Regulatory domain selectivity in the cell-type specific PKN-dependence of cell migration

The mammalian protein kinase N (PKN) family of Serine/Threonine kinases comprises three isoforms, which are targets for Rho family GTPases. Small GTPases are major regulators of the cellular cytoskeleton, generating interest in the role(s) of specific PKN isoforms in processes such as cell migration and invasion. It has been reported that PKN3 is required for prostate tumour cell invasion but not PKN1 or 2. Here we employ a cell model, the 5637 bladder tumour cell line where PKN2 is relatively highly expressed, to assess the potential redundancy of these isoforms in migratory responses. It is established that PKN2 has a critical role in the migration and invasion of these cells. Furthermore, using a PKN wild-type and chimera rescue strategy, it is shown that PKN isoforms are not simply redundant in supporting migration, but appear to be linked through isoform specific regulatory domain properties to selective upstream signals. It is concluded that intervention in PKNs may need to be directed at multiple isoforms to be effective in different cell types.     

Efficacy of genetically modified Bt toxins against insects with different genetic mechanisms of resistance

Transgenic crops that produce Bacillus thuringiensis (Bt) toxins are grown widely for pest control, but insect adaptation can reduce their efficacy. The genetically modified Bt toxins Cry1AbMod and Cry1AcMod were designed to counter insect resistance to native Bt toxins Cry1Ab and Cry1Ac. Previous results suggested that the modified toxins would be effective only if resistance was linked with mutations in genes encoding toxin-binding cadherin proteins. Here we report evidence from five major crop pests refuting this hypothesis. Relative to native toxins, the potency of modified toxins was >350-fold higher against resistant strains of Plutella xylostella and Ostrinia nubilalis in which resistance was not linked with cadherin mutations. Conversely, the modified toxins provided little or no advantage against some resistant strains of three other pests with altered cadherin. Independent of the presence of cadherin mutations, the relative potency of the modified toxins was generally higher against the most resistant strains.     

Evaluation of the anti-inflammatory effect of milk fermented by a strain of IL-10-producing Lactococcus lactis using a murine model of Crohn's disease

Interleukin-10 (IL-10) is the most important anti-inflammatory cytokine at intestinal level, and its absence is involved in inflammatory bowel diseases. However, oral treatment with IL-10 is difficult because of its low survival in the gastrointestinal tract and systemic treatments lead to undesirable side effects. The aim of this paper was to evaluate the anti-inflammatory effect of the administration of milks fermented by Lactococcus lactis strains that produce IL-10 under the control of the xylose-inducible expression system using a trinitrobenzenesulfonic acid-induced colitis murine model. Mice that received milks fermented by L. lactis strains producing IL-10 in the cytoplasm (Cyt strain) or secreted to the product (Sec strain) showed lower damage scores in their large intestines, decreased IFN-γ levels in their intestinal fluids and lower microbial translocation to liver, compared to mice receiving milk fermented by the wild-type strain or those not receiving any treatment. The results obtained in this study show that the employment of fermented milks as a new form of administration of IL-10-producing L. lactisis effective in the prevention of inflammatory bowel disease in a murine model.     

Neuroprotective role of PrPC against kainate-induced epileptic seizures and cell death depends on the modulation of JNK3 activation by GluR6/7-PSD-95 binding

Cellular prion protein (PrP(C)) is a glycosyl-phosphatidylinositol-anchored glycoprotein. When mutated or misfolded, the pathogenic form (PrP(SC)) induces transmissible spongiform encephalopathies. In contrast, PrP(C) has a number of physiological functions in several neural processes. Several lines of evidence implicate PrP(C) in synaptic transmission and neuroprotection since its absence results in an increase in neuronal excitability and enhanced excitotoxicity in vitro and in vivo. Furthermore, PrP(C) has been implicated in the inhibition of N-methyl-d-aspartic acid (NMDA)-mediated neurotransmission, and prion protein gene (Prnp) knockout mice show enhanced neuronal death in response to NMDA and kainate (KA). In this study, we demonstrate that neurotoxicity induced by KA in Prnp knockout mice depends on the c-Jun N-terminal kinase 3 (JNK3) pathway since Prnp(o/o)Jnk3(o/o) mice were not affected by KA. Pharmacological blockage of JNK3 activity impaired PrP(C)-dependent neurotoxicity. Furthermore, our results indicate that JNK3 activation depends on the interaction of PrP(C) with postsynaptic density 95 protein (PSD-95) and glutamate receptor 6/7 (GluR6/7). Indeed, GluR6-PSD-95 interaction after KA injections was favored by the absence of PrP(C). Finally, neurotoxicity in Prnp knockout mice was reversed by an AMPA/KA inhibitor (6,7-dinitroquinoxaline-2,3-dione) and the GluR6 antagonist NS-102. We conclude that the protection afforded by PrP(C) against KA is due to its ability to modulate GluR6/7-mediated neurotransmission and hence JNK3 activation.     

