Chemoenzymatic galactosialylation with integrated cofactor regeneration

As a precursor for the chemical synthesis of sialylated oligosaccharides, the trisaccharide glycoside Neu5Ac (2-8)Gal(1-4)GlcNAc(1-O)-pent-4-ene was synthesized starting from GlcNAc(1-O)-pent-4-ene, UDP-glucose andN-acetylneuraminic acid in a one pot reaction employing galactosyltransferase and (2-6)sialyl-transferase in a complete cofactor regeneration system.Abbreviations Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5-monophosphosialate - CMP cytidine 5-monophosphate - CDP cytidine 5-diphosphate - CTP cytidine 5-triphosphate - Gal galactose - GlcNAc N-acetylglucosamine - UDP uridine 5-diphosphate - UDP-Glc uridine-5-diphosphoglucose - UDP-Gal uridine-5-diphosphogalactose - PEP phosphoenolpyruvate     

Evidence that α-dihydrograyanotoxin II does not bind to the sodium gate

Summary The basis for the ability of -dihydrograyanotoxin II (-2HG-II) to promote Na⁺ conductance in axons was sought. The apparent binding of tritiated -2HG-II to neural and other preparations was studied, using equilibrium dialysis, with lobster axon membranes,Torpedo electroplax, housefly head, and rat brain, liver and kidney. In every case the binding was nonsaturating and was suggested to involve nonspecific partitioning into the tissue. Supporting evidence was the similarity of extent of binding in all tissues and its relative insensitivity to neuropharmacological agents. -2HG-II did not affect the Na⁺ conductance of phospholipid bilayers, nor did it permit transport of²²Na into a bulk organic phase. It was concluded that -2HG-II did not bind to the sodium gate, but possibly to a sodium permease present at a frequency of less than one per ² of cell membrane.     

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.     

Cytochemical studies on the localization of 5′-nucleotidase in the acinar cells of the rat salivary glands

Summary The cytochemical localization of 5-nucleotidase (5-AMPase), and its validity, were investigated in parotid and submandibular acinar cells of a rat. Biochemical determinations showed that adequate treatment with glutaraldehyde could minimize the loss of enzymatic activity, and that 5-AMPase and non-specific alkaline phosphatase (-GPase) possessed different pH optima.The cytochemical distribution of the reaction products from the 5-AMPase activity was distinct from those of -GPase. 5-AMPase activity was localized on the surface membranes of acinar, ductal and myoepithelial cells of both salivary glands. -GPase activity was evenly distributed on the entire plasma membranes of myoepithelial cells and on the basal plasmalemma of acinar cells. The reaction products, which appeared on the luminal and lateral plasma membranes of the acinar cells, were presumed to reflect the presence of 5-AMPase, while those on the myoepithelial surface and basal plasma membranes of the acinar cells demonstrated both 5-AMPase and -GPase.The results indicate that 5-AMPase activity can be utilized as a reliable marker enzyme of plasma membranes in the salivary acinar cells.     

Die bursa- und schwanzstrukturen und ihre aberrationen bei den strongylina (Nematoda) morphologische studien zum problem der pluri-und paripotenzerscheinungen

