A high-throughput method for enzyme kinetic studies

A simple and flexible setup for conducting drug metabolism studies is described in this report. A heating block was designed for the Multimek liquid handler platform for incubation of multiple samples at 37 degrees C in a 96-well format. This setup enables the rapid performance of drug metabolism experiments on a large number of samples. In this report, the authors present the validation of the system by 1) showing reproducible and consistent determination of the in vitro half-life of midazolam in every well across the entire plate and 2) determination of metabolic parameter values of midazolam, testosterone, diclofenac, warfarin, and dextromethorphan and inhibition parameter values of quinidine and ketoconazole, all comparable to literature values. In addition, the authors demonstrate the application of the setup to determining the metabolic stability of a set of proprietary compounds, the inhibition of activity of cytochrome P450 (CYP) enzymes, and the conduct of a single combination experiment that can simultaneously determine the metabolic stability and CYP inhibition activity. Overall, the system represents a simple, high-throughput and useful tool for drug metabolism screening in drug discovery.     

Theaflavins induced apoptosis of LNCaP cells is mediated through induction of p53, down-regulation of NF-kappa B and mitogen-activated protein kinases pathways

Prostate cancer (PCA), the most frequently diagnosed malignancy in men, represents an excellent candidate disease for chemoprevention studies because of its particularly long latency period, high rate of mortality and morbidity. Infusion of black tea and its polyphenolic constituents have been shown to possess antineoplastic effects in androgen dependent PCA in both in vivo and in vitro models including transgenic animals. In the present study, we report that black tea polyphenol, Theaflavins (TF)-induced apoptosis in human prostate carcinoma, LNCaP cells is mediated via modulation of two related pathways: up-regulation of p53 and down-regulation of NF-kappa B activity, causing a change in the ratio of pro-and antiapoptotic proteins leading to apoptosis. The altered expression of Bcl-2 family member proteins triggered the release of cytochrome-C and activation of initiator capsase 9 followed by activation of effector caspase 3. Furthermore, TF also affected the protein expression of mitogen activated protein kinases (MAPK) pathways. Our results demonstrated that TF treatment resulted in down-regulation of phospho-extracellular signal-regulated protein kinase (Erk1/2) and phospho-p38 MAPK expressions. We conclude that TF induces apoptosis in LNCaP cells by shifting the balance between pro-and antiapoptotic proteins and down-regulation of cell survival pathways leading to apoptosis. Further extending this work, we also showed that TF induces apoptosis in androgen independent PCA cell line, PC-3 through caspases and MAPKs mediated pathways. Thus, effect of TF on PCA cell lines seems to be irrespective of their androgen status.     

Insights into eukaryotic evolution from transmembrane domain lengths

Biological membranes, comprised of proteins anchored by their trans-membrane domains (TMDs) creating a semi-permeable phase with lipid constituents, serve as ‘checkposts’ for not only intracellular trafficking in eukaryotic cells but also for material transactions of all living cells with external environments. Hydropathy (or hydrophobicity) plots of ‘bitopic’ proteins (i.e. having single alpha-helical TMDs) are routinely utilized in biochemistry texts for predicting their TMDs. The number of amino acids (i.e. TMD length) embedded as alpha-helices may serve as indicators of thickness of biological membranes in which they reside under assumptions that are universally applied for fixing window sizes for identifying TMDs using hydropathy plots. In this work we explore variations in thickness of different eukaryotic biological membranes (reflected by TMD lengths of their resident proteins) over evolutionary time scales. Rigorous in silico analyses of over 23,000 non-redundant membrane proteins residing in different subcellular locations from over 200 genomes of fungi, plants, non-mammalian vertebrates and mammals, reveal that differences in plasma membrane and organellar TMD lengths have decreased over time (scales) of eukaryotic cellular evolution. While earlier work has indicated decreasing differences in TMD lengths with increasing ‘perceived’ organismal complexity, this work is the first report on TMD length variations as a function of evolutionary time of eukaryotic cellular systems. We report that differences in TMD lengths of bitopic proteins residing in plasma membranes and other intra-cellular locations have decreased with evolutionary time, suggesting better/more avenues of intracellular trafficking in the emergence of eukaryotic organisms.     

