Glial uptake of neurotransmitter glutamate from the extracellular fluid studied in vivo by microdialysis and (13)C NMR

Glial uptake of neurotransmitter glutamate (GLU) from the extracellular fluid was studied in vivo in rat brain by (13)C NMR and microdialysis combined with gas-chromatography/mass-spectrometry. Brain GLU C5 was (13)C enriched by intravenous [2,5-(13)C]glucose infusion, followed by [(12)C]glucose infusion to chase (13)C from the small glial GLU pool. This leaves [5-(13)C]GLU mainly in the large neuronal metabolic pool and the vesicular neurotransmitter pool. During the chase, the (13)C enrichment of whole-brain GLU C5 was significantly lower than that of extracellular GLU (GLU(ECF)) derived from exocytosis of vesicular GLU. Glial uptake of neurotransmitter [5-(13)C]GLU(ECF) was monitored in vivo through the formation of [5-(13)C,(15)N]GLN during (15)NH(4)Ac infusion. From the rate of [5-(13)C,(15)N]GLN synthesis (1.7 +/- 0.03 micromol/g/h), the mean (13)C enrichment of extracellular GLU (0.304 +/- 0.011) and the (15)N enrichment of precursor NH(3) (0.87 +/- 0.014), the rate of synthesis of GLN (V'(GLN)), derived from neurotransmitter GLU(ECF), was determined to be 6.4 +/- 0.44 micromol/g/h. Comparison with V(GLN) measured previously by an independent method showed that the neurotransmitter provides 80-90% of the substrate GLU pool for GLN synthesis. Hence, under our experimental conditions, the rate of 6.4 +/- 0.44 micromol/g/h also represents a reasonable estimate for the rate of glial uptake of GLU(ECF), a process that is crucial for protecting the brain from GLU excitotoxicity.     

Refsum's disease: a peroxisomal disorder affecting phytanic acid alpha-oxidation

Refsum's disease (hereditary motor sensory neuropathy type IV, heredopathia atactica polyneuritiformis) is an autosomal recessive disorder the clinical features of which include retinitis pigmentosa, blindness, anosmia, deafness, sensory neuropathy, ataxia and accumulation of phytanic acid in plasma- and lipid-containing tissues. The transport and biochemical pathways of phytanic acid metabolism have recently been defined with the cloning of two key enzymes, phytanoyl-CoA 2-hydroxylase (PAHX) and 2-hydroxyphytanoyl-CoA lyase, together with the confirmation of their localization in peroxisomes. PAHX, an iron(II) and 2-oxoglutarate-dependent oxygenase is located on chromosome 10p13. Mutant forms of PAHX have been shown to be responsible for some, but not all, cases of Refsum's disease. Certain cases have been shown to be atypical mild variants of rhizomelic chondrodysplasia punctata type 1a. Other atypical cases with low-plasma phytanic acid may be caused by alpha-methylacyl-CoA racemase deficiency. A sterol-carrier protein-2 (SCP-2) knockout mouse model shares a similar clinical phenotype to Refsum's disease, but no mutations in SCP-2 have been described to-date in man. This review describes the clinical, biochemical and metabolic features of Refsum's disease and shows how the biochemistry of the alpha-oxidation pathway may be linked to the regulation of metabolic pathways controlled by isoprenoid lipids, involving calcineurin or the peroxisomal proliferator activating alpha-receptor.     

Ethanol-dependent induction of bilirubin UDP-glucuronosyl-transferase in rat liver is mediated by Kupffer cells

Recently we have reported that bilirubin UDP-glucuronosyltransferase (UGT1A1) is induced in rat liver by chronic ethanol treatment. Several studies have shown that Kupffer cells play a central role in the mediation of various hepatic effects of chronic alcohol consumption. In the present work, the participation of Kupffer cells in the ethanol dependent induction of UGT1A1 was investigated. A group of rats was pretreated with gadolinium chloride, a known Kupffer-cell-depleting agent. We compared the effect of chronic ethanol ingestion on UGT1A1 expression in the liver of normal and gadolinium chloride treated rats. The effect of ethanol on bilirubin glucuronidation was completely prevented in Kupffer cell deficient rats. The western and northern blot analyses showed that the increase of both the protein and mRNA of UGT1A1 was prevented in these animals. These results suggest that Kupffer cells play a major role in the mediation of ethanol-stimulated induction of UGT1A1 in liver parenchymal cells.     

