Isolation and characterization of a Forssman antigen-binding lectin from velvet bean (Mucuna derringiana) seeds

A Forssman antigen (GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer)-binding lectin has been purified from velvet bean (Mucuna derringiana) seeds by a combination of affinity chromatography and reversed phase HPLC. This lectin agglutinates both native and trypsin-treated sheep erythrocytes as well as trypsinized rabbit erythrocytes, but neither native rabbit nor human erythrocytes, irrespective of blood group type. SDS-PAGE and gel filtration chromatography reveal the lectin to be a homodimer consisting of two 54 kDa subunits linked by non-covalent bonds. The results obtained by quantitative precipitation, haemagglutination inhibition and TLC overlay assays indicate that theMucuna lectin specifically recognizes Forssman antigen and Forssman disaccharide (GalNAc1-3GalNAc)-related structures. Abbreviations: The abbreviations and the trivial names used are: AH, 6-aminohexyl; BSA, bovine serum albumin; Cer, ceramide; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PBS, 10mm phosphate-buffered saline, pH 7,2, containing 0.15m NaCl; PMSF, phenyl methyl sulfonyl fluoride; SDS, sodium dodecyl sulphate; TFA, trifluoroacetic acid; TBS, 20mm tris-buffered saline, pH 7.2; TLC, thin-layer chromatography; A disaccharide, GalNAc1-3Gal; A trisaccharide, GalNAc1-3[Fuc1-2]Gal; Forssman disaccharide, GalNAc1-3GalNAc; CDH (ceramide dihexoside or lactosyl ceramide) Gal1-4Glc1-1Cer (LacCer); CTH (ceramide trihexoside or globotriosyl ceramide), Gal1-4Gal1-4Glc1-1Cer (GbOse₃Cer or Gb₃); globoside (globotetraosyl ceramide), GalNAc1-3Gal1-4Gal1-4Glc1-1Cer (GbOse₄Cer or Gb₄); Forssman antigen (globopentaosyl ceramide), GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer (GbOse₅Cer).     

Spatiotemporal expression patterns of sialoglycoconjugates during nephron morphogenesis and their regional and cell type-specific distribution in adult rat kidney

The expression of 2,6- and 2,3-linked sialic acids on N-glycans was studied in embryonic, postnatal, and adult rat kidney. Histochemistry and blotting using Polyporus squamosus and Sambucus nigra lectins for 2,6-linked sialic acids and the Maackia amurensis lectin for 2,3-linked sialic acids were performed and sialyltransferase activity was assayed. N-glycans with 2,6- and 2,3-linked sialic acid were differently expressed in the two embryonic anlagen and early stages of nephron. Metanephrogenic mesenchyme was positive for 2,3-linked sialic acid but not for the 2,6-linked one, which became detectable initially in the proximal part of S-shaped bodies. Collecting ducts were positive for 2,6-linked sialic acid, whereas 2,3-linked sialic acid was restricted to their ampullae. Although positive in embryonic kidney, S1 and S2 of proximal tubules became unreactive for 2,3-linked sialic acid in postnatal and adult kidneys. In adult kidney, intercalated but not principal cells of collecting ducts were reactive for 2,3-linked sialic acid. In contrast, 2,6-linked sialic acids were detected in all cells of adult kidney nephron. Blot analysis revealed a different but steady pattern of bands reactive for 2,6- and 2,3-linked sialic acid in embryonic, postnatal, and adult kidney. Activity of 2,6 and 2,3 sialyltransferases was highest in embryonic kidney and decreased over postnatal to adult kidney with the activity of 2,6 sialyltransferase always being three to fourfold that of 2,3 sialyltransferase. Thus, 2,6- and 2,3-linked sialic acids are differently expressed in embryonic anlagen and mesenchyme-derived early stages of nephron and show regional and cell type-specific differences in adult kidney.     

