Comparing Two Intestinal Porcine Epithelial Cell Lines (IPECs): Morphological Differentiation, Function and Metabolism.

The pig shows genetical and physiological resemblance to human, which predestines it as an experimental animal model especially for mucosal physiology. Therefore, the intestinal epithelial cell lines 1 and J2 (IPEC-1, IPEC-J2)--spontaneously immortalised cell lines from the porcine intestine--are important tools for studying intestinal function. A microarray (GeneChip Porcine Genome Array) was performed to compare the genome wide gene expression of IPECs. Different significantly up-regulated pathways were identified, like "lysosome", "pathways in cancer", "regulation of actin cytoskeleton" and "oxidative phosphorylation" in IPEC-J2 in comparison to IPEC-1. On the other hand, "spliceosome", "ribosome", "RNA-degradation" and "tight junction" are significantly down-regulated pathways in IPEC-J2 in comparison to IPEC-1. Examined pathways were followed up by functional analyses. ATP-, oxygen, glucose and lactate-measurement provide evidence for up-regulation of oxidative phosphorylation in IPEC-J2. These cells seem to be more active in their metabolism than IPEC-1 cells due to a significant higher ATP-content as well as a higher O2- and glucose-consumption. The down-regulated pathway "ribosome" was followed up by measurement of RNA- and protein content. In summary, IPEC-J2 is a morphologically and functionally more differentiated cell line in comparison to IPEC-1. In addition, IPEC-J2 cells are a preferential tool for in vitro studies with the focus on metabolism.

Substrate flexibility and reaction specificity of tropinone reductase-like short-chain dehydrogenases.

Annotations of protein or gene sequences from large scale sequencing projects are based on protein size, characteristic binding motifs, and conserved catalytic amino acids, but biochemical functions are often uncertain. In the large family of short-chain dehydrogenases/reductases (SDRs), functional predictions often fail. Putative tropinone reductases, named tropinone reductase-like (TRL), are SDRs annotated in many genomes of organisms that do not contain tropane alkaloids. SDRs in vitro often accept several substrates complicating functional assignments. Cochlearia officinalis, a Brassicaceae, contains tropane alkaloids, in contrast to the closely related Arabidopsis thaliana. TRLs from Arabidopsis and the tropinone reductase isolated from Cochlearia (CoTR) were investigated for their catalytic capacity. In contrast to CoTR, none of the Arabidopsis TRLs reduced tropinone in vitro. NAD(H) and NADP(H) preferences were relaxed in two TRLs, and protein homology models revealed flexibility of amino acid residues in the active site allowing binding of both cofactors. TRLs reduced various carbonyl compounds, among them terpene ketones. The reduction was stereospecific for most of TRLs investigated, and the corresponding terpene alcohol oxidation was stereoselective. Carbonyl compounds that were identified to serve as substrates were applied for modeling pharmacophores of each TRL. A database of commercially available compounds was screened using the pharmacophores. Compounds identified as potential substrates were confirmed by turnover in vitro. Thus pharmacophores may contribute to better predictability of biochemical functions of SDR enzymes.

Enterococcus faecium NCIMB 10415 supplementation affects intestinal immune-associated gene expression in post-weaning piglets.

In a Salmonella challenge study of weaned piglets supplemented with the probiotic Enterococcus faecium NCIMB 10415 (SF68), we observed a delayed, post-infection proliferative response of purified blood mononuclear cell fractions towards Salmonella antigens. In order to clarify this observation, we examined the patterns of immune-associated gene expression in long-term feeding trials of both pre- and post-weaning piglets. Piglets supplemented with E. faecium NCIMB 10415 showed a post-weaning dysregulation in the expression patterns of both pro- and anti-inflammatory cytokine expression in intestinal tissues and spleen. Piglets of the supplemented group showed significantly reduced levels of IL-8, IL-10 and the co-stimulatory molecule CD86 mRNA expression in ileal Peyer's patches. The expression of CTLA4, an inhibitor of T-cell activation/proliferation, showed similar levels of expression in all tissues examined, particularly in ileal Peyer's patches post-weaning where IL-8, IL-10 and CD86 transcript levels were significantly reduced relative to control animals. Blood serum cytokine protein levels showed elevated TGFß in pre-weaning piglets which, together with IL-6, may have suppressed IFNγ production in the probiotic-fed animals. In a second Salmonella challenge study, post-weaning, E. faecium-fed animals showed significantly elevated levels of IL-8 gene expression in mesenteric lymph nodes, but reduced levels in the spleen. At early times post-infection, the probiotic-fed group showed similar levels of IL-10, CD86 and CTLA4 mRNA expression as the control animals in intestinal Peyer's Patches, despite high relative levels of IL-8 expression in mesenteric lymph nodes. The sum of the observations suggests that supplementation of pre-weaning piglets with E. faecium affects intestinal immune-associated gene expression, which is aggravated post-weaning when the animals receive increased levels of the probiotic in feed. We suggest the post-weaning reductions in gene expression may delay the host response to infections, and provide pathogenic bacteria such as Salmonella with a "window of opportunity", leading to the increased bacterial loads and shedding observed in challenge trials. Possible mechanisms explaining these effects of E. faecium NCIMB 10415 are discussed.

