Membrane potential changes during transport of hexoses in Lemna gibba G1

The membrane potential (pd) of duck weed (Lemna gibba G1) proved to be energy dependent. At high internal ATP levels of 74 to 105 nmol ATP g^-1 FW, pd was between -175 and -265 mV. At low ATP levels of 23 to 46 nmol ATP g^-1 FW, pd was low, about -90 to -120 mV at pH 5.7, but -180 mV at pH 8. Upon addition of glucose in the dark or by light energy the low pd recovered to the high values. The active component of the pd was depolarized by the addition of hexoses in the dark and in the light. Hexose-dependent depolarization of the pd (= pd) followed a saturation curve similar to active hexose influx kinetics. Depolarization of the pd recovered in the dark even in the presence of the hexoses and with a 10fold enhancement in the light. Depolarization and recovery could be repeated several times with the same cell. Glucose uptake caused a maximum depolarization of 133 mV, fructose uptake half that amount, sucrose had the same effect as glucose. During 3-O-methylglucose and 2-deoxyglucose uptake the depolarizing effect was only slightly lower. The pd remained unchanged in the presence of mannitol. The glucose dependent pd and especially the rate of pd recovery proved to be pH-dependent between pH 4 and pH 8. It was independent of the presence of 1 mM KCl. Although no pH could be measured in the incubation medium, these results can be best explained by a H⁺-hexose cotransport mechanism powered by active H⁺ extrusion at the plasmalemma.Abbreviations LD longday - SD shortday - pd membrane electropotential difference - pd maximum membrane potential depolarization - L light - D dark - FW fresh weight - d days of culture of Lemna gibba - 1X perfusing solution without sugar, see methods     

Stereospecificity and electrogenicity of amino acid transport in Riccia fluitans

In the aquatic liverwort Riccia fluitans, membrane depolarization (_m), change in membrane conductance (g_m), and current-voltage (I-V) characteristics in the presence of different amino acids as well as the uptake of ¹⁴C-labeled amino acids were measured. L-isomers of the tested amino acids generate larger electrical effects (_m, g_m) than D-isomers, and the I-V characteristics show that the positive electrical inward-current of 20 mA m^-2 generated by 0.5 mM D-serine is only about 50% of the current generated by adding 0.5 mM L-serine. Whereas - and -amino acids rapidly depolarize the membrane to the same extend, with -aminobutyric acid (-AB) and dipeptides no significant electrical effects have been measured. The uptake kinetics of ¹⁴C-labeled amino acids display three components: (I) A saturable high-affinity component with K_s-values of 48 M D-alanine, 12 M -aminoisobutyric acid (AIB), 9 M L-alanine, 8 M L-proline, and 6 M L-serine, respectively; (2) an apparently linear low-affinity component, and (3) an also linear but unspecific component at concentrations >20 times the given K_s-value. Uptake of ¹⁴C-labeled AIB can be inhibited competitively by all tested neutral amino acids, the L-isomers being more effective than the D-isomers, as well as by ammonium or methylamine. Vice versa, AIB competitively inhibits uptake of L-serine and L-alanine. It is concluded that an uncharged stereospecific carrier for the investigated amino acids exists in the plasmalemma of Riccia fluitans. Accumulation ratios of about 50 suggest secondary active transport driven by a transmembrane electro-chemical gradient (mainly _m) which is generated by the electrogenic proton pump. It is suggested that this carrier binds to the amino group forming either a charged binary complex with positively charged amines (Felle 1980), or an uncharged complex with -AB or dipeptides, whereas electrogenic transport of - and -amino acids is mediated by a ternary carrier complex, probably charged by a proton.Symbols and Abbreviations _m membrane potential (mV) - E_co equilibrium potential (mV) of the transport system - g_m membrane (slope) conductance (Sm^-2) - g_m change in g_m - I-V curve current-voltage curve - AIB -aminoisobutytric acid - -AB -aminobutyric acid     

Glycerol-, inositol-, and reducing end hexose-containing oligosaccharides in human urine

