Kaposi's sarcoma-associated herpesvirus forms a multimolecular complex of integrins (alphaVbeta5, alphaVbeta3, and alpha3beta1) and CD98-xCT during infection of human dermal microvascular endothelial cells, and CD98-xCT is essential for the postentry stage of infection

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface heparan sulfate (HS) and α3β1 integrin during the early stages of infection of human dermal microvascular endothelial cells (HMVEC-d) and human foreskin fibroblasts (HFF), and these interactions are followed by virus entry overlapping with the induction of preexisting host cell signal pathways. KSHV also utilizes the amino acid transporter protein xCT for infection of adherent cells, and the xCT molecule is part of the cell surface heterodimeric membrane glycoprotein CD98 (4F2 antigen) complex known to interact with α3β1 and αVβ3 integrins. KSHV gB mediates adhesion of HMVEC-d, CV-1, and HT-1080 cells and HFF via its RGD sequence. Anti-αV and -β1 integrin antibodies inhibited the cell adhesion mediated by KSHV-gB. Variable levels of neutralization of HMVEC-d and HFF infection were observed with antibodies against αVβ3 and αVβ5 integrins. Similarly, variable levels of inhibition of virus entry into adherent HMVEC-d, 293 and Vero cells, and HFF was observed by preincubating virus with soluble α3β1, αVβ3, and αVβ5 integrins, and cumulative inhibition was observed with a combination of integrins. We were unable to infect HT1080 cells. Virus binding and DNA internalization studies suggest that αVβ3 and αVβ5 integrins also play roles in KSHV entry. We observed time-dependent temporal KSHV interactions with HMVEC-d integrins and CD98/xCT with three different patterns of association and dissociation. Integrin αVβ5 interaction with CD98/xCT predominantly occurred by 1 min postinfection (p.i.) and dissociated at 10 min p.i., whereas α3β1-CD98/xCT interaction was maximal at 10 min p.i. and dissociated at 30 min p.i., and αVβ3-CD98/xCT interaction was maximal at 10 min p.i. and remained at the observed 30 min p.i. Fluorescence microscopy also showed a similar time-dependent interaction of αVβ5-CD98. Confocal-microscopy studies confirmed the association of CD98/xCT with α3β1 and KSHV. Preincubation of KSHV with soluble heparin and α3β1 significantly inhibited this association, suggesting that the first contact with HS and integrin is an essential element in subsequent CD98-xCT interactions. Anti-CD98 and xCT antibodies did not block virus binding and entry and nuclear delivery of viral DNA; however, viral-gene expression was significantly inhibited, suggesting that CD98-xCT play roles in the post-entry stage of infection, possibly in mediating signal cascades essential for viral-gene expression. Together, these studies suggest that KSHV interacts with functionally related integrins (αVβ3, α3β1, and αVβ5) and CD98/xCT molecules in a temporal fashion to form a multimolecular complex during the early stages of endothelial cell infection, probably mediating multiple roles in entry, signal transduction, and viral-gene expression.     

Synthesis of novel steroidal agonists,partial agonists,and antagonists for the glucocorticoid receptor

Adverse effects of glucocorticoids could be limited by developing new compounds that selectively modulate anti-inflammatory activity of the glucocorticoid receptor (GR). We have synthesized a novel series of steroidal GR ligands, including potent agonists, partial agonists and antagonists with a wide range of effects on inhibiting secretion of interleukin-6. Some of these new ligands were designed to directly impact conformational stability of helix-12, in the GR ligand-binding domain (LBD). These compounds modulated GR activity and glucocorticoid-induced gene expression in a manner that was inversely correlated to the degree of inflammatory response. In contrast, compounds designed to directly modulate LBD epitopes outside helix-12, led to dissociated levels of GR-mediated gene expression and inflammatory response. Therefore, these new series of compounds and their derivatives will be useful to dissect the ligand-dependent features of GR signaling specificity.     

