Locking and unlocking of ribosomal motions

During the ribosomal translocation, the binding of elongation factor G (EF-G) to the pretranslocational ribosome leads to a ratchet-like rotation of the 30S subunit relative to the 50S subunit in the direction of the mRNA movement. By means of cryo-electron microscopy we observe that this rotation is accompanied by a 20 A movement of the L1 stalk of the 50S subunit, implying that this region is involved in the translocation of deacylated tRNAs from the P to the E site. These ribosomal motions can occur only when the P-site tRNA is deacylated. Prior to peptidyl-transfer to the A-site tRNA or peptide removal, the presence of the charged P-site tRNA locks the ribosome and prohibits both of these motions.     

Non-timber forest products (NTFPs): a viable option for biodiversity conservation and livelihood enhancement in central Himalaya

The present study aims to document detail information of some of the selected wild edible having enormous potential for livelihood enhancement and socio-economic development by making a variety of value added products. To this end, some of the wild edibles of central Himalaya were selected and prioritized for harnessing their economic potential along with their detail information in terms of distribution, ethnobiology, phenophases and appropriate time of harvesting so as to make communities well aware about the resource availability and their harvesting period round the year. The cost-benefit analysis of each value added product prepared from selected wild edibles was worked out in detail and these analyses revealed that total monetary output, as well as the net return, is very high for all value added products prepared. Since wild edible fruits or other edible parts can be collected from wild free of cost except labour is involved in collection of these wild edibles bio-resources. In addition, information on a participatory action research framework & approaches for promoting participatory conservation of these wild edible species were also highlighted for appropriate management of these resources. The present attempt provides a practical example of sustainable utilization of wild edibles, their potential in livelihood improvement of local people, distribution and phenophases and availability in natural conditions, participatory conservation of these wild edibles may help policy planners at the regional and national levels to link livelihood/socio-economic development with conservation.     

Decon2LS: An open-source software package for automated processing and visualization of high resolution mass spectrometry data

Background  

Data generated from liquid chromatography coupled to high-resolution mass spectrometry (LC-MS)-based studies of a biological sample can contain large amounts of biologically significant information in the form of proteins, peptides, and metabolites. Interpreting this data involves inferring the masses and abundances of biomolecules injected into the instrument. Because of the inherent complexity of mass spectral patterns produced by these biomolecules, the analysis is significantly enhanced by using visualization capabilities to inspect and confirm results. In this paper we describe Decon2LS, an open-source software package for automated processing and visualization of high-resolution MS data. Drawing extensively on algorithms developed over the last ten years for ICR2LS, Decon2LS packages the algorithms as a rich set of modular, reusable processing classes for performing diverse functions such as reading raw data, routine peak finding, theoretical isotope distribution modelling, and deisotoping. Because the source code is openly available, these functionalities can now be used to build derivative applications in relatively fast manner. In addition, Decon2LS provides an extensive set of visualization tools, such as high performance chart controls.     

Identification and characterization of the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana

Despite the importance of riboflavin as the direct precursor of the cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), the physiologically relevant catalyst dephosphorylating the riboflavin biosynthesis pathway intermediate 5‐amino‐6‐ribitylamino‐2,4(1H,3H) pyrimidinedione 5′‐phosphate (ARPP) has not been characterized from any organism. By using as the query sequence a previously identified plastidial FMN hydrolase AtcpFHy1 (At1g79790), belonging to the haloacid dehalogenase (HAD) superfamily, seven candidates for the missing ARPP phosphatase were found, cloned, recombinantly expressed, and purified. Activity screening showed that the enzymes encoded by AtcpFHy1, At4g11570, and At4g25840 catalyze dephosphorylation of ARPP. AtcpFHy1 was renamed AtcpFHy/PyrP1, At4g11570 and At4g25840 were named AtPyrP2 and AtGpp1/PyrP3, respectively. Subcellular localization in planta indicated that AtPyrP2 was localized in plastids and AtGpp1/PyrP3 in mitochondria. Biochemical characterization of AtcpFHy/PyrP1 and AtPyrP2 showed that they have similar K_m values for the substrate ARPP, with AtcpFHy/PyrP1 having higher catalytic efficiency. Screening of 21 phosphorylated substrates showed that AtPyrP2 is specific for ARPP. Molecular weights of AtcpFHy/PyrP1 and AtPyrP2 were estimated at 46 and 72 kDa, suggesting dimers. pH and temperature optima for AtcpFHy/PyrP1 and AtPyrP2 were ~7.0–8.5 and 40–50°C. T‐DNA knockout of AtcpFHy/PyrP1 did not affect the flavin profile of the transgenic plants, whereas silencing of AtPyrP2 decreased accumulation of riboflavin, FMN, and FAD. Our results strongly support AtPyrP2 as the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana. The identification of this enzyme closes a long‐standing gap in understanding of the riboflavin biosynthesis in plants.     