Ten previously unreported oligosaccharides have been purified from the urines of human subjects using a combination of gel filtration, ion exchange, and thin-layer chromatographies. Their structures were determined by direct probe mass spectrometry, methylation analysis, and proton NMR spectroscopy of the permethylated oligosaccharide alditols.On the basis of composition, the oligosaccharides could be divided into three groups. Five oligosaccharides containing glycerol were characterized as glucosyl1-1glycerol; glucosyl1-1glycerol; galactosyl1-1glycerol; glucosyl-1-1(fucosyl-1-2)glycerol and/or fucosyl-1-1(glucosyl-1-2)glycerol; and glucosyl-1-1(galactosyl-1-2)glycerol or galactosyl-1-1(glucosyl-1-2)glycerol. Four inositol-containing oligosaccharides were characterized as galactosyl1 (fucosyl1)inositol,N-acetylgalactosaminyl1 (fucosyl1)inositol, fucosyl1-2galactosyl1 (N-acetylgalactosaminyl1)inositol and fucosyl1-2galactosyl1-4-N-acetylglucosaminyl1(N-acetylgalactosaminyl1)inositol. Finally, galactosyl1-3(fucosyl1-2)galactosyl1-6galactosyl1-4(fucosyl1-3)glucose, an oligosaccharide with glucose at its reducing end, was tentatively identified. The significance and possible origins of the carbohydrate structures are discussed.     

Hormonal regulation of gene expression in the “slender” mutant of barley (Hordeum vulgare L.)

The slender mutant of barley resembles a normal barley plant treated with high doses of gibberellic acid (GA₃). Expression of GA₃-regulated and abscisic acid (ABA)-regulated mRNAs was studied in the endosperm and roots of mutant and wild-type (WT) plants.Production of -amylase (EC 3.2.1.1) by WT embryoless half-grains was dependent on the presence of GA₃, and was prevented by ABA. In contrast, -amylase was produced by half-grains of the slender mutant in the absence of added GA₃, although it was still reduced by ABA. The spectrum of -amylase mRNAs in slender embryoless half-grains incubated in the absence of added GA₃ was the same as in WT endosperm half-grains incubated in the presence of GA₃. These results indicate that the endosperm of the slender mutant exhibits similar properties to WT endosperm treated with GA₃.In roots the expression of an ABA-inducible mRNA was similar in slender and WT seedlings either treated with exogenous ABA or exposed to dehydration. This result, and the effect of ABA on -amylase production by the endosperm, indicate that the slender plants retain sensitivity to ABA.Abbreviations ABA abscisic acid - AMV avian myeloblastosis virus - GA gibberellin - GA₁ gibberellin A₁ - GA₃ gibberellic acid - WT wild-type     

The formation of peptides from the 2′(3′)-glycyl ester of a nucleotide

Summary We have synthesized 2(3)-O-(glycyl)-adenosine-5-(O-methylphos-phate), an analogue of the 3-terminus of aminoacylated tRNA. A 0.4M solution of this compound maintained at pH 8.2, yields 5.5% of diglycine and 11.5% of diketopiperazine, in addition to the hydrolysis products glycine and adenosine-5-(O-methylphosphate). Under the same conditions, glycine ethyl ester reacts much more slowly, but ultimately gives similar yields of diglycine and diketopiperazine.The aminolysis of 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) by free glycine is relatively inefficient, but serine reacts 20 times more rapidly and yields up to 50% of N-glycylserine. The prebiotic significance of these reactions is discussed.Abbreviations MepA adenosine-5-(O-methylphosphate) - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-gly 2,3-O-(bis-glycyl)-adenosine-5-(O-methylphosphate) - DKP diketopiperazine - gly Et glycine ethyl ester - gly-ser N-glycylserine - O-gly-ser O-glycylserine - O-(gly)-gly-ser O-(glycyl)-glycylserine - Boc-gly N-tert-butyloxycarbonylglycine - MepA-Boc-gly 2(3)-O-(Boc-glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-Boc-gly 2,3-O-(bis-Boc-glycyl)-adenosine-5(O-methylphosphate) - (gly)₂ diglycine - (gly)₃ triglycine     

Light-induced membrane potential changes of epidermal and mesophyll cells in growing leaves of Pisum sativum

