Structure of a new nonasaccharide isolated from human milk: VI2Fuc,V4Fuc,III3Fuc-p-lacto-N-hexaose

The structure of a new nonasaccharide isolated from human milk has been investigated. By using methylation analysis, FAB-MS and¹H-and¹³C-NMR spectroscopy as basic methods of structural investigation, this oligosaccharide was identified as VI²--Fuc,V⁴-Fuc,III³--Fuc-p-lacto-n-hexaose: Fuc1-2Gal1-3[Fuc1-4]GlcNAc1-3Gal1-4[Fuc1-3]GlcNAc1-3Gal1-4Glc.Abbreviations COSY correlation spectroscope - DP degree of polymerisation - FAB-MS fast atom bombardment-mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - GLC gas-liquid chromatography     

Ganglioside lactones:1H-NMR determination of the inner ester position of GD1b-ganglioside lactone naturally occurring in human brain or produced by chemical synthesis

The complete definition of the chemical structure of G_D1b-ganglioside (G_D1b) lactone isolated from human brain has been given by means of spectrometric and spectroscopic analyses. G_D1h lactone contains a single ester linkage involving the external sialic acid carboxyl group and the C-9 hydroxyl group of the internal sialic acid unit. A synthetic lactone of G_D1b prepared treating G_D1b with glacial acetic acid characterized in the same way showed an identical chemical structure.Abbreviations: Ganglioside nomenclature is according to Svennerholm [16] and the IUPAC-IUB Recommendations [17] G_M1 G_M1-ganglioside, II³NeuAc-GgOse₄Cer, Gal1-3GalNac1-4[NeuAc2-3]Gal1-4Glc1-1Cer - G_D1b G_D1b-ganglioside, II³(NeuAc)₂GgOse₄Cer, Gal1-3GalNAc1-4[NeuAc2-8NeuAc2-3]Gal1-4Glc1-1Cer - G_D1b lactone G_D1b-L, Gal1-3GalNAc1-4[NeuAc(1-9)2-8NeuAc2-3]Gal1-4Glc1-1Cer - Cer ceramide - FAB-MS fast atom bombardment-mass spectrometry - ¹H-NMR proteon nuclear magnetic resonance - 1D-NMR one dimensional NMR - 2D-COSY two dimensional correlated spectroscopy - DMSO-d₆ deuterated dimethylsulfoxide     

Presence of hybridizing DNA sequences homologous to bovine acidic and basicβ-crystallins in all classes of vertebrates

Summary The eye lens-crystallins in cow and chicken are encoded by a family of at least six genes. In order to assess the distribution of the corresponding genes among other vertebrates we hybridized -crystallin sequences (A2, A3/A1, A4, B1, B2, B3), isolated from a bovine lens cDNA library, to Southern blots on whichEcoR1-digested chromosomal DNA was blotted from different vertebrate species. These included human, chimpanzee, calf, rat, pigeon, duck, monitor lizard, toad, trout, and lamprey. Positive hybridization signals were found in the representatives of virtually all classes of vertebrates. The basic B-crystallins gave hybridization signals in more species than the acidic A ones. In monitor lizard and toad the weakest hybridization signals for basic crystallin probes were found. For acidic crystallin probes the distribution pattern was more simple; among cold-blooded vertebrates a signal for A2 was found in trout and lamprey, for A4 in trout, and for A3/A1 only in toad. The results demonstrate that the duplications leading to the -crystallin gene family occurred before or during the earliest stages of vertebrate evolution.     

Peptide des β-Alanins als Ersatz für die freie Aminosäure bei pantothensäurebedürftigen Hefezellen und ihr Verhalten gegenüber dem β-Alanin-Antagonisten Asparagin

Zusammenfassung Pantothensäurebedürftige Hefezellen können ihren Bedarf an diesem Vitamin nicht allein aus -Alanin decken, sondern auch aus Benzoyl--Alanin, -Alanyl-d,l-Norleucin und -Alanyl-l-Histidin. Der Antagonist Asparagin hemmt die Verwertung dieser Peptide genauso wie diejenige der freien Aminosäure. Durch höhere Konzentrationen an -Alanin oder -Alanyl-d,l-Norleucin läßt sich die Hemmwirkung nicht allein kompensieren, es kommt sogar zu einer Förderung des Hefewachstums. Der Antagonist wird dann zum Synergisten.