Zusammenfassung In der Einleitung ist das Ziel der Arbeit in den wesentlichsten Punkten herausgestellt.Die Bursastrukturen (Bursavelum und Rippen bzw. Papillen) der parasitischen Strongylina lassen sich von den entsprechenden Bildungen der freilebenden Rhabditina, vor allem der Gattung Rhabditis, ableiten und in ihren Einzelgliedern homologisieren.Die im Laufe der Phylogenie bei den Strongylina auftretenden strukturellen Transformationen lassen sich auf einige wenige, relativ einfache morphogenetische Grundvorgänge zurückführen, die da sind: Wachstumsallometrien, Materialkompensationen, Organverschmelzungen und Spaltungen (Fissationen), Rudimentationen und ähnliche Vorgänge.Innerhalb der Strongylina Bursa ist ein Gefälle der Wachstumsgradienten feststellbar, das sich vom Zentrum der Bursa sowohl nach distal als auch proximalwärts abschwdcht. Zunehmende Förderung der zentral gelegenen Organe (Rippen) führt zu entsprechender Reduktion der peripheren Bursastrukturen, was vor allem im terminalen Schwanzabschnitt auffällt und zur Ausbildung des oft nur noch als Rudiment vorhandenen Dorsalrippenkomplexes führt. Letzterer entspricht in seiner Gesamtheit der Schwanzspitze der peloderen Rhabditiden mit den Papillen 9 und 10.Die bei Rhabditis moist getrennten Papillen 7 und 8 sind bei allen Strongylina zu einer Rippe (Externodorsal-Rippe) verschmolzen, die jedoch in manchen Aberrationen durch Abspaltung eines akzessorischen Astes ihre wahre Natur (als Verschmelzungsprodukt) zu erkennen gibt (Atavismus).Da dieselben Transformationsvorgänge innerhalb der Strongylina mehrfach unabhängig voneinander wirksam geworden sind, treten bestimmte Strukturformen als Parallelbildungen in verschiedenen phylogenetischen Union auf (polytope Entstehung).Zahlreich untersuchte Bildungsabweichungen (Aberrationen), deren Bedeutung für die Morphologie kurz umrissen wird, erschöpfen sich in den gleichen strukturellen Transformationstypen, die auch bei der Evolution der verschiedenen Union der Strongylina nachweisbar sind. Die Aberrationen führen daher häufig zu Atavismen oder zu Parallelvariationen (homologe Variationen").Die Zahl der Umwandlungsmbglichkeiten (Potenzen) der Bursastrukturen innerhalb der Strongylina ist beschränkt (Paripotenz im Sinne Haeckers). Bestimmte Arten (und Entwicklungshnien) haben jeweils nur bestimmte Potenzen realisiert. Andere können jedoch latent (virtuell) im Kryptotypus vorhanden sein, ohne normalerweise in Erscheinung. zu treten. In bestimmten Aberrationen können sie jedoch plötzlich realisiert werden, so ihr latentes Vorhandensein demonstrierend (Pluripotenz).Wie lange bestimmte Potenzen in einer Gruppe erhalten bleiben konnen, verdeutlichen auch die Schwanzhocker weiblicher Nematoden, als zum Bauplan der Nematoden gehbrende Bildungen. Die Potenz zur Ausbildung dieser Strukturen kommt offensichtlich sehr vielen Nematoden-Arten zu, wird jedoch nur in relativ wenigen Fällen, aber innerhalb der verschiedenen Gruppen bald hier, bald dort (disjunkte Verbreitung), realisiert. Es handelt sich bei den Schwanzhöckern um rudimentäre Organe, die bei keiner Nematoden-Art mehr voll ausgebildet erhalten sind. Ihre Rudimentation beruht zum Teil auf Materialentzug, als Folge von Unkonstruktionen der Schwanzregion, wobei die Adultstadien zuerst betroffen werden (Aphanisie nach Sewertzoff).Bei den in Chiropteren parasitierenden Strongylacanthinae haben sich Schwanzhöcker noch bei allen Arten erhalten, was ein offensichtlich archaisches Merkmal darstellt. Bei anderen Nematoden, denen sie nur im Larvalstadium zukommen, treten sie wohl durch Fötalisation in seltenen Fällen auch bei den adulten Stadien wieder auf.Alle speziellen Bursaformen der Strongylina lassen sich durch relativ wenige und einfache Transformationsvorgänge aus einem durch Abstraktion gewonnenen diagrammatischen Typus ableiten (Prinzip der variablen Proportionen" nach Troll).Die typisierten Umwandlungsvorgänge decken sich weitgehend mit den von Remane allgemein gefaßten strukturellen Typen der Realmutationen. Da sie bei den beobachteten Aberrationen, deren Entstehung auf dem Wege über Realmutationen sehr wahrscheinlich ist, in homologer Weise auftreten, kann das innerhalb der Strongylina zu beobachtende Evolutionsphänomen auf Realmutationen zurückgeführt warden.Obwohl sich die untersuchten strukturellen Transformationen in dem systematisch relativ wait gefaßten Rahmen einer Unterordnung abspielen (transspezifische Evolution nach Rensch), handelt es sich bei der von uns bevorzugten Terminologie (nach Woltereck und Remane), unter Berücksichtigung des Charakters der Umwandlungen, doch nur um Vorgänge, die in den Bereich der Mikroevolution fallen.     

