Nucleic Acids Res

The HIV-1 viral infectivity factor (Vif) is required for productive infection of non-permissive cells, including most natural HIV-1 targets, where it counteracts the antiviral activities of the cellular cytosine deaminases APOBEC-3G (A3G) and A3F. Vif is a multimeric protein and the conserved proline-rich domain (161)PPLP(164) regulating Vif oligomerization is crucial for its function and viral infectivity. Here, we expressed and purified wild-type Vif and a mutant protein in which alanines were substituted for the proline residues of the (161)PPLP(164) domain. Using dynamic light scattering, circular dichroism and fluorescence spectroscopy, we established the impact of these mutations on Vif oligomerization, secondary structure content and nucleic acids binding properties. In vitro, wild-type Vif formed oligomers of five to nine proteins, while Vif AALA formed dimers and/or trimers. Up to 40% of the unbound wild-type Vif protein appeared to be unfolded, but binding to the HIV-1 TAR apical loop promoted formation of beta-sheets. Interestingly, alanine substitutions did not significantly affect the secondary structure of Vif, but they diminished its binding affinity and specificity for nucleic acids. Dynamic light scattering showed that Vif oligomerization, and interaction with folding-promoting nucleic acids, favor formation of high molecular mass complexes. These properties could be important for Vif functions involving RNAs

Aquaporin gene expression and apoplastic water flow in bur oak (Quercus macrocarpa) leaves in relation to the light response of leaf hydraulic conductance

It has previously been shown that hydraulic conductance in bur oak leaves (Quercus macrocarpa Michx.), measured with the high pressure flow meter technique (HPFM), can significantly increase within 30 min following exposure to high irradiance. The present study investigated whether this increase could be explained by an increase in the cell-to-cell pathway and whether the response is linked to changes in the transcript level corresponding to aquaporin genes. Four cDNA sequences showing high similarity to members of the aquaporin gene family from other plant species were characterized from bur oak leaves and the expression levels of these cDNA sequences were examined in leaves by quantitative real-time PCR (QRT-PCR). No change was found in the relative transcript abundance corresponding to these four putative aquaporin genes in leaves with light-induced high hydraulic conductance (exposed to high irradiance) compared to leaves with low hydraulic conductance (exposed to low irradiance). However, in sun leaves that were exposed to different light levels prior to leaf collection (full sunlight, shade, and covered with aluminium foil for 16 h), the relative transcript levels of two of the putative aquaporin genes increased several-fold in shaded leaves compared to the sun-exposed or covered leaves. When the leaves were pressure-infiltrated with the apoplastic tracer dye trisodium 3-hydroxy-5,8,10-pyrenetrisulphonate (PTS3, 0.02%), there was no change in the PTS3 concentration of leaf exudates collected in ambient light or in high irradiance, but there was a small apoplastic acidification. There was also no change in PTS3 concentration between the leaves infiltrated under high irradiance with 0.02% PTS3 or with 0.1 mM HgCl2 in 0.02% PTS3. The results suggest that the putative aquaporin genes that were identified in the present study probably do not play a role in the light responses of hydraulic conductance at the transcript level, but they may function in regulating water homeostasis in leaves adapted to different light conditions. In addition, it is shown that high irradiance induced changes in the pH of the apoplast and that there does not appear to be a significant shift to the cell-to-cell mediated water transport in bur oak leaves exposed to high irradiance as measured by the apoplastic tracer dye

