Induction of trefoil factor (TFF)1, TFF2 and TFF3 by hypoxia is mediated by hypoxia inducible factor-1: implications for gastric mucosal healing.

BACKGROUND AND PURPOSE: Mucosal microcirculation is compromised during gastric damage induced by non-steroidal anti-inflammatory drugs, such as aspirin. Consequently, oxygen supply to epithelial cells is decreased. The trefoil factor (TFF) peptides are involved in mechanisms of defence and repair in the gastrointestinal tract but their regulation at sites of gastric injury is unknown. EXPERIMENTAL APPROACH: Hypoxia and expression of TFF genes and peptides were measured in the damaged stomach of aspirin-treated rats. In a human gastric cell line (AGS cells), the effects of hypoxia and of hypoxia inducible factor (HIF)-1 (through transient transfection of HIF-1alpha siRNA or over-expression of HIF-1alpha) on TFF gene expression were evaluated. KEY RESULTS: Hypoxyprobe immunostaining, up-regulation of TFF2 (1.9-fold) and TFF3 (1.8-fold) and a non-significant increase of TFF1 (1.5-fold) mRNA were observed in the damaged stomach of aspirin-treated rats, compared with control animals. Hypoxia (3% O(2), 16 h) induced mRNA for TFF1 (5.8-fold), TTF2 (9.1-fold) and TFF3 (9.3-fold) in AGS cells, an effect mediated by HIF-1, as transient transfection of HIF-1alpha siRNA reduced the effects of hypoxia. Over-expression of HIF-1alpha by transfection in non-hypoxic epithelial cells produced a similar pattern of TFF induction to that observed with hypoxia and transactivated a TFF1 reporter construct. CONCLUSIONS AND IMPLICATIONS: Hypoxia inducible factor-1 mediated the induction of TFF gene expression by hypoxia in gastric epithelial cells. Low oxygen levels and up-regulation of TFF gene expression in the damaged stomach of aspirin-treated rats suggest that hypoxia induced expression of TFF genes at sites of gastric injury.

Purification and Characterization of a Population of EGFP-Expressing Cells from the Developing Pancreas of a Neurogenin3/EGFP Transgenic Mouse.

Neurogenin 3 (ngn3) is a basic helix loop helix transcription factor that is transiently expressed in the developing mouse pancreas with peak expression around E15. In mice lacking the ngn3 gene the endocrine cells of the pancreas fail to develop suggesting that the ngn3-positive cell may represent a progenitor cell for the endocrine pancreas. In order to purify and characterize this cell in detail we have generated a transgenic mouse, in which the ngn3 promoter drives expression of enhanced green fluorescent protein (EGFP). In the E15.5 embryo EGFP was expressed in the dorsal and ventral pancreas, the duodenum, and lower intestine as well as in the brain. This pattern of expression was in keeping with the known expression profile of the endogenous ngn3 gene. Within the pancreas EGFP was localized in close proximity to cells that stained positive for ngn3, insulin, and glucagon, but was absent from regions of the pancreas that stained positive for amylase. EGFP was also present in the pancreas at E18.5, although there was no detectable expression of ngn3. At this stage EGFP did not colocalize with any of the hormones or exocrine markers. EGFP(+) cells were FACS purified (96%) from the E15 pancreas yielding approximately 10,000 cells or 1.6% of the total pancreatic cells from one litter. RT/PCR analysis confirmed that the purified cells expressed EGFP, ngn3, insulin, glucagon, somatostatin and pancreatic polypeptide. The ability to purify ngn3(+) cells provides an invaluable source of material for charactering in detail their properties.

Production of gene-targeted sheep by nuclear transfer from cultured somatic cells.

It is over a decade since the first demonstration that mouse embryonic stem cells could be used to transfer a predetermined genetic modification to a whole animal. The extension of this technique to other mammalian species, particularly livestock, might bring numerous biomedical benefits, for example, ablation of xenoreactive transplantation antigens, inactivation of genes responsible for neuropathogenic disease and precise placement of transgenes designed to produce proteins for human therapy. Gene targeting has not yet been achieved in mammals other than mice, however, because functional embryonic stem cells have not been derived. Nuclear transfer from cultured somatic cells provides an alternative means of cell-mediated transgenesis. Here we describe efficient and reproducible gene targeting in fetal fibroblasts to place a therapeutic transgene at the ovine alpha1(I) procollagen (COL1A1) locus and the production of live sheep by nuclear transfer.

