Proteomic analysis of glutamine-treated human intestinal epithelial HCT-8 cells under basal and inflammatory conditions

Glutamine (Gln) promotes intestinal growth and maintains gut structure and function, especially in situations of injury and during inflammation. Several mechanisms could contribute to Gln protective effects on gut. Proteomics enable us to characterize differentially expressed proteins in tissues in response to modifications of the biological or nutritional environment. Gln effects on the human intestinal epithelial HCT-8 cell line proteome were assessed under basal and proinflammatory conditions. The 2-DE gels were obtained and compared. Proteins were identified by MS and using databases. About 1200 spots were detected in both 2- and 10-mM Gln concentrations. Under basal conditions, 24 proteins were differentially expressed in response to Gln. Half of these proteins were implicated in protein biosynthesis or proteolysis and 20% in membrane trafficking. Under proinflammatory conditions, 27 proteins were up- or down-regulated by Gln 10 mM. From these proteins, 40% were involved in protein biosynthesis or proteolysis, 16% in membrane trafficking, 8% in cell cycle and apoptosis mechanisms and 8% in nucleic acid metabolism. This study provides the first holistic picture of proteome modulation by Gln in a human enterocytic cell line under basal and proinflammatory conditions, and supports further evaluation of nutritional modulation of intestinal proteome in humans.     

Neuroembryopathic effect of trichloroacetic acid in rats exposed during organogenesis

BACKGROUND: Halogenated hydrocarbons such as trichloroacetic acid (TCA) are among the most common water supply contaminants in the world. This study examines the effect of TCA on the developing brain of the Charles Foster rat. METHODS: Adult pregnant rats were placed in the test group and exposed to various concentration of TCA (i.e., 1000, 1200, 1400, 1600, and 1800 mg/kg body weight [b.w.]) by oral gavage throughout the period of organogenesis from Gestation Day (GD) 6-15 of gestation. Trichloroacetic acid was administered in the form of trichloroacetate, which is reduced to TCA in the body. The control mother rats were administered an equal volume of distilled water. Fetal brains were examined for their external and histological malformation. RESULTS: On GD 19, TCA administration led to an initial increase of brain weight at 1000 mg/kg b.w. and then a weight reduction after TCA doses of 1200 mg/kg b.w. and over. The brain of the formalin-fixed fetuses at 1000 and 1200 mg/kg b.w. showed hydrocephalus with breech of the ependymal lining, altered choroids plexus architecture, and increased apoptosis. At doses of 1400 mg/kg b.w. and above, the brain showed not only enhanced apoptosis of the neuronal cells, but extravasation of erythrocytes within the cortical parenchyma, vacuolation of the neuropil, and multiple cavity formation. CONCLUSION: With an increase in dose of TCA i.e., 1200 mg/kg b.w. and above, there is enhanced apoptosis, leading to increased neuronal death, which consequently led to the reduction in the brain weight as compared to controls. The fetal central nervous system is susceptible to the toxic effect of TCA.     

Cellular isoform of the prion protein PrPc in human intestinal cell lines: genetic polymorphism at codon 129, mRNA quantification and protein detection in lipid rafts

The cellular isoform of the normal prion protein PrP(c), encoded by the PRNP gene, is expressed in human intestinal epithelial cells where it may represent a potential target for infectious prions. We have sequenced the PRNP gene in Caco-2 and HT-29 parental and clonal cell lines, and found that these cells have a distinct polymorphism at codon 129. HT-29 cells are homozygous Met/Met, whereas Caco-2 cells are heterozygous Met/Val. The 129Val variant was also detected in Caco-2 mRNAs. Real-time PCR quantifications revealed that PrP(c) mRNAs were more expressed in HT-29 cells than in Caco-2 cells. These data were confirmed by studying the expression of PrP(c) in plasma membranes and lipid rafts prepared from these cells. Overall, these results may be important in view of using human intestinal cell lines Caco-2 and HT-29 as cellular in vitro models to study the initial steps of prion propagation after oral inoculation.     

The role of reactive oxygen and nitrogen species in cellular iron metabolism

The catalytic role of iron in the Haber-Weiss chemistry, which results in propagation of damaging reactive oxygen species (ROS), is well established. In this review, we attempt to summarize the recent evidence showing the reverse: That reactive oxygen and nitrogen species can significantly affect iron metabolism. Their interaction with iron-regulatory proteins (IRPs) seems to be one of the essential mechanisms of influencing iron homeostasis. Iron depletion is known to provoke normal iron uptake via IRPs, superoxide and hydrogen peroxide are supposed to cause unnecessary iron uptake by similar mechanism. Furthermore, ROS are able to release iron from iron-containing molecules. On the contrary, nitric oxide (NO) appears to be involved in cellular defense against the iron-mediated ROS generation probably mainly by inducing iron removal from cells. In addition, NO may attenuate the effect of superoxide by mutual reaction, although the reaction product—peroxynitrite—is capable to produce highly reactive hydroxyl radicals.     

