Metabolic Enzyme Alterations and Astrocyte Dysfunction in a Murine Model of Alexander Disease With Severe Reactive Gliosis

Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAP^Tg;Gfap^+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAP^Tg;Gfap^+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAP^Tg;Gfap^+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAP^Tg;Gfap^+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAP^Tg;Gfap^+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap⁺ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAP^Tg;Gfap^+/R236H mice. Last, to determine whether the findings in GFAP^Tg;Gfap^+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAP^Tg;Gfap^+/R236H mice.     

Three-dimensional structure of α-conotoxin EI determined by 1H NMR spectroscopy H resonance assignments of α-conotoxin EI

q8ukx84336712840/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-Conotoxin EI is an 18-residue peptide (RDOCCYHPTCNMSNPQIC; 4–10, 5–18) isolated from the venom of Conus ermineus, the only fish-hunting cone snail of the Atlantic Ocean. This peptide targets specifically the nicotinic acetylcholine receptor (nAChR) found in mammalian skeletal muscle and the electric organ Torpedo, showing a novel selectivity profile when compared to other q8ukx84336712840/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-conotoxins. The 3D structure of EI has been determined by 2D-NMR methods in combination with dynamical simulated annealing protocols. A total of 133 NOE-derived distances were used to produce 13 structures with minimum energy that complied with the NOE restraints. The structure of EI is characterized by a helical loop between Thr⁹ and Met¹² that is stabilized by the Cys⁴-Cys¹⁰ disulfide bond and turns involving Cys⁴-Cys⁵ and Asn¹⁴-Pro¹⁵. Other regions of the peptide appear to be flexible. The overall fold of EI is similar to that of other q8ukx84336712840/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">4/7-conotoxins (PnIA/B, MII, EpI). However, unlike these other q8ukx84336712840/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">4/7-conotoxins, EI targets the muscular type nAChR. The differences in selectivity can be attributed to differences in the surface charge distribution among these q8ukx84336712840/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">4/7-conotoxins. The implications for binding of EI to the muscular nAChR are discussed with respect to the current NMR structure of EI.     

Fromβ-lactams toα- andβ-amino acid derived peptides

Summary The potential ofq3183/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-lactams as intermediates for the access toq3183/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">- andq3183/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-amino acid-derived peptides is shortly reviewed, with major focus on the technologies developed in our group. The two general strategies lie, on one side, in the oxidative ring expansion of 3-hydroxyq3183/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-lactams toN-carboxyq3183/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-amino acid anhydrides or Leuch's anhydrides and subsequent coupling withq3183/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-amino acid esters and, on the other side, in the nucleophilic ring opening ofN-Boc-q3183/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-lactams. Both approaches have been successfully applied to the synthesis ofq3183/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">,q3183/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-diamino acid,q3183/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-amino-q3183/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-hydroxy acid, polyhydroxylatedq3183/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-amino acid,q3183/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">,q3183/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-disubstitutedq3183/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-amino acid,q3183/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-amino acid,q3183/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-amino-q3183/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-hydroxy acid andq3183/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">,q3183/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-disubstitutedq3183/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-amino acid derived peptides. Because of the mild reaction conditions needed for the above transformations and the highly stereoselective procedures employed for the construction of the startingq3183/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-lactam ring, the whole process allows the production of optically pure final products.     

Complex between α-Chymotrypsin and poly(ethylene glycol) catalytically active in organic media

q43n081422685005/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-Chymotrypsin and poly(ethylene glycol) were mixed in an aqueous phosphate buffer solution and lyophilized. The preparation was readily soluble in anhydrous isooctane. The polymer/enzyme complex, i.e., poly(ethylene glycol)/q43n081422685005/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-chymotrypsin, was catalytically active in anhydrous isooctane even when the molecular ratio of them was unity.     

Synthesis of N<Subscript>ɛ</Subscript>-protected-L-lysine and γ-benzyl-L-glutamate N-carboxyanhydrides (NCA) by carbamoylation and nitrosation

Summary. This paper reports on an original process to synthesize N-carboxyanhydrides, which consists of nitrosating N-carbamoylamino acids with a NO/O₂ gas mixture in acetonitrile. The synthesis of several N-carbamoylamino acids of L-lysine was described using potassium cyanate in water. The latter were then nitrosated to yield the corresponding NCA with more or less efficiency. Indeed, the NCA carrying an acid-sensitive protecting group led to a partial deprotection to give the L-lysine NCA salt. The NCA of N_{q/xxlarge603.gif" alt="epsiv" align="BASELINE" BORDER="0">}-trifluoroacetyl-L-lysine, N_{q/xxlarge603.gif" alt="epsiv" align="BASELINE" BORDER="0">}-benzyloxycarbonyl-L-lysine and q/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">-benzyl-L-glutamate were successfully synthesized with satisfactory yields. Their polymerizability was compared to that of the N_{q/xxlarge603.gif" alt="epsiv" align="BASELINE" BORDER="0">}-trifluoroacetyl-L-lysine NCA initiated by n-hexylamine in N,N-dimethylformamide. It also showed that this new process of NCA synthesis could be applied to the synthesis of polypeptides and more generally to the protein chemistry.     

NTPDase and 5′-Nucleotidase Activities in Synaptosomes of Hippocampus and Serum of Rats Subjected to Homocysteine Administration

Patients with homocystinuria, an inborn error of metabolism, present neurological dysfunction and commonly experience frequent thromboembolic complications. The nucleoside triphosphate diphosphohydrolase (NTPDase) and 5'-nucleotidase enzymes regulate the nucleotide/nucleoside ratio in the central nervous system and in the circulation and are thought to be involved in these events. Thus, the current study investigated the effect of homocysteine administration on NTPDase and 5'-nucleotidase activities, in the synaptosomal fraction of rat hippocampus, and on nucleotidase activities in rat serum. Twenty-nine-day-old Wistar rats were divided in two groups: group I (control), animals received 0.9% saline; group II (homocysteine-treated), animals received one single subcutaneous injection of homocysteine (0.6 micromol/g). Rats were killed 1 h after the injection. NTPDase and 5'-nucleotidase activities from brain and serum were significantly increased in the homocysteine-treated group. Results show that, in hippocampus, ATP and ADP hydrolysis increased by 20.5% and 20%, respectively, and AMP hydrolysis increased by 48%, when compared to controls. In serum, ATP and ADP hydrolysis increased 136% and 107%, respectively, and AMP hydrolysis increased 95%, in comparison to controls. The current data strongly indicate that in vivo homocysteine administration alters the activities of the enzymes involved in nucleotide hydrolysis, both in the central nervous system and in the serum of adult rats.     

Suggestive evidence of association of C-159T functional polymorphism of the <Emphasis Type="Italic">CD14</Emphasis> gene with atopic asthma in northern and northwestern Indian populations

CD14 is a lipopolysaccharide receptor known to be an important modulator of Th1–Th2 response during early childhood. Genetic association studies of the CD14 gene with asthma and atopic disorders have shown positive as well as negative results in different ethnic populations. The aim of this study was to test for association of C-159T functional promoter polymorphism with atopic asthma and serum IgE levels in northern and northwestern Indian populations. DNA was assayed for the CD14 C-159T polymorphism in a case-control study involving atopic asthmatics (n=187) and healthy normal controls (n=227), and in a family-based association study of 106 trios. The case-control study showed an association at the genotypic (P=0.0146) as well as the allelic level (P=0.0048). Moreover, we observed a deviation of allelic transmission from random proportions (P=0.024) in the transmission disequilibrium test analysis. When we analyzed our results for serum total IgE levels, against this polymorphism, we observed a difference at the genotypic (P=0.0026) as well as at the allelic level (P=0.0016) in a case-control study, whereas no association in the quantitative transmission disequilibrium test analysis was obtained. These findings provide suggestive evidence of association of the CD14 gene locus with atopic asthma in northern and northwestern Indian populations.     

Association of β<Subscript>2</Subscript>-microglobulin with the α<Subscript>3</Subscript> domain of H-2D<Superscript>b</Superscript> heavy chain

MHC class I molecules are heterotrimeric complexes composed of heavy chain, q45r23a5jee5u/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂-microglobulin (q45r23a5jee5u/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂m) and short peptide. This trimeric complex is generated in the endoplasmic reticulum (ER), where a peptide loading complex (PLC) facilitates transport from the cytosol and binding of the peptide to the preassembled ER resident heavy chain/q45r23a5jee5u/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂m dimers. Association of mouse MHC class I heavy chain with q45r23a5jee5u/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂m is characterized by allelic differences in the number and/or positions of amino acid interactions. It is unclear, however, whether all alleles follow common binding patterns with minimal contributions by allele-specific contacts, or whether essential contacts with q45r23a5jee5u/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂m are different for each allele. While searching for the PLC binding site in the q45r23a5jee5u/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">₃ domain of the mouse MHC class I molecule H-2D^b, we unexpectedly discovered a site critical for binding mouse, but not human, q45r23a5jee5u/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂m. Interestingly, amino acids in the corresponding region of another MHC class I heavy chain allele do not make contacts with the mouse q45r23a5jee5u/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂m. Thus, there are allelic differences in the modes of binding of q45r23a5jee5u/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂m to the heavy chain of MHC class I.     

Fluorescence spectroscopic study of α-chymotrypsin relevant to the enantioselectivity for optical resolution of amino acid esters in organic solvents

The fluorescence emission wavelength of alpha-chymotrypsin (CT) correlated with its enantioselectivity (E value) for the resolution of DL-tyrosine ethyl ester. The changes in the E value of the CT due to the changes in the solvent composition were closely related to its fluorescence properties (delta lambda(em)), which were most probably associated with the structural modification of the enzyme. A linear relationship was established between E value and delta lambda(em) in aqueous acetonitrile with high correlation coefficients (r = 0.94).