Contribution of “substrate shuttles” in the transport of extramitochondrial reducing equivalents by hepatic mitochondria from chronic alcohol-fed mice

Effects of chronic alcohol treatment have been investigated on the rates of extramitochondrial NADH utilization by hepatic mitochondria in the presence or absence of “malate-aspartate shuttle,” oxidation of ethanol, α-glycerophosphate, and the activity of succinic dehydrogenase, along with the changes in the intrahepatic distribution of aspartate aminotransferase. The rates of blood alcohol clearance, hepatic alcohol dehydrogenase activity, and NADPH-dependent microsomal ethanol oxidation were also studied after different time intervals of alcohol withdrawal from chronically alcohol-fed animals. Hepatic mitochondria from chronically ethanol-fed mice (ethanol withheld 20 hr before sacrifice) utilized extramitochondrial NADH at rates 25–40% higher than the corresponding pair-fed controls. Addition of malateaspartate shuttle components to mitochondria from control and ethanol-fed groups resulted in 70 and 90% stimulation of NADH utilization, respectively. Mitochondria from both groups showed respiratory control upon ADP addition (state 3). Preincubation with amino-oxyacetate or hydrazine, which inhibit aspartate aminotransferase activity, prevented the stimulatory effect of malate-aspartate shuttle on NADH utilization. Mitochondria from livers of chronic ethanol-fed mice in the presence of reconstituted malate-aspartate shuttle showed 30–40% higher utilization of ethanol than the corresponding pair-fed control animals. The rate of mitochondrial α-glycerophosphate utilization by alcohol-fed animals was significantly higher than the control group. Succinic dehydrogenase activity measured as an index of mitochondrial permeability in the absence of Ca²⁺ showed 85% higher activity in alcoholtreated group than the control animals. Chronic ethanol feeding for 4 weeks resulted in an increase in the activity of hepatic aspartate aminotransferase in the cytoplasmic fraction and a corresponding decrease in the mitochondrial fraction. Alcohol withdrawal from chronic alcohol-fed animals resulted in a decrease in the blood alcohol clearance rate after 10 days. Furthermore, a lack of correlation was observed between the rates of blood alcohol clearance and the activity of hepatic alcohol dehydrogenase on one hand, and between the rates of blood alcohol clearance and the microsomal ethanol-oxidizing activity on the other.     

Two ellagitannins from the stem bark of Caesalpinia pulcherrima

Two ellagitannins have been isolated from the stem bark of C. pulcherrima. These tannins have been assigned structures with glucose as the carbohydrate core, esterified with two galloyl and one hexahydroxydiphenoyl group and with a galloyl, a hexahydroxydiphenoyl and a m-digalloyl group, respectively.     

Floral ontogeny inMazus pumilus (Scrophulariaceae)

InMazus pumilus, all the floral appendages are initiated in acropetal sequence in the second cell layer (except stamens) of the floral primordium by periclinal divisions. The actinomorphic calyx tube is formed due to zonal growth. The zygomorphy in corolla is evident from the inception of petal primordia which arise sequentially as independent units in order of one anterior, a pair of anterio-lateral followed by a pair of posterio-lateral. Later these primordia exhibit differential growth because of which zygomorphy becomes more pronounced. The upper corolla tube is formed by interprimordial growth and lower corolla tube by zonal growth. Stamens are initiated in the third layer of the floral apex. Unlike sepals and petals, in the development of stamens (4) underlying cells of corpus also contribute. Posterior stamen is absent. The stamens become epipetalous because of interprimordial and zonal growth in the common region below the bases of petals as well as stamens. The two carpel primordia arise as crescent shaped structures which become continuous due to interprimordial growth. The ovary is formed by a ring of zonal meristem. The style develops later between stigma and ovary because of intercalary growth. The residual apex grows vertically along with the ovary and forms the septum of the ovary. All the floral appendages exhibit similar pattern of histogenesis and early growth suggesting thereby the appendicular nature of these appendages.     