β-Glucanases in developing cotton (Gossypium hirsutum L.) fibres

Cotton fibres possess several -glucanase activities which appear to be associated with the cell wall, but which can be partially solubilised in buffers. The main activity detected was that of an exo-(13)--d-glucanase (EC 3.2.1.58) but which also had the characteristics of a -glucosidase (EC 3.2.1.21). Endo-(13)--d-glucanase activity (EC 3.2.1.39) and much lower levels of (14)--d-glucanase activity were also detected. The exo-(13)--glucanase showed a maximum late on (40 days post-anthesis) in the development of the fibres, whereas the endo-(13)--glucanase activity remained constant throughout fibre development. The -glucanase complex associated with the cotton-fibre cell wall also functions as a transglucosylase introducing, inter alia, (16)--glucosyl linkages into the disaccharide cellobiose to give the trisaccharide 4-O--gentiobiosylglucose.Abbreviations CMC carboxymethylcellulose - ONPG o-nitrophenyl--d-glucopyranoside - TLC thin-layer chromatography Presented at the Third Cell Wall Meeting held in Fribourg in 1984     

Purification and characterization of flavone-specific β-glucuronidase from callus cultures of Scutellaria baicalensis Georgi

-Glucuronidase from callus cultures of Scutellaria baicalensis Georgi was purified to apparent homogeneity by fractionated ammonium-sulfate precipitation and chromatography on diethylaminoethyl-cellulose, hydroxylapatite and baicalin-conjugated Sepharose 6B. A 650-fold purification was obtained by this purification system. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified protein migrated as a single band with a molecular mass of 55 kDa. We determined that the native enzyme has a molecular mass of 230 kDa using gel-filtration chromatography. These results suggested that the enzyme exists as a homotetramer composed of four identical 55-kDa subunits. The enzyme showed a broad pH optimum between 7.0 and 8.0. The K _m values were 9 M, 10 M, 30 M and 40 M for luteolin 3 -O--d-glucuronide, baicalin, wogonin 7-O--d-glucoronide and oroxlin 7-O--d-glucuronide, respectively. The enzyme was most active with flavone 7-O--d-glucuronides.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - pI isoelectric point - R _t retention time     

Observations on the cell-wall compositions of the bracket fungi Laetiporus sulphureus and Piptoporus betulinus

Homogenized tissues and their alkali-soluble and alkali-insoluble fractions of fruiting bodies of the basidiomycetes Laetiporus sulphureus and Piptoporus betulinus were investigated using X-ray diffraction, infrared spectrometry and chemical methods. The presence of (13)--d-glucan, (13)--d-glucan and chitin was established. The relative amounts of these polysaccharides were different in the two species and differences were also found between context and trama. The proportion of (13)--d-glucan was exceptionally high in the context of L. sulphureus (about 78%). In addition, the trama of both species contained a substance resembling a cyclic wax by its X-ray pattern and solubility properties. The substances identified are considered to belong to the hyphal wall     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

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.     

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     

Production of β-fructofuranosidase byAspergillus japonicus

A newly isolated strain, MU-2, which produces very high -fructofuranosidase activity, was identified asAspergillus japonicus. For enzyme production by the strain, sucrose at 20% (w/v) was the best carbon source and yeast extract at 1.5 to 3% (w/v) the best nitrogen source. Total enzymatic activity and cell growth were at maximum after 48 h, at 1.57×10⁴ U/flask and 0.81 g dry cells/flask, respectively. The optimum pH value of the enzymatic reaction was between 5.0 and 5.5 and the optimum temperature 60 to 65°C. The enzyme produced 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose by fructosyl-transferring activity. The strain was found to be very useful for industrial production of -fructofuranosidase.     

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     

Benzyl-N-acetyl-α-d-galactosaminide inhibits the sialylation and the secretion of mucins by a mucin secreting HT-29 cell subpopulation

