Pyridoxal 5′-phosphate binds to a lysine residue in the adenosine 3′-phosphate 5′-phosphosulfate recognition site of glycolipid sulfotransferase from human renal cancer cells

In the course of characterization of glycolipid sulfotransferase from human renal cancer cells, the manner of inhibition of sulfotransferase activity with pyridoxal 5-phosphate was investigated. Incubation of a partially purified sulfotransferase preparation with pyridoxal 5-phosphate followed by reduction with NaBH₄ resulted in an irreversible inactivation of the enzyme. When adenosine 3-phosphate 5-phosphosulfate was co-incubated with pyridoxal 5-phosphate, the enzyme was protected against this inactivation. Furthermore, pyridoxal 5-phosphate was found to behave as a competitive inhibitor with respect to adenosine 3-phosphate 5-phosphosulfate with aK _i value of 287 µm. These results suggest that pyridoxal 5-phosphate modified a lysine residue in the adenosine 3-phosphate 5-phosphosulfate-recognizing site of the sulfotransferase.     

Interactions of Aβ(1–40) with Glycerophosphocholine and Intact Erythrocyte Membranes: Fluorescence and Circular Dichroism Studies

Deposition of amyloid peptide in human brain in the form of senile plaques is a neuropathological hallmark of Alzheimers disease (AD). Levels of a phospholipid breakdown product, glycerophosphocholine (GPC), also increase in AD brain. The effect of GPC on amyloid (1–40) peptide (A) aggregation in PBS buffer was investigated by circular dichroism and fluoresence spectroscopy; interactions of A and GPC with the intact erythrocyte membrane was examined by fluoresence spectroscopy. Fluorescamine labeled A studies indicate GPC enhances A aggregation. CD spectroscopy reveals that A in the presence of GPC adopts 14% more -sheet structure than does A alone. Fluorescamine anisotropy measurements show that GPC and A interact in the phospholipid head-group region of the erythrocyte membrane. In summary, both soluble A and GPC insert into the phospholipid head-group region of the membrane where they interact leading to -sheet formation in soluble A which enhances A aggregation.     

Purification of (1→3)-β-glucan endohydrolase isoenzyme II from germinated barley and determination of its primary structure from a cDNA clone

A (13)--D-glucan 3-glucanonydrolase (EC 3.2.1.39) of apparent M _r 32 000, designated GII, has been purified from germinated barley grain and characterized. The isoenzyme is resolved from a previously purified isoenzyme (GI) on the basis of differences in their isoelectric points; (13)--glucanases GI and GII have pI values of 8.6 and 10.0, respectively. Comparison of the sequences of their 40 NH₂-terminal amino acids reveals 68% positional identity. A 1265 nucleotide pair cDNA encoding (13)--glucanase isoenzyme GII has been isolated from a library prepared with mRNA of 2-day germinated barley scutella. Nucleotide sequence analysis of the cDNA has enabled the complete primary structure of the 306 amino acid (13)--glucanase to be deduced, together with that of a putative NH₂-terminal signal peptide of 28 amino acid residues. The (13)--glucanase cDNA is characterized by a high (G+C) content, which reflects a strong bias for the use of G or C in the wobble base position of codons. The amino acid sequence of the (13)--glucanase shows highly conserved internal domains and 52% overall positional identity with barley (13, 14)--glucanase isoenzyme EII, an enzyme of related but quite distinct substrate specificity. Thus, the (13)--glucanases, which may provide a degree of protection against microbial invasion of germinated barley grain through their ability to degrade fungal cell wall polysaccharides, appear to share a common evolutionary origin with the (13, 14)--glucanases, which function to depolymerize endosperm cell walls in the germinated grain.     

