Identification of a novel penicillin-binding protein from Helicobacter pylori.

The Helicobacter pylori genome encodes four penicillin-binding proteins (PBPs). PBPs 1, 2, and 3 exhibit similarities to known PBPs. The sequence of PBP 4 is unique in that it displays a novel combination of two highly conserved PBP motifs and an absence of a third motif. Expression of PBP 4, but not PBP 1, 2, or 3, is significantly increased during mid- to late-log-phase growth.

Surface localization of Helicobacter pylori urease and a heat shock protein homolog requires bacterial autolysis.

Helicobacter pylori is a gram-negative bacterium which causes chronic gastritis and is associated with peptic ulcer disease, gastric carcinoma, and gastric lymphoma. The bacterium is characterized by potent urease activity, thought to be located on the outer membrane, which is essential for survival at low pH. The purpose of the present study was to investigate mechanisms whereby urease and HspB, a GroEL homolog, become surface associated in vitro. Urease, HspB, and catalase were located almost exclusively within the cytoplasm in fresh log-phase cultures assessed by cryo- immunoelectron microscopy. In contrast, significant amounts of surface-associated antigen were observed in older or subcultured preparations concomitantly with the appearance of significant amounts of extracellular antigen, amorphous debris, and membrane fragments. By use of a variety of biochemical methods, a significant fraction of urease and HspB was associated with the outer membrane in subcultured preparations of H. pylori. Taken together, these results strongly suggest that H. pylori cells undergo spontaneous autolysis during culture and that urease and HspB become surface associated only concomitant with bacterial autolysis. By comparing enzyme sensitivity to flurofamide (a potent, poorly diffusible urease inhibitor) in whole cells with that in deliberately lysed cells, we show that both extracellular and intracellular urease molecules are active enzymatically. Autolysis of H. pylori is an important phenomenon to recognize since it likely exerts significant effects on the behavior of H. pylori. Furthermore, the surface properties of H. pylori must be unique in promoting adsorption of cytoplasmic proteins.

Localization of bFGF-like proteins as punctate inclusions in the preseptation myocardium of the chicken embryo.

Immunocytochemistry has been employed to map the appearance of bFGF-like proteins in precardiac and preseptation myocardial cells between stages 6 and 15 of chicken embryogenesis. Stage 6 embryos exhibited no staining, with the exception of a subtle signal in endoderm cells. At subsequent stages, staining was observed only in cells of the developing myocardium, first appearing at the time of heart tube fusion (stage 9+) as punctate cytoplasmic aggregates. While the expression of bFGF-like antigen was temporally similar to that of myosin heavy chain, their staining patterns differed in that bFGF-like proteins were nonsarcomeric and did not extend into the inflow or outflow tracts. Western blotting of heparin agarose affinity-isolated proteins from stage 15 hearts revealed an antigen migrating at approximately 19 kDa. In contrast with the unique localization of bFGF-like proteins in myocardial cells, FGF receptor (FGFR) staining was widely distributed in the embryo; however, concentrated deposits of FGFR were detected in endothelial and myocardial cells, which diminished in the myocardium but not in the endothelium by stage 15. These results suggest that FGF-like proteins may have autocrine and/or paracrine functions during early cardiac morphogenesis.

Enrichment of ubiquitinated histone H2A in a low salt extract of micrococcal nuclease-digested myotube nuclei.

We recently demonstrated that ubiquitinated histone H2A (uH2A) declines 2.5-fold during terminal skeletal muscle differentiation, coincident with reductions in transcriptional activity (Wunsch, A. M., Haas, A. L., and Lough, J. (1987) Dev. Biol. 119, 85-93). To assess whether this indicates an association of uH2A with transcriptionally active genes, we have used micrococcal nuclease digestion and salt extraction to fractionate myotube nuclei. An oligonucleosomal fraction obtained by micrococcal nuclease digestion and extraction in low salt (100 mM NaCl) comprising only 25% of the nuclear DNA contained 90% of the total uH2A, as revealed by Western blotting. Further fractionation of this 100 mM salt extract by sucrose gradient centrifugation revealed that virtually all of the uH2A was localized in monomer to heptamer-sized oligonucleosomes. A second ubiquitinated species of 57-kDa (u57) was also localized in the 100 mM salt extract. In contrast, an 18-kD band (u18) was associated with fractions that were resistant to micrococcal nuclease digestion and salt extraction. Although micrococcal nuclease recognized a unique structural feature of active myotube chromatin, as evidenced by the appearance of hybridized skeletal alpha-actin sequences as a smear rather than the nucleosomal ladder exhibited by inactive and bulk sequences, neither the skeletal alpha-actin gene nor the inactive alpha D-globin gene was exclusively localized in the 100 mM salt fraction. Moreover, further fractionation of the 100 mM salt extract on a sucrose gradient failed to separate active from inactive genes. These findings suggest that uH2A is localized in a fraction of myotube chromatin which, although nuclease-sensitive and relatively soluble, is not enriched in active or inactive genes.