β-Glucuronidase activity in human T and B lymphocytes and the Tμ and Tγ subpopulations

Summary The -glucuronidase staining characteristics of isolated T cell populations and the T and T enriched fractions derived of them were studied. T lymphocytes were obtained from purified T lymphocytes by ox-IgG rosette sedimentation. The rosette-forming cells in the pellet were referred to as T lymphocytes, whereas the lymphocytes in the interface were referred to as T depleted or T lymphocytes. B cells were studied on rosetted mononuclear cells with either mouse erythrocytes or with Staphylococcus Aureus (Cowan I) bacteria, after a preceeding polyvalent anti-human Ig treatment of the cells. While B cells showed mostly no reactivity, T and T cells were respectively characterised by a dot-like and granular pattern of reactivity. These findings are in agreement with those observed by others after -naphthyl-acetate esterase or acid phosphatase staining. Within the T lymphocyte fraction, the T non-, non lymphocytes seemed to have a granular pattern of reactivity. The same staining pattern was found in non-B, non-T lymphocytes.     

Cr(VI) increases tyrosine phosphorylation through reactive oxygen species-mediated reactions

While Cr (VI)containing compounds are well established carcinogens, the mechanisms of their action remain to be investigated. In this study we show that Cr (VI) causes increased tyrosine phosphorylation in human lung epithelial A549 cells in a timedependent manner. Nacetylcysteine (NAC), a general antioxidant, inhibited Cr (VI)induced tyrosine phosphorylation. Catalase, a scavenger of H₂O₂, sodium formate and aspirin, scavengers of hydroxyl radical (OH), also inhibited the increased tyrosine phosphorylation induced by Cr (VI). SOD, an inhibitor of superoxide radical (O₂ ^–), caused less inhibition. ESR study shows that incubation of Cr (VI) with the A549 cells generates OH radical. The generation of radical was decreased by addition of catalase and sodium formate, while SOD did not have any inhibitory effect. Oxygen consumption measurements show that addition of f Cr (VI) to A549 cells resulted in enhanced molecular oxygen consumption. These results indicate that Cr (VI) can induce an increase in tyrosine phosphorylation. H₂O₂ and OH radicals generated during the process are responsible for the increased tyrosine phosphorylation induced by Cr (VI).     

Problems in estimating growth rates of marine phytoplankton from short-term14C assays

Growth rate estimates () of phytoplankton populations that were sampled from nitrogen-limited continuous cultures and then incubated for short durations in batch culture with added¹⁴C-HCO₃ ^– were significantly different than steady-state growth rates () for 3 of 5 marine phytoplankton species. Two diatoms,Thalassiosira weissflogii andChaetoceros simplex, displayed virtually identical growth rates (=) over a wide range of, whereas for a third diatom,Phaeodactylum tricornutum, was overestimated by an average of 40% compared to. In contrast, was underestimated by the¹⁴C technique for the two remaining species: up to 40% at a steady-state of 1.0 day^–1 for the chlorophyteDunaliella tertiolecta and up to 100% at of 1.4 day^–1 for the haptophytePavlova lutheri. For the latter two species the divergence between and appeared to increase with increasing steady-state. A simple model of labeled and total carbon flow between the aqueous phase and cellular biomass was constructed to demonstrate that respiration was negligible when=, but was significant when>. In the cases in which<, a rapid physiological alteration presumably took place once the steady state was disturbed and cells were placed in the incubation chambers, which perhaps was related to the nutritional state of the cultures at the time of sampling. Questions thus are raised regarding our ability to measure accurately primary productivity from shipboard experiments with confined samples of phytoplankton from nutrient-impoverished waters that probably are less hardy than the laboratory cultures used in these studies.     

