Genotypic control of pollen plant formation in Nicotiana tabacum L.

Summary Tobacco plants (Nicotiana tabacum L.) of four varieties (Badischer Burley, White Burley, Techne, Kupchunos) were raised at different temperatures and daylengths and the effect of genotype on embryogenic pollen grain formation in situ and on pollen plant formation in anther and pollen cultures from these plants was studied. Genotype controlled embryogenic pollen grain and pollen plant formation by defining productivity under standard growth conditions (long days at 24 °C). Kupchunos was the most productive variety, followed by White Burley, Techne, and Badischer Burley. Furthermore, genotype defined which environmental factor was able to affect embryogenic pollen grain and pollen plant formation and also to which degree. In anther cultures, in addition to these effects, genotype controlled the formation of (an) inhibitory substance(s) in the anther wall in interaction with the plant growth conditions. In Badischer Burley and Techne, inhibitor action could be prevented by isolation of the pollen after one week of anther culture. Finally, direct pollen cultures in Badischer Burley and Techne produced embryos were only when the pollen was isolated from nearly mature anthers, while in White Burley and Kupchunos, embryos also produced at earlier stages and at higher yields. This indicated that genotype controls the time when the embryogenic pollen grains become ready to divide. The results are discussed in relation to strategies to overcome recalcitrance of species and genotypes.     

Nuclear Overhauser effect investigation on GM1 ganglioside containingN-glycolyl-neuraminic acid (II3Neu5GcGgOse4Cer)

The conformational properties of the oligosaccharide chain of GM1 ganglioside containingN-glycolyl-neuraminic acid, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-were found to fix the relative orientitation of the three saccharides, while the glycosidic linkage of the terminal -Gal-was found to be quite mobile as the -Gal-(1-3)--GalNAc-disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containingN-acetyl-neuraminic acid and neuraminic acid [1].Abbreviations Ganglioside nomenclature is in accordance with Svennerholm [23] and the IUPAC-IUB Recommendations [24] GM3(Neu5Ac) II³Neu5AcLacCer, -Neu5Ac-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM3(Neu5Gc) II³Neu5GcLacCer, -Neu5Gc-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Ac) II³Neu5AcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Gc) II³Neu5GcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu) II³NeuGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu-(2-3)]--Glc-(1-1)-Cer - GD1a IV³Neu5AcII³Neu5AcGgOse₄Cer, -Neu5Ac-(2-3)--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GalNAc-GD1a IV⁴GalNAcIV³Neu5AcII³Neu5AcGgOse₄Cer, -GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - Neu neuraminic acid - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolyl-neuraminic acid - Cer ceramide     

Analysis of the effect of rye chromosomes 4R, 6R and telomeric heterochromatin on 7R on homologous chromosome pairing in pentaploid hybrids (AABBR,AABBD)

Summary Meiotic pairing in Triticum turgidum cv. Ma (4x) with a mean chiasmata frequency of 27.16 per cell was compared with chiasmata frequencies in its hybrids with several triticale strains, Chinese Spring wheat and its addition lines for Imperial rye chromosomes 4R and 6R. In hybrids between Ma and x Triticosecale cv. Rosner the chiasmata frequency was marginally reduced by an average of 1.25%, by 8.8% in hybrids with x Triticosecale cv. DRIRA HH and by 6.7% with DRIRA EE (lacking 90% telomeric heterochromatin from chromosome arm 7RL). In pentaploid hybrids between Ma and T. aestivum cv. Chinese Spring the reduction was an average of 10.30%, while addition lines with rye chromosome 6R reduced chiasmata frequencies by an average of 7.4% and rye addition line for 4R showed the greatest depression in chiasmata frequency in hybrids by a 25.04% reduction. An interchange difference involving long chromosome segments was observed between Ma and Rosner.Contribution No. 819 Ottawa Research Station     

Genetic analysis of resistance to whitebacked planthopper in twenty-one varieties of rice,Oryza sativa L.

Summary The inheritance of resistance to whitebacked planthopper Sogatella furcifera (Horvath) was studied in 21 rice varieties. Reactions of F₁; F₂ and F₃ progenies of the crosses of 21 resistant varieties with the susceptible variety TN 1 revealed that a single dominant gene governs resistance in Mushkan 41, Santhi, Siahnakidar 195, SM2-34, Tirisurkh 251, Zirijowaian 245, 18, 24A, 39, 76 S, 78, 180, 213 B, 267, 293, CI 6037-4, NP97, S39 JKW and Bansphul. In varieties 65 and 274 A, resistance is governed by one dominant and one recessive gene which segregate independently of each other. Tests for allelism with the Wbph 1 gene originally identified in N 22 revealed that the dominant gene present in all the test varieties is the same as Wbph 1. Further studies are required to determine the allelic relationships of the recessive gene found in varieties 65 and 274 A.     

Negative-ion fast atom bombardment tandem mass spectrometry for characterization of sulfated unsaturated disaccharides from heparin and heparan sulfate

Negative-ion fast atom bombardment tandem mass spectrometry has been used in the characterization of non-, mono-, di- and trisulfated disaccharides from heparin and heparan sulfate. The positional isomers of the sulfate group of monosulfated disaccharides were distinguished from each other by negative-ion fast atom bombardment tandem mass spectra, which provide an easy way of identifying the positional isomers. This fast atom bombardment collision induced dissociation mass spectrometry/mass spectrometry technique was also applied successfully to the characterization of di- and trisulfated disaccharides.Abbreviations FABMS fast atom bombardment mass spectrometry - CID collision induced dissociation - MIKE mass analysed ion kinetic energy - MS/MS mass spectrometry/mass spectrometry - HPLC high performance liquid chromatography - UA d-gluco-4-enepyranosyluronic acid - CS chondroitin sulfate - DS dermatan sulfate - HA hyaluronan - Hep heparin - HS heparan sulfate - UA(14) GlcNAc 2-acetamido-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose - UA(14)GlcNAc6S 2-acetamido-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcNAc 2-acetamido-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcNAc6S 2-acetamido-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(14)GlcN6S 2-amino-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcN 2-amino-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcN6S 2-amino-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(14)GlcNS 2-deoxy-2-sulfamino-4-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose - UA(14)GlcNS6S 2-deoxy-2-sulfamino-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcNS 2-deoxy-2-sulfamino-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcNS6S 2-deoxy-2-sulfamino-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(13)GalNAc 2-acetamido-2-deoxy-3-O-(-d-Gluco-4-enepyranosyluronic acid)-d-galatose - UA(13)GalNAc4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA(13)GalNAc6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA2S(13)GalNAc 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-galactose - UA2S(13)GalNAc4S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA2S(13)GalNAc6S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA(13)GalNAcDiS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose - UA(13)GlcNAc 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose.     

Isolation and characterization of a multienzyme complex containing DNA replicative enzymes from mitochondria ofS. cerevisiae

A 40 S multienzyme complex containing mtDNA polymerase was isolated from mitochondria ofS. cerevisiae by density gradient centrifugation and by gel filtration chromatography. Besides DNA polymerase, RNA polymerase, primase, 35 exonuclease and an ATPase activities were found to be associated with it. The presence of some of these enzymes were confirmed by Western blot. This high molecular weight multienzyme complex containing DNA has most of the attributes of a putative replisome.Abbreviations BCIP 5 bromo 4 chloro 3 indolyl phosphate - mtDNA mitochondrial DNA - NBT Nitroblue tetrazolium - PBS phosphate buffered saline     

Sialyltransferase activity in FR3T3 cells transformed withras oncogene: decreased CMP-Neu5Ac:Galβ1-3GalNAc α-2,3-sialyltransferase

We have investigated the activity of CMP-Neu5Ac:Gal\1-3GalNAc -2,3-sialyltransferase (EC 2.4.99.4) in FR3T3 cells transformed by the Ha-ras oncogene in which we have previously demonstrated the higher expression of the -galactosidase -2,6-sialyltransferase (EC 2.4.99.1) [21]. We demonstrate that the presence of the activatedras gene decreases the activity of this specific -2,3-sialyltransferase fourfold. According to the kinetic parameters and to mixing experiments, we can assume that this decreased enzymatic activity reflects a decrease in the number of activeO-glycan -2,3-sialyltransferase polypeptides inras-transformed cells. However, no change in the binding of Peanut agglutinin was observed on the cell surface ofras-transformed FR3T3 suggesting that no change in the sialylation ofO-glycan core 1 appeared in these cells, although the activity of the -2,3-sialyltransferase was decreased.Abbreviations -2,3-ST(O) CMP-Neu5Ac:Gal1-3GalNAc-R -2,3-sialyltransferase - -2,3-ST(N/O) CMP-Neu5Ac:Gal1-3/4GlcNAc-R -2,3-sialyltransferase - -2,6-ST(N) CMP-Neu5Ac:Gal1-4GlcNAc-R -2,6-sialyltransferase - -2,6-ST(O)I CMP-Neu5Ac:R-GalNAc(1-O)Ser -2,6-sialyltransferase - -2,6-ST(O)II CMP-Neu5Ac:Neu5Ac2-3Gal1-3GalNAc-R -2,6-sialyltransferase - ASFet asialofetuin - FR3T3 Fisher rat fibroblast - FRras Ha-ras-transfected FR3T3 fibroblasts - NaCl/P_i sodium phosphate 10mm, NaCl 0.15m, pH 7.4, buffer - pNp p-nitrophenol     

Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Lex determinant

A defined set of oligosaccharides and glycopeptides containing -linked fucose were used to examine the specificity of the immobilized fucose-binding lectin Lotus tetragonolobus agglutinin (LTA1), also known as lotus lectin. Glycans containing the Lewis x determinant (Lex) Gal1-4[Fuc1-3]GlcNAc1-3-R were significantly retarded in elution from high density LTA-Emphaze columns. The lectin also bound the fucosylated lacdiNAc trisaccharide GalNAc1-4[Fuc1-3]GlcNAc. The lectin did not bind glycans containing either sialylLex or VIM-2 determinants, nor did it bind the isomeric Lea, Gal1-3[Fuc1-4]GlcNAc-R. Although 2-fucosyllactose Fuc1-2Gal1-4Glc) was retarded in elution from the columns, larger glycans containing the H-antigen Fuc1-2Gal1-3(4)GlcNAc-R interacted poorly with immobilized LTA. Our results demonstrate that immobilized LTA is effective in isolating glycans containing the Lex antigen and is useful in analyzing specific fucosylation of glycoconjugates. Abbreviations: LTA, Lotus tetragonolobus agglutinin; UEA-1, Ulex europaeus agglutinin-I; LNT, AAL, Aleuria aurantia agglutinin; Gal1-3GlcNAc1-3Gal1-3Glc; LNnT, Gal1-4GlcNAc1-3Gal1-3Glc; Lex, Lewis x antigen; Lea, Lewis a antigen; GDPFuc, guanosine 5-diphosphate--L-fucose     

11 S seed storage proteins fromHelianthus species (Compositae): Biochemical,size and charge heterogeneity

The seeds of 19 sunflower species were compared on the basis of their protein contents and the relative proportions of their protein fractions. The globulin content varied from 50% to about 70% and the albumin content from 18% to 35% according to the species. The level of intermediateMr polypeptides showed a great variability (9.6 to 24.3%). Comparative studies onMr polymorphism were carried out by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of non reduced and/or reduced samples using both mono- and bidimensional procedures. Polypeptide constituents of helianthinin were compared including both number and molecular size (cultivatedH. annuus was used as a standard). Studies focused on differences observed between the major two (Mr 38 000), (Mr 32 000) and (Mr 25 500), (21 000) polypeptides families constituting the main A, B, and C subunits. and polypeptides analyses permit to discriminate easilyH. petiolaris from the other species. Charge polymorphism was studied using isoelectric focusing (IEF) and IEF-PAGE in mono and bidimensional procedures in the presence or absence of 2-mercaptoethanol (2-ME). Only a specific ₄ polypeptide enables an easy discrimination betweenH. petiolaris and all the other species. Detailed nomenclature of the , and , polypeptides constituting the different helianthinin globulin subunits is given via the results of pI andMr analyses. Monodimensional IEF patterns of the more basic albumins (pI > 8.0) appear to provide a more valuable approach to identifying specific protein markers.     

Control of glycoprotein synthesis: substrate specificity of rat liver UDP-GlcNAc:Manα3R β2-N-acetylglucosaminyl-transferase I using synthetic substrate analogues

UDP-GlcNAc: Man3R 2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) is the key enzyme in the synthesis of complex and hybrid N-glycans. Rat liver GlcNAc-T I has been purified more than 25,000-fold (M _r 42,000). TheV _max for the pure enzyme with [Man6(Man3)Man6](Man3)Man4GlcNAc4GlcNAc-Asn as substrate was 4.6 µmol min^–1 mg^–1. Structural analysis of the enzyme product by proton nuclear magnetic resonance spectroscopy proved that the enzyme adds anN-acetylglucosamine (GlcNAc) residue in 1–2 linkage to the Man3Man-terminus of the substrate. Several derivatives of Man6(Man3)Man-R, a substrate for the enzyme, were synthesized and tested as substrates and inhibitors. An unsubstituted equatorial 4-hydroxyl and an axial 2-hydroxyl on the -linked mannose of Man6(Man3)Man-R are essential for GlcNAc-T I activity. Elimination of the 4-hydroxyl of the 3-linked mannose (Man) of the substrate increases theK _M 20-fold. Modifications on the 6-linked mannose or on the core structure affect mainly theK _M and to a lesser degree theV _max, e.g., substitutions of the Man6 residue at the 2-position by GlcNAc or at the 3- and 6-positions by mannose lower theK _M, whereas various other substitutions at the 3-position increase theK _M slightly. Man6(Man3)4-O-methyl-Man4GlcNAc was found to be a weak inhibitor of GlcNAc-T I.Abbreviations BSA Bovine serum albumin - Bn benzyl - Fuc, F l-fucose - Gal, G d-galactose - GalNAc, GA N-acetyl-d-galactosamine - Glc d-glucose - GlcNAc, Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man, M d-mannose - mco 8-methoxycarbonyl-octyl, (CH₂)₈ COOOCH₃ - Me methyl - MES 2-(N-morpholino)ethanesulfonate - NMR nuclear magnetic resonance - PMSF phenylmethylsulfonylfluoride - pnp p-nitrophenyl - SDS sodium dodecyl sulfate - T transferase - Tal d-talose - Xyl d-xylose; - {0, 2 + F} Man6 (GlcNAc2Man3) Man4GlcNAc4 (Fuc6) GlcNAc - {2, 2} GlcNAc2Man6 (GlcNAc2Man3) Man4GlcNAc4GlcNAc; M₅-glycopeptide, Man6 (Man3) Man6 (Man3) Man4 GlcNAc4GlcNAc-Asn Enzymes: GlcNAc-transferase I, EC 2.4.1.101; GlcNAc-transferase II, EC 2.4.1.143; GlcNAc-transferase III, EC 2.4.1.144; GlcNAc-transferase IV, EC 2.4.1.145; GlcNAc-transferase V, UDP-GlcNAc: GlcNAc2 Man6-R (GlcNAc to Man) 6-GlcNAc-transferase; GlcNAc-transferase VI, UDP-GlcNAc: GlcNAc6(GlcNAc2) Man6-R (GlcNAc to Man) 4-GlcNAc-transferase; Core 1 3-Gal-transferase, EC 2.4.1.122; 4-Gal-transferase, EC 2.4.1.38; 3-Gal-transferase, UDP-Gal: GlcNAc-R 3-Gal-transferase; blood group i 3-GlcNAc-transferase, EC 2.4.1.149; blood group I 6-GlcNAc-transferase, UDP-GlcNAc: GlcNAc3Gal-R (GlcNAc to Gal) 6-GlcNAc-transferase.     

Continuous production of α,α-trehalose by immobilised fungal trehalose phosphorylase

Immobilisation of trehalose phosphorylase from Schizophyllum commune by adsorption onto anion-exchange materials stabilised the enzyme activity at 30°C by approx. 35-fold. Immobilised and free enzymes showed similar pH-dependence of activity but different inactivation behavior above 30°C. A fixed-bed enzyme reactor produced ,-trehalose at a stable substrate conversion of 80% with a productivity of 2.6 g l–1 h–1 for 72 h. Inhibition of trehalose phosphorylase by phosphate limited the productivity of a direct conversion of starch into ,-trehalose.     

Gene structure of the ‘large’ sialidase isoenzyme fromClostridium perfringens A99 and its relationship with other clostridialnanH proteins

Clostridium perfringens possesses two sialidase isoenzymes of different molecular weight. Almost 90% of the gene encoding the large form was found on a 3.1 kb chromosomal fragment (Sau3AI) of strain A99 by hybridization with probes developed from the N-terminal protein sequence and from commonly conserved sialidase motifs (Asp-boxes), whereas the remaining 3-terminal part was detected on a 2.1 kb fragment (Hind III) of chromosomal DNA. After combination of both fragments, the resultingE. coli clones expressed sialidase activity, the properties of the recombinant sialidase corresponding with those of the wild type enzyme. The entire chromosomal fragment of 3665 bp encompasses the complete sialidase gene of 2082 bp corresponding to 694 amino aids, from which a molecular weight of 72956 for the mature protein can be deduced. The first 41 amino acids are mostly hydrophobic and probably represent a signal peptide. The sialidase structural gene follows a non-coding region with an inverted repeat and a ribosome-binding site. Upstream from the regulatory region, another open reading frame (ORF) was detected. The 3-terminus of the sialidase structural gene is directly followed by a further ORF of unknown function, which possibly encodes a putative permease or the acylneuraminate pyruvate-lyase involved in sialic acid catabolism. The primary structure of the large isoenzyme is very similar to the sialidase ofClostridium septicum (55% identical amino acids), whereas the homology with the small form of the same species is comparatively low (26%).     

UDP-galactose:lactosylceramide α-galactosyltransferase activity in human placenta

The activity of UDP-Gal: LacCer galactosyltransferase in human placenta was studied by using crude homogenate and Triton CF-54 extract as the source of enzyme. Transfer of galactose to lactosylceramide was optimal in the presence of 0.1% Triton CF-54 and Mn²⁺ at pH 6.3, and the reaction product was susceptible to -galactosidase.Abbreviations LacCer lactosylceramide (Gal1-4Glc1-1Cer) - Gb₃ globotriaosylceramide (Gal1-4Gal1-4Glc1-1Cer) - Gb₄ globoside (GalNAc1-3Gal1-4Gal1-4Glc1-1Cer) - TLC thin-layer chromatography - GC/MS gas chromatography/mass spectrometry - NMR nuclear magnetic resonance - EDTA ethylenediamine tetraacetic acid - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate     

Essential role of the small subunit of thermostable glucose dehydrogenase from <Emphasis Type="Italic">Burkholderia cepacia</Emphasis>

The co-expression in Escherichia coli of the -subunit and the catalytic -subunit of the thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia sp. SM4 produced 12.7 U GDH activity mg^–1 protein. A 47-amino acid, twin-arginine translocase signal peptide was identified at the amino terminus of the -subunit. The expression of the -subunit in the absence of the -subunit or the -subunit signal peptide failed to produce any detectable GDH protein or activity. The -subunit may be a chaperone-like component that assists folding of the -subunit polypeptide to the active form and its translocation to the periplasm.     

Contribution to the knowledge of the enzymatic activity of yeasts of clinical interest

The determination of the enzymatic activity of the yeasts has been applied to the identification of species, specially that ofCandida albicans. In order to know its usefulness in species of clinical interest, we have tested the commercial system API ZYM (Bio Mérieux) on 500 isolated strains of different organic samples, belonging to eight genera and twenty species. All the strains showed positivity to Phosphatase alcaline, Esterase (C4), Esterase lipase (C8), Leucine arylamidase and Phosphatase acid, and negativity to Lipase (C14), Trypsin, Chymotrypsin, -galactosidase, -glucoronidase, -manosidase and -fucosidase. Fourteen enzymatic activity patterns were obtained considering the substrates with variable results for the whole of the strains: Valine arylamidase, Cystine arylamidase, Naphthol-AS-BI-phosphohydrolase, -galactosidase, -glucosidase, -glucosidase and N-acetyl--glucosaminidase. In the majority of the species, the enzymatic profile did not have very specific results since it is usually shared by more than one species.C. albicans is that which presents the greatest number of enzymatic variations, some of these are similar to those of other common clinical species, such asCandida krusei, Candida parapsilosis andCandida tropicalis. This system is proposed as a rapid method for identification and as an epidemiological marker of medically important yeasts.Abbreviations AGL -glucosidase - BGA -galactosidase - BGL -glucosidase - CAA Cystine arylamidase - NAG N.Acetyl--glucosaminidase - PHO Naphthol-AS-BI-phosphohydrolase - VAA Valine arylamidase     

Effect of photoperiod on in vitro tuberization of potato (Solanum tuberosum L.)

Single-node cuttings of potato cultivars Jemseg, Katahdin, Russet Burbank and Superior were cultured on a multiplication medium containing MS salts and no growth regulators. Cultures were exposed to 8 h (SD) and 16 h (LD) photoperiodic regimes. The subsequent plantlets were excised and single node cuttings from each photoperiodic regime were placed under SD or LD on a second medium containing growth regulators which promoted tuberization. Production of microtubers was strongly influenced by genotype and by photoperiodic treatments. Superior produced stunted plantlets and some microtubers under SD conditions in the multiplication medium. The number of microtubers formed by Jemseg was not influenced by photoperiod. However, Katahdin and Russet Burbank formed fewer microtubers under LD-LD conditions compared to LD-SD, SD-SD and SD-LD regimes. Compared with the other regimes, LD-SD photoperiod generally promoted microtuber formation with larger diameters and significantly (p<0.05) greater fresh weight. The intensity of the tuberization stimulus was affected by daylength, and this was characterized by microtubers with secondary tubers, the growth of more than one axillary microtuber, and microtubers subtended by stolons. The maturity group of the potato cultivars and photoperiodic regime in vitro strongly influenced the production of microtubers. These results can be employed to adapt light regimes for multiplication and tuberization to the specific requirements for cultivars from different maturity groups, and thus increase the efficiency of potato multiplication protocols.     

Synthesis ofNeoglycoproteins containing the 3,6-di-O-methyl-β-d-glucopyranosyl epitope and their use in serodiagnosis of leprosy

A stratagem for the synthesis ofneoglycoproteins suitable for the selective serodiagnosis of leprosy is described in which synthetic 3,6-di-O-methyl--d-glucopyranose, the epitope of phenolic glycolipid I fromMycobacterium leprae, was used. Condensation of 8-methoxycarbonyloctanol with the acetobromo derivative of 3,6-di-O-methylglucose gave 8-methoxycarbonyloctyl 2,4-di-O-acetyl-3,6-di-O-methyl--d-glucopyranoside in 65% yield, and with absolute stereospecificity for the anomer. The deacylated product was converted to the crystalline hydrazide and coupled to bovine gamma globulin, bovine serum albumin and poly-d-lysinevia intermediate acyl azide formation to produce the 8-carbonyloctyl 3,6-di-O-methyl--d-glucopyranosyl polypeptides. Theneoglycoproteins were highly sensitive in ELISA and emulated the specificity of the native glycolipid in analysis of sera from patients throughout the spectrum of leprosy and from different geographical regions. The 8-carbonyloctyl 3,6-di-O-methyl--d-glucopyranoside-bovine serum albumin was also synthesized and shown to have about one-half the activity of the -linkedneoglycoprotein. A different synthetic approach produced the 8-carbonyloctyl 4-O-(3,6-di-O-methyl--d-glucopyranosyl)--l-rhamnopyranoside-bovine serum albumin which was also highly sensitive and specific for the serodiagnosis of leprosy. The presence of the second sugar unit, similar to that in the native glycolipid but for the absence ofO-methyl groups, seemed to provide a probe with greater felicity for the serological detection of tuberculoid leprosy.Thus, the results indicate that highly sensitive and specific antigen probes for the serodiagnosis of leprosy can be constructed based only on the terminal one or two sugars of phenolic glycolipid I, and the synthetic approach leads to the formation of haptens with absolute stereospecificity.Nomenclature BGG bovine gamma globulin - PGL-I phenolic glycolipid I - PDL poly-d-lysine - PBS phophate-buffered saline - 3,6-Me₂-Glc-Link-BSA 8-carbonyloctyl 3,6-di-O-methyl-glucopyranoside-bovine senalbumin - 3,6-Me₂-Glc-Rha-Link-BSA 8-carbonyloctyl 4-O-(3,6-di-O-methyl--d-glucopyranosyl)--l-rhan pyranoside-BSA     

The inflammatory cytokines tumor necrosis factor α and interleukin-1β stimulate phosphatidylcholine secretion in primary cultures of rat type II pneumocytes

Tumor necrosis factor and interleukin-1 increase surfactant secretion in type II pneumocytes in a time- and dose-dependent manner. This stimulatory effect was additive to that of lipopolysaccharide, suggesting that cytokines and lipopolysaccharide may exert their actions through different signal transduction pathways. Tumor necrosis factor and interleukin-1 did not modify the increase on phosphatidylcholine secretion induced by the direct protein kinase C activator tetradecanoylphorbol 13-acetate, whereas this effect was inhibited by the protein kinase C inhibitors bisindolylmaleimide (2 × 10-6M) and 1-(5-isoquinolinylsulphonyl)-2-methyl piperazone (10-4M). In addition, the stimulatory effect of tumor necrosis factor and interleukin-1 was not suppressed by the intracellular Ca2+ chelator BAPTA (5 × 10-6M) or by KN-62 (3 × 10-5M), a specific inhibitor of Ca2+-calmodulin-dependent protein kinase. These results suggest that tumor necrosis factor or interleukin-1 stimulate phosphatidylcholine secretion via protein kinase C activation in a Ca2+ -independent manner.     

The degradation of amyloid beta as a therapeutic strategy in Alzheimer's disease and cerebrovascular amyloidoses

The deposition of 4-kDa amyloid peptide in the brain is a prominent feature of several human diseases. Such process is heterogeneous in terms of causative factors, biochemical phenotype, localization and clinical manifestations. Amyloid accumulates in the neuropil or within the walls of cerebral vessels, and associates with dementia or stroke, both hereditary and sporadic. Amyloid is normally released by cells as soluble monomeric-dimeric species yet, under pathological conditions, it self-aggregates as soluble oligomers or insoluble fibrils that may be toxic to neurons and vascular cells. Lowering amyloid levels may be achieved by inhibiting its generation from the amyloid -precursor protein or by promoting its clearence by transport or degradation. We will summarize recent findings on brain proteases capable of degrading amyloid with a special focus on those enzymes for which there is genetic, transgenic or biochemical evidence suggesting that they may participate in the proteolysis of amyloid in vivo. We will also put in perspective their possible utilization as therapeutic agents in amyloid diseases.     

Quantitative resistance to barley leaf stripe (Pyrenophora graminea) is dominated by one major locus

A major gene underlying quantitative resistance of barley against Pyrenophora graminea, a seedborne pathogen causing leaf stripe, was mapped with molecular markers in a barley doubled haploid (DH) population derived from the cross Proctor x Nudinka. This quantitative trait locus (QTL) accounts for r ²= 58.5% and was mapped on barley chromosome 1, tightly linked to the naked gene. A second resistance QTL accounting for 29.3% of the variation in the trait was identified on the P arm of barley chromosome 2. Another two minor QTLs were detected by further analysis. None of the QTLs was found in the barley chromosome 2 Vada region studied by Giese et al. (1993).