We have analysed the mucins synthesized by the HT-29 MTX cell subpopulation, derived from the HT-29 human colon carcinoma cells through a selective pressure with methotrexate (Lesuffleuret al., 1990,Cancer Res 50: 6334–43), in the presence of benzyl-N-acetyl--galactosaminide (GalNAc-O-benzyl), which is a potential competitive inhibitor of the 1,3-galactosyltransferase that synthesizes the T-antigen. The main observation was a 13-fold decrease in the sialic acid content of mucins after 24 h of exposure to 5mm GalNAc-O-benzyl. This effect was accompanied by an increased reactivity of these mucins to peanut lectin, testifying to the higher amount of T-antigen. The second observation was a decrease in the secretion of the mucins by GalNAc-O-benzyl treated cells. The decrease in mucin sialyation was achieved through thein situ -galactosylation of GalNAc-O-benzyl into Gal1–3GalNAc-O-benzyl, which acts as a competitive substrate of Gal1–3GalNAc 2,3-sialyltransferase, as shown by the intracellular accumulation of NeuAc2–3Gal1–3GalNAc-O-benzyl in treated cells.Abbreviations BSM bovine submaxillary mucin - MTX methotrexate - PBS sodium phosphate 10mm, NaCl 0.15m, pH 7.4 buffer - pNp p-nitrophenol - TBS Tris/HCl 10mm, NaCl 0.15m, pH 7.4 buffer Enzymes: CMP-NeuAc: Gal1–3/4GlcNAc 2,3-sialyltransferase, ST3(N), EC 2.4.99.6; CMP-NeuAc: Gal1–4GlcNAc 2,6-sialyltransferase, ST6(N), EC 2.4.99.1; CMP-NeuAc: Gal1–3GalNAc 2,3-sialyltransferase, ST3(O), EC 2.4.99.4; CMP-NeuAc: R-GalNAc1-O-Ser 2,6-sialyltransferase, ST6(O)-I, EC 2.4.99.3; CMP-NeuAc: NeuAc2–3Gal1–3GalNAc 2,6-sialyltransferase, ST6(O)-II, EC 2.4.99.7; UDP-GlcNAc: Gal1–3GalNAc-R·(GlcNAc to GalNAc) 1,6-N-acetylglucosaminyltransferase, EC 2.4.1.102; UDP-GlcNAc: GalNAc-R 1,3-N-acetylglucosaminyltransferase, EC 2.4.1.147; UDP-Gal: GalNAc-R 1,3-galactosyltransferase, EC 2.4.1.122.     

Purification and properties ofβ-fructofuranosidase from Aspergillus japonicus

-Fructofuranosidase fromAspergillus japonicus, which produces 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose, was purified to homogeneity by fractionation with calcium acetate and ammonium sulphate and chromatography with DEAE-Cellulofine and Sephadex G-200. Its molecular size was estimated to be about 304,000 Da by gel filtration. The enzyme was a glycoprotein which contained about 20% (w/w) carbohydrate. Optimum pH for the enzymatic reaction was 5.5 to 6. The enzyme was stable over a wide pH range, from pH 4 to 9. Optimum reaction temperature for the enzyme was 60 to 65°C and it was stable below 60°C. The K_m value for sucrose was 0.21m. The enzyme was inhibited by metal ions, such as those of silver, lead and iron, and also byp-chloromercuribenzoate.     

Über die Eignung von Naphthyl-α-l-fucosiden zur Untersuchung der α-l-Fucosidasen

Zusammenfassung Verglichen mit 1- und 2-Naphthyl--d-glucosid,--d-galactosid,--d-glucuronid,--d-N-acetylglucosaminid,--d-glucosid,--d-galactosid und--d-mannosid werden 1- und 2-Naphthyl--l-fucosid schneller oder im gleichen Ausmaß von Homogenaten verschiedener Rattenorgane hydrolysiert. Trotzdem fällt der histochemische Nachweis der -l-Fucosidasen methodenunabhängig im Gegensatz zu dem der anderen Glykosidasen überwiegend negativ aus. Ursache dafür ist die massive Hemmung der -l-Fucosidase durch Aldehydfixation und Diazoniumsalze; die Inhibitionsrate liegt bei 90% bzw. zwischen 85 und 98%; die - und -d-Glucosidase, - und -d-Galactosidase, -d-Mannosidase, -d-Glucuronidase sowie -d-N-Acetylglucosaminidase werden durch Aldehydfixation oder Kuppler höchstens zu 70% gehemmt. Daher können 1- und 2-Naphthyl--l-fucosid für die histochemische Darstellung der -l-Fucosidase nicht einschränkungslos empfohlen werden. Kleine Mengen Dimethylformamid hemmen die meisten Glykosidasen nicht.Für biochemische Messungen der -l-Fucosidase eignet sich speziell 1-Naphthyl--l-fucosid und läßt sich an Stelle von p-Nitrophenyl--l-fucosid werwenden. Bei der fluorometrischen Untersuchung der -l-Fucosidase in Rattenorganen mit dem 2-Naphthylderivat ergeben sich bemerkenswerte Aktivitätsunterschiede.

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Suitability of naphthyl--l-fucosides for the investigation of -l-fucosidases

Summary In comparison with 1- and 2-naphthyl -d-glucoside, -d-galactoside, -d-glucuronide, -d-N-acetylglucosaminide, -d-glucoside, -d-galactoside and -d-mannoside 1- and 2-naphthyl -l-fucoside are hydrolyzed more quickly or to the same extent by homogenates prepared from freezedried cryostate sections of various rat organs. Nevertheless, when the fucosides are employed for the histochemical demonstration of -l-fucosidase mostly negative data were obtained independent on the method used, whereas all other naphthyl glycosides deliver positive results. The reasons for these discrepancies are the marked inhibition of -l-fucosidase by aldehyde fixation and diazonium salts. Then, -l-fucosidase activity is suppressed to 90% and between 85 and 98% respectively; the inhibition of - and -d-glucosidase, - and -d-galactosidase, -d-mannosidase, -d-glucuronidase and -d-N-acetylglucosaminidase by the fixative or coupling reagent does not exceed 70%. Therefore 1- and 2-naphthyl -l-fucoside cannot be recommended in general for histochemical purposes. Small amounts of dimethylformamide do not influence the activity of most of the glycosidases investigated.For biochemical measurements, however, especially 1-naphthyl -l-fucoside represents a suitable alternative in a fluorometric procedure instead of p-nitrophenyl -l-fucoside used for the photometric evaluation of -l-fucosidase. With the fluorometric method the enzyme was measured in rat organs, which posses remarkably different activities of -l-fucosidase.

     

Physiological comparison of d-cysteine desulfhydrase of Escherichia coli with 3-chloro-d-alanine dehydrochlorinase of Pseudomonas putida CR 1-1

d-Cysteine desulfhydrase of Escherichia coli W3110 trpED102/F trpED102 was physiologically characterized. It was found to be located in the cytosolic fraction, as 3-chloro-d-alanine dehydrochlorinase is. d-Cysteine desulfhydrase catalyzed not only the ,-elimination reaction of O-acetyl-d-serine to form pyruvate, acetic acid and ammonia, but also the -replacement reaction of O-acetyl-d-serine with sulfide to form d-cysteine. However, these reactions appeared not to proceed in vivo. No other activity of d-cysteine synthesis from O-acetyl-d-serine and sulfide was detected in a crude cell extract of E. coli which was immunotitrated with antibodies raised against the purified d-cysteine desulfhydrase. Although d-cysteine desulfhydrase catalyzes the degradation (,-elimination reaction) of 3-chloro-d-alanine, which is an effective antibacterial agent, E. coli W3110 trpED102/F trpED102 did not show resistance against 3-chloro-d-alanine. Therefore, d-cysteine desulfhydrase does not contribute to 3-chloro-d-alanine detoxification in vivo.     

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

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

Genetic linkage maps of two apple cultivars (<Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">domestica</Emphasis> Borkh.) based on AFLP and microsatellite markers

Genetic linkage maps for two apple cultivars were constructed using AFLP and SSR markers and the pseudo-testcross mapping strategy. The F₁-mapping population was produced by crossing the cultivar Braeburn to the cultivar Telamon and consisted of 257 individuals. Out of the 182 AFLP primer combinations screened, a total of 48 were selected. Using these, 463 AFLP markers segregating 1:1 in the progeny were identified, of which 231 were heterozygous in Telamon and 232 in Braeburn. Eighty-five AFLP markers present in both cultivars (3:1 segregation) were scored in the whole mapping population. Twenty-one SSR primer pairs were tested, which clearly screened 23 loci (some multi-locus markers). This resulted in the identification of 3 loci heterozygous only in Telamon (1:2:1), 5 loci heterozygous only in Braeburn (1:2:1) and 15 loci which were heterozygous in both cultivars (1:1:1:1). Two linkage maps were produced. The Telamon map comprised 259 markers (242 AFLPs and 17 SSRs) divided into 17 linkage groups. The total map length was 1039 cM with a marker density of 4.0 cM. At = 0.05, 8.9% of the mapped loci showed distorted segregation. The Braeburn map consisted of 264 markers (245 AFLPs and 19 SSRs) mapped on 17 linkage groups and spanning 1245 cM. The average distance between two markers was 4.7 cM and segregation distortion was observed for 18.6% of the mapped markers ( = 0.05). Fourty-six markers common to both maps (32 AFLPs and 14 SSRs) allowed the identification of 16 homologous linkage groups. The seventeenth pair of homologous linkage groups from Telamon and Braeburn was identified by 2 SSR markers which were in common to the genetic linkage maps of Fiesta and Discovery, two other apple cultivars.     

Proliferation and germination of somatic embryos from embryogenic suspension cultures in <Emphasis Type="Italic">Phœnix dactylifera</Emphasis>

The present study demonstrates a procedure for the rapid development of a high number of somatic embryos from embryogenic suspension culture. This method might be efficient for mass propagation of Phnix dactylifera L. Embryogenic callus placed in liquid medium with 10^–5M ABA yielded an average 72 embryos per 100ml of culture medium within 2months, while those placed on solid medium yielded an average of 33, 20 and 16 embryos per 100ml of culture medium respectively for 10^–7, 10^–6 and 10^–5 M ABA after 4months. The combination of 2,4-DIchlorophenoxyacetic acid (2,4-D) (4.5×10^–7M), glutamine (6.7×10^–4M), and ABA (10^–5M) (L8 liquid medium) showed a beneficial effect on somatic embryos production compared to 2,4-D and glutamine alone, while this combination significantly (p<0.05) increased the accumulation of storage proteins (144 and 138mgg^–1 DW respectively for Jihel and Bousthami noir cultivars) in somatic embryos. The somatic embryos which underwent maturation on medium containing only 4.5×10^–7M 2,4-D and 10^–5M ABA (L6 liquid medium) accumulated more sugars (292 and 265mgg^–1 DW respectively for Jihel and Bousthami noir) than those matured on any other liquid medium. Histological studies revealed that somatic embryos (developed in L6 and L8 liquid media) accumulated less reserve compounds (proteins and sugars) than zygotic embryos. The addition of activated charcoal (0.25 and 0.5gl^–1) and phytagel^® (2.5gl^–1) to the germination medium may be useful for enhancing the germination of Phnix dactyliferasomatic embryos.     

Enzymatic β-galactosidation of β-xylopyranosides

Summary The disaccharides formed by enzymatic transfer of the -D-galactopyranosyl residue fromo-nitrophenyl -d-galactopyranoside to -d-xylopyranosides have been identified. The influence of different factors on the yields of the disaccharides obtained was evaluated. Significant changes in selectivity were observed when -galactosidase fromE. coli was used instead of -galactosidase fromA. oryzae.     

Synthesis of an H type 2 and a Y (Ley) glycoside from thioglycoside intermediates

The trisaccharide 2-(p-trifluoroacetamidophenyl)ethyl 2-acetamido-2-deoxy-4-O-[2-O-(-l-fucopyranosyl)--d-galactopyranosyl]--d-glucopyranoside 1 and the tetrasaccharide 2-(p-trifluoroacetamidophenyl)ethyl 2-acetamido-2-deoxy-3-O-(-l-fucopyranosyl)-4-O-[2-O-(-l-fucopyranosyl)--d-galactopyranosyl]--d-glucopyranoside 2 were synthesized. Thioglycosides, suitably protected, activated directly with methyl trifluoromethanesulfonate or dimethyl(methylthio)sulfonium tetrafluoroborate or activated after bromine treatment with halophilic reagents, were used as glycosyl donors in the construction of the glycosidic linkages.Abbreviations DMTSB dimethyl(methylthio)sulfonium tetrafluoroborate - Phth phthaloyl - MBn p-methoxybenzyl - ClBn p-chlorobenzyl     

Meiotic studies ofElymus nutans andE. jacquemontii (Poaceae,Triticeae) and their hybrids withPseudoroegneria spicata and seventeenElymus species

Hybridizations ofElymus nutans andE. jacquemontii were carried out with one species ofPseudoroegneria (S genome), and 20Elymus species, each containing either of the SH, SY, SYH, or SYW genomes. Chromosome configurations were analysed at metaphase I of the two target taxa and their interspecific hybrids. It is concluded that (i)E. nutans is an allohexaploid containing the SYH genomes, andE. jacquemontii is an allotetraploid having the SY genomes; (ii) the genomic affinity is associated with the geographic distance between the species studied; (iii) minor genomic structural rearrangements have occurred within the hexaploid taxon ofE. nutans.