Re-engineering specificity in 1,3-1, 4-β-glucanase to accept branched xyloglucan substrates

Family 16 carbohydrate active enzyme members Bacillus licheniformis 1,3-1,4-β-glucanase and Populus tremula x tremuloides xyloglucan endotransglycosylase (XET16-34) are highly structurally related but display different substrate specificities. Although the first binds linear gluco-oligosaccharides, the second binds branched xylogluco-oligosaccharides. Prior engineered nucleophile mutants of both enzymes are glycosynthases that catalyze the condensation between a glycosyl fluoride donor and a glycoside acceptor. With the aim of expanding the glycosynthase technology to produce designer oligosaccharides consisting of hybrids between branched xylogluco- and linear gluco-oligosaccharides, enzyme engineering on the negative subsites of 1,3-1,4-β-glucanase to accept branched substrates has been undertaken. Removal of the 1,3-1,4-β-glucanase major loop and replacement with that of XET16-34 to open the binding cleft resulted in a folded protein, which still maintained some β-glucan hydrolase activity, but the corresponding nucleophile mutant did not display glycosynthase activity with either linear or branched glycosyl donors. Next, point mutations of the 1,3-1,4-β-glucanase β-sheets forming the binding site cleft were mutated to resemble XET16-34 residues. The final chimeric protein acquired binding affinity for xyloglucan and did not bind β-glucan. Therefore, binding specificity has been re-engineered, but affinity was low and the nucleophile mutant of the chimeric enzyme did not show glycosynthase activity to produce the target hybrid oligosaccharides. Structural analysis by X-ray crystallography explains these results in terms of changes in the protein structure and highlights further engineering approaches toward introducing the desired activity.     

Recent trends in butterfly populations from north-east Spain and Andorra in the light of habitat and climate change

Although butterfly declines have been reported across Europe, no assessment based on detailed quantitative data has ever been made for any extensive area in the Mediterranean Basin. In 1994, a Butterfly Monitoring Scheme was launched in Catalonia (NE Spain), and in 2005 a similar, albeit much smaller, scheme started in the neighbouring Pyrenean country of Andorra. Here we provide a first thorough assessment of butterfly trends in both areas for the last 15 years. Several patterns emerged, above all a worrying decline of a substantial part of the fauna. It was also evident that habitat specialists are experiencing greater declines than habitat generalists, thereby butterfly communities becoming progressively dominated by common species. However, habitat indicators based on characteristic species also revealed that trends are actually associated with habitat types, grassland and scrub specialists declining strongly but woodland specialists showing a marginal increase. These differences are certainly related to profound landscape changes, mainly a dramatic reduction of semi-natural grasslands and open Mediterranean scrub, and a major increase in woodlands. The general effect of climatic warming on butterfly populations was investigated by using the temperature community index (CTI) approach. The thermal structure of butterfly communities remained very stable over time, except in one case where, contrary to the expectations, a significant negative trend in the CTI was noted. However, this surprising result can be explained by taking into account the above-reported pattern of butterfly communities becoming dominated by common species, characterized by low thermal indices in comparison with declining Mediterranean specialists.     

Capture myopathy in little bustards after trapping and marking

Four little bustards (Tetrax tetrax) (one adult and three juvenile males), captured with leg nooses and fitted with a backpack radiotag, died after capture. The first bird was found after 16 days with its left foot caught in the harness and died after 1 day. The other birds showed symptoms of capture myopathy after release, such as the difficulty or inability to fly and/or walk. They died after 5, 6, and 8 days, respectively. At necropsy, muscles affected in all cases were those from the legs, and these were diffusely pale and dull, with a soft friable texture. Microscopically these muscles had multiple foci of myofiber fragmentation, loss of striation, and necrosis; a mononuclear cell infiltrate was observed in muscle from two birds. These findings suggest the little bustard is susceptible to capture myopathy and that caution should be exercised during its capture and handling.     

S 23521 Decreases Food Intake and Body Weight Gain in Diet‐Induced Obese Rats

Objective: To investigate the effect of S 23521, a new glucagon‐like peptide‐1‐(7‐36) amide analogue, on food intake and body weight gain in obese rats, as well as on gene expression of several proteins involved in energy homeostasis. Research Methods and Procedures: Lean and diet‐induced obese rats were treated with either S 23521 or vehicle. S 23521 was given either intraperitoneally (10 or 100 μg/kg) or subcutaneously (100 μg/kg) for 14 and 20 days, respectively. Because the low‐dose treatment did not affect food intake and body weight, the subcutaneous treatment at high dose was selected to test the effect on selected end‐points. Results: Treated obese rats significantly decreased their cumulative energy intake in relation to vehicle‐treated counterparts (3401 ± 65 vs. 3898 ± 72 kcal/kg per 20 days; p < 0.05). Moreover, their body weight gain was reduced by 110%, adiposity was reduced by 20%, and plasma triglyceride levels were reduced by 38%. The treatment also improved glucose tolerance and insulin sensitivity of obese rats. Regarding gene expression, no changes in uncoupling protein‐1, uncoupling protein‐3, leptin, resistin, and peroxisome proliferator‐activated receptor (PPAR)‐γ were observed. Discussion: S 23521 is an effective glucagon‐like peptide‐1‐(7‐36) amide analogue, which induced a decrease in energy intake, body weight, and adiposity in a rat model of diet‐induced obesity. In addition, the treatment also improved glucose tolerance and insulin sensitivity of obese rats. These results strongly support S 23521 as a putative molecule for the treatment of obesity.     

