High similarity between flanking regions of different microsatellites detected within each of two species of Lepidoptera: Parnassius apollo and Euphydryas aurinia

Microsatellite flanking regions have been compared in two butterfly species. Several microsatellite flanking regions showed high similarity to one another among different microsatellites within a same species, but very few similarities were found between species. This can be the consequence of either duplication/multiplication events involving large regions containing microsatellites or of microsatellites imbedded in minisatellite regions. The multiplication of microsatellites might also be linked to mobile elements. Furthermore, crossing over between nonhomologous microsatellites can lead to the exchange of the flanking regions between microsatellites. The same phenomenon was observed in both studied butterfly species but not in Aphis fabae (Hemiptera), which was screened at the same time using the same protocol. These findings might explain, at least partially, why microsatellite isolation in Lepidoptera has been relatively unsuccessful so far.     

Construction of avian adenovirus CELO recombinants in cosmids

The avian adenovirus CELO is a promising vector for gene transfer applications. In order to study this potentiality, we developed an improved method for construction of adenovirus vectors in cosmids that was used to engineer the CELO genome. For all the recombinant viruses constructed by this method, the ability to produce infectious particles and the stability of the genome were evaluated in a chicken hepatocarcinoma cell line (LMH cell line). Our aim was to develop a replication-competent vector for vaccination of chickens, so we first generated knockout point mutations into 16 of the 22 unassigned CELO open reading frames (ORFs) to determine if they were essential for virus replication. As the 16 independent mutant viruses replicated in our cellular system, we constructed CELO genomes with various deletions in the regions of these nonessential ORFs. An expression cassette coding for the enhanced green fluorescent protein (eGFP) was inserted in place of these deletions to easily follow expression of the transgene and propagation of the vector in cell monolayers. Height-distinct GFP-expressing CELO vectors were produced that were all replication competent in our system. We then retained the vector backbone with the largest deletion (i.e., 3.6 kb) for the construction of vectors carrying cDNA encoding infectious bursal disease virus proteins. These CELO vectors could be useful for vaccination in the chicken species.     

Ammonium transport by the colonic H+-K+-ATPase expressed in Xenopus oocytes

Functional expression of the rat colonicH⁺-K⁺-ATPasewas obtained by coexpressing its catalytic -subunit and the₁-subunit of theNa⁺-K⁺-ATPasein Xenopus laevis oocytes. We observedthat, in oocytes expressing the rat colonicH⁺-K⁺-ATPasebut not in control oocytes (expressing₁ alone),NH₄Cl induced a decrease in⁸⁶Rb uptake and the initial rateof intracellular acidification induced by extracellularNH₄Cl was enhanced, consistentwith NH⁺₄ influx via the colonicH⁺-K⁺-ATPase.In the absence of extracellularK⁺, only oocytes expressing thecolonicH⁺-K⁺-ATPasewere able to acidify an extracellular medium supplemented withNH₄Cl. In the absence ofextracellular K⁺ and in thepresence of extracellular NH⁺₄, intracellular Na⁺ activity in oocytes expressingthe colonicH⁺-K⁺-ATPasewas lower than that in control oocytes. A kinetic analysis of⁸⁶Rb uptake suggests thatNH⁺₄ acts as a competitive inhibitor of thepump. Taken together, these results are consistent withNH⁺₄ competition forK⁺ on the external site of thecolonicH⁺-K⁺-ATPaseand with NH⁺₄ transport mediated by this pump.

     

The virotoxin model of HIV-1 enteropathy: Involvement of GPR15/Bob and galactosylceramide in the cytopathic effects induced by HIV-1 gp120 in the HT-29-D4 intestinal cell line

BACKGROUND: Malabsorption and diarrhea are common, serious problems in AIDS patients, and are in part due to the incompletely understood entity HIV enteropathy. Our prior in vitro work has shown that increased transepithelial permeability and glucose malabsorption, similar to HIV enteropathy, are caused by HIV surface protein gp120, although the mechanism remains unclear. RESULTS: We studied the effects of HIV surface protein gp120 on the differentiated intestinal cell line HT-29-D4, specifically the effects on microtubules, transepithelial resistance, and sodium glucose cotransport. gp120 induced extensive microtubule depolymerization, an 80% decrease in transepithelial resistance, and a 70% decrease in sodium-dependent glucose transport, changes closely paralleling those of HIV enteropathy. The effects on transepithelial resistance were used to study potential inhibitors. Neutralizing antibodies to GPR15/Bob but not to CXCR4 (the coreceptor allowing infection with these HIV strains) inhibited these effects. Antibodies to galactosylceramide (GalCer) and a synthetic analog of GalCer also inhibited the gp120-induced changes, suggesting the involvement of GalCer-enriched lipid rafts in gp120 binding to intestinal epithelial cells. CONCLUSION: We conclude that direct HIV infection and gp120-induced cytopathic effects are distinct phenomena. While in vivo confirmation is needed to prove this, gp120 could be a virotoxin significantly contributing to HIV enteropathy.     

