Ribosomal DNA ITS-1 Intergenic Spacer Polymorphism in Triatomines (Triatominae, Heteroptera)

The length polymorphism of ribosomal DNA ITS-1 intergenic spacer was analyzed in eight species of triatomines belonging to Triatoma, Rhodnius, and Panstrongylus genera. The analyzed species were Rhodnius domesticus, R. neivai, R. robustus, Triatoma brasiliensis, T. infestans, T. vitticeps, Panstrongylus megistus, and P. herreri. These insects are vectors of Chagas' disease, one of the most prominent public health problems among South American countries. This work allowed the differentiation between species of the Triatomini and Rhodniini tribes through the analysis of ITS-1 length polymorphism by PCR and RFLP techniques. The species of the Triatoma and Panstrongylus genera presented an amplified ITS-1 fragment between 600 and 1000 bp, whereas Rhodnius presented a less variable ITS-1 length fragment, around 300 bp, which could reflect the monophyletic origin of the Rhodniini tribe. Species belonging to this genus were further differentiated by RFLP with HaeIII and AluI endonucleases. Our results corroborate the hypothesis of polyphyletic origin in this group of insects and contribute to knowledge about evolutionary relationships in triatomines.     

Amelioration of radiation-induced fibrosis: inhibition of transforming growth factor-beta signaling by halofuginone

Radiation-induced fibrosis is an untoward effect of high dose therapeutic and inadvertent exposure to ionizing radiation. Transforming growth factor-beta (TGF-beta) has been proposed to be critical in tissue repair mechanisms resulting from radiation injury. Previously, we showed that interruption of TGF-beta signaling by deletion of Smad3 results in resistance to radiation-induced injury. In the current study, a small molecular weight molecule, halofuginone (100 nm), is demonstrated by reporter assays to inhibit the TGF-beta signaling pathway, by Northern blotting to elevate inhibitory Smad7 expression within 15 min, and by Western blotting to inhibit formation of phospho-Smad2 and phospho-Smad3 and to decrease cytosolic and membrane TGF-beta type II receptor (TbetaRII). Attenuation of TbetaRII levels was noted as early as 1 h and down-regulation persisted for 24 h. Halofuginone blocked TGF-beta-induced delocalization of tight junction ZO-1, a marker of epidermal mesenchymal transition, in NMuMg mammary epithelial cells and suggest halofuginone may have in vivo anti-fibrogenesis characteristics. After documenting the in vitro cellular effects, halofuginone (intraperitoneum injection of 1, 2.5, or 5 microg/mouse/day) efficacy was assessed using ionizing radiation-induced (single dose, 35 or 45 Gy) hind leg contraction in C3H/Hen mice. Halofuginone treatment alone exerted no toxicity but significantly lessened radiation-induced fibrosis. The effectiveness of radiation treatment (2 gray/day for 5 days) of squamous cell carcinoma (SCC) tumors grown in C3H/Hen was not affected by halofuginone. The results detail the molecular effects of halofuginone on the TGF-beta signal pathway and show that halofuginone may lessen radiation-induced fibrosis in humans.     

Nonmuscle myosin heavy-chain gene MYH14 is expressed in cochlea and mutated in patients affected by autosomal dominant hearing impairment (DFNA4)

Myosins have been implicated in various motile processes, including organelle translocation, ion-channel gating, and cytoskeleton reorganization. Different members of the myosin superfamily are responsible for syndromic and nonsyndromic hearing impairment in both humans and mice. MYH14 encodes one of the heavy chains of the class II nonmuscle myosins, and it is localized within the autosomal dominant hearing impairment (DFNA4) critical region. After demonstrating that MYH14 is highly expressed in mouse cochlea, we performed a mutational screening in a large series of 300 hearing-impaired patients from Italy, Spain, and Belgium and in a German kindred linked to DFNA4. This study allowed us to identify a nonsense and two missense mutations in large pedigrees, linked to DFNA4, as well as a de novo allele in a sporadic case. Absence of these mutations in healthy individuals was tested in 200 control individuals. These findings clearly demonstrate the role of MYH14 in causing autosomal dominant hearing loss and further confirm the crucial role of the myosin superfamily in auditive functions.     

Up-regulation of the Kv3.4 potassium channel subunit in early stages of Alzheimer's disease

Gene expression throughout the different stages of Alzheimer's disease was analysed in samples from cerebral cortex. The gene encoding the voltage-gated potassium channel Kv3.4 was already overexpressed in early stages of the disease, and in advanced stages Kv3.4 was present at high levels in neurodegenerative structures. This subunit regulates delayed-rectifier currents, which are primary determinants of spike repolarization in neurones. In unique samples from a patient with Alzheimer's disease whose amount of amyloid plaques was decreased by beta amyloid immunization, Kv3.4 was overexpressed. The channel subunit was expressed in the neuropil, in the remaining conventional plaques in the frontal cortex and in collapsed plaques in the orbitary cortex. Therefore, amyloid deposition in plaques does not seem to be responsible for the increase in Kv3.4 levels. Nevertheless, Kv3.4 up-regulation is related to amyloid pathology, given that transgenic mice with the Swedish mutation of amyloid precursor protein showed increased expression of Kv3.4. Up-regulation of voltage-gated potassium channel subunits alters potassium currents in neurones and leads to altered synaptic activity that may underlie the neurodegeneration observed in Alzheimer's disease. Thus, Kv3.4 likely represents a novel therapeutic target for the disease.     

