Bacopasaponins with cytotoxic activity against human breast cancer cells in vitro

Molecular Biology Reports - Globally, breast cancer is a serious concern that exhibits a persistent rise in its incidence and related mortality even after significant advancement in the field of...     

Effect of tertiary amine local anesthetics on G protein-coupled receptor lateral diffusion and actin cytoskeletal reorganization

Although widely used clinically, the mechanism underlying the action of local anesthetics remains elusive. Direct interaction of anesthetics with membrane proteins and modulation of membrane physical properties by anesthetics are plausible mechanisms proposed, although a combination of these two mechanisms cannot be ruled out. In this context, the role of G protein-coupled receptors (GPCRs) in local anesthetic action is a relatively new area of research. We show here that representative tertiary amine local anesthetics induce a reduction in two-dimensional diffusion coefficient of the serotonin_1A receptor, an important neurotransmitter GPCR. The corresponding change in mobile fraction is varied, with tetracaine exhibiting the maximum reduction in mobile fraction, whereas the change in mobile fraction for other local anesthetics was not appreciable. These results are supported by quantitation of cellular F-actin, using a confocal microscopic approach previously developed by us, which showed that a pronounced increase in F-actin level was induced by tetracaine. These results provide a novel perspective on the action of local anesthetics in terms of GPCR lateral diffusion and actin cytoskeleton reorganization.     

Extracellular matrix proteome of chickpea (Cicer arietinum L.) illustrates pathway abundance, novel protein functions and evolutionary perspect

The extracellular matrix (ECM) or cell wall is a dynamic system and serves as the first line mediator in cell signaling to perceive and transmit extra- and intercellular signals in many pathways. Although ECM is a conserved compartment ubiquitously present throughout evolution, a compositional variation does exist among different organisms. ECM proteins account for 10% of the ECM mass, however, comprise several hundreds of different molecules with diverse functions. To understand the function of ECM proteins, we have developed the cell wall proteome of a crop legume, chickpea (Cicer arietinum). This comprehensive overview of the proteome would provide a basis for future comparative proteomic efforts for this important crop. Proteomic analyses revealed new ECM proteins of unknown functions vis-à-vis the presence of many known cell wall proteins. In addition, we report here evidence for the presence of unexpected proteins with known biochemical activities, which have never been associated with ECM.     

The affinity of copper binding to the prion protein octarepeat domain: evidence for negative cooperativity

The prion protein (PrP) binds Cu(2+) in its N-terminal octarepeat domain, composed of four or more tandem PHGGGWGQ segments. Previous work from our laboratory demonstrates that copper interacts with the octarepeat domain through three distinct coordination modes at pH 7.4, depending upon the precise ratio of Cu(2+) to protein. Here, we apply both electron paramagnetic resonance (EPR) and fluorescence quenching to determine the copper affinity for each of these modes. At low copper occupancy, which favors multiple His coordination, the octarepeat domain binds Cu(2+) with a dissociation constant of 0.10 (+/-0.08) nM. In contrast, high copper occupancy, involving coordination through deprotonated amide nitrogens, exhibits a weaker affinity characterized by dissociation constants in the range of 7.0-12.0 microM. Decomposition of the EPR spectra reveals the proportions of all coordination species throughout the copper concentration range and identifies significant populations of intermediates, consistent with negative cooperativity. At most copper concentrations, the Hill coefficient is less than 1.0 and approximately 0.7 at half copper occupancy. These findings demonstrate that the octarepeat domain is responsive to a remarkably wide copper concentration range covering approximately 5 orders of magnitude. Consideration of these findings, along with the demonstrated ability of the protein to quench copper redox activity at high occupancy, suggests that PrP may function to protect cells by scavenging excess copper.     

Polyphenolics and Energy Content in Phytoplankton: Evidence from a Freshwater Lake

Investigation was carried out in the ancient man-made, shallow, freshwater lake, Lake Krishnasayer, at Burdwan (23°16′N, 87°54′E), India, to note the variations in physico-chemical parameters, nutrients, phytoplankton communities, concentration of phenols and energy content in phytoplanktons during January–December 2006. The lake was represented by 16, 15, 2 and 17 genera of Cyanophyceae, Chlorophyceae, Euglenophyceae and Bacillariophyceae, respectively with significant variation in relative abundance as was revealed through Discriminant Analysis. Wilk’s λ values indicate that nitrate, phosphate, dissolved chlorides and silica, and phenol content were significant (P < 0.05) predictor for such differences. PCA analysis yielded three components explaining more than 68 % of the variations in the data on physico-chemical parameters, nutrients, phenol concentration and energy content of total phytoplankton. The interactions of the environmental variables contribute significantly to the observed variations in the phenol and energy content of the phytoplankton. However, the phenomenon of temporal variation in the total phytoplankton and energy content is accounted as a function of nitrate, phosphate-phosphorous, silicate and organic carbon levels. It is apparent that this quality of phytoplanktons varied with the seasons and thereby influencing the amount of phenol and energy content in the engulfing phytoplanktons. Therefore, adequate allocation is needed regarding nutritional status of phytoplanktons, in respect to the dietary need of aquaculture programme in practice.     

Structure and function of the repressor of bacteriophage lambda

Summary By mutagenizing a cIts (cI857) lysogen, a mutant has been isolated with a wild-type phenotype. This mutant phage lysogenizes with low efficiency and produces a low burst. Though the initial rates of repressor synthesis in Escherichia coli after infection with wild-type and mutant are the same, the maximum level of repressor that is synthesized in the latter case is only about 30% of that synthesized in the former. Virulent plates on the lysogen of mutant with slightly less efficiency producing very tiny plaques. Operator-binding studies made in vitro with purified mutant and wild-type repressors show that the binding curve of the former repressor is a rectangular hyperbola while that of the latter is sigmoid. The half-lives of the complexes of mutant and wild-type repressors with right operator are 133 and 27 min, respectively. All these results suggest that the mutant repressor possibly has a higher affinity for the operators. This mutant has been named cIha (ha=high affinity).     

Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.     

Identification of Arabidopsis rat mutants

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various “reverse genetic” approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.