Helicobacter pylori infection activates NF-kappa B in gastric epithelial cells.

BACKGROUND & AIMS: Helicobacter pylori adheres to gastric epithelial cells and stimulates interleukin (IL)-8 production. This may be instrumental in neutrophil infiltration of the gastric epithelium that characterizes H. pylori gastritis. This study examined the molecular mechanisms leading to H. pylori-induced epithelial cell IL-8 production. METHODS: Electrophoretic mobility shift analyses for NF-kappa B were performed on cell and nuclear extracts from H. pylori-infected AGS and Kato III human gastric epithelial cells. RESULTS: H. pylori infection activated the transcription factor NF-kappa B and induced nuclear translocation of both NF-kappa B p50/p65 heterodimers and p50 homodimers. Nuclear translocation of NF-kappa B (30 minutes) was followed by increased IL-8 messenger RNA (1 hour) and protein levels (4 hours) consistent with NF-kappa B up-regulation of IL-8 gene transcription. Pretreatment of AGS cells with PDTC, which blocks NF-kappa B activation, inhibited H. pylori-induced increases in IL-8 production by 90%. Immunohistochemical studies using a monoclonal antibody that recognizes the I-kappa B binding region of p65 showed activated NF-kappa B in gastric epithelial cells of patients with H. pylori gastritis. CONCLUSIONS: H. pylori infection activates NF-kappa B in gastric epithelial cells in vitro and in vivo. NF-kappa B is a transcriptional regulator of IL-8 production, and its activation after bacterial infection may be an important defense response in gastrointestinal epithelial cells.

Rabbit sucrase-isomaltase contains a functional intestinal receptor for Clostridium difficile toxin A.

The intestinal effects of Clostridium difficile toxin A are inidated by toxin binding to luminal enterocyte receptors. We reported previously that the rabbit ileal brush border (BB) receptor is a glycoprotein with an alpha-d-galactose containing trisaccharide in the toxin-binding domain (1991. J. Clin. Invest. 88:119-125). In this study we characterized the rabbit ileal BB receptor for this toxin. Purified toxin receptor peptides of 19 and 24 amino acids showed 100% homology with rabbit sucrase-isomaltase (SI). Guinea pig receptor antiserum reacted in Western blots with rabbit SI and with the purified toxin receptor. Antireceptor IgG blocked in vitro binding of toxin A to rabbit ileal villus cell BB. Furthermore, anti-SI IgG inhibited toxin A-induced secretion (by 78.1%, P < 0.01), intestinal permeability (by 80.8%, P < 0.01), and histologic injury (P < 0.01) in rabbit ileal loops in vivo. Chinese hamster ovary cells transfected with SI cDNA showed increased intracellular calcium increase in response to native toxin (holotoxin) or to a recombinant 873-amino acid peptide representing the receptor binding domain of toxin A. These data suggest that toxin A binds specifically to carbohydrate domains on rabbit ileal SI, and that such binding is relevant to signal transduction mechanisms that mediate in vitro and in vivo toxicity.

Genes for Xenopus laevis U3 small nuclear RNA.

Genomic Southern blots showed there are only 14 to 20 copies of U3 snRNA genes per somatic genome in Xenopus laevis, unlike the highly repetitive, tandem arrangement of other snRNA genes in this organism. Sequencing of two U3 snRNA genes from lambda clones of a genomic library revealed striking similarity upstream, but much more divergence downstream. Consensus motifs common to other U snRNA genes were also found: a distal sequence element (DSE, octamer motif at -222 to -215), a proximal sequence element (PSE, at -62 to -52) and a 3' Box (15 or 16 bp downstream of the U3 genes). The DSE of mammals also has an inverted CCAAT motif specific for U3 snRNA genes, and we find this is conserved in the amphibian U3 snRNA genes. The Xenopus inverted CCAAT motif is exactly one helical turn further upstream of the octamer motif than its mammalian counterpart, suggesting interaction of putative transcription factors bound to these motifs. Mutation of the inverted CCAAT motif and part of an adjacent Sp1 site greatly depresses transcription of the mutant U3 snRNA gene in Xenopus oocytes, implying a role in transcriptional efficiency. Electrophoretic mobility shift assays implicate transcription factor binding to this region.

Cell and species distribution of prolactin-inducible annexin I mRNA.

The major prolactin-induced gene in the Columbid cropsac (cp35) is a unique member of the annexin (lipocortin/calpactin) gene family, most closely related to mammalian annexin I. Because no other annexins are known to be regulated by a specific hormonal signal, we have analyzed the distribution of annexin I mRNAs which hybridize to cp35 cDNA by comparing several tissue and cell systems. In addition we have used in situ hybridization to locate the expression of cp35 mRNA in the cropsac. Of nine separate organs extracted only cropsac, spleen, trachea, intestine, and lung expressed easily detectable levels of annexin I mRNA. Heart, liver, kidney, and skeletal muscle did not consistently express detectable annexin I. Prolactin (PRL) injection had no measurable effect on the mRNAs expressed in any of the tissues other than cropsac. Mammalian cell lines which respond to PRL (COMMA-D, HC11) were probed for expression of cp35-hybridizing mRNAs. These cell lines contained high levels of annexin I mRNA, but the mRNA level was not stimulated by PRL. Lactating mouse mammary gland did not contain measurable RNAs for either annexin I or II. In situ hybridization of cropsac sections showed that high-level expression of annexin I (cp35) mRNA was localized in the differentiating layer of the cropsac mucosal epithelium after PRL stimulation. It was not abundant in either the proliferating layer or the outermost desquamating layer of cells. These experiments argue that mRNAcp35 expression is a unique component of the PRL-induced differentiation response of cropsac and that closely related mRNAs are expressed in some, but not all, other tissues of the pigeon.

