BAD,a Proapoptotic Protein,Escapes ERK/RSK Phosphorylation in Deguelin and siRNA-Treated HeLa Cells

This study has been undertaken to explore the therapeutic effects of deguelin and specific siRNAs in HeLa cells. The data provided clearly show the silencing of ERK 1/2 with siRNAs and inhibition of ERK1/2 with deguelin treatment in HeLa cells. Additionally, we are providing information that deguelin binds directly to anti-apoptotic Bcl-2, Bcl-xl and Mcl-1 in the hydrophobic grooves, thereby releasing BAD and BAX from dimerization with these proteins. This results in increased apoptotic activity through the intrinsic pathway involved in rupture of mitochondrial membrane and release of cytochrome C. Evidence for inhibition of ERK1/2 by deguelin and escape of BAD phosphorylation at serine 112 through ERK/RSK pathway has been further fortified by obtaining similar results by silencing ERK 1/2 each with specific siRNAs. Increase in BAD after treatment with deguelin or siRNAs has been interpreted to mean that deguelin acts through several alternative pathways and therefore can be used as effective therapeutic agent.     

Phylogenetic patterns and genetic diversity of Indian Tinospora species based on chloroplast sequence data and cytochrome P450 polymorphisms

Based on morphology, the species status and taxonomic affinities of three species of Tinospora (T. cordifolia, T. sinensis, and T. crispa) with ranges in India, have been questioned. To evaluate species delimitation and population structure among 40 accessions of the three species, a relatively new marker, cytochrome P450, was used. Five out of nine primers generated polymorphisms with 39 out of 47 bands polymorphic. The P450 binary data, when analyzed using distance methods, strongly supported the monophyly of each Indian species and were congruent with previous RAPD work. To further investigate the status of these species, we combined P450 and RAPD data. The resulting unrooted phylogram highly supports the monophyly of each species but with little population structure within each species. To understand the phylogenetic placement of the three Indian Tinospora species within Menispermaceae, chloroplast atpB and rbcL sequence data for a large sampling of the family were analyzed using likelihood and parsimony methods. The resulting phylogenies highly support the Indian Tinospora species as part of a clade (expanded Tinosporeae), consisting of diverse Menispermaceae from around the world. The three Indian species are monophyletic and are most closely related to Tinospora species from Australia (T. esiangkara and T. smilacina).     

Phosphoproteome sequence analysis and significance: Mining association patterns around phosphorylation sites utilizing MAPRes

Phosphorylation, one of the most common protein post‐translational modifications (PTMs) on hydroxyl groups of S/T/Y is catalyzed by kinases and involves the presence or absence of certain amino acid residues in the vicinity of the phosphorylation sites. Using MAPRes, we have analyzed the substrate proteins of Phospho.ELM 7.0 and found that there are both general and specific requirements for the presence or absence of particular amino acids in the vicinity of phosphorylated S/T/Y for both of the phosphorylation data, whether or not kinase information was taken into account. Patterns extracted by MAPRes for kinase‐specific data have been utilized to find the consensus sequence motifs for various kinases required to catalyze the process of phosphorylation on S/T/Y. These consensus sequences for different kinase groups, families, and individual members are consistent with those described earlier with some novel consensus reported for the first time. A comparison study for the patterns mined by MAPRes with the results of existing prediction methods was performed by searching for these patterns in the vicinity of phosphorylation sites predicted by different available method. This comparison resulted in 87–98% conformity with the results of the predictions by available methods. Additionally, the patterns mined by MAPRes for substrate sites included 61 kinases, the highest number analyzed so far. J. Cell. Biochem. 108: 64–74, 2009. © 2009 Wiley‐Liss, Inc.     

Molecular characterization of bacterial population in the forest soil of Kashmir,India

The bacterial diversity in the forest soil of Kashmir, India was investigated by 16S rDNA-dependent molecular phylogeny. Small subunit rRNA (16S rDNA) from forest soil metagenome were amplified by polymerase chain reaction (PCR) using primers specific to the domain bacteria. 30 unique phylotypes were obtained by PCR based RFLP of 16S rRNA genes using endonucleases Hae 111 and Msp 1, which were most suitable to score the genetic diversity. The use of 16S rRNA analysis allowed identification of several bacterial populations in the soil belonging to the following phyla: Firmicutes (33.3%), Bacteroidetes (13.3%), Proteobacterium (6.6%), Planctomycete (3.3%), and Deferribacteraceae (3.3%) in addition to the others that were not classified, beyond Archaea domain, However, 36.6% of the retrieved bacterial sequences could not be grouped with any phylum/lineage. The large amount of unclassified clone sequence could imply that novel groups of bacteria were present in the forest soil.     

A PCR-based method, with internal control, for the detection of Banana bunchy top virus in banana

Banana bunchy top disease is a major constraint to banana production in most regions where this crop is grown. The disease is caused by Banana bunchy top virus (BBTV), a multicomponent, single-stranded DNA virus of the family Nanoviridae. We have designed primers to a conserved region of the master replication-associated protein that are useful for the polymerase chain reaction (PCR)-mediated detection of BBTV. In addition, primers to banana genomic sequence are used as an internal control, overcoming the uncertainty (owing to false-negatives) inherent in PCR diagnostics. Together these primer sets are a valuable tool in the effort to control BBTV, particularly in screening micropropagated banana plantlets for the absence of virus before release to farmers.     

