Smooth muscle actin determines mechanical force-induced p38 activation

The mitogen-activated protein kinase p38 is activated by mechanical force, but the cellular elements that mediate force-induced p38 phosphorylation are not defined. As alpha-smooth muscle actin (SMA) is an actin isoform associated with force generation in fibroblasts, we asked if SMA participates in the activation of p38 by force. Tensile forces (0.65 pn/mum(2)) generated by magnetic fields were applied to collagen-coated magnetite beads bound to Rat-2 cells. Immunoblotting showed that p38alpha was the predominant p38 isoform. Analysis of bead-associated proteins demonstrated that SMA enrichment of collagen receptor complexes required the alpha2beta1 integrin. SMA was present almost entirely as filaments. Swinholide depolymerized SMA filaments and blocked force-induced p38 phosphorylation and force-induced increases of SMA. Knockdown of SMA (70% reduction) using RNA interference did not affect beta-actin but inhibited force-induced p38 phosphorylation by 50%. Inhibition of Rho kinase blocked SMA filament assembly, force-induced increases of SMA, and force-induced p38 activation. Force application increased SMA content and enhanced the association of phosphorylated p38 with SMA filaments. Blockade of p38 phosphorylation by SB203586 abrogated force-induced increases of SMA. In cells transfected with SMA promoter-beta-galactosidase fusion constructs, co-transfection with constitutively active p38 or MKK6 increased SMA promoter activity by 2.5-3-fold. Dominant negative p38 blocked force-induced activation of the SMA promoter. In SMA negative cells, there was no force-induced p38 phosphorylation. We conclude that force-induced p38 phosphorylation is dependent on an SMA filament-dependent pathway that uses a feed-forward amplification loop to synergize force-induced SMA expression with p38 activation.     

In silico evidence for DNA polymerase-beta's substrate-induced conformational change

Structural information for mammalian DNA pol-beta combined with molecular and essential dynamics studies have provided atomistically detailed views of functionally important conformational rearrangements that occur during DNA repair and replication. This conformational closing before the chemical reaction is explored in this work as a function of the bound substrate. Anchors for our study are available in crystallographic structures of the DNA pol-beta in "open" (polymerase bound to gapped DNA) and "closed" (polymerase bound to gapped DNA and substrate, dCTP) forms; these different states have long been used to deduce that a large-scale conformational change may help the polymerase choose the correct nucleotide, and hence monitor DNA synthesis fidelity, through an "induced-fit" mechanism. However, the existence of open states with bound substrate and closed states without substrates suggest that substrate-induced conformational closing may be more subtle. Our dynamics simulations of two pol-beta/DNA systems (with/without substrates at the active site) reveal the large-scale closing motions of the thumb and 8-kDa subdomains in the presence of the correct substrate--leading to nearly perfect rearrangement of residues in the active site for the subsequent chemical step of nucleotidyl transfer--in contrast to an opening trend when the substrate is absent, leading to complete disassembly of the active site residues. These studies thus provide in silico evidence for the substrate-induced conformational rearrangements, as widely assumed based on a variety of crystallographic open and closed complexes. Further details gleaned from essential dynamics analyses clarify functionally relevant global motions of the polymerase-beta/DNA complex as required to prepare the system for the chemical reaction of nucleotide extension.     

Solvation energy density occlusion approximation for evaluation of desolvation penalties in biomolecular interactions

