Two structurally discrete GH7-cellobiohydrolases compete for the same cellulosic substrate fiber

Background: Cellulose consisting of arrays of linear beta-1,4 linked glucans, is the most abundant carbon-containing polymer present in biomass. Recalcitrance of crystalline cellulose towards enzymatic degradation is widely reported and is the result of intra-and inter-molecular hydrogen bonds within and among the linear glucans. Cellobiohydrolases are enzymes that attack crystalline cellulose. Here we report on two forms of glycosyl hydrolase family 7 cellobiohydrolases common to all Aspergillii that attack Avicel, cotton cellulose and other forms of crystalline cellulose. Results: Cellobiohydrolases Cbh1 and CelD have similar catalytic domains but only Cbh1 contains a carbohydrate-binding domain (CBD) that binds to cellulose. Structural superpositioning of Cbh1 and CelD on the Talaromyces emersonii Cel7A 3-dimensional structure, identifies the typical tunnel-like catalytic active site while Cbh1 shows an additional loop that partially obstructs the substrate-fitting channel. CelD does not have a CBD and shows a four amino acid residue deletion on the tunnel-obstructing loop providing a continuous opening in the absence of a CBD. Cbh1 and CelD are catalytically functional and while specific activity against Avicel is 7.7 and 0.5 U. mg prot-1, respectively specific activity on pNPC is virtually identical. Cbh1 is slightly more stable to thermal inactivation compared to CelD and is much less sensitive to glucose inhibition suggesting that an open tunnel configuration, or absence of a CBD, alters the way the catalytic domain interacts with the substrate. Cbh1 and CelD enzyme mixtures on crystalline cellulosic substrates show a strong combinatorial effort response for mixtures where Cbh1 is present in 2: 1 or 4: 1 molar excess. When CelD was overrepresented the combinatorial effort could only be partially overcome. CelD appears to bind and hydrolyze only loose cellulosic chains while Cbh1 is capable of opening new cellulosic substrate molecules away from the cellulosic fiber. Conclusion: Cellobiohydrolases both with and without a CBD occur in most fungal genomes where both enzymes are secreted, and likely participate in cellulose degradation. The fact that only Cbh1 binds to the substrate and in combination with CelD exhibits strong synergy only when Cbh1 is present in excess, suggests that Cbh1 unties enough chains from cellulose fibers, thus enabling processive access of CelD.

General biochemical and immunological characteristics of the venom from Peruvian scorpion Hadruroides lunatus

This communication describes the general biochemical properties and some immunological characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic to mice, the LD50 determined was 0.1 mg/kg and 21.55 mg/kg when the venom was injected intracranial or intraperitoneally, respectively. The soluble venom displayed proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two peptides with phospholipasic activity were isolated to homogeneity and their molecular masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were produced in rabbits. Western blotting analysis showed that most of the protein content of this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity with venom antigens from the new World-scorpions Tityus serrulatus and Centruroides sculpturatus venoms; however, a weaker reactivity was observed against the venom antigens from the old World-scorpion Androctonus australis Hector. (C) 2012 Elsevier Ltd. All rights reserved.

Comparison of microsatellites and isozymes in genetic diversity studies of Oryza glumaepatula (Poaceae) populations