Analysis of ascorbate peroxidase genes expressed in resistant and susceptible wheat lines infected by the cereal cyst nematode, Heterodera avenae

Changes in ascorbate peroxidase (APX) enzyme activity in response to nematode (Heterodera avenae) attack were studied in roots of three hexaploid wheat lines carrying Cre2, Cre5, or Cre7 nematode resistance genes and the susceptible Triticum aestivum cv. Anza. A spectrophotometric analysis was carried out with root extracts of infected plants 4, 7, 11, and 14 days after nematode inoculation using uninfected plant as control. APX induction in infected resistant genotypes was similar and higher than in the susceptible control. The introgression wheat/Aegilops ventricosa H-93-8 line, carrying the Cre2 gene, and its parental line H-10-15 as susceptible control were used to analyze whether this increase of activity was correlated with the induction of APX gene expression. Genes encoding cytosolic forms of APX were induced in roots of both lines in response to nematode infection. This induction took place both earlier and with greater intensity in the resistant line than in the susceptible one, and it was also higher in the root area at the site of nematode attachment.     

On/off TLR signaling decides proinflammatory or tolerogenic dendritic cell maturation upon CD1d-mediated interaction with invariant NKT cells

Invariant NKT (iNKT) cells play an effector/adjuvant function during antimicrobial and antitumoral immunity and a regulatory role to induce immune tolerance and prevent autoimmunity. iNKT cells that differentially modulate adaptive immunity do not bear a unique phenotype and/or specific cytokine secretion profile, thus opening questions on how a single T cell subset can exert opposite immunological tasks. In this study, we show that iNKT cells perform their dual roles through a single mechanism of action relying on the cognate interaction with myeloid dendritic cells (DCs) and leading to opposite effects depending on the presence of other maturation stimuli simultaneously acting on DCs. The contact of murine purified iNKT cells with immature autologous DCs directly triggers the tolerogenic maturation of DCs, rendering them able to induce regulatory T cell differentiation and prevent autoimmune diabetes in vivo. Conversely, the interaction of the same purified iNKT cells with DCs, in the presence of simultaneous TLR4 stimulation, significantly enhances proinflammatory DC maturation and IL-12 secretion. The different iNKT cell effects are mediated through distinct mechanisms and activation of different molecular pathways within the DC: CD1d signaling and activation of the ERK1/2 pathway for the tolerogenic action, and CD40-CD40L interaction and NF-κB activation for the adjuvant effect. Our data suggest that the DC decision to undergo proinflammatory or tolerogenic maturation results from the integration of different signals received at the time of iNKT cell contact and could have important therapeutic implications for exploiting iNKT cell adjuvant/regulatory properties in autoimmune diseases, infections, and cancer.     

Adenylyl cyclase type-VIII activity is regulated by G(betagamma) subunits

The Ca2+-activated adenylyl cyclase type VIII (AC-VIII) has been implicated in several forms of neural plasticity, including drug addiction and learning and memory. It has not been clear whether Gi/o proteins and G-protein coupled receptors regulate the activity of AC-VIII. Here we show in intact mammalian cell system that AC-VIII is inhibited by mu-opioid receptor activation and that this inhibition is pertussis toxin sensitive. Moreover, we show that G(betagamma) subunits inhibit AC-VIII activity, while constitutively active alphai/o subunits do not. Different Gbeta isoforms varied in their efficacies, with Gbeta1gamma2 or Gbeta2gamma2 being more efficient than Gbeta3gamma2 and Gbeta4gamma2, while Gbeta5 (transfected with gamma2) had no effect. As for the Ggamma subunits, Gbeta1 inhibited AC-VIII activity in the presence of all gamma subunits tested except for gamma5 that had only a marginal activity. Moreover, cotransfection with proteins known to serve as scavengers of Gbetagamma dimers, or to reduce Gbetagamma plasma membrane anchorage, markedly attenuated the mu-opioid receptor-induced inhibition of AC-VIII. These results demonstrate that Gbetagamma (originating from agonist activation of these receptors) and probably not Galphai/o subunits are involved in the agonist inhibition of AC-VIII.     

