA reacts strongly with antibodies directed against the genus-specific epitope of Chlamydia, whereas lipopolysaccharide from parental E. coli K-12 does not. Our studies prove that Kdo attachment during lipid A biosynthesis is essential for cell growth and accounts for the conditional lethality associated with mutations in Kdo biosynthesis.

Protein-interaction modules that organize nuclear function: FF domains of CA150 bind the phosphoCTD of RNA polymerase II.

An approach for purifying nuclear proteins that bind directly to the hyperphosphorylated C-terminal repeat domain (CTD) of RNA polymerase II was developed and used to identify one human phosphoCTD-associating protein as CA150. CA150 is a nuclear factor implicated in transcription elongation. Because the hyperphosphorylated CTD is a feature of actively transcribing RNA polymerase II (Pol II), phosphoCTD (PCTD) binding places CA150 in a location appropriate for performing a role in transcription elongation-related events. Several recombinant segments of CA150 bound the PCTD. Predominant binding is mediated by the portion of CA150 containing six FF domains, compact modules of previously unknown function. In fact, small recombinant proteins containing the fifth FF domain bound the PCTD. PCTD binding is the first specific function assigned to an FF domain. As FF domains are found in a variety of nuclear proteins, it is likely that some of these proteins are also PCTD-associating proteins. Thus FF domains appear to be compact protein-interaction modules that, like WW domains, can be evolutionarily shuffled to organize nuclear function.