Phosphorylation of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) by Proline-Directed Protein Kinases and Its Dephosphorylation

Abstract: Excitatory amino acid (EAA) neurotransmitters may play a role in the pathophysiology of traumatic injury to the CNS. Although NMDA receptor antagonists have been reported to have therapeutic efficacy in animal models of brain injury, these compounds may have unacceptable toxicity for clinical use. One alternative approach is to inhibit the release of EAAs following traumatic injury. The present study examined the effects of administration of a novel sodium channel blocker and EAA release inhibitor, BW1003C87, or the NMDA receptor-associated ion channel blocker magnesium chloride on cerebral edema formation following experimental brain injury in the rat. Animals (n = 33) were subjected to fluid percussion brain injury of moderate severity (2.3 atm) over the left parietal cortex. Fifteen minutes after injury, the animals received a constant infusion of BW1003C87 (10 mg/kg, i.v.), magnesium chloride (300 µmol/kg, i.v.), or saline over 15 min (2.75 ml/kg/15 min). In all animals, regional tissue water content in brain was assessed at 48 h after injury, using the wet weight/dry weight technique. In saline-treated control animals, fluid percussion brain injury produced significant regional brain edema in injured left parietal cortex ( p < 0.001), the cortical area adjacent to the site of maximal injury ( p < 0.001), left hippocampus ( p < 0.001), and left thalamus ( p = 0.02) at 48 h after brain injury. Administration of BW1003C87 15 min postinjury significantly reduced focal brain edema in the cortical area adjacent to the site of maximal injury ( p < 0.02) and left hippocampus ( p < 0.01), whereas magnesium chloride attenuated edema in left hippocampus ( p = 0.02). These results suggest that excitatory neurotransmission may play an important role in the pathogenesis of posttraumatic brain edema and that pre- or post-synaptic blockade of glutamate receptor systems may attenuate part of the deleterious sequelae of traumatic brain injury.     

A comparison of screening methods for antioxidant activity in seaweeds

The inhibition of lipid peroxidation and radical scavenging effects were studied to evaluate the antioxidant activity for extracts of 17 species of seaweed. The antioxidant effect was evaluated by determination of lipoxygenase activity and by α, α-diphenyl-β-picrylhydrazyl (DPPH) decolorization. Lipoxygenase activity was depressed in the presence of aqueous and ethanol extracts of 4 algal species; Sargassum species had the highest antioxidant activity of all the species examined. The ethanol extracts of one Sargassum species showed competitive inhibition with the substrate. The same species also showed radical scavenging activity in the DPPH decolorization test. Comparison of these results shows no relationship between enzyme inhibition and radical scavenging activity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Induction of proliferation and Ig production in human B leukemic cells by anti-immunoglobulins and T cell factors

The proliferation and differentiation of human leukemic B cells (B-CLL cells) with anti-Ig and T cell-derived helper factors are described. Stimulation of B-CLL cells with anti-Ig and T helper factors could induce proliferation as well as differentiation into IgM- and IgG-producing cells. Neither anti-Ig nor T helper factors alone could induce any proliferation and/or differentiation of B-CLL cells. Not only whole molecules of anti-Ig but also F(ab')2 fragments could induce proliferation and differentiation of B-CLL cells in the presence of T helper factors, but monovalent Fab' fragments were not effective. Induction of both IgM and IgG with the same idiotype was confirmed by immunofluorescent and SDS-PAGE analysis. By employing an IL 2-dependent cytotoxic T cell line and a TRF-responsive B cell line, T cell factors were separated into a fraction with IL2 activity but no TRF activity and a fraction with TRF activity but no IL 2 activity by chromatofocusing. Anti-Ig and IL 2 fraction could induce proliferation of B-CLL cells, but TRF fraction was not effective for the induction of proliferation in anti-IG-stimulated cells. For IgM and IgG production, anti-Ig and both IL 2 and TRF fractions were required. Depletion of IL 2 fraction in the first 2 days' culture inhibited Ig production, whereas the absence of TRF fraction in the first 2 days did not show any inhibitory effect on Ig production.     

Conversion by rat brain preparation of lignoceric acid to ceramides and cerebrosides containing both alpha-hydroxy and nonhydroxy fatty acids. Effects of compounds which affect sphingolipid metabolism.

