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 α1(I)
procollagen (COL1A1) locus and the production of live sheep by nuclear
transfer.
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‐1α siRNA or over‐expression of HIF‐1α) 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% O2, 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‐1α siRNA reduced the effects of hypoxia. Over‐expression of HIF‐1α 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.
Abstract
The behavior of gelatin hydrogels is influenced by the charges located on the amino acid side chains throughout the gelatin molecules. The presence and distribution of ionisable side chains ...influences the surface activity of gelatin and ultimately determines the material properties. Herein, we report the influence of pH on mechanical properties as studied by texture analysis supported by data from polarimetry, zeta potential, pH titrations and NMR experiments. When adjusted to more extreme pH values (pH 2 and 12), softer gelatin blocks were observed. However, at pH values close to the isoelectric point (pH 5–10), the material is firmer. This behavior is related to the helical content. At pH 2 and pH 12 the surface of the gelatin carries a net charge, positive and negative, respectively, that inhibits the formation of tight helices and lowers the physical crosslink network density. Chemical shift perturbations were observed for the acidic amino acids glutamic and aspartic acid, under acidic pH, where their peaks shifted to higher ppm. Intense amide signals were observed at acidic pH but diminished with increasing pH. This was due to an increase in the rate of chemical exchange between the solvent and peptide amide protons as the pH increases.
Galactose- alpha 1, 3-galactose ( alpha 1,3Gal) is the major xenoantigen causing hyperacute rejection in pig-to-human xenotransplantation. Disruption of the gene encoding pig alpha ...1,3-galactosyltransferase ( alpha 1,3GT) by homologous recombination is a means to completely remove the alpha 1,3Gal epitopes from xenografts. Here we report the disruption of one allele of the pig alpha 1,3GT gene in both male and female porcine primary fetal fibroblasts. Targeting was confirmed in 17 colonies by Southern blot analysis, and 7 of them were used for nuclear transfer. Using cells from one colony, we produced six cloned female piglets, of which five were of normal weight and apparently healthy. Southern blot analysis confirmed that these five piglets contain one disrupted pig alpha 1,3GT allele.
Exposure to limiting oxygen in cells and tissues induce the stabilization and transcriptional activation of the hypoxia-inducible factor 1 alpha (HIF-1α) protein, a key regulator of the hypoxic ...response. Reactive oxygen species (ROS) generation has been implicated in the stabilization of HIF-1α during this response, but this is still a matter of some debate. In this study we utilize a mitochondria-targeted antioxidant, mitoubiquinone (MitoQ), and examine its effects on the hypoxic stabilization of HIF-1α. Our results show that under conditions of reduced oxygen (3% O
2), MitoQ ablated the hypoxic induction of ROS generation and destabilized HIF-1α protein. This in turn led to an abrogation of HIF-1 transcriptional activity. Normoxic stabilization of HIF-1α, on the other hand, was unchanged in the presence of MitoQ suggesting that ROS were not involved. This study strongly suggests that mitochondrial ROS contribute to the hypoxic stabilization of HIF-1α.
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.
Cellular response to limiting oxygen levels is managed, in part, by the transcription factor hypoxia-inducible factor 1 (HIF-1), and the prolyl hydroxylase (PHD) family of oxygen-requiring enzymes. ...In the process of analyzing the expression of
PHD3, we observed the presence of two alternatively processed
PHD3 transcripts, designated
PHD3Δ1 and
PHD3Δ4 . The expression of both
PHD3 and
PHD3Δ1 was observed in all tissues and cell lines tested, although the expression of the novel
PHD3Δ4 appeared to be restricted to primary cancer tissues. The function of
PHD3Δ4 was assessed in transfection experiments showing a preserved prolyl hydroxylase activity. We would submit that
PHD3 variants generated by alternative splicing may be intrinsically involved in the complex system of oxygen sensing.
In different mammalian species, in vitro culture and manipulation can lead to aberrant fetal and peri-natal development. It has been postulated that these diverse abnormalities are caused by ...epigenetic alterations and that these could affect genes that are regulated by genomic imprinting. To explore this hypothesis relative to somatic cell nuclear transfer in sheep, we investigated whether the ovine
H19-IGF2 and
IGF2R loci are imprinted and analysed their DNA methylation status in cloned lambs. A comparison between parthenogenetic and control concepti established that imprinting at these two growth-related loci is evolutionarily conserved in sheep. As in humans and mice,
IGF2R and
H19 comprise differentially methylated regions (DMRs) that are methylated on one of the two parental alleles predominantly. In tongue tissue from 12 out of 13 cloned lambs analysed, the DMR in the second intron of
IGF2R had strongly reduced levels of DNA methylation. The DMR located upstream of the ovine
H19 gene was found to be similarly organised as in humans and mice, with multiple CTCF binding sites. At this DMR, however, aberrant methylation was observed in only one of the cloned lambs. Although the underlying mechanisms remain to be determined, our data indicate that somatic cell nuclear transfer procedures can lead to epigenetic deregulation at imprinted loci.
The associations of high job demands, low decision latitude and job strain with cardiovascular disease (CVD) risk factors among 2665 black and white working men and women were examined in the ...Coronary Artery Risk Development in Young Adults study—a large, prospective, multi-center study of the development of CVD risk factors in young adults aged 18–30 years at baseline (1985–1986). Multiple linear and multiple logistic regression were used in cross-sectional analyses to examine the associations of job demands, decision latitude and job strain with blood pressure, total serum cholesterol, alcohol use and cigarette consumption. Inverse associations with risk factors were found for high job demands, low decision latitude and job strain. Few associations supported the hypotheses that high job demands, low decision latitude or job strain are associated with increased levels of CVD risk factors. We discuss possible explanations for these findings, including methodologic, age and gender differences between studies. In addition, we discuss the validity of job strain measures for women and minority workers.
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