Synthetic substrate analogues for UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme

UDP-GlcNAc: Man1-6R (1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complexN-glycans. We have tested a series of synthetic analogues of the substrate Man1-6(GlcNAc1-2Man1-3)Man-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attachesN-acetylglucosamine in 1-2 linkage to the 2-OH of the Man1-6 residue. The 2-deoxy analogue is a competitive inhibitor (K _i=0.13mm). The 2-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3-, 4- and 6-OH groups are not essential for binding or catalysis since the 3-, 4- and 6-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3-position to pentyl and substituted pentyl groups causes competitive inhibition (K _i=1.0–2.5mm). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3-O-(4,4-azo)pentyl group and a 3-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man1-6 residue are essential for binding although the 2- and 3-OH face the catalytic site of the enzyme. The 4-OH group of the Man-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind. This contrasts with GlcNAc-T I which cannot bind to the 4-deoxy-Man- substrate analogue. The data are compatible with our previous observations that a bisectingN-acetylglucosamine at the 4-OH position prevents both GlcNAc-T I and GlcNAc-T II catalysis. However, in the case of GlcNAc-T II, the bisectingN-acetylglucosamine prevents binding due to steric hindrance rather than to removal of an essential OH group. The 3-OH of the Man1-3 is an essential group for GlcNAc-T II since the 3-deoxy derivative does not bind to the enzyme. The trisaccharide GlcNAc1-2Man1-3Man-O-octyl is a good inhibitor (K _i=0.9mm). The above data together with previous studies indicate that binding of the GlcNAc1-2Man1-3Man- arm of the branched substrate to the enzyme is essential for catalysis. Abbreviations: GlcNAc-T I, UDP-GlcNAc:Man1-3R (1-2)-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GlcNAc-T II, UDP-GlcNAc:Man1-6R (1-2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143); MES, 2-(N-morpholino)ethane sulfonic acid monohydrate.     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.     

A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C₁₈ cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar G_M3-ganglioside is simple and rapid relative to previously described methods. Recovery of G_M3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (G_M3 synthase; ST-1), the transfer of [¹⁴C] sialic acid from CMP-[¹⁴C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations G_M3 G_M3-ganglioside - II³NeuAc-LacCer NeuAc2-3Gal1-4Glc1-1Cer - G_D1a G_D1a-ganglioside, IV³NeuAc, II³NeuAc-GgOse₄Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer - G_D3 G_D3-ganglioside, II³(NeuAc)₂LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer - GgOse₄Cer asialo-G_M1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer - FucG_MI fucosyl-G_MI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer - ST-1 G_M3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase - LacCer lactosylceramide, Gal1-4Glc1-1Cer - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride     

Steroid Polyols from the Far Eastern Starfish Henricia sanguinolenta and H. leviuscula leviuscula

Two new asterosaponins, (20R)-3-O--D-(2-O-methylxylopyranosyl)-24-propylcholest-4-ene-3,6,8,15,16,29-hexaol (sanguinoside A) and (20R,24S)-3-O--D-(2,3,4-tri-O-methylxylopyranosyl)-5-cholestane-3,4,6,8,15,24-hexaol (sanguinoside B), were isolated from two species of Pacific Far Eastern Starfish Henricia sanguinolenta and H. leviuscula leviuscula, collected in the Sea of Okhotsk. Both glycosides contain aglycones with pentahydroxysteroid nuclei of similar structures, which are substituted at the 3-hydroxy group with differently methylated -D-xylosyl residues. Sanguinoside A has an unusual structure of its aglycone side chain, whereas sanguinoside B has a unique permethylated carbohydrate chain. In addition, laevisculoside G, a known glycoside, was identified in the H. leviuscula starfish. The structures of the isolated glycosides were established by interpreting their spectral data and by comparing their spectral characteristics with those of known compounds.     

The carotenoids of Flavobacterium strain R1560

The main carotenoid of Flavobacterium strain R1560 has been identified as (3R,3R)-zeaxanthin. Also present were small amounts of 15-cis-phytoene, phytofluene, -carotene (7,8,7,8-tetrahydro-, -carotene plus 7,8,11,12-tetrahydro-, -carotene), neurosporene, lycopene, -zeacarotene, -carotene, -carotene, -cryptoxanthin, rubixanthin, 3-hydroxy--zeacarotene and several apo-carotenals. Zeaxanthin production was inhibited by nicotine (10 mM), and lycopene and rubixanthin accumulated. The biosynthesis of zeaxanthin is discussed in terms of pathways and also of half-molecule reaction sequences. The presence of zeaxanthin may be a characteristic of a group of Flavobacterium species, and may thus be useful in the taxonomic classification of these organisms.     

