Regulation of exocytosis in electrically permeabilized insulin-secreting cells. Evidence for Ca2+ dependent and independent secretion

The regulation of insulin secretion from RINm5F cells exposed to high voltage discharge has been investigated. Electron microscopy revealed that the overall structure of the cells was preserved after permeabilization. In this preparation insulin release was stimulated by Ca²⁺ (EC₅₀=2.4 M). The stable GTP analogue GTPS enhanced secretion both at intermediate (nano- to micromolar) and vanishingly low (<10 pM) Ca²⁺ concentrations. At optimal Ca²⁺ (10 M) the effect of GTPS was greatly reduced. We investigated whether the secretory response to GTP analogues was mediated by any of three enzyme systems regulated by GTP-binding proteins, i.e. generation of cyclic AMP by adenylate cyclase, of diacylglycerol by phospholipase C and of arachidonic acid by phospholipase A₂. The involvement of these messenger systems could be excluded as (i) cyclic AMP only had minor, Ca²⁺ dependent effects, (ii) phospholipase C was not activated in the absence of Ca²⁺ and insulin secretion due to the phorbol ester TPA displayed a different Ca²⁺ dependency, (iii) arachidonic acid did not elicit Ca²⁺ independent insulin secretion. These results, taken together with the finding that insulin secretion due to Ca²⁺ or TPA is attenuated by the inhibitory guanine nucleotide GDPS, suggest the existence of a regulatory site in exocytosis which is sensitive to guanine nucleotides.Abbreviations InsP₃ inositol trisphosphate - Ptd-InsP₂ phosphatidylinositol 4,5-bisphosphate - GTPS guanosine 5-(3-O-thio)triphosphate - GDPS guanosine 5-(2-O-thio)diphosphate - Gpp(NH)p guanyl-5-yl imidodiphosphate - TPA 12-O-tetradecanoylphorbol-13-acetate - OAG 1-oleoyl-2-acetylglycerol - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - EGTA (ethylenebis(oxyethylenenitrilo)tetraacetic acid - DAG diacylglycerol - [Ca²⁺]_i cytosolic free Ca²⁺ concentration     

High affinity insulin binding in the human placenta insulin receptor requires αβ heterodimeric subunit interactions

Summary Insulin binding to human placenta membranes treated at pH 7.6 or 8.5 in the presence or absence of 2.0mm DTT for 5 min, followed by the simultaneous removal of the DTT and pH adjustment to pH 7.6, displayed curvilinear (heterogeneous) insulin binding plots when analyzed by the method of Scatchard. However, Triton X-100 solubilization followed by Bio-Gel A-1.5m gel filtration chromatography of the placenta membranes previously treated with DTT at pH 8.5 generated a nearly straight line (homogeneous) Scatchard plot.¹²⁵I-insulin affinity crosslinking studies coupled with Bio-Gel A-1.5m gel filtration chromatography demonstrated that the alkaline pH and DTT treatment of placenta membranes followed by detergent solubilization generated an heterodimeric insulin receptor complex from the ₂₂ heterotetrameric disulfide-linked state. The ability of alkaline pH and DTT to produce a functional heterodimeric insulin receptor complex was found to be time dependent with maximal formation and preservation of tracer insulin binding occurring at 5 min. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of placenta membranes can result in the formation of a functional heterodimeric insulin receptor complex. (ii) the heterodimeric complex displays homogeneous insulin binding. (iii) the insulin receptor membrane environment maintains the ₂₂ association state, which displays heterogeneous insulin binding, despite reduction of the critical domains that are responsible for the covalent interaction between the heterodimers.Abbreviations used are ATP adenosine 5-triphosphate - DTT dithiothreitol - SDS sodium dodecyl sulfate - DSS disuccinimidyl suberate - NEM N-ethylmaleimide - IGF-I insulin-like growth factor-I - EDTA ethylenediaminetetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

Localization and synthesis of an insulin-binding region on human insulin receptor

Seven regions of the subunit of human insulin receptor (HIR) were synthesized and examined for their ability to bind radioiodinated insulin. A peptide representing one of these regions (namely, residues 655–670) exhibited a specific binding activity for insulin. In quantitative radiometric titrations, the binding curves of¹²⁵I-labeled insulin to adsorbents of peptide 655–670 and of purified placental membrane were similar or superimposable. The binding of radioiodinated insulin to peptide or to membrane adsorbents was completely inhibited by unlabeled insulin, and the inhibition curves indicated that the peptide and the membrane on the adsorbents had similar affinities. Synthetic peptides that were shorter (peptide 661–670) or longer (peptide 651–670) than the region 655–670 exhibited lower insulin-binding activity. It was concluded that an insulin-binding region in the HIR subunit resides within residues 655–670. The results do not rule out the possibility that other regions of the subunit may also participate in binding of HIR to insulin, with the region described here forming a face within a larger binding site.     