HIV-1 Tat reprograms immature dendritic cells to express chemoattractants for activated T cells and macrophages

Immature dendritic cells are among the first cells infected by retroviruses after mucosal exposure. We explored the effects of human immunodeficiency virus-1 (HIV-1) and its Tat transactivator on these primary antigen-presenting cells using DNA microarray analysis and functional assays. We found that HIV-1 infection or Tat expression induces interferon (IFN)-responsive gene expression in immature human dendritic cells without inducing maturation. Among the induced gene products are chemokines that recruit activated T cells and macrophages, the ultimate target cells for the virus. Dendritic cells in the lymph nodes of macaques infected with simian immunodeficiency virus (SIV) have elevated levels of monocyte chemoattractant protein 2 (MCP-2), demonstrating that chemokine induction also occurs during retroviral infection in vivo. These results show that HIV-1 Tat reprograms host dendritic cell gene expression to facilitate expansion of HIV-1 infection.     

Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells

The improvement of non-viral-based gene delivery systems is of prime importance for the future of gene and antisense therapies. We have previously described a peptide-based gene delivery system, MPG, derived from the fusion peptide domain of HIV-1 gp41 protein and the nuclear localisation sequence (NLS) of SV40 large T antigen. MPG forms stable non-covalent complexes with nucleic acids and improves their delivery. In the present work, we have investigated the mechanism through which MPG promotes gene delivery. We demonstrate that cell entry is independent of the endosomal pathway and that the NLS of MPG is involved in both electrostatic interactions with DNA and nuclear targeting. MPG/DNA particles interact with the nuclear import machinery, however, a mutation which affects the NLS of MPG disrupts these interactions and prevents nuclear delivery of DNA. Nevertheless, we show that this mutation yields a variant of MPG which is a powerful tool for delivery of siRNA into mammalian cells, enabling rapid release of the siRNA into the cytoplasm and promoting robust down-regulation of target mRNA. Taken together, these results support the potential of MPG-like peptides for therapeutic applications and suggest that specific variations in the sequence may yield carriers with distinct targeting features.     

Resveratrol-induced apoptosis is associated with Fas redistribution in the rafts and the formation of a death-inducing signaling complex in colon cancer cells

Resveratrol, a polyphenol found in grape skin and various other food products, may function as a cancer chemopreventive agent for colon and other malignant tumors and possesses a chemotherapeutic potential through its ability to trigger apoptosis in tumor cells. The present study analyses the molecular mechanisms of resveratrol-induced apoptosis in colon cancer cells, with special attention to the role of the death receptor Fas in this pathway. We show that, in the 10-100 microm range of concentrations, resveratrol activates various caspases and triggers apoptosis in SW480 human colon cancer cells. Caspase activation is associated with accumulation of the pro-apoptotic proteins Bax and Bak that undergo conformational changes and relocalization to the mitochondria. Resveratrol does not modulate the expression of Fas and Fas-ligand (FasL) at the surface of cancer cells, and inhibition of the Fas/FasL interaction does not influence the apoptotic response to the molecule. Resveratrol induces the clustering of Fas and its redistribution in cholesterol and sphingolipid-rich fractions of SW480 cells, together with FADD and procaspase-8. This redistribution is associated with the formation of a death-inducing signaling complex (DISC). Transient transfection of either a dominant-negative mutant of FADD, E8, or MC159 viral proteins that interfere with the DISC function, decreases the apoptotic response of SW480 cells to resveratrol and partially prevents resveratrol-induced Bax and Bak conformational changes. Altogether, these results indicate that the ability of resveratrol to induce the redistribution of Fas receptor in membrane rafts may contribute to the molecule's ability to trigger apoptosis in colon cancer cells.