Immuno capture PCR for rapid and sensitive identification of pathogenic Bacillus anthracis

Immuno capture PCR (IPCR) is a technique capable of detecting the pathogens with high specificity and sensitivity. Rapid and accurate detection of Bacillus anthracis was achieved using anti-EA1 antibodies to capture the cells and two primer sets targeting the virulence factors of the pathogen i.e., protective antigen (pag) and capsule (cap) in an IPCR format. Monoclonal antibodies specific to B. anthracis were generated against extractable antigen 1 protein and used as capture antibody onto 96 well polystyrene plates. Following the binding of the pathogen, the DNA extraction was carried out in the well itself and further processed for PCR assay. We compared IPCR described here with conventional duplex PCR using the same primers and sandwich ELISA using the monoclonal antibodies developed in the present study. IPCR was capable of detecting as few as 10 and 100 cfu ml^?1 of bacterial cells and spores, respectively. IPCR was found to be 2–3 logs more sensitive than conventional duplex PCR and the sandwich ELISA. The effect of other bacteria and any organic materials on IPCR was also analyzed and found that this method was robust with little change in the sensitivity in the presence of interfering agents. Moreover, we could demonstrate a simple process of microwave treatment for spore disruption which otherwise are resistant to chemical treatments. Also, the IPCR could clearly distinguish the pathogenic and nonpathogenic strains of B. anthracis in the same assay. This can help in saving resources on unnecessary decontamination procedures during false alarms.     

Centrobin-tubulin interaction is required for centriole elongation and stability

Centrobin is a daughter centriole protein that is essential for centrosome duplication. However, the molecular mechanism by which centrobin functions during centriole duplication remains undefined. In this study, we show that centrobin interacts with tubulin directly, and centrobin-tubulin interaction is pivotal for the function of centrobin during centriole duplication. We found that centrobin is recruited to the centriole biogenesis site via its interaction with tubulins during the early stage of centriole biogenesis, and its recruitment is dependent on hSAS-6 but not centrosomal P4.1-associated protein (CPAP) and CP110. The function of centrobin is also required for the elongation of centrioles, which is likely mediated by its interaction with tubulin. Furthermore, disruption of centrobin-tubulin interaction led to destabilization of existing centrioles and the preformed procentriole-like structures induced by CPAP expression, indicating that centrobin-tubulin interaction is critical for the stability of centrioles. Together, our study demonstrates that centrobin facilitates the elongation and stability of centrioles via its interaction with tubulins.     

Purification and partial characterization of a toxin produced by Ganoderma lucidum,the coconut Ganoderma disease pathogen

Abstract

An extracellular, hydrophilic, thermostable phytotoxin was purified to homogeneity from culture fluids of Ganoderma lucidum. The phytotoxin was purified by solvent extraction, gel filtration on Sephadex G-75. Toxicity was evaluated with detached leaf sheath and electrolyte leakage bioassays. Purified phytotoxin induced visible symptoms of the disease, when applied to coconut leaves, fronds and roots even at a low concentration. The toxin is a glycoprotein with carbohydrate as the major component. The importance of the carbohydrate moiety for toxic activity was indicated by inactivation of toxic compounds after periodate oxidation. The toxin caused lesions on a number of other monocots and dicots and proved to be non-host specific.     

Proteomic identification of glyceraldehyde 3-phosphate dehydrogenase as an inhibitory target of hydrogen peroxide in Arabidopsis.

Hydrogen peroxide (H2O2) is now recognised as a key signalling molecule in eukaryotes. In plants, H2O2 is involved in regulating stomatal closure, gravitropic responses, gene expression and programmed cell death. Although several kinases, such as oxidative signal-inducible 1 (OXI1) kinase and mitogen-activated protein kinases are known to be activated by exogenous H2O2, little is known about the proteins that directly react with H2O2. Here, we utilised a proteomic approach, using iodoacetamide-based fluorescence tagging of proteins in conjunction with mass spectrometric analysis, to identify several proteins that might be potential targets of H2O2 in the cytosolic fraction of Arabidopsis thaliana, the most prominent of which was cytosolic glyceraldehyde 3-phosphate dehydrogenase (cGAPDH; EC 1.2.1.12). cGAPDH from Arabidopsis is inactivated by H2O2 in vitro, and this inhibition is reversible by the subsequent addition of reductants such as reduced glutathione (GSH). It has been suggested recently that Arabidopsis GAPDH has roles outside of its catalysis as part of glycolysis, while in other systems this includes that of mediating reactive oxygen species (ROS) signalling. Here, we suggest that cGAPDH in Arabidopsis might also have such a role in mediating ROS signalling in plants.     

Neutrophilic phagocytosis by tumor cells. A feature to consider in the differential diagnosis in fine needle aspiration smears

OBJECTIVE: To analyze neutrophilic phagocytosis by tumor cells in fine needle aspirate (FNA) smears from different types of tumor. STUDY DESIGN: A retrospective review of a total of 7 cases showing prominent neutrophilic phagocytosis by tumor cells in FNA smears during the period July 2003-December 2004. RESULTS: This feature was seen in malignant fibrous histiocytoma and poorly differentiated renal cell carcinoma in addition to giant cell carcinoma of the lung. CONCLUSION: Neutrophilic phagocytosis by tumor cells is seen in FNA smears and on cytomorphology. The differential diagnoses should include both pleomorphic sarcomas and carcinomas.     