β-Ether cleavage of the lignin model compound 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether and derivatives by Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized 4-ethoxy-3-methoxyphenyl-glycerol--guaiacyl ether (V) in low nitrogen, stationary cultures under which conditions the ligninolytic enzyme system is expressed. 4-Ethoxy-3-methoxyphenylglycerol XIII, guaicol and 4-ethoxy-3-methoxybenzyl alcohol (II) were isolated as metabolic products. Exogenously added XIII was rapidly converted to 4-ethoxy-3-methoxybenzyl alcohol indicating that it is an intermediate in the metabolism of V. P. chrysosporium also metabolized 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-3-hydroxypropane VI. The degradation pathway for this dimer also included initial -ether cleavage and -hydroxylation of the diol product 1-(4-ethoxy-3-methoxyphenyl) 2,3 dihydroxypropane (XI) to yield the triol XIII which was cleaved at the , bond to yield 4-ethoxy-3-methoxybenzyl alcohol. Finally P. chrysosporium also cleaved the dimer 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1-hydroxypropane (VIII) at the -ether linkage yielding 1-(4-ethoxy-3-methoxyphenyl) 1,2 dihydroxypropane (IX) which was subsequently cleaved at the , bond to yield II. All of the results indicate that oxidative -ether cleavage is an important initial reaction in the metabolism of -aryl ether lignin substructure dimeric compounds. Metabolities were identified after comparison with chemically synthesized standards by gas liquid chromatography-mass spectrometry.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC thin layer chromatography     

Water stress-induced oxidative damage and antioxidant responses in micropropagated banana plantlets

Oxidative injury and antioxidant responses were investigated in two banana genotypes (Musa AAA Berangan and Musa AA Mas) subjected to 40 % PEG-induced water stress. PEG treatment resulted in oxidative injury, as expressed in increased lipid peroxidation and reduced membrane stability index, in both cultivars; however, greater oxidative injury was detected in Mas. Under PEG treatment, catalase activity and glutathione reductase activity were enhanced in both cultivars, but were higher in Mas. Ascorbate peroxidase activity was enhanced in Berangan under water stress, but was unaffected in Mas. Meanwhile, superoxide dismutase activity was inhibited in both cultivars under water stress, but higher activity was detected in Berangan. Higher ascorbate peroxidase and superoxide dismutase activities were associated with greater protection against water stress-induced oxidative injury.     

Monocyte-derived dendritic cells that capture dead tumor cells secrete IL-12 and TNF-α through IL-12/TNF-α/NF-κB autocrine loop

This study focused on the question of how monocyte-derived dendritic cells (Mo-DCs) that capture dead tumor cells (Mo-DCs-Tum) secrete interleukin 12 (IL-12) and tumor necrosis factor (TNF-). Mo-DCs-Tum showed higher secretions of IL-12 and TNF- than were shown by Mo-DCs. Enhanced nuclear factor-kappa B (NF-B) activation was also induced in Mo-DCs-Tum within 6 h. The NF-B inhibitor, pyrrolidine dithiocarbamate (PDTC), suppressed both IL-12 and TNF- secretions from Mo-DCs-Tum. Administration of recombinant TNF- or IL-12 enhanced IL-12 or TNF- secretion respectively in Mo-DCs-Tum. Addition of anti-TNF- or anti-IL-12 neutralizing antibody decreased NF-B activation and IL-12 or TNF- secretion in Mo-DCs-Tum. These results suggest that TNF- or IL-12 secretion induces NF-B activation, and it stimulates further TNF- and IL-12 secretions, i.e., an IL-12/TNF-/NF-B autocrine loop, in Mo-DCs-Tum. Thus, Mo-DCs-Tum secrete a large amount of IL-12 and TNF- through accelerated NF-B activation induced by the IL-12/TNF-/NF-B autocrine loop.     

Ecto-ATPase/phosphatase activity in the olfactory sensilla of the spiny lobster,Panulirus argus: localization and characterization

