Actomyosin Pulls to Advance the Nucleus in a Migrating Tissue Cell

The cytoskeletal forces involved in translocating the nucleus in a migrating tissue cell remain unresolved. Previous studies have variously implicated actomyosin-generated pushing or pulling forces on the nucleus, as well as pulling by nucleus-bound microtubule motors. We found that the nucleus in an isolated migrating cell can move forward without any trailing-edge detachment. When a new lamellipodium was triggered with photoactivation of Rac1, the nucleus moved toward the new lamellipodium. This forward motion required both nuclear-cytoskeletal linkages and myosin activity. Apical or basal actomyosin bundles were found not to translate with the nucleus. Although microtubules dampen fluctuations in nuclear position, they are not required for forward translocation of the nucleus during cell migration. Trailing-edge detachment and pulling with a microneedle produced motion and deformation of the nucleus suggestive of a mechanical coupling between the nucleus and the trailing edge. Significantly, decoupling the nucleus from the cytoskeleton with KASH overexpression greatly decreased the frequency of trailing-edge detachment. Collectively, these results explain how the nucleus is moved in a crawling fibroblast and raise the possibility that forces could be transmitted from the front to the back of the cell through the nucleus.     

Chemical modification of ascorbic acid and evaluation of its lipophilic derivatives as inhibitors of secretory phospholipase A2 with anti-inflammatory activity

This investigation aims to evaluate the antitumor and antioxidant potential of Chrysaora quinquecirrha (sea nettle) nematocyst venom on Ehrlich ascites carcinoma (EAC) tumor model. Tumor was induced in mice by intraperitoneal injection of EAC cells. The antitumor effect of sea nettle nematocyst venom (SNV) peptide was evaluated by assessing in vitro cytotoxicity, survival time, hematological, and antioxidant parameters. Intraperitoneal injection of SNV peptide increased the survival time of the EAC-bearing mice. The SNV peptide brought back the altered levels of the hematological and antioxidant parameters in a dose dependent manner in EAC-bearing mice. The results were comparable to that of the result obtained from the animals treated with the standard drug 5-fluorouracil (20 mg/kg bw). Thus, present study revealed that SNV peptide possessed significant antitumor and antioxidant activity.     

Rapid biodegradation and decolorization of Direct Orange 39 (Orange TGLL) by an isolated bacterium <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain BCH

A newly isolated novel bacterium from sediments contaminated with dyestuff was identified as Pseudomonas aeruginosa strain BCH by 16S rRNA gene sequence analysis. The bacterium was extraordinarily active and operative over a wide rage of temperature (10–60°C) and salinity (5–6%), for decolorization of Direct Orange 39 (Orange TGLL) at optimum pH 7. This strain was capable of decolorizing Direct Orange 39; 50 mg l⁻¹ within 45 ± 5 min, with 93.06% decolorization, while maximally it could decolorize 1.5 g l⁻¹ of dye within 48 h with 60% decolorization. Analytical studies as, UV–Vis spectroscopy, FTIR, HPLC were employed to confirm the biodegradation of dye and formation of new metabolites. Induction in the activities of lignin peroxidases, DCIP reductase as well as tyrosinase was observed, indicating the significant role of these enzymes in biodegradation of Direct Orange 39. Toxicity studies with Phaseolus mungo and Triticum aestivum revealed the non-toxic nature of degraded metabolites.     

Isolation and characterization of alkalotolerant bacteria and optimization of process parameters for decolorization and detoxification of pulp and paper mill effluent by Taguchi approach

