1H- and13C-NMR spectroscopic studies of lipid extracts of the red algaGracilaria longa

Lipid extracts of the red algaGracilaria longa were studied by¹H- and¹³C-NMR spectroscopy. Peaks in the¹³C-NMR spectra attributable to sterols, chlorophylls and carotenoids allowed free and acylated cholesterol, chlorophylla and lutein to be identified as the most abundant components of these classes. A content of 0.5 ± 0.1 μmoles of total cholesterol/g wet alga was estimated from the¹H-NMR spectrum, which also allowed the determination of the phosphatidylcholine/total lipid molar ratio (9.5 ± 0.5%). The¹³C-NMR spectroscopic experiments provided information on the position of the double bonds on the fatty acid residues. A comparison between NMR spectra of lipid extracts obtained for wet and dried alga showed that the alga undergoes both a dramatic peroxidation and some glycolipid degradation during the drying process.     

Occurrence of Prorocentrum lima,a DSP toxin-producing species from the Atlantic coast of Canada

A species of Prorocentrum (Dinophyta, Prorocentrales), isolated from a phytoplankton net sample from the Atlantic coast of Nova Scotia, has been brought into unialgal culture. The sample was collected at an aquaculture site immediately following an incident of diarrhetic shellfish poisoning (DSP) due to the consumption of contaminated mussels. This clonal isolate has been identified as P. lima, based on its morphological characteristics. Analysis of the culture extract, using high performance liquid chromatography (HPLC) with fluorescence detection, indicated the presence of the DSP toxins, okadaic acid (OA) and dinophysistoxin-1 (DTX-1).     

Interleukin-1 inhibits PGE2 binding to macrophage-like P388D1 cells by a cyclic AMP-independent process

The interaction between interleukin IL-1α and PGE₂ on P388D₂ on cells has been investigated. Preincubation of murine macrophage-like cells, P388D₁, with IL-1α (0–73 pM) reduced the binding of PGE₂ to these cells in a concentration-dependent manner. Scatchard analysis showed that IL-1α decreased the PGE₂ binding by lowering both the high and low affinity receptor binding capacities (from 0.31 ± 0.02 to 0.12 ± 0.01 fmol/10⁶ cells for the high affinity receptor binding sites and from 2.41 ± 0.12 to 1.51 ± 0.21 fmol/10⁶ cells for the low affinity receptor binding sites). However, the dissociation constants of the receptor of the IL-1α-treated cells remained unchanged. Inhibition of PGE₂ binding IL-1α did not involve changes in either protein phosphorylation or intracellular cyclic AMP levels. Our data clearly show that IL-1α inhibits the binding of PGE₂ to monocytes/macrophages and may thereby counter the immunosuppressive actions of PGE₂.     

A semiempirical study of the prototropic tautomerism of hypoxanthine

The total and relative energies, bond order matrices and localized MOs for the eight possible tautomers of hypoxanthine (HYP) have been calculated, with full geometry optimization, using both AM1 and MNDO methods. The AM1 relative energies show that HYP(9,1), HYP(7,1) and HYP (9,10) are the predominant species at room temperature, the two former being in larger concentration that the latter. The calculated IR spectra for these species agree well with the reported spectrum in an isolated matrix, which has been interpreted in terms of the presence of these three tautomeric forms. The MNDO method does not predict the right order, and the more stable tautomer would be HYP(9,10). The calculated structure for the HYP(9,1) species shows that the molecule is essentially planar. The bond distances compare well with those of hypoxanthine hydrochloride and guanine and also correlate well with the calculated bond orders. The proton affinities for the three more stable tautomers have also been calculated. For HYP(9,1) the prefered site of protonation is N7, whereas for HYP(7,1) the protonation occurs rather at N9. These results agree well with¹⁵N and¹³C NMR studies in DMSO.     

Direct observation by X-ray analysis of the tetrahedral "intermediate" of aspartic proteinases.

We report the X-ray analysis at 2.0 A resolution for crystals of the aspartic proteinase endothiapepsin (EC 3.4.23.6) complexed with a potent difluorostatone-containing tripeptide renin inhibitor (CP-81,282). The scissile bond surrogate, an electrophilic ketone, is hydrated in the complex. The pro-(R) (statine-like) hydroxyl of the tetrahedral carbonyl hydrate is hydrogen-bonded to both active-site aspartates 32 and 215 in the position occupied by a water in the native enzyme. The second hydroxyl oxygen of the hydrate is hydrogen-bonded only to the outer oxygen of Asp 32. These experimental data provide a basis for a model of the tetrahedral intermediate in aspartic proteinase-mediated cleavage of the amide bond. This indicates a mechanism in which Asp 32 is the proton donor and Asp 215 carboxylate polarizes a bound water for nucleophilic attack. The mechanism involves a carboxylate (Asp 32) that is stabilized by extensive hydrogen bonding, rather than an oxyanion derivative of the peptide as in serine proteinase catalysis.     