Summary Electrophysiological studies have shown that the olfactory organ (antennule) of the spiny lobster, Panulirus argus, has chemoreceptors that are selectively excited by adenine nucleotides in seawater. Biochemical studies have revealed that these same nucleotides can be rapidly dephosphorylated by ectoenzymes associated with the olfactory sensilla (aesthetascs). In this study the distribution of ecto-ATPase/phosphatase activity within aesthetascs was determined cytochemically and the nature of the adenine-nucleotide dephosphorylating activity was dissected biochemically. Cytochemically, the distribution of ATP-dephosphorylating activity was similar to that shown previously for AMP and -glycerol phosphate; i.e., cerium phosphate reaction product was specifically localized to the transitional zone where the sensory dendrites develop cilia and branch to form the outer dendritic segments. Unlike the dephosphorylation of AMP and -glycerol phosphate, Mg²⁺ or Ca²⁺ was required for ecto-ATPase/phosphatase activity. Biochemical measures of both AMP-and ATP-dephosphorylating activity within aesthetascs corroborated the cytochemical evidence that these activities are localized to the transitional zone. A major portion of the AMP dephosphorylation (about 67%) derives from nonspecific alkaline phosphatase activity that is insensitive to levamisole and L-bromotetramisole. In contrast, nonspecific phosphatase activity accounted for a much smaller part of the ATP dephosphorylation (about 15%). Ectoenzymatic activity in the transitional zone may be an important means of removing excitatory/inhibitory nucleotides from this region.Abbreviations ADP Adenosine 5-diphosphate - AMP adenosine 5-monophosphate - AMPCP , -methylene ADP - ASW artificial seawater - ATP adenosine 5-triphosphate - -GP -glycerol phosphate - EM electron microscopy     

Tumor necrosis factor α modifies resistance to interferon α in vivo: First clinical data

Summary Patients with Philadelphia-positive chronic-phase chronic myelogenous leukemia (CML) resistant to interferon (IFN) were treated in a phase I/II study with recombinant human tumor necrosis factor to overcome IFN resistance. Doses of 40, 80, 120 or 160 µg/m² TNF were given as 2-h infusions on 5 consecutive days every 3 weeks. IFN (4 × 10⁶ IU/m² s.c., daily) treatment was continued. Six patients were treated, completing 1–24 (median, 12) treatment cycles. Five of the six patients achieved partial hematological remission, while the remaining patient had to stop treatment because of WHO grade 4 thrombocytopenia following the first TNF cycle. No complete hematologic remission or cytogenetic improvement was seen. Side-effects were similar to those described for both substances alone. Maximum tolerable TNF doses usually varied between 80 µg/m² and 160 µg/m². To examine possible pathways of TNF activity in these patients, interferon receptor status and (2–5)-oligoadenylate synthetase levels were examined in peripheral blood mononuclear cells. Both parameters remained unchanged during TNF treatment. These preliminary data point to significant clinical efficacy of additionally applyed TNF in IFN-resistant CML patients.     

Characterisation of a developmentally related polypeptide with glutelin solubility characteristics from Lupinus albus L.

