Differential regulation of threonine and tyrosine phosphorylations on protein kinase Cdelta by G-protein-mediated pathways in platelets

Phosphorylation of activation loop threonine (Thr(505)) and regulatory domain tyrosine (Tyr(311)) residues are key regulators of PKC (protein kinase C) delta function in platelets. In the present study, we show that G(q) and G(12/13) pathways regulate the Thr(505) and Tyr(311) phosphorylation on PKCdelta in an interdependent manner. DiC8 (1,2-dioctanoylglycerol), a synthetic analogue of DAG (diacylglycerol), caused Thr(505), but not Tyr(311), phosphorylation on PKCdelta, whereas selective activation of G(12/13) pathways by the YFLLRNP peptide failed to cause phosphorylation of either residue. However, simultaneous activation by DiC8 and YFLLRNP resulted in Thr(505) and Tyr(311) phosphorylation on PKCdelta. In addition, we found that the activation of SFKs (Src family tyrosine kinases) is essential for G(12/13)-mediated Tyr(311) phosphorylation of PKCdelta. These results were confirmed using G(q)-deficient mouse platelets. Finally, we investigated whether Thr(505) phosphorylation is required for Tyr(311) phosphorylation. A T505A PKCdelta mutant failed to be phosphorylated at Tyr(311), even upon stimulation of both G(q) and G(12/13) pathways. We conclude that (i) PKCdelta binding to DAG, downstream of G(q) pathways, and its translocation results in Thr(505) phosphorylation, (ii) G(12/13) pathways activate SFKs required for the phosphorylation of Tyr(311) on Thr(505)-phosphorylated PKCdelta, and (iii) Thr(505) phosphorylation is a prerequisite for Tyr(311) phosphorylation on PKCdelta.     

Phosphorylation of multifunctional nucleolar protein nucleophosmin (NPM1) by aurora kinase B is critical for mitotic progression

The functional association of NPM1 with Aurora kinases is well documented. Surprisingly, although NPM1 is a well characterized phosphoprotein, it is unknown whether it is a substrate of Aurora kinases. We have found that Aurora kinases A and B can phosphorylate NPM1 at a single serine residue, Ser125, in vitro and in vivo. Phosphorylated-S125-NPM1 (pS125-NPM1) localizes to the midbody region during late cytokinesis where it colocalizes with Aurora B. The overexpression of mutant (S125A) NPM1 resulted in the deregulation of centrosome duplication and mitotic defects possibly due to cytokinesis failure. These data suggest that Aurora kinase B-mediated phosphorylation of NPM1 plays a critical role during mitosis, which could have wider implications in oncogenesis.     

Heat Shock Protein 90 Positively Regulates Chikungunya Virus Replication by Stabilizing Viral Non-Structural Protein nsP2 during Infection

BackgroundThe high morbidity and socio-economic loss associated with the recent massive global outbreak of Chikungunya virus (CHIKV) emphasize the need to understand the biology of the virus for developing effective antiviral therapies.ConclusionHsp90 positively regulates Chikungunya virus replication by stabilizing CHIKV-nsP2 through its interaction during infection. The study highlights the possible molecular mechanism of GA mediated inhibition of CHIKV replication and differential effect of this drug on S 27 and DRDE-06, which will be informative for developing effective anti-CHIKV therapies in future.     

Cytoskeletal Control of Antigen-Dependent T Cell Activation

1. Download high-res image (160KB)
2. Download full-size image

     

Accumulation of a Natural Substrate of Laccase in Gills of Pleurotus florida During Sporulation

During sporulation, laccase activity of Pleurotus florida decreased to a minimum level in spite of increase in the number of isozymes. An endogenous laccase substrate was detected especially in the gill structure of the sporophore, which competitively inhibited oxidation of guaiacol by the enzyme during in vitro assay. Appearance of the laccase substrate in the gill structure may be linked with the sporulation phenomenon. Received: 21 January 2000 / Accepted: 2 May 2000     

Plasmodium yoelii: novel rhoptry proteins identified within the body of merozoite rhoptries in rodent Plasmodium malaria

The biogenesis, organization and function of the rhoptries are not well understood. Antisera were prepared to synthetic peptides prepared as multiple antigenic peptides (MAPs) obtained from a Plasmodium yoelii merozoite rhoptry proteome analysis. The antisera were used in immunofluorescence and immunoelectron microscopy of schizont-infected erythrocytes. Twenty-seven novel rhoptry proteins representing proteases, metabolic enzymes, secreted proteins and hypothetical proteins, were identified in the body of the rhoptries by immunoelectron microscopy. The merozoite rhoptries contain a heterogeneous mixture of proteins that may initiate host cell invasion and establish intracellular parasite development.     

A novel nitric oxide producing probiotic patch and its antimicrobial efficacy: preparation and in vitro analysis

Microbial and fungal infections are a significant consideration in the etiology of all wounds. Numerous antimicrobial and antifungal formulations have been developed with varying degrees of efficacy and stability. Here, we report a nitric oxide producing probiotic adhesive patch device and investigate its antimicrobial and antifungal efficacy in vitro. This probiotic patch utilizes the metabolic activity of immobilized lactic acid bacteria, glucose, and nitrite salts for the production of gaseous nitric oxide (gNO), which is used as an antimicrobial agent against bacterial and fungal pathogens. Results show that application of gNO-producing probiotic patches to cultures of E. coli, S. aureus, P. aeruginosa, MRSA, T. mentagrophytes, and T. rubrum resulted in complete cell death at between 4 and 8 h, and application to cultures of A. baumannii, resulted in fewer than ten colonies detected per milliliter at 6 h. These results demonstrate that a gNO-producing probiotic patch device containing bacteria, glucose, and nitrite salts can produce sufficient levels of gNO over a therapeutically relevant duration to kill common bacterial and fungal wound pathogens in humans.     

Recombinant Baculovirus as a Highly Potent Vector for Gene Therapy of Human Colorectal Carcinoma: Molecular Cloning,Expression, and In Vitro Characterization

Present therapeutic strategies for most cancers are restricted mainly to the primary tumors and are also not very effective in controlling metastatic states. Alternatively, gene therapy can be a potential option for treating such cancers. Currently mammalian viral-based cancer gene therapy is the most popular approach, but the efficacy has been shown to be quite low in clinical trials. In this study, for the first time, the insect cell-specific baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been evaluated as a vector for gene delivery to colorectal cancer cells. Experiments involving factorial design were employed to study the individual and combined effects of different parameters such as multiplicity of infection (MOI), viral incubation time and epigenetic factors on transduction efficiency. The results demonstrate that baculovirus gene delivery system holds immense potential for development of a new generation of highly effective virotherapy for colorectal, as well as other major carcinomas (breast, pancreas, and brain), and offers significant benefits to traditional animal virus-based vectors with respect to safety concerns.