Characterization of a novel mouse recombinant processing {alpha}-mannosidase

In previous work (Herscovics et al., J. Biol. Chem., 269, 9864–9871),a novel mouse      

A mathematical model of sialylation of N-linked oligosaccharides in the trans-Golgi network

A mathematical model is developed of the compartmentalized sialylationof N-linked oligosaccharides in order to understand and predictthe outcome of sialylation reactions. A set of assumptions arepresented, including Michaelis-Menten-type dependency of reactionrate on the concentration of the glycoprotein substrate. Theresulting model predicts the heterogeneous outcome of a posttranslationaloligosaccharide biosynthesis step, a critical aspect that isnot accounted for in the modeling of the cotranslational attachmentof oligosaccharides to glycosylation sites (Shelikoff et al.,Biotech. Bioeng., 50, 73–90, 1996) or general models ofthe secretion process (Noe and Delenick, J. Cell Sci, 92, 449–459,1989). In the steady-state for the likely case where the concentrationof substrate is much less than the K_m of the sialyltransferase,the model predicts that the extent of sialylation, x, will dependupon the enzyme concentration, enzyme kinetic parameters andsubstrate residence time in the reaction compartment. The valueof x predicted by the model using available literature datais consistent with the values of x that have been recently determinedfor the glycoproteins CD4 (Spellman et al, Biochemistry, 30,2395–2406, 1991) and t-PA (Spellman et al, J. Biol Chem.,264, 14100–14111, 1989) secreted by Chinese hamster ovarycells. For the unsaturated case, the model also predicts thatx is independent of the concentration of secreted glycoproteinin the Golgi. The general modeling approach outlined in thisarticle may be applicable to other glycosylation reactions andposttranslational modifications. model sialylation N-linked glycosylation     

Carbohydrate structures of a normal counterpart of the carcinoembryonic antigen produced by colon epithelial cells of normal adults

Normal Faecal antigen-2 (NFA-2) and non-specific crossreactingantigen-2 (NCA-2), cross-reacting with anticarcinoembryonicantigen (CEA) antibodies, were found in normal human faececand meconium, respectively. Because NFA-2, NCA-2 and CEA areconsidered as the same gene products, NFA-2 and NCA-2 shouldbe normal counterparts of CEA produced by colon epithelial cellsof normal adults and fetuses, respectively. Comparison of sugarchain structures of these three antigens is indispensable inorder to unravel the stnaural alteration induced by malignanttransformation and development of colon epithelial cells. Thesugar chain structures of CEA (Yamashita,K. et al., Cancer Res.,47,3451–3459,1987) and NCA-2 (Yarnashita,K. et al., J.Bid Chem, 264,17873-17881,1989) were previously reported. Inthis paper, the structures of the oligosaccharides releasedfrom four NFA-2 samples by hydrazinolysis were studied by meansof lectin-affinity column chromatography, endo- and em-glycosidasedigestion, methylation analysis, hydrazinolys-nitrous acid deaminationand electrospray ionization mass spectrometry. NFA-2 contains24–27 mol of N-linked sugar chains/molecule, which issimilar to NCA-2 (27 mol) and CEA (24–27 mol). In contrastto CEA, which contains {small tilde} 8% high-mannose-type sugarchains all sugar chains of NFA-2 are mono- to tetra-antennarycomplex-type chains having four types of tri-mannosyl cores,with or without bisecting N-acetylglucosamine and fucose residues.The structures of their outer chain moieties comprise Galß1     

Clearance of picoplankton-sized partides and formation of rapidly sinking aggregates by the pteropod, Limacina reiroversa

Findings from experiments showed that the web-feeding euthecosomatouspteropod, Limacina retroversa, can produce rapidly sinking,mucous aggregates. It is suggested that, by adhesion, theseaggregates scavenged picoplankton-sized particles, which werethus effectively cleared from the medium. In contrast, Calanusfinmarchicus was not able to clear these particles in our experiments.Sedimentation velocities of 10 aggregates measured in vivo wereup to 1000 m day^–1, with an average of {small tilde}300m day^–1 (not including two aggegates with neutral buoyancy).Mean velocities measured for feces of C.finmarchicus, Calanushyperboreus and Thyssnoessa sp. were consider ably lower. Wesuggest that the sedimentation of L retroversa aggregates wasthe source of mucous flocs collected in sediment traps (Bathmannet al., Deep-Sea Res., 38,1341–1360,1991) and at the seafloor at 1200 m depth in the southern Norwegian Sea. This processmay be an important mediator of sedimentation to the deep sea,when these pteropods are present in surface waters in largeabundance.     

