Membrane association of glutathione S-transferase mGSTA4-4, an enzyme that metabolizes lipid peroxidation products.

Lipid peroxidation products have signaling functions and at higher concentrations are toxic and may trigger cell death. The compounds are metabolized predominantly by glutathione S-transferases exemplified by mGSTA4-4, an enzyme highly efficient in glutathione conjugation of 4-hydroxyalkenals, and possessing glutathione peroxidase activity toward phospholipid hydroperoxides. mGSTA4-4 belongs to the predominant group of "canonical" glutathione S-transferases that are soluble and generally localized in the cytoplasm. The intracellular localization of mGSTA4-4 was examined in hepatocytes of normal mouse liver and in transfected HepG2 cells by fluorescence microscopy and digital deconvolution. mGSTA4-4 was found to be predominantly localized at or near the plasma membrane in transfected HepG2 cells, as well as in hepatocytes endogenously expressing the protein. In vitro, mGSTA4-4 associated with liposomes, and this interaction was potentiated when the liposomes contained negatively charged phospholipids. Mutating lysine 115 to glutamic acid resulted in a loss of the plasma membrane targeting of mGSTA4-4 as well as in a significant reduction of its binding to liposomes in vitro. These data suggest preferential targeting of mGSTA4-4 to the plasma membrane that may contain the major substrate(s) for this enzyme. Lysine 115 is critically important for the membrane association of mGSTA4-4, most likely by entering into an electrostatic interaction with negatively charged phospholipid headgroups.     

Pentoxifylline functions as an adjuvant in vivo to enhance T cell immune responses by inhibiting activation-induced death

Modalities for inducing long-lasting immune responses are essential components of vaccine design. Most currently available immunological adjuvants empirically used for this purpose cause some inflammation, limiting clinical acceptability. We show that pentoxifylline (PF), a phosphodiesterase (PDE) inhibitor in common clinical use, enhances long-term persistence of T cell responses, including protective responses to a bacterial immunogen, Salmonella typhimurium, via a cAMP-dependent protein kinase A-mediated effect on T cells if given to mice for a brief period during immunization. PF inhibits activation-mediated loss of superantigen-reactive CD4 as well as CD8 T cells in vivo without significantly affecting their activation, and inhibits activation-induced death and caspase induction in stimulated CD4 as well as CD8 T cells in vitro without preventing the induction of activation markers. Consistent with this ability to prevent activation-induced death in not only CD4 but also CD8 T cells, PF also enhances the persistence of CD8 T cell responses in vivo. Thus, specific inhibition of activation-induced T cell apoptosis transiently during immune priming is likely to enhance the persistence of CD4 and CD8 T cell responses to vaccination, and pharmacological modulators of the cAMP pathway already in clinical use can be used for this purpose as immunological adjuvants.     

A liquid synchronized-growth culture assay for the identification of true positive and negative yeast three-hybrid transformants

AIMS: To develop a simple and easy-to-use assay for detection of truely positive transformants from a yeast three-hybrid assay. METHODS AND RESULTS: The yeast three-hybrid system is a new powerful system for studying RNA-protein interactions in vivo. There are, however, many reports from investigators about the difficulty in distinguishing a positive from a negative result due to the hard-to-detect differences between truely positive transformants and the negative ones. A liquid synchronous-growth culture approach has been described for all positive and negative transformants and their growth densities have been compared to each other at fixed intervals of incubation times. We have designed a simple yet effective procedure to assay for positive and negative RNA-protein interactions based on liquid culture analysis of synchronously growing yeast cells. Results obtained from this new procedure clearly show differences in positive and negative transformants after a 24-h incubation of synchronously growing transformants in liquid culture. CONCLUSIONS: The procedure mentioned in this report shows clearly the differences between positive and negative results from a three-hybrid system. SIGNIFICANCE AND IMPACT OF THE STUDY: The method proposed here is a clear advantage over existing methods based on measuring growth on restrictive growth medium plates by the naked eye. This method will have substantial usage with investigators using the yeast three-hybrid studies for RNA-protein interactions.     