Proteins from Lupinus albus L. cv. Rio Maior seeds were fractionated according to solubility criteria. Patterns of concanavalin A (ConA)-binding polypeptides from the different classes, albumins, globulins, glutelins and prolamins, were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands of apparent molecular masses of 29 and 23.5 kDa with glutelin solubility characteristics bound the lectin. The 23.5-kDa band was separated by two-dimensional electrophoresis into two components: one glycosylated and heterogeneous with an isoelectric point of approx. 10 (designated as G23) and another, not detected with ConA, precipitating in the first dimension. The amino acid and hexosamine analysis of G23 showed that it is particularly rich in Gly (11.2%), Glx (10.0%), Ser (9.0%), Leu (8.2%), Asx (7.5%), and Pro (6.7%) and that it has a considerable content of the sulphur-containing amino acids Met (2.0%) and Cys (5.8%) and contains glucosamine. The determined N-terminal amino acid sequence of G23 was: ¹KG(R)V⁵KGTGD¹⁰(T)PXXV¹⁵XLY(N)R²⁰T, and this had no significant similarity to any of the amino acid sequences contained in the data bank SWISS-PROT 26. The glycoprotein G23 was completely deglycosylated with peptide-N-glycosidase F, yielding a homogeneous 21-kDa polypeptide composed of approximately 191 amino acids. The structures of the major N-linked neutral oligosaccharides of G23, determined by exoglycosidase sequencing, were as follows: Man2Man6(Man3) Man6(Man2Man2Man3)Man4GlcNAc4GlcNAc (13%); ± Man2Man6(Man3)Man6(± Man2 Man2 Man3)Man4GlcNAc4GlcNAc (29%); Man6(Man3) Man6(Man2Man3)Man4GlcNAc4GlcNAc (13%); Man6(Man3)Man6(Man3)Man4GlcNAc4GlcNAc (16%); Man6(Man3)(Xyl2)Man4GlcNAc 4GlcNAc (28%). Changes in G23 abundance during seed development, germination and seedling growth were monitored with a specific antibody. The glycoprotein G23 started to accumulate appreciably during seed formation between the 40th and the 50th days after anthesis and was detected following seed imbibition, until the 9th day in cotyledons, the 2nd day in roots and the 4th day in hypocotyls and leaves.Abbreviations ConA concanavalin A - Endo H endo-N-acetyl--d-glucosaminidase H - GlcNAc N-acetylglucosamine - gu glucose unit - IEF isoelectric focusing - Man mannose - NEPHGE non-equilibrium pH gradient electrophoresis - PNGase F peptide-N-glycosidase F - PVDF polyvinylidenedifluoride - Xyl xylose We thank Geoffrey Guile (Oxford Glycobiology Institute, Oxford, UK) for help with HPLC separations and amino acid and hexosamine analysis, Terry Butters (Oxford Glycobiology Institute) for providing the exoglycosidases and advice in their use, Manuela Regala (Instituto de Tecnologia Química e Biológica Oeiras, Portugal) and Paula Veríssimo (University of Coimbra, Portugal) for determining the N-terminal amino acid sequence of G20 and G23 and Dr. Jorge Lampreia (Universidade Nova de Lisboa, Lisbon, Portugal) for the computerised search of the SWISS-PROT data bank. Lupinus albus seeds were provided by Dr. João Neves Martins (Instituto Superior de Agronomia Lisbon, Portugal). We also thank J. Romão (Instituto Gulbenkian de Ciência, Oeiras, Portugal) for technical assistance in antibody production. This work was supported by Junta National de Investigação Científica e Tecnológica, Portugal.     

Adoptions expérimentales de larves entre des fourmis de genres différents (II):Myrmica laevinodis Nylander etAnergates atratulus Schenck

Résumé Nous avons fait élever des larves d'Anergates atratulus par des ouvrières deMyrmica laevinodis à 22°C. Pour y parvenir, il n'est pas utile de faire hivernerensemble les larves d'Anergates et les ouvrières deMyrmica. La présence de larves autochtones n'empêche pas lesMyrmica d'élever des larves d'Anergates. Dans toutes les expériences lesMyrmica ont été soumises au fridavant de recevoir des larves d'Anergates. Aucune reine deMyrmica n'a été utilisée dans ces expériences.Sur les 64 larves d'Anergates que nous avons utilisées, 38 se sont transformées en imagos. C'est au début de l'adoption et au moment des métamorphoses que périrent la plupart des 26Anergates perdus. Les femelles vécurent en général 2 ou 3 jours et cherchèrent très tôt à quitter le nid natal. Les mâles vécurent 2 à 3 semaines.

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Summary Larvae ofAnergates atratulus were experimentally reared by workers ofMyrmica laevinodis, at 22°C. An overwintering of both larvae ofAnergates and workers ofMyrmica is not necessary for the success of that experiment. The presence of larvae ofMyrmica does not keep theMyrmica from rearing larvae ofAnergates. The workers ofMyrmica have been cooled, in all the experiments, before receiving larvae ofAnergates. No queen ofMyrmica have been used in that experiments.38 of the 64 larvae ofAnergates used became imagos. Most of the 26 lostAnergates died at the beginning of the adoption and during the metamorphosis. The females lived generally 2 or 3 days and tried, very early, to leave their native nest. The males lived 2 or 3 weeks.

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Anergates atratulus Myrmica laevinodis, 22 . bmecme Anergates Myrmica. Myrmica Anergates. Myrmica Anergates. Myrmica . 64 Anergates , 38 . 26 Anergates 2 3 . 2 3 .