Social and cultural dimensions of hygiene in Cambodian health care facilities

<p>Abstract</p> <p>Background</p> <p>The frequency of bloodborne pathogen healthcare-associated infections is thought to be high in developing Southeast Asian Countries. The underlying social-cultural logics contributing to the risks of transmission are rarely studied. This report provides some insights on the social and cultural factors that shape hygiene practices in Cambodian health care settings.</p> <p>Methods</p> <p>We conducted qualitative surveys in various public and private health facilities in Phnom Penh, the capital city and in provinces. We observed and interviewed 319 participants, health care workers and patients, regarding hygiene practices and social relationships amongst the health care staff and with patients. We also examined the local perceptions of hygiene, their impact on the relationships between the health care staff and patients, and perceptions of transmission risks. Data collection stem from face to face semi-structured and open-ended interviews and focus group discussions with various health care staffs (i.e. cleaners, nurses, midwives and medical doctors) and with patients who attended the study health facilities.</p> <p>Results</p> <p>Overall responses and observations indicated that hygiene practices were burdened by the lack of adequate materials and equipements. In addition, many other factors were identified to influence and distort hygiene practices which include (1) informal and formal social rapports in hospitals, (2) major infection control roles played by the cleaners in absence of professional acknowledgment. Moreover, hygiene practices are commonly seen as an unessential matter to be devoted to low-ranking staff.</p> <p>Conclusion</p> <p>Our anthropological findings illustrate the importance of comprehensive understanding of hygiene practices; they need to be considered when designing interventions to improve infection control practices in a Cambodian medical setting.</p

A peptide corresponding to the neuropilin-1-binding site on VEGF165 induces apoptosis of neuropilin-1-expressing breast tumour cells

There is increasing evidence that vascular endothelial growth factor (VEGF) has autocrine as well as paracrine functions in tumour biology. Vascular endothelial growth factor-mediated cell survival signalling occurs via the classical tyrosine kinase receptors Flt-1, KDR/Flk-1 and the more novel neuropilin (NP) receptors, NP-1 and NP-2. A 24-mer peptide, which binds to neuropilin-1, induced apoptosis of murine and human breast carcinoma cells, whereas a peptide directed against KDR had no effect. Both anti-NP1 and anti-KDR peptides induced endothelial cell apoptosis. Confocal microscopy using 5-(6)-carboxyfluorescein-labelled peptides showed that anti-NP1 bound to both tumour and endothelial cells, whereas anti-KDR bound endothelial cells only. This study demonstrates that NP-1 plays an essential role in autocrine antiapoptotic signalling by VEGF in tumour cells and that NP1-blockade induces tumour cell and endothelial cell apoptosis. Specific peptides can therefore be used to target both autocrine (tumour cells) and paracrine (endothelial cells) signalling by VEGF

HSPG-Binding Peptide Corresponding to the Exon 6a-Encoded Domain of VEGF Inhibits Tumor Growth by Blocking Angiogenesis in Murine Model

Vascular endothelial growth factor VEGF165 is a critical element for development of the vascular system in physiological and pathological angiogenesis. VEGF isoforms have different affinities for heparan sulphate proteoglycan (HSPG) as well as for VEGF receptors; HSPGs are important regulators in vascular development. Therefore, inhibition of interactions between VEGF and HSPGs may prevent angiogenesis. Here, we demonstrate that an HSPG-binding synthetic peptide, corresponding to exon 6a-encoded domain of VEGF gene, has anti-angiogenic property. This 20 amino acids synthetic peptide prevents VEGF165 binding to several different cell types, mouse embryonic sections and inhibits endothelial cell migration, despite its absence in VEGF165 sequence. Our in vivo anti-tumor studies show that the peptide inhibits tumor growth in both mouse Lewis-Lung Carcinoma and human Liposarcoma tumor-bearing animal models. This is the first evidence that a synthetic VEGF fragment corresponding to exon 6a has functional antagonism both in vitro and in vivo. We conclude that the above HPSG binding peptide (6a-P) is a potent inhibitor of angiogenesis-dependent diseases

Developmental pattern of aquaporin expression in barley (Hordeum vulgare L.) leaves