Existence and uniqueness of a sharp travelling wave in degenerate non-linear diffusion Fisher-KPP equations

In this paper we use a dynamical systems approach to prove the existence of a unique critical value c ^* of the speed c for which the degenerate density-dependent diffusion equation u _ct = [D(u)u _x ] _x + g(u) has: 1. no travelling wave solutions for 0 < c < c ^*, 2. a travelling wave solution u(x, t) = (x - c ^* t) of sharp type satisfying (– ) = 1, () = 0 ^*; '(^*–) = – c ^*/D'(0), '(^*+) = 0 and 3. a continuum of travelling wave solutions of monotone decreasing front type for each c > c ^*. These fronts satisfy the boundary conditions (– ) = 1, '(– ) = (+ ) = '(+ ) = 0. We illustrate our analytical results with some numerical solutions.     

Fromβ-lactams toα- andβ-amino acid derived peptides

Summary The potential of-lactams as intermediates for the access to- and-amino acid-derived peptides is shortly reviewed, with major focus on the technologies developed in our group. The two general strategies lie, on one side, in the oxidative ring expansion of 3-hydroxy-lactams toN-carboxy-amino acid anhydrides or Leuch's anhydrides and subsequent coupling with-amino acid esters and, on the other side, in the nucleophilic ring opening ofN-Boc--lactams. Both approaches have been successfully applied to the synthesis of,-diamino acid,-amino--hydroxy acid, polyhydroxylated-amino acid,,-disubstituted-amino acid,-amino acid,-amino--hydroxy acid and,-disubstituted-amino acid derived peptides. Because of the mild reaction conditions needed for the above transformations and the highly stereoselective procedures employed for the construction of the starting-lactam ring, the whole process allows the production of optically pure final products.     

Primary structure and expression of mRNAs encoding basic chitinase and 1,3-β-glucanase in potato

Infection of potato leaves (Solanum tuberosum L. cv. Datura) by the late blight fungus Phytophthora infestans, or treatment with fungal elicitor leads to a strong increase in chitinase and 1,3--glucanase activities. Both enzymes have been implicated in the plant's defence against potential pathogens. In an effort to characterize the corresponding genes, we isolated complementary DNAs encoding the basic forms (class I) of both chitinase and 1,3--glucanase, which are the most abundant isoforms in infected leaves. Sequence analysis revealed that at least four genes each are expressed in elicitor-treated leaves. The structural features of the potato chitinases include a hydrophobic signal peptide at the N-terminus, a hevein domain which is characteristic of class I chitinases, a proline- and glycine-rich linker region which varies among all potato chitinases, a catalytic domain, and a C-terminal extension. The potato 1,3--glucanases also contain a N-terminal hydrophobic signal peptide and a C-terminal extension, the latter comprising a potential glycosylation site. RNA blot hybridization experiments showed that basic chitinase and 1,3--glucanase are strongly and coordinately induced in leaves in response to infection, elicitor treatment, ethylene treatment, or wounding. In addition to their activation by stress, both types of genes are regulated by endogenous factors in a developmental and organ-specific manner. Appreciable amounts of chitinase and 1,3--glucanase mRNAs were found in old leaves, stems, and roots, as well as in sepals of healthy, untreated plants, whereas tubers, root tips, and all other flower organs (petals, stamen, carpels) contained very low levels of both mRNAs. In young leaves and stems, chitinase and 1,3--glucanase were differentially expressed. While chitinase mRNA was abundant in these parts of the plant, 1,3--glucanase mRNA was absent. DNA blot analysis indicated that in potato, chitinase and 1,3--glucanase are encoded by gene families of considerable complexity.     

