Evidence of β-carotene 7,8(7′,8′) oxygenase (β-cyclocitral,crocetindial generating) in Microcystis

A -carotene oxygenase is described which occurs in the Cyanobacterium Microcystis. It cleaves -carotene and zeaxanthin specifically at the positions 7,8 and 7,8, while echinenone and myxoxanthophyll are not affected. The oxidative cleavage of -carotene leads to the formation of -cyclocitral and crocetindial and that of zeaxanthin to hydroxy--cyclocitral and crocetindial in nearly stoichiometric amounts. Oxidant is dioxygen as has been demonstrated by high incroporation (86%) of ¹⁸O₂ into -cyclocitral. -Carotene oxygenase is membrane bound, sensitive to sulfhydryl reagents, antioxidants and chelating agents. Iron seems to be an essential part of the enzyme activity. Cofactors necessary for the reaction could not be detected.Abbreviations TLC thin layer-chromatography - PIPES piperazine-N,N-bis-(2-ethanesulfonate) Na - TES 2{[tris-(hydroxymethyl)-methyl]-amino} ethanesulfonic acid Dedicated to Professor G. Drews on occasion of his 60th birthday     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

A comparative assessment of TLC overlay technique and microwell adsorption assay in the examination of influenza A and Sendai virus specificities towards oligosaccharides and sialic acid linkages of gangliosides

Influenza A and Sendai viruses bind toneolacto-series gangliosides isolated from human granulocytes. Differences in receptor specificity of influenza viruses A/PR/8/34 (H1N1), A/X-31 (H3N2), and parainfluenza Sendai virus (HNF1, Z-strain) were determined by two direct solid phase binding assays: the overlay technique, which combines high-resolution in the separation of gangliosides on thin-layer chromatograms with direct binding; and the microwell adsorption assay as a convenient binding assay which is performed in microtitre wells to estimate the avidity of binding to an isolated ganglioside. Both methods were applied for comparative binding studies. Viruses were found to exhibit specificity for oligosaccharides and sialic acids as well as for chain length of the neutral carbohydrate backbone, whereas differing fatty acids (C_24:1 and C_16:0) in the ceramide portion had no impact on virus adsorption. Terminal sialyloligosaccharides Neu5Ac2-3Gal1-4Glc-R of G_M3, and Neu5Ac2-3Gal1-4GlcNAc-R as well as Neu5Ac2-6Gal1-4GlcNAc-R ofneolacto-series gangliosides with nLcOse₄Cer and nLcOse₆Cer backbone, exhibited significant specific receptor activity towards the different viruses. To compare the data revealed from both test systems, values of virus binding were ascertained by a non-parametric statistical approach based on rank correlation. The rank correlation coefficientr _s was calculated according to Spearman from each virus binding towards G_M3, IV³Neu5Ac-nLcOse₄Cer, IV⁶Neu5Ac-nLcOse₄Cer and VI³Neu5Ac-nLcOse₆SCer. The rank correlation coefficients 0.74, 0.95 and 0.92, which were determined for A/PR/8/34 (H1N1), A/X-31 (H3N2) and Sendai virus (HNF1, Z-strain), respectively, indicated that both assays generate highly correlated experimental data. Based on these results, analyses of virus binding on thin-layer chromatograms as well as in microwells were found equivalent tools for ganglioside receptor studies. Abbreviations: BSA, bovine serum albumin; GSL(s), glycosphingolipids; HPTLC, high performance thin-layer chromatography; PBS, phosphate buffered saline; Neu5Ac,N-acetylneuraminic acid [35];r _s = rank correlation coefficient according to Spearman. The designation of the glycosphingolipids follows the IUPAC-IUB recommendations [36]. LacCer or lactosylceramide, Gal1-4Glc1-1Cer; lacto-N-neotetraosylceramide or nLcOse₄Cer, Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; lacto-N-norhexaosylceramide or nLcOse₆Cer, Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; G_M3 (according to Svennerholm [37]) or II³Neu5AcLacCer.     

Gibberellins play a role in the interaction between imidazole fungicides and cytokinins in Araceae

