Cathepsin V, but not cathepsins L, B and K, may release angiostatin-like fragments from plasminogen

Abstract Cathepsin V is a lysosomal cysteine peptidase highly expressed in corneal epithelium; however, its function in the eye is still unknown. Here, we describe the capability of cathepsin V to hydrolyze plasminogen, which is also expressed in human cornea at levels high enough to produce physiologically relevant amounts of angiostatin-related molecules. The co-localization of these two proteins suggests an important role for the enzyme in the maintenance of corneal avascularity, essential for optimal visual performance. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of plasminogen digestion by cathepsin V revealed the generation of three major products of 60, 50 and 40 kDa, which were electrotransferred to polyvinylidene difluoride membranes and excised for characterization. NH(2)-terminal amino acid sequencing of these fragments revealed the sequences EKKVYL, TEQLAP and LLPNVE, respectively. These data are compatible with cleavage sites at plasminogen F94-E95, S358-T359 and V468-L469 peptide bonds generating fragments of the five-kringle domains. In contrast, we did not detect any plasminogen degradation by cathepsins B, K and L. Using a Matrigel assay, we confirmed the angiogenesis inhibition activity on endothelial cells caused by plasminogen processing by cathepsin V. Our results suggest a novel physiological role for cathepsin V related to the control of neovascularization in cornea.     

Differences in endothelin receptor types in the vasculature of Bothrops jararaca (Viperidae) and Oxyrhopus guibei (Colubridae) snakes

Endothelins (ETs) are vasoactive peptides evolutionary well conserved that exert their effects through two specific receptors (ET(A) and ET(B)) widely distributed in all vertebrates. In snakes, the presence and function of endothelins and their receptors are still scarcely described. We have recently demonstrated the presence of ET(A) and ET(B2) receptors in the snake Bothrops jararaca (Bj). In the present work we showed that distinctively from Bj, the vascular contraction induced by endothelin in Oxyrhopus guibei (Og) snake is mediated only by ET(A) receptors. Selective ET(B) agonists (SRTX-c and IRL(1620)) and antagonists (IRL(1038) and BQ(788)) were ineffective in Og preparations of isolated aorta. We also showed that ET-1 response on Og arterial blood pressure was monophasic hypertensive as opposed to biphasic (hypotension followed by hypertension) in Bj. Furthermore, we characterized the relaxing properties of endothelin receptor ET(B1) in pre-contracted aorta preparations. We showed that IRL(1620) induced relaxation of pre-contracted Bj aorta but was ineffective in relaxing Og preparations. IRL(1620) relaxing effect on Bj aorta was abolished by l-NAME, indicating involvement of NO release, and was reduced by selective ET(B) antagonists. Our findings suggest that Og snake has a more primitive spectrum of ET receptors (only ET(A) receptor) than Bj (presence of ET(A), ET(B1) and ET(B2) receptors).     

Integration of DIGE and bioinformatics analyses reveals a role of the antiobesity agent tungstate in redox and energy homeostasis pathways in brown adipose tissue

Our previous results demonstrated that tungstate decreased weight gain and adiposity in obese rats through increased thermogenesis and lipid oxidation, suggesting that brown adipose tissue was one of the targets of its antiobesity effect. To identify potential targets of tungstate, we used DIGE to compare brown adipose tissue protein extracts from the following experimental groups: untreated lean, tungstate-treated lean, untreated obese, and tungstate-treated obese rats. To distinguish direct targets of tungstate action from those that are secondary to body weight loss, we also included in the analysis an additional group consisting of obese rats that lose weight by caloric restriction. Hierarchical clustering of analysis of variance and t test contrasts clearly separated the different experimental groups. DIGE analysis identified 20 proteins as tungstate obesity direct targets involved in Krebs cycle, glycolysis, lipolysis and fatty acid oxidation, electron transport, and redox. Protein oxidation was decreased by tungstate treatment, confirming a role in redox processes; however, palmitate oxidation, as a measure of fatty acid beta-oxidation, was not altered by tungstate, thus questioning its putative function in fatty acid oxidation. Protein network analyses using Ingenuity Pathways Analysis highlighted peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) as a potential target. We confirmed by real time PCR that indeed tungstate up-regulates PGC-1alpha, and its major target, uncoupling protein 1, was also increased as shown by Western blot. These results illustrate the utility of proteomics and bioinformatics approaches to identify targets of obesity therapies and suggest that in brown adipose tissue tungstate modulates redox processes and increases energy dissipation through uncoupling and PGC-1alpha up-regulation, thus contributing to its overall antiobesity effect.     