Phosphate-buffered saline-based nucleofection of primary endothelial cells

Although various nonviral transfection methods are available, cell toxicity, low transfection efficiency, and high cost remain hurdles for in vitro gene delivery in cultured primary endothelial cells. Recently, unprecedented transfection efficiency for primary endothelial cells has been achieved due to the newly developed nucleofection technology that uses a combination of novel electroporation condition and specific buffer components that stabilize the cells in the electrical field. Despite superior transfection efficiency and cell viability, high cost of the technology has discouraged cardiovascular researchers from liberally adopting this new technology. Here we report that a phosphate-buffered saline (PBS)-based nucleofection method can be used for efficient gene delivery into primary endothelial cells and other types of cells. Comparative analyses of transfection efficiency and cell viability for primary arterial, venous, microvascular, and lymphatic endothelial cells were performed using PBS. Compared with the commercial buffers, PBS can support equally remarkable nucleofection efficiency to both primary and nonprimary cells. Moreover, PBS-mediated nucleofection of small interfering RNA (siRNA) showed more than 90% knockdown of the expression of target genes in primary endothelial cells. We demonstrate that PBS can be an unprecedented economical alternative to the high-cost buffers or nucleofection of various primary and nonprimary cells.     

In-vivo dose measurements with MOSFET dosimeters during MV portal imaging

BackgroundThe purpose of this study was to investigate the feasibility of MOSFET dosimeter in measuring eye dose during 2D MV portal imaging for setup verification in radiotherapy.Materials and methodsThe in-vivo dose measurements were performed by placing the dosimeters over the eyes of 30 brain patients during the acquisition of portal images in linear accelerator by delivering 1 MU with the field sizes of 10 × 10 cm² and 15 × 15 cm².ResultsThe mean doses received by the left and right eyes of 10 out of 30 patients when both eyes were completely inside the anterior portal field were found to be 2.56 ± 0.2 cGy and 2.75 ± 0.2, respectively. Similarly, for next 10 patients out of the same 30 patients the mean doses to left and right eyes when both eyes were completely out of the anterior portal fields were found to be 0.13 ± 0.02 cGy and 0.17 ± 0.02 cGy, respectively. The mean doses to ipsilateral and contralateral eye for the last 10 patients when one eye was inside the anterior portal field were found to be 3.28 ± 0.2 cGy and 0.36 ± 0.1 cGy, respectively.ConclusionThe promising results obtained during 2D MV portal imaging using MOSFET have shown that this dosimeter is well suitable for assessing low doses during imaging thereby enabling to optimize the imaging procedure using the dosimetric data obtained. In addition, the documentation of the dose received by the patient during imaging procedure is possible with the help of an in-built software in conjunction with the MOSFET reader module.     

Whole-Genome Sequences of Two Clinical Isolates of Mycobacterium tuberculosis from Kerala, South India

We report the annotated genome sequence of two clinical isolates of Mycobacterium tuberculosis isolated from Kerala, India.     

Efficient direct plant regeneration from immature leaf roll explants of sugarcane (<Emphasis Type="Italic">Saccharum officinarum</Emphasis> L.) using polyamines and assessment of genetic fidelity by SCoT markers

A rapid and efficient method for in vitro direct plant regeneration from immature leaf roll explants of Saccharum officinarum L. (sugarcane) cv. Co 86032 was developed by the application of exogenous polyamines (PA). The effect of explant source from apical meristems and pre-culture of explants in the dark on shoot regeneration was studied. Adventitious shoot regeneration occurred on the proximal regions of immature leaf roll explants when pre-incubated in the dark for 2 wk and the regeneration response was decreased from the middle to distal end. A higher number of direct shoots (130 primary shoots explant^?1) and multiple shoots (657 secondary shoots explant^?1), were obtained with a combination of spermidine (103.27 μM), spermine (49.42 μM), and putrescine (31.04 μM) along with plant growth regulators. Shoot induction was increased up to twofold and multiplication was increased up to threefold in the medium supplemented with PA. Profuse rooting was observed in putrescine (93.12 μM), spermidine (68.84 μM), and spermine (24.71 μM), with mean number of 57 roots. A twofold increase in the number of roots was observed in medium supplemented with PA with respect to control cultures, which facilitated the successful transplantation and acclimatization process of in vitro propagated sugarcane plants. Histology and scanning electron microscopy analyses supported adventitious direct shoot regeneration from immature leaf roll explants. The genetic stability of in vitro regenerated plants was confirmed using start codon targeted polymorphism marker system.     