Light transiently depolarizes the membrane of growing leaf cells. The ionic basis for changes in cell membrane electrical potentials in response to light has been determined separately for growing epidermal and mesophyll cells of the argenteum mutant of pea (Pisum sativum L.). In mesophyll cells light induces a large, transient depolarization that depends on the external Cl^– concentration, is unaffected by changes in the external Ca²⁺ or K⁺ concentration, is stimulated by K⁺-channel blockers tetraethylammonium (TEA⁺) and Ba²⁺, and is inhibited by 3-(3-4-dichlorophenyl)-1,1-dimethylurea (DCMU). In isolated epidermal tissue, light induces a small, transient depolarization followed by a hyperpolarization of the membrane potential. The depolarization is enhanced by increasing the external Ca²⁺ concentration and by addition of Ba²⁺, and is not sensitive to DCMU. Epidermal cells in contact with mesophyll display a depolarization resembling the response of the underlying mesophyll cells. The light-induced depolarization in mesophyll cells seems to be mediated by an increased efflux of Cl^– while the membrane-potential changes in epidermal strips reflect changes in the fluxes of Ca²⁺ and in the activity of the proton-pumping ATPase.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - CCCP carbonylcyanide m-chlorophenylhydrazone - DCMU 3-(3-4-dichlorophenyl)-1,1-dimethylurea - LID _e light-induced depolarization in epidermal cells - LID _m light-induced depolarization in mesophyll cells - LIH light-induced hyperpolarization - TEA⁺ tetraethylammonium Ecotrans paper #43. This research was supported by National Science Foundation grants DCB-8903744 and MCB-9220110 to E.V.     

Effects of abscisic acid analogues on abscisic acid-induced gene expression in barley aleurone protoplasts: Relationship between structure and function of the abscisic acid molecule

The plant hormone abscisic acid (ABA) mediates gene expression in barley aleurone protoplasts. In order to elucidate the essential functional groups of the ABA molecule, the specificity of a number of ABA analogues for inducing ABA-regulated gene (e.g., RAB, BASI) expression in barley aleurone protoplasts was studied. These analogues have modifications at three different positions of the ABA molecule: (a) the 1-hydroxyl group (1-deoxy ABA), (b) the carboxyl group (ABA-methyl ester or ABA-glucose ester), and (c) both the 1-hydroxyl and 4-carbonyl groups (-ionylidene acetic acid). The importance of the different putative functional groups was analyzed. The dose-response analysis of ABA analogues upon the induction gene expression showed the following order: ABA > ABA methyl ester > 1-deoxy ABA > ABA glucose ester > -ionylidene acetic acid > --ionone.     

An abscisic acid analog inhibits abscisic acid-induced freezing tolerance and protein accumulation, but not abscisic acid-induced sucrose uptake in a bromegrass (Bromus inermis Leyss) cell culture

The application of abscisic acid (ABA), either as a racemic mixture or as optically resolved isomers, increases freezing tolerance in a bromegrass (Bromus inermis Leyss) cell culture and induces the accumulation of several heat-stable proteins. Two stereoisomers of an ABA analog, 23 dihydroacetylenic abscisyl alcohol (DHA), were used to study the role of ABA-induced processes in the acquisition of freezing tolerance in these cells. Freezing tolerance was unchanged in the presence of (–) DHA (LT₅₀ -9°C), and no increase in heat-stable protein accumulation was detected; however, the (+) enantiomer increased the freezing tolerance (LT₅₀ -13°C) and induced the accumulation of these polypeptides. All three forms of ABA increased freezing tolerance in the bromegrass cells, although (–) ABA was less effective than either (+) or (±) ABA when added at equal concentrations. Cells pretreated with 20 or 50 M (–) DHA displayed lower levels of freezing tolerance following the addition of 2.5, 7.5 or 25 M (±) ABA. Full freezing tolerance could be restored by increasing the concentration of (±) ABA to > 25 M. Pretreatment of cells with (–) DHA (20 or 50 M) had no effect on freezing tolerance when 25 M (+) ABA was added. The induction of freezing tolerance by 25 M (–) ABA was completely inhibited by the presence of 20 M (–) DHA. The accumulation of ABA-responsive heat-stable proteins was inhibited by pretreatment with 20 M (–) DHA in cells treated with 2.5 or 7.5M (+) ABA, and in cells treated with 25 M (–) ABA. The accumulation of these polypeptides was restored when (±) or (+) ABA was added at a concentration of 25 M. The analysis of proteins which cross-reacted with a dehydrin antibody revealed a similar inhibitory pattern as seen with the other ABA-responsive proteins. The effects of the various isomers of ABA and DHA on cell osmolarity and sucrose uptake was also investigated. In both cases, (±) and (+) ABA had pronounced effects on the parameters measured, whereas (–) ABA treated cells gave substantially different results. In both sucrose uptake and cell osmolarity, DHA had no significant effect on the results obtained following (±) or (+) ABA treatment. Maximum freezing tolerance was only observed in cells when both heat-stable protein accumulation and sucrose uptake were observed.Abbreviations ABA abscisic acid - DHA 2,3 dihydroacetylenicabscisyl alcohols - DMSO dimethyl sulfoxide - LT₅₀ temperature at which 50% of cells are killed The authors would like to acknowledge the technical assistance of Angela Bollman, Bruce Ewan and Angela Shaw. This work was supported by grants from the Natural Science and Engineering Research Council of Canada to L.V.G. and N.H.L., and a grant from the University of Saskatchewan to R.W.W.     