Modulation of radiation-induced changes in the xanthine oxidoreductase system in the livers of mice by its inhibitors

The xanthine oxidoreductase (XOD) system, which consists of xanthine dehydrogenase (XDH) and xanthine oxidase (XO), is one of the major sources of free radicals in biological systems. The XOD system is present predominantly in the normal tissues as XDH. In damaged tissues, XDH is converted into XO, the form that generates free radicals. Therefore, the XO form of the XOD system is expected to be found mainly in radiolytically damaged tissue. In this case, XO may catalyze the generation of free radicals and potentiate the effect of radiation. Inhibition of the XOD system is likely to attenuate the detrimental effects of ionizing radiation. We have examined this possibility using allopurinol and folic acid, which are known inhibitors of the XOD system. Swiss albino mice (7-8 weeks old) were given single doses of allopurinol and folic acid (12.5-50 mg/kg) intraperitoneally and irradiated with different doses of gamma radiation at a dose rate of 0.023 Gy/s. The XO and XDH activities as well as peroxidative damage and lactate dehydrogenase (LDH) were determined in the liver. An enhancement of the activity of XO and a simultaneous decrease in the activity of XDH were observed at doses above 3 Gy. The decrease in the ratio XDH/XO and the unchanged total activity (XDH + XO) suggested the conversion of XDH into XO. The enhanced activity of XO may potentiate radiation damage. The increased levels of peroxidative damage and the specific activity of LDH in the livers of irradiated mice supported this possibility. Allopurinol and folic acid inhibited the activities of XDH and XO, decreased their ratio (XDH/XO), and lowered the levels of peroxidative damage and the specific activity of LDH. These results suggested that allopurinol and folic acid have the ability to inhibit the radiation-induced changes in the activities of XDH and XO and to attenuate the detrimental effect of this conversion, as is evident from the diminished levels of peroxidative damage and the decreased activity of LDH.     

Heat modifiability of outer membrane protein of Pasteurella multocida serotype B:2

Outer membrane proteins (OMP) are generally porins, functioning as molecular sieves assisting in the transmembrane transportation. Heat modifiable characteristics of OMP from P. multocida B: 2 have been explored to know their basic characteristics on event of temperature rise. A major band of 32 kDa and two minor bands of approximately 39 and approximately 28 kDa were found to be heat modifiable. It is suggested that boiling at 100 degrees C in presence of beta mercaptoethanol for 5 min is sufficient for characterisation of OMP by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis.     

The Substrate Utilization and Concentration of 14C Photosynthates in Citronella under Fe Deficiency

Changes in the utilization pattern of primary substrate, viz. [U-14C] acetate, 14CO2 and [U-14C] saccharose, and the contents of 14C fixation products in photosynthetic metabolites (sugars, amino acids, and organic acids) were determined in Fe-deficient citronella in relation to the essential oil accumulation. There was an overall decrease in photosynthetic efficiency of the Fe-deficient plants as evidenced by lower levels of incorporation into the sugar fraction and essential oil after 14CO2 had been supplied. When acetate and saccharose were fed to the Fe-deficient plants, despite a higher incorporation of label into sugars, amino acids, and organic acids, there was a lower incorporation of these metabolites into essential oils than in control plants. Thus, the availability of precursors and the translocation to a site of synthesis/accumulation, severely affected by Fe deficiency, is equally important for the essential oil biosynthesis in citronella.     

Involvement of Microtubules in the Regulation of Bcl2 Phosphorylation and Apoptosis through Cyclic AMP-Dependent Protein Kinase

The Bcl2 family of proteins plays a significant role in regulation of apoptosis. In this study, the microtubule-damaging drugs paclitaxel, vincristine, and vinblastine induced Bcl2 hyperphosphorylation and apoptosis in MCF-7 and MDA-MB-231 cells and reduced Bcl2-Bax dimerization. Paclitaxel or vincristine induced increased expression of Bax, while overexpression of Bcl2 in these cell lines counteracted the effects of low doses of these drugs. In addition, paclitaxel- and vincristine-induced activation of cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]) induced Bcl2 hyperphosphorylation and apoptosis, which were blocked by the PKA inhibitor Rp diastereomers of cAMP (Rp-cAMP). This finding suggests that activation of PKA due to microtubule damage is an important event in Bcl2 hyperphosphorylation and induction of apoptosis. These microtubule-damaging drugs caused growth arrest in G₂-M phase of the cell cycle and had no effect on p53 induction, suggesting that hyperphosphorylation mediated inactivation of Bcl2 and apoptosis without the involvement of p53. By comparison, the DNA-damaging drugs methotrexate and doxorubicin had no effect on Bcl2 hyperphosphorylation but induced p53 expression. Interestingly, paclitaxel or vincristine induced activation of caspase 3 and cleavage of poly(ADP-ribose) polymerase downstream of Bcl2 hyperphosphorylation. These data suggest that there may be a signaling cascade induced by agents that disrupt or damage the cytoskeleton that is distinct from (i.e., p53 independent), but perhaps related to (i.e., involves kinase activation and leads to apoptosis), the cellular response to DNA damage.