In calculations involving many displacements of an interacting pair of biomolecules, such as brownian dynamics, the docking of a substrate/ligand to an enzyme/receptor, or the screening of a large number of ligands as prospective inhibitors for a particular receptor site, there is a need for rapid evaluation of the desolvation penalties of the interacting pair. Although continuum electrostatic treatments with distinct dielectric constants for solute and solvent provide an account of the electrostatics of solvation and desolvation, it is necessary to re-solve the Poisson equation, at considerable computational cost, for each displacement of the interacting pair. We present a new method that uses a formulation of continuum electrostatic solvation in terms of the solvation energy density and approximates desolvation in terms of the occlusion of this density. We call it the SEDO approximation. It avoids the need to re-solve the Poisson equation, as desolvation is now estimated by an integral over the occluded volume. Test calculations are presented for some simple model systems and for some real systems that have previously been studied using the Poisson equation approach: MHC class I protein-peptide complexes and a congeneric series of human immunodeficiency virus type 1 (HIV-1) protease--ligand complexes. For most of the systems considered, the trends and magnitudes of the desolvation component of interaction energies obtained using the SEDO approximation are in reasonable correlation with those obtained by re-solving the Poisson equation. In most cases, the error introduced by the SEDO approximation is much less than that of the often-used test-charge approximation for the charge-charge components of intermolecular interactions. Proteins 2001;43:12-27.     

Chromodomain protein Swi6-mediated role of DNA polymerase alpha in establishment of silencing in fission Yeast

Although DNA replication has been thought to play an important role in the silencing of mating type loci in Saccharomyces cerevisiae, recent studies indicate that silencing can be decoupled from replication. In Schizosaccharomyces pombe, mating type silencing is brought about by the trans-acting proteins, namely Swi6, Clr1-Clr4, and Rhp6, in cooperation with the cis-acting silencers. The latter contain an autonomous replication sequence, suggesting that DNA replication may be critical for silencing in S. pombe. To investigate the connection between DNA replication and silencing in S. pombe, we analyzed several temperature-sensitive mutants of DNA polymerase alpha. We find that one such mutant, swi7H4, exhibits silencing defects at mat, centromere, and telomere loci. This effect is independent of the checkpoint and replication defects of the mutant. Interestingly, the extent of the silencing defect in the swi7H4 mutant at the silent mat2 locus is further enhanced in absence of the cis-acting, centromere-proximal silencer. The chromodomain protein Swi6, which is required for silencing and is localized to mat and other heterochromatin loci, interacts with DNA polymerase alpha in vivo and in vitro in wild type cells. However, it does not interact with the mutant pol alpha and is delocalized away from the silent mat loci in the mutant. Our results demonstrate a role of DNA polymerase alpha in the establishment of silencing. We propose a recruitment model for the coupling of DNA replication with the establishment of silencing by the chromodomain protein Swi6, which may be applicable to higher eukaryotes.     

Effects of various media and supplements on laccase production by some white rot fungi

White rot fungi produce three main extracellular enzymes involved in ligninolysis; laccase, lignin peroxidase and manganese peroxidase. Though all white rot fungi do not produce all three enzymes, laccase occupies an important place in ligninolysis. The present paper reports its production by some white rot fungi; Daedalea flavida, Phlebia brevispora, Phlebia radiata and Polyporus sanguineus under different nutritional conditions. Of the various basal media tested, mineral salts malt extract broth proved to be the best medium for laccase production. Sugarcane bagasse proved to be the best laccase inducer among the various supplements added to different media.     

Refolded outer membrane protein A of Escherichia coli forms ion channels with two conductance states in planar lipid bilayers

Outer membrane protein A (OmpA), a major structural protein of the outer membrane of Escherichia coli, consists of an N-terminal 8-stranded beta-barrel transmembrane domain and a C-terminal periplasmic domain. OmpA has served as an excellent model for studying the mechanism of insertion, folding, and assembly of constitutive integral membrane proteins in vivo and in vitro. The function of OmpA is currently not well understood. Particularly, the question whether or not OmpA forms an ion channel and/or nonspecific pore for uncharged larger solutes, as some other porins do, has been controversial. We have incorporated detergent-purified OmpA into planar lipid bilayers and studied its permeability to ions by single channel conductance measurements. In 1 M KCl, OmpA formed small (50-80 pS) and large (260-320 pS) channels. These two conductance states were interconvertible, presumably corresponding to two different conformations of OmpA in the membrane. The smaller channels are associated with the N-terminal transmembrane domain, whereas both domains are required to form the larger channels. The two channel activities provide a new functional assay for the refolding in vitro of the two respective domains of OmpA. Wild-type and five single tryptophan mutants of urea-denatured OmpA are shown to refold into functional channels in lipid bilayers.     

