The HIV-1 Integrase Mutations Y143C/R Are an Alternative Pathway for Resistance to Raltegravir and Impact the Enzyme Functions

Resistance to HIV-1 integrase (IN) inhibitor raltegravir (RAL), is encoded by mutations in the IN region of the pol gene. The emergence of the N155H mutation was replaced by a pattern including the Y143R/C/H mutations in three patients with anti-HIV treatment failure. Cloning analysis of the IN gene showed an independent selection of the mutations at loci 155 and 143. Characterization of the phenotypic evolution showed that the switch from N155H to Y143C/R was linked to an increase in resistance to RAL. Wild-type (WT) IN and IN with mutations Y143C or Y143R were assayed in vitro in 3′end-processing, strand transfer and concerted integration assays. Activities of mutants were moderately impaired for 3′end-processing and severely affected for strand transfer. Concerted integration assay demonstrated a decrease in mutant activities using an uncleaved substrate. With 3′end-processing assay, IC₅₀ were 0.4 µM, 0.9 µM (FC = 2.25) and 1.2 µM (FC = 3) for WT, IN Y143C and IN Y143R, respectively. An FC of 2 was observed only for IN Y143R in the strand transfer assay. In concerted integration, integrases were less sensitive to RAL than in ST or 3′P but mutants were more resistant to RAL than WT.     

CDK13, a Kinase Involved in Pre-mRNA Splicing,Is a Component of the Perinucleolar Compartment

The perinucleolar compartment (PNC) is a subnuclear stucture forming predominantly in cancer cells; its prevalence positively correlates with metastatic capacity. Although several RNA-binding proteins have been characterized in PNC, the molecular function of this compartment remains unclear. Here we demonstrate that the cyclin–dependent kinase 13 (CDK13) is a newly identified constituent of PNC. CDK13 is a kinase involved in the regulation of gene expression and whose overexpression was found to alter pre-mRNA processing. In this study we show that CDK13 is enriched in PNC and co-localizes all along the cell cycle with the PNC component PTB. In contrast, neither the cyclins K and L, known to associate with CDK13, nor the potential kinase substrates accumulate in PNC. We further show that CDK13 overexpression increases PNC prevalence suggesting that CDK13 may be determinant for PNC formation. This result linked to the finding that CDK13 gene is amplified in different types of cancer indicate that this kinase can contribute to cancer development in human.     

Genome plasticity in Lactococcus lactis

Comparative genome analyses contribute significantly to our understanding of bacterial evolution and indicate that bacterial genomes are constantly evolving structures. The gene content and organisation of chromosomes of lactic acid bacteria probably result from a strong evolutionary pressure toward optimal growth of these microorganisms in milk. The genome plasticity of Lactococcus lactis was evaluated at inter- and intrasubspecies levels by different experimental approaches. Comparative genomics showed that the lactococcal genomes are not highly plastic although large rearrangements (a.o. deletions, inversions) can occur. Experimental genome shuffling using a new genetic strategy based on the Cre-loxP recombination system revealed that two domains are under strong constraints acting to maintain the original chromosome organisation: a large region around the replication origin, and a smaller one around the putative terminus of replication. Future knowledge of the rules leading to an optimal genome organisation could facilitate the definition of new strategies for industrial strain improvement.     

Cre-loxP recombination system for large genome rearrangements in Lactococcus lactis

We have used a new genetic strategy based on the Cre-loxP recombination system to generate large chromosomal rearrangements in Lactococcus lactis. Two loxP sites were sequentially integrated in inverse order into the chromosome either at random locations by transposition or at fixed points by homologous recombination. The recombination between the two chromosomal loxP sites was highly efficient (approximately 1 x 10(-1)/cell) when the Cre recombinase was provided in trans, and parental- or inverted-type chromosomal structures were isolated after removal of the Cre recombinase. The usefulness of this approach was demonstrated by creating three large inversions of 500, 1,115, and 1,160 kb in size that modified the lactococcal genome organization to different extents. The Cre-loxP recombination system described can potentially be used for other gram-positive bacteria without further modification.     

Demodicosis in a mule deer (Odocoileus hemionus hemionus) from Saskatchewan, Canada

Infestation of deer with Demodex spp. mites has been described in white-tailed deer (Odocoileus virginianus) and in Columbian black-tailed deer (Odocoileus hemionus columbianus) in North America, as well as in four species of deer in Europe. We describe Demodex sp. infestation in an adult female mule deer (Odocoileus hemionus hemionus) with skin lesions found dead near Saskatoon, Saskatchewan, Canada. This is believed to be the first report of demodicosis in mule deer.     

