Characterization of the P protein-binding domain on the 10-kilodalton protein of Borna disease virus

The Borna disease virus (BDV) is the prototype member of the Bornaviridae, and it replicates in the cell nucleus. The BDV p24P and p40N proteins carry nuclear localization signals (NLS) and are found in the nuclei of infected cells. The BDV p10 protein does not have an NLS, but it binds with P and/or N and is translocated to the nucleus. Hence, p10 may play a role in the replication of BDV in the cell nucleus. Here, we show that the P-binding domain is located in the N terminus of p10 and that S(3) and L(16) are important for the interaction.     

SorCS2 Controls Functional Expression of Amino Acid Transporter EAAT3 and Protects Neurons from Oxidative Stress and Epilepsy-Induced Pathology

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Optimization of nitrilase production from Alcaligenes faecalis MTCC 10757 (IICT-A3): effect of inducers on substrate specificity

Microbial nitrilases are biocatalysts of interest and the enzyme produced using various inducers exhibits altered substrate specificity, which is of great interest in bioprocess development. The aim of the present study is to investigate the nitrilase-producing Alcaligenes faecalis MTCC 10757 (IICT-A3) for its ability to transform various nitriles in the presence of different inducers after optimization of various parameters for maximum enzyme production and activity. The production of A. faecalis MTCC 10757 (IICT-A3) nitrilase was optimum with glucose (1.0%), acrylonitrile (0.1%) at pH 7.0. The nitrilase activity of A. faecalis MTCC 10757 (IICT-A3) was optimum at 35 °C, pH 8.0 and the enzyme was stable up to 6 h at 50 °C. The nitrilase enzyme produced using different inducers was investigated for substrate specificity. The enzyme hydrolyzed aliphatic, heterocyclic and aromatic nitriles with different substitutions. Acrylonitrile was the most preferred substrate (~40 U) as well as inducer. Benzonitrile was hydrolyzed with almost twofold higher relative activity than acrylonitrile when it was used as an inducer. The versatile nitrilase-producing A. faecalis MTCC 10757 (IICT-A3) exhibits efficient conversion of both aliphatic and aromatic nitriles. The aromatic nitriles, which show not much or no affinity towards nitrilase from A. faecalis, are hydrolyzed effectively with this nitrilase-producing organism. Studies are in progress to exploit this organism for synthesis of industrially important compounds.     

Studies on N2-fixing bacteria associated with the salt-tolerant grass,Leptochloa fusca (L.) Kunth

Summary N₂-fixing bacteria were isolated from the rhizosphere of naturally grown salt tolerant grass (Leptochloa fusca). A broad spectrum of diazotrophs was found to be associated with the roots ofL. fusca. the systematic position of the three isolates, NIAB-1, C-2 and Iso-2 was determined by morphological, biochemical and mol % (G+C) DNA contents. Two isolates were identified asKlebsiella pneumoniae (NIAB-1) andBeijerinckia sp. (Iso-2).¹⁵N enrichment studies confirmed the nitrogen fixing ability of the isolates. The effects of different levels of combined nitrogen (NO ₃ ^– & NH ₄ ⁺ ), pH (5.5–9.0) and salt (NaCl) on nitrogenase activity of the isolates were determined at various time intervals. All isolates exhibited nitrogenase activity even in the presence of 5 mmol/l NO ₃ ^– or NH ₄ ⁺ in a semi-solid medium after 24 h of growth. Maximum nitrogenase activity was observed at alkaline pH and all isolates were able to tolerate up to 3% NaCl in the medium.

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Resumen Se han aíslado bacterias fijadoras de N₂ en la rizosfera del hábitat natural de la graminea halófilaLeptochloa fusca. Un amplio espectro de diazotrofos se encontró asociado con las raíces deL. fusca. La posición sistemática de tres aíslados: NIAB-1, C-2 y Iso-2 se determinó utilizando sus características morfológicas, bioquímicas y el % (G+C) molar del ADN. El aíslado NIAB-1 se identificó comoKlebsiella pneumoniae y el aíslado Iso-2 comoBeijerinckia. sp. Estudios mediante¹⁵N confirmaron la habilidad fijadora de N₂ de los aíslados. Se determinaron periodicamente los efectos de distintos niveles de nitrógeno combinado (NO₃ ^– y NH₄ ⁺), pH (5.5–9.0) y sal (NaCl) en la actividad nitrogenásica de los aíslados. Todas las cepas aísladas mostraron actividad nitrogenásica incluso en presencia de 5mmol/l de NO₃ ^– y NH₄ ⁺ en un medio semisólido desqués de 24 h. de crecimiento. La actividad nitrogenásica máxima se observó a pH alcalino y todos loa aíslados eran capaces de tolerar hasta 3% de NaCl en el medio.