Moonlighting protein in Starkeyomyces koorchalomoides: Characterization of dihydrolipoamide dehydrogenase as a protein acetyltransferase utilizing acetoxycoumarin as the acetyl group donor

In this report we have identified for the first time a transacetylase (TAase) in a mesophilic fungi Starkeyomyces koorchalomoides catalyzing the transfer of acetyl group from polyphenolic acetate (PA) to a receptor protein glutathione S-transferase (GST). An elegant assay procedure was established for TAase based on its ability to mediate inhibition of GST by 7,8-diacetoxy-4-methylcoumarin (DAMC), a model PA. Utilizing this assay procedure, S. koorchalomoides TAase was purified to homogeneity. TAase was found to have MW of 50 kDa. The purified enzyme exhibited maximum activity at 45 °C at pH 6.8. The N-terminal sequence of purified fungal TAase (ANDASTVED) showed identity with corresponding N-terminal sequence of dihydrolipoamide dehydrogenase (LADH), a mitochondrial matrix enzyme and an E3 component of pyruvate dehydrogenase complex (PDHC). TAase was found to have all the properties of LADH and avidly interacted with the anti-LADH antibody. TAase catalyzed acetylation of GST by DAMC was identified by LC–MS/MS and a single lysine residue (Lys-113) was found to be acetylated. Further, recombinant LADH from Streptococcus pneumoniae lacking lipoyl domain was found to exhibit little TAase activity, suggesting the role of lipoyl domain in the TAase activity of LADH. These observations bear evidence for the protein acetyltransferase activity of LADH. Such an activity of LADH can be attributed as a moonlighting function of the enzyme.     

Response surface optimization for enhanced production of cellulases with improved functional characteristics by newly isolated Aspergillus niger HN-2

Fungi isolated from partially decayed wood log samples showing characteristic diversity for spore colour, colony morphology and arrangement of spores were assessed for cellulolytic enzyme production. Isolates showing a cellulolytic index of ≥2.0 were assayed for filter paper (FP) cellulase and β-glucosidase (BGL) production. Molecular characterization confirmed the identity of the selected cellulolytic isolate as a strain of Aspergillus niger (A. niger HN-2). Addition of 2 % (w/v) urea enhanced FP and BGL activity by about 20 and 60 %, respectively. Validation studies conducted at parameters (29 °C, pH 5.4, moisture content 72 % and 66 h) optimized through response surface methodology in a solid-state static tray fermentation resulted in FP, BGL, cellobiohydrolase I (CBHI), endoglucanase (EG), xylanase activity and protein content of 25.3 FPU/g ds, 750 IU/g ds, 13.2 IU/g ds, 190 IU/g ds, 2890 IU/g ds and 0.9 mg/ml, respectively. In comparison, A. niger N402 which is a model organism for growth and development studies, produced significantly lower FP, BGL, CBHI, EG, xylanase activity and protein content of 10.0 FPU/g ds, 100 IU/g ds, 2.3 IU/g ds, 50 IU/g ds, 500 IU/g ds and 0.75 mg/ml, respectively under the same process conditions as were used for A. niger HN-2. Process optimization led to nearly 1.8- and 2.2-fold increase in FP and BGL activity, respectively showing promise for cellulase production by A. niger HN-2 at a higher scale of operation. Zymogram analysis revealed two isoforms each for EG and cellobiohydrolase and three isoforms for BGL. Crude cellulase complex produced by A. niger HN-2 exhibited thermostability under acidic conditions showing potential for use in biofuel industry.