Isolation and characterization of the Cryptococcus neoformans MATa pheromone gene

Cryptococcus neoformans is a heterothallic basidiomycete with two mating types, MATa and MATalpha. The mating pathway of this fungus has a number of conserved genes, including a MATalpha-specific pheromone (MFalpha1). A modified differential display strategy was used to identify a gene encoding the MATa pheromone. The gene, designated MFa1, is 42 amino acids in length and contains a conserved farnesylation motif. MFa1 is present in three linked copies that span a 20-kb fragment of MATa-specific DNA and maps to the MAT-containing chromosome. Transformation studies showed that MFa1 induced filament formation only in MATalpha cells, demonstrating that MFa1 is functionally conserved. Sequence analysis of the predicted Mfa1 and Mfalpha1 proteins revealed that, in contrast to other fungi such as Saccharomyces cerevisiae, the C. neoformans pheromone genes are structurally and functionally conserved. However, unlike the MFalpha1 gene, which is found in MATalpha strains of both varieties of C. neoformans, MFa1 is specific for the neoformans variety of C. neoformans.     

Comparative studies of genetic diversity in kenaf (Hibiscus cannabinus L.) varieties based on analysis of agronomic and RAPD data

Kenaf (Hibiscus cannabinus L.) is a fiber crop classified in the genus Hibiscus (Malvaceae), and has a great potential for its multipurpose utilization, in addition to its traditional usage. Varietal identification of kenaf is always problematic and knowledge on genetic diversity of kenaf varieties is also limited, which significantly hindered our effective utilization and conservation of the valuable kenaf germplasm. In order to find a proper method for identifying kenaf varieties and studying their variation, morpho-agronomic characters and random amplified polymorphic DNA (RAPD) markers were analyzed among 14 kenaf varieties commonly used in Japan. Data from morphological analysis showed that the included kenaf varieties could be divided into three major groups. The characters, such as middle stem diameter, whole stalk weight, and days to 50% flowering, are highly responsible for the variation of the kenaf varieties, but it is difficult to identify individual varieties merely by the morpho-agronomic characters. On the other hand, clearly separation of the kenaf varieties was achieved based on the RAPD variation patterns. Genetic relationship of the kenaf varieties can also be traced through the analysis of RAPD and morph-agronomic variation. It is concluded from the present study that RAPD analysis is an effective tool in identifying of kenaf varieties and determining their genetic relationships, particularly when combined with the analysis of morpho-agronomic characters.     

Regulation of vasodilator-stimulated phosphoprotein phosphorylation and interaction with Abl by protein kinase A and cell adhesion

Members of the vasodilator-stimulated phosphoprotein (VASP) family are important regulators of actin cytoskeletal dynamics whose functions and protein-protein interactions are regulated by phosphorylation by the cAMP-dependent protein kinase (PKA). Herein, we show that phosphorylation of VASP is dynamically regulated by cellular adhesion to extracellular matrix. Detachment of cells stimulated PKA activity and induced PKA-dependent phosphorylation of VASP and the related murine-Enabled (Mena) protein. VASP and Mena were rapidly dephosphorylated immediately following reattachment but showed an intermediate level of phosphorylation during active cell spreading. This pattern correlated closely with adhesion-dependent changes in PKA activity. The in vivo interaction of VASP with the Abl tyrosine kinase, shown here for the first time, was readily apparent in adherent cells, lost following cellular detachment, and induced upon reattachment to matrix. Importantly, inhibition of PKA activity prevented phosphorylation of VASP and dissociation of VASP-Abl complexes after cellular detachment, whereas activation of PKA completely eliminated the co-immunoprecipitation of Abl activity with VASP. These data establish a new biochemical link between cell adhesion and regulation of VASP proteins and provide the first demonstration of a regulated interaction between VASP and Abl in mammalian cells.     

Analysis of the structural basis of specificity of inhibition of the Abl kinase by STI571

STI571, a selective inhibitor of Bcr-Abl, has been a successful therapeutic agent in clinical trials for chronic myelogenous leukemia. Chronic phase chronic myelogenous leukemia patients treated with STI571 have durable responses; however, most responding blast phase patients relapse despite continued therapy. Co-crystallization studies of Abl kinase and an STI571-related compound identify specific amino acid residues as critical to STI571 binding, one of which, T315, has been characterized as an acquired Thr to Ile mutation in relapsed patients. Other studies, however, suggest that mutations other than these predicted contact points are capable of conferring STI571 resistance in relapsed patients. Using a variety of models of STI571 binding to the Abl kinase, we have performed an extensive mutational analysis of sites that might alter the sensitivity of the Abl kinase to STI571. Although mutation of many of the predicted contact points between Abl and STI571 result in a kinase-inactive protein, additional mutations that render the Abl kinase less sensitive to STI571 demonstrate a broad range of possibilities for clinical resistance that are now becoming evident.