Targeting Dyrk1A with AAVshRNA attenuates motor alterations in TgDyrk1A, a mouse model of Down syndrome

Genetic-dissection studies carried out with Down syndrome (DS) murine models point to the critical contribution of Dyrk1A overexpression to the motor abnormalities and cognitive deficits displayed in DS individuals. In the present study we have used a murine model overexpressing Dyrk1A (TgDyrk1A mice) to evaluate whether functional CNS defects could be corrected with an inhibitory RNA against Dyrk1A, delivered by bilateral intrastriatal injections of adeno-associated virus type 2 (AAVshDyrk1A). We report that AAVshDyrk1A efficiently transduced HEK293 cells and primary neuronal cultures, triggering the specific inhibition of Dyrk1A expression. Injecting the vector into the striata of TgDyrk1A mice resulted in a restricted, long-term transduction of the striatum. This gene therapy was found to be devoid of toxicity and succeeded in normalizing Dyrk1A protein levels in TgDyrk1A mice. Importantly, the behavioral studies of the adult TgDyrk1A mice treated showed a reversal of corticostriatal-dependent phenotypes, as revealed by the attenuation of their hyperactive behavior, the restoration of motor-coordination defects, and an improvement in sensorimotor gating. Taken together, the data demonstrate that normalizing Dyrk1A gene expression in the striatum of adult TgDyrk1A mice, by means of AAVshRNA, clearly reverses motor impairment. Furthermore, these results identify Dyrk1A as a potential target for therapy in DS.     

Role of the AAA protease Yme1 in folding of proteins in the intermembrane space of mitochondria

The vast majority of mitochondrial proteins are synthesized in the cytosol and transported into the organelle in a largely, if not completely, unfolded state. The proper function of mitochondria thus depends on folding of several hundreds of proteins in the various subcompartments of the organelle. Whereas folding of proteins in the mitochondrial matrix is supported by members of several chaperone families, very little is known about folding of proteins in the intermembrane space (IMS). We targeted dihydrofolate reductase (DHFR) as a model substrate to the IMS of yeast mitochondria and analyzed its folding. DHFR can fold in this compartment, and its aggregation upon heat shock can be prevented in an ATP-dependent manner. Yme1, an AAA (ATPases associated with diverse cellular activities) protease of the IMS, prevented aggregation of DHFR. Analysis of protein aggregates in mitochondria lacking Yme1 revealed the presence of a number of proteins involved in the establishment of mitochondrial ultrastructure, lipid metabolism, protein import, and respiratory growth. These findings explain the pleiotropic effects of deletion of YME1 and suggest an important role for Yme1 as a folding assistant, in addition to its proteolytic function, in the protein homeostasis of mitochondria     

Size diversity and species diversity relationships in fish assemblages of Western Palearctic lakes

Body size, coupled with abundance and taxonomy, may help to understand the mechanisms shaping community structure. Since the body size of fish is closely related to their trophic niche, size diversity (based on individual body size) of fish communities may capture intraspecific variations in fish trophic niches that are not detected by species diversity. Thus, the relationship between size diversity and species diversity may help to integrate variation at both intraspecific and interspecific levels. We studied the relationship between species diversity and size diversity as a measure of the degree of overlap in size among species and thereby the potential overlap in niches in a community. We hypothesized that the relationship between size diversity and species would be different across the European continent due to different levels of size overlap in fish communities. The data were derived from samplings of fish communities using standardised benthic gill nets in 363 lakes. At the continental scale, size diversity increased with species diversity; at the ecoregion scale, the slope of the relation changed across the continent, with the greatest mismatch occurring in northern Europe where communities comprised only one or a few species, but each of which exhibited a great range in size. There was an increase in slope towards the south with significant relations for four out of six ecoregions. The steeper size diversity‐species diversity slope at lower latitudes is attributable to a lower overlap in fish size and thus likely to finer niche separation. Our results also suggest that size diversity is not a strong surrogate for species diversity in European lake fish communities. Thus, particularly in fish communities composed of few species, measuring size diversity may help to detect potential functional variation which may be neglected by measuring species diversity alone.     

Evolutionary combinatorial chemistry, a novel tool for SAR studies on peptide transport across the blood-brain barrier. Part 2. Design, synthesis and evaluation of a first generation of peptides.

The use of high-throughput methods in drug discovery allows the generation and testing of a large number of compounds, but at the price of providing redundant information. Evolutionary combinatorial chemistry combines the selection and synthesis of biologically active compounds with artificial intelligence optimization methods, such as genetic algorithms (GA). Drug candidates for the treatment of central nervous system (CNS) disorders must overcome the blood-brain barrier (BBB). This paper reports a new genetic algorithm that searches for the optimal physicochemical properties for peptide transport across the blood-brain barrier. A first generation of peptides has been generated and synthesized. Due to the high content of N-methyl amino acids present in most of these peptides, their syntheses were especially challenging due to over-incorporations, deletions and DKP formations. Distinct fragmentation patterns during peptide cleavage have been identified. The first generation of peptides has been studied by evaluation techniques such as immobilized artificial membrane chromatography (IAMC), a cell-based assay, log Poctanol/water calculations, etc. Finally, a second generation has been proposed.