Ala258Phe substitution in Bacillus sp. YX-1 glucose dehydrogenase improves its substrate preference for xylose

Bacillus sp. YX-1 glucose dehydrogenase (BsGDH) with good solvent resistance catalyzes the oxidation of β-d-glucose to d-glucono-1,5-lactone. Xylose is a recyclable resource from hemicellulase hydrolysis. In this work, to improve the preference of BsGDH for xylose, we designed seven mutants inside or adjacent to the substrate binding pocket using site-directed mutagenesis. Among all mutants, Ala258Phe mutant displayed the highest activity of 7.59 U mg⁻¹ and nearly 8-folds higher k_cat/K_m value towards xylose than wild-type BsGDH. The kinetic constants indicated that the A258F mutation effectively altered the transition state. By analysis of modeled protein structure, Ala258Phe created a space to facilitate the reactivity towards xylose. A258F mutant retained good solvent resistance in glycol, ethyl caprylate, octane, decane, cyclohexane, nonane, etc. as with BsGDH. This work provides a protein engineering approach to modify the substrate stereo-preference of alcohol dehydrogenase and a promising enzyme for cofactor regeneration in chiral catalysis.     

Biology and host preference of Selitrichodes neseri: A potential biological control agent of the Eucalyptus gall wasp,Leptocybe invasa

Selitrichodes neseri (Hymenoptera: Eulophidae) is a parasitoid of the invasive gall-forming wasp Leptocybe invasa (Hymenoptera: Eulophidae), which has caused serious damage to Eucalyptus plantations in many parts of the world. S. neseri is a recently discovered parasitoid considered to be a potentially important biological control agent of L. invasa. The aim of this study was to provide the first basic data on the biology of S. neseri, which is essential for its application in biological control. S. neseri was shown to be a biparental ectoparasitoid. Observation from dissected galls indicated that the parasitoid developed on late larvae, pupae and callow adults, although development did occur in a range of gall ages. Observed nominal parasitism in captivity ranged from 9.7% to 71.8%. Adult S. neseri specimens, fed with honey-water and galled Eucalyptus leaves, survived an average of 26 days at 26 °C. The average developmental time from oviposition to emergence was 19.3 days ± 0.2 days. There was no pre-oviposition period. A single female produced a maximum of thirty-nine offspring, with a maximum of ten per day. Dissection of the ovaries showed that twelve ovarioles were present. The sex ratio of S. neseri observed in this study was 1:3.43 males:females. Galls of native insects most closely related to L. invasa and to galls of similar morphology to L. invasa-induced galls, were not suitable for S. neseri oviposition. S. neseri showed considerable potential as a biological control agent of L. invasa due to its relatively short developmental time, long adult life span when supplemented with carbohydrates, ability to utilize a range of gall ages and the fact that it has a high level of host specificity.     

Meligethes aeneus oviposition preferences,larval parasitism rate and species composition of parasitoids on Brassica nigra,Raphanus sativus and Eruca sativa compared with on Brassica napus

The trap crop strategy is based on host plant discrimination by pests and their parasitoids, which may respond differently to various host plant cues, thus affecting their respective population distributions. We conducted a three-year study to compare the responses of the most damaging pest of oilseed rape (Brassica napus L.), the pollen beetle (Meligethes aeneus Fab.), and its hymenopteran parasitoids to various potential trap crops: Brassica nigra L., Raphanus sativus var. olifera Pers. and Eruca sativa Mill. with that to B. napus. We recorded their abundance, oviposition preferences and the species composition of the parasitoids.Our results show that oviposition rates of the pollen beetle and its parasitoids as well the species composition of the parasitoids varies with plant species. We discuss the potential of these plant species, especially B. nigra, to enhance the natural control of the beetle by fostering several parasitoid species. The species composition of the parasitoids on different host plants compared with on B. napus is presented for the first time. In addition to trapping pests, the trap crops could also act as parasitoid banks, enhancing natural control of the pest through providing suitable hosts for natural enemies, without increasing the population growth of the next generation of pests.     