Selection and implantation of yeast strains of genus Saccharomyces at a winery regulated by Appellation Contrôlée "Chacolí de Vizcaya/Bizkaiko Txakolina"

The white wine Chacolía de Vizcaya/Bizkaiko Txakolina is characteristic from The Basque Country region and regulated under Appellation Contr?lée standards (BOPV 14/6/94). The objective of this study was the identification and selection of autochthonous yeast strains, to improve the conditions used to maintain the typical characteristics of this region wines. Yeasts identified as Saccharomyces bayanus isolated around these fields from 1996 to 1998, were subjected to a selective procedure based on enological characteristics and fermentative behaviour. Three of the selected strains were used to inoculate, at winery scale, two grape juice varieties accepted by the Appellation Contr?lée (Hondarrabi Zuri and Folle Blanche). The inoculated strains on the respective vinifications was followed by restriction fragment length polymorphism of mitochondrial DNA (REAmt) method with AluI enzyme, due to their specificity, short outcome, and technological simplicity compared with other molecular typing methods such as: chromosomal karyotyping analyzed by pulsed field gel electrophoresis, Random Amplified Polymorphic DNA-PCR (RAPD-PCR) and restriction fragment length polymorphism using the infrequently cutting enzyme SfiI (REA infrequent). This study demonstrated that strains with different phenotypic traits could show indistinguishable restriction patterns with REAmt, but could be discriminated using other typing methods such as RAPD-PCR, which although showing low reproducibility could be used as complementary to REAmt. Our results demonstrate that in spite of using autochthonous selected strains, the inoculation of musts with a particular strain do not guarantee its predominance and driving fermentation features. Of all yeast strains studied, strain no. 2 showed the best results in sensory testing and at the implantation process. Therefore, it could be used with commercial purposes for the production of Chacolí de Vizcaya/Bizkaiko Txakolina, especially when using musts from Folle Blanche.     

A DNA vaccine encoding CCL4/MIP-1beta enhances myocarditis in experimental Trypanosoma cruzi infection in rats

Chagas' disease, caused by Trypanosoma cruzi, is a major cause of cardiovascular disease in Latin America. Exacerbated inflammation disproportional to parasite load characterizes chronic myocardial lesions in chagasic patients. Chemokines and their receptors are expected to account for the renewed inflammatory processes after the inoculation of the parasite, but their potential unique functions are far from being clear. Herein, we evaluated the effect of a DNA vaccine encoding CCL4/MIP-1beta, a CC-chemokine, in T. cruzi-elicited myocarditis in rats. Holtzman rats were given intramuscularly cardiotoxin and the CCL4/MIP-1beta DNA-containing plasmid (100microg) was delivered in this muscular site four times. Fourteen days after last immunization, animals were inoculated with a myotropical CL-Brener T. cruzi clone. Peak of parasitism was observed at day 15 after infection, preceding the peak of myocardial inflammation at day 20. Myocarditis was still intense at day 30, but the inflammatory infiltrates showed a more focal distribution. The expression of CCL2/MCP-1 and CCL4/MIP-1beta correlated closely with the kinetics of myocardial inflammation. The CCL4/MIP-1beta DNA vaccine induced an increase of the levels of the anti-CCL4/MIP-1beta observed in T. cruzi-infected animals. This was associated with an exacerbation of myocardial inflammation and fibrosis, although alterations in parasitemia and myocardial parasitism were not observed. Our data suggest that CCL4/MIP-1beta plays a role in preventing excessive inflammation and pathology rather than in controlling parasite replication.     

Biodegradation of chlorinated alkanes and their commercial mixtures by Pseudomonas sp. strain 273

The biodegradation of chlorinated alkanes was studied under oxic conditions with the objective of identifying favorable and unfavorable intramolecular chlorination sequences with respect to the enzymes studied. Several dehalogenating bacterial strains were screened for their ability to degrade middle-chain polychlorinated alkanes as well as a commercial mixture. Of the organisms tested, the most promising was Pseudomonas sp. strain 273, which possesses an oxygenolytic dehalogenase. The effects of carbon chain length (C₆–C₁₆), halogen position, and overall chlorine content (14–61% w/w) were examined using both commercially available compounds and molecules synthesized in our laboratory. The effects of co-substrates, solvents, and inducing agents were also studied. The results with pure chlorinated alkanes showed that the relative positions of the chlorine atoms strongly influenced the total amount of dehalogenation achieved. The greatest dehalogenation yields were associated with terminally chlorinated alkanes. The α- and α,ω-chlorinated compounds yielded similar results. Vicinal chlorination had the most dramatic impact on degradation. When present on both ends or at the center of the molecule, no dehalogenation was detected. Although partial dehalogenation of 1,2-dichlorodecane was observed, it was likely due to a combination of β-oxidation and an abiotic mechanism. Cereclor S52 was appreciably dehalogenated in shake flasks only when 1,10-dichlorodecane was present as a co-substrate and after increasing the oil surface area through mechanical emulsification, demonstrating the importance of abiotic factors in degrading commercial polychlorinated alkane mixtures.