Arginine deficiency in preconfluent intestinal Caco-2 cells modulates expression of proteins involved in proliferation, apoptosis, and heat shock response

Arginine is classified as a conditionally essential amino acid required exogenously during catabolic disease states and periods of rapid growth, both characterized by increased arginine utilization. Arginine plays an important role in the intestine, where it is extensively metabolized, and enhances its immune-supportive function and mucosal repair. Cell proliferation is important for the latter process. This study aimed for a better molecular insight in the response to arginine deprivation/supplementation of preconfluent and 5-day-confluent, differentiated Caco-2 intestinal cells. The potential of citrulline to counteract the effects of arginine deprivation was investigated in preconfluent cells. 2-DE combined with MALDI-TOF-MS and the antibody microarray technology were applied. Evidence is provided that arginine deficiency modulates the protein expression profiles of preconfluent Caco-2 cells differently than that of postconfluent differentiated cells. In preconfluent cells, certain proteins changed in direct response to arginine deficiency, whereas other proteins did not, but instead responded during the recovery phase after an arginine/citrulline resupplementation. The protein changes suggest that arginine deprivation decreases cell proliferation and heat shock protein expression, and enhances the cells susceptibility to apoptosis. These processes are critical for proper cell function, and hence a state of arginine deficiency can be detrimental for intestinal cells which proliferate actively in vivo.     

Comparative proteomic analysis of cell lines and scrapings of the human intestinal epithelium

Background

The homologues of human disease genes are expected to contribute to better understanding of physiological and pathogenic processes. We made use of the present availability of vertebrate genomic sequences, and we have conducted the most comprehensive comparative genomic analysis of the prion protein gene PRNP and its homologues, shadow of prion protein gene SPRN and doppel gene PRND, and prion testis-specific gene PRNT so far.

Results

While the SPRN and PRNP homologues are present in all vertebrates, PRND is known in tetrapods, and PRNT is present in primates. PRNT could be viewed as a TE-associated gene. Using human as the base sequence for genomic sequence comparisons (VISTA), we annotated numerous potential cis-elements. The conserved regions in SPRNs harbour the potential Sp1 sites in promoters (mammals, birds), C-rich intron splicing enhancers and PTB intron splicing silencers in introns (mammals, birds), and hsa-miR-34a sites in 3'-UTRs (eutherians). We showed the conserved PRNP upstream regions, which may be potential enhancers or silencers (primates, dog). In the PRNP 3'-UTRs, there are conserved cytoplasmic polyadenylation element sites (mammals, birds). The PRND core promoters include highly conserved CCAAT, CArG and TATA boxes (mammals). We deduced 42 new protein primary structures, and performed the first phylogenetic analysis of all vertebrate prion genes. Using the protein alignment which included 122 sequences, we constructed the neighbour-joining tree which showed four major clusters, including shadoos, shadoo2s and prion protein-likes (cluster 1), fish prion proteins (cluster 2), tetrapode prion proteins (cluster 3) and doppels (cluster 4). We showed that the entire prion protein conformationally plastic region is well conserved between eutherian prion proteins and shadoos (18–25% identity and 28–34% similarity), and there could be a potential structural compatibility between shadoos and the left-handed parallel beta-helical fold.

Conclusion

It is likely that the conserved genomic elements identified in this analysis represent bona fide cis-elements. However, this idea needs to be confirmed by functional assays in transgenic systems.     

Changes in growth and nutrient uptake in Brassica oleracea exposed to atmospheric ammonia

BACKGROUND AND AIMS: Plant shoots form a sink for NH3, and are able to utilize it as a source of N. NH3 was used as a tool to investigate the interaction between foliar N uptake and root N uptake. To what extent NH3 can contribute to the N budget of the plant or can be regarded as a toxin, was investigated in relation to its concentration and the N supply in the root environment. METHODS: Brassica oleracea was exposed to 0, 4 and 8 microL L(-1) NH3, with and without nitrate in the nutrient solution. Growth, N compounds, nitrate uptake rate, soluble sugars and cations were measured. KEY RESULTS: In nitrate-sufficient plants, biomass production was not affected at 4 microL L(-1) NH3, but was reduced at 8 microL L(-1) NH3. In nitrate-deprived plants, shoot biomass was increased at both concentrations, but root biomass decreased at 8 microL L(-1) NH3. The measured nitrate uptake rates agreed well with the plant's N requirement for growth. In nitrate-sufficient plants nitrate uptake at 4 and 8 microL L(-1) NH3 was reduced by 50 and 66 %, respectively. CONCLUSIONS: The present data do not support the hypothesis that NH3 toxicity is caused by a shortage of sugars or a lack of capacity to detoxify NH3. It is unlikely that amino acids, translocated from the shoot to root, are the signal metabolites involved in the down-regulation of nitrate uptake, since no relationship was found between changes in nitrate uptake and root soluble N content of NH3-exposed plants.     

Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: Electrostatic charging for section attachment and implementation of an anti-contamination glove box

Cryo-electron tomography of vitreous cryo-sections is the most suitable method for exploring the 3D organization of biological samples that are too large to be imaged in an intact state. Producing good quality vitreous cryo-sections, however, is challenging. Here, we focused on the major obstacles to success: contamination in and around the microtome, and attachment of the ribbon of sections to an electron microscopic grid support film. The conventional method for attaching sections to the grid has involved mechanical force generated by a crude stamping or pressing device, but this disrupts the integrity of vitreous cryo-sections. Furthermore, attachment is poor, and parts of the ribbon of sections are often far from the support film. This results in specimen instability during image acquisition and subsequent difficulty with aligning projection images.Here, we have implemented a protective glove box surrounding the cryo-ultramicrotome that reduces the humidity around and within the microtome during sectioning. We also introduce a novel way to attach vitreous cryo-sections to an EM grid support film using electrostatic charging. The ribbon of vitreous cryo-sections remains in place during transfer and storage and is devoid of stamping related artefacts. We illustrate these improvements by exploring the structure of putative cellular 80S ribosomes within 50 nm, vitreous cryo-sections of Saccharomyces cerevisiae.     

Novel markers for tying-up in horses by proteomics analysis of equine muscle biopsies

The aim of the study was to identify new biomarkers for acute tying-up in horses. Skeletal muscle biopsies were taken from 3 horses suffering from acute tying-up and 3 healthy horses. We performed 2D gel electrophoresis and mass spectrometry for identification of proteins that are differentially expressed in tying-up. 2D gel electrophoresis of skeletal muscle sequential extracts yielded more than 350 protein spots on each gel, of which 14 were differentially expressed more than two-fold (p < 0.05). In-gel digestion followed by peptide mass fingerprinting enabled identification of three significantly increased proteins: alpha actin, tropomyosin alpha chain and creatine kinase M chain (CKM). CKM was represented by multiple spots probably due to posttranslational modification, one of which appeared to be unique for tying-up. Since changes in the rates of synthesis and degradation of proteins are likely to lead to pathological conditions, identification of differentially expressed proteins in acute tying-up might result in the finding of more specific diagnostic markers and in new hypotheses for the common mechanisms that result in this condition. Our findings point to a specific isoform of CKM as a novel biomarker for tying-up suggesting that altered energy distribution within muscle cells is part of the disease etiology.     

Potential protein markers for nutritional health effects on colorectal cancer in the mouse as revealed by proteomics analysis

It is suggested that colorectal cancer might be prevented by changes in diet, and vegetable consumption has been demonstrated to have a protective effect. Until now, little is known about the effects of vegetable consumption at the proteome level. Therefore, the effect of increased vegetable intake on the protein expression in the colonic mucosa of healthy mice was studied. Aim was to identify the proteins that are differentially expressed by increased vegetable consumption and to discriminate their possible role in the protection against colorectal cancer. Mice were fed four different vegetable diets, which was followed by analysis of total cellular protein from colonic mucosal cells by a combination of 2-DE and MS. We found 30 proteins that were differentially expressed in one or more diets as compared to the control diet. Six could be identified by MALDI-TOF MS: myosin regulatory light chain 2, carbonic anhydrase I, high-mobility group protein 1, pancreatitis-associated protein 3, glyceraldehyde-3-phosphate dehydrogenase and ATP synthase oligomycin sensitivity conferral protein. Alterations in the levels of these proteins agree with a role in the protection against colon cancer. We conclude that these proteins are suitable markers for the health effect of food on cancer. The observed altered protein levels therefore provide support for the protective effects of vegetables against colorectal cancer.     

Iroquois genes influence proximo-distal morphogenesis during rat lung development

Lung development is a highly regulated process directed by mesenchymal-epithelial interactions, which coordinate the temporal and spatial expression of multiple regulatory factors required for proper lung formation. The Iroquois homeobox (Irx) genes have been implicated in the patterning and specification of several Drosophila and vertebrate organs, including the heart. Herein, we investigated whether the Irx genes play a role in lung morphogenesis. We found that Irx1-3 and Irx5 expression was confined to the branching lung epithelium, whereas Irx4 was not expressed in the developing lung. Antisense knockdown of all pulmonary Irx genes together dramatically decreased distal branching morphogenesis and increased distention of the proximal tubules in vitro, which was accompanied by a reduction in surfactant protein C-positive epithelial cells and an increase in beta-tubulin IV and Clara cell secretory protein positive epithelial structures. Transmission electron microscopy confirmed the proximal phenotype of the epithelial structures. Furthermore, antisense Irx knockdown resulted in loss of lung mesenchyme and abnormal smooth muscle cell formation. Expression of fibroblast growth factors (FGF) 1, 7, and 10, FGF receptor 2, bone morphogenetic protein 4, and Sonic hedgehog (Shh) were not altered in lung explants treated with antisense Irx oligonucleotides. All four Irx genes were expressed in Shh- and Gli(2)-deficient murine lungs. Collectively, these results suggest that Irx genes are involved in the regulation of proximo-distal morphogenesis of the developing lung but are likely not linked to the FGF, BMP, or Shh signaling pathways.