Resveratrol inhibits type II phosphatidylinositol 4-kinase: a key component in pathways of phosphoinositide turn over

Resveratrol has anti-inflammatory, cardio protective and cancer chemopreventive properties. The molecular targets for resveratrol in early signaling cascades are not well understood. Resveratrol inhibits type II PtdIns 4-kinase but not PtdIns 3-kinase activity in vitro. Resveratrol directly binds to the enzyme with a Kd of 7.2 microM. Kinetic studies show that resveratrol competes with PtdIns binding. Inhibition of PtdIns 4-kinase activity by resveratrol/phenylarsine oxide reduces Jurkat cell adhesion to matrigel/fibronectin coated surfaces, suggesting a role for type II PtdIns 4-kinase in lymphocyte infiltration to the sites of inflammation.     

FcepsilonRI cross-linking activates a type II phosphatidylinositol 4-kinase in RBL 2H3 cells

Crosslinking of FcepsilonRI on rat basophilic leukemia (RBL 2H3) cells leads to an increase in Phosphatidylinositol 4-kinase activity. This increase in Ptdlns 4-kinase activity is strongly correlated with its tyrosyl phosphorylation state. Characterization of the enzyme activity in anti phosphotyrosine immunoprecipitates suggests it as a type II Ptdlns 4-kinase. Membrane cholesterol depletion studies showed a reduction in type II Ptdlns 4-kinase activity suggesting that lipid rafts play an important role in activation of the enzyme. The enzyme activity was inhibited by resveratrol. In situ inhibition of type II Ptdlns 4-kinase activity showed a reduction in beta-hexosaminidase release upon FcepsilonRI cross-linking. These studies suggest that a type II Ptdlns 4-kinase is an integral component of FcepsilonRI mediated signal transduction mechanisms.     

Ultraviolet light-induced unscheduled DNA-synthesis in isolated neurons of rat brain of different ages

DNA-repair capacity, by incorporation in vitro of [3H]thymidine into DNA of isolated neuronal cells and splenic lymphocytes of rat was studied as a function of age. The incubations were carried out both in the presence and absence of hydroxyurea (HU), a known inhibitor of replicative DNA synthesis. The results indicate that neurons, unlike lymphocytes, obtained from adult and old animals offer a good model system to measure the DNA-repair process without any possible interference of DNA replicative synthesis. Further, the 'spontaneous' DNA repair by unscheduled DNA synthesis (UDS) in old neurons remained unchanged as compared to the adult level. However, the response of aging neurons, in contrast to that of young and adult neurons or of lymphocytes of any age, to a mutagenic challenge like UV light is limited. It is suggested that this lack of responsive DNA-repair against a given damage may lead to a general metabolic deterioration and senescence.     

A type II phosphatidylinositol 4-kinase associates with T cell receptor zeta chain in Con A stimulated splenic lymphocytes through tyrosyl phosphorylation-dependent mechanisms

T cells show rapid reorganization of cytoskeleton in response to antigenic stimulation. The molecular mechanisms by which TCR-CD3 regulates actin cytoskeleton are not well defined. Here we show that a type II PtdIns 4-kinase associates with cytoskeletal fraction in splenic lymphocytes in response to Con A. Protein tyrosyl phosphorylation of type II PtdIns 4-kinase appears to be the mechanism for its association with cytoskeleton. Over-lay blots suggest that the enzyme binds to TCR-CD3 zeta chain in the cytoskeletal fraction. Anti-TCR-CD3 zeta antibodies competitively inhibit PtdIns 4-kinase association with TCR-CD3 zeta chain. Immunodepletion of TCR-CD3 zeta decreases PtdIns 4-kinase activity in the cytoskeletal fraction with a concomitant increase in PtdIns 4-kinase activity in anti-TCR-CD3 zeta immunoprecipitates. We propose that the association of type II PtdIns 4-kinase with TCR-CD3 zeta chain may bring the enzyme into close proximity of actin and a possible regulation of actin polymerization through localized production of PtdIns4P and PtdIns(4,5)P2.     

Biomarkers,metabonomics, and drug development: Can inborn errors of metabolism help in understanding drug toxicity?

