Sialylation of N-Carbohydrate Chains of Glycoproteins with Trans-Sialidase from Trypanosoma cruzi

2,3-Sialylation of the lactosamine type N-glycans with trans-sialidase from Trypanosoma cruzi is reported. Trans-sialidase (160 kDa, pI 5.35–5.65) and its catalytic fragment (70 kDa, pI 6.0–6.3) were isolated from T. ruzi cells and immobilized on ConA-Sepharose. The resulting preparation retained its activity for several months and was repeatedly used for obtaining mono-, di-, tri-, and tetrasialylated 7-amino-4-metylcoumarin-labeled oligosaccharides with various numbers of antennas and for 2,3-sialylation of glycans within glycoproteins and neoglycoconjugates.     

Phylogenetic relationship of zeins and coixins as determined by immunological cross-reactivity and Southern blot analysis

Zeins from Zea mays L cv. Maya and coixins from Coix lacryma-jobi L. cv. Adlay were fractionated to obtain -, -, and -zein and -, -, and -coixin. The -coixins were composed of 4 polypeptide classes of 27 kDa (C1), 25 kDa (C2), 17 kDa (C4) and 15 kDa (C5) with solubility properties very similar to those of the 22 kDa and 19 kDa -zeins. Like the -zeins, the C1 and C2 -coixins corresponded to 80% of total Coix prolamins. The fraction corresponding to -coixin contained only one protein band of 22 kDa (C3). This coixin fraction has solubility properties similar to those of -zein and represents 15% of the total coixin. The -zein fraction was composed of a major 17 kDa protein band, while the -coixin fraction consisted of a mixture of - and -coixins.Polyclonal antibodies raised against C1 recognized C1 and C2 and cross-reacted strongly with the 22 kDa -zein, as did C4 and C5 antisera. The antiserum against -coixin showed strong cross-reaction with -zein. The homology between coixins and zeins was further investigated by using Southern hybridization analyses. The genomic DNA of maize and Coix were digested with several restriction enzymes and probed with cDNA clones representing 19 and 22 kDa -zeins as well as the 28 and 16 kDa -zeins. The Coix genome showed complex cross-hybridization sequences with the 22 kDa -zein cDNA, while no cross-hybridization was observed with the 19 kDa cDNA clone. The cDNA clone representing the 28 kDa -zein cross-hybridized with only one band of Coix genomic DNA, in contrast to the three bands observed in maize. This same Coix sequence also cross-hybridized with the cDNA clone representing the 16 kDa -zein. The relevance of these findings are discussed in the context of the origin of zein and coixin genes.     

Influence of the nitrogen level on root growth and morphology of two potato varieties differing in nitrogen acquisition

Two potato (Solanum tuberosum L.) cultivars (Astrid and Bodenkraft) differing in their nitrogen acquisition from the soil (Hunnius, 1981) were used in nutrient solutions to study the effect of increasing concentrations of nitrate (0.05; 0.5; 5.0 mol m^-3) particularly on root growth and morphology. In each variety increasing nitrogen concentrations stimulated shoot growth more than root growth. At all nitrate concentrations, the variety with higher nitrogen acquisition (Astrid) had a significantly larger root system. The larger root system of Astrid compared to Bodenkraft was particularly evident when surface area and total length of the roots, instead of root dry weight were used as parameters. The results stress the importance of root length and surface area for nitrogen acquisition from soils.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Cryptomonad biliprotein: Phycocyanin-645 from a Chroomonas species

