IL-6 stimulates osteoclast-like multinucleated cell formation in long term human marrow cultures by inducing IL-1 release

IL-6 enhances the differentiation of pluripotent hematopoietic stem cells but predominantly affects the differentiation of hematopoietic cells in the granulocyte-macrophage lineage. We have previously shown that multinucleated cells (MNC) with many features of the osteoclast phenotype form in long term human marrow cultures. Addition of rhIL-6 (10 to 100 pg/ml) to these cultures significantly increased MNC formation, with greater than 80% of the MNC expressing an Ag that cross-reacts with the mAb 23c6. This antibody preferentially binds to osteoclasts. rhIL-6 did not enhance MNC formation in marrow cultures treated with 1,25 dihydroxyvitamin D3, a potent stimulator of MNC formation, but significantly increased the percentage of MNC that cross-reacted with the 23c6 mAb. Addition of antihuman IL-1 to cultures treated with rhIL-6 totally inhibited the increase in MNC formation stimulated by rhIL-6. In contrast, anti-IL-1 did not affect MNC formation stimulated by 1,25 dihydroxyvitamin D3. Further, conditioned media from marrow cultures exposed to rhIL-6 contained elevated levels of IL-1 beta (500 pg/ml compared to 23 pg/ml in control cultures 15 h after IL-6 addition). These results suggest that the capacity of rhIL-6 to stimulate formation of MNC which cross-react with 23c6 is mediated by induction of release of IL-1 beta.     

Construction of an ordered cosmid collection of the Escherichia coli K-12 W3110 chromosome.

A cosmid library of the Escherichia coli K-12 W3110 chromosome was constructed in which clones were assigned to locations on the chromosome map by hybridization and genetic marker complementation tests. Approximately 70% of the genome was represented by this library. The identified clones can be maintained in the homologous system and would facilitate genetic studies of E. coli.     

Mechanism of the stimulatory effect of cyclodextrins on lankacidin-producing Streptomyces

Summary Gel-filtration analysis of a mixture of cyclodextrin (CyD) and lankacidin C showed that -CyD had strong, -CyD weak and -CyD no affinity for lankacidin C. Lankacidin C production activity, which was assayed by measuring the incorporation of l-[methyl-¹⁴C-]methionine into the lankacidin molecule, was the greatest with cells grown in the presence of -CyD, less with -CyD and the least with -CyD. Lankamycin and T-2636M, which are by-products in lankacidin C fermentation, were not included by -CyD and their production was not stimulated by -CyD. It was apparent that the stimulatory effect of CyD was closely related to the formation of an inclusion complex between CyD and the antibiotic. Lankacidin C biosynthesis was repressed by preincubating cells with lankacidin C, while the repressive effect of lankacidin C was abrogated by the inclusion by -CyD. Thus, abrogation of feed-back repression seems to be a main mechanism of the effect of CyD. However, -CyD, which had no affinity for lankacidin C, stimulated the production to the least extent and exhibited a complementary effect on the stimulation by -CyD or -CyD. -CyD also caused a change in cell morphology and cell-surface hydrophobicity. It was assumed that the modification of the cell surface is a secondary mechanism of the effect of CyD.The second report of the stimulatory effect of cyclodextrins on lankacidin fermentationOffprint requests to: H. Sawada     

Analysis of the Epitopes Recognized by Mouse Monoclonal Antibodies Directed to Yersinia enterocolitica Heat-Shock Protein 60

To determine amino acid sequences of the epitopes recognized by monoclonal antibodies (mAbs) 3C8 and 5C3 directed against Yersinia enterocolitica heat-shock protein (HSP60), a dot blot analysis was perfomed using synthesized peptides of Y. enterocolitica HSP60 such as peptides p316-342, p327-359, p340-366, p316-326, p316-321, p319-323, and p321-326 which represent positions of amino acids in Y. enterocolitica HSP60. The dot blot analysis revealed that 5C3 mAb reacted with p316-342, p316-326 and p321-326, and 3C8 mAb p316-342 and p316-326. These results indicate that the epitopes recognized by the mAbs were associated with eleven amino acids, Asp Leu Gly Gln Ala Lys Arg Val Val Ile Asn, of p316-326. The sequence homology between p316-326 of Y. enterocolitica HSP60 and the rest of the HSP60 family suggests that the five amino acids of Lys, Arg, Val, Ile and Asn, which are highly conserved in the HSP60 family, might be related with the epitope recognized by 3C8. In contrast, it was also demonstrated that three amino acids of Leu, Gly and Val, which are not well conserved in the HSP60 family, might be related to the epitope recognized by 5C3.     

A two-domain mechanism for group A streptococcal adherence through protein F to the extracellular matrix.