Application of "omics" technology during drug discovery and development is rapidly evolving. This review evaluates the current status and future role of "metabonomics" as a tool in the drug development process to reduce the safety-related attrition rates and bridge the gaps between preclinical and clinical, and clinical and market. Particularly, the review looks at the knowledge gap between the pharmaceutical industry and pediatric hospitals, where metabonomics has been successfully applied to screen and treat newborn babies with inborn errors of metabolism. An attempt has been made to relate the clinical pathology associated with inborn errors of metabolism with those of drug-induced pathology. It is proposed that extending the metabonomic biomarkers used in pediatric hospitals, as "advanced clinical chemistry" for preclinical and clinical drug development, is immediately warranted for better safety assessment of drug candidates. The latest advances in mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy should help replace the traditional approaches of laboratory clinical chemistry and move the safety evaluation of drug candidates into the new millennium.     

RAGs’ eye view of the immunoglobulin heavy chain gene locus

The immunoglobulin heavy chain (IgH) gene locus is activated at a precise stage of B lymphocyte development to undergo gene rearrangements that assemble the functional gene. In this review we summarize our current understanding of the chromatin state of the IgH as it appears just prior to the initiation of V(D)J recombination, and the implications of this structure for regulation of recombination. We also examine the role of the intron enhancer, Eμ, in establishing the pre-rearrangement chromatin structure. The emerging picture shows that the IgH locus consists of independently regulated domains, each of which requires multiple levels of epigenetic changes to reach the fully activated state.     

Oncogenic NRAS rapidly and efficiently induces CMML- and AML-like diseases in mice

Activating mutations in RAS, predominantly NRAS, are common in myeloid malignancies. Previous studies in animal models have shown that oncogenic NRAS is unable to induce myeloid malignancies effectively, and it was suggested that oncogenic NRAS might only act as a secondary mutation in leukemogenesis. In this study, we examined the leukemogenicity of NRAS using an improved mouse bone marrow transduction and transplantation model. We found that oncogenic NRAS rapidly and efficiently induced chronic myelomonocytic leukemia (CMML)- or acute myeloid leukemia (AML)- like disease in mice, indicating that mutated NRAS can function as an initiating oncogene in the induction of myeloid malignancies. In addition to CMML and AML, we found that NRAS induced mastocytosis in mice. This result indicates that activation of the RAS pathway also plays an important role in the pathogenesis of mastocytosis. The mouse model for NRAS leukemogenesis established here provides a system for further studying the molecular mechanisms in the pathogenesis of myeloid malignancies and for testing relevant therapies.     

Critical care issues in adult liver transplantation

Over the last decade, liver transplantation has become an operational reality in our part of the world. As a result, clinicians working in an intensive care unit are more likely to be exposed to these patients in the immediate postoperative period, and thus, it is important that they have a working knowledge of the common complications, when they are likely to occur, and how to deal with them. The main focus of this review is to address the variety of critical care issues in liver transplant recipients and to impress upon the need to provide favorable circumstances for the new liver to start functioning and maintain the function of other organs to aid in this process.     

Interaction of monoclonal antibodies with cuticular antigens of filarial parasites, Brugia malayi and Wuchereria bancrofti

Monoclonal antibodies (mAbs) have been prepared against excretory-secretory-metabolic (ESM) antigens of microfilariae (mf) of Wuchereria bancrofti (WbmfESM) and against third stage larvae (L3) of Brugia malayi (BmL3), and purified from ascites fluids with ammonium sulphate. Both antibodies were of the IgM type and did not react with phosphorycholine. The mAb against BmL3 (F46) reacted in ELISA with antigens of L3 of B. malayi, B. pahangi and W. bancrofti and of adults of B. malayi. The mAb raised against wbmfESM (F32) resembled F46 in this respect, though with a lower titer towards the antigens, and in addition reacted with the ESM-antigens of mf and of L3 of W. bancrofti. F46 was able to detect L3 antigens of filarial parasites in spiked serum samples with a detection limit of 8-16 ng in absolute amount. The antibody was found to label the cuticular portion of L3 and adults of the lymphatic parasites, and not the epicuticular surface, in immunoelectron microscopic studies. The antibody recognized a 36 kDa component of the beta-mercaptoethanol extracts of B. pahangi-adults in Western blot analysis.