Regulation of plasminogen activators in human thyroid follicular cells and their relationship to differentiated function

Human thyroid cells in culture take up and organify (125)I when cultured in TSH (acting through cAMP) and insulin. They also secrete urokinase (uPA) and tissue-type (tPA) plasminogen activators (5-100 IU/10(6)cells/day). TSH and insulin both decreased secreted PA activity (PAA), uPA and tPA protein and their mRNAs. Autocrine fibroblast growth factor increased secreted PAA and inhibited thyroid cell (125)I uptake. Epidermal growth factor (EGF) and the protein kinase C (PKC) activator, TPA significantly increased PAA and inhibited thyroid differentiated function, (TPA > EGF). For TPA, effects were rapid, increased PAA secretion and decreased (125)I uptake being seen at 4 h whereas for EGF, a 24 h incubation was required. qRT-PCR showed significantly increased mRNA expression of uPA with lesser effects on tPA. Aprotinin, which inhibits PAA, increased (125)I uptake but did not abrogate the effects of TPA and EGF. The MEKK inhibitor, PD98059 partially reversed the effects of EGF and TPA on PAA, and largely reversed the effects of EGF but not TPA on differentiated function. PKC inhibitors bisindoylmaleimide 1, and the specific PKCbeta inhibitor, LY379196 completely reversed the effects of TPA on (125)I uptake and PAA whereas EGF effects were unaffected. TPA inhibited follicle formation and this effect was blocked by LY379196 but not PD98059. We conclude that in thyroid cells, MAPK activation inversely correlates with (125)I uptake and directly correlates with PA expression, in contrast to the effects of cAMP. TPA effects on iodide metabolism, dissolution of follicles and uPA synthesis are mediated predominantly through PKCbeta whereas EGF exerts its effects through MAPK but not PKCbeta.     

Bioactive chemical constituents of Caesalpinia bonduc (Fabaceae)

Phytochemical studies on the ethanolic extracts of Caesalpinia bonduc (Fabaceae) yielded two new homoisoflavonoids, caesalpinianone, and 6-O-methylcaesalpinianone along with five known natural products, namely, hematoxylol, stereochenol A, 6′-O-acetylloganic acid, 4′-O-acetylloganic acid, and 2-O-β-d-glucosyloxy-4-methoxybenzenepropanoic acid. Structures of these compounds were elucidated with the aid of extensive NMR spectral studies. All of these compounds exhibited different levels of glutathione S-transferase (GST) inhibitory and antifungal activities.     

Chronic spinal cord injury impairs primary antibody responses but spares existing humoral immunity in mice

Spinal cord injury (SCI) results in immune depression. To better understand how injury inhibits humoral immunity, the effects of chronic thoracic SCI on B cell development and immune responses to thymus-independent type 2 and thymus-dependent Ags were determined. Mice received complete crush injury or control laminectomy at either thoracic level 3, which disrupts descending autonomic control of the spleen, or at thoracic level 9, which conserves most splenic sympathetic activity. Although mature B cell numbers were only mildly reduced, bone marrow B cell production was transiently but profoundly depressed immediately after injury. Despite the return of normal B cell production 4 wk after SCI, mice receiving thoracic level 3 injury showed a significant reduction in their ability to mount primary thymus-independent type 2 or thymus-dependent immune responses. The latter were marked by decreases in germinal center B cells as well as class-switched high-affinity Ab-secreting cells. Importantly, injury did not affect affinity maturation per se, pre-existing B cell memory, or secondary humoral immune responses. Taken together, these findings show that chronic high thoracic SCI impairs the ability to mount optimal Ab responses to new antigenic challenges, but spares previously established humoral immunity.     

Influence of oxidatively modified LDL on monocyte-macrophage differentiation

Cellular cytoskeletal remodeling reflects alterations in local biochemical and mechanical changes in terms of stress that manifests relocation of signaling molecules within and across the cell. Although stretching due to load and chemical changes by high homocysteine (HHcy) causes cytoskeletal re-arrangement, the synergism between stretch and HHcy is unclear. We investigated the contribution of HHcy in cyclic stretch-induced focal adhesion (FA) protein redistribution leading to cytoskeletal re-arrangement in mouse aortic endothelial cells (MAEC). MAEC were subjected to cyclic stretch (CS) and HHcy alone or in combination. The redistribution of FA protein, and small GTPases were determined by Confocal microscopy and Western blot techniques in membrane and cytosolic compartments. We found that each treatment induces focal adhesion kinase (FAK) phosphorylation and cytoskeletal actin polymerization. In addition, CS activates and membrane translocates small GTPases RhoA with minimal effect on Rac1, whereas HHcy alone is ineffective in both GTPases translocation. However, the combined effect of CS and HHcy activates and membrane translocates both GTPases. Free radical scavenger NAC (N-Acetyl-Cysteine) inhibits CS and HHcy-mediated FAK phosphorylation and actin stress fiber formation. Interestingly, CS also activates and membrane translocates another FA protein, paxillin in HHcy condition. Cytochalasin D, an actin polymerization blocker and PI3-kinase inhibitor Wortmannin inhibited FAK phosphorylation and membrane translocation of paxillin suggesting the involvement of PI3K pathway. Together our results suggest that CS- and HHcy-induced oxidative stress synergistically contribute to small GTPase membrane translocation and focal adhesion protein redistribution leading to endothelial remodeling.