Fibulins: physiological and disease perspectives

The fibulins are a family of proteins that are associated with basement membranes and elastic extracellular matrix fibres. This review summarizes findings from studies of animal models of fibulin deficiency, human fibulin gene mutations, human tumours and injury models that have advanced our understanding of the normal and pathological roles of members of this formerly obscure family.     

Inhibition of hepatitis B virus in mice by RNA interference

Hepatitis B virus (HBV) infection substantially increases the risk of chronic liver disease and hepatocellular carcinoma in humans. RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral mechanism. Here we show that RNAi can be applied to inhibit production of HBV replicative intermediates in cell culture and in immunocompetent and immunodeficient mice transfected with an HBV plasmid. Cotransfection with plasmids expressing short hairpin RNAs (shRNAs) homologous to HBV mRNAs induced an RNAi response. Northern and Southern analyses of mouse liver RNA and DNA showed substantially reduced levels of HBV RNAs and replicated HBV genomes upon RNAi treatment. Secreted HBV surface antigen (HBsAg) was reduced by 94.2% in cell culture and 84.5% in mouse serum, whereas immunohistochemical detection of HBV core antigen (HBcAg) revealed >99% reduction in stained hepatocytes upon RNAi treatment. Thus, RNAi effectively inhibited replication initiation in cultured cells and mammalian liver, showing that such an approach could be useful in the treatment of viral diseases.     

PepT1-mediated fMLP transport induces intestinal inflammation in vivo

In the presentstudy, the effect of H⁺/peptide transporter(PepT1)-mediatedN-formylmethionyl-leucyl-phenylalanine (fMLP)transport on inflammation in vivo in the rat small intestine, whichexpresses high PepT1 levels, and in the rat colon, which does notexpress PepT1, were investigated using myeloperoxidase (MPO) activity and histological analysis. We found that 10 µM fMLP perfusion in thejejunum for 4 h significantly increased MPO activity and alteredthe architecture of jejunal villi. In contrast, 10 µM fMLP perfusionin the colon for 4 h did not induce any inflammation. In addition,we have shown that 50 mM Gly-Gly alone did not affect basal MPOactivity but completely inhibited the MPO activity induced by 10 µMfMLP in the jejunum. Together, these experiments demonstrate that1) the differential expression of PepT1 between the small intestine and the colon plays an important role inepithelial-neutrophil interactions and 2) the inhibition offMLP uptake by jejunal epithelial cells (expressing PepT1) reduces theneutrophil ability to move across the epithelium, in agreement with ourpreviously published in vitro study. This report constitutes the firstin vivo study showing the implication of a membrane transporter (PepT1)in intestinal inflammation.

     

A role for the Psi-U mismatch in the recognition of the 5' splice site of yeast introns by the U1 small nuclear ribonucleoprotein particle

The U1 small nuclear ribonucleoprotein particle (snRNP)/5' splice site (5'SS) interaction in yeast is essential for the splicing process and depends on the formation of a short RNA duplex between the 5' arm of U1 snRNA and the 1st intronic nucleotides. This RNA/RNA interaction is characterized by the presence of a mismatch that occurs with almost all yeast introns and concerns nucleotides 4 on the pre-mRNA (a U) and 5 on U1 snRNA (a Psi). The latter nucleotide is well conserved from yeast to vertebrates, but its role in yeast and the significance of the associated mismatch in the U1 snRNA/5'SS interaction have never been fully explained. We report here that the presence of this mismatch is a determinant of stability that mainly affects the off rate of the interaction. To our knowledge this is the first report assigning a function to this noncanonical interaction. We also performed SELEX (systematic evolution of ligands by exponential enrichment) experiments by immunoprecipitating U1 snRNP and the associated RNA. The artificial phylogeny derived from these experiments allows the isolation of the selective pressure due to U1 snRNP binding on the 5'SS of yeast introns.     

Putative effect of Helicobacter pylori and gastritis on gastric acid secretion in cat

Helicobacter pylori may increase or inhibit gastric acid. We studied acid variations and plasma gastrin in cats harboring Helicobacter felis, harboring H. pylori, or free of gastric pathogens with reference to thioperamide (H(3) receptor antagonist) and SR-27417A (PAF receptor antagonist). In cats harboring H. felis, gastric mucosa were histologically normal. After H. felis eradication, pentagastrin-stimulated acid secretion was increased (40%) compared with the situation before eradication. Thioperamide abolished this inhibitory effect of H. felis, whereas SR-27417A did not. Basal and meal-stimulated plasma gastrin levels were not affected by eradication therapy. Acid secretion was inhibited (-80%) in week 3, increased from weeks 5 to 9, and remained constant for up to 42 weeks after H. pylori infection. SR-27417A had no effect on acid secretion before week 8 but inhibited it thereafter, and thioperamide increased it (20%) only before week 7 in those cats. Helicobacter inhibits gastric acid via an H(3) receptor pathway. Inflammatory mediators are thus involved in adaptation to the inhibitory effects of H. pylori on acid secretion.     