Detection and partial characterization of two antigenically distinct β-amylases in developing kernels of wheat

A -amylase (EC 3.2.1.2) was identified in the outer pericarp (P) of developing seeds of wheat (Triticum aestivum L.) and compared with the well known -amylase which is synthesized during seed development in the starchy endosperm (E). The enzyme P already exists in the tissues before anthesis and vanishes at the time when E starts to accumulate. The isoelectric-focusing patterns of P and E are very similar. The relative molecular weight (M_r) of P is slightly higher than that of E (66 and 64.5 kDa, respectively). Both P and E exhibit common epitopes in addition to epitopes specific for each of them. The two enzymes were identified in small amounts in the green tissues of the developing seeds (inner pericarp and testa). No antigenic difference was detected between P and the -amylases of roots and leaves.Abbreviations P pericarp -amylase - E endosperm -amylase - IS1 anti--amylase immune serum - IS2 anti- and anti- amylase immune serum - IS3 anti- amylase immune serum - IEF isoelectric focusing - IgG immunoglobulin G The authors thank Dr. P. Ziegler (Universität Bayreuth, FRG) for stimulating discussion and for useful suggestions during the writing of the text. The authors thank Miss C. Mayer for her skillful technical assistance.     

Carbohydrates of influenza virus. Structure of the oligosaccharides linked to asparagines 406 and 478 in the hemagglutinin of fowl plague virus,strain dutch

Fowl plague virus, strain Dutch, was metabolically labeled withd-[2-³H]mannose, or withd-[6-³H]glucosamine, and the small subunit (HA₂; 0.8 mg in total) of the viral hemagglutinin was isolated by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis. After proteolytic digestion, the radioactive oligosaccharides were sequentially liberated from the glycopeptides by treatment with different endo--N-acetylglucosaminidases and with peptide:N-glycosidase or, finally, by hydrazinolysis. In this manner, four groups of glycans could be obtained by consecutive gel filtrations and were subfractionated by HPLC. The structures of the individual oligosaccharides were analyzed by micromethylation, by acetolysis or by digestion with exoglycosidases. The major species amongst the high mannose glycans at Ans-406 of the viral glycopolypeptide were found to be Man1-2Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNac1-4GlcNAc and Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNAc1-4GlcNAc, while the complex glycans at Asn-478 are predominantly GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc (lacking, in part, one of the outerN-acetylglucosamine residues) and GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc.Abbreviation BSA bovine serum albumin - endo D (F,H) endo--N-acetyl-d-glucosaminidase D (F,H) - HA hemagglutinin (HA₁, large subunit of HA - HA₂ small subunit - FPV fowl plague virus - PNGase F peptide:N-glycosidase F - SDS sodium dodecylsulfate     

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     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Subcellular localization and topology of β(1→4)galactosyltransferase that elongates β(1→4)galactan side chains in rhamnogalacturonan I in potato

The subcellular localization and topology of rhamnogalacturonan I (RG-I) (14)galactosyltransferase(s) ([14]GalTs) from potato (Solanum tuberosum L.) were investigated. Using two-step discontinuous sucrose step gradients, galactosyltransferase (GalT) activity that synthesized 70%-methanol-insoluble products from UDP-[¹⁴C]Gal was detected in both the 0.5 M sucrose fraction and the 0.25/1.1 M sucrose interface. The former fraction contained mainly soluble proteins and the latter was enriched in Golgi vesicles that contained most of the UDPase activity, a Golgi marker. By gel-filtration analysis, products of 180–2,000 Da were found in the soluble fraction, whereas in the Golgi-enriched fraction the products were larger than 80 kDa and could be digested with rhamnogalacturonan lyase and (1,4)endogalactanase to yield smaller rhamnogalacturonan oligomers, galactobiose and galactose. The endogalactanase requires (14)galactans with at least three galactosyl residues for cleavage, indicating that the enzyme(s) present in the 0.25/1.1 M Suc interface transferred one or more galactosyl residues to pre-existing (14)galactans producing RG-I side chains in total longer than a trimer. Thus, the (14)GalT activity that elongates (14)-linked galactan on RG-I was located in the Golgi apparatus. This (14)GalT activity was not reduced after treatment of the Golgi vesicles with proteinase, but approximately 75% of the activity was lost after treatment with proteinase in the presence of Triton X-100. In addition, the (14)GalT activity was recovered in the detergent phase after treatment of Golgi vesicles with Triton X-114. Taken together, these observations supported the view that the RG-I (14)GalT that elongates (14)galactan was mainly located in the Golgi apparatus and integrated into the membrane with its catalytic site facing the lumen.Abbreviations GalT Galactosyltransferase - (14)GalT (14)-Galactosyltransferase - H ⁺ -ATPase Proton ATPase - HG Homogalacturonan - HSP70 ER resident Bip - mMDH Mitochondrial malate dehydrogenase - RG-I Rhamnogalacturonan I - RG-II Rhamnogalacturonan II - RGP Reversibly glycosylated polypeptide - RG-Lyase Rhamnogalacturonan lyase - Suc Sucrose - UDPase Uridine-5-diphosphatase     