Structure of the gene encoding columbid annexin Icp35.

The cp35 gene, encoding an annexin I (AnxI) cropsac 35-kDa protein (cp35) from the pigeon, consists of 13 exons and twelve introns. The borders of exons 2-13 were mapped by comparison with the known cDNA sequence. A 5-kb sequence containing exons 1, 2, and 3, and 1.4 kb of 5'-flanking DNA, is presented. The transcription start point was mapped by S1 nuclease protection. The region of the cp35 mRNA sequence, which we had previously shown to be profoundly different from mammalian anxI, is located in the first half of exon 3. Whereas human anxI is known to be single copy, Southern analysis of pigeon genomic DNA and genomic clones demonstrated multiple anxI genes in the pigeon, diverging significantly in their 5'-termini. Pigeon vimentin, on the other hand, is encoded by a single-copy gene as it is in other birds and mammals. These experiments have demonstrated that the cp35 mRNA is transcribed from its individual gene and is not a product of alternative processing of the pigeon homolog of mammalian anxI. We speculate that the diversification of anxI genes in Columbid birds allowed the recruitment of one of these genes (cp35) for unique regulation by prolactin in the absence of post-translational regulation via residues encoded by exons 2 and 3.

Biosynthetic precursors of cartilage chondroitin sulfate proteoglycan.

Early steps in the biosynthesis of chondroitin sulfate proteoglycan (CSPG) and collagenous cartilage matrix molecules were examined by the comparison of products translated in mRNA-directed cell-free reactions and those synthesized by intact cartilage cells. RNA isolated from embryonic chicken sterna was used to direct cell-free translation reactions. Chicken sternal chondrocytes in culture were pulse-labeled with [35S]-methionine. The CSPG core protein was identified by immunoprecipitation. The Mr of the cartilage cell-synthetized core protein was determined to be 370K, approximately 10-15K greater than that of the comparable cell-free translation product. Experimental results strongly support the view that the observed difference in Mr reflects the cotranslational addition of mannose-rich, N-asparagine-linked oligosaccharides to the cell-synthesized core protein: 1) the cell-synthesized product was labeled with [3H]-mannose and precipitated by concanavalin A-sepharose beads; 2) the incorporated [3H]-mannose could be subsequently removed by digestion with endoglycosidase H (Endo H); 3) the Mr of the cell-synthesized core protein was reduced by Endo H digestion to that of the comparable cell-free translation product; 4) the core protein synthesized by tunicamycin-treated chondrocytes (inhibited in their ability to add N-asparagine-linked mannose-rich oligosaccharides to proteins) was comparable in electrophoretic mobility to that of the core protein cell-free translation product; and 5) the core protein translated in microsome-coupled cell-free reactions had an Mr 8-10K greater than that of the core protein translated in the absence of microsomes. For the purpose of examining biosynthetic intermediates, chondrocytes were labeled continuously or pulse-chase labeled for varying times. No biosynthetic CSPG intermediates migrating between the core protein and the CSPG monomer were detected. However, a band of 355Kdal appeared to share certain characteristics with the 307Kdal core protein (including its immunoprecipitability with CSPG antibodies), and a 340Kdal band was noted. Type II procollagen and other collagenase-sensitive products of 205Kdal and 110Kdal were observed among translation and chondrocyte-synthesized products. In chondrocytes, all three products exhibited labeling or chase time-dependent increases in Mr which were accelerated by ascorbate supplements and inhibited by the addition of alpha, alpha'-dipyridyl. These results suggest that the observed time-dependent increases in Mr are a consequence of collagen hydroxylation. The 110Kdal and 205Kdal collagenous proteins may be related to the minor collagens recently described in cartilage.

Glyceraldehyde-3-phosphate dehydrogenase from Drosophila melanogaster. Identification of two isozymic forms encoded by separate genes.

The enzyme glyceraldehyde-3-phosphate dehydrogenase from Drosophila melanogaster has been purified, and these preparations contain two subunits forms which have molecular weights of 37,000 and 35,500, respectively. Each subunit is found in crude extracts, and two activity bands are seen in nondenaturing acrylamide gels. Translation of Drosophila poly(A)-containing RNA results in two products which are precipitable with anti-glyceraldehyde-3-phosphate dehydrogenase serum. Two recombinant DNA clones have been isolated from a genomic library of Drosophila DNA. Each of these clones has the ability to hybrid select mRNAs which translate into both subunit forms. These clones have been genetically characterized by in situ hybridization and restriction mapping. One clone hybridizes to region 13F and the other to region 43E of the Drosophila cytogenetic map. Therefore, it appears that the Drosophila melanogaster genome contains two unlinked genes for glyceraldehyde-3-phosphate dehydrogenase; one of them encodes a protein of 37,000 daltons, the other a protein of 35,500 daltons.