The properties of phycocyanin-645 from the fresh water cryptomonad Chroomonas spec. were investigated after the pigment was isolated and purified by a combination of differential ammonium sulphate fractionation, gel filtration chromatography and ammonium sulphate gradient elution.Phycocyanin-645 is characterized by absorption maxima at 645 nm, 584 nm, 369 nm, 275 nm and shoulders at 340 nm and 620 nm. The CD spectrum has a negative maximum at 645 nm and a positive maximum at 584 nm with a shoulder at 610 nm.The fluorescence emission spectrum is asymmetrical and shows a maximum at 660 nm and a shoulder at approximately 715 nm. The molecular weight of the native phycocyanin-645, estimated by gel filtration, is 45000 for all multiple pigment forms below.Phycocyanin-645 is heterogenous in charge as revealed by isoelectric focusing with pIs at 7.03, 6.17, 5.75, 5.25 and 4.88, respectively, the main bands lying at pI 7.03 and pI 6.17. This was confirmed by polyacrylamide gel electrophoresis; five pigment components differing in mobility were found. We propose the term multiple pigment forms for these five phycocyanin-645 modifications.Calibrated SDS gel electrophoresis shows phycocyanin-645 to consist of three subunits, two light chains (₁, ₂), having molecular weights of 9200 and 10400, respectively, and one heavy chain (), having a molecular weight of 15500. Suggesting a 1:1:2 ratio between the subunits, the quaternary structure of the pigment molecule is _{1 1}-_{2 1}.Abbreviations PC-645 phycocyanin-645 - C-PC C-phycocyanin - SDS sodium dodecyl sulphate - pI isoelectric point - mol. wt. molecular weight     

Wheat chromosome 2D carries genes controlling the activity of two DNA-degrading enzymes

DNA-degrading enzymes of 24.0 kDa and 27.0 kDa were observed to have different activities in two common wheat (Triticum aestivum L.) cultivars, Wichita and Cheyenne. A substrate-based SDS-PAGE assay revealed that these two enzymes were much more active in Wichita than in Cheyenne. Genes controlling the activities of these two enzymes were localized on chromosome 2D by testing DNA-degrading activities in reciprocal chromosome substitution lines between Wichita and Cheyenne. While the allele on Wichita chromosome 2D stimulated the activities of the 24.0- and 27.0-kDa enzymes in Cheyenne, the allele on Cheyenne chromosome 2D did not reduce the activities of the 24-kDa and 27-kDa enzymes in Wichita. Whether these genes code for the DNA-degrading enzymes themselves or for factors that regulate the enzyme activities remains unknown.This work was supported in part by USDA-Competitive Research Grants Office grant No. 90-37140-5426 to P.S.B. Contribution from Agricultural Research Division, University of Nebraska. Journal Series Number 10304     

Fine root biomass,production and turnover in a fertilized <Emphasis Type="Italic">Larix leptolepis</Emphasis> plantation in central Korea

The effects of fertilization [control (C), 200kgNha^–1+25kgP ha^–1 (LNP) and 400kgNha^–1+ 50kgP ha^–1 (HNP)] on fine root dynamics were examined in a 40-year-old Larix leptolepis plantation in central Korea. The average fine root biomass during the growing season for C, LNP and HNP was 957, 934 and 814kgha^–1, respectively, whereas the fine root production for C, LNP and HNP was 2103, 2131 and 2066kgha^–1, respectively. Nitrogen and P inputs into the soil via fine root turnover for C, LNP and HNP were 23.0 and 1.2, 23.3 and 1.2 and 22.6 and 1.2kgha^–1, respectively. There were no significant differences in fine root biomass, production and N and P inputs through fine root turnover between the fertilization treatments during the first growing season after fertilization.     