Genetic analysis of brown planthopper resistance in twenty varieties of rice,Oryza saliva L.

Summary The inheritance of resistance to brown planthopper, Nilaparvata lugens (Stol.), of 20 rice cultivars was studied. Single dominant genes that are allelic to Bph 3 condition the resistance in cultivars Ptb 19, Gangala (Acc. 7733), Gangala (Acc. 15207), Horana Mawee, Kuruhondarwala, Mudu Kiriyal and Muthumanikam. Single recessive genes that are allelic to bph 4 govern the resistance in cultivars Gambada Samba, Heenhoranamawee, Hotel Samba, Kahata Samba, Kalukuruwee, Lekam Samba, Senawee, Sulai, Thirissa and Vellai Illankali. The resistance in Ptb 33, Sudu Hondarwala, and Sinna Sivappu is governed by one dominant and one recessive gene which segregate independently of each other. The dominant resistance genes in these cultivars appear allelic to either Bph 1 or Bph 3. Similarly, the recessive genes in these cultivars seem allelic to either bph 2 or bph 4. Further investigations are needed to conclusively determine the allelic relationships of resistance genes in Ptb 33, Sudu Hondarwala and Sinna Sivappu.     

Hexagonally ordered “rosettes” of particles in the plasma membrane ofMicrasterias denticulata Bréb. and their significance for microfibril formation and orientation

Summary Cells ofMicrasterias denticulata Bréb. at the stage of secondary wall formation have been studied by freeze-etching. It was found that the plasma membrane exhibits oval areas in which arrays of membrane particles occur. These particles form rosettes which are arranged in a hexagonally ordered lattice with a center to center spacing of 25 nm. Nearly the same periodicities can be found between microfibrils. It is concluded that the rosettes probably together with the thickened area of the plasma membrane below them represent the apparatus for the production and orientation of microfibrils. The hypothesis suggesting the incorporation of membrane templates functional in microfibril formation, originally advanced byKiermayer andDobberstein (1973) has received further support.     

Abscisic acid, temperature and salinity interactions on growth and some mineral elements in Carthamus plants

Growth and contents of sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), chloride (Cl), phosphorus (P) and sulphur (S) in shoot and root tissues of Carthamus tinctorius plants were measured at combinations of four nutrient solution osmotic potentials (_s=0, -0.3, -0.6 and -0.9 MPa) induced by NaCl and CaCl treatments, three constant temperatures (T) ranging from 15 to 35°C and four abscisic acid (ABA) concentrations (0,10,50 and 100 mg L^–1). Unstressed and stressed plants grown in optimal temperature conditions (25°C) maintained higher growth rates (dry mass production) than plants grown under low and high temperatures (15 and 35°C respectively). Shoot and root growth (dry mass production) were largely inhibited by salinity but the magnitude of growth inhibition was temperature dependent. Safflower plants respond to salinity stress by increases in Ca, Cl and to a lesser extent Na in their shoots and roots and by a decrease in the ratio of fresh to dry weight. The ratio of K/Na was decreased progressively on salinization. With stressed plants, ABA application reduced the toxicity of salt treatment, improved K uptake under salinity, effectively increased K/Na ratio and helped the plants to avoid Na toxicity and sometimes enhanced growth. The effect of ABA on the growth was more pronounced at optimum temperature (25°C). The association between the internal mineral element concentrations was largely affected by ABA application and temperature change but a wide fluctuation in response was noticed. The effects of single factors (_s, T and ABA) on the growth and mineral contents were statistically significant. Also, bifactorial (_s× T, _s × ABA and T × ABA) and three factorial (_s × T × ABA) interactions significantly affected the parameters. Further statistical treatment of the data (coefficient of determination ²) led to four important findings: (1) Salinity (_s) was dominant in affecting Ca and Cl contents in both shoot and root as well as root Na content. (2) Temperature (T) had a dominant effect on growth, shoot K, Mg, P, S and root P, and S contents (3) The share of _s × T × ABA interaction was dominant for root Na and Mg contents. (4) The single factors and their interactions had a dual role in their subsidiary effects.Abbreviations ABA abscisic acid - _s osmotic potential - ² coefficient of determination - F.wt fresh weight - d.m. dry matter - T temperature - MPa mega pascal - SAR sodium adsorption ratio - P phosphorus - S sulphur     

