A retrospective overview of enterovirus infection diagnosis and molecular epidemiology in the public hospitals of Marseille, France (1985-2005)

Human enteroviruses (HEV) are frequent human pathogens and, associated in particular with large outbreaks of aseptic meningitis. Here, we have compiled a database of clinical HEV isolates from the Public Hospitals of Marseille, from 1985 to 2005. Amongst 654 isolates that could be characterized by complete sequencing of the VP1 gene, 98% belonged to species HEV-B; the most frequently isolated serotypes were Echovirus E30, E11, E7, E6 and E4. The high incidence of E30 and the recent emergence of E13 are consistent with reports worldwide and peak HEV isolation occurred mostly in the late spring and summer months. The proportion of echoviruses has decreased across the years, while that of coxsackieviruses has increased. Stool (the most frequent sample type) allowed detection of all identified serotypes. MRC5 (Human lung fibroblasts) cell line was the most conducive cell line for HEV isolation (84.9% of 10 most common serotype isolates, 96.3% in association with BGM (Buffalo green monkey kidney cells)). Previous seroneutralization-based serotype identification demonstrated 55.4% accuracy when compared with molecular VP1 analysis. Our analysis of a large number of clinical strains over 20 years reinforced the validity of VP1 serotyping and showed that comparative p-distance scores can be coupled with phylogenetic analysis to provide non-ambiguous serotype identification. Phylogenetic analysis in the VP1, 2C and 3D regions also provided evidence for recombination events amongst clinical isolates. In particular, it identified isolates with dissimilar VP1 but almost identical nonstructural regions.     

Host specificity and population dynamics of a sponge-endosymbiotic bivalve

We assessed the host-use pattern of the sponge-endosymbiotic bivalve Vulsella vulsella and its demographic consequences in an inland sea in Okinawa Island, Japan. Vulsella vulsella utilized only one massive globular sponge species Spongia sp. as a host, and no Spongia sp. without V. vulsella were found. Individual sponges contained 9-248 live bivalves and 0-222 dead bivalves. The densities of live and dead bivalves in individual sponges were approximately constant irrespective of sponge size, indicating that available space is very scarce inside each sponge. The size distribution of bivalves was skewed to small, young individuals less than 30 mm in shell height, although the estimated largest possible size was 106 mm. The bivalve population at each sampling date was composed of three yearly cohorts, and recruitment of juveniles occurred in the summer. The bivalves became sexually mature as males within one year after recruitment and changed sex from male to female as they grew. The size and sex distributions of the bivalve were largely similar among sponges regardless of sponge size, suggesting that the recruitment, growth, longevity, and sex change of the bivalve were strictly regulated, probably by the high water temperature and strong waves generated by typhoons in summer months.     

Aminothiazole derivatives with antidegenerative activity on cartilage

A series of 2-dialkylamino-N-(4-substituted thiazolyl-2)acetamides and 3-dialkylamino-N-(4-substituted thiazolyl-2)propionamides were synthesized and evaluated for their anti-inflammatory activity. Encouraging results led us to investigate the effect of these compounds on NO production and GAGs release. Their effects were evaluated in vitro on the metabolism of pig cartilage, treated with IL-1beta. The amount of glycosaminoglycans (GAGs) and the production of nitric oxide (NO) in the culture medium were determined. The results, obtained, showed that all compounds, in the presence of IL-1beta, blocked the cartilage breakdown, with different behavior. A quantitative structure-activity relationship (QSAR) study was performed.     

RefZ Facilitates the Switch from Medial to Polar Division during Spore Formation in Bacillus subtilis

During sporulation, Bacillus subtilis redeploys the division protein FtsZ from midcell to the cell poles, ultimately generating an asymmetric septum. Here, we describe a sporulation-induced protein, RefZ, that facilitates the switch from a medial to a polar FtsZ ring placement. The artificial expression of RefZ during vegetative growth converts FtsZ rings into FtsZ spirals, arcs, and foci, leading to filamentation and lysis. Mutations in FtsZ specifically suppress RefZ-dependent division inhibition, suggesting that RefZ may target FtsZ. During sporulation, cells lacking RefZ are delayed in polar FtsZ ring formation, spending more time in the medial and transition stages of FtsZ ring assembly. A RefZ-green fluorescent protein (GFP) fusion localizes in weak polar foci at the onset of sporulation and as a brighter midcell focus at the time of polar division. RefZ has a TetR DNA binding motif, and point mutations in the putative recognition helix disrupt focus formation and abrogate cell division inhibition. Finally, chromatin immunoprecipitation assays identified sites of RefZ enrichment in the origin region and near the terminus. Collectively, these data support a model in which RefZ helps promote the switch from medial to polar division and is guided by the organization of the chromosome. Models in which RefZ acts as an activator of FtsZ ring assembly near the cell poles or as an inhibitor of the transient medial ring at midcell are discussed.     