Repair of ultraviolet-light damaged ColE1 factor carrying Escherichia coli genes for guanine synthesis.

Summary Hybrid ColE1 plasmids called ColE1-cos-guaA or ColE1-cos-gal can be efficiently transduced into various E. coli K-12 cells through packaging into phage particles. Using these plasmids, repair of ultraviolet-light (UV) damaged ColE1 DNAs was studied in various UV sensitive E. coli K-12 mutants. (1) The host mutations uvrA and uvrB markedly reduced host-cell reactivation of UV-irradiated ColE1-cos-guaA. (2) Pre-existing hybrid ColE1 plasmids had no effect on the frequency of phage-mediated transduction of another differentially marked hybrid ColE1 DNAs. (3) ColE1-cos-guaA and ColE1-cos-gal DNAs could temporarily but not stably co-exist in E. coli K-12 recA cells. (4) The presence of ColE1-cos-gal in uvrB cells promoted the repair of super-infected UV-irradiated ColE1-cos-guaA about 7-fold. (5) The same ColE1-cos-gal plasmid in a uvrB recA double mutant did not have this promoting effect. These results indicate that the effect of resident hybrid ColE1 plasmids is manifested by the host recA ⁺ gen function(s) and suggest that ColE1 plasmid itself provides no recA ⁺-like functions.     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

Biosynthesis of the storage polysaccharide from the snail Biomphalaria glabrata,identification and specificity of a branching β1→6 galactosyltransferase

Adult snails synthesize in their albumen glands a storage polysaccharide called galactan which is utilized by the developing embryos. With [6-³H]-uridine 5diphosphogalactose the incorporation of labelled d-galactose into the polysaccharide can be traeed in freshly removed glands maintained in a bathing buffer. After centrifugation of homogenized glands, galactosyltrasferase activity is only found in the insoluble fraction. Chaps extracts of this material retain almost all of their activity and can be used for comparison of the incorporation rates into different native galactans or in various oligosaccharides. A highly efficient -(16) galactosyltransferase was detected when methyl 3-O-(-d-galactopyranosyl)--d-galactopyranoside was offered as acceptor. The substitution at the penultimate residue resulted in a branched trisaccharide as demonstrated by ¹H-NMR-spectroscopy and permethylation analysis of the reaction product. Comparable results were obtained with various oligosaccharides containing an internal galactose unit glycosidically linked 13. Attempts to separate and purify the various enzymes involved resulted in the isolation of a fraction which is able to transfer d-Gal exclusively to native galactan, but not to oligosaccharides. A further fraction was obtained from a different resin with activity for native galactan and 6-O-(-d-galactopyranosyl)-d-galactopyranose. but without any for methyl-3-O-(-d-galactopyranosyl)--d-galactopyranose. It is thus concluded that at least three different enzymes are involved in the biosynthesis of this snail galactan.Abbreviation Gal galactose - glc gas-liquid chromatography - Gro glycerol - tlc thin layer chromatography     

Structural diversity of O-polysaccharides and serological classification of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">garcae</Emphasis> and other strains of genomospecies 4

Novel O-serotypes were revealed among Pseudomonas syringae pv. garcae strains by using a set of mouse monoclonal antibodies specific to the lipopolysaccharide O-polysaccharide. Structural studies showed that the O-polysaccharide of P. syringae pv. garcae NCPPB 2708 is a hitherto unknown linear L-rhamnan lacking strict regularity and having two oligosaccharide repeating units I and II, which differ in the position of substitution in one of the rhamnose residues and have the following structures: I:3)--L Rha (12)-- L Rha (12)--L-Rha-(13)--L Rha (1;II: 2)--L-Rha-(13) -L-Rha-(12)--L-Rha-(13)--L Rha (1.The branched O-polysaccharides of P. syringae pv. garcae ICMP 8047 and NCPPB 588^T have the same L-rhamnan backbone with repeating units I and II and a lateral chain of 14)- or 13)-linked residues of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc). Several monoclonal antibody epitopes associated with the L-rhamnan backbone or the lateral -D-Fuc3NAc residues were characterized.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 777–789.Original Russian Text Copyright © 2004 by Ovod, Zdorovenko, Shashkov, Kocharova, Knirel.     