Meiotic studies ofElymus nutans andE. jacquemontii (Poaceae,Triticeae) and their hybrids withPseudoroegneria spicata and seventeenElymus species

Hybridizations ofElymus nutans andE. jacquemontii were carried out with one species ofPseudoroegneria (S genome), and 20Elymus species, each containing either of the SH, SY, SYH, or SYW genomes. Chromosome configurations were analysed at metaphase I of the two target taxa and their interspecific hybrids. It is concluded that (i)E. nutans is an allohexaploid containing the SYH genomes, andE. jacquemontii is an allotetraploid having the SY genomes; (ii) the genomic affinity is associated with the geographic distance between the species studied; (iii) minor genomic structural rearrangements have occurred within the hexaploid taxon ofE. nutans.     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

Phylogenetic relationship of zeins and coixins as determined by immunological cross-reactivity and Southern blot analysis

Zeins from Zea mays L cv. Maya and coixins from Coix lacryma-jobi L. cv. Adlay were fractionated to obtain -, -, and -zein and -, -, and -coixin. The -coixins were composed of 4 polypeptide classes of 27 kDa (C1), 25 kDa (C2), 17 kDa (C4) and 15 kDa (C5) with solubility properties very similar to those of the 22 kDa and 19 kDa -zeins. Like the -zeins, the C1 and C2 -coixins corresponded to 80% of total Coix prolamins. The fraction corresponding to -coixin contained only one protein band of 22 kDa (C3). This coixin fraction has solubility properties similar to those of -zein and represents 15% of the total coixin. The -zein fraction was composed of a major 17 kDa protein band, while the -coixin fraction consisted of a mixture of - and -coixins.Polyclonal antibodies raised against C1 recognized C1 and C2 and cross-reacted strongly with the 22 kDa -zein, as did C4 and C5 antisera. The antiserum against -coixin showed strong cross-reaction with -zein. The homology between coixins and zeins was further investigated by using Southern hybridization analyses. The genomic DNA of maize and Coix were digested with several restriction enzymes and probed with cDNA clones representing 19 and 22 kDa -zeins as well as the 28 and 16 kDa -zeins. The Coix genome showed complex cross-hybridization sequences with the 22 kDa -zein cDNA, while no cross-hybridization was observed with the 19 kDa cDNA clone. The cDNA clone representing the 28 kDa -zein cross-hybridized with only one band of Coix genomic DNA, in contrast to the three bands observed in maize. This same Coix sequence also cross-hybridized with the cDNA clone representing the 16 kDa -zein. The relevance of these findings are discussed in the context of the origin of zein and coixin genes.     

Expression of the β-subunit of β-conglycinin in seeds of transgenic plants

Soybean (Glycine max (L.) Merr.) seeds contain the storage protein -conglycinin, encoded by a multigene family. -Conglycinin consists of three subunits; , , and . A genomic clone for a -subunit of -conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this -subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The -subunit expressed in seeds of petunia and tobacco was recognized by anti--conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the -subunit were produced. There was approximately a twofold variation in the accumulation of the -subunit protein in the mature seeds of transgenic petunia plants, each containing a single -subunit gene. However, the level of protein accumulation in mature seeds and the amount of -subunit mRNA in developing seeds was not correlated. Accumulation of the -subunit protein in transgenic seeds was less than the -subunit protein that accumulated in transgenic petunia seeds containing a single -subunit gene and less than the amount of the -subunit in mature soybean seeds which contain 8–13 -subunit genes. In transgenic tobacco plants, the accumulation of the -subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.Abbreviations kb kilobase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

The histochemical distribution of hydroxysteroid dehydrogenases in the human foetal membranes at term

Summary The histochemical distribution of various hydroxysteroid dehydrogenases in human, term, foetal membranes has been investigated using the tetrazolium dye, Nitro-B.T.The trophoblastic layer was the most active, showing 3-, 3-, 11-, 16- and 17-hydroxysteroid dehydrogenase activities, a pattern of activity similar to that of the placental villous trophoblast.The amniotic epithelium showed weak 3-, 3-, 16- and 17-hydroxysteroid dehydrogenase activity; weak 3- and 3-hydroxysteroid dehydrogenase activity was noted in the connective tissue layers.All activity demonstrated was N.A.D.-linked.     