Endogenous abscisic acid and 2-trans-abscisic acid in alternate bearing ‘Wilking’ mandarin trees

The relationship of abscisic acid (ABA) and 2-trans-abscisic acid (t-ABA) to alternate bearing has been examined in Wilking mandarin (Citrus reticulata Blanco) trees. Leaves, stems and buds of trees loaded with fruit (on trees) had 4.3, 6.0 and 2.2 fold higher ABA levels than the corresponding organs from off trees. Leaves had higher ABA levels than stems and buds in both on and off trees. t-ABA was non-detectable in Wilking leaf, stem and bud tissue. Amounts of t-ABA not exceeding 40% of the ABA content, were found in Shamouti and Valencia orange buds and in Wilking fruit peel.The elevated levels of ABA in on tree organs may reflect a stress imposed by the fruit overload.     

Molecular mapping of a new gene Rrs4 CI 11549 for resistance to barley scald (Rhynchosporium secalis)

Doubled haploid (DH) progeny from a cross between the scald susceptible barley (Hordeum vulgare L.) cultivar Ingrid and the resistant accession CI 11549 (Nigrinudum) was evaluated for resistance in the pathogen Rhynchosporium secalis (Oudem) J.J. Davis. Two linked and incompletely dominant loci confer resistance CI 11549 against isolate 4004. One is an allele at the complex Rrs1 locus on chromosome 3H close to the centromere; the other is located 22 cM distally on the long arm. The latter locus is designated Rrs4. In BC₃-lines into Ingrid from CI 2222 (another Nigrinudum) resistance seems governed by one locus close to the telomeric region of chromosome 7H, probably allelic to Rrs2. In neither case did we find any trace of the recessive gene rh8 reported to be present in Nigrinudum. Various resistance donors of Ethiopian origin designated as Nigrinudum, Jet or Abyssinian were identical to a great extent with respect to markers, but differed in resistance to different isolates of scald or in barley yellow dwarf virus (BYDV) resistance. The implications for their use as differentials in scald tests and screening of germplasm collections are discussed.     

Uptake of methylamine via an inducible,energy-dependent transport system in the facultative methylotroph Arthrobacter P1

Cytoplasmic membrane vesicles were prepared by a lysozyme-salt treatment from Arthrobacter P1 grown on methylamine as the carbon and energy source. In the presence of an ascorbate-phenazine methosulphate electron donor system, these vesicles accumulated methylamine in unmodified form by an inducible transport system. This system has a high affinity for methylamine (K_app=20–25 M). The effect of the ionophores valinomycin and nigericin combined with membrane potential () and pH-gradient (pH) measurements demonstrated that methylamine uptake is electrogenic and driven by the . Optimal activity is observed at pH 6.5 and 30°C. Methylamine uptake was not affected by the presence of ammonium ions but was inhibited by the primary amines ethylamine (competitively), propylamine, butylamine and benzylamine. In addition, formaldehyde and acetate, at a concentration of 1 mM, inhibited methylamine uptake almost completely. These compounds were shown to be non-competitive inhibitors. A strong inhibition observed in the presence of plumbagin could be relieved by addition of dithiothreitol. This indicates that the oxidation-reduction state of, probably, carrier dithiol-disulfide-groups is an important factor in methylamine translocation in Arthrobacter P1.     