Identification of (Na,K)ATPase inhibitor in brine shrimp,Artemia salina,as long-chain fatty acids

Summary An inhibitory activity to (Na,K)ATPase was found in cell extracts of the brine shrimp, Artemia salina, irrespective of its developmental stages. Organic solvent extraction together with gas chromatographic analysis reveals that the inhibitory activity is due to long-chain, non-esterified fatty acids and their derivatives. Unsaturated fatty acids, especially with cis-configuration, are more effective in inhibition than saturated ones.Abbreviations ATPase adenosine triphosphatase - EDTA ethylenediamine-tetraacetate - TLC thin-layer chromatography     

1H NMR investigation of reduced copper-cobalt superoxide dismutase

Human copper-cobalt superoxide dismutase in the reduced form has been investigated through ¹H NMR techniques. The aim is to monitor the structural properties of this derivative and to compare them with those of reduced and oxidized native superoxide dismutases. The observed signals of the cobalt ligands have been assigned as well as the signals of the histidines bound to copper(I). The latter signals experience little pseudocontact shifts which allow a rough orientation of the magnetic susceptibility tensor in the molecular frame. The connectivities indicate that, although the histidine bridge is broken in the reduced form, the interproton distances between ligands of both ions are essentially the same.Abbreviations WEFT water eliminated Fourier transform - NOE nuclear Overhauser effect - NOESY NOE spectroscopy - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - SOD superoxide dismutase - E₂Co(II)SOD SOD with empty copper site (E=empty) and with cobalt(II) in the Zinc(II) site Offprint requests to: I. Bertini     

Different oligosaccharides accumulate in the brain and urine of a cat with α-mannosidosis: structure determination of five brain-derived and seventeen urinary oligosaccharides

Five brain-derived and 17 urinary oligomannose-type oligosaccharides were isolated by ion-exchange chromatography on Mono Q or Dowex, followed by HPLC on Lichrosorb-NH₂ from a Persian cat suffering from -mannosidosis. The structures ofthe carbohydrate chains were determined by 500- or 600-MHz¹H-NMR spectroscopy. Different oligosaccharide patterns were found in brain and urine. 99% of the urinary oligosaccharides possess an (1-6)-linked mannose residue attached to -mannose, whereas only 5% of the brain-derived oligosaccharides contain such a residue. Furthermore, of the urinary carbohydrate chains 71% end with Man1-4GlcNAc1-4GlcNAc and 29% end with Man1-4GlcNAc, whereas the corresponding amounts are 23% and 77%, respectively, for the brain-derived oligosaccharides.Abbreviations MLEV-17 composite pulse devised by M. Levitt - HOHAHA homonuclear Hartman-Hahn spectroscopy - TPPI time-proportional phase incrementation - 2D two dimensional - GlcNAc N-acetylglucosamine - Man mannose - Fuc fucose     

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     

Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism

Thyrotropin-R eleasing hormone (TRH)-degrading pyroglutamyl peptidase I(PGP I) and prolyl endopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively.After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membranebound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme.Gelfiltration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 µM and 235 µM, and a Vmax of 1.49 and 8.80 pmoUmin/µg protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 ± 0.9, 22.5 ± 11.1 and 28.7 ± 14.6pg/10⁶ cells, respectively. When cells have been incubated for 2 to 72 hours with a P. E. inhibitor (Z-Gly-Pro-CHN₂) at 5 × 10^–7M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.These data suggest that TRH is not degraded by cytosolic, unspecific PGP I and PE enzymes in RINm 5F. The finding that these cells contain 10 and 13 times more His-Pro-Diketopiperazine and acid TRH than TRH may be an indirect evidence for the existence of another precursor than TRH for these two peptides or of the possibility that TRH can be degraded by other peptidases.Abbreviations TRH Thyrotropin-Releasing Hormone or Thyroliberin - His-Pro-DKP Histidyl-ProlineDiketopiperazine - TRH-OH acid TRH or deamidated TRH - LH-RH Luteinizing Hormone-Releasing Hormone - Z-Gly-Pro-CHN₂ N-benzyloxycarboxyl-Gly-Pro-diazomethylketone - PGP Pyroglutamyl Peptidase, PGP I (EC 3.4.19.3) and PGP II (EC 3.4.19.-) - PE Prolyl Endopeptidase or post-proline cleaving enzyme (EC 3.4.21.26)