Synthesis of Aminoethyl Glycosides of the Carbohydrate Chains of Glycolipids Gb3, Gb4, and Gb5

4-O-Glycosylation of 2-azidoethyl 2,3,6-tri-O-benzoyl-4-O-(2,3,6-tri-O-benzoyl--D-galactopyranosyl)--D-glucopyranoside with ethyl 2,3,4,6-tetra-O-benzyl- and ethyl 3-O-acetyl-2,4,6-tri-O-benzyl-1-thio--D-galactopyranoside in the presence of methyl trifluoromethanesulfonate led to trisaccharide 2-azidoethyl (2,3,4,6-tetra-O-benzyl--D-galactopyranosyl)-(14)-(2,3,6-tri-O-benzoyl--D-galactopyranosyl)-(14)-2,3,6-tri-O-benzoyl--D-glucopyranoside and its 3"-O-acetylated analogue, 2-azidoethyl (3-O-acetyl-2,4,6-tri-O-benzyl--D-galactopyranosyl)-(14)-(2,3,6-tri-O-benzoyl--D-galactopyranosyl)-(14)-2,3,6-tri-O-benzoyl--D-glucopyranoside in yields of 85 and 83%, respectively. Deacetylation of the latter compound and subsequent glycosylation with 4-trichloroacetamidophenyl 3,4,6-tri-O-acetyl-2-deoxy-1-thio-2-trichloroacetamido--D-galactopyranoside and 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-1-thio-2-trichloroacetamido--D-galactopyranoside in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid resulted in the corresponding selectively protected derivatives of the tetrasaccharide GalNAc(13)Gl(14)Gal(14)Glc-OH₂CH₂N₃ and the pentasaccharide Gal(13)GalNAc(13)Gl(14)Gal(14)Glc-OH₂CH₂N₃ in 88 and 73% yields, respectively. Removal of O-protecting groups, substitution of acetyl group for the N-trichloroacetyl group, and reduction of the aglycone azide group resulted in the target 2-aminoethyl globo-tri-, -tetra-, and -pentasaccharide, respectively.     

Protocol-dependence of equivalent circuit parameters of toad urinary bladder

Summary Determinations of current-voltage relationships are widely employed in the characterization of epithelial sodium transport. In order to determine the protocol dependence of transport parameters in the toad urinary bladder, studies were carried out in the presence and absence of amiloride, an inhibitor of active sodium transport. With symmetric positive and negative perturbations of the transepithelial electrical potential difference (0±100 mV) for 30 sec, the amiloride-sensitive current-voltage (i ^a -) relationship was near linear over the range –75+100 mV, indicating constancy of the conductance ^a and the apparent electromotive force E _Na, lumped parameters of the standard electrical equivalent circuit model of the active transport system. With a reverse protocol (±1000 mV) or 15 min perturbations thei ^a - relationships were highly nonlinear. Nonlinearity reflected voltage dependence of parameters: perturbations that increased active transport decreased E _Na and increased ^a, as evaluated from 10 sec perturbations of ; slowing of active transport produced the converse changes. These effects are usefully analyzed in both quasi-steady states and true steady states by means of a detailed equivalent circuit incorporating the significant ionic currents across each plasma membrane. Precise understanding of the significance of ^a and E _Na will require characterization of the partial ionic conductances on perturbation of .     

A suitable method for construction and cloning hybrid plasmids containing EcoRI-fragments of E. coli genome.

Summary A convenient procedure for the isolation of specificEcoRI-fragments ofE. coli genome and their amplification on Km-resistance plasmid vector CK 11 is described. The hybrid molecules were constructedin vitro usingEcoRI-digestion, followed by ligation. Then appropriatedE. coli strain was transformed with ligated DNA mixture and hybrid plasmids CK 11-arg ⁺, CK 11-his ⁺, CK 11-thr ⁺ and CK 11-leu ⁺ containing loci ofE. coli genome were selected by molecular cloning. The hybrid plasmids obtained consisted of oneEcoRI-fragment of initial plasmid CK 11 and one respective specific portion ofE. coli genome.     