The dynamics of replication licensing in live Caenorhabditis elegans embryos

Accurate DNA replication requires proper regulation of replication licensing, which entails loading MCM-2-7 onto replication origins. In this paper, we provide the first comprehensive view of replication licensing in vivo, using video microscopy of Caenorhabditis elegans embryos. As expected, MCM-2-7 loading in late M phase depended on the prereplicative complex (pre-RC) proteins: origin recognition complex (ORC), CDC-6, and CDT-1. However, many features we observed have not been described before: GFP-ORC-1 bound chromatin independently of ORC-2-5, and CDC-6 bound chromatin independently of ORC, whereas CDT-1 and MCM-2-7 DNA binding was interdependent. MCM-3 chromatin loading was irreversible, but CDC-6 and ORC turned over rapidly, consistent with ORC/CDC-6 loading multiple MCM-2-7 complexes. MCM-2-7 chromatin loading further reduced ORC and CDC-6 DNA binding. This dynamic behavior creates a feedback loop allowing ORC/CDC-6 to repeatedly load MCM-2-7 and distribute licensed origins along chromosomal DNA. During S phase, ORC and CDC-6 were excluded from nuclei, and DNA was overreplicated in export-defective cells. Thus, nucleocytoplasmic compartmentalization of licensing factors ensures that DNA replication occurs only once.     

A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C

CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.     

The spinal basis of opioid tolerance and physical dependence: Involvement of calcitonin gene-related peptide, substance P, and arachidonic acid-derived metabolites

Chronic opioid use in the management of pain is limited by development of analgesic tolerance and physical dependence. The mechanisms underlying tolerance-dependence are not entirely clear, however, recent evidence suggests that spinal adaptations leading to increased activity of sensory neuropeptides (calcitonin gene-related peptide (CGRP), substance P) and their downstream signaling messengers derived from metabolism of arachidonic acid: prostaglandins (PG), lipoxygenase (LOX) metabolites, and endocannabinoids, plays an important role in this phenomenon. In this communication we review the evidence implicating these factors in the induction and expression of opioid tolerance and physical dependence at the spinal level.     

Threonine 308 within a putative casein kinase 2 site of the cytoplasmic tail of leukotriene B(4) receptor (BLT1) is crucial for ligand-induced,G-protein-coupled receptor-specific kinase 6-mediated desensitization

Desensitization of G-protein-coupled receptors may involve phosphorylation of serine and threonine residues. The leukotriene B(4) (LTB(4)) receptor (BLT1) contains 14 intracellular serines and threonines, 8 of which are part of consensus target sequences for protein kinase C (PKC) or casein kinase 2. In this study, we investigated the importance of PKC and GPCR-specific kinase (GRK) phosphorylation in BLT1 desensitization. Pretreatment of BLT1-transfected COS-7 cells with PKC activators caused a decrease of LTB(4)-induced inositol phosphate (IP) accumulation. This reduction was prevented with the PKC inhibitor, staurosporine, and not observed in cells expressing a BLT1 deletion mutant (G291stop) lacking the cytoplasmic tail. Moreover LTB(4)-induced IP accumulation was significantly inhibited by overexpression of GRK2, GRK5, and especially GRK6, in cells expressing wild type BLT1 but not in those expressing G291stop. GRK6-mediated desensitization correlated with increased phosphorylation of BLT1. The G319stop truncated BLT1 mutant displayed functional characteristics comparable with wild type BLT1 in terms of desensitization by GRK6, but not by PKC. Substitution of Thr(308) within a putative casein kinase 2 site to proline or alanine in the full-length BLT1 receptor prevented most of GRK6-mediated inhibition of LTB(4)-induced IP production but only partially affected LTB(4)-induced BLT1 phosphorylation. Our findings thus suggest that Thr(308) is a major residue involved in GRK6-mediated desensitization of BLT1 signaling.