Apical cell protrusions cause vertical deformation of the soft cancer nucleus

Breast cancer nuclei have highly irregular shapes, which are diagnostic and prognostic markers of breast cancer progression. The mechanisms by which irregular cancer nuclear shapes develop are not well understood. Here we report the existence of vertical, apical cell protrusions in cultured MDA-MB-231 breast cancer cells. Once formed, these protrusions persist over time scales of hours and are associated with vertically upward nuclear deformations. They are absent in normal mammary epithelial cells (MCF-10A cells). Microtubule disruption enriched these protrusions preferentially in MDA-MB-231 cells compared with MCF-10A cells, whereas inhibition of nonmuscle myosin II (NMMII) abolished this enrichment. Dynamic confocal imaging of the vertical cell and nuclear shape revealed that the apical cell protrusions form first, and in response, the nucleus deforms and/or subsequently gets vertically extruded into the apical protrusion. Overexpression of lamin A/C in MDA-MB-231 cells reduced nuclear deformation in apical protrusions. These data highlight the role of mechanical stresses generated by moving boundaries, as well as abnormal nuclear mechanics in the development of abnormal nuclear shapes in breast cancer cells.     

Propensity of <Emphasis Type="Italic">Withania somnifera</Emphasis> to Attenuate Behavioural,Biochemical, and Histological Alterations in Experimental Model of Stroke

The present study was designed to evaluate the beneficial effects of Withania somnifera (WS) pre-supplementation on middle cerebral artery occlusion (MCAO) model of ischemic stroke. Ischemic stroke was induced in the rats by inserting intraluminal suture for 90 min, followed by reperfusion injury for 24 h. The animals were assessed for locomotor functions (by neurological deficit scores, narrow beam walk and rotarod test), cognitive and anxiety-like behavioural functions (by morris water maze and elevated plus maze test). MCAO animals showed significant impairment in locomotor and cognitive functions. Neurobehavioural changes were accompanied by decreased acetylcholinesterase activity, increased oxidative stress in terms of enhanced lipid peroxidation and lowered thiol levels in the MCAO animals. In addition, MCAO animals had cerebral infarcts and the presence of pycnotic nuclei. Single-photon emission computerized tomography (SPECT) of MCAO animals revealed a cerebral infarct as a hypoactive area. On the other hand, pre-supplementation with WS (300 mg/kg body weight) for 30 days to MCAO animals was effective in restoring the acetylcholinesterase activity, lipid peroxidation, thiols and attenuated MCAO induced behavioural deficits. WS significantly reduced the cerebral infarct volume and ameliorated histopathological alterations. Improved blood flow was observed in the SPECT images from the brain regions of ischemic rats pre-treated with WS. The results of the study showed a protective effect of WS supplementation in ischemic stroke and are suggestive of its potential application in stroke management.     

Biomass production,nutrient cycling,and carbon fixation by Salicornia brachiata Roxb.: A promising halophyte for coastal saline soil rehabilitation

In order to increase our understanding of the interaction of soil-halophyte (Salicornia brachiata) relations and phytoremediation, we investigated the aboveground biomass, carbon fixation, and nutrient composition (N, P, K, Na, Ca, and Mg) of S. brachiata using six sampling sites with varying characteristics over one growing season in intertidal marshes. Simultaneously, soil characteristics and nutrient concentrations were also estimated. There was a significant variation in soil characteristics and nutrient contents spatially (except pH) as well as temporally. Nutrient contents in aboveground biomass of S. brachiata were also significantly differed spatially (except C and Cl) as well as temporally. Aboveground biomass of S. brachiata ranged from 2.51 to 6.07 t/ha at maturity and it was positively correlated with soil electrical conductivity and available Na, whereas negatively with soil pH. The K/Na ratio in plant was below one, showing tolerance to salinity. The aboveground C fixation values ranged from 0.77 to 1.93 C t/ha at all six sampling sites. This study provides new understandings into nutrient cycling—C fixation potential of highly salt-tolerant halophyte S. brachiata growing on intertidal soils of India. S. brachiata have a potential for amelioration of the salinity due to higher Na bioaccumulation factor.     