     

Complete protection against melanoma in absence of autoimmune depigmentation after rejection of melanoma cells expressing α(1,3)galactosyl epitopes

The major barrier for xenotransplantation in humans is the presence of (1–3) Galactosyl epitopes (Gal) in xenogeneic tissue and the vast quantities of natural antibodies (Ab) produced by humans against this epitope. The binding of anti-Gal Ab to cells expressing Gal triggers a complement-mediated hyperacute rejection of target cells. The hyperacute rejection of whole cancer cells, modified to express Gal epitopes, could be exploited as a new cancer vaccine to treat human cancers. We tested this hypothesis in Galactosyltransferase knockout (GT KO) mice which, like humans, do not express Gal on their cell surfaces and can produce anti-Gal Ab. Forty-five percent of mice with preexisting anti-Gal Ab rejected Gal positive melanoma cells (B16Gal). These mice remained tumor-free for more than 90 days. The majority of control mice injected with B16Null, Gal negative cells succumbed to melanoma. The rejection of B16Gal induced strong long-lasting antitumor immunity against B16Null measured by the expansion of cytotoxic T lymphocytes. In addition, mice rejecting B16Gal were protected against melanoma since they survived a second rechallenge with B16Null. Protected mice developed antitumor immunity in the absence of autoimmune depigmentation (vitiligo). These results show that rejection of Gal positive melanoma cells can efficiently boost the immune response to other tumor associated antigens present in Gal negative melanoma cells. This study supports the concept of a novel anticancer vaccine to treat human malignancies.     

Sialidase of swine influenza A viruses: variation of the recognition specificities for sialyl linkages and for the molecular species of sialic acid with the year of isolation

The sialidase of swine influenza A viruses of N1 and N2 subtypes, isolated from 1930 to 1992, was studied for substrate specificity with ganglio-series, lacto-series type II and GM3 gangliosides containing Neu5Ac2-3Gal, Neu5Gc2-3Gal and Neu5Ac2-6Gal linkages. All viral sialidases tested showed that the activity for hydrolysing substrates with Neu5Ac2-3Gal was higher than the activities with Neu5Gc2-3Gal and Neu5Ac2-6Gal linkages. When GM1b, GM3 and sialylparagloboside were used as substrates, the earliest strain (A/Wisconsin/15/30 H1N1, isolated in 1930) showed the activity ratio of Neu5Ac2-6Gal to Neu5Ac2-3Gal to be 0.13:0.2, and the ratio Neu5Gc2-3Gal/Neu5Ac2-3Gal to be 0.19:0.37, while those strains isolated from 1978 to 1992 exhibited ratios of 0.29:0.58 for Neu5Ac2-6Gal/Neu5Ac2-3Gal and 0.51:0.76 for Neu5Gc2-3Gal/Neu5Ac2-3Gal. The above results indicate that the substrate specificities of sialidases from swine influenza A viruses towards sialyl linkages and the molecular species of sialic acid are related to the year of isolation, i.e. strains isolated after 1978 exhibited higher activity towards Neu5Ac2-6Gal and Neu5Gc2-3Gal linkages when compared with strains isolated in an earlier year, 1930.Abbreviation Neu5Ac 5-N-acetylneuraminic acid - Neu5Gc 5-N-glycolyneuraminic acid - Gal d-galactose - Glc d-glucose - Cer Ceramide - II³(Neu5Ac)Lac Neu5Ac2-3Gal1-4Glc - GM3(Neu5Ac2-3Gal) Neu5Ac2-3Gal1-4Glc1-Cer - GM3(Neu5Gc2-3Gal) Neu5Gc2-3Gal1-4Glc1-Cer - GM1b(Neu5Ac2-3Gal) Neu5Ac2-3Gal1-3GalNac1-4Gal1-4Glc1-Cer - GMlb(Neu5Gc2-3Gal) Neu5Gc2-3Gal1-3GalNAc1-4Gal1-4Glc1-Cer - IV³(Neu5Ac)nLc4Cer Neu5Ac2-3Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - IV³(Neu5Gc)nLc4Cer Neu5Gc2-3Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - IV⁶(Neu5Ac)nLc4Cer Neu5Ac2-6Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - TDC taurodeoxycholate.     