Catalytic antibodies from HIV-infected patients specifically hydrolyzing viral integrase suppress the enzyme catalytic activities

Human immunodeficiency virus type 1 integrase (IN) catalyzes integration of a DNA copy of the viral genome into the host genome. It was shown previously that IN preincubation with various oligodeoxynucleotides (ODNs) induces formation of dimers and oligomers of different gyration radii; only specific ODNs stimulate the formation of catalytically active dimers. Here we have shown that preincubation of IN with specific and nonspecific ODNs leads to a significant and comparable decrease in its hydrolysis by chymotrypsin, while nonspecific ODNs protect the enzyme from the hydrolysis by trypsin worse than specific ODNs; all ODNs had little effect on the IN hydrolysis by proteinase K. In contrast to canonical proteweases, IgGs from HIV‐infected patients specifically hydrolyze only IN. While d(pT)_n markedly decreased the IgG‐dependent hydrolysis of IN, d(pA)_n and d(pA)_n?d(pT)_n demonstrated no detectable protective effect. The best protection from the hydrolysis by IgGs was observed for specific single‐ and especially double‐stranded ODNs. Although IN was considerably protected by specific ODNs, proteolytic IgGs and IgMs significantly suppressed both 3′‐processing and integration reaction catalyzed by IN. Since anti‐IN IgGs and IgMs can efficiently hydrolyze IN, a positive role of abzymes in counteracting the infection cannot be excluded. Copyright © 2011 John Wiley & Sons, Ltd.     

Acquisition and maintenance of resistance to viruses in eukaryotic phytoplankton populations

Viruses are known to play a key role in the regulation of eukaryotic phytoplankton population densities; however, little is known about the mechanisms of how they interact with their hosts and how phytoplankton populations mediate their regulations. Viruses are obligate parasites that depend on host cell machinery for their dissemination in the environment (most of the time through host cell lysis that liberates many new particles). But viruses also depend on a reliable host population to carry on their replication before losing their viability. How do hosts cells survive when they coexist with their viruses? We show that clonal lines of three picoeukaryotic green algae (i.e. Bathycoccus sp., Micromonas sp., Ostreococcus tauri) reproducibly acquire resistance to their specific viruses following a round of infection. Our observations show that two mechanisms of resistance may operate in O. tauri. In the first resistant type, viruses can attach to their host cells but no new particles develop. In the second one, O. tauri acquires tolerance to its virus and releases these viruses consistently. These lines maintained their resistance over a 3‐year period, irrespective of whether or not they were re‐challenged with new viral inoculations. Co‐culturing resistant and susceptible lines revealed resistance to be associated with reduced host fitness in terms of growth rate.     

Regulation of lipolytic activity by long-chain acyl-coenzyme A in islets and adipocytes

Intracellular lipolysis is a major pathway of lipid metabolism that has roles, not only in the provision of free fatty acids as energy substrate, but also in intracellular signal transduction. The latter is likely to be particularly important in the regulation of insulin secretion from islet beta-cells. The mechanisms by which lipolysis is regulated in different tissues is, therefore, of considerable interest. Here, the effects of long-chain acyl-CoA esters (LC-CoA) on lipase activity in islets and adipocytes were compared. Palmitoyl-CoA (Pal-CoA, 1-10 microM) stimulated lipase activity in islets from both normal and hormone-sensitive lipase (HSL)-null mice and in phosphatase-treated islets, indicating that the stimulatory effect was neither on HSL nor phosphorylation dependent. In contrast, we reproduced the previously published observations showing inhibition of HSL activity by LC-CoA in adipocytes. The inhibitory effect of LC-CoA on adipocyte HSL was dependent on phosphorylation and enhanced by acyl-CoA-binding protein (ACBP). In contrast, the stimulatory effect on islet lipase activity was blocked by ACBP, presumably due to binding and sequestration of LC-CoA. These data suggest the following intertissue relationship between islets and adipocytes with respect to fatty acid metabolism, LC-CoA signaling, and lipolysis. Elevated LC-CoA in islets stimulates lipolysis to generate a signal to increase insulin secretion, whereas elevated LC-CoA in adipocytes inhibits lipolysis. Together, these opposite actions of LC-CoA lower circulating fat by inhibiting its release from adipocytes and promoting fat storage via insulin action.