MicroRNAs and their role in viral infection

Recently, a class of about 22 nucleotides (nt) small RNA has been discovered in many eukaryotes, termed microRNAs (miRNAs), which have a variety of functions. Many recent findings have demonstrated that viruses can also encode their own miRNAs. Meanwhile, other findings reveal a relationship between host miRNA and viral infection. These findings suggest a tight relationship between host and viral infection via miRNA pathway. This article introduces the miRNAs encoded by viruses and reviews the advances of the interaction of the mammalian host miRNAs and viral infection.     

Structure, stability, and chaperone function of alphaA-crystallin: role of N-terminal region

Small heat shock protein alphaA-crystallin, the major protein of the eye lens, is a molecular chaperone. It consists of a highly conserved central domain flanked by the N-terminal and C-terminal regions. In this article we studied the role of the N-terminal domain in the structure and chaperone function of alphaA-crystallin. Using site directed truncation we raised several deletion mutants of alphaA-crystallin and their protein products were expressed in Escherichia coli. Size exclusion chromatography of these purified proteins showed that deletion from the N-terminal beyond the first 20 residues drastically reduced the oligomeric association of alphaA-crystallin and its complete removal resulted in a tetramer. Chaperone activity of alphaA-crystallin, determined by thermal and nonthermal aggregation and refolding assay, decreased with increasing length of deletion and little activity was observed for the tetramer. However it was revealed that N-terminal regions were not responsible for specific recognition of natural substrates and that low affinity substrate binding sites existed in other part of the molecule. The number of exposed hydrophobic sites and the affinity of binding hydrophobic probe bis-ANS as well as protein substrates decreased with N-terminal deletion. The stability of the mutant proteins decreased with increase in the length of deletion. The role of thermodynamic stability, oligomeric size, and surface hydrophobicity in chaperone function is discussed. Detailed analysis showed that the most important role of N-terminal region is to control the oligomerization, which is crucial for the stability and in vivo survival of this protein molecule.     

The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

In mouse intestine, caveolae and caveolin‐1 (Cav‐1) are present in smooth muscle (responsible for executing contractions) and in interstitial cells of Cajal (ICC; responsible for pacing contractions). We found that a number of calcium handling/dependent molecules are associated with caveolae, including L‐type Ca²⁺ channels, Na⁺‐Ca²⁺ exchanger type 1 (NCX1), plasma membrane Ca²⁺ pumps and neural nitric oxide synthase (nNOS), and that caveolae are close to the peripheral endo‐sarcoplasmic reticulum (ER‐SR). Also we found that this assemblage may account for recycling of calcium from caveolar domains to SR through L‐type Ca ⁺ channels to sustain pacing and contractions. Here we test this hypothesis further comparing pacing and contractions under various conditions in longitudinal muscle of Cav‐1 knockout mice (lacking caveolae) and in their genetic controls. We used a procedure in which pacing frequencies (indicative of functioning of ICC) and contraction amplitudes (indicative of functioning of smooth muscle) were studied in calcium‐free media with 100 mM ethylene glycol tetra‐acetic acid (EGTA). The absence of caveolae in ICC inhibited the ability of ICC to maintain frequencies of contraction in the calcium‐free medium by reducing recycling of calcium from caveolar plasma membrane to SR when the calcium stores were initially full. This recycling to ICC involved primarily L‐type Ca²⁺ channels; i.e. pacing frequencies were enhanced by opening and inhibited by closing these channels. However, when these stores were depleted by block of the sarco/endoplasmic reticulum Ca²⁺‐ATPase (SERCA) pump or calcium release was activated by carbachol, the absence of Cav‐1 or caveolae had little or no effect. The absence of caveolae had little impact on contraction amplitudes, indicative of recycling of calcium to SR in smooth muscle. However, the absence of caveolae slowed the rate of loss of calcium from SR under some conditions in both ICC and smooth muscle, which may reflect the loss of proximity to store operated Ca channels. We found evidence that these channels were associated with Cav‐1. These changes were all consistent with the hypothesis that a reduction of the extracellular calcium associated with caveolae in ICC of the myenteric plexus, the state of L‐type Ca²⁺ channels or an increase in the distance between caveolae and SR affected calcium handling.     

Relationship between interaction sites in the gut, hydrophobicity, mucosal immunomodulating capacities and cell wall protein profiles in indigenous and exogenous bacteria

AIMS: To investigate whether there is a relationship between interaction sites in the gut, hydrophobicity, mucosal immunomodulating capacities and cell wall protein profiles in lactobacilli, bifidobacteria and enterococci. METHODS AND RESULTS: Hydrophobicity, cell wall protein profiles and sites of interaction in the gut (by using fluorescein isothiocyanate-labelled bacteria) were determined for Lactobacillus casei, L. acidophilus, L. fermentum, Bifidobacterium bifidum, B. animalis and Enterococcus faecalis. We also determined the number of immunoglobulin (Ig)A+, tumour necrosis factor (TNF)alpha+, interleukin (IL)-6+ and IL-10+ cells after oral administration of the above bacteria to BALB/c mice. All strains assessed were found to interact with the sites of induction of the immune response in the gut. No correlation with hydrophobicity was observed. When some strains at certain doses were administered to mice, bacterial translocation to liver was observed. The oral administration of indigenous (104 cells day(-1)) and exogenous (107 cells day(-1)) bifidobacteria and lactobacilli for 5 consecutive days activated the systemic and intestinal mucosal immune response in a strain-specific way, independently whether the strain was indigenous or exogenous in relation to the host. The differences in the immunopotentiating capacity of the various strains might be related to the differences in their cell wall protein profiles. CONCLUSIONS: Indigenous bacteria activated the mucosal immune response at a dose significantly smaller than the one required for probiotic exogenous bacteria. However, probiotic exogenous bacteria can be used at high concentrations in fermented dairy products with a great impact on the immune system, favouring its immunomodulation. SIGNIFICANCE AND IMPACT OF THE STUDY: The immunomodulation capacity of probiotic bacteria is strain specific and independent of the specificity of the host. The ability of certain strains to down-regulate the production and release of IL-6 by IL-10 may have potential implications in their use in cases in which cytokine deregulation or excessive production at the mucosal level can be the cause of tissue damage.     

Inhibition of ileal brush-border chloride conductance by specific antibody

Summary Antibody raised in mice was used in attempting to identify proteins responsible for the conductive chloride transport that can be measured in porcine ileal brush border membrane vesicles. Ileal brush-border membrane vesicle protein from pig was separated into five different molecular mass fractions by preparative SDS polyacrylamide disc gel electrophoresis. Separated protein fractions were used to immunize mice. Antibody was screened for reactivity with antigen by Western blotting, and for effects on conductive chloride transport in ileal brush border membrane vesicles. Immunization with brush-border protein from fraction I proteins (>110 kDa) produced polyclonal antisera which specifically inhibited the conductive component of chloride uptake by ileal brush border vesicle preparations. Western blotting of the antigen showed the presence of several protein species of molecular mass >100 kDa that were recognized by immune serum. Spleen cells from a mouse producing antiserum that inhibited conductive chloride transport were fused with a myeloma cell line. The resulting hybridoma colonies produced antibody that reacted with at least seven distinct protein bands by Western blot assay and inhibited chloride conductance in brush-border membrane vesicles.