Influence of growth and environmental conditions on cell surface hydrophobicity of Pseudomonas fluorescens in non-specific adhesion

The relative cell surface hydrophobicity (CSH) of 18 soil isolates of Pseudomonas fluorescens, determined by phase exclusion, hydrophobic interaction chromatography (HIC), electrostatic interaction chromatography (ESIC), and contact angle, revealed large degrees of variability. Variation in the adhesion efficiency to Macrophomina phaseolina of the hyphae/sclerotia of these isolates was also examined. Two such isolates with maximum (32.8%; isolate 12-94) and minimum (12%; isolate 30-94) CSH were selected for further study. Early- to mid-log exponential cells of these isolates were more hydrophobic than those in stationary phase, and the CSH of these isolates was also influenced by fluctuations in temperatures and pH. Isolate 12-94 exhibited high CSH (32.3%) at 30 degrees C, compared to lower values (28-24%) in the higher temperature range (35-40 degrees C). Increasing concentrations of either Zn2+, Fe3+, K+, and Mg2+ in the growth medium were associated with the increased CSH. Trypsin, pepsin, and proteinase K (75 to 150 micrograms.mL-1) reduced the CSH of isolate 12-94 cells. CSH was reduced, following exposure to DTT, SDS, Triton X-100, or Tween 80. Prolonged exposure of cells to starvation (60 days) also caused a significant decline in CSH. Several protein bands (18, 21, 23, 26 kDa) of the outer cell membrane were absent in 60-day starved cells compared to unstarved cells. In conclusion, our findings demonstrate that CSH of P. fluorescens isolates may contribute to nonspecific attachment/adhesion onto M. phaseolina hyphae/sclerotia, and the efficiency of adhesion is regulated by growth and other environmental conditions.     

Extraction,profiling and bioactivity analysis of volatile glucosinolates present in oil extract of <Emphasis Type="Italic">Brassica juncea</Emphasis> var. <Emphasis Type="Italic">raya</Emphasis>

Cruciferous vegetables are rich source of glucosinolates (GSLs), which in presence of myrosinase enzyme cause hydrolytic cleavage and result in different hydrolytic products like isothiocyanates, thiocyanates, nitriles and epinitriles. The GSLs hydrolytic products are volatile compounds, which are known to exhibit bioactivities like antioxidant, fungicidal, bioherbicidal and anticancer. Among the Brassicaceae family, Brassica juncea is very well known for high content of GSLs. In the present study, the isolation of volatile oil of B. juncea var. raya was done by hydrodistillation method using clevenger apparatus and further there extraction was done by solvents ethyl acetate and dichloromethane. The volatile compounds present in the extract were analysed by gas chromatography/gas chromatography–mass spectrometry (GC/GC–MS). Fatty acid esters, sulphur and/or nitrogen compounds, carbonyl compounds and some other volatile compounds were also identified. Besides the analytical studies, the extracts were analysed for their bioactivities including radical scavenging activity by using DNA nicking assay and cytotoxic effect using different human cancer cell lines viz. breast (MCF-7 and MDA-MB-231), prostate (PC-3), lung (A-549), cervix (HeLa) and colon (HCT116) by MTT assay. The oil extracts were efficiently able to reduce the increase of cancer cells in a dose-dependent manner. Among all cell lines, the most effective anticancer activity was observed in case of breast (MCF-7) cancer cell line. So, MCF-7 cells were used for further mechanistic studies for analysing the mechanism of anticancer activity. Confocal microscopy was done for analysing morphological changes in the cells and the images confirmed the features typical of apoptosis. For evaluating the mode of cell death, spectrofluorometric determination of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) was done. The volatile oil extract treated MCF-7 cells had a significant increase in number of ROS, also there was a rise in percentage of cells with increased disruption of MMP. So, the present study marks necessary indication that B. juncea (raya) oil extracts significantly induces apoptosis in all the above mentioned cancer cells lines through a ROS-mediated mitochondrial pathway and thus play a remarkable role in death of cancer cells.