Streptococcus pyogenes binds to the extracellular matrix (ECM) and a variety of host cells and tissues, causing diverse human diseases. Protein F, a S.pyogenes adhesin that binds fibronectin (Fn), contains two binding domains. A repeated domain (RD2) and an additional domain (UR), located immediately N-terminal to RD2. Both domains are required for maximal Fn binding. In this study, we characterize RD2 and UR precisely and compare their functions and binding sites in Fn. The minimal functional unit of RD2 is of 44 amino acids, with contributions from two adjacent RD2 repeats flanked by a novel 'MGGQSES' motif. RD2 binds to the N-terminal fibrin binding domain of Fn. UR contains 49 amino acids, of which six are from the first repeat of RD2. It binds to Fn with higher affinity than RD2, and recognizes a larger fragment that contains fibrin and collagen binding domains. Expression of UR and RD2 independently on the surface-exposed region of unrelated streptococcal protein demonstrates that both mediate adherence of the bacteria to the ECM. We describe here a mechanism of adherence of a pathogen that involves two pairs of sites located on a single adhesin molecule and directed at the same host receptor.     

Peroxyoxalate chemiluminescent assay for oxidase activities based on detecting enzymatically formed hydrogen peroxide

A sensitive peroxyoxalate chemiluminescent (PO-CL) assay for activities of oxidases (uricase, choline oxidase, cholesterol oxidase and xanthine oxidase) which catalyse a formation of hydrogen peroxide was developed using 4,4′-oxalyl-bis[(trifluoromethylsulphonyl)imino]trimethylene-bis(4-methylmorpholinium)trifluoromethanesulphonate as a chemiluminogenic reagent and 2,4,6,8-tetramorpholinopyrimido[5,4-d]pyrimidine as a fluorophore. The standard curve for hydrogen peroxide was linear over the range 1 × 10^?7-1 × 10^?4 mol/L. Relative standard deviations for oxidase assays were 5.1–12.7% (n = 10). Detection limits were 1 × 10^?3 U/mL for uricase, 5 × 10^?4 U/mL for choline oxidase, 5 × 10^?3 U/mL for cholesterol oxidase and 5 × 10^?4 U/mL xanthine oxidase (sample to blank ratio, 3).     

Isolation of Serratia marcescens mutants which could overproduce and excrete Escherichia coli alkaline phosphatase and beta-lactamase

Serratia marcescens mutants, which excrete Escherichia coli alkaline phosphatase (APase) encoded by the plasmid-bearing phoA gene, were isolated after mutagenesis by N-methyl--nitro-N-nitrosoguanidine. These mutants produced two to four times as much APase as did the parent strain under a phosphate-limiting condition, and more than 70% of the enzyme was released into the culture medium. In addition, overproduction and excretion of beta-lactamase was observed in these mutants.     

Thermotropic phase properties of 1,2-di-O-tetradecyl-3-O-(3-O-methyl- beta-D-glucopyranosyl)-sn-glycerol.

The hydration properties and the phase structure of 1,2-di-O-tetradecyl-3-O(3-O-methyl-beta-D-glucopyranosyl)-sn-glycerol (3-O-Me-beta-D-GlcDAIG) in water have been studied via differential scanning calorimetry, 1H-NMR and 2H-NMR spectroscopy, and x-ray diffraction. Results indicate that this lipid forms a crystalline (Lc) phase up to temperatures of 60-70 degrees C, where a transition through a metastable reversed hexagonal (Hll) phase to a reversed micellar solution (L2) phase occurs. Experiments were carried out at water concentrations in a range from 0 to 35 wt%, which indicate that all phases are poorly hydrated, taking up < 5 mol water/mol lipid. The absence of a lamellar liquid crystalline (L alpha) phase and the low levels of hydration measured in the discernible phases suggest that the methylation of the saccharide moiety alters the hydrogen bonding properties of the headgroup in such a way that the 3-O-Me-beta-D-GlcDAIG headgroup cannot achieve the same level of hydration as the unmethylated form. Thus, in spite of the small increase in steric bulk resulting from methylation, there is an increase in the tendency of 3-O-Me-beta-D-GlcDAIG to form nonlamellar structures. A similar phase behavior has previously been observed for the Acholeplasma laidlawii A membrane lipid 1,2-diacyl-3-O-(6-O-acyl-alpha-D-glucopyranosyl)-sn-glycerol in water (Lindblom et al. 1993. J. Biol. Chem. 268:16198-16207). The phase behavior of the two lipids suggests that hydrophobic substitution of a hydroxyl group in the sugar ring of the glucopyranosylglycerols has a very strong effect on their physicochemical properties, i.e., headgroup hydration and the formation of different lipid aggregate structures.