Different susceptibilities of complex-, hybrid- and high-mannose-type α1- inhibitor and α1-acid glycoprotein to endo-β-N-acetylglucosaminidase F and peptide:N-glycosidase F

Endo--N-acetylglucosaminidase F (endo F, EC 3.2.1.96) and peptide:N-glycosidase F (PNGase F, EC 3.2.2.18) fromFlavobacterium meningosepticum were used for the deglycosylation of ₁-proteinase inhibitor and ₁-acid glycoprotein carrying oligosaccharide side chains of the complex-, high-mannose- and hybrid-type. High-mannose-and hybrid-type glycoproteins were obtained by the incubation of rat hepatocyte primary cultures with 1-deoxymannojirimycin or swainsonine, respectively. It was found that endo F cleaves hybrid- and high-mannose-type ₁-proteinase inhibitor and ₁-acid glycoprotein at pH 4.5 as well as at pH 8.5 in the presence or absence of 1% octyl--d-glucopyranoside. Complex-type ₁-proteinase inhibitor or ₁-acid glycoprotein were not cleaved by endo F even in the presence of octyl--d-glucopyranoside.PNGase F was found to cleave complex-, hybrid- and high-mannose-type oligosaccharide side chains of ₁-proteinase inhibitor and ₁-acid glycoprotein at pH 4.5 and pH 8.5 in the presence of 0.75% octyl--d-glucopyranoside. The deglycosylation of both protein substrates was very poor without detergents.Abbreviations Endo F endo--N-acetylglucosaminidase F (EC 3.2.1.96) - PNGase F peptide:N-glycosidase F (EC 3.2.2.18) Dedicated to Prof. Dr. Wolfgang Gerok on the occasion of his 60th birthday     

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.     

Nucleotide sequence of the chloroplast gene responsible for triazine resistance in canola

Summary The nucleotide sequence for the psbA gene from a triazine resistant cultivar of B. napus (cv Triton) has been determined. This gene encodes an open reading frame of 353 amino acids that is highly homologous to other higher plant psbA genes at both the nucleotide and amino acid levels. As has been found for other triazine resistant psbA genes, the Triton psbA contains an A to G nucleotide change which results in a serine to glycine amino acid substitution at position 264. The B. napus psbA gene also has a G insertion at position –9 resulting in a ribosome binding site sequence (AGGA) just before the initial methionine and suggesting that the entire open reading frame is translated. A large (72 bp) insertion is also found upstream of the B. napus psbA gene which resembles a similar insertion in the mustard psbA. The uncloneable nature of the entire gene is further investigated through reconstruction experiments and the implications discussed.     

The assignment of a Thinopyrum distichum (Thunb.) Löve-derived translocation to the long arm of wheat chromosome 7D using endopeptidase polymorphisms

Summary Endopeptidase zymograms of the translocation line Indis revealed the presence of several major and minor bands that had differential expression in coleoptile and seed tissues. While Indis lacks Ep-D1a, which is present in the parental cultivar Inia 66, it also may not express any of the Th. distichum bands. The Indis zymogram was found to be identical to that of an isogenic line of Inia 66 possessing Lr19. Since the absence of an Ep-D1a product appears to be linked to the 7DL translocation, it is possible to use the null condition as a marker for both the Lr19 or Indis translocations. The Indis translocation also did not show recombination with the cn-D1 chlorophyl mutant on 7DL, confirming that a part of 7D was involved. The results of a telocentric mapping experiment involving the 7D telosomes indicated that in Indis a chromosome segment from Th. distichum replaced a large section of 7DL of Inia 66.     

Inheritance and linkage of isozyme loci in almond

The segregation of seven isozyme marker genes was investigated using eight controlled crosses in almond. The cultivar Nonpareil was the maternal parent in all crosses. Pollination was achieved using eight different cultivars, and a total of 3200 individual kernels were assessed. For each isozyme the goodness-of-fit test was used to test for departure from the expected frequencies assuming Mendelian inheritance. Given a higher than expected number of significant results for individual isozymes, independent segregation between pairs of isozymes was tested using the chi-square statistic on the resulting two-way contingency tables. In all crosses a highly significant association (P value< 0.001) was observed between (1) the AAT- 1 and IDH isozymes loci and (2) the LAP-1 and PGM-2 isozymes loci, which leads to the conclusion that the respective isozyme pairs are linked.In addition, a significant association (P value < 0.001) was observed between LAP-1 and GPI-2 when the pollen sources were Fritz, Mission, or Price, but this could not be tested for the remaining five pollen sources, Carmel, Grant, Keane, Ne plus Ultra, Peerless, because they are homozygous at these loci. If LAP-1 is linked with GPI-2 and PGM-2, it might be expected that we should find evidence of linkage between GPI-2 and PGM-2. The lack of a significant association between these two isozymes suggests that LAP-1 is located centrally on the chromosome. These three pairs of linked loci are the first to be reported in almond.     

