A Tyrosine Residue Along with a Glutamic Acid of the Omega-Like Loop Governs the Beta-Lactamase Activity of MSMEG_4455 in <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>

Mycobacterial beta-lactamases are involved in exerting beta-lactam resistance, though many of these proteins remain uncharacterized. Here, we have characterized MSMEG_4455 of Mycobacterium smegmatis as a beta-lactamase using molecular, biochemical and mutational techniques. To elucidate its nature in vivo and in vitro, and to predict its structure–function relationship in silico analysis is done. The MSMEG_4455 is cloned and expressed ectopically in a beta-lactamase deficient Escherichia coli mutant to establish the in vivo beta-lactamase like nature via minimum inhibitory concentration (MIC) determination. Likewise the in vivo results, purified soluble form of MSMEG_4455 showed beta-lactam hydrolysis pattern similar to group 2a penicillinase. In silico analyses of MSMEG_4455 reveal glutamic acid (E)193 and tyrosine (Y)194 of omega-like loop might have importance in strengthening hydrogen bond network around the active-site, though involvement of tyrosine is rare for beta-lactamase activity. Accordingly, these residues are mutated to alanine (A) and phenylalanine (F), respectively. The mutated proteins have partially lost their ability to exert beta-lactamase activity both in vivo and in vitro. The Y194F mutation had more prominent effect on the enzymatic activity. Therefore, we infer that Y194 is the key for beta-lactamase activity of MSMEG_4455.     

Theoretical and experimental study of polycyclic aromatic compounds as β-tubulin inhibitors

In this work, through a docking analysis of compounds from the ZINC chemical library on human β-tubulin using high performance computer cluster, we report new polycyclic aromatic compounds that bind with high energy on the colchicine binding site of β-tubulin, suggesting three new key amino acids. However, molecular dynamic analysis showed low stability in the interaction between ligand and receptor. Results were confirmed experimentally in in vitro and in vivo models that suggest that molecular dynamics simulation is the best option to find new potential β-tubulin inhibitors.

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Graphical abstract Bennett’s acceptance ratio (BAR) method

     

Landscape control of nitrous oxide emissions during the transition from conservation reserve program to perennial grasses for bioenergy

Future liquid fuel demand from renewable sources may, in part, be met by converting the seasonally wet portions of the landscape currently managed for soil and water conservation to perennial energy crops. However, this shift may increase nitrous oxide (N₂O) emissions, thus limiting the carbon (C) benefits of energy crops. Particularly high emissions may occur during the transition period when the soil is disturbed, plants are establishing, and nitrate and water accumulation may favor emissions. We measured N₂O emissions and associated environmental drivers during the transition of perennial grassland in a Conservation Reserve Program (CRP) to switchgrass (Panicum virgatum L.) and Miscanthus x giganteus in the bottom 3‐ha of a watershed in the Ridge and Valley ecoregion of the northeastern United States. Replicated treatments of CRP (unconverted), unfertilized switchgrass (switchgrass), nitrogen (N) fertilized switchgrass (switchgrass‐N), and Miscanthus were randomized in four blocks. Each plot was divided into shoulder, backslope, and footslope positions based on the slope and moisture gradient. Soil N₂O flux, soil moisture, and soil mineral nitrogen availability were monitored during the growing season of 2013, the year after the land conversion. Growing season N₂O flux showed a significant vegetation‐by‐landscape position interaction (P < 0.009). Switchgrass‐N and Miscanthus treatments had 3 and 6‐times higher cumulative flux respectively than the CRP in the footslope, but at other landscape positions fluxes were similar among land uses. A peak N₂O emission event, contributing 26% of the cumulative flux, occurred after a 10.8‐cm of rain during early June. Prolonged subsoil saturation coinciding with high mineral N concentration fueled N₂O emission hot spots in the footslopes under energy crops. Our results suggest that mitigating N₂O emissions during the transition of CRP to energy crops would mostly require a site‐specific management of the footslopes.     

Strain-induced orientation response of endothelial cells: effect of substratum adhesiveness and actin-myosin contractile level

Endothelial cells subjected to cyclic stretching change orientation so as to be aligned perpendicular to the direction of applied strain in a magnitude and time-dependent manner. Although this type of response is not the same as motility, it could be governed by motility-related factors such as substratum adhesiveness and actin-myosin contractile level. To examine this possibility, human aortic endothelial cells (HAEC) were uniaxially, cyclically stretched on silicone rubber membranes coated with various concentrations of fibronectin, collagen type IV and laminin to produce differing amounts of adhesiveness (measured using a radial flow detachment assay). Cells were subjected to 10% pure cyclic uniaxial stretching for three hours at a rate of 10%/sec. Time-lapse images revealed that cells underwent large morphological changes without moving. For each type of protein there was a parabolic dependence on initial adhesiveness with optimal cell orientation occurring at very similar adhesive strengths. The effect of actin-myosin contractile level was examined by stretching cells treated with different doses of 2,3-butanedione monoxime (BDM) and Blebbistatin. Each drug induced a dose-dependent decrease in orientation angles after three hours of cyclic stretching. Furthermore, cell and stress fiber orientations were tightly coupled for untreated and Blebbistatin-treated cells but were uncoupled for BDM-treated cells. Even though orientation response to cyclic stretching is not a spontaneous motile response, it is determined, in large part, by the same factors that affect spontaneous motility--the cell-substratum adhesiveness and actin-myosin contractile level.     