Diversity of the mouse T cell receptor Cγ1 gene: structural analysis in C57BL/Ka

We have isolated an unusual T cell receptor chain cDNA clone (7.1) from a library made from RNA derived from adult thymus of C57BL/Ka mice. This cDNA clone corresponds to the appropriately processed C1 constant region exons preceded by 1.5 kb of J-C1 intron. The 7.1 coding region is extremely homologous to the C1 gene of BALB/c mice, differing at the protein level by a single deletion (alanine 139) and a single substitution. This latter change eliminates the sole N-linked sugar attachment site, providing a basis for strain-specific glycosylation patterns. The J-C1 intronic region contains two DNA segments (termed J1 and J2) that are highly reminiscent of joining (J) segments; both have potentially functional recombination and donor splice sequences flanking an open reading frame. Northern analysis suggests that 7.1 may be derived from a large, variable region-containing precursor.     

Induction of antifreeze protein production by juvenile hormone in larvae of the beetle,Dendroides canadensis

Summary Larvae of the beetleDendroides canadensis accumulate protein antifreezes during the winter.D. canadensis which were collected in the early fall, prior to the initiation of cold hardening processes, were treated with either 3.3 or 6.6 g juvenile hormone I topically in acetone and maintained for 21 days under normally non-inductive acclimation conditions (16 light/8 dark, 20 °C). Hormone treated animals significantly elevated the levels of antifreeze protein in their hemolymph compared to those of acetone treated and untreated controls or animals measured on the day of collection. D. canadensis treated with the anti-JH compound precocene II (P2) in acetone for 24 h at a concentration of 20 g/cm² (a dose below LD₅₀ for behavioral survival) and then maintained under acclimation conditions conducive to antifreeze protein production (8 light/16 dark, 20 °C) for 2 weeks failed to elevate levels of antifreeze. Acetone treated control animals accumulated a significant concentration of antifreeze protein.D. canadensis were also treated with 20 and 150 g/cm² P2 (a dose below the LD₅₀ for gross survival) followed by acclimation to short (8 h) photoperiod at 10 °C. All animals receiving the higher P2 dosage failed to elevate antifreezes while only 42.9% of the individuals treated with the lower dosage initiated antifreeze protein production. In contrast, over 80% of untreated and 70% of acetone treated controls responded to these inductive acclimation conditions by elevating antifreeze concentrations.These results indicate that juvenile hormone participates in the seasonal control of antifreeze protein production inDendroides canadensis. Since this species does not enter a diapause state prior to or throughout the winter this is the first evidence establishing a direct hormonal mechanism involved with insect cold hardiness.     

Gene and primary structures of dye-linked <Emphasis Type="SmallCaps">l</Emphasis>-proline dehydrogenase from the hyperthermophilic archaeon<Emphasis Type="Italic"> Thermococcus profundus</Emphasis> show the presence of a novel heterotetrameric amino acid dehydrogenase complex