Uptake and metabolism of BuCast: a glycoprotein processing inhibitor and a potential anti-HIV drug

We have previously shown (Sunkara et al., 1989; Taylor et al.,1991) that 6-o-butanoyl castanospermine (BuCast) was a 30–50-foldbetter inhibitor of HIV syncytia formalion than castanospermine(Cast). Radiolabeled Cast and BuCast were used to study theuptake and metabolism of these compounds in cultured cells andin mice. BuCast was preferentially taken up by cells comparedto Cast. Approximately 30–50-fold higher radioactivitywas found in cells treated with BuCast compared to cells treatedwith Cast during the initial 4–6h of labeling. HPLC analysisshowed that once BuCast was taken up by cells, it was rapidlyconverted to Cast. Mice given oral doses of BuCast had 5–10-foldhigher levels of Cast in the plasma and tissues as comparedto Cast treated mice. However, when the compounds were giveni.v., the levels of plasma and tissue radioactivity obtainedfrom Cast or BuCast were equivalent suggesting rapid conversionof BuCast to Cast in the blood. In mice orally treated withBuCast, HPLC analysis suggested that only Cast was found inthe plasma and tissues. With multiple dosing of mice, additiveresults were obtained, suggesting that multiple doses may beused to obtain higher concentrations of the compound in thetarget cells. These data suggest that the lipophilic propertiesof butanoyl side chain on the Cast ring makes BuCast significantlybetter absorbed, and this may help to alleviate some of thegut toxicity associated with Cast treatment. HIV AIDS glycoproteins inhibitors     

An improved method for the structural profiling of keratan sulfates: analysis of keratan sulfates from brain and ovarian tumors

A previously developed method for the structural fingerprinting of keratan sulfates (Brown et al., Glycobiology, 5, 311-317, 1995) has been adapted for use with oligosaccharides fluorescently labeled with 2-aminobenzoic acid following keratanase II digestion. The oligosaccharides are separated by high-pH anion-exchange chromatography on a Dionex AS4A-SC column. This methodology permits quantitative analysis of labeled oligosaccharides which can be detected at the sub-nanogram ( approximately 100 fmol) level. Satisfactory calibration of this method can be achieved using commercial keratan sulfate standards. Keratan sulfates from porcine brain phosphocan and human ovarian tumors have been examined using this methodology, and their structural features are discussed.     

Regulation of Ferricyanide Reduction by Epidermal Tissue

Ferricyanide reduction by epidermal strips of Commelina communisis stimulated by plasmolysis, and this stimulation is partiallyreversed by re-hydration. The reduction of ferricyanide takesplace only in the guard cells. KCN stimulates ferricyanide reduction, indicating that NADHserves as an electron donor. Catalase, which inhibits NAD(P)Hdriven oxygen uptake [Askerlund et al. (1987) Physiol. Plant.71: 9–19., Pantoja and Willmer (1988) Planta 174: 44–50.]enhances ferricyanide reduction and this stimulation is evidentmainly in the non-plasmolysed tissues. Differences between thenon-pretreated tissues and the plasmolysed ones are discussed. (Received July 11, 1989; Accepted November 2, 1989)     

Organization of a human UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase gene and a related processed pseudogene

We have previously characterized a cDNA that encodes a fulllength human UDP-GalNAc:polypeptide, N-acetyl galactosaminyltransferase(GalNAc-transferase) (J.A. Meurer et al., J. Biochem., 118,568–574, 1995). The present report describes the characterizationof the corresponding human GalNAc-transferase gene and a relatedpseudogene. Two human genomic libraries,     

Quantal Response of Seed Germination in Seven Genera of Cruciferae to White Light of Varying Photon Flux Density and Photoperiod