Production of β-fructofuranosidase byAspergillus japonicus

A newly isolated strain, MU-2, which produces very high -fructofuranosidase activity, was identified asAspergillus japonicus. For enzyme production by the strain, sucrose at 20% (w/v) was the best carbon source and yeast extract at 1.5 to 3% (w/v) the best nitrogen source. Total enzymatic activity and cell growth were at maximum after 48 h, at 1.57×10⁴ U/flask and 0.81 g dry cells/flask, respectively. The optimum pH value of the enzymatic reaction was between 5.0 and 5.5 and the optimum temperature 60 to 65°C. The enzyme produced 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose by fructosyl-transferring activity. The strain was found to be very useful for industrial production of -fructofuranosidase.     

Normal expression of the viral gene N interferes with growth of bacteriophage lambda in Escherichia coli 15T-.

Summary Growth of and of some lambdoid phages is considerably inhibited on strain 3057 derived from E. coli 15T^-. Mutants of which overcome this inhibition map in gene N. Some of these hty mutants are temperature sensitive for growth on E. coli K12. Thus plating of on strain 3057 allows one to isolate temperature sensitive N mutants. The hty mutants produce less than normal N activity as judged by their low efficiency of plating on a nus ^- host and by the extended latent period of some of them on normal hosts. The inability of strain 3057 to propagate can be at least partially reversed by addition of thymidine to the medium and the growth difference between hty and in 3057 increases with decreasing thymidine concentration. The amount of DNA produced by in 3057 at low thymidine concentration is lower than that produced by hty under the same conditions. Only a small percentage of the DNA produced by in 3057 is packaged into viable phage particles. This suggests that not only produces less DNA in 3057 than hty but that an important part of the DNA in 3057 is in a form which can not be packaged or which is noninfective for other reasons. A hypothesis is discussed that hty mutations enable to grow on E. coli 15T^- at low thymidine concentration because they lead to reduction in the number of single strand nicks in the DNA by reducing the intracellular endonuclease activity. Under permissive conditions conditional lethal N mutants are favored for growth on 3057 over N ⁺ which confirms the idea that N activity or the activity of a gene under N control interferes with growth in 3057 at low thymidine concentration.     

Reaction centers from three herbicide resistant mutants of Rhodobacter sphaeroides 2.4.1: Kinetics of electron transfer reactions

Electron transfer rates were measured in RCs from three herbicide-resistant mutants with known amino acid changes to elucidate the structural requirements for last electron transfer. The three herbicide resistant mutants were IM(L229) (Ile-L229 Met), SP(L223) (Ser-L223 Pro) and YG(L222) (Tyr-L222 Gly). The electron transfer rate D⁺Q_A ^-Q_BD⁺Q_AQ_B (k _AB) is slowed 3 fold in the IM(L229) and YG(L222) RCs (pH 8). The stabilization of D⁺Q_AQ_B ^- with respect to D⁺Q_AQ_B ^- (pH 8) was found to be eliminated in the IM(L229) mutant RCs (G⁰ 0 meV), was partially reduced in the SP(L223) mutant RCs (G⁰=–30 meV), and was unaltered in the YG(L222) mutant RCs (G⁰=–60 meV), compared to that observed in the native RCs (G⁰=–60 meV). The pH dependences of the charge recombination rate D⁺Q_AQ_B ^-DQ_AQ_B (k _BD) and the electron transfer from Q_A ^- (k _QA ^-Q_A) suggest that the mutations do not affect the protonation state of Glu-L212 nor the electrostatic interactions of Q_B and Q_B ^- with Glu-L212. The binding affinities of UQ₁₀ for the Q_B site were found in order of decreasing values to be native IM(L229) > YG(L222) SP(L223). The altered properties of the mutant RCs are used to deduce possible structural changes caused by the mutations and are dicscussed in terms of photosynthetic efficiency of the herbicide resistant strains.Abbreviations Bchl bacteriochlorophyll - Bphe bacteriopheophytin - cholate 3,7,12-trihydroxycholanic acid - D donor (bacteriochlorophyll dimer) - EDTA ethylenediamine tetraacetic acid - Fe²⁺ non-heme iron atom - LDAO lauryl dimethylamine oxide - PS II photosystem II - Q_A and Q_B primary and secondary quinone acceptors - RC bacterial reaction center - Tris tris(hydroxymethyl)aminomethane - UQ₀ 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50     

Negative-ion fast atom bombardment tandem mass spectrometry for characterization of sulfated unsaturated disaccharides from heparin and heparan sulfate

Negative-ion fast atom bombardment tandem mass spectrometry has been used in the characterization of non-, mono-, di- and trisulfated disaccharides from heparin and heparan sulfate. The positional isomers of the sulfate group of monosulfated disaccharides were distinguished from each other by negative-ion fast atom bombardment tandem mass spectra, which provide an easy way of identifying the positional isomers. This fast atom bombardment collision induced dissociation mass spectrometry/mass spectrometry technique was also applied successfully to the characterization of di- and trisulfated disaccharides.Abbreviations FABMS fast atom bombardment mass spectrometry - CID collision induced dissociation - MIKE mass analysed ion kinetic energy - MS/MS mass spectrometry/mass spectrometry - HPLC high performance liquid chromatography - UA d-gluco-4-enepyranosyluronic acid - CS chondroitin sulfate - DS dermatan sulfate - HA hyaluronan - Hep heparin - HS heparan sulfate - UA(14) GlcNAc 2-acetamido-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose - UA(14)GlcNAc6S 2-acetamido-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcNAc 2-acetamido-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcNAc6S 2-acetamido-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(14)GlcN6S 2-amino-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcN 2-amino-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcN6S 2-amino-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(14)GlcNS 2-deoxy-2-sulfamino-4-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose - UA(14)GlcNS6S 2-deoxy-2-sulfamino-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcNS 2-deoxy-2-sulfamino-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcNS6S 2-deoxy-2-sulfamino-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(13)GalNAc 2-acetamido-2-deoxy-3-O-(-d-Gluco-4-enepyranosyluronic acid)-d-galatose - UA(13)GalNAc4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA(13)GalNAc6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA2S(13)GalNAc 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-galactose - UA2S(13)GalNAc4S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA2S(13)GalNAc6S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA(13)GalNAcDiS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose - UA(13)GlcNAc 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose.     

Synthesis of Aminoethyl Glycosides of the Ganglioside GM1 and Asialo-GM1 Oligosaccharide Chains

4-O-Glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)--D-glucopyranoside with a disaccharide donor, 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-1-thio-2-trichloroacetamido--D-galactopyranoside, in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid resulted in a tetrasaccharide, 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, in 69% yield. The complete removal of O-protecting groups in the tetrasaccharide, the replacement of N-trichloroacetyl by N-acetyl group, and the reduction of the aglycone azide group to amine led to the target aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of asialo-GM₁ ganglioside in 72% overall yield. Selective 3-O-glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,6-di-O-benzyl--D-galactopyranosyl)--D-glucopyranoside with thioglycoside methyl (ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero--D-galacto-2-nonulopyranosyl)oate in acetonitrile in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid afforded 2-azidoethyl [methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)oate]-(2 3)-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, the selectively protected derivative of the oligosaccharide chain of GM₃ ganglioside, in 79% yield. Its 4-O-glycosylation with a disaccharide glycosyl donor, (4-trichloroacetophenyl-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl) 1-thio-2-trichloroacetamido--D-galactopyranoside in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid gave 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-{[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)onate]-(2 3)}-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside in 85% yield. The resulting pentasaccharide was O-deprotected, its N-trichloroacetyl group was replaced by N-acetyl group, and the aglycone azide group was reduced to afford in 85% overall yield aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)-[-D-Neu5Ac-(2 3)]--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of GM₁ ganglioside.     

Long-range physical mapping of the alpha-amylase-1 (alpha-Amy-1) loci on homoeologous group 6 chromosomes of wheat

Summary Long-range physical maps of the small multigene family of the malt -amylase genes (-Amy-1) located on the long arms of wheat chromosomes 6A (the -Amy-A1 locus) and 6B (-Amy-B1) were generated by pulsed-field gel electrophoresis analysis. By using three methylation-sensitive rare-cutter restriction endonucleases, NotI, NruI and MluI, and an -Amy-1 cDNA probe and four gene-specific genomic probes from the -Amy-B1 locus, the size of the -Amy-B1 locus was estimated to be about 700 kb and of the -Amy-B1 locus to be about approximately 4300 kb. These two maps indicate clustering of GC-rich and C-methylation-sensitive restriction enzyme recognition sites. At least five regions reminiscent of CpG islands are apparent in -Amy-B1, and three in -Amy-A1. Correlation between recombination frequency and physical distance within the -Amy-B1 locus suggests that 1 cM approximates to 1 Mb in physical distance.     