Prediction of the Secondary Structure Contents of Globular Proteins Based on Three Structural Classes

The prediction of the secondary structural contents (those of -helix and -strand) of a globular protein is of great use in the prediction of protein structure. In this paper, a new prediction algorithm has been proposed based on Chou's database [Chou (1995), Proteins 21, 319–344]. The new algorithm is an improved multiple linear regression method, taking into account the nonlinear and coupling terms of the frequencies of different amino acids and the length of the protein. The prediction is also based on the structural classes of proteins, but instead of four classes, only three classes are considered, the class, class, and the mixed + and / class or simply the class. Thus the ambiguity that usually occurs between + proteins and / proteins is eliminated. A resubstitution examination for the algorithm shows that the average absolute errors are 0.040 and 0.035 for the prediction of -helix content and -strand content, respectively. An examination of cross-validation, the jackknife analysis, shows that the average absolute errors are 0.051 and 0.045 for the prediction of -helix content and -strand content, respectively. Both examinations indicate the self-consistency and the extrapolating effectiveness of the new algorithm. Compared with other methods, ours has the merits of simplicity and convenience for use, as well as high prediction accuracy. By incorporating the prediction of the structural classes, the only input of our method is the amino acid composition and the length of the protein to be predicted.     

Synthesis of Gal-β-(1→4)-GlcNAc-β-(1→6)-[Gal-β- (1→3)]-GalNAc-α-OBn oligosaccharides bearing O-methyl or O-sulfo groups at C-3 of the Gal residue: specific acceptors for Gal: 3-O-sulfotransferases

Our recent studies have revealed the existence of two distinct Gal: 3-O-sulfotransferases capable of acting on the C-3 position of galactose in a Core 2 branched structure, e.g., Gal14GlcNAc16(Gal13)GalNac1OBenzyl as acceptor to give 3-O-sulfoGal14GlcNAc13(Gal13)GalNAc1OB 20 and Gal14GlcNAc16(3-O-sulfoGal13)GalNAc1OB 23. We herein report the synthesis of these two compounds and also that of other modified analogs that are highly specific acceptors for the two sulfotransferases. Appropriately protected 1-thio-glycosides 7, 8, and 10 were employed as glycosyl donors for the synthesis of our target compounds.     

Evolution of the variable gene segments and recombination signal sequences of the human T-cell receptor α/δ locus

The T-cell receptor (TCR) and loci are particularly interesting because of their unique genomic structure, in that the gene segments for each locus are interspersed. The origin of this remarkable gene segment arrangement is obscure. In this report, we investigated the evolution of the TCR and variable loci and their respective recombination signal sequences (RSSs). Our phylogenetic analyses divided the and variable gene segments into two major groups each with distinguishing motifs in both the framework and complementarity determining regions (CDRs). Sequence analyses revealed that TCR variable segments share similar CDR2 sequences with immunoglobulin light chain variable segments, possibly revealing similar evolutionary histories. Maximum likelihood analysis of the region on Chromosome 14q11.2 containing the loci revealed two possible ancestral TCR / variable segments, TRDV2 and TRAV1-1/1-2, respectively. Maximum parsimony revealed different evolutionary patterns between the variable segment and RSS of the same variable gene arguing for dissimilar evolutionary origins. Two models could account for this difference: a V(D)J recombination activity involving embedded heptamer-like motifs in the germline genome, or, more plausibly, an unequal sister chromatid crossing-over. Either mechanism would have resulted in increased diversity for the adaptive immune system.     