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Summary The -alanine containing peptides benzoyl--alanine, -alanyl-d,l-norleucine and -alanyl-l-histidine can substitute for the amino acid -alanine in a pantothenic acid requiring yeast. Asparagine, an antagonist of -alanine, affects these peptides in a similar manner. In combination with an overdose of -alanine or -alanyl-d,l-norleucine, asparagine is no longer an antagonist but becomes a synergist.

     

A general strategy for the isolation of carbohydrate chains fromN-,O-glycoproteins and its application to human chorionic gonadotrophin

For the structural analysis of the carbohydrate chains ofN-,O-glycoproteins a straightforward strategy was developed based on the cleavage of theN-linked chains with immobilized peptide-N ⁴-(N-acetyl--glucosaminyl) asparagine amidase-F (PN-Gase-F) fromFlavobacterium meningosepticum, followed by alkaline borohydride treatment of the remainingO-glycoprotein material. This methodology was applied to the isolation of the Asn- and Ser-linked carbohydrate chains of human chorionic gonadotrophin. The structures of the isolated oligosaccharides were verified by 500-MHz¹H-NMR spectroscopy. The Asn-linked sugar chains were shown to be: NeuAc2-3Gal1-4GlcNAc1-2Man1-6[NeuAc2-3Gal1-4GlcNAc1-2Man1-3]Man 1-4GlcNAc1-4[Fuc1-6]_0-1GlcNAc and Man1-6[NeuAc2-3Gal1-4GlcNAc1-2Man 1-3]Man1-4GlcNAc1-4GlcNAc. Also some minor constituents occurred. The structures of the Ser-linked oligosaccharides were established in the form of their oligosaccharide-alditols as: NeuAc2-3Gal1-3[NeuAc2-6]GalNAc, NeuAc2-3Gal 1-3GalNAc and NeuAc2-3Gal1-3[NeuAc2-3Gal1-4GlcNAc1-6]GalNAc.Abbreviations hCG human chorionic gonadotrophin - hCG- -subunit - hCG- -subunit - ElA enzyme immunoassay - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F (EC 3.5.1.52) - SDS sodium dodecyl sulphate - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - Man mannose - Gal galactose - Fuc fucose     

Primary structure of the hemoglobin beta-chain of rose-ringed parakeet (Psittacula krameri)

The primary structure of Rose-ringed Parakeet hemoglobin -chain was established, completing the analysis of this hemoglobin. Comparisons with other avian -chains show variations smaller than those for the corresponding -chains. There are 11 amino acid exchanges in relationship to the only other characterized psittaciform -chain, and a total of 35 positions are affected by differences among all avian -chains analyzed (versus 61 for the -chains). At three positions, the Psittacula -chain has residues unique to this species. Three ₁₁ contacts are modified, by substitutions at positions 51, 116, and 125.     

Role of βγ-Dimers of GTP-Binding Proteins in Processes of Hormonal Signal Transduction

This review summarizes and analyzes data on structural and functional peculiarities of - and -subunits of heterotrimeric G proteins and their participation in -dimer in processes of hormonal signal transduction to the cell. The molecular mechanisms are discussed, which are responsible for formation of the -dimer complex and its interaction with the wide spectrum of signal and structural proteins, such as G protein -subunit, serpentine type receptors, kinases of G protein-coupled receptors, adenylyl cyclase, phospholipase C2, phosphatidyl-inositol-3-kinase, Raf-kinase, potassium and calcium channels, microtubular and RGS-proteins. The characteristics of - and -subunits are given for living organisms of different phylogenetic levels (lower eukaryotes, fungi, plants, vertebrates, and invertebrates). It is concluded that the -dimer is the most important regulatory link in various signal systems to carry out switch from one signal cascade to another and thereby to determine the final cellular response to the signal impulse.     