Phosphorylation of profilin regulates its interaction with actin and poly (L-proline)

Activation of bovine platelets with thrombin and phorbol 12,13-dibutyrate (PDBu) resulted in phosphorylation of profilin on serine. The phosphorylation was inhibited when platelets were pretreated with the PI 3-kinase inhibitor, LY294002, indicating that profilin phosphorylation is a downstream event with respect to PI 3-kinase activation. Phosphorylation of profilin resulted in significant decrease in actin polymerization (16.5%), indicating an increased affinity of phosphoprofilin towards actin. The critical actin monomer concentration (Cc) increased to 260 nM in the presence of phosphoprofilin in comparison with 200 nM in the presence of profilin. The interaction of phosphoprofilin with phosphatidylinositol 4,5-bisphosphate [PI (4,5)-P2] and poly (L-proline) (PLP) was examined by monitoring the quenching of tryptophan fluorescence. Scatchard plot and binding isotherm data obtained revealed no difference in PI (4,5)-P2 binding between profilin and phosphoprofilin (Kd=20.4 microM), while poly (L-proline)-binding studies indicated a sixfold decrease (27.34 microM for profilin and 4.73 microM for phosphoprofilin) in Kd with phosphoprofilin. In vivo studies with platelets indicated an increased association of p85alpha, the regulatory subunit of PI 3-kinase with phosphoprofilin over profilin. Overall, the data presented conclude that profilin phosphorylated under in vivo conditions and phosphorylation depends upon activation of PI 3-kinase. Phosphoprofilin exhibited increased affinity to poly (L-proline) sequences both in vitro and in vivo.     

Adriamycin induced myocardial failure in rats: Protective role of Centella asiatica

Generation of reactive oxygen species and mitochondrial dysfunction has been implicated in adriamycin induced cardiotoxicity. Mitochondrial dysfunction is characterized by the accumulation of oxidized lipids, proteins and DNA, leading to disorganization of mitochondrial structure and systolic failure. The present study was aimed to evaluate the efficacy of Centella asiatica on the mitochondrial enzymes; mitochondrial antioxidant status in adriamycin induced myocardial injury. Adriamycin (2.5 mg/kg body wt., i.p.) induced mitochondrial damage in rats was assessed in terms of decreased activities (p< 0.05) of cardiac marker enzymes (lactate dehydrogenase, creatine phosphokinase, amino transferases), TCA cycle enzymes (isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, respiratory marker enzymes (NADH-dehydrogenase, cytochrome-C-oxidase), mitochondrial antioxidant enzymes (GPx, GSH, SOD,CAT) and increased (p< 0.05) level of lipid peroxidation. Mitochondrial damage was confirmed by transmission electron microscopic examination. Pre-co-treatment with aqueous extract of Centella asiatica (200 mg/kg body wt, oral) effectively counteracted the alterations in mitochondrial enzymes and mitochondrial defense system. In addition, transmission electron microscopy study confirms the restoration of cellular normalcy and accredits the cytoprotective role of Centella asiatica against adriamycin induced myocardial injury. Our results demonstrated elevated oxidative stress and mitochondrial dysfunction in adriamycin treated rats. Moreover, on the basis of our findings it may be concluded that the aqueous extract of C. asiatica not only possesses antioxidant properties but it may also reduce the extent of mitochondrial damage