The scaffolding protein EBP50 regulates microvillar assembly in a phosphorylation-dependent manner

The mechanisms by which epithelial cells regulate the presence of microvilli on their apical surface are largely unknown. A potential regulator is EBP50/NHERF1 (ERM-binding phosphoprotein of 50 kD/Na(+)-H(+) exchanger regulatory factor), a microvillar scaffolding protein with two PDZ domains followed by a C-terminal ezrin-binding domain. Using RNAi and expression of RNAi-resistant EBP50 mutants we systematically show that EBP50 is necessary for microvillar assembly and requires that EBP50 has both a functional first PDZ domain and an ezrin-binding site. Expression of mutants mimicking Cdc2 or PKC phosphorylation are nonfunctional in microvillar assembly. Biochemical analysis reveals that these mutants are defective in PDZ1 accessibility when PDZ2 is occupied, and can be rendered functional in vivo by additional mutation of PDZ2. EBP50 is not necessary for mitotic cell microvilli, and PKC activation causes a rearrangement of microvilli on cells due to phosphorylation-dependent loss of EBP50 function. Thus, EBP50 is a critical factor that regulates microvilli assembly and whose activity is regulated by signaling pathways and occupation of its PDZ2 domain.     

Opioid elevation of intracellular free calcium: possible mechanisms and physiological relevance

Opioid receptors are seven transmembrane domain Gi/G0 protein-coupled receptors, the activation of which stimulates a variety of intracellular signalling mechanisms including activation of inwardly rectifying potassium channels, and inhibition of both voltage-operated N-type Ca2+ channels and adenylyl cyclase activity. It is now apparent that like many other Gi/G0-coupled receptors, opioid receptor activation can significantly elevate intracellular free Ca2+ ([Ca2+]i), although the mechanism underlying this phenomenon is not well understood. In some cases opioid receptor activation alone appears to elevate [Ca2+]i, but in many cases it requires concomitant activation of Gq-coupled receptors, which themselves stimulate Ca2+ release from intracellular stores via the inositol phosphate pathway. Given the number of Ca2+-sensitive processes known to occur in cells, there are therefore a myriad of situations in which opioid receptor-mediated elevations of [Ca2+](i) may be important. Here, we review the literature documenting opioid receptor-mediated elevations of [Ca2+]i, discussing both the possible mechanisms underlying this phenomenon and its potential physiological relevance.     

High-activity radio-iodine labeling of conventional and stealth liposomes

A new method to label preformed liposomes with high activities of radiohalogenated compounds has been developed. It uses activated esters of simple synthetic molecules that may be readily halogenated, such as Bolton-Hunter reagent (BH), and arginine-containing liposomes. BH, in the form of an activated ester, crosses the liposome membrane to react with arginine inside the liposomes, as demonstrated by thin-layer chromatography and by the fact that saline-containing liposomes, or hydrolyzed BH or the water soluble sulfo-BH afforded only marginal encapsulation yields. Under optimized conditions, between 37 and 55 degrees C, 62 +/- 4% (mean +/- SD) of radiolabeled BH were consistently encapsulated in the liposomes within 30 min. In molar amounts, this corresponds to a mean of 56 nmol of BH per micromol of lipids. Based on achievable specific activity, up to 2.8 GBq of iodine-131 could be entrapped per micromol of lipids. Leakage of radioactivity was very low, with less than 5% of the encapsulated activity released within 6 days at 4 degrees C in phosphate-buffered saline and less than 50% within 24 h in human serum at 37 degrees C. The labeling stability, and the fact that both conventional and PEGylated liposomes can be readily labeled with high doses of radioactivity, will make this technique useful for in vivo targeting applications, such as tumor detection (using iodine-123 or iodine-124) or therapy (with iodine-131 or astatine-211).     

Translocation and Spread of Piscivorous Fishes in the Burdekin River, North-eastern Australia

The distribution of the biogeographically distinctive fish fauna of the Burdekin River, north-eastern Australia, is largely determined by the presence of a large waterfall located at the lower quarter of the river’s length. Downstream of the falls, assemblages are characterised by the presence of piscivorous fishes whereas such species are largely absent from upstream reaches. Sleepy cod (Oxyeleotris lineolatus), a large piscivorous gudgeon, was first introduced into the upper reaches of the Burdekin River in 1980 and other releases, both official and unofficial, have occurred subsequently. The population remained small and restricted to the site of introduction for a decade, but expanded in size and distribution after the occurrence of a large flood and entry into a prolonged period of drought. This gudgeon is now present in every tributary system of the Burdekin Basin. Despite the occurrence of substantial temporal variation in fish abundance due to a highly variable flow regime, negative impacts on one species, a small gudgeon (Mogurnda adspersa), are evident. Both deliberate and accidental releases of other species into the upper Burdekin River have also occurred, often to satisfy recreational fishing demand. Such species are typified by large size and piscivorous habit, characteristics alien and inimical to the native fish fauna. It is hypothesised that these piscivorous species may have even greater impact than O. lineolatus in some tributary systems of the upper Burdekin River.     

Analysis of hepatitis C virus RNA dimerization and core-RNA interactions

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3′-untranslated region (3′-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623–2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3′-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.     

Exposure of Mongolian gazelles (Procapra gutturosa) to foot and mouth disease virus

Foot and mouth disease is a highly contagious acute viral disease that affects most ruminant and porcine species. During 2001, 33 serum samples were collected from Mongolian gazelles (Procapra gutturosa) in the Eastern Steppe of Mongolia. Samples were tested for antibodies to seven subtypes of foot-and-mouth-disease virus (FMDV). Antibodies were detected in 67% of the animals, and serologic results indicated exposure to FMDV-O. This virus was present in domestic animal populations in Mongolia from 2000 to 2002, and it is likely that the antibodies to FMDV detected in these gazelles resulted from spillover of virus from domestic animal sources.     

EnP1 and EnP2, two proteins associated with the Encephalitozoon cuniculi endospore, the chitin-rich inner layer of the microsporidian spore wall

Microsporidia are obligate intracellular parasites forming environmentally resistant spores that harbour a rigid cell wall. This wall comprises an outer layer or exospore and a chitin-rich inner layer or endospore. So far, only a chitin deacetylase-like protein has been shown to localize to the Encephalitozoon cuniculi endospore and either one or two proteins have been clearly assigned to the exospore in two Encephalitozoon species: SWP1 in E. cuniculi, SWP1 and SWP2 in Encephalitozoon intestinalis. Here, we report the identification of two new spore wall proteins in E. cuniculi, EnP1 and EnP2, the genes of which are both located on chromosome I (ECU01_0820 and ECU01_1270, respectively) and have no known homologue. Detected by immunoscreening of an E. cuniculi cDNA library, enp1 is characterized by small-sized 5' and 3' untranslated regions and is highly expressed throughout the whole intracellular cycle. The encoded basic 40 kDa antigen displays a high proportion of cysteine residues, arguing for a significant role of disulfide bridges in spore wall assembly. EnP2 is a 22 kDa serine-rich protein that is predicted to be O-glycosylated and glycosylated phosphatidyl inositol-anchored. Although having been identified by mass spectrometry of a dithiothreitol-soluble fraction, this protein contains only two cysteine residues. Mouse polyclonal antibodies were raised against EnP1 and EnP2 recombinant proteins produced in Escherichia coli Our immunolocalisation data indicate that EnP1 and EnP2 are targeted to the cell surface as early as the onset of sporogony and are finally associated with the chitin-rich layer of the wall in mature spores.