Identification of two RAPD markers tightly linked with the Nicotiana debneyi gene for resistance to black root rot of tobacco

Linkage of randomly amplified polymorphic DNA (RAPD) markers with a single dominant gene for resistance to black root rot (Chalara elegans Nag Raj and Kendrick; Syn. Thielaviopsis basicola [Berk. and Broome] Ferraris) of tobacco (Nicotiana tabacum L.), which was transferred from N. debneyi Domin, was investigated in this study. There were 2594 repeatable RAPD fragments generated by 441 primers on DNAs of Delgold tobacco, a BC₅F₈ near isogenic line (NIL) carrying the resistance gene in a Delgold background, and PB19, the donor parent of the resistance gene. Only 7 of these primers produced eight RAPD markers polymorphic between Delgold and PB19, indicating there are few RAPD polymorphisms between them despite relatively dissimilar pedigrees. Five of the eight RAPD markers were not polymorphic between Delgold and the NIL. All of these markers proved to be unlinked with the resistance gene in F₂ linkage tests. Of the remaining three RAPD markers polymorphic between Delgold and the NIL, two were shown to be strongly linked with the resistance gene; one in coupling and the other in repulsion. Application of the two RAPDs in the elimination of linkage drag associated with the N. debneyi resistance gene and marker-assisted selection for the breeding of new tobacco cultivars with the resistance gene is discussed.     

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.     

Subcellular localization and topology of β(1→4)galactosyltransferase that elongates β(1→4)galactan side chains in rhamnogalacturonan I in potato

The subcellular localization and topology of rhamnogalacturonan I (RG-I) (14)galactosyltransferase(s) ([14]GalTs) from potato (Solanum tuberosum L.) were investigated. Using two-step discontinuous sucrose step gradients, galactosyltransferase (GalT) activity that synthesized 70%-methanol-insoluble products from UDP-[¹⁴C]Gal was detected in both the 0.5 M sucrose fraction and the 0.25/1.1 M sucrose interface. The former fraction contained mainly soluble proteins and the latter was enriched in Golgi vesicles that contained most of the UDPase activity, a Golgi marker. By gel-filtration analysis, products of 180–2,000 Da were found in the soluble fraction, whereas in the Golgi-enriched fraction the products were larger than 80 kDa and could be digested with rhamnogalacturonan lyase and (1,4)endogalactanase to yield smaller rhamnogalacturonan oligomers, galactobiose and galactose. The endogalactanase requires (14)galactans with at least three galactosyl residues for cleavage, indicating that the enzyme(s) present in the 0.25/1.1 M Suc interface transferred one or more galactosyl residues to pre-existing (14)galactans producing RG-I side chains in total longer than a trimer. Thus, the (14)GalT activity that elongates (14)-linked galactan on RG-I was located in the Golgi apparatus. This (14)GalT activity was not reduced after treatment of the Golgi vesicles with proteinase, but approximately 75% of the activity was lost after treatment with proteinase in the presence of Triton X-100. In addition, the (14)GalT activity was recovered in the detergent phase after treatment of Golgi vesicles with Triton X-114. Taken together, these observations supported the view that the RG-I (14)GalT that elongates (14)galactan was mainly located in the Golgi apparatus and integrated into the membrane with its catalytic site facing the lumen.Abbreviations GalT Galactosyltransferase - (14)GalT (14)-Galactosyltransferase - H ⁺ -ATPase Proton ATPase - HG Homogalacturonan - HSP70 ER resident Bip - mMDH Mitochondrial malate dehydrogenase - RG-I Rhamnogalacturonan I - RG-II Rhamnogalacturonan II - RGP Reversibly glycosylated polypeptide - RG-Lyase Rhamnogalacturonan lyase - Suc Sucrose - UDPase Uridine-5-diphosphatase     

Effects of root herbivory by an insect on a foliar-feeding species,mediated through changes in the host plant

Summary The effects of root herbivory by larvae of the scarabaeid, Phyllopertha horticola, on the growth of Capsella bursa-pastoris were examined. Individuals of Aphis fabae were reared on the leaves to determine what effect, if any, root feeding has on the performance of this insect. The experiment was conducted under two watering regimes (low and high). Low watering and root feeding caused water stress in the plants and this was reflected in a reduction in vegetative biomass and an increase in the proportion of material allocated to reproduction. Supplying plants with ample water in the high treatment enabled the water stress caused by root herbivory to be offset, but not completely overcome. Low watering and root feeding caused an increase in aphid weight and growth rate, while root feeding also increased fecundity and adult longevity. These effects are attributed to an improvement in food quality, measured by total soluble nitrogen, and caused by amino acid mobilization due to the water stress. The implications of these results in agricultural and ecological situations are discussed.     

The ability of acidic pH,growth inhibitors,and glucose to increase the proton motive force and energy spilling of amino acid-fermenting <Emphasis Type="Italic">Clostridium sporogenes</Emphasis> MD1 cultures

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (p) was only –25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZpH) and the electrical membrane potential () increased. The p was –125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but increased to –70 mV. Protein synthesis inhibitors also significantly increased . Non-growing, arginine-energized cells had a of –80 mV at pH 6.7 or pH 4.7, but was not detected if the F₁F₀ ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large p to grow, and p appears to serve as a mechanism of ATP dissipation or energy spilling.Mandatory disclaimer: Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

Purification and characterization of cyclic 3′5′-nucleotide phosphodiesterase D3 from Sinorhizobium fredii MAR-1

The cyclic 35-nucleotide phosphodiesterase D3 was purified from Sinorhizobium fredii MAR-1. The native enzyme had a molecular weight of approximately 44.5kDa and a subunit molecular weight of approximately 21kDa as judged by SDS-gel electrophoresis. The pH optimum of the enzyme for the hydrolysis of cyclic AMP was approximately 6.0 with both acetate and Tris-maleate buffers. The optimum temperature for hydrolysing cyclic AMP was approximately 50C. No metal ion was required for activity and EDTA up to 2.5mM did not markedly affect the enzyme. However, methylxanthines, adenine and adenosine as well as 5-AMP, ATP, ADP and metal ions like Zn²⁺, Fe²⁺, Pb²⁺, Al³⁺ and Fe³⁺, were strongly inhibitory at 2.5mM.The D3 enzyme could hydrolyse both cyclic AMP and cyclic GMP with the apparent K _m for cyclic AMP of approximately 0.23M.     

sym 18. A novel gene conditioning altered strain specificity in Pisum sativum cv. ‘Sparkle’

An induced mutant of pea Pisum sativum cv. Sparkle forms few nodules with R. leguminosarum bv. viciea from temperate regions, exemplified by strain PRE, but nodulates normally with some rhizobia from Middle East soils, exemplified by strain TOM. The mutant gene is not an allele of sym2, found in the primitive cultivar Afghanistan. Mutant line E54 has a specificity similar to Afghanistan but forms more nodules with temperate strains, especially PF₂ which nodulates Afghanistan only poorly. The new phenotype is conditioned by gene sym18, which can act as recessive or semi-dominant depending on the rhizobial strain. Also sym18 is distinguished from sym 2 by its location on a different linkage group. Sym18 was mapped 9cM from k on linkage group II.