Modulation of cytokine responses of murine CD8+ αβ intestinal intraepithelial lymphocytes by IL-4 and IL-12

The immune responses of the intestine mucosa feature the noninflammatory type, such as IgA production and oral tolerance. Th2 type cytokines have been implicated in the induction of these noninflammatory responses. In the present study, cytokine responses of CD8+ and CD4+ TCR+ intestinal intraepithelial lymphocyte ( iIEL) subsets to TCR stimulation under the influence of IL-12, IL-4, or CD28 costimulation were examined. IL-12 enhanced production of IL-10 and IFN- by the CD8+ iIEL significantly but only marginally affected the CD8+ subset, whereas IL-4 induced IL-4, IL-5, and IL-10 production and augmented TGF- production by both subsets. CD28 costimulation induced production of Th2 cytokines by CD4+ iIEL in the absence of exogenous IL-4. Unlike lymph node CD4+ cells, the CD28 costimulation-induced Th2 differentiation of CD4+ iIEL was not inhibited by IFN-. These results demonstrate active cytokine production by CD4+, CD8+, as well as CD8+ iIEL. The Th2-skewed cytokine profile of CD8+ iIEL and the IFN--resistance of Th2 differentiation of the CD4+ iIEL suggest that both iIEL subsets contribute to the induction of noninflammatory mucosal immune responses.     

Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Lex determinant

A defined set of oligosaccharides and glycopeptides containing -linked fucose were used to examine the specificity of the immobilized fucose-binding lectin Lotus tetragonolobus agglutinin (LTA1), also known as lotus lectin. Glycans containing the Lewis x determinant (Lex) Gal1-4[Fuc1-3]GlcNAc1-3-R were significantly retarded in elution from high density LTA-Emphaze columns. The lectin also bound the fucosylated lacdiNAc trisaccharide GalNAc1-4[Fuc1-3]GlcNAc. The lectin did not bind glycans containing either sialylLex or VIM-2 determinants, nor did it bind the isomeric Lea, Gal1-3[Fuc1-4]GlcNAc-R. Although 2-fucosyllactose Fuc1-2Gal1-4Glc) was retarded in elution from the columns, larger glycans containing the H-antigen Fuc1-2Gal1-3(4)GlcNAc-R interacted poorly with immobilized LTA. Our results demonstrate that immobilized LTA is effective in isolating glycans containing the Lex antigen and is useful in analyzing specific fucosylation of glycoconjugates. Abbreviations: LTA, Lotus tetragonolobus agglutinin; UEA-1, Ulex europaeus agglutinin-I; LNT, AAL, Aleuria aurantia agglutinin; Gal1-3GlcNAc1-3Gal1-3Glc; LNnT, Gal1-4GlcNAc1-3Gal1-3Glc; Lex, Lewis x antigen; Lea, Lewis a antigen; GDPFuc, guanosine 5-diphosphate--L-fucose     

α-and β-Glycosidases in maize roots

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for -glucosidase, 4.4 for -galactosidase, 6.4 for -glucosidase and 6.0 for -galactosidase. The -glucosidase showed 4-fold higher activity than the -galactosidase. The distribution of the -glucosidase activity was signifcantly different from that of the -galactosidase, -glucosidase and -galactosidase.Abbreviations -Glu -glucosidase - -Gal -galactosidase - -Glu -glucosidase - -Gal -galactosidase     

Comparative Modulation by 3α,5α and 3β,5β Neurosteroids of GABA Binding Sites During Avian Central Nervous System Development