The study of the genetic structure of wild plant populations is essential for their management and conservation. Several DNA markers have been used in such studies, as well as isozyme markers. In order to provide a better comprehension of the results obtained and a comparison between markers which will help choose tools for future studies in natural populations of Oryza glumaepatula, a predominantly autogamous species, this study used both isozymes and microsatellites to assess the genetic diversity and genetic structure of 13 populations, pointing to similarities and divergences of each marker, and evaluating the relative importance of the results for studies of population genetics and conservation. A bulk sample for each population was obtained, by sampling two to three seeds of each plant, up to a set of 50 seeds. Amplified products of eight SSR loci were electrophoresed on non-denaturing polyacrylamide gels, and the fragments were visualized using silver staining procedure. Isozyme analyses were conducted in polyacrylamide gels, under a discontinuous system, using six enzymatic loci. SSR loci showed higher mean levels of genetic diversity (A=2.83, p=0.71, A(P)=3.17, H-o=0.081, H-e=0.351) than isozyme loci (A=1.20, p=0.20, A(P)=1.38, H-o=0.006, H-e=0.056). Interpopulation genetic differentiation detected by SSR loci (R-ST=0.631, equivalent to F-ST=0.533) was lower than that obtained with isozymes (F-ST=0.772). However, both markers showed high deviation from Hardy-Weinberg expectations (F-IS=0.744 and 0.899, respectively for SSR and isozymes). The mean apparent outcrossing rate for SSR ((t) over bar (a)=0.14) was higher than that obtained using isozymes ((t) over bar (a)=0.043), although both markers detected lower levels of outcrossing in Amazonia compared to the Pantanal. The migrant number estimation was also higher for SSR (Nm=0.219) than isozymes (Nm=0.074), although a small number for both markers was expected due to the mode of reproduction of this species, defined as mixed with predominance of self fertilization. No correlation was obtained between genetic and geographic distances with SSR, but a positive correlation was found between genetic and geographic distances with isozymes. We conclude that these markers are divergent in detecting genetic diversity parameters in O. glumaepatula and that microsatellites are powerful for detecting information at the intra-population level, while isozymes are more powerful for inter-population diversity, since clustering of populations agreed with the expectations based on the geographic distribution of the populations using this marker. Rev. Biol. Trop. 60 (4): 1463-1478. Epub 2012 December 01.

cDNA-AFLP analysis of Psidium guajava L. cultivars under water stress and mechanical injury: methodological implications

Studies involving amplified fragment length polymorphism (cDNA-AFLP) have often used polyacrylamide gels with radiolabeled primers in order to establish best primer combinations, to analyze, and to recover transcript-derived fragments. Use of automatic sequencer to establish best primer combinations is convenient, because it saves time, reduces costs and risks of contamination with radioactive material and acrylamide, and allows objective band-matching and more precise evaluation of transcript-derived fragments intensities. This study aimed at examining the gene expression of commercial cultivars of P. guajava subjected to water and mechanical injury stresses, combining analyses by automatic sequencer and fluorescent kits for polyacrylamide gel electrophoresis. Firstly, 64 combinations of EcoRI and MseI primers were tested. Ten combinations with higher number of polymorphic fragments were then selected for transcript-derived fragments recovering and cluster analysis, involving 45 saplings of P. guajava. Two groups were obtained, one composed by the control samplings, and another formed by samplings undergoing stress, with no clear distinction between stress treatments. The results revealed the convenience of using a combination of automatic sequencer and fluorescent kits for polyacrylamide gel electrophoreses to examine gene expression profiles. The Unweighted Pair Group Method with Arithmetic Mean analysis using Euclidean distances points out a similar induced response mechanism of P. guajava undergoing water stress and mechanical injury.

In Vitro Characterization of Chitosan Gels for Buccal Delivery of Celecoxib: Influence of a Penetration Enhancer

Celecoxib (Cx) shows high efficacy in the treatment of osteoarthritis and rheumatoid arthritis as a result of its high specificity for COX-2, without gastrolesivity or interference with platelet function at therapeutic concentrations. Besides of anti-inflammatory effects, Cx also has a potential role for oral cancer chemoprevention. For these conditions, oral administration in long-term treatment is a concern due to its systemic side effects. However, local application at the site of injury (e.g., buccal inflammation conditions or chemoprevention of oral cancer) is a promising way to reduce its toxicity. In this study, the in vitro characterization of mucoadhesive chitosan (CHT) gels associated to AzoneA (R) was assessed to explore the potential buccal mucosal administration of Cx in this tissue. Rheological properties of gels were analyzed by a rheometer with cone-plate geometry. In vitro Cx release and permeability studies used artificial membranes and pig cheek mucosa, respectively. Mucoadhesion were measured with a universal test machine. CHT gels (3.0%) containing 2.0% or 3.0% Az showed more appropriate characteristics compared to the others: pH values, rheology, higher amount of Cx retained in the mucosa, and minimal permeation through mucosa, besides the highest mucoadhesion values, ideal for buccal application. Moreover, the flux (J) and amounts of drug released decreased with increased CHT and Az concentrations. CHT gels (3.0%) associated with 2.0% or 3.0% Az may be considered potential delivery systems for buccal administration of Cx.