Dye-linked l-proline dehydrogenase catalyzes the oxidation of l-proline in the presence of artificial electron acceptors such as 2, 6-dichloroindophenol and ferricyanide. The enzyme from the hyperthermophilic archaeon Thermococcus profundus was purified and characterized for the first time in archaea by Sakuraba et al. in 2001. In this study, cloning and sequencing analyses of the gene encoding the enzyme and functional analysis of the subunits were performed. The gene formed an operon that consisted of four genes, pdhA, pdhB, pdhF, and pdhX, which are tandemly arranged in the order of pdhA-F-X-B. SDS-PAGE analysis of the purified recombinant enzyme showed four different bands corresponding to (54 kDa), (43 kDa), (19 kDa), and (8 kDa) subunits encoded by pdhA, pdhB, pdhF, and pdhX, respectively, and the molecular ratio of these subunits was determined to be equal. This indicates that the enzyme consists of a heterotetrameric structure. Functional analysis of each subunit revealed that the subunit catalyzed the dye-linked l-proline dehydrogenase reaction by itself and that, unexpectedly, the subunit exhibited dye-linked NADH dehydrogenase activity. This is the first example showing the existence of a bifunctional dye-linked l-proline/NADH dehydrogenase complex. On the basis of genome analysis, similar gene clusters were observed in the genomes of Pyrococcus horikoshii, Pyrococcus abyssi, Pyrococcus furiosus, and Archaeoglobus fulgidus. These results indicate that the dye-linked l-proline dehydrogenase is a novel type of heterotetrameric amino acid dehydrogenase that might be widely distributed in the hyperthermophilic archaeal strain.Communicated by K. Horikoshi     

Uptake of sewage derived nitrogen by<Emphasis Type="Italic"> Ulva rigida</Emphasis> (Chlorophyceae) in Bahía Nueva (Golfo Nuevo,Patagonia, Argentine)

Uptake kinetics of nitrogen derived from sewage–seawater mixtures (2.5–20% v/v effluent) were determined in the laboratory for Ulva rigida (Chlorophyceae) native from Bahía Nueva (Golfo Nuevo, Patagonia, Argentine). In terms of nitrogen concentration, experimental enrichment levels varied between 53.7 and 362.3M of ammonium and between 0.77 and 6.21M of nitrate+nitrite. Uptake rates were fitted to the Michaelis–Menten equation, with the following kinetic parameters: ammonium: V_max = 591.2molg^–1h^–1, K _s=262.3M, nitrate+nitrite: V _max=12.9molg^–1h^–1, K _s=3.5M). Both nutrients were taken up simultaneously, but ammonium incorporation was faster in all cases. The results show a high capability of Ulva rigida to remove sewage-derived nitrogen from culture media. In the field, most of the nitrogen provided by the effluent would be tied up in algal biomass, supporting low nitrogen levels found at a short distance away from the source.     

AKIN<Emphasis Type="Italic">β</Emphasis>3, a plant specific SnRK1 protein,is lacking domains present in yeast and mammals non-catalytic <Emphasis Type="Italic">β</Emphasis>-subunits

The SNF1/AMPK/SnRK1 heterotrimeric kinase complex is involved in the adaptation of cellular metabolism in response to diverse stresses in yeast, mammals and plants. Following a model proposed in yeast, the kinase targets are likely to bind the complex via the non-catalytic -subunits. These proteins currently identified in yeast, mammals and plants present a common structure with two conserved interacting domains named Kinase Interacting Sequence (KIS) and Association with SNF1 Complex (ASC), and a highly variable N-terminal domain. In this paper we describe the characterisation of AKIN3, a novel protein related to AKIN subunits of Arabidopsis thaliana, containing a truncated KIS domain and no N-terminal extension. Interestingly the missing region of the KIS domain corresponds to the glycogen-binding domain (-GBD) identified in the mammalian AMPK1. In spite of its unusual features, AKIN3 complements the yeast sip1sip2gal83 mutant. Moreover, interactions between AKIN3 and other AKIN complex subunits from A. thaliana were detected by two-hybrid experiments and in vitro binding assays. Taken together these data demonstrate that AKIN3 is a -type subunit. A search for -type subunits revealed the existence of 3-type proteins in other plant species. Furthermore, we suggest that the AKIN3-type subunits could be plant specific since no related sequences have been found in any of the other completely sequenced genomes. These data suggest the existence of novel SnRK1 complexes including AKIN3-type subunits, involved in several functions among which some could be plant specific.     