Aquaporins are multifunctional membrane channels which belong to the family of major intrinsic proteins (MIPs) and are best known for their ability to facilitate the movement of water. In the present study, earlier results from microarray experiments were followed up. These experiments had suggested that, in barley (Hordeum vulgare L.), aquaporin family members are expressed in distinct patterns during leaf development. Real-time PCR and in situ hybridization were used to analyse the level and tissue-distribution of expression of candidate aquaporins, focusing on plasma membrane and tonoplast intrinsic proteins (PIPs, TIPs). Water channel function of seven aquaporins, whose transcripts were the most abundant and the most variable, was tested through expression in yeast and, in part, through expression in oocytes. All PIP1 and PIP2 subfamily members changed in expression during leaf development, with expression being much higher or lower in growing compared with mature tissue. The same applied to those TIPs which were expressed at detectable levels. Specific roles during leaf development are proposed for particular aquaporins

Caveolin-1 Plays a Crucial Role in Inhibiting Neuronal Differentiation of Neural Stem/Progenitor Cells via VEGF Signaling-Dependent Pathway

In the present study, we aim to elucidate the roles of caveolin-1(Cav-1), a 22 kDa protein in plasma membrane invaginations, in modulating neuronal differentiation of neural progenitor cells (NPCs). In the hippocampal dentate gyrus, we found that Cav-1 knockout mice revealed remarkably higher levels of vascular endothelial growth factor (VEGF) and the more abundant formation of newborn neurons than wild type mice. We then studied the potential mechanisms of Cav-1 in modulating VEGF signaling and neuronal differentiation in isolated cultured NPCs under normoxic and hypoxic conditions. Hypoxic embryonic rat NPCs were exposed to 1% O2 for 24 h and then switched to 21% O2 for 1, 3, 7 and 14 days whereas normoxic NPCs were continuously cultured with 21% O2. Compared with normoxic NPCs, hypoxic NPCs had down-regulated expression of Cav-1 and up-regulated VEGF expression and p44/42MAPK phosphorylation, and enhanced neuronal differentiation. We further studied the roles of Cav-1 in inhibiting neuronal differentiation by using Cav-1 scaffolding domain peptide and Cav-1-specific small interfering RNA. In both normoxic and hypoxic NPCs, Cav-1 peptide markedly down-regulated the expressions of VEGF and flk1, decreased the phosphorylations of p44/42MAPK, Akt and Stat3, and inhibited neuronal differentiation, whereas the knockdown of Cav-1 promoted the expression of VEGF, phosphorylations of p44/42MAPK, Akt and Stat3, and stimulated neuronal differentiation. Moreover, the enhanced phosphorylations of p44/42MAPK, Akt and Stat3, and neuronal differentiation were abolished by co-treatment of VEGF inhibitor V1. These results provide strong evidence to prove that Cav-1 can inhibit neuronal differentiation via down-regulations of VEGF, p44/42MAPK, Akt and Stat3 signaling pathways, and that VEGF signaling is a crucial target of Cav-1. The hypoxia-induced down-regulation of Cav-1 contributes to enhanced neuronal differentiation in NPCs

Algal MIPs, high diversity and conserved motifs

<p>Abstract</p> <p>Background</p> <p>Major intrinsic proteins (MIPs) also named aquaporins form channels facilitating the passive transport of water and other small polar molecules across membranes. MIPs are particularly abundant and diverse in terrestrial plants but little is known about their evolutionary history. In an attempt to investigate the origin of the plant MIP subfamilies, genomes of chlorophyte algae, the sister group of charophyte algae and land plants, were searched for MIP encoding genes.</p> <p>Results</p> <p>A total of 22 MIPs were identified in the nine analysed genomes and phylogenetic analyses classified them into seven subfamilies. Two of these, Plasma membrane Intrinsic Proteins (PIPs) and GlpF-like Intrinsic Proteins (GIPs), are also present in land plants and divergence dating support a common origin of these algal and land plant MIPs, predating the evolution of terrestrial plants. The subfamilies unique to algae were named MIPA to MIPE to facilitate the use of a common nomenclature for plant MIPs reflecting phylogenetically stable groups. All of the investigated genomes contained at least one <it>MIP </it>gene but only a few species encoded MIPs belonging to more than one subfamily.</p> <p>Conclusions</p> <p>Our results suggest that at least two of the seven subfamilies found in land plants were present already in an algal ancestor. The total variation of MIPs and the number of different subfamilies in chlorophyte algae is likely to be even higher than that found in land plants. Our analyses indicate that genetic exchanges between several of the algal subfamilies have occurred. The PIP1 and PIP2 groups and the Ca²⁺gating appear to be specific to land plants whereas the pH gating is a more ancient characteristic shared by all PIPs. Further studies are needed to discern the function of the algal specific subfamilies MIPA-E and to fully understand the evolutionary relationship of algal and terrestrial plant MIPs.</p