Neurosteroids are endogenous Central Nervous System (CNS) compounds which act mainly by allosteric modulation of the GABA_A receptor complex. The presence of a 3-hydroxyl group and a 5-hydrogen atom have been found to be essential structural requirements for biological activity in mammals. In the present work we report the enhancing activity on [³H]GABA binding to its receptor sites in chick optic lobe produced by progesterone metabolites 3-hydroxy,5-pregnan-20-one (3,5-P) and 3-hydroxy,5-pregnan-20-one (3,5-P). Both steroids were found able to enhance [³H]GABA binding along ontogeny, displaying a similar profile at early developmental stages, while in adulthood 3,5-P had greater potency (EC₅₀ 0.22 M) and enhancing effect (E_max: 122%). In adult synaptic membranes, the two compounds displayed a complex interaction with the GABA_A receptor, disclosed by a Schild plot with slope below one and an incomplete displacement of 3,5-P by its 3,5 isomer. Such complexity could be related to the steroidogenic profile in avian CNS, with 5-reduced progesterone metabolites present since early development, while 3,5-P is found only in adulthood. Bearing in mind differences between avian and mammalian steroidogenic profiles and the relevance of 5-steroids in early avian development, we propose that 3,5-P, instead of the classical potent 3,5-steroids, may be the endogenous modulator of GABAergic activity in developing avian brain.     

UDP-GlcNAc: Galβ3GalNAc-Mucin: (GlcNAc → GalNAc) β6-N-Acetylglucosaminyltransferase and UDP-GlcNAc: Galβ3(GlcNAcβ6) GalNAc-Mucin (GlcNAc → Gal)β3-N-Acetylglucosaminyltransferase from Swine Trachea Epithelium

Summary Two specific -N-acetylglucosaminyltransferases involved in the branching and elongation of mucin oligosaccharide chains, namely, a 1,6 N-acetylglucosaminylsaminyltransferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3GalNAc-Mucin to yield Gal3(GlcNAc6)GalNAc-Mucin and a 3-N-acetylglucosaminyl transferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3(GlcNAC6)GalNAc-mucin to yield GlcNAc3Gal3 (GlcNAc6)GalNAc-Mucin were purified from the microsomal fraction of swine trachea epithelium. The 1,6-N-acetylglucosaminyltransferase was purified about 21,800-fold by procedures which included affinity chromatography on DEAE columns containing bound asialo Cowper's gland mucin glycoprotein with Gal1,3GalNAc side chains. The apparent molecular weight estimated by gel filtration was found to be about 60 Kd. The purified enzyme showed a high specificity for Gal1,3GalNAc chains and the most active substrates were mucin glycoproteins containing these chains. The apparent Km of the 6-glucosaminyltrans-ferase for Cowper's gland mucin glycoprotein containing Gal1,3GalNAc chains was 0.53 µM; for UDP-N-acetylglucosamine, 12 µM; and for Gal 1,3GalNAc NO₂ø, 4 mM. The activity of the 6-glucosaminyltransferase was dependent on the extent of glycosylation of the Gal3GalNAc chains in Cowper's gland mucin glycoprotein.The best substrate for the partially purified 3-Glucosaminyltransferase was Cowper's gland mucin glycoprotein containing Gal1,3(GlcNAc6)GalNAc side chains. This enzyme showed little or no activity with intact sialylated Cowper's gland mucin glycoprotein or derivatives of this glycoprotein containing GalNAc or Gal1,3GalNAc side chains.The radioactive oligosaccharides formed by these enzymes in large scale reaction mixtures were released from the mucin glycoproteins by treatment with alkaline borohydride, isolated by gel filtration on Bio-Gel P-6 and characterized by methylation analysis and sequential digestion with exoglycosidases. The oligosaccharide products formed by the 6- and 3-glucosaminyltransferases were shown to be Gal3(GlcNAC6) GalNAc and GlcNAc3 Gal3(GlcNAC6)GalNAc respectively.Taken collectively, these results demonstrate that swine trachea epithelium contains two specific N-acetylglucosaminyltransferases which catalyze the initial branching and elongation reactions involved in the synthesis of O-linked oligosaccharide chains in respiratory mucin glycoproteins. The first enzyme a 6-glucosaminyltransferase converts Gal3GalNAc chains in mucin glycoproteins to Gal3(GlcNAc6)GalNAc chains. This product is the substrate for a second 3-glucosaminyltransferase which converts the Gal3(GlcNAc6)GalNAc chains to GlcNAc3Gal(GlcNAc6)GalNAc chains in the glycoprotein. The 3-glucosaminyltransferase did not utilize Gal3GalNAc chains as a substrate and this results in an ordered sequence of addition of N-acetylglucosamine residues to growing oligosaccharide chains in tracheal mucin glycoproteins.Abbreviations NeuNAc N-acetylneuraminic acid - GalNAcol N-acetylgalactosaminitol - CGMG Cowper's gland mucin glycoprotein - GalNAc-CGMG Cowper's gland mucin glycoprotein containing GalNAc side chains O-glycosidically linked to serine or threonine - Gal3GalNAc-CGMC Cowper's gland mucin glycoprotein containing Gal3GalNAc side chains - MES 2-(N-morpholino) Ethane Sulfonic acid - PBS Phosphate Buffered Saline     