Phagostimulation of the mediterranean fruit fly, Ceratitis capitata by ribonucleotides and related compounds

Females of the medfly, Ceratitis capitata, prefer sucrose solutions containing ribonucleotides to sucrose solutions without them. The order of preference for the nucleotides was: 5GMP>GTP>5CMP>5IMP >dGMP>5UMP>5AMP>5XMP=ATP=2 & 3GMP=RP>3AMP.2AMP, guanosine, inosine, adenine and 5TMP produced no significant stimulation. Females sterilized by irradiation showed reduced attraction to 5GMP as compared to non-irradiated females.Optimal molecular configuration for phagostimulation includes: phosphorylation at the 5 position of the ribose, free hydroxyl groups at 2 and 3 on the ribose, and an NH₂ group at the 2 position of the aromatic ring of purine.It is proposed that the 5GMP in yeast hydrolyzate can be used as a measure of the suitability of the hydrolyzate as a bait.

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Résumé La femelle de la mouche méditerranéenne des fruits, Ceratitis capitata, préfère les solutions de sucrose contenant des ribonucléotides aux simples solutions de sucrose. Lórdre de préférence pour les nucléotides est le suivant: 5GMP>GTP>5CMP>5IMP >dGMP>5UMP>5AMP>5XMP=ATP =2 & 3GMP=RP>3AMP.Le 2AMP, la guanosine, l'inosine, l'adénine et le 5TMP provoquent une stimulation significative. Les femelles montrent aprés stérilisation par irradiation une attirance réduite pour le 5GMP par comparaison avec les femelles non-irradiées.La configuration moléculaire optimale pour la phagostimulation comprend: la phosphorylation en position 5 du ribose; des groupes hydroxyles libres en 2 et 3 sur le ribose; et un groupe NH₂ en position 2 sur le noyau aromatique.Nous proposons que le 5GMP dans l'hydrolysat de levure puisse être utilisé pour mesurer la capacité de l'hydrolysat comme appât.

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Abbreviations 5AMP Adenosine 5-monophosphate - 3AMP Adenosine 3-monophosphate - 2AMP Adenosine 2-monophosphate - dAMP 2-deoxyadenosine 5-monophosphate - ADP Adenosine 5-diphosphate - ATP Adenosine 5-triphosphate - 5GMP Guanosine 5-monophosphate - 2GMP Guanosine 2-monophosphate - 3GMP Guanosine 3-monophosphate - dGMP 2-deoxyguanosine 5-monophosphate - GDP Guanosine 5-diphosphate - GTP Guanosine 5-triphosphate - 5IMP Inosine 5-monophosphate - IDP Inosine 5-diphosphate - ITP Inosine 5-triphosphate - 5XMP Xanthosine 5-monophosphate - 5CMP Cytidine 5-monophosphate - dCMP 2 deoxycytidine 5-monophosphate - CTP Cytidine 5-triphosphate - 5UMP Uridine 5-monophosphate - 5TMP Thymidine 5-monophosphate - RP Ribose 5 monophosphate     

Purification and characterization of α(2-6)-sialyltransferase from human liver

A Gal1-4GlcNAc (2-6)-sialyltransferase from human liver was purified 34 340-fold with 18% yield by dye chromatography on Cibacron Blue F3GA and cation exchange FPLC. The enzyme preparation was free of other sialyltransferases. It did not contain CMP-NeuAc hydrolase, protease, or sialidase activity, and was stable at –20°C for at least eight months. The donor substrate specificity was examined with CMP-NeuAc analogues modified at C-5 or C-9 of theN-acetylneuraminic acid moiety. Affinity of the human enzyme for parent CMP-NeuAc and each CMP-NeuAc analogue was substantially higher than the corresponding Gal1-4GlcNAc (2-6)-sialyltransferase from rat liver.Abbreviations FPLC fast protein liquid chromatography - NeuAc 5-N-acetyl-d-neuraminic acid - 9-amino-NeuAc 5-acetamido-9-amino-3,5,9-trideoxy-d-glycero-2-nonulosonic acid - 9-acetamido-NeuAc 5,9-diacetamido-3,5,9-trideoxy-d-glycero--d-2-nonulosonic acid - 9-benzamido-NeuAc 5-acetamido-9-benzamido-3,5,9-trideoxy-d-glycero--d-galacto-2-nonulosonic acid - 9-fluoresceinyl-NeuAc 9-fluoresceinylthioureido-NeuAc - 5-formyl-Neu 5-formyl--d-neuraminic acid - 5-aminoacetyl-Neu 5-aminoacetyl--d-neuraminic acid - CMP-NeuAc cytidine-5-monophospho-N-acetylneuraminic acid - G_M1 Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc-ceramide - ST sialyltransferase - DTE 1,4-dithioerythritol Enzyme: Gal1-4GlcNAc (2-6)-sialyltransferase, EC 2.4.99.1.     