The response of the germination of seeds of Barbarea vema (Mill.)Aschers, Brassica chinensis L., Brassica juncea (L.) Czern.& Coss., Brassica oleracea L. var. gongylodes L., Camelinasaliva (L.) Crantz, Eruca saliva Mill., Lepidium sativum L.,Nasturtium officinale R. Br., and Rorippa palustris (L.) Besserto white fluorescent light of different photon flux densitiesapplied for different daily durations in a diurnal alternatingtemperature regime of 20 °C/30 °C (16 h/8 h) was quantifiedby linear relations between probit percentage germination andthe logarithm of photon dose, the product of photon flux densityand duration. The low energy reaction, in which increasing dosepromotes germination, was detected in all the seed populationsbut in Barbarea vema and Brassica Juncea the lowest photon doseapplied (10^–5–2 and 10^{–5 7} mol m^–2 d^–1,respectively) was sufficient to saturate the response. Comparisons,where possible, between photoperiods demonstrated reciprocity,i.e. germination was proportional to photon dose irrespectiveof photoperiod, for the low energy reaction in Brassica oleracea(1 min d^–1 to 1 h d^–1), Camelina saliva (1 min d^–1to 8 h d^–1), Eruca saliva (1 min d^–1 to 24 h d^–1),Lepidium sativum (I min d^–1 to 8 h d^–1) and Rorippapalustris (1 min d^–1 to 8 h d^–1), but not in Brassicachinensis and Nasturtium officinale. The high irradiance reaction,in which increasing dose inhibits germination, was detectedin Barbarea vema, Brassica chinensis, Brassica juncea, Brassicaoleracea, and Camelina saliva. The minimum dose at which inhibitionwas detected was lO^–0–3 mol m^–2 d^–1.These results are discussed in the context of devising optimallight regimes for laboratory tests intended to maximize germination The response of germination to photon dose was also quantifiedwith 3 x 10^–4 M GA₂, co-applied (Brassica chinensis, Camelinasaliva, and Lepidium sativum) and with 2 x 10^–2 M potassiumnitrate co-applied (Brassica chinensis). In the latter casepotassium nitrate had no effect in the dark and inhibited germinationin the light, but GA₂, promoted germination substantially inall three species. Variation amongst seeds in the minimum photondose required to stimulate germination was not affected by co-applicationof GA₂, in Brassica chinensis and Camelina saliva, whereas seedsof Lepidium salivum showed a narrower distribution of sensitivitiesto the low energy reaction in the presence of GA₂ Barbarea vema (Mill.) Aschers, Brassica chinensis L., Brassica juncea (L.) Czern. & Coss., Brassica oleracea L. var. gongylodes L., Camelina saliva (L.) Crantz, Eruca saliva Mill., Lepidium satiaum L., Nasturtium officinale R. Br., Rorippa palustris (L.) Besser, Cruciferae, light, gibberellic acid, seed germination, seed dormancy     

Field Measurements of the Gas Exchange of Woody Plant Species in Simulated Sunflecks

The photosynthetic responses of eight tree and shrub speciesto simulated sunflecks was measured in the field. The net carbonexchange (NCE) of Corylus avellana and Ulmus glabra increasedwith irradiance up to the maximum irradiance of 230 µmolm^–2 s^–1. The NCE of Fraxinux excelsior, Hedera helixand the sun and shade forms of Rhododendron ponticum saturatedat about 120 µmol m^–2 s^–1 whereas the NCEof Ilex aquifolium, Daphne laureola and Fagus sylvatica hadeffectively saturated at 27 µmol m^–2 s^–1. In all cases the quantum efficiency of NCE could be predictedfrom measurements of chlorophyll fluorescence and the maximumvalue for NCE from measurements of stomatal conductance. Therelationships were combined into a model for predicting NCE/irradiancecharacteristics. Corylus avellana L., Daphne laureola L., Fagus sylvatica L., Fraxinus excelsior L., Hedera helix L., Ilex aquifolium L., Prunus laurocerasus L., Rhododendron ponticum L., Ulmus glabra Huds., gas exchange, stomatal resistance, water use efficiency, chlorophyll fluorescence, quantum efficiency     

Direct Somatic Embryoid Formation on Immature Embryos of Trifolium repens, T. pratense and Medicago sativa, and Rapid Clonal Propagation of T. repens

By direct somatic embryogenesis in vitro a clone of asepticplantlets can be raised from a single immature embryo of Trifoliumrepens (white clover) within about 6 weeks of pollination. Embryoidsare induced directly from intact zygotic embryonic tissue ona culture medium containing 0·025 or 0·05 mg 1^–1BAP and 1·0 g 1^–1 yeast extract. Similar directsomatic embryogenesis has also been achieved for Trifolium pratense(red clover) and Medicago sativa (lucerne). Applications ofembryo propagation by direct somatic embryogenesis are discussed,particularly in relation to multiple screening of host genotypesfor analysis of host/pathogen and legume/Rhizobium interactions. Trifolium repens L., Trifolium pratense L., Medicago sativa L., clover, lucerne, tissue culture, embryoid, somatic embryogenesis, legumes     

The Partitioning of Nitrate Assimilation Between Root and Shoot of a Range of Temperate Cereals and Pasture Grasses