Four different bacterial strains were isolated from pulp and paper mill sludge in which one alkalotolerant isolate (LP1) having higher capability to remove color and lignin, was identified as Bacillus sp. by 16S RNA sequencing. Optimization of process parameters for decolorization was initially performed to select growth factors which were further substantiated by Taguchi approach in which seven factors, % carbon, % black liquor, duration, pH, temperature, stirring and inoculum size, at two levels, applying L-8 orthogonal array were taken. Maximum color was removed at pH 8, temperature 35°C, stirring 200 rpm, sucrose (2.5%), 48 h, 5% (w/v) inoculum size and 10% black liquor. After optimization 2-fold increase in color and lignin removal from 25–69% and 28–53%, respectively, indicated significance of Taguchi approach in decolorization and delignification of lignin in pulp and paper mill effluent. Enzymes involved in the process of decolorization of effluent were found to be xylanase (54 U/ml) and manganese peroxidase (28 U/ml). Treated effluent was also evaluated for toxicity by Comet assay using Saccharomyces cerevisiae MTCC 36 as model organism, which indicated 58% reduction after treatment by bacterium.     

Prophylactic and therapeutic use of antibodies for protection against respiratory infection with Francisella tularensis

The role of Abs in protection against respiratory infection with the intracellular bacterium Francisella tularensis is not clear. To investigate the ability of Abs to clear bacteria from the lungs and prevent systemic spread, immune serum was passively administered i.p. to naive mice before intranasal F. tularensis live vaccine strain infection. It was found that immune serum treatment provided 100% protection against lethal challenge while normal serum or Ig-depleted immune serum provided no protection. Protective efficacy was correlated with increased clearance of bacteria from the lung and required expression of FcgammaR on phagocytes, including macrophages and neutrophils. However, complement was not required for protection. In vitro experiments demonstrated that macrophages were more readily infected by Ab-opsonized bacteria but became highly efficient in killing upon activation by IFN-gamma. Consistent with this finding, in vivo Ab-mediated protection was found to be dependent upon IFN-gamma. SCID mice were not protected by passive Ab transfer, suggesting that T cells but not NK cells serve as the primary source for IFN-gamma. These data suggest that a critical interaction of humoral and cellular immune responses is necessary to provide sterilizing immunity against F. tularensis. Of considerable interest was the finding that serum Abs were capable of conferring protection against lethal respiratory tularemia when given 24-48 h postexposure. Thus, this study provides the first evidence for the therapeutic use of Abs in Francisella-infected individuals.     

Isolation and Characterization of Dibenzofuran-Degrading <Emphasis Type="Italic">Serratia marcescens</Emphasis> from Alkalophilic Bacterial Consortium of the Chemostat

Alkalophilic bacterial consortium developed by continuous enrichment in the chemostat in presence of 4-chlorosalicylic acid as sole source of carbon and energy contained six bacterial strains, Micrococcus luteus (csa101), Deinococcus radiothilus (csa102), csa103 (Burkholderia gladioli), Alloiococcus otilis (csa104), Micrococcus diversus (csa105), Micrococcus luteus (csa106), identified by the Biolog test method. The strains were tested for utilization of organic compounds in which one of the strains (csa101) had higher potency to utilize dibenzofuran (DF) as sole carbon and energy source identified as Serratia marcescens on the basis of 16S rDNA. The degradation of DF by bacterial strain proceeded through an oxidative route as indicated by 2,2′3-trihydroxybiphenyl, 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid, salicylic acid, and catechol, which was identified by gas chromatography–mass spectrometry.     

beta2-Adrenergic receptor agonists stimulate L-type calcium current independent of PKA in newborn rabbit ventricular myocytes