Observations on sporozoite detection in naturally infected sibling species of the <Emphasis Type="Italic">Anopheles culicifacies</Emphasis> complex and variant of <Emphasis Type="Italic">Anopheles stephensi</Emphasis> in India

Sporozoites were detected in naturally infected sibling species of the primary rural vector Anopheles culicifacies complex in two primary health centres (PHCs) and a variant of the urban vector Anopheles stephensi in Mangalore city, Karnataka, south India while carrying out malaria outbreak investigations from 1998–2006. Sibling species of An. culicifacies were identified based on the banding patterns on ovarian polytene chromosomes, and variants of An. stephensi were identified based on the number of ridges on the egg floats. Sporozoites were detected in the salivary glands by the dissection method. Of the total 334 salivary glands of An. culicifacies dissected, 17 (5.08%) were found to be positive for sporozoites. Of the 17 positive samples, 11 were suitable for sibling species analysis; 10 were species A (an efficient vector) and 1 was species B (a poor vector). Out of 46 An. stephensi dissected, one was sporozoite positive and belonged to the type form (an efficient vector). In malaria epidemiology this observation is useful for planning an effective vector control programme, because each sibling species/variant differs in host specificity, susceptibility to malarial parasites, breeding habitats and response to insecticides.     

Development of hydrogels for regenerative engineering

The aim of regenerative engineering is to restore complex tissues and biological systems through convergence in the fields of advanced biomaterials, stem cell science, and developmental biology. Hydrogels are one of the most attractive biomaterials for regenerative engineering, since they can be engineered into tissue mimetic 3D scaffolds to support cell growth due to their similarity to native extracellular matrix. Advanced nano‐ and micro‐technologies have dramatically increased the ability to control properties and functionalities of hydrogel materials by facilitating biomimetic fabrication of more sophisticated compositions and architectures, thus extending our understanding of cell‐matrix interactions at the nanoscale. With this perspective, this review discusses the most commonly used hydrogel materials and their fabrication strategies for regenerative engineering. We highlight the physical, chemical, and functional modulation of hydrogels to design and engineer biomimetic tissues based on recent achievements in nano‐ and micro‐technologies. In addition, current hydrogel‐based regenerative engineering strategies for treating multiple tissues, such as musculoskeletal, nervous and cardiac tissue, are also covered in this review. The interaction of multiple disciplines including materials science, cell biology, and chemistry, will further play an important role in the design of functional hydrogels for the regeneration of complex tissues.     

Chronic cholesterol depletion increases F-actin levels and induces cytoskeletal reorganization via a dual mechanism

Previous work from us and others has suggested that cholesterol is an important lipid in the context of the organization of the actin cytoskeleton. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol is rarely addressed in the literature. In this work, we explored the signaling crosstalk between cholesterol and the actin cytoskeleton by using a high-resolution confocal microscopic approach to quantitatively measure changes in F-actin content upon cholesterol depletion. Our results show that F-actin content significantly increases upon chronic cholesterol depletion, but not during acute cholesterol depletion. In addition, utilizing inhibitors targeting the cholesterol biosynthetic pathway at different steps, we show that reorganization of the actin cytoskeleton could occur due to the synergistic effect of multiple pathways, including prenylated Rho GTPases and availability of membrane phosphatidylinositol 4,5-bisphosphate. These results constitute one of the first comprehensive dissections of the mechanistic basis underlying the interplay between cellular actin levels and cholesterol biosynthesis. We envision these results will be relevant for future understating of the remodeling of the actin cytoskeleton in pathological conditions with altered cholesterol.     

Biaxial incremental homeostatic elastic moduli of coronary artery: two-layer model

The detailed mechanical properties of various layers of the coronary artery are important for understanding the function of the vessel. The present article is focused on the determination of the incremental modulus in different layers and directions in the neighborhood of the in vivo state. The incremental modulus can be defined for any material subjected to a large deformation if small perturbations in strain lead to small perturbations of stresses in a linear fashion. This analysis was applied to the porcine coronary artery, which was treated as a two-layered structure consisting of an inner intima-media layer and an outer adventitia layer. We adopted a theory based on small-perturbation experiments at homeostatic conditions for determination of incremental moduli in circumferential, axial, and cross directions in the two layers. The experiments were based on inflation and axial stretch. We demonstrate that under homeostatic conditions the incremental moduli are layer- and direction dependent. The incremental modulus is highest in the circumferential direction. Furthermore, in the circumferential direction, the media is stiffer than the whole wall, which is stiffer than the adventitia. In the axial direction, the adventitia is stiffer than the intact wall, which is stiffer than the media. Hence, the coronary artery must be treated as a composite, nonisotropic body. The data acquire physiological relevance in relation to coronary artery health and disease.