Nitrate reductase activity (NRA, in vivo assay) and nitrate(NO^-₃) content of root and shoot and NO^-₃ and reduced nitrogencontent of xylem sap were measured in five temperate cerealssupplied with a range of NO^-₃ concentrations (0·1–20mol m^–3) and three temperate pasture grasses suppliedwith 0·5 or 5 0 mol m^–3 NO^-₃ For one cereal (Hordeumvulgare L ), in vitro NRA was also determined The effect ofexternal NO^-₃ concentration on the partitioning of NO^-₃ assimilationbetween root and shoot was assessed All measurements indicatedthat the root was the major site of NO3 assimilation in Avenasatwa L, Hordeum vulgare L, Secale cereale L, Tnticum aestivumL and x Triticosecale Wittm supplied with 0·1 to 1·0mol m^–3 NO^-₃ and that for all cereals, shoot assimilationincreased in importance as applied NO^-₃ concentration increasedfrom 1.0 to 20 mol m^–3 At 5.0–20 mol m^–3 NO3,the data indicated that the shoot played an important if notmajor role in NO^-₃ assimilation in all cereals studied Measurementson Lolium multiflorum Lam and L perenne L indicated that theroot was the main site of NO^-₃ assimilation at 0.5 mol m^–3NO^-₃ but shoot assimilation was predominant at 5.0 mol m^–3NO^-₃ Both NRA distribution data and xylem sap analysis indicatedthat shoot assimilation was predominant in Dactylis glomerataL supplied with 0.5 or 5.0 mol m^–3 NO^-₃ Avena sativa L., oats, Hordeum vulgare L., barley, Secale cereale L., rye, x Triticosecale Wittm., triticale, Triticum aestivum L., wheat, Dactylis glomerata L., cocksfoot, Lolium multiflorum Lam., Italian ryegrass, Lolium perenne L., perennial ryegrass, nitrate, nitrate assimilation, nitrate reductase activity, xylem sap     

Gloeotrichia echinulata, a colonial cyanobacterium with a unique phosphorus uptake and life strategy

Epilimnetic colonies of Gloeotrichia echinulata were harvestedfrom 200–300 I of water in Lake Erken with filtrationthrough appropriate plankton nets (200 or 70 µm). Phosphateuptake characteristics, phosphorus (P) status and photosynthesisof the colonies were determined twice a week during July andAugust 1991. Phosphate uptake was analysed according to thesimple force- flow relationship of Falkner et al. (Arch. Microbiol.,152, 353–361, 1989). The threshold concentration of Puptake below which uptake ceases for energetic reasons, wasconstantly much higher than the epilimnetic soluble reactivephosphorus (SRP) concentration, so that the planktonic colonieswere unable to acquire any Pin the epilimnion. Neither did organicP seem to be a source of P for planktonic colonies. Gloeotrichiaechinulata has a unique life strategy in comparison to othercommon genera of bloom-forming cynanobacteria. Its P assimilationand growth are completely separated both in time and space;growth is preceded by benthic P assimilation. Epilimnetic growthwas based solely on internally stored P and growth rates fittedthe Droop model well. Depletion of stored P restricts the lengthof the planktonic phase to 15–20 days under ‘optimal’growth conditions. Wind-induced surface drift seemed to be themost important loss factor from the epilimnion. Massive recruitmentof P-rich benthic colonies accounted for two-thirds of totalnet internal P loading observed between mid-July and mid-August(3.8 mg P m^–2 day^–1).     

Amitriptyline reduces olfactory neuron adenylyl cyclase activity

Adenylyl cyclase plays an important role in olfactory signaltransduction. Recently, a novel type III adenylyl cyclase hasbeen localized in olfactory neurons (Pfeuffer et al., 1989;Bakalyar and Reed, 1990). Because amitriptyline (AMI), a tricyclicantidepressant, appears to have an inhibitory effect on adenylylcyclase activity in other in other neuronal tissue (Yamaokaet al., 1988; Wong et al., 1991), we measured the effect ofAMI on forskolin-stimulated adenylyl cyclase activity in membranepreparations of olfactory mucosa from adult rats. In the presenceof 5'-guanylyl-imidodiphosphate, AMI (0.5–8.0 µM)inhibited forskolin-stimulated adenylyl cyclase activity ina dose-dependent manner. To determine whether this effect wasspecific for olfactory neurons, as opposed to other cells inthe olfactory epithelium, rats were unilaterally bulbectomizedin order to reduce selectively the number of olfactory neuronson the side ipsilateral to the bulbectomy. In membrane preparationsfrom unilaterally bulbectomized animals we saw significantlylower adenylyl cyclase activity in ipsilateral olfactory mucosa,compared with adenylyl cyclase activity from non-bulbectomizedmucosa. These results indicate that AMI inhibition of adenylylcyclase activity is primariy localized in olfactory neurons.     