A Comparison of the Lipopolysaccharides and O-Specific Polysaccharides of Azospirillum brasilense Sp245 and Its Omegon-Km Mutants KM018 and KM252

The lipopolysaccharides (LPSs) extracted from the outer membrane of Azospirillum brasilense Sp245 and its Omegon-Km mutants KM018 and KM252 with a hot aqueous solution of phenol were found to differ in the content of carbohydrates, glucosamine, and total phosphorus and in the proportion of octadecenoic and hexadecanoic acids in the lipid moieties of the LPSs. The carbohydrate moieties of the LPSs were heterogeneous in charge. The analysis of the O-specific polysaccharides (O-PSs) of the mutants KM018 and KM252 by gas–liquid chromatography, IR spectroscopy, and NMR spectroscopy showed that they are composed of the same linear pentasugar repeating units 2)--D-Rhap-(1 3)--D-Rhap-(1 3)--D-Rhap-(1 2)--D-Rhap-(1 2)--D-Rhap-(1 as the O-PSs of the parent strain Sp245. The reported differences in the biological activity of the LPSs of the parent and mutant strains can be due to their different chemical composition.     

Purification and immunocytochemical localization of kafirins inSorghum bicolor (L. Moench) endosperm

Summary Kafirins are the storage proteins of sorghum and are found in protein bodies in the seed endosperm. They have been classified as -, -, and -kafirins according to differences in molecular weight, solubility, and structure. The kafirins were purified, amino acid composition was determined, and immunolocalization methods were used to determine the organization of the protein bodies and distribution of kafirins throughout the endosperm. All three groups of kafirins were low in lysine. -Kafirins and -kafirins were relatively high in cysteine, and -kafirins were relatively high in methionine. Transmission electron microscopy showed that protein bodies in the peripheral endosperm were spheroid with concentric rings and few darkly stained inclusions. In contrast, protein bodies of the central endosperm were irregularly shaped with a higher proportion of darkly stained material. The light staining regions of the protein bodies are composed primarily of -kafirins with minor portions of - and -kafirins. The dark staining regions, however, are composed primarily of - and -kafirins. Immunoelectron microscopy showed that protein bodies in the peripheral endosperm contain predominantly a-kafirin with minor amounts of - and -kafirin. Central endosperm protein bodies are also predominantly -kafirin, but have a higher proportion of -kafirin and -kafirin than the peripheral endosperm protein bodies.Abbreviations GAR-HRP Goat anti-rabbit horseradish peroxidase - IgG immunoglobulin G - 2-ME 2-mercaptoethanol - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TBS Tris buffer saline - TBS-T Tris buffer saline with Tween - TBS-T-B Tris buffer saline with Tween and bovine serum albumin - TCA trichloroacetic acid - UV ultraviolet     

Somatic embryogenesis,plant regeneration and somaclonal variation in barley

In vitro culture of immature embryo and young leaf tissues was carried out with five cultivars of barley, Hordeum vulgare. Two cultivars (Albacete and Porthos) responded poorly from both types of explants, while the three others (Dissa, Golden Promise and Ingrid) produced a high frequency of embryogenic callus from these explants (25–60%). For Dissa and Ingrid, young leaf explants were slightly better than immature embryo explants for embryogenic callus induction, while immature embryo cultures of Golden Promise responded better than young leaf explants. Thus, there appears to be a significant genotype × explant interaction in the initiation of embryogenic callus in barley.Some phenotypic variants were detected among the regenerated plants of Golden Promise and Ingrid, most originating by epigenetic changes. Only in one case was the variant phenotype heritable, probably due to a mutation in the chloroplast DNA. Mitotic alteractions were not detected. Consequently, somaclonal variation did not appear to be a very frequent event in plants regenerated from 1- to 6- month-old cultures of barley.     