Early development of the self-fertilizing mangrove killifish <Emphasis Type="Italic">Rivulus marmoratus</Emphasis> reared in the laboratory

The mangrove killifish Rivulus marmoratus was reared at 25°±1°C and 17ppt salinity from 0 to 100 days after hatching (DAH), and its early development was described by examining growth and morphometric parameters, meristic characters (vertebral and fin-ray counts), bone-cartilage development, and pigmentation. Growth was isometric for preanal length, head length, snout length, body depth, pectoral-fin length, dorsal-fin length, anal-fin length, and caudal-peduncle depth. Negative allometric growth was observed in eye diameter and gape size. Meristic counts (mean±SD) for vertebrae (34.2±0.4) and dorsal- (8.6±0.5), anal- (11.4±0.5), and caudal-fin rays (30.2±0.8) were complete at 0 DAH (n=5), whereas pectoral-fin rays and pelvic-fin rays were complete by 30 DAH (14.5±0.4, n=5) and 60 DAH (4.2±0.8, n=5). Full ossification of meristic elements proceeded in the following sequence: vertebrae (by 30 DAH), caudal-, dorsal-, and anal-fin rays (by 60 DAH), pectoral-fin rays (between 60 DAH and 100 DAH), and pelvic-fin rays (by 100 DAH). Both morphological characters and meristic counts indicate that this species can be considered to be a juvenile after 9.8mm in standard length (20 DAH).     

Theβ-subunit of human chorionic gonadotropin containsN-glycosidic trisialo tri- and tri′-antennary carbohydrate chains

TheN-linked carbohydrate chains of the-subunit of highly purified urinary human chorionic gonadotropin have been re-investigated. The oligosaccharides were released enzymatically by peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F, and fractionated by a combination of FPLC and HPLC. As a result of the application of improved fractionation methods, apart from the earlier reported carbohydrate chains, also small amounts of trisialo tri- and tri-antennary oligosaccharides were found. The primary structures of the latter carbohydrate chains have been determined by 500-MHz¹H-NMR spectroscopy to beAbbreviations hCG human chorionic gonadotropin - hCG- -subunit - hCG- -subunit - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F (E.C. 3.5.1.52) - endo-F endo--N-acetylglucosaminidase-F (E.C. 3.2.1.96) - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - CBB coomassie brilliant blue R 250 - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - Man mannose - Gal galactose - Fuc fucose     

Beta-1,3-glucanase from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. Comparison with beta-1,3-glucanases of marine and terrestrial mollusks

-1,3-Glucanase (Lu) was isolated from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. A comparative study of some properties of -1,3-glucanase Lu and -1,3-glucanases with different action types—endo--1,3-glucanase from crystalline style of the marine mollusk Spisula sachalinensis (LIV) and exo--1,3-glucanase from the terrestrial snail Eulota maakii (LII)—was performed. It was found that -1,3-glucanase Lu hydrolyzes laminaran with a high yield of glucose in the reaction products. The enzyme hydrolyzes substrates with retention of the glycosidic bond configuration, is able to cleave modified substrates, and exhibits transglycosylating activity. All properties of -1,3-glucanase from S. intermedius were more similar to those of the endo--1,3-glucanase from the marine mollusk (LIV) than exo--1,3-glucanase LII from the terrestrial snail. The differences in the effect of LIV and Lu on laminaran are probably related to the functions of -1,3-glucanase Lu from sea urchin eggs (which, in contrast to LIV, is not a digestive enzyme).     

A 500-MHz1H-NMR study on theN-linked carbohydrate chain of bromelain

The 500-MHz¹H-NMR characteristics of theN-linked carbohydrate chain Man1-6[Xyl1-2]Man1-4GlcNAc1-4[Fuc1-3]GlcNAc1-NAsn of the proteolytic enzyme bromelain (EC 3.4.22.4) from pineapple stem were determined for the oligosaccharide-alditol and the glycopeptide, obtained by hydrazinolysis and Pronase digestion, respectively. The¹H-NMR structural-reporter-groups of the (1–3)-linked fucose residue form unique sets of data for the alditol as well as for the glycopeptide.