Biomass, feeding and production of Noctiluca scintillans in the Seto Inland Sea, Japan

The abundance and biomass of the large heterotrophic dinoflagellateNoctiluca scintillans, together with the changes in its potentialprey items, were monitored in the Seto Inland Sea, Japan, duringsummer 1997 (17 July-11 August). Growth and grazing rates ofNscintillans fed natural plankton populations were also measuredeight and seven times, respectively, during the survey period.The abundance and biomass of N scintillans averaged over thewater column (19 m) were in the range 1–345 cells 1^–1(temporalaverage = 93 cell1^–1) and 0.1–49.6 µg C l^–1(temporalaverage = 13.8 µg C l^–1; three times higher thanthat of calanoid copepods during the same period). Noctilucascintillans populations followed the changes in phytoplankton:N.scintillans biomass was increasing during the period of diatomblooms and was at a plateau or decreasing during periods oflow chlorophyll a. The growth rates of N.scintillans (µ)were also consistent with the wax and wane of the N.scintillanspopulation: N.scintillans showed highest growth rates duringdiatom blooms. A simple relationship between µ and chlorophylla concentration was established, and the production of N.scintillanswas estimated using this relationship and the measured biomass.The estimated production averaged over the water column wasin the range >0.1–5.2 µg C l^–1 day^–1(temporalaverage = 1.4 µg C l^–1 day^–1; 64% of the productionof calanoid copepods during the same period). Diatom clearancerates by N.scintillans were in the range 0.10–0.35 mlcell^–1 day^–1, and the phytoplankton population clearanceby N.scintillans was >12% day^–1. Thus, although thefeeding pressure of N.scintillans on phytoplankton standingstock was low, N.scintillans was an important member of themesozooplank-ton in terms of biomass and production in the SetoInland Sea during summer.     

Bioluminescence of sound-scattering layers in the Gulf of Maine

Submersible-based investigations of bioluminescence were conductedin sound-scattering layers (SSLs) in the Gulf of Maine, usingintensified video and dual-beam acoustic methods. Stimulatedbioluminescence in the SSLs was high (3–41 µW sr^–1m^–3 while spontaneous bioluminescence was not detected.The average intensity of individual bioluminescent sources inthe SSLs was 30–200 times greater than the intensity oflight emitters outside the SSLs. The two brightest sources ofbioluminescence were identified as the euphausiid, Meganyctiphanesnorvegica and the cydippid ctenophore, Euplokamis sp. Meganyctiphanesnorvegica formed a SSL within 50 m of the bottom during theday and migrated to the uppermost 30 m of the water column atnight, forming a near-surface SSL. Euplokamis sp., which producedexceptionally intense and long-lasting bioluminescent secretions,occurred within the near-bottom SSL in concentrations up to7 m^–3. Our findings indicate that traditional methodsof identifying the primary light emitters in a region, basedon light measurements from net- or pump-captured organisms,may have underestimated the significant in situ bioluminescencepotential of euphausiids and gelatinous zooplankton. ³ Present address: NOAA/NURP/R-OR2, Silver Spring, MD 20910,USA     

Exogenous ABA as a Modulator of the Response of Sorghum to High Salinity

Treatment of Sorghum bicolor (L.) Moench, cv. 610, with abscisicacid (ABA) during the first week of sahnization with 150 molm^–3 NaCl induced enhancement of growth and acceleratedadaptation to high salinity (300 mol m^–3 NaCl) Adaptationis defined as the development of the ability of the plant tosurvive, grow, and set seeds upon exposure to a NaCl concentrationwhich is lethal for the unadapted plant In the absence of ABAthe saline pretreatment requires 20 d for the development ofadaptation (Amzallag et al., 1990), with ABA treatment the sameresult is achieved within approximately one week The exposureof the plants to non-lethal salinity (150 mol m^–3 NaCl)apparently triggers a transient sensitivity to ABA lasting forabout 8 to 10 d following the beginning of sahnization Thisperiod coincides with an increase in leaf PEP carboxylase activitywhich seems to occur faster if the plants are treated with ABA.Exogenous ABA-induced enhancement of growth and control of shootNa⁺ concentration, occur at a lower ABA concentration (10 mmolm^–3) than the induction of adaptation to salinity whichoc curs at 40 mmol m^–3 or above. The lowered shoot Na⁺concentration which is induced by a low ABA concentration isnot sufficient to induce survival of the plants in high salinity(300 mol m^–3 NaCl). Key words: Growth, adaptation to salinity, ABA