Structures of asparagine linked oligosaccharides of immunoglobulins (IgY) isolated from egg-yolk of Japanese quail

Structures of the Asn linked oligosaccharides of quail egg-yolk immunoglobulin (IgY) were determined in this study. Asn linked oligosaccharides were cleaved from IgY by hydrazinolysis and labelled withp-aminobenzoic acid ethyl ester (ABEE) afterN-acetylation. The ABEE labelled oligosaccharides were then fractionated by a combination of Concanavalin A-agarose column chromatography and anion exchange, normal phase and reversed phase HPLC before their structures were determined by sequential exoglycosidase digestion, methylation analysis, HPLC, and 500 MHz¹H-NMR spectroscopy. Quail IgY contained only neutral oligosaccharides of the following categories: the glucosylated oligomannose type (0.6%, Glc1-3Glc1-3Man₉GlcNAc₂; 35.6%, Glc1-3Man_7–9GlcNAc₂). oligomannose type (15.0%, with the structure Man_5–9GlcNAc₂) and biantennary complex type with core structures of-Man1-3(-Man1-6)Man1-4GlcNAc1-4GlcNAc (9.9%),-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4GlcNAc (25.1%) and-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc (11.4%). Although never found in mammalian proteins, glucosylated oligosaccharides (Glc₁Man_7–9GlcNAc₂) have been located previously in hen IgY.Abbreviations IgG, IgM, IgA, IgY immunoglobulin G, M, A and Y, respectively - ABEE p-aminobenzoic acid ethyl ester     

Ultrastructure and behaviour of the spindle pole body of the aphid-pathogenic fungusErynia neoaphidis

Summary A detailed account of the ultrastructure and behaviour of the spindle pole body (SPB) of the entomophthoraceous fungusErynia neoaphidis is presented for the first time.The SPB consists of extranuclear (ENC) and intranuclear (INC) components. The ENC is a saucepan-shaped structure which lies in a pocket of the nuclear envelope. It is composed of a forked, fibrillar handle and a shallow, cylindrical pan. The pan has a wall of two layers, both of which are thickened with a regular periodicity so that they appear to be beaded. It is postulated that the pan is formed from rough endoplasmic reticulum and that it synthesizes the amorphous, electron-dense material coating the ENC.The INC is a saucer-shaped, electron-dense plaque in which the ends of the spindle microtubules terminate. During metaphase, a clear zone separates the INC from the nuclear envelope and persists until telophase. The roles of the amorphous, electron-dense material and the clear zone as well as the method of SPB replication are discussed.     

Production and characterization of antibodies to the β-(1→6)-galactotetraosyl group and their interaction with arabinogalactan-proteins

Rabbit antisera were raised against -(16)-galactotetraose coupled to bovine serum albumin (Gal₄-BSA). The antisera reacted with arabinogalactan-proteins (AGPs) isolated from seeds, roots, or leaves of radish (Raphanus sativus L.) as revealed by immunodiffusion analysis. Extensive removal of -l-arabinofuranosyl residues from these AGPs enhanced the formation of precipitin with the antisera. The antisera did not react with such other polysaccharides as soybean arabinan-4-galactan, -(14)-galactan, and -(13)-galactan, indicating their high specificity toward the consecutive -(16)-galactosyl side chains of AGPs. The antibodies were purified by affinity chromatography on a column of immobilized -(16)-galactotetraose as ligand. The specificity of the antibodies toward consecutive (16)-linked -galactosyl residues was confirmed by enzyme-linked immunosorbent assay for hapten inhibition against Gal₄-BSA as antigen, which revealed that -(16)-galactotriose and-tetraose were potent inhibitors, while -(13)-or -(14)-galactobioses and -trioses were essentially unreactive. Electron-microscopic observation of immunogold-stained tissues demonstrated that AGPs were localized in the middle lamella as well as at the plasma membrane of primary roots of radish. Agglutination of protoplasts prepared from cotyledons occurred with the antibodies, supporting the evidence for localization of AGPs in the plasma membrane. The antibody-mediated agglutination was inhibited by addition of AGPs or -(16)-galactotetraose.Abbreviations AGP arabinogalactan-protein - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate - Gal₃-BSA -(16)-galactotriose coupled to BSA - Gal₄-BSA -(16)-galactotetraose coupled to BSA - Ig immunoglobulin - 4-Me-GlcpA 4-O-methyl-d-glucopyranosyluronic acid - M_r relative molecular mass The authors wish to thank Dr. J. Ohnishi of Department of Biochemistry, Saitama University, for his help in preparing protoplasts.