Preparation of a series of sulfated tetrasaccharides from shark cartilage chondroitin sulfate D using testicular hyaluronidase and structure determination by 500 MHz1H NMR spectroscopy

Six tetrasaccharide fractions were isolated from shark cartilage chondroitin sulfate D by gel filtration chromatography followed by HPLC on an amine-bound silica column after exhaustive digestion with testicular hyaluronidase. Their structures were determined unambiguously by one- and two-dimensional 500 MHz¹H NMR spectroscopy in conjunction with HPLC analysis of chondroitinase AC-II digests of the tetrasaccharides. One fraction was found to contain two tetrasaccharide components. All the seven tetrasaccharides shared the common core structure GlcA1-3GalNAc1-4GlcA1-3GalNAc with various sulfation profiles. Four were disulfated comprising of two monosulfated disaccharide units GlcA1-3GalNAc(4-sulfate) and/or GlcA1-3GalNAc(6-sulfate), whereas the other three were hitherto unreported trisulfated tetrasaccharides containing a disulfated disaccharide unit GlcA(2-sulfate)1-3GalNAc(6-sulfate) and a monosulfated disaccharide unit GlcA1-3GalNAc(4-or 6-sulfate). These sulfated tetrasaccharides were demonstrated to serve as appropriate acceptor substrates for serum -N-acetylgalactosaminyltransferase, indicating their usefulness as authentic oligosaccharide substrates or probes for the glycobiology of sulfated glycosaminoglycans.Abbreviations NFU National formulary unit - COSY correlation spectroscopy - HOHAHA homonuclear Hartmann-Hahn - 1D or 2D one- or two-dimensional - IdoA l-iduronic acid - GlcA d-gluco-4-enepyranosyluronic acid - Di-0S GlcA1-3GalNAc - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-diS _d GlcA(2-sulfate)1-3GalNAc(6-sulfate) - Di-diS_E GlcA1-3GalNAc(4, 6-disulfate) - U G, U, 2S, 4S, and 6S represent GlcA, GalNAc, GlcA, 2-O-sulfate, 4-O-sulfate, and 6-O-sulfate, respectively     

Formation of nucleoside 5′-phosphoramidates under potentially prebiological conditions

Summary Adenosine 5-phosphoramidates form when solutions containing adenosine 5-polyphosphates p_nA (n 3) or P₁, P₂-diadenosine 5-diphosphate and amines are allowed to dry out. Mg ions catalyze these reactions. We have studied systems containing ammonia, imidazole, glycine, ethylenediamine and histamine. The yields of adenosine 5-phosphoramidates range from 10–50 % based on the nucleotide. The prebiotic significance of the reactions is discussed.Abbreviations Im imidazole - hist histamine - gly glycine - en ethylenediamine - CDI 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride - EDTA ethylenediaminetetraacetic acid - A adenosine - P_n (n = 1, 2 ) linear polyphosphate containing n phosphate residues - p_nA adenosine 5-polyphosphate containing n phosphate residues - ADP adenosine 5-diphosphate - ATP adenosine 5-triphosphate - AppA P₁, P₂-diadenosine 5-diphosphate - gly-pA adenylyl-(5N)-glycine - ImpA adenosine 5-phosphorimidazolide - NH₂-pA adenosine 5-phosphoramidate - en-pA adenylyl-(5N)-ethylenediamine - hist (NH) - pA adenosine 5-phospho-[2-(4-imidazolyl)-ethylamide] - hist(Im)-pA adenosine 5-phospho-[4-(2-aminoethyl)-imidazolide] - enP_1,2 phosphoramidates of ethylenediamine derived from H₃PO₄ and H₄P₂O₇