Anomeric preference of glucose utilization in rat erythrocytes

The -anomer of glucose relative to the -anomer was more rapidly metabolized into lactate by rat erythrocytes at 37° C (/ ratio = ca. 1.3): the amounts of - and -D-glucose metabolized into lactate during 3 min were 0.21 and 0.27 mol/gHb, respectively. Also, the transport of -D-glucose into erythrocytes was more rapid than that of -D-glucose: the amounts of - and -D-glucose transported into erythrocytes during 3 min were approximately 3.5 and 5.0 mol/gHb, respectively. Glucose phosphorylation by rat erythrocyte hexokinase (i.e., a possible rate-limiting step in glycolysis) occurred at higher velocities with the -anomer than with the a-anomer (/ ratio = 1.28). The Km value of hexokinase for either anomer of glucose was 53 M. The glucose concentrations in erythrocytes incubated with - and -D-glucose reached about 1 mM in 1 min, indicating that hexokinase is almost completely saturated with glucose within less than 1 min. The results suggest that glucose phosphorylation and glucose transport are major and minor determinants, respectively, for the anomeric preference of glucose utilization in rat erythrocytes.     

Synthesis of disaccharide derivatives employing β-N-acetyl- d-hexosaminidase, β-d-galactosidase and β-d-glucuronidase

-N-Acetyl-d-hexosaminidase from Aspergillus oryzae catalysed the stereo- and regiospecific formation of the 6-O-benzylated disaccharide derivatives GalNAc1-3(6- OBn)Gal-SEt and GlcNAc1-3(6-OBn)Gal-SEt, which were obtained in transglycosylation reactions employing ethyl 6- O-benzyl-1-thio--d-galactopyranoside as acceptor. Preparative amounts of the chitobiose derivative GlcNAc1- 3GlcNAc-OPhNO2-p was prepared as well. - N-Acetyl-d-hexosaminidase from bovine testes catalysed the specific synthesis of GlcNAc1-3(6-OBn)GlcNH2-SEt and GalNAc1-3(6-OBn)GlcNH2-SEt, employing ethyl 2-amino-6-O-benzyl-2-deoxy-1-thio--d-glucopyranoside as acceptor. -d-Glucuronidase from E. coli was found to catalyse the formation of GlcA1-3(6-OBn)GlcNH2- SEt employing the same acceptor.     

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.     

Uptake and metabolism of 6-benzyladenine in shoot cultures of a range of species

Shoots of a range of species were cultured in vitro on a medium containing 2.22 M benzyladenine (BA). After 25 days in culture the amount of BA taken up differed between species. The conjugates of BA present in the plant tissues were identified by HPLC. The pattern of BA metabolism varied considerably between species as did the actual levels of each conjugate. Benzyladenine accumulated in Hippeastrum, Alstroemeria and Ficus with little conjugation. In Musa, Rhododendron and Fuchsia BA also accumulated along with its ribosyl and glucoside conjugates. In contrast, very low levels of BA were detected in Gerbera and Dendranthema, but various glucosides did accumulate.Abbreviations BA 6-benzyladenine - [3G]BA-3- D-glucopyranosyl-BA - [7G]BA 7--D-glucopyranosyl-BA - [9G]BA 9--D-glucopyranosyl-BA - [9R-G]BA 9--D-(ribosylglucoside)-BA - [9R]BA 9--ribofuranosyl-BA - HPLC high performance liquid chromatography - PAR photo-synthetically active radiation - TEAB triethylammonium bicarbonate     