Death receptor-associated pro-apoptotic signaling in aged skeletal muscle

Tumor necrosis factor-alpha (TNF-α) is elevated in the serum as a result of aging and it promotes pro-apoptotic signaling upon binding to the type I TNF receptor. It is not known if activation of this apoptotic pathway contributes to the well-documented age-associated decline in muscle mass (i.e. sarcopenia). We tested the hypothesis that skeletal muscles from aged rodents would exhibit elevations in markers involved in the extrinsic apoptotic pathway when compared to muscles from young adult rodents, thereby contributing to an increased incidence of nuclear apoptosis in these muscles. The plantaris (fast) and soleus (slow) muscles were studied in young adult (5–7 mo, n=8) and aged (33 mo, n=8) Fischer₃₄₄ × Brown Norway rats. Muscles from aged rats were significantly smaller while exhibiting a greater incidence of apoptosis. Furthermore, muscles from aged rats had higher type I TNF receptor and Fas associated death domain protein (FADD) mRNA, protein contents for FADD, BCL-2 Interacting Domain (Bid), FLICE-inhibitory protein (FLIP), and enzymatic activities of caspase-8 and caspase-3 than muscles from young adult rats. Significant correlations were observed in the plantaris muscle between caspase activity and muscle weight and the apoptotic index, while similar relationships were not found in the soleus. These data demonstrate that pro-apoptotic signaling downstream of the TNF receptor is active in aged muscles. Furthermore, our data extend the previous demonstration that type II fibers are preferentially affected by aging and support the hypothesis that type II fiber containing skeletal muscles may be more susceptible to muscle mass loses via the extrinsic apoptotic pathway.     

Heritability of Phenols in the Resistance of Theobroma cacao against Phytophthora megakarya, the Causal Agent of Black Pod Disease

The black pod disease caused by Phytophthora megakarya is responsible for 80% of the cocoa production loss in Cameroon. To assess the resistance of cocoa plants against this pathogen, necrotic lesions, phenolic content and qualitative alteration of phenolics were conducted in ICS84 and ICS95 clones (two Trinitario introduced from Trinidad) and their hybrids (families F30 and F25) derived from reciprocal cross breeding between these two parental clones after inoculation. The existence of strong hybrid vigour has been shown. Ninety percentage of the hybrid's genotypes manifested a positive heterosis effect for the development of lesion size. This suggests the existence of hybrid vigour with a genetic additive effect. F3086, F2509, F2552 and F2586 hybrids were characterized by localized lesions. Those hybrids genotypes can be considered as elite clones. In relation to analysis of total phenolics and lesion size, no maternal effect was detected in the transmission of these characters. A significant and negative correlation (r = −0.683) (P < 0.01) has been observed between necrosis evolution and phenolics accumulation. The values of the heritability of lesion size and the total phenolic content in offsprings did not permit to show the maternal effect. Qualitative analyses of phenolics showed high flavonones content in cocoa leaves. Qualitative analyses of phenolics in ICS84, ICS95 clones and their hybrids showed a modification of the phenolics profiles, notably concerning apigenin and luteolin derivatives due to the inoculation. These compounds, along with others that were not identified, could have a role in the reaction and mechanism of defence of cocoa against P. megakarya.     

Microfilament-dependent modulation of cytoplasmic protein binding to TNFalpha mRNA AU-rich instability element in human lymphoid cells

Cytoplasmic proteins with binding capability to AU-rich instability determinant sequences (ARE) of tumour necrosis factor alpha (TNFalpha) mRNA 3' untranslated region (3'UTR) were assessed in human lymphoid cells. In vitro label transfer experiments using wild type as well as mutant sequences in which the 70 nucleotide-long AUUUA pentamer-containing portion of the 3'UTR had been deleted conferred binding specificity to five major activities of 22/25-, 38/40-, 50-, 60- and 80-kDa proteins in cytoplasmic extracts of peripheral blood mononuclear cells (PBMCs). Cytochalasin-induced disarrangement of the F-actin-based microfilament system led to a Triton X-100-insoluble to soluble redistribution of these binding activities. No such changes were observed in Jurkat tumour cells. Combination of in vivo UV-crosslinking and in vitro label transfer experiments revealed considerable differences in RNA association between proteins of the same cell type as well as between proteins of identical molecular weight (Mw) derived from either PBMCs or Jurkat cells. Our findings may explain some aspects of differential regulation of interleukin 2 (IL-2) and TNFalpha mRNA stability upon microfilament disruption in human PBMCs observed in an earlier study. These results also suggest that the physical state of cytoplasmic structural environment might contribute to important regulatory processes regarding key elements of eukaryotic mRNA metabolism, such as modulation of stability. Finally, these data highlight the possibility that the often observed disorganization of the cytoskeleton in tumour cells may partly be responsible for the maintenance of the neoplastic state, a phenomenon that potentially involves ARE-AUBP interactions.     

Mitochondrial Uncoupling: Role of Uncoupling Protein Anion Carriers and Relationship to Thermogenesis and Weight Control “The Benefits of Losing Control”

Uncoupling proteins, a subgroup of the mitochondrial anion transporter superfamily, have beenidentified in prokaryotes, plants, and mammalian cells. Evolutionary conservation of thesemolecules reflects their importance as regulators of two critical mitochondrial functions, i.e.,ATP synthesis and the production of reactive oxygen species (ROS). Although the amino acidsequences of the three mammalian uncoupling proteins, UCP1, UCP2 and UCP3, are verysimilar, each homolog is the product of a unique gene and important differences have beendemonstrated in their tissue-specific expression and regulation. UCP1 and UCP3 appear to bekey regulators of energy expenditure, and hence, nonshivering thermogenesis, either in brownadipose tissue (UCP1) or skeletal muscle (UCP3). UCP2 is expressed more ubiquitously,although generally at low levels, in many tissues. There is conflicting evidence about itsimportance as a regulator of resting metabolic rate. However, evidence suggests that thishomolog might modulate the mitochondrial generation of ROS in some cell types, includingmacrophages and hepatocytes. While the induction of various uncoupling protein homologsprovides adaptive advantages, both to the organism (e.g., thermogenesis) and to individual cells(e.g., reduced ROS), increased uncoupling protein activity also increases cellular vulnerability tonecrosis by compromising the mitochondrial membrane potential. This narrow risk—benefitmargin necessitates tight control of uncoupling protein activity in order to preserve cellularviability and much remains to be learned about the regulatory mechanisms involved.