Gender differences in anaerobic power tests

The purpose of this study was to determine if the differences in anaerobic power between males and females could be accounted for by differences in body composition, strength, and neuromuscular function. A total of 82 untrained men and 99 women took part in the study. Body composition, somatotype, isometric strength, neuromuscular function were measured, and four anaerobic power tests performed. The men were significantly different from the women on all strength, power, and neuromuscular measurements except reaction time and on all anthropometric and somatotype dimensions except ectomorphy. Strength and anthropometric dimensions were similarly related to anaerobic power values within each sex. Relative fat (%fat) exerted different degrees of influence on sprint and jump performances in each sex. Removing the influence of anthropometric, strength, and neuromuscular differences by analysis of covariance reduced, but did not remove, the significant differences between the sexes. Therefore, factors other than lean body mass, leg strength, and neuromuscular function may be operating in short-term, explosive power performances to account for the differences between the sexes. The task-specific nature of anaerobic power tests and the relatively large influence of anthropometric factors on power production were confirmed.     

Metformin promotes Mycobacterium tuberculosis killing and increases the production of human β-defensins in lung epithelial cells and macrophages

Diabetes has been associated with an increased risk of developing tuberculosis. The reasons related to the increased susceptibility to develop TB in type 2 diabetes mellitus (T2DM) individuals, has not been completely elucidated. However, this susceptibility has been attributed to several factors including failures and misfunctioning of the immune system. In the present study, we aimed to determine the role of anti-hyperglycemic drugs such as glyburide, insulin, and metformin to promote the killing of mycobacteria through the regulation of innate immune molecules such as host defense peptides (HDP) in lung epithelial cells and macrophages. Our results showed that metformin reduces bacillary loads in macrophages and lung epithelial cells which correlates with higher production of β-defensin-2, -3 and -4. Since β-defensins are crucial molecules for controlling Mycobacterium tuberculosis growth, the present results suggest that the use of metformin would be the first choice in the treatment for T2DM2, in patients within tuberculosis-endemic areas.     

Study of thiol proteases of normal human skin fibroblasts

The protease activity of cultured normal human skin fibroblasts was studied using the synthetic fluorigenic peptides, the modified protein 4-methylumbelliferyl-casein, the thiol inhibitors and the affinity for concanavalin A-Sepharose. The majority of the activity to N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methyl-coumarin and N-a-benzyloxycarbonyl-L-arginyl-arginyl-7-amido-4-methylcoumarin had a pH optimum of 6.0, and was thiol-dependent and inhibited by leupeptin and antipain. The activity toward N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methylcoumarin represents both cathepsin B and cathepsin L, whereas the activity towards 4-methylumbelliferyl-casein represent only cathepsin L. Cathepsin H could not be detected when assayed with L-arginine-7-amido-4-methylcoumarin substrate. Cathepsin D was present in comparatively small amounts when assayed with 4-methylumbelliferyl-casein. Activity towards 4-methylumbelliferyl-casein had pH optima at 3 and 6 and was stimulated by dithiothreitol. A proportion of the activity at pH 6.0 was not dependent on thiols and not inhibited by leupeptin, and had the general characteristics of a carboxyl proteinase. Over 70 per cent of the activity was in the lysosomal fraction and showed structure-linked latency. All the detectable protein emerged from the immobilized concanavalin A column and the fractions eluted by alpha-methyl-D-mannoside were significantly hydrolysed the synthetic peptides. Only that fraction which bound to concanavalin A was active towards 4-methylumbelliferyl-casein. Cathepsin B had no affinity for concanavalin A-Sepharose due to the absence of glycoprotein content, unlike cathepsin L which showed a strong affinity for concanavalin A-Sepharose.     