Enzymic synthesis of lacto-N-triose II and its positional analogues

N-acetylhexosaminidase fromNocardia orientalis catalysed the synthesis of lacto-N-triose II glycoside (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OMe,3) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OMe (4) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OMe (5) throughN-acetylglucosaminyl transfer fromN,N-diacetylchitobiose (GlcNAc₂) to methyl -lactoside. The enzyme formed the mixture of trisac-charides3, 4 and5 in 17% overall yield based on GlcNAc₂, in a ratio of 20:21:59. Withp-nitrophenyl -lactoside as an acceptor, the enzyme also producedp-nitrophenyl -lacto-N-trioside II (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p,6) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p (7) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OC₆H₄NO₂-p (8). In this case, when an inclusion complex ofp-nitrophenyl lactoside acceptor with -cyclodextrin was used, the regioselectivity of glycosidase-catalysed formation of trisaccharide glycoside was substantially changed. It resulted not only in a significant increase of the overall yield of transfer products, but also in the proportion of the desired compound6.Abbreviations GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1-4)-2-acetamido-2-deoxy-d-glucose - NAHase N-acetylhexosaminidase - -CD -cyclodextrin     

Regulation ofN-acetylglucosaminyltransferase V by protein kinases

When 7721 human hepatocarcinoma cells were treated with 100nm phorbol-12-myristate-13-acetate (PMA), the activity ofN-acetylglucosaminyltransferase V(GnT-V) in the cells varied in accordance with the activity of membranous protein kinase C (PKC), but not with that of cytosolic PKC. Quercetin, a non-specific inhibitor of Ser/Thr protein kinase, andd-sphingosine and staurosporine, two specific inhibitors of PKC, blocked the activation of membranous PKC and GnT-V by PMA. Among the three inhibitors, quercetin was least effective. The inhibitory rates of quercetin and staurosporine toward membranous PKC and GnT V were proportional to the concentrations of the two inhibitors. The activities of GnT V and membranous protein kinase A (PKA) were also induced in parallel by dibutyryl cAMP (db-cAMP) and this induction was blocked by a specific PKA inhibitor. When cell free preparations of 7721 cells and human kidney were treated with alkaline phosphatase (ALP) to remove the phosphate groups, the GnT V activities were decreased. These results suggest that GnT V may be activated by membranous PKC or PKA, indirectly or directly, via phosphorylation of Ser/Thr residues.Abbreviations UDP uridine diphospho- - GnT N-acetylglucosaminyltransferase - GlcNAc Gn N-acetylglucosamine - M mannose - PMA phorbol-12-myristate-13-acetate - PKC protein kinase C - PKA protein kinase A - cAMP adenosine 3, 5-cyclic monophosphate - db-cAMP dibutyryl cAMP - TPK tyrosine protein kinase - MES 2-[N-morpholino]ethanesulfonic acid - DTT dithiothreitol - PMSF phenylmethylsulfonyl fluoride - EDTA ethylene diamine tetraacetic acid - EGTA glycol-bis-(-aminoethyl) etherN,N,N,N-tetraacetic acid - PA 2-aminopyridine - ALP alkaline phosphatase - C₂C₂ GlcNAc1-2 Man1-6(GlcNAc1-2Man1-3)ManR - C_2,4C₂ GlcNAc1-2Man1-6(GlcNAc1-4[GlcNAc1-2]Man1-3)ManR - C₂C_2,6 GlcNAc1-6[GlcNAc1-2]Man1-6(GlcNAc1-2Man1-3)ManR - C_2,4C_2,6 GlcNAc1-6[GlcNAc1-2]Man1-6(GlcNAc1-4[GlcNAc1-2]Man1-3)ManR where R=1-4GlcNAc1-4GlcNAcAsnX - Gn₂M₃Gn₂-PA C₂C₂ where R=1-4GlcNAc1-4GlcNAc-PA - Gn₃M₃Gn₂-PA C₂C_2,6 where R=1-4GlcNAc1-4GlcNAc-PA     

Binding of the galactose-specificPseudomonas aeruginosa lectin,PA-I,to glycosphingolipids and other glycoconjugates

The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact