Synthetic approaches to 6-substituted 9-(3-azido-3,4-dideoxy-β-d,l-erythro-pentopyranosyl)purines

Methyl 3-azido-2-O-benzoyl-3,4-dideoxy-β-dl-erythro-pentopyranoside (6) was synthesized through two routes in five steps from methyl 2,3-anhydro-4-deoxy-β-dl-erythro-pentopyranoside (1). The first route proceeded via selective azide displacement of the 3-tosyloxy group of methyl 4-deoxy-2,3-di-O-tosyl-α-dl-threo-pentopyranoside, followed by detosylation and benzoylation. The second route consisted, with a better overall yield, in the azide displacement of the mesyloxy group of methyl O-benzoyl-4-deoxy-3-O-methylsulfonyl-α-dl-threo-pentopyranoside (10), obtained by benzylate opening of 1, followed by benzoylation, debenzylation, and mesylation. Compound 6 was transformed into its glycosyl chloride, further treated by 6-chloropurine to give the nucleoside 9-(3-azido-2-O-benzoyl-3,4-dideoxy-β-dl-erythro-pentopyranosyl)-6-chloropurine (13). When treated with propanolic ammonia, 13 yielded 9-(3-azido-3,4-dideoxy-β-dl-erythro-pentopyranosyl)adenine.     

Production of interleukin-1 alpha and interleukin-2 by mononuclear cells from children with growth delay in relation to the degree of growth hormone deficiency: effects of substitutive treatment

Interleukin-1 alpha (IL-1 alpha) and interleukin-2 (IL-2) levels were measured by radioimmunoassay in samples of conditioned medium from mononuclear cells taken from 20 normal subjects (14 adults ranging in age from 20 to 45 years and 6 children ranging in age from 3 to 11 years) and from 49 children with growth delay. Cultures were performed with 10(6) cells/ml in medium containing 1% normal human serum and 4.8 g/l phytohemagglutinin M. The incubation was performed for 48 h in an atmosphere containing 5% CO2. In normal subjects, the production of IL-1 alpha was 38.5 +/- 9.8 fmol/ml of conditioned medium (mean +/- SEM) in 14 adults and 41.6 +/- 3.0 fmol/ml in 6 children. The production of IL-2 was 46.9 +/- 6.5 and 57.3 +/- 10.5 fmol/ml, respectively. In the 16 patients with growth hormone (GH) deficiency studied before treatment, the production of ILs was significantly decreased in relation to the degree of deficiency. We observed a positive correlation between the production of IL-1 alpha and the values of insulin-like growth factor I but not with serum GH values. IL-1 alpha production was normalized after 15 days of substitutive GH therapy and IL-2 was normalized after 3 months of therapy. In 10 other patients with GH deficiency (4 with total and 6 with partial isolated GH deficiency) studied after long-term GH treatment (5 months or more), the mean of IL production was not significantly different from that of GH-deficient children treated for 3 months.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP stimulates inositol phosphates accumulation and calcium mobilization in a primary culture of rat aortic myocytes

The effects of extracellular ATP on phosphoinositide metabolism and intracellular Ca2+ concentration were studied in a primary culture of rat aortic myocytes. ATP increases the level of inositol phosphates, the putative second messenger for Ca2+ mobilization. No saturation of inositol phosphates accumulation is obtained (up to 10(-2) M ATP). Under the same conditions, ATP rapidly mobilizes intracellular Ca2+ in fura-2 loaded myocytes. The mobilization of intracellular Ca2+ is dose-dependent (maximal at 10(-4) M ATP), and is not affected by addition of EGTA. It is concluded that the receptors mediating the cytosolic increase of Ca2+ are of the P2-purinoceptor subtype. The physiological functions of these receptors are not presently known.     

Separation of chitosomes and secretory vesicles from the “slime” variant of Neurospora crassa

Cells from the slime variant of Neurospora crassa were broken in isotonic conditions by use of triethanolamine buffer plus EDTA. After removal of large membranous structures by low-speed centrifugation, chitosomes and secretory vesicles were separated by means of gel filtration, precipitation of membranous contaminants with Concanavalin A, and centrifugation in sucrose or glycerol gradients. Polypeptidic composition of fractions enriched in secretory vesicles or chitosomes was found to be distinct. By these criteria we concluded that chitosomes and secretory vesicles represent different populations of microvesicles. Both microvesicular populations appeared free of endoplasmic reticulum and vacuolar contaminants as demonstrated by determination of appropriate enzymatic markers.Abbreviations ER Endoplasmic reticulum - UDP-GlcNAc uridine-5-diphosphate N-acetyl glucosamine - GlcNAc N-acetyl glucosamine - SDS sodium dodecyl sulfate - PMSF phenyl methyl sulfonyl fluoride - EDTA ethylene diamino tetraacetic acid Investigador Nacional de Mexico. On leave from the Centro de Investigacion y Estudios Avanzados (IPN), and the Universidad de Guanajuato, Gto., Mexico     

Effects of phospholipases,proteases and neuraminidase on γ-hydroxybutyrate binding sites

Summary -Hydroxybutyric acid (GHB) is a natural compound of mammalian brain synthesized from GABA. The characteristics of its synthesis, transport, release, distribution and turnover, in addition to the presence of a high affinity binding site for this substance in brain are in favor of a modulator role for GHB. The effects of hydrolytic enzymes on the specific binding capacity of GHB have been studied in the present work. Phospholipases A₂ and C, neuraminidase and Pronase markedly decrease GHB binding to crude synaptosomal membranes from rat brain. This effect is time and enzyme concentration dependent. Trypsin, under the conditions employed, is less active. The inhibitory effects of phospholipases is correlated with phospholipid hydrolysis. Lysophospholipids, in the absence of bovine fatty acid free serum albumin partially inhibit GHB binding. The action of neuraminidase has been followed by sialic acid release and modifications of the ganglioside profile. The effects of phospholipase C and of neuraminidase are completely different to those on GABA binding sites. These results represent further data concerning the molecular existence of specific GHB binding sites on rat brain membranes.Abbreviations GHB -hydroxybutyrate - LPC L--lysophosphatidylcholine - LPE Lysophosphatidylethanolamine - PC Phosphatidylcholine - PE Phosphatidylethanolamine - BSA Bovine Serum Albumin     

Peptide-N 4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity could explain the occurrence of extracellular xylomannosides in a plant cell suspension

We have previously isolated mannoside and xylomannoside oligosaccharides with one or two terminal reducingN-acetylglucosamine residues from the extracellular medium of white campion (Silene alba) suspension culture. We have now demonstrated the presence of peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity in cell extracts as well in the culture medium that could explain the production of those compounds. An additional xylomannoside, (GlcNAc)Man₃(Xyl)GlcNAc(Fuc)GlcNAc, was characterized, and¹H- and¹³C-NMR assignments for the oligosaccharide Man₃(Xyl)GlcNAc(Fuc)GlcNAc were obtained using homonuclear and heteronuclear spectroscopy (COSY).Abbreviations Endo endo--N-acetylglucosaminidase - Fuc fucose - GlcNAc N-acetylglucosamine - Man mannose - NMR nuclear magnetic resonance - PNGase peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase - Xyl xylose     

Parenteral supplementation with zinc in surgical patients corrects postoperative serum-zinc drop

Zinc has been known for a long time to facilitate wound healing. But, so far, supplementation trials in patients treated by major severity surgery gave either partial or controversial results. In a double-blind, randomized study including 30 patients, we show that zinc supplements (30 mg/d for 3 d) administered by a drip correct postoperative drop of serum zinc, that this correction concerns the available part of serum zinc (i.e., zinc that is bound to compounds other than alpha-2 macroglobulin in serum), and that this supplementation can improve clinical wound healing. Possible influence of increased urinary output after the intervention is discussed, and we found that serum cortisol remains stable when zinc/albumin ratio is stable, and increases sharply when the same ratio drops. Cortisol, therefore, seems to play a major role in zinc redistribution after surgery.     

Structures of the α(1-3)-galactose-containing asparagine-linked glycans of a Lewis lung carcinoma cell subline resistant toAleuria aurantia agglutinin: elucidation by1H-NMR spectroscopy

Four bi-antennary glycan fractions of theN-acetyllactosamine-type, derived from a Lewis lung carcinoma (LL₂) cell subline resistant to theAleuria aurantia agglutinin were studied by 400 MHz¹H-NMR spectroscopy. By this method, their antennae were found to be terminated either by (2-3 or 6)-linkedN-acetylneuraminic acid or (1-3)-linked galactose residues. The primary structure of glycans of these four glycopeptide or derived oligosaccharide-alditols has been determined in full detail.Abbreviations NAc N-acetyl group - NGc N-glycolyl group - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - Man mannose - Gal galactose - Fuc fucose - Con A concanavalin A - LCA Lens culinaris agglutinin - AAA Aleuria aurantia agglutinin - WGA Wheat germ agglutinin - RCA II Ricinus communis agglutinin II - PBS phosphate buffered saline, 0.01m Na₂HPO₄/0.14m NaCl, pH 7.2 - HPLC high performance liquid chromatography - EMEM Eagle's Minimal Essential Medium - Lec^R lectin resistant - MG -methylglycoside     

Expression of platelet glycoprotein Ib alpha in HEL cells

We have previously shown that platelet glycoprotein Ib is expressed in a minority of cells of the human leukemic cell line HEL (Tabilio, A., Rosa, J. P., Testa, U., Kieffer, N., Nurden, A. T., Del Canizo, M. C., Breton-Gorius, J., and Vainchenker, W. (1984) EMBO J. 3, 453-459). In this report, we have selected a stable HEL subclone with increased expression of glycoprotein (GP) Ib as assessed by 6 different monoclonal antibodies in order to investigate the biochemical characteristics of this glycoprotein. A single polypeptide chain of apparent Mr = 60,000 was precipitated under reducing and nonreducing conditions by a specific polyclonal anti-platelet glycocalicin antibody and two anti-GPIb alpha monoclonal antibodies (AN51 and AP1), both from surface-labeled and metabolically labeled HEL cells. We were unable to demonstrate the presence of a polypeptide corresponding to the beta subunit of GPIb or GPIX which is closely associated with GPIb. Competitive immunoprecipitation performed in the presence of an excess amount of cold platelet glycocalicin completely displaced the Mr = 60,000 polypeptide. Synthesis of N-linked oligosaccharide chains on this Mr = 60,000 polypeptide was inhibited by the antibiotic tunicamycin, and a shift of the apparent Mr from 60,000 to 48,000 was observed. O-Linked oligosaccharide chains identical to platelet GPIb hexasaccharides were deficient or incomplete since no peanut agglutinin binding to the Mr = 60,000 polypeptide was observed after neuraminidase treatment of HEL cells. Thus, our results provide evidence that the Mr = 60,000 polypeptide expressed on the surface membrane of HEL cells is closely related to platelet GPIb and corresponds to an incompletely or abnormally O-glycosylated GPIb alpha subunit.