A mannose-specific lectin from Vicia villosa seeds

A lectin specific to mannose has been purified from Vicia villosa seed by (NH₄)₂SO₄ fractionation, GalNAc-Sepharose and Man-Sepharose affinity chromatography. It was defined as VVL_M, which showed a single band on an acidic-PAGE stained with Coosmassie brilliant blue. The molecular weight of VVL_M was 50 kDa as determined by gel filtration on Biogel P-100 column. The VVL_M molecule consists of 2 distinct subunits with apparent molecular weight of 30 kDa and 22kDa determined by SDS-PAGE. VVL_M has at least four isolectins with similar haemagglutinating activity. Its extinction coefficient is calculated as A_1cm¹ = 16.4 at 280 nm. Sugars could not be detected phenol-sulfuric acid method. The circular dichroism analysis at far UV indicated that VVL_M was a β-sheet-rich protein, and gave no α-helix, 69% β-sheet, 14% β-turn by Provencher and Glockner method. The lectin was inhibited by α-methyl-d-mannose at 12.5 mM and glucose or GlcNAc at 50 mM. The carbohydrate binding specificity of VVL_M was investigated by using affinity chromatography on a VVL_M-Sepharose column. Among various Asn-linked oligosaccharides, core structure Manα1→3(Manα1→6)Manβ1→4GlcNAcβ1→4GlcNAc_OT were found to have high affinity for VVL_M-Sepharose. The antisera of VVL_M did not produce precipitin line with VVL_G in agar double diffusion plate indicating so serological relationship between VVL_M and VVL_G. However VVL_M showed similar immunodeterminants of some other lectins of mannose specificity such as Con A, PSL, LCA and VFL.     

Fatty Acid Specificity in Lipase-Catalyzed Synthesis of Glucoside Esters

The fatty acid specificity of the B-lipase derived from Candida antarctica was investigated in the synthesis of esters of ethyl D-glucopyranoside. The specificity was almost identical with respect to straight-chain fatty acids with 10 to 18 carbon atoms. However, lower fatty acids such as hexanoic and octanoic acid and the unsaturated 9-cis-octadecenoic acid were found to be poor substrates of the enzyme. As a consequence of this selectivity, these fatty acids were accumulated in the unconverted fraction when ethyl D-glucopyranoside was esterified with an excess of a mixture of fatty acids. This accumulation can reduce the overall effectiveness of the process as the activity of the lipase was found to be reduced when exposed to high concentrations of short-chain fatty acids. Finally, using a simplified experimental set-up, the specificity of the C. antarctica B-lipase was compared to the specificity of lipases derived from C. rugosa, Mucor miehei, Humicola, and Pseudomonas. Apart from the C. rugosa lipase, which exhibited a very poor performance, all the enzymes showed a very similar specificity with respect to fatty acids longer than octanoic acid while only the C. antarctica B-lipase showed activity towards sort-chain fatty acids.     

Ephelides are More Related to Pigmentary Constitutional Host Factors than Solar Lentigines

Ephelides and solar lentigines are benign pigmented spots, which are currently associated with an increased risk of skin cancer. These two pigmented spots are known to be discriminated by their clinical, histological, and electron microscopic characteristics, even though occasional misclassification can occur because of their similarity. It has also been questioned whether these spots are not one and the same. In this study, we have attempted to differentiate between these two pigmented spots with the use of a standardized protocol for clinical examinations on 272 healthy volunteers, paying particular consideration to their pigmentary and constitutional host factors. We found that solar lentigines 1) are more prevalent than ephelides, 2) increase in prevalence and number with higher age, and 3) are most prevalent on the trunk and occur more frequently in males than in females. A trend is also observed whereby ephelides 1) loose their prevalence with age, 2) become equally distributed on the face, arms, and trunk, and 3) occur more frequently in females. An intimate association of ephelides, but not solar lentigines, has been found with hair color and skin type. All of these findings are in agreement with most of those reported in the literature, supporting the view that ephelides and solar lentigines are different types of pigmented lesions.     

Effects of chemical modification of lysine residues in trypsin

Chemical modifications are a simple method to identify and modify functional determinants of enzymes. In the case of serine proteases, it is possible to induce characteristics which are advantageous for peptide synthesis. In this work, we investigated the influence of guanylation and succinylation of lysine residues on the S′-subsite specificity, the catalytic behavior and stability of trypsin. We have found, that succinylation leads to an about 10-fold better acceptance of basic residues in P1′, whereas guanylation shows no remarkable effects. Furthermore, guanylation enhances, succinylation reduces the general enzyme–substrate interactions in P2′. The structural fundamentals of these specificity changes are discussed. The catalytic behavior of trypsin was not influenced by guanylation and succinylation but an enhancement of the stability against autolytic processes by introducing additional negative charges into the protein was observed.