A chronic oral exposure of pigs with deoxynivalenol partially prevents the acute effects of lipopolysaccharides on hepatic histopathology and blood clinical chemistry.

Lipopolysaccharides (LPS), a cell wall component of gram-negative bacteria, and deoxynivalenol (DON), a prevalent Fusarium-derived contaminant of cereal grains, are each reported to have detrimental effects on the liver. A potentiating toxic effect of the combined exposure was reported previously in a mouse model and hepatocytes in vitro, but not in swine as the most DON-susceptible species. Thus, pigs were fed either a control diet (CON) or a Fusarium contaminated diet (DON, 3.1mg DON/kg diet) for 37 days. At day 37 control pigs were infused for 1h either with physiological saline (CON_CON), 100µg/kg BW DON (CON_DON), 7.5µg/kg BW LPS (CON_LPS), or both toxins (CON_DON/LPS) and Fusarium-pigs with saline (DON_CON) or 7.5µg/kg BW LPS (DON_LPS). Blood samples were taken before and after infusion (-30, +30, +60, +120, and +180min) for clinical blood chemistry. Pigs were sacrificed at +195min and liver histopathology was performed. LPS resulted in higher relative liver weight (p<0.05), portal, periportal and acinar inflammation (p<0.05), haemorrhage (p<0.01) and pathological bilirubin levels (CON_CON 1.0µmol/L vs. CON_LPS 5.4µmol/L, CON_DON/LPS 8.3µmol/L; p<0.001). DON feeding alleviated effects of LPS infusion on histopathology and blood chemistry to control levels, whereas DON infusion alone had no impact.

The plasma clearance of the Fusarium toxin deoxynivalenol (DON) is decreased in endotoxemic pigs.

The plasma elimination kinetics of the Fusarium toxin deoxynivalenol (DON) was investigated in male castrated pigs (40.4±3.7 kg) when infused intravenously either alone (100 µg/kg/h, n=6) or together with lipopolysaccharides (LPS, 7.5 µg/kg/h, n=6). The maximum DON concentration after one hour of infusion was significantly higher by 61% in the DON+LPS Group compared to pigs infused with DON alone. The area under the plasma DON concentration vs. time curve of the DON+LPS Group was approximately twice as high as that of the DON Group after 24h while the initial (0.63 vs. 0.6 h) and terminal half-lifes (2.97 vs. 2.30 h) remained uninfluenced. The apparent volume of distribution and the plasma clearance were significantly lower for the DON+LPS Group compared to the DON Group (2.14 vs. 1.45 L/kg and 11.9 vs. 5.87 mL/kg/min). Glucuronidated DON seemed to persist longer in the DON+LPS Group. In conclusion, clearance of DON was decreased during an LPS induced acute phase reaction in pigs. Whether the higher plasma DON concentrations in endotoxemic pigs are due to a hemodynamically associated longer persistence of the DON glucuronide or because of an altered glucuronidation activity needs to be examined further.

Evolution of putrescine N-methyltransferase from spermidine synthase demanded alterations in substrate binding.

Putrescine N-methyltransferase (PMT) catalyses S-adenosylmethionine (SAM)-dependent methylation of putrescine in tropane alkaloid biosynthesis. PMT presumably evolved from the ubiquitous spermidine synthase (SPDS). SPDS protein structure suggested that only few amino acid exchanges in the active site were necessary to achieve PMT activity. Protein modelling, mutagenesis, and chimeric protein construction were applied to trace back evolution of PMT activity from SPDS. Ten amino acid exchanges in Datura stramonium SPDS dismissed the hypothesis of facile generation of PMT activity in existing SPDS proteins. Chimeric PMT and SPDS enzymes were active and indicated the necessity for a different putrescine binding site when PMT developed.