Retinal cytoarchitecture in Some Mountain-stream Teleosts of India

In this study, the cytoarchitecture of retina in 10 mountain-stream teleosts (seven cyprinid and three loach species) of India was examined by conventional light microscopy. The mountain-streams are shallow, cold water bodies having high turbidity in the Monsoon and clear water in the colder months. The incumbent fishes are, thus, periodically exposed to low and bright light of the streams, respectively. It was of particular interest to see whether their retina would reflect adaptations to the changing photic environments of the mountain streams. The retina of the cyprinids possess multiple cone types, a moderate cone density, high convergence ratios and prominent retinomotor responses. Triple cones occur only in the danio, Danio aequipinnatus; other cyprinids possess miniature, short and long single cones, and double or twin cones. In general, the cones are bulky in appearance. Cone droplets (ellipsosomes) are found in Garra lamta and G. gotyla gotyla. All cone types, including the miniature single cones, undergo elongation during dark adaptation. The common retinal features in the loaches are the bulky cones, ellipsosomes, low rod density and low convergence ratios. There is no area-like specialization of the retina in any of the species examined. No relationship is obvious between cone pattern and ambient light or food habit in any of those teleosts studied; row pattern is present throughout the retina of the surface-dwelling, total insectivores (cyprinids: Danio aequipinnatus and Barilius spp.), whereas square mosaic pattern is present in the bottom-dwelling total herbiovores (Garra spp.) as well as in the insectivores (loaches: Nemacheilus spp.). The retinal features have suggested that the cyprinids are well-adapted to both clear and turbid water (ellipsosomes, multiple cone types, bulky cones, high rod count and high convergence ratios); the loaches, on the other hand, are better adapted to clear water of the mountain streams.     

Immunohistochemical Localization of GFAP and Glutamate Regulatory Proteins in Chick Retina and Their Levels of Expressions in Altered Photoperiods

Moderate to intense light is reported to damage the chick retina, which is cone dominated. Light damage alters neurotransmitter pools, such as those of glutamate. Glutamate level in the retina is regulated by glutamate–aspartate transporter (GLAST) and glutamine synthetase (GS). We examined immunolocalization patterns and the expression levels of both markers and of glial fibrillary acidic protein (GFAP, a marker of neuronal stress) in chick retina exposed to 2000 lux under 12-h light:12-h dark (12L:12D; normal photoperiod), 18L:6D (prolonged photoperiod), and 24L:0D (constant light) at post-hatch day 30. Retinal damage (increased death of photoreceptors and inner retinal neurons and Müller cell hypertrophy) and GFAP expression in Müller cells were maximal in 24L:0D condition compared to that seen in 12L:12D and 18L:6D conditions. GS was present in Müller cells and GLAST expressed in Müller cell processes and photoreceptor inner segments. GLAST expression was decreased in 24L:0D condition, and the expression levels between 12L:12D and 18L:6D, though increased marginally, were statistically insignificant. Similar was the case with GS expression that significantly decreased in 24L:0D condition. Our previous study with chicks exposed to 2000 lux reported increased retinal glutamate level in 24L:0D condition. The present results indicate that constant light induces decreased expressions of GLAST and GS, a condition that might aggravate glutamate-mediated neurotoxicity and delay neuroprotection in a cone-dominated retina.     

Metabolic diversity in the grains of Indian varieties of rice

The aim of the present work was to analyze metabolic diversity in 26 different indica varieties of rice grains. Seventy-six metabolites could be identified in the methanol extracts of each of the rice varieties analyzed by gas chromatography-mass spectrometry. These metabolites included 9 sugars/sugar alcohols, 17 amino acids/derivatives, 18 fatty acids, 5 free phenolic acids and 19 other organic acids, 3 phytosterols, 5 other constituents. Cluster analyses to extract information for similarity and differences in metabolites unveiled diversity in metabolite profile. Two hierarchical clusters were generated based on the metabolite contents of the rice varieties. The first cluster (cluster I) consisted of one variety only. The second cluster again segregated into four clusters (clusters II, III, IV and V). Very distinct differences were visible amongst the clusters with respect to their sugars/sugar alcohols, organic acid, amino acid and fatty acid, phenol, and sterol profiles. Metabolites determine nutritional quality, taste, aroma. This and future efforts on the metabolomic information would help biochemists and nutritionists to better understand the nutritional quality of such grains at varietal level and correlating metabolites and long term human health related issues.     

Alpha-amylase in the serum and tissues of rabbits and the effect of vitamin C on its activity

The activity of total alpha-amylase was determined by Caraway's method in the serum and organs of 25 rabbits after administration of physiological salt solution or of vitamin C. The activity in the serum was 4658 U/l, 80% of which was constituted by thermolabile amylase. Among the tissues studied, the highest activity of this enzyme was found in the parotid and pancreas -375.6 U/g and 294.8 U/g respectively. The activity was six times lower in the wall of small intestine, and ten times lower in the liver. The activity of alpha-amylase in the rabbit serum was found to be higher than that in man and it varied under the influence of vitamin C in relation to the dose and administration time. The diurnal oscillations of alpha-amylase activity in serum indicate that the determination of the activity of this enzyme for research or diagnostic purposes should be performed at the same time of day.