Exploring the three PIPs and three TIPs of grapevine for transport of water and atypical substrates through heterologous expression in aqy-null yeast

Aquaporins are membrane channels that facilitate the transport of water and other small molecules across the cellular membranes. We examined the role of six aquaporins of Vitis vinifera (cv. Touriga nacional) in the transport of water and atypical substrates (other than water) in an aqy-null strain of Saccharomyces cerevisiae. Their functional characterization for water transport was performed by stopped-flow fluorescence spectroscopy. The evaluation of permeability coefficients (Pf) and activation energies (Ea) revealed that three aquaporins (VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2) are functional for water transport, while the other three (VvTnPIP1;4, VvTnPIP2;3 and VvTnTIP4;1) are non-functional. TIPs (VvTnTIP1;1 and VvTnTIP2;2) exhibited higher water permeability than VvTnPIP2;1. All functional aquaporins were found to be sensitive to HgCl2, since their water conductivity was reduced (24–38%) by the addition of 0.5 mM HgCl2. Expression of Vitis aquaporins caused different sensitive phenotypes to yeast strains when grown under hyperosmotic stress generated by KCl or sorbitol. Our results also indicate that Vitis aquaporins are putative transporters of other small molecules of physiological importance. Their sequence analyses revealed the presence of signature sequences for transport of ammonia, boron, CO2, H2O2 and urea. The phenotypic growth variations of yeast cells showed that heterologous expression of Vitis aquaporins increased susceptibility to externally applied boron and H2O2, suggesting the contribution of Vitis aquaporins in the transport of these speciesinfo:eu-repo/semantics/publishedVersio

Viruses Associated with Ovarian Degeneration in Apis mellifera L. Queens

Queen fecundity is a critical issue for the health of honeybee (Apis mellifera L.) colonies, as she is the only reproductive female in the colony and responsible for the constant renewal of the worker bee population. Any factor affecting the queen's fecundity will stagnate colony development, increasing its susceptibility to opportunistic pathogens. We discovered a pathology affecting the ovaries, characterized by a yellow discoloration concentrated in the apex of the ovaries resulting from degenerative lesions in the follicles. In extreme cases, marked by intense discoloration, the majority of the ovarioles were affected and these cases were universally associated with egg-laying deficiencies in the queens. Microscopic examination of the degenerated follicles showed extensive paracrystal lattices of 30 nm icosahedral viral particles. A cDNA library from degenerated ovaries contained a high frequency of deformed wing virus (DWV) and Varroa destructor virus 1 (VDV-1) sequences, two common and closely related honeybee Iflaviruses. These could also be identified by in situ hybridization in various parts of the ovary. A large-scale survey for 10 distinct honeybee viruses showed that DWV and VDV-1 were by far the most prevalent honeybee viruses in queen populations, with distinctly higher prevalence in mated queens (100% and 67%, respectively for DWV and VDV-1) than in virgin queens (37% and 0%, respectively). Since very high viral titres could be recorded in the ovaries and abdomens of both functional and deficient queens, no significant correlation could be made between viral titre and ovarian degeneration or egg-laying deficiency among the wider population of queens. Although our data suggest that DWV and VDV-1 have a role in extreme cases of ovarian degeneration, infection of the ovaries by these viruses does not necessarily result in ovarian degeneration, even at high titres, and additional factors are likely to be involved in this pathology