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Résumé Des bactéries fixatrices de l'azote ont été isolées à partir de la rhizosphère de l'herbe halotoléranteLeptochloa fusca développée dans les conditions naturelles. Il a été constaté qu'un large spectre de diazotrophes est associé aux racines de la plante. La position taxonomique de 3 souches isolées, NIAB-1, C-2 et Iso-2, a été déterminée par des critères morphologiques et biochimiques et par le pourcentage de (G+C) de l'ADN. Deux souches on été identifiées commeKlebsiella pneumoniae (NIAB-1) etBeijerinckia sp. (Iso-2). Les études d'enrichissement en¹⁵N ont confirmé l'aptitude des souches à fixer l'azote. les effets de différents niveaux d'azote combiné (NO₃ ^– et NH₄ ⁺), de pH (5.5–9.0) et de sel (NaCl) sur l'activité nitrogénasique des souches ont été déterminés à divers intervalles de temps. Toutes les souches présentent une activité nitrogènase après 24 h de croissance en milieu semi-solide, et cela même en présence de 5 mmol/l de NO₃ ^– ou NH₄ ⁺. L'activité nitrogènase maximum est observée à pH alcalin, et toutes les souches tolèrent jusqu'à 3% de NaCl dans le milieu.

     

Marker Gene Controversy in Transgenic Plants

Biosafety implications of selectable marker genes that are integrated into the transgenic plants are discussed. In the laboratory, selectable marker genes are used at two stages to distinguish transformed cells out of a large population of nontransformed cells: 1) initial assembly of gene cassettes is generally done in E. coli on easily manipulatable plasmid vectors that contain the selectable marker genes which often code for antibiotic inactivating enzymes, and 2) Then the gene cassettes are inserted into the plant genome by various transformation methods. For selection of transformed plant cells, antibiotic and herbicide resistance genes are widely used. Consequently, transgenic plants can end up with DNA sequences of selectable markers that are functional in E. coli and plants. The potential for horizontal gene transfer of selectable markers from transgenic plants to other organisms both in the environment and in the intestine of humans and animals is evaluated. Mechanisms and consequences of the transfer of marker genes from plants to other organisms is examined. Strategies to avoid marker genes in plants are discussed. It is possible to avoid the use of controversial selectable markers in the construction of transgenic plants.     

Citric acid production from sugar cane molasses by 2-deoxyglucose-resistant mutant strain ofAspergillus niger

Citric acid production from sugar cane molasses byAspergillus niger NIAB 280 was studied in a batch cultivation process. A maximum of 90 g/L total sugar was utilized in citric acid production medium. From the parental strainA. niger, mutant strains showing resistance to 2-deoxyglucose in Vogal's medium containing molasses as a carbon source were induced by γ-irradiation. Among the new series of mutant strains, strain RP7 produced 120 g/L while the parental strain produced 80 g/L citric acid (1.5-fold improvement) from 150 g/L of molasses sugars. The period of citric acid production was shortened from 10 d for the wild-type strain to 6–7 d for the mutant strain. The efficiency of substrate uptake rate with respect to total volume substrate consumption rate,Q _s (g per L per h) and specific substrate consumption rate,q _s (g substrate per g cells per h) revealed that the mutant grew faster than its parent. This indicated that the selected mutant is insensitive to catabolite repression by higher concentrations of sugars for citric acid production. With respect to the product yield coefficient (Y _p/x), volume productivity (Q _p) and specific product yields (q _p), the mutant strain is significantly (p≤0.05) improved over the parental strain.     

Expression, purification, and characterization of a structurally disordered and functional C-terminal autoinhibitory domain (AID) of the 70 kDa 40S ribosomal protein S6 kinase-1 (S6K1)

S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (HM; T389) peptide regions of its catalytic kinase domain (residues 1-398). In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID; residues 399-502), which prevents T-loop and HM phosphorylation; and autoinhibition is relieved on multi-site Ser-Thr phosphorylation of the AID (S411, S418, T421, and S424). Interestingly, 66 of the 104 C-terminal AID amino acid residues were computer predicted to exist in structurally disordered peptide regions, begetting interest as to how such dynamics could be coupled to autoregulation. To begin addressing this issue, we developed and optimized protocols for efficient AID expression and purification. Consistent with computer predictions, aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in (1)H-(15)N HSQC NMR spectra, indicated purified recombinant AID to be largely unfolded. Yet, trans-addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1. Using an identical purification protocol, similar protein yields of a tetraphospho-mimic mutant AID(D(2)ED) construct were obtained; and this construct displayed only weak inhibition of PDK1-catalyzed T229 phosphorylation. Purification of the structurally 'disordered' and functional C-terminal AID and AID(D(2)ED) constructs will facilitate studies aimed to understand the role of conformational plasticity and protein phosphorylation in modulating autoregulatory domain-domain interactions.     

Community-Based Network Study of Protein-Carbohydrate Interactions in Plant Lectins Using Glycan Array Data

Lectins play major roles in biological processes such as immune recognition and regulation, inflammatory responses, cytokine signaling, and cell adhesion. Recently, glycan microarrays have shown to play key roles in understanding glycobiology, allowing us to study the relationship between the specificities of glycan binding proteins and their natural ligands at the omics scale. However, one of the drawbacks in utilizing glycan microarray data is the lack of systematic analysis tools to extract information. In this work, we attempt to group various lectins and their interacting carbohydrates by using community-based analysis of a lectin-carbohydrate network. The network consists of 1119 nodes and 16769 edges and we have identified 3 lectins having large degrees of connectivity playing the roles of hubs. The community based network analysis provides an easy way to obtain a general picture of the lectin-glycan interaction and many statistically significant functional groups.