Binding of Amyloid Beta-Protein to Intracellular Brain Proteins in Rat and Human

Amyloid beta-protein (A), in its soluble form, is known to bind several circulatory proteins such as apolipoprotein (apo) E, apo J and transthyretin. However, the binding of A to intracellular proteins has not been studied. We have developed an overlay assay to study A binding to intracellular brain proteins. The supernatants from both rat and human brains were found to contain several proteins that bind to A 1–40 and A 1–42. No major difference was observed in the A binding-proteins from brain supernatants of patients with Alzheimer's disease and normal age-matched controls. Binding studies using shorter amyloid beta-peptides and competitive overlay assays showed that the binding site of A to brain proteins resides between 12–28 amino acid sequence of A. The presence of several intracellular A-binding (AB) proteins suggests that these proteins may either protect A from its fibrillization or alternatively promote A polymerization. Identification of these proteins and their binding affinities for A are needed to assess their potential role in the pathogenesis of Alzheimer's disease.     

Variability of bacterial gene-directed enzyme production in human genetically deficient cells

Summary Human -galactosidase-deficient skin fibroblasts from a patient with generalized gangliosidosis (GM₁-gangliosidosis type I) were treated with phage plac DNA, coding for Escherichia coli -galactosidase (-D-galactoside galactohydrolase, EC 3.2.1.23). New -galactosidase activity detected in cell extracts of phage DNA-treated GM₁-gangliosidosis fibroblasts continued to vary considerably from one experiment to another. It behaved like the E. coli z-gene product upon immunochemical and physicochemical investigation. In some experiments the antigenic behavior of resultant -galactosidase activity in plac DNA-treated cells resembled that of mutant E. coli -galactosidase. Among the factors and variables that may be responsible for the variation in the results obtained here and elsewhere, low physical binding between prokaryotic mRNA sequences and fibroblast ribosomal RNA could play a part connected with effective translation. This hypothesis is discussed under the aspect of a comparison of the ribosomal binding site of lac z mRNA with the 3-terminus of the eukaryotic 18s ribosomal RNA, which shows limited possibilities for base-pairing interactions.More extensive possibilities for forming Watson-Crick base pairs between their initiation site and the eukaryotic ribosomal binding site exist for other prokaryotic messengers, such as those of Q-replicase, f 1-coat protein, or UDPG-4-epimerase.     

Conformational change of a synthetic amyloid analogue des[Ala21,3O]A42 upon binding to octyl glucoside micelles

The secondary structure of a synthetic amyloid fragment des [Ala^21,30]A42 was studied by circular dichroism and Fourier transformed infrared spectroscopy. Measurements were performed in trifluoroethanol/water and octyl -d-glucopyranoside solutions. The spectra of the peptide in trifluoroethanol indicate a high percentage of a-helical structure. However, in octyl glucoside, at and above the critical micelle concentration, the peptide adopts a -sheet conformation. Secondary structure analysis yields a predominant (> 70 %) -sheet content. Our data suggest that the peptide backbone or polar side groups of des[Ala^21,30]A42 interact with the sugar-coated surface of micelles, which promotes an a to conformational transition.     

Enhancement of enzymatic hydrolysis of triolein in aqueous solution by cyclodextrin derivatives

Summary Based on the ability of -cyclodextrin and its partially methylated derivatives to form inclusion complexes, the hydrolysis of triolein by pancreatic lipase in aqueous solution can be increased up to 7-fold. -Cyclodextrin, dimethyl--cyclodextrin and trimethyl--cyclodextrin accelerated the lipolysis 3-7-fold in accordance with their solubility in water.