Nocardia bhagyanesis sp. nov., a novel actinomycete isolated from the rhizosphere of Callistemon citrinus (Curtis), India

A novel actinomycete strain, designated VRC07^T, was isolated from a Callistemon citrinus rhizosphere sample collected from Hyderabad, India. Its taxonomic status was determined by using polyphasic approach. It is a Gram-positive, aerobic, non-motile, weakly acid-fast strain. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain VRC07^T is a member of the genus Nocardia. The highest levels of 16S rRNA gene sequence similarity was found between the strains Nocardia niwae W9241^T (99.6 %), Nocardia amikacinitolerans W9988^T (99.3 %) and Nocardia arthritidis IFM 10035^T (98.9 %); similarity to other type strains of the genus Nocardia was below 98.7 %. The organism had chemical and morphological features consistent with its classification in the genus Nocardia such as meso-diaminopimelic acid as the diagnostic diamino acid in the cell wall peptidoglycan. Arabinose and galactose as the diagnostic sugars. Diagnostic polar lipids were phosphatidylinositol, diphosphatidylglycerol, and phosphatidylglycerol. The predominant menaquinone was MK-8(H_{4, ω-cycl}). The major fatty acids were C_16:0, C_18:0, C_{18:1 w9c}, C_{18:0 10-methyl TBSA} and sum in feature 3 (16:1 w7c/16:1 w6c). The G+C content of the genomic DNA was 68.5 mol%. The DNA–DNA relatedness data, together with phenotypic differences clearly distinguished the isolate from its closest relatives. On the basis of these phenotypic and genotypic data, the isolate represents a novel species, for which the name Nocardia bhagyanesis sp. nov., is proposed. The type strain is VRC07^T (=KCTC 29209^T = MTCC 11725^T = ATCC BAA-2548).     

Growth and development of catla (Catla catla) fed with different levels of diet containing Spirogyra sp

The effect of Spirogyra sp. incorporated into diet formulations on the growth and body composition of Indian major carp, catla (Catla catla) was investigated in a 45 days feeding trial. Spirogyra dry powder was mixed with different feed ingredients in different amounts (0%, 10%, 25%, 37% and 40% of the total feed). Carps fed with Spirogyra demonstrated higher feed conversion ratio. The study also revealed a direct relationship between the amount of Spirogyra in the diet, and muscle protein and fat contents in the fish. In general, this study demonstrated the benefits of incorporating Spirogyra into carp feeds.     

Subcloning,expression, purification,and characterization of recombinant human leptin-binding domain

A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding.     

Akt signaling pathway in pacing-induced heart failure

Marked changes in energy substrate utilization occur during the progression of congestive heart failure (CHF) where fatty acid utilization, as the primary source of cardiac energy, is severely diminished, oxidative phosphorylation is down-regulated, and glucose uptake and utilization increase. Neither the signaling events or the molecular basis for the shift in substrate utilization have yet been elucidated. This study was designed to examine in the canine model of paced-induced CHF, the potential role of the Akt pathway in signaling the metabolic transitions central to progression to heart failure. Myocardial Akt levels were elevated in early heart failure (after 1–2 weeks of pacing) accompanied by increased levels of oxidative stress, cytokine tumor necrosis factor- (TNF-) and free fatty acid accumulation, reduced activity levels of mitochondrial respiratory complexes III and V and apoptosis initiation. At severe heart failure (3–4 weeks of pacing), there was significant further increase in myocardial apoptosis, with pronounced decline in myocardial Akt kinase activity. At this later stage, there were no further changes in free fatty acid accumulation, complex V activity or in oxidative stress levels indicating that these changes primarily occurred in the earlier stage of evolving heart failure. In contrast, during severe heart failure, both the reduction in complex III activity and increase in TNF- level became more pronounced. Our data provide critical support for the hypothesis that the Akt signaling pathway is a contributory element in the early signaling events leading to the progression of pacing-induced heart failure, accompanying the shift in substrate utilization. (Mol Cell Biochem 268: 103–110, 2005)     

Small molecule scaffolds that disrupt the Rev1-CT/RIR protein-protein interaction

Translesion synthesis (TLS) is a DNA damage tolerance mechanism that allows replicative bypass of DNA lesions, including DNA adducts formed by cancer chemotherapeutics. Previous studies demonstrated that suppression of TLS can increase sensitivity of cancer cells to first-line chemotherapeutics and decrease mutagenesis linked to the onset of chemoresistance, marking the TLS pathway as an emerging therapeutic target. TLS is mediated by a heteroprotein complex consisting of specialized DNA polymerases, including the Y-family DNA polymerase Rev1. Previously, we developed a screening assay to identify the first small molecules that disrupt the protein–protein interaction between the C-terminal domain of Rev1 (Rev1-CT) and the Rev1-interacting region (RIR) present in multiple DNA polymerases involved in TLS. Herein we report additional hit scaffolds that inhibit this key TLS PPI. In addition, through a series of biochemical, computational, and cellular studies we have identified preliminary structure–activity relationships and determined initial pharmacokinetic parameters for our original hits.     