Calcium‐permeable AMPA receptors and silent synapses in cocaine‐conditioned place preference

Exposure to cocaine generates silent synapses in the nucleus accumbens (NAc), whose eventual unsilencing/maturation by recruitment of calcium‐permeable AMPA‐type glutamate receptors (CP‐AMPARs) after drug withdrawal results in profound remodeling of NAc neuro‐circuits. Silent synapse‐based NAc remodeling was shown to be critical for several drug‐induced behaviors, but its role in acquisition and retention of the association between drug rewarding effects and drug‐associated contexts has remained unclear. Here, we find that the postsynaptic proteins PSD‐93, PSD‐95, and SAP102 differentially regulate excitatory synapse properties in the NAc. Mice deficient for either of these scaffold proteins exhibit distinct maturation patterns of silent synapses and thus provided instructive animal models to examine the role of NAc silent synapse maturation in cocaine‐conditioned place preference (CPP). Wild‐type and knockout mice alike all acquired cocaine‐CPP and exhibited increased levels of silent synapses after drug‐context conditioning. However, the mice differed in CPP retention and CP‐AMPAR incorporation. Collectively, our results indicate that CP‐AMPAR‐mediated maturation of silent synapses in the NAc is a signature of drug–context association, but this maturation is not required for establishing or retaining cocaine‐CPP.     

Single-chain Fv phage display propensity exhibits strong positive correlation with overall expression levels

Background

Progress in generating comprehensive EST libraries and genome sequencing is setting the stage for reverse genetic approaches to gene function studies in the blacklegged tick (Ixodes scapularis). However, proving that RNAi can work in nervous tissue has been problematic. Developing an ability to manipulate gene expression in the tick synganglia likely would accelerate understanding of tick neurobiology. Here, we assess gene silencing by RNA interference in the adult female black-legged tick synganglia.

Results

Tick β-Actin and Na⁺-K⁺-ATPase were chosen as targets because both genes express in all tick tissues including synganglia. This allowed us to deliver dsRNA in the unfed adult female ticks and follow a) uptake of dsRNA and b) gene disruption in synganglia. In vitro assays demonstrated total disruption of both tick β-Actin and Na⁺-K⁺-ATPase in the synganglia, salivary glands and midguts. When dsRNA was microinjected in unfed adult female ticks, nearly all exhibited target gene disruption in the synganglia once ticks were partially blood fed.

Conclusion

Abdominal injection of dsRNA into unfed adult female ticks appears to silence target gene expression even in the tick synganglia. The ability of dsRNA to cross the blood-brain barrier in ticks suggests that RNAi should prove to be a useful method for dissecting function of synganglia genes expressing specific neuropeptides in order to better assess their role in tick biology.     

Citral derived amides as potent bacterial NorA efflux pump inhibitors

Monoterpene citral and citronellal have been used as starting material for the preparation of 5,9-dimethyl-deca-2,4,8-trienoic acid amides and 9-formyl-5-methyl-deca-2,4,8-trienoic acid amides. The amides on bioevaluation as efflux pump inhibitors (EPIs) against Staphylococcus aureus 1199 and NorA overexpressing S. aureus 1199B bacteria resulted in the identification of several of these as potent EPIs. Many of these amides have been shown to possess potency higher or equivalent to known EPIs such as reserpine, verapamil, carsonic acid, and piperine. In this communication, we report a convenient synthesis of alkenyl amides, their bioevaluation and identification as efflux pump inhibitors against S. aureus.     

Development of gfp Vectors for Expression in Listeria monocytogenes and Other Low G+C Gram Positive Bacteria

The gfp (green fluorescent protein) gene has previously been used to construct a variety of reporter plasmids for Gram-positive bacteria for bacterial localization and gene expression studies. When a native red-shifted gfp variant (gfp3) was cloned into an expression vector using the P _xyn promoter and used to transform the soil-borne pathogen Listeria monocytogenes, only a small proportion of the population was seen to fluoresce when examined by epifluorescence microscopy. When the P _xyn promoter was replaced with the P _xylA promoter, with accompanying modification of the translation initiation region of the gfp3 gene, a homogeneously fluorescent population of cells was obtained. When expressed in other Gram-positive organisms, such as Staphylococcus aureus and Bacillus subtilis, the translationally enhanced gene also resulted in high-level and homogeneous GFP production within the bacterial population. High-level expression of these reporter constructs in L. monocytogenes was evaluated to determine if it had any detrimental biological effect during intracellular infection of eukaryotic cell lines. The gfp3 ⁺ Listeria were found to invade equally as well as the wild-type cells; showing that these expression systems can be used to monitor the bacterium in natural environments. Based on these results, similar translationally enhanced vectors were also developed using unstable GFP3 variants, which retain their short-half life characteristics in L. monocytogenes and therefore can be used as a sensitive monitor of gene expression.     

Steroid biotransformation by different strains ofMicrococcus sp.

A strain ofMicrococcus sp. was isolated for its capability of side chain degradation of cholesterol. This strain was characterized and identified asMicrococcus roseus. It was found to be the best strain for the production of androsta-1,4-diene-3, 17-dione and androst-4-ene-3, 17-dione compared with otherMicrococcus strains.