Molecular cloning and characterization of a Candida albicans gene coding for cytochrome c haem lyase and a cell wall-related protein.

Immunoscreening of a Candida albicans cDNA library with a monoclonal antibody (mAb 4C12) recognizing an epitope present in high-molecular-weight mannoprotein (HMWM) components specific for the mycelial cell walls (a 180 kDa component and a poly-dispersed 260 kDa species) resulted in the isolation of the gene CaCYC3 encoding for cytochrome c haem lyase (CCHL). The CaCYC3 gene was transcribed preferentially in mycelial cells in which two mRNA transcripts of 0.8 and 1 kb were found. The nucleotide and the deduced amino acid sequences of this gene displayed 45% homology and 46% identity, respectively, to the Saccharomyces cerevisiae CYC3 gene and shared common features with other reported genes encoding for CCHL. The CaCYC3 gene restored the respiratory activity when transformed in a S. cerevisiae cyc3- mutant strain. A C. albicans CYC3 null mutant was constructed after sequential disruption using the hisG::URA3::hisG ('ura-blaster') cassette. Null mutant cells were unable to use lactate as a sole carbon source and had a reduced ability to form germ tubes. Western immunoblotting analysis of subcellular fractions from wild-type and null mutant strains demonstrated the presence of two gene products, a 33kDa mitochondrial protein and a 40 kDa cell wall-associated moiety reacting with antibodies against CCHL, in both yeast cells and germ tubes. mAb 4C12 still reacted with the CaCYC3 null mutant (by immunofluorescence and immunoblotting) but showed an altered pattern of immunoreactivity against cell wall HMWM species, indicating a relationship between these moieties and the CaCYC3 gene products. The results suggest that the CaCYC3 gene encodes two proteins, one targeted to the mitochondria and the other to the cell wall.

Cloning and expression of two chitin deacetylase genes of Saccharomyces cerevisiae.

Chitin deacetylase (EC 3.5.1.41), which hydrolyses the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin, has been demonstrated in crude extracts from sporulating Saccharomyces cerevisiae. Two S. cerevisiae open reading frames (ORFs), identified by the Yeast Genome Project, have protein sequence homology to a chitin deacetylase from Mucor rouxii. Northern blot hybridizations show each ORF was transcribed in diploid cells after transfer to sporulation medium and prior to formation of asci. Each ORF was cloned in a vector under transcriptional control of the GAL 1, 10 promoter and introduced back into haploid strains of S. cerevisiae. Chitin deacetylase activity was detected by in vitro assays from vegetative cells grown in galactose. Chemical analysis of these cells also demonstrated the synthesis of chitosam in vivo. Both recombinant chitin deacetylases showed similar qualitative and quantitative activities toward chitooligosaccharides in vitro. A diploid strain deleted to both ORFs, when sporulated, did not show deacetylase activity. The mutant spores were hypersensitive to lytic enzymes (Glusulase or Zymolyase).

Molecular cloning of a third chitinase gene (CHT1) from Candida albicans.

Here we report the complete nucleotide sequence of a third chitinase gene (CHT1) from the dimorphic human pathogen Candida albicans. The deduced amino acid (aa) sequence of Cht1 consists of 416 aa and displays 36% protein sequence similarity to chitinases Cht2 and Cht3, from C. albicans. Interestingly the domain structure of Cht1 is truncated when compared to the other chitinases of C. albicans and lacks a Ser/Thr-rich region.

Molecular cloning and characterization of chitinase genes from Candida albicans.

Chitinase (EC 3.2.1.14) is an important enzyme for the remodeling of chitin in the cell wall of fungi. We have cloned three chitinase genes (CHT1, CHT2, and CHT3) from the dimorphic human pathogen Candida albicans. CHT2 and CHT3 have been sequenced in full and their primary structures have been analyzed: CHT2 encodes a protein of 583 aa with a predicted size of 60.8 kDa; CHT3 encodes a protein of 567 aa with a predicted size of 60 kDa. All three genes show striking similarity to other chitinase genes in the literature, especially in the proposed catalytic domain. Transcription of CHT2 and CHT3 was greater when C. albicans was grown in a yeast phase as compared to a mycelial phase. A transcript of CHT1 could not be detected in either growth condition.