Influence of co-inoculation of bacteria-cyanobacteria on crop yield and C–N sequestration in soil under rice crop

The performance of three selected bacterial strains—PR3, PR7 and PR10 (Providencia sp., Brevundimonas sp., Ochrobacterium sp.) and three cyanobacterial strains CR1, CR2 and CR3 (Anabaena sp., Calothrix sp., Anabaena sp.), and their combinations was evaluated in a pot experiment with rice variety Pusa-1460, comprising 51 treatments along with recommended fertilizer controls. Highest yield enhancement of 19.02% was recorded in T12 (CR2), over control, while significant enhancement in nitrogen fixing potential was recorded in treatments involving combination of bacterial-cyanobacterial strains—T37 (PR3 + CR1 + CR3) and T21 (PR7 + CR1). Organic carbon was significantly increased in all microbe-inoculated treatments, which could be correlated with microbial biomass carbon values and activities of all the enzymes tested in our study. Also, panicle weight and plant biomass were highly correlated with soil microbial carbon. Comparative evaluation revealed the superior performance of strains CR2, CR1 (both Anabaena sp.) and PR10 (Ochrobacterium sp.) in increasing the growth and grain yield of rice and improving soil health, besides N (nitrogen) savings of 40–80 kg ha⁻¹. The study for the first time illustrated the positive effects of co-inoculation of bacterial and cyanobacterial strains for integrated nutrient management of rice crop.     

Human mitochondrial function during cardiac growth and development

Little information is presently available concerning mitochondrial respiratory and oxidative phosphorylation function in the normal human heart during growth and development. We investigated the levels of specific mitochondrial enzyme activities and content during cardiac growth and development from the early neonatal period (10-20 days) to adulthood (67 years). Biochemical analysis of enzyme specific activities and content and mitochondrial DNA (mtDNA) copy number was performed with left ventricular tissues derived from 30 control individuals. The levels of cytochrome c oxidase (COX) and complex V specific activity, mtDNA copy number and COX subunit II content remained unchanged in contrast to increased citrate synthase (CS) activity and content. The developmental increase in CS activity paralleled increasing CS polypeptide content, but was neither related to overall increases in mitochondrial number nor coordinately regulated with mitochondrial respiratory enzyme activities. Our findings of unchanged levels of cardiac mitochondrial respiratory enzyme activity during the progression from early childhood to older adult contrasts with the age-specific regulation found with CS, a Krebs cycle mitochondrial enzyme.     

Rescue of photoreceptor degeneration by curcumin in transgenic rats with P23H rhodopsin mutation

The P23H mutation in the rhodopsin gene causes rhodopsin misfolding, altered trafficking and formation of insoluble aggregates leading to photoreceptor degeneration and autosomal dominant retinitis pigmentosa (RP). There are no effective therapies to treat this condition. Compounds that enhance dissociation of protein aggregates may be of value in developing new treatments for such diseases. Anti-protein aggregating activity of curcumin has been reported earlier. In this study we present that treatment of COS-7 cells expressing mutant rhodopsin with curcumin results in dissociation of mutant protein aggregates and decreases endoplasmic reticulum stress. Furthermore we demonstrate that administration of curcumin to P23H-rhodopsin transgenic rats improves retinal morphology, physiology, gene expression and localization of rhodopsin. Our findings indicate that supplementation of curcumin improves retinal structure and function in P23H-rhodopsin transgenic rats. This data also suggest that curcumin may serve as a potential therapeutic agent in treating RP due to the P23H rhodopsin mutation and perhaps other degenerative diseases caused by protein trafficking defects.     

Study of the Interaction between Bacteriophage T4 asiA and Escherichia coli ς70, Using the Yeast Two-Hybrid System: Neutralization of asiA Toxicity to E. coli Cells by Coexpression of a Truncated ς70 Fragment

The interaction of T4 phage-encoded anti-sigma factor, asiA, and Escherichia coli sigma(70) was studied by using the yeast two-hybrid system. Truncation of sigma(70) to identify the minimum region involved in the interaction showed that the fragment containing amino acid residues proximal to the C terminus (residues 547 to 603) was sufficient for complexing to asiA. Studies also indicated that some of the truncated C-terminal fragments (residues 493 to 613) had higher affinity for asiA as judged by the increased beta-galactosidase activity. It is proposed that the observed higher affinity may be due to the unmasking of the binding region of asiA on the sigma protein. Advantage was taken of the increased affinity of truncated sigma(70) fragments to asiA in designing a coexpression system wherein the toxicity of asiA expression in E. coli could be neutralized and the complex of truncated sigma(70) and asiA could be expressed in large quantities and purified.     

Cloning, overexpression, and characterization of a serine/threonine protein kinase pknI from Mycobacterium tuberculosis H37Rv

Protein phosphorylation-dephosphorylation is the principal mechanism for translation of external signals into cellular responses. Eukaryotic-like serine/threonine kinases have been reported to play important roles in bacterial development and/or virulence. The PknI protein is one of the 11 eukaryotic-like serine/threonine kinases in Mycobacterium tuberculosis H37Rv. From the bioinformatic studies, PknI protein has been shown to have an N-terminal cytoplasmic domain followed by a transmembrane region and an extracellular C-terminus suggestive of a sensor molecule. In this study, we have cloned, overexpressed, and characterized the entire coding region and the cytoplasmic domain of PknI as a fusion protein with an N-terminal histidine tag, and used immobilized metal affinity chromatography for purification of recombinant proteins. The purified recombinant proteins were found to be functionally active through in vitro phosphorylation assay and phosphoamino acid analysis. In vitro kinase assay of both proteins revealed that PknI is capable of autophosphorylation and showed manganese-dependent activity. Phosphoamino acid analysis indicated phosphorylation at serine and threonine residues. Southern blot analysis with genomic DNA highlighted the conserved nature of pknI among the various mycobacterial species. In silico analysis revealed a close homology of PknI to Stk1 from Streptococcus agalactiae, shown to have a role in virulence and cell segregation of the organism.