Glutamine decreases interleukin-8 and interleukin-6 but not nitric oxide and prostaglandins e(2) production by human gut in-vitro

BACKGROUND: Glutamine modulates cytokine production in various tissues but its effects on the production of other inflammatory mediators such as eicosanoids and nitric oxide have not been investigated in human gut. AIM: To evaluate the influence of glutamine on interleukin (IL)-8, IL-6, nitric oxide and prostaglandin E(2) production by human gut. METHODS: Ten fasted volunteers received either enteral glutamine or isonitrogenous amino acids over 6 h in a cross-over design. Series of duodenal biopsies were frozen or cultured for 24 h with 0.5 or 5 mM of glutamine or amino acids. IL-6, IL-8 and PGE(2) were measured in culture media by ELISA and nitrites by Griess assay. mRNA levels for IL-6, IL-8, Cyclooxygenase-2 and NO synthase-2 were assessed in biopsies by RT-PCR. Results in percent, (median [range]) were compared by Wilcoxon test. RESULTS: Glutamine decreased IL-8 and IL-6 in-vitro production: 63 [2-173] vs 100 [19-177] and 37 [5-489] vs 100 [33-431], both P<0.05. IL-8 mRNA level also decreased in biopsies cultured with 5 mM glutamine: 26 [13-142] vs 92 [34-215], P<0.05. Nitrites and PGE(2) concentrations were not significantly affected by glutamine. CONCLUSION: Glutamine has a specific inhibitory effect on pro-inflammatory cytokine production in the gut and may contribution to the modulation of intestinal inflammation.     

Acute hypoxemia does not increase the oxidative stress in resting and contracting muscle in humans

In healthy humans sustaining static handgrip at 60% of maximal voluntary contraction (MVC) until exhaustion, we measured the venous blood concentration of reduced ascorbic acid (RAA) and thiobarbituric acid reactive substances (TBARS), respectively, used as markers of the post-exercise oxidative stress and lipid peroxidation. Measurements were conducted in normoxemia, then during a 30-min period of hypoxemia (PaO 2 =56 mmHg) produced by inhalation of an hypoxic gas mixture. Compared to normoxemia, hypoxemia did not significantly modify the resting concentrations of TBARS and RAA, and did not affect the consumption of ascorbic acid after 60% MVC but suppressed the post-exercise TBARS increase. We conclude that acute hypoxemia does not modify the production of oxygen free radicals after strenuous static efforts and even seems to attenuate the lipid peroxidation.     

Sensitivity of whole body protein synthesis to amino acid administration during short-term bed rest

We tested the hypothesis that a reduced stimulation of whole-body protein synthesis by amino acid administration represents a major mechanism for the bed rest-induced loss of lean body mass. Healthy young subjects and matched controls were studied on the last day of a 14-day bed rest or ambulatory period, as part of the overall protocol "Short-term Bed Rest - Integrated Physiology" set up by the German Aerospace Centre (DLR) in co-operation with the European Space Agency. A balanced mixture of essential and non-essential amino acids was intravenously infused in the postabsorptive state for 3 hours at the rate of 0.1 g/kg/hour. The oxidative and non-oxidative (i.e., to protein synthesis) disposal of the infused leucine was determined by stable isotope and mass spectrometry techniques. The clearance of total infused amino acids tended to be greater (P=0.07) in the ambulatory group than in the bed rest group. When leucine clearance was partitioned between its oxidative and non-oxidative (i.e., to protein synthesis) components, the results indicated that the oxidative disposal was not statistically different in the bed rest and in the ambulatory groups. In contrast, the non-oxidative leucine disposal (i.e., to protein synthesis) was about 20% greater (P<0.01) in the ambulatory group than in the bed rest group. In conclusion, these preliminary data suggest that 14-day bed rest impairs the ability to utilise exogenous amino acids for protein synthesis.     

The family of toxin-related ecto-ADP-ribosyltransferases in humans and the mouse

ADP-ribosyltransferases including toxins secreted by Vibrio cholera, Pseudomonas aerurginosa, and other pathogenic bacteria inactivate the function of human target proteins by attaching ADP-ribose onto a critical amino acid residue. Cross-species polymerase chain reaction (PCR) and database mining identified the orthologs of these ADP-ribosylating toxins in humans and the mouse. The human genome contains four functional toxin-related ADP-ribosyltransferase genes (ARTs) and two related intron-containing pseudogenes; the mouse has six functional orthologs. The human and mouse ART genes map to chromosomal regions with conserved linkage synteny. The individual ART genes reveal highly restricted expression patterns, which are largely conserved in humans and the mouse. We confirmed the predicted extracellular location of the ART proteins by expressing recombinant ARTs in insect cells. Two human and four mouse ARTs contain the active site motif (R-S-EXE) typical of arginine-specific ADP-ribosyltransferases and exhibit the predicted enzyme activities. Two other human ARTs and their murine orthologues deviate in the active site motif and lack detectable enzyme activity. Conceivably, these ARTs may have acquired a new specificity or function. The position-sensitive iterative database search program PSI-BLAST connected the mammalian ARTs with most known bacterial ADP-ribosylating toxins. In contrast, no related open reading frames occur in the four completed genomes of lower eucaryotes (yeast, worm, fly, and mustard weed). Interestingly, these organisms also lack genes for ADP-ribosylhydrolases, the enzymes that reverse protein ADP-ribosylation. This suggests that the two enzyme families that catalyze reversible mono-ADP-ribosylation either were lost from the genomes of these nonchordata eucaryotes or were subject to horizontal gene transfer between kingdoms.