Selective stimulation of beta(2)-adrenergic receptors (ARs) in newborn rabbit ventricular myocardium invokes a positive inotropic effect that is lost during postnatal maturation. The underlying mechanisms for this age-related stimulatory response remain unresolved. We examined the effects of beta(2)-AR stimulation on L-type Ca(2+) current (I(Ca,L)) during postnatal development. I(Ca,L) was measured (37 degrees C; either Ca(2+) or Ba(2+) as the charge carrier) using the whole-cell patch-clamp technique in newborn (1 to 5 days old) and adult rabbit ventricular myocytes. Ca(2+) transients were measured concomitantly by dialyzing the cell with indo-1. Activation of beta(2)-ARs (with either 100 nM zinterol or 1 microM isoproterenol in the presence of the beta(1)-AR antagonist, CGP20712A) stimulated I(Ca,L) twofold in newborns but not in adults. The beta(2)-AR-mediated increase in Ca(2+) transient amplitude in newborns was due exclusively to the augmentation of I(Ca,L). Zinterol increased the rate of inactivation of I(Ca,L) and increased the Ca(2+) flux integral. The beta(2)-AR inverse agonist, ICI-118551 (500 nM), but not the beta(1)-AR antagonist, CGP20712A (500 nM), blocked the response to zinterol. Unexpectedly, the PKA blockers, H-89 (10 microM), PKI 6-22 amide (10 microM), and Rp-cAMP (100 microM), all failed to prevent the response to zinterol but completely blocked responses to selective beta(1)-AR stimulation of I(Ca,L) in newborns. Our results demonstrate that in addition to the conventional beta(1)-AR/cAMP/PKA pathway, newborn rabbit myocardium exhibits a novel beta(2)-AR-mediated, PKA-insensitive pathway that stimulates I(Ca,L). This striking developmental difference plays a major role in the age-related differences in inotropic responses to beta(2)-AR agonists.     

Ultrasensitive detection of cortisol with enzyme fragment complementation technology using functionalized nanowire

Cortisol is a member of the glucocorticoid hormone family and a key metabolic regulator. Increased intracellular cortisol levels have been implicated in type 2 diabetes, obesity, and metabolic syndrome. Cortisol is an important bio-marker of stress and its detection is also important in sports medicine. However, rapid methods for sensitive detection of cortisol are limited. Functionalized gold nanowires were used to enhance the sensitivity and selectivity of cortisol detection. Gold nanowires are used to improve the electron transfer between the electrodes. Moreover, the large surface to volume ratio, small diffusion time and high electrical conductivity and their aligned nature will enhance the sensitivity and detection limit of the biosensor several fold. The biosensor was fabricated using, aligned gold (Au) nanowires to behave as the working electrode, platinum deposited on a silicon chip to function as the counter electrode, and silver/silver chloride as reference electrode. The gold nanowires were coupled with cortisol antibodies using covalent linkage chemistry and a fixed amount of 3alpha-hydroxysteroid dehydrogenase was introduced into the reaction cell during each measurement to convert (reduce) ketosteroid into hydroxyl steroid. Furthermore, the micro-fluidic, micro-fluid part of the sensor was fabricated using micro-electro-mechanical system (MEMS) technology to have better control on liquid flow over Au nanowires to minimize the signal to noise ratio. The biosensor was characterized using SEM, AFM and FTIR technique. The response curve of the biosensor was found to be linear in the range of 10-80 microM of cortisol. Moreover, the presence of hydrocortisone is sensitively detected in the range of 5-30 microM. It is concluded that the functionalized gold nanowires with micro-fluidic device using enzyme fragment complementation technology can provide an easy and sensitive assay for cortisol detection in serum and other biological fluids.     

Structure–sequence features based prediction of phosphosites of serine/threonine protein kinases of Mycobacterium tuberculosis

Elucidation of signaling events in a pathogen is potentially important to tackle the infection caused by it. Such events mediated by protein phosphorylation play important roles in infection, and therefore, to predict the phosphosites and substrates of the serine/threonine protein kinases, we have developed a Machine learning-based approach for Mycobacterium tuberculosis serine/threonine protein kinases using kinase-peptide structure–sequence data. This approach utilizes features derived from kinase three-dimensional-structure environment and known phosphosite sequences to generate support vector machine (SVM)-based kinase-specific predictions of phosphosites of serine/threonine protein kinases (STPKs) with no or scarce data of their substrates. SVM outperformed the four machine learning algorithms we tried (random forest, logistic regression, SVM, and k-nearest neighbors) with an area under the curve receiver-operating characteristic value of 0.88 on the independent testing dataset and a 10-fold cross-validation accuracy of ~81.6% for the final model. Our predicted phosphosites of M. tuberculosis STPKs form a useful resource for experimental biologists enabling elucidation of STPK mediated posttranslational regulation of important cellular processes.