Sugar conformation of a stereospecific 2′-R or 2′-S deuterium-labeled DNA decamer studied with proton-proton J coupling constants

The sugar conformation of a DNA decamer was studied with proton-proton ³J coupling constants. Two samples, one comprising stereospecifically labeled 2-R-²H for all residues and the other 2-S-²H, were prepared by the method of Kawashima et al. [J. Org. Chem. (1995) 60, 6980–6986; Nucleosides Nucleotides (1995) 14, 333–336], the deuterium labeling being highly stereospecific 99% for all 2-²H, 98% for 2-²H of A, C, and T, and 93% for 2-²H of G). The ³J values of all H1-H2 and H1-H2 pairs, and several H2-H3 and H2-H3 pairs were determined by line fitting of 1D spectra with 0.1–0.2 Hz precision. The observed J coupling constants were explained by the rigid sugar conformation model, and the sugar conformations were found to be between C3-exo and C2-endo with _m values of 26° to 44°, except for the second and 3 terminal residues C2 and C10. For the C2 and C10 residues, the lower fraction of S-type conformation was estimated from J_H1H2 and J_H1H2 values. For C10, the N–S two-site jump model or Gaussian distribution of the torsion angle model could explain the observed J values, and 68% S-type conformation or C1-exo conformation with 27° distribution was obtained, respectively. The differences between these two motional models are discussed based on a simple simulation of J-coupling constants.     

Effects of supplied cinnamic acids and biosynthetic intermediates on the anthocyanins accumulated by wild carrot suspension cultures

Anthocyanins isolated and characterized from the wild carrot suspension cultures used here were 3-O--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D<-galactopyranosylcyanidin (1), 3-O-[-D- xylopyranosyl-(12)--D-galactopyranosyl]cyanidin (2), 3-O-(6-O-sinapoyl)--D-glucopyranosyl-(16)-[-D- xylopyranosyl-(12)-]-D-galactopyranos ylcyanidin (3), 3-O-(6-O-feruoyl)--D-glucopyranosyl-(16)-[- D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (4), 3-O-(6-O-coumaroyl)--D-glucopyranosyl-(16)- [-D-xylopyranosyl-(12)-]-D-galactopyrano sylcyanidin (5), 3-O-[6-O-(3,4,5-trimethoxycinnamoyl)]-- D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (6), 3-O-[6-O-(3,4-dime- thoxycinnamoyl)]--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (7), 3-O-[(6-O-sinapoyl)--D-glucopyranosyl-(16)--D-galactopyranosyl]cyanidin (8), and 3-O-(-D-galactopyranosyl)cyanidin (9). Except when cinnamic acids were provided in the culture medium, the major anthocyanin present in the two clones examined was 2. When the naturally occurring and some non-naturally occurring cinnamic acids were provided individually in the medium, 1 and 2 were minor components and the anthocyanin acylated with the supplied cinnamic acid, namely 3, 4, 5, 6, or 7 was the major anthocyanin present in the tissue. When caffeic acid was provided the major anthocyanin in the tissue was 4, thereby suggesting that the caffeic acid was methylated before its use in anthocyanin biosynthesis. Other cinnamic acids supplied had limited effects on the anthocyanins accumulated and appeared not to result in the accumulation of new anthocyanins by the tissue. Thus the tissue can use some but not all analogues of sinapic acid to acylate anthocyanins. Additional anthocyanins were detected in extracts of the wild carrot tissue cultures using mass spectrometry (both MS/MS and HPLC/MS). The additional compounds detected have also been found in cultures of black carrot, an Afghan cultivar of Daucus carota ssp. sativa and the flowers of wild carrot giving no evidence for qualitative differences in the anthocyanins synthesized by subspecies, cell cultures from subspecies, or clones from cell cultures. There are major differences in the amounts of individual anthocyanins found in cultures from different subspecies and in different clones from cell cultures. Here anthocyanins without acyl groups were usually found in the tissues and their accumulation is discussed. On the basis of the structures of the isolated anthocyanins, a likely pathway from cyanidin to the accumulated anthocyanins is proposed and discussed.Abbreviations Sin sinapoyl - Fer feruoyl - 4-Coum. 4-coumaroyl - 3,4-MeO₂Cin 3,4-dimethoxyeinnamoyl - 3,4,5-MeO₃Cin 3,4,5-trimethoxycinnamoyl - Cya cyanidin