CTL escape mediated by proteasomal destruction of an HIV-1 cryptic epitope

Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.     

Genotipificación de aislamientos clínicos de Aspergillus flavus y su relación con aislamientos ambientales de un centro oncohematológico

BackgroundDuring 4 months, and while conducting an environmental sampling of air, 2 cases of aspergillosis by Aspergillus flavus (A. flavus) were diagnosed at an oncohematological center in Buenos Aires, Argentina.AimsThe aim of this study was to know the variability and the genetic relationship between the clinical and environmental isolates, obtained in the oncohematological center.MethodsTwo genotyping techniques of different discriminatory power (RAPD and AFLP) were used. A genetic similarity matrix was calculated using Jaccard method and was the basis for the construction of a dendrogram by UPGMA. The level of genetic variability was assessed by measuring the percentage of polymorphic loci, number of effective allele, expected heterocygozity and association index test (I_A).ResultsThe dendrogram reveals that the A. flavus isolates recovered from the patients were not genetically related to those gotten from the rooms occupied by the patients. The environmental isolates had higher values of genetic diversity than the clinical isolates. The I_A estimated for all the isolates suggest that recombination events occurred.ConclusionsPatients 1 and 2 were not infected with isolates from the nosocomial environment. Clinical and environmental isolates of A. flavus showed high genetic variability among them.     

Clomipramine and benznidazole association for the treatment of acute experimental Trypanosoma cruzi infection

Alternative strategies are being designed to identify candidates among drugs already available on the market that could be used in combination to improve the efficacy of Chagas disease treatment. This work evaluates the effect of the association of clomipramine (CLO) with benznidazole (BZN) for the treatment of experimental Chagas disease in the acute stage, in Swiss albino mice infected with Trypanosoma cruzi Tulahuen strain. Infected mice were treated with CLO 5 mg/kg/day and BZN 50 and 100 mg/kg/day, each separately or together. Efficacy of the treatment was evaluated through parasitemia, survival, electrocardiography, histopathological studies, serological and PCR assays at 90 days post-infection (dpi). All treatments significantly (P < 0.05) reduced mortality and decreased parasitemia. Histopathological analysis of liver and kidneys of mice treated with CLO and the drug combination showed less injury than mice treated only with BZN. The lower dose of BZN (50 mg/kg/day) combined with CLO showed the same efficacy as the habitual dose of BZN (100 mg/kg/day) combined with CLO. The therapeutic results from the combination of BZN with CLO presented lesser side effects than the treatment with BZN.     

Using choice experiments to understand public demand for the conservation of nature: A case study in a protected area of Chile

We conducted a choice experiment for the economic valuation of benefits of components of biodiversity that are provided by the natural systems protected in the Peñuelas Lake National Reserve, located in the Mediterranean zone of Chile. The Mediterranean zone of central Chile is one of the world's 34 biodiversity hotpots. Furthermore, we estimated the economic benefits of the water supply provided by the reserve. Unlike most of the previous studies on willingness to pay (WTP) for conservation, part of the data that we produce refers to: (a) public WTP for unpopular species of fauna (an endemic amphibian currently in danger of extinction); and (b) public WTP for flora present at the Reserve (endemic orchid species). Specifically the attributes of the study were the following: existence of endemic orchid species; chances of observing animals with scenic attraction; additional protection for an endemic amphibian; and, availability of drinkable water in the future. A rate of entry to the area was incorporated to estimate WTP for additional protection for the selected attributes. WTP data were obtained from a representative sample of Chilean tourists that visit the area. Factors influencing the visitors’ WTP were also explored.Three hundred and four Chilean visitors of the reserve were randomly selected for interviews. Multinomial Logit and Random Parameter Logit models results show that visitors are willing to pay to protect the selected attributes. Marginal mean WTP/visitor for the single levels of variation of the attributes range from about $1.7 per visitor per visit for securing the existence of five species of endemic orchids to about $8.9 for guaranteeing the availability of drinkable water for 50 years. The analysis of visitors’ WTP for different levels of protection allowed an improved understanding of the sensitivity of the participants to the scope of the information provided.