Litterfall dynamics and nitrogen use efficiency in two evergreen temperate rainforests of southern Chile

Abstract In unpolluted regions, where inorganic nitrogen (N) inputs from the atmosphere are minimal, such as remote locations in southern South America, litterfall dynamics and N use efficiency of tree species should be coupled to the internal N cycle of forest ecosystems. This hypothesis was examined in two evergreen temperate forests in southern Chile (42°30′S), a mixed broad‐leaved forest (MBF) and a conifer forest (CF). Although these forests grow under the same climate and on the same parental material, they differ greatly in floristic structure and canopy dynamics (slower in the CF). In both forests, biomass, N flux, and C/N ratios of fine litterfall were measured monthly from May 1995 to March 1999. There was a continuous litter flux over the annual cycle in both forests, with a peak during autumn in the CF. In the MBF, litterfall decreased during spring. In both forests, the C/N ratios of litterfall varied over the annual cycle with a maximum in autumn. Annual litterfall biomass flux (Mean ± SD = 3.3 ± 0.5 vs 2.0 ± 0.5 Mg ha^?1) and N return (34.8 ± 16 vs 9.1 ± 2.8 kg N ha^?1) were higher in the MBF than in the CF. At the ecosystem level, litterfall C/N was lower in the MBF (mean C/N ratio = 60.1 ± 15, n = 3 years) suggesting decreased N use efficiency compared with CF (mean C/N ratio = 103 ± 19.6, n = 3 years). At the species level, subordinated (subcanopy) tree species in the MBF had significantly lower C/N ratios (<50) of litterfall than the dominant trees in the CF and MBF (>85). The litterfall C/N ratio and percentage N retranslocated were significantly correlated and were lower in the MBF. The higher net N mineralization in soils of the MBF is related to a lower N use efficiency at the ecosystem and species level.     

Screening HIV‐1 antigenic peptides as receptors for antibodies and CD4 in allosteric nanosensors

We have analyzed the suitability of six antigenic peptides from several HIV‐1 structural proteins (namely gp41, gp120, p17, and p24), as anti‐HIV‐1 antibody receptors in an allosteric enzymatic biosensor. These peptides were inserted in a solvent‐exposed surface of Escherichia coli (E. coli) beta‐galactosidase by means of conventional recombinant DNA technology. The resulting enzymes were tested to allosterically respond to sera from HIV‐1‐infected individuals. Only stretches from gp41 and gp120 envelope proteins were able to transduce the molecular contact signal in the presence of immunoreactive sera. Intriguingly, the enzyme displaying the CD4 binding site segment KQFINMWQEVGKAMYAPP was activated by soluble CD4, suggesting that it produces conformational modifications on the allosteric enzyme as those occurring during antibody‐promoted induced fit. This fact is discussed in the context of the design of smart protein drugs and markers targeted to CD4⁺ cells. Copyright © 2009 John Wiley & Sons, Ltd.     

Cell-associated acid β-xylosidase production by Penicillium sclerotiorum

In recent decades, β-xylosidases have been used in many processing industries. In this work, the study of xylosidase production by Penicillium sclerotiorum and its characterization are reported. Optimal production was obtained in medium supplemented with oat spelts xylan, pH 5.0, at 30 °C, under stationary condition for six days. The optimum activity temperature was 60 °C and unusual optimum pH 2.5. The enzyme was stable at 50 and 55 °C, with half-life of 240 and 232 min, respectively. High pH stability was verified from pH 2.0 to 4.0 and 7.5. The β-xylosidase was strongly inhibited by divalent cations, sensitive to denaturing agents SDS, EDTA and activated by thiol-containing reducing agents. The apparent V_max and K_m values was 0.48 μmol PNXP min^?1 mg^?1 protein and 0.75 mM, respectively. The enzyme was xylose tolerant with a K_i of 28.7. This enzyme presented interesting characteristics for biotechnological process such as animal feed, juice and wine industries.     

A recombinant thioredoxin-glutathione reductase from Fasciola hepatica induces a protective response in rabbits

Antioxidant systems are fundamental components of host–parasite interactions, and often play a key role in parasite survival. Here, we report the cloning, heterologous expression, and characterization of a thioredoxin glutathione reductase (TGR) from Fasciola hepatica. The deduced polypeptide sequence of the cloned open reading frame (ORF) confirmed the experimental N-terminus previously determined for a native F. hepatica TGR showing thioredoxin reductase (TR) activity. The sequence revealed the presence of a fusion between a glutaredoxin (Grx) and a TR domain, similar to that previously reported in Schistosoma mansoni and Echinococcus granulosus. The F. hepatica TGR sequence included an additional redox active center (ACUG; U being selenocysteine) located at the C-terminus. The addition of a recombinant selenocysteine insertion sequence (SECIS) element in the Escherichia coli expression vector, or the substitution of the native selenocysteine by a cysteine, indicated the relevance of this unusual amino acid residue for the activity of F. hepatica TGR. Rabbit vaccination with recombinant F. hepatica TGR reduced the worm burden by 96.7% following experimental infection, further supporting the relevance of TGR as a promising target for anti Fasciola treatments.     

Galvanic vestibular stimulation improves the results of vestibular rehabilitation

Here, we present findings from a three-step investigation of the effect of galvanic vestibular stimulation (GVS) in normal subjects and in subjects undergoing vestibular rehabilitation (VR). In an initial study, we examined the body sway of 10 normal subjects after one minute of 2 mA GVS. The effect of the stimulation lasted for at least 20 minutes in all subjects and up to two hours in 70% of the subjects. We then compared a group of patients who received conventional VR (40 patients) with a group that received a combination of VR and GVS. Results suggest a significant improvement in the second group. Finally, we attempted to establish the optimal number of GVS sessions and to rule out a placebo effect. Fifteen patients received "systematic" GVS: five sessions, once a week. Five patients received "nonsystematic" galvanic stimulation in a sham protocol, which included two stimulations of the clavicle. These data were analyzed with Fisher's exact test and indicated that the best results were obtained after three sessions of GVS and no placebo effect was observed.