Three synthetic compounds which affect sphingolipid metabolism in vitro were examined for their effects on the synthesis in a rat brain system of nonhydroxyceramide, hydroxyceramide, nonhydroxycerebroside, and hydroxycerebroside from [1-14C]ignoceric acid. These compounds are N-octanoyl-D-threo-p-nitrophenylaminopropanediol (Compound I), N-(2,3-epoxydecanoyl)-norephedrine (Compound II), and N-decyl-N'-glucosylthiourea (Compound III). In the presence of up to 0.5 mM Compound I, only the hydroxyceramide fromation increased, while the synthesis of the other three lipids decreased. Compound II strongly inhibited the formation of all four lipids at all concentrations tested (0.1 to 1 mM). Compound III increased the synthesis of both ceramides approximately 2-fold at 1 mM, but the conversion of lignoceric acid to both cerebrosides remained relatively unchanged. Several significant conclusions from these observations are discussed.     

LPS-or 8Br-cyclic AMP-induced production of T cell-activating factor(s) in macrophage tumor cell line J774.1.

The macrophage tumor cell line J774.1 replaced the function of normal macrophages in the induction of polyclonal killer T cells with 2-mercaptoethanol. J774.1 does not normally release soluble factor(s) which we have shown to be responsible for the differentiation of T cells to killer T cells. However, stimulation of J774.1 with LPS induced soluble factor(s) for T cell activation. An optimum concentration of LPS for the production of soluble factor(s) was 1 to 10 microgram/ml, which completely inhibited growth of the tumor cells. The production of soluble factor(s) was observed within 6 hr after LPS stimulation and reached its maximum level at 24 hr. Incubation of the cell line with 8Br-cyclic AMP and theophylline induced soluble factor(s), suggesting that LPS stimulation induced an increase in intracellular cyclic AMP which leads to the synthesis of soluble factor(s).     

Chemical modification and 1H-NMR studies on the receptor-binding region of human interleukin 6

Oxidation of the Met residues of human interleukin 6 (IL-6) molecule has been performed. Reactivity of Met for the oxidation reaction was found to decrease in the order of Met50, Met118, Met185, Met162, and Met68. Chemical modifications involving oxidation and carboxypeptidase A digestion of IL-6 have led to the assignments of the methyl proton resonances of Met162 and Met185, respectively. The hydroxynitrobenzyl chromophore attached to Trp158 in the IL-6 molecule showed a different absorption spectrum when the labeled IL-6 was bound to the soluble IL-6 receptor. This result indicates that Trp158 is near the receptor-binding region in IL-6. On the basis of the 1H-NMR and chemical modification data, it has been concluded that Trp158 is in spatial proximity to Met162, His165 and Met185. The receptor-binding activity decreased with an increase in the number of oxidized Met residues. Of these five Met residues, Met162 was the residue in which the receptor-binding activity decreased in the most parallel degree with that of the oxidation reaction.     

Protein kinase C in rat brain synaptosomes. Beta II-subspecies as a major isoform associated with membrane-skeleton elements.

A small fraction (approximately 5%) of protein kinase C (PKC) in the adult rat brain synaptosomes is tightly associated with Triton X-100-insoluble components (most likely membrane-skeleton elements), and is solubilized only after denaturation with sodium dodecyl sulfate. The kinase domain of this PKC can be released as a soluble form after limited proteolysis with calpain, whereas the regulatory domain which binds phorbol ester remains insoluble. The PKC in this fraction was identified as the beta II-subspecies or its related molecule. Presumably, this enzyme subspecies is responsible for the phosphorylation of a major PKC substrate protein, growth-associated protein-43, which is located in nerve endings as well as in growth cones in association with the membrane-skeleton elements.     

CNTF and LIF act on neuronal cells via shared signaling pathways that involve the IL-6 signal transducing receptor component gp130.

Ciliary neurotrophic factor (CNTF) has a variety of actions within the nervous system. While some of the actions of leukemia inhibitory factor (LIF) on neurons resemble those of CNTF, LIF also has broad actions outside of the nervous system that in many cases mimic those of interleukin-6 (IL-6). Comparison of the tyrosine phosphorylations and gene activations induced by CNTF and LIF in neuron cell lines reveals that they are indistinguishable and also very similar to signaling events that characterize LIF and IL-6 responses in hematopoietic cells. We provide a basis for the overlapping actions of these three factors by demonstrating that the shared CNTF and LIF signaling pathways involve the IL-6 signal transducing receptor component gp130. Thus, the receptor system for CNTF is surprisingly unlike those used by the nerve growth factor family of neurotrophic factors, but is instead related to those used by a subclass of hematopoietic cytokines.