Imidazole fungicides such as imazalil, prochloraz, and triflurnizole and the triazole growth retardant paclobutrazol promote the shoot-inducing effect of exogenous cytokinins in Araceae, such as Spathiphyllum floribundum Schott and Anthurium andreanum Schott. The mechanism of their action could partially be based on the inhibition of gibberellic acid (GA) biosynthesis, because administration of GA₃ inhibits the phenomenon completely in S. floribundum. Not only is the suppression of GA biosynthesis involved, but also the metabolism of endogenous cytokinins is significantly altered. Although the balance between isopentenyladenine, zeatin, dihydrozeatin, and their derivatives was shifted to distinguished directions by administration of BA and/or imazalil and/or GA₃, no correlation between these changes in metabolic pathways and the number of shoots could be found. The metabolism of BA was not significantly altered by adding imazalil to the micropropagation medium of S. floribundum.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - [9R-5P]DHZ 9--d-ribofuranosyl-dihydrozeatin-monophosphate - [9R-5P]iP 6-isopentenyl-9--d-ribofuranosyladenine-monophosphate - [9R-5P]Z 9--d-ribofuranosyl-zeatin-monophosphate - [9G]BA 6-benzyl-9--d-glucopyranosyladenine - [9G]DHZ 9--d-glucopyranosyl-dihydrozeatin - [9G]iP 6-isopentenyl-9--d-glucopyranosyladenine - [9G]Z 9--d-glucopyranosyl-zeatin - [9R]BA 6-benzyl-9--d-ribofuranosyladenine - [9R]DHZ 9--d-ribofuranosyl-dihydrozeatin - [9R]iP 6-isopentenyl-9--d-ribofuranosyladenine - [9R]Z 9--d-ribofuranosyl-zeatin - BA 6-benzyladenine - DHZ dihydrozeatin - ES⁺ LC-MS/MS HPLC coupled Electrospray Tandem Mass Spectrometry - f.m. fresh mass - mT 6-(3-hydroxybenzyl)adenine - IMA imazalil - iP isopentenyladenine - NAA 1-naphthalene acetic acid - NFT Nutrient Film Technique - (OG)[9R]DHZ O--glucopyranosyl-9--d-ribofuranosyl-dihydrozeatin - (OG)[9R]Z O--d-glucopyranosyl-9--d-ribofuranosyl-zeatin - (OG)DHZ O--d-glucopyranosyl-dihydrozeatin - (OG)Z O--d-glucopyranosyl-zeatin - PAR Photosynthetic Active Radiation - PBZ paclobutrazol - PRO prochloraz - TDZ thidiazuron - TRI triflurnizole - Z zeatin     

Identification of beta subunit of the rhesus monkey chorionic gonadotropin (rmCG)

Chorionic gonadotropin (CG) is a placental derived hormone that plays a crucial role in successful implantation and establishment of early pregnancy in the primates. The rhesus monkey was chosen as a model to understand the feasibility of developing human DNA immuno-contraceptive. The coding region of rhesus monkey CG -subunit (rmCG) was isolated by the TDRT-PCR method. The nucleotide sequence including the leader peptide was 499 nucleotide long and encoded 166 amino acids. In comparing with the previous known primates CG -subunits, the rmCG was the highest degree of homology with baboon CG -subunit at the deduced amino acid sequence (94%), 79.5% homology with human CG -subunit and 70.4% homology with marmoset monkey CG -subunit. The eukaryotic expression vector pCMV4-rmCG inserted full-coding cDNA sequence of rmCG was constructed, and the expression of rmCG -subunit in HeLa cells transient expressing system in vitro and BALB/c mice in vivo was determined. The results demonstrated that the recombinant PCMV4-rmCG eukaryotic expression vector could express rmCG -subunit in vitro and in vivo.     

Abnormal subcellular distribution of β-glucuronidase in mice with a genetic alteration in enzyme structure

Liver -glucuronidase is structurally altered in inbred strain PAC so that a peptide subunit with a more basic isoelectric point, GUS-SN, is produced. This allele of -glucuronidase was transferred to strain C57BL/6J by 12 backcross matings to form the congenic line B6 · PAC-Gus ⁿ. Liver -glucuronidase activity was halved in males of the congenic strain compared to normal males. The lowered activity was specifically accounted for by a decrease in the lysosomal component. There was no alteration in the concentration of microsomal activity. This alteration in the subcellular distribution of -glucuronidase in Gus ⁿ/Gus ⁿ mice was confirmed by two independent gel electrophoretic systems which separate microsomal and lysosomal components. -Glucuronidase activity was likewise approximately halved in mutant spleen, lung, and brain, organs which contain exclusively or predominantly lysosomal -glucuronidase. The loss of liver lysosomal -glucuronidase activity was shown by immunotitration to be due to a decrease in the number of -glucuronidase molecules in lysosomes of the congenic strain. The Gus ⁿ structural alteration likely causes the lowered lysosomal -glucuronidase activity since the two traits remain in congenic animals. Heterozygous Gus ⁿ/Gus ^b animals had intermediate levels of liver -glucuronidase. Also, the effect was specific, in that three other lysosomal enzymes were not reproducibly lower in Gus ⁿ/Gus ⁿ mice. Gus ⁿ is, therefore, an unusual example of a mutation which causes a change in the subcellular distribution of a two-site enzyme.This work was supported by National Institutes of Health Grants GM-33559 and GM-33160 and National Science Foundation Grant PCM-8215808.     

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     

Theβ-subunit of human chorionic gonadotropin containsN-glycosidic trisialo tri- and tri′-antennary carbohydrate chains

TheN-linked carbohydrate chains of the-subunit of highly purified urinary human chorionic gonadotropin have been re-investigated. The oligosaccharides were released enzymatically by peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F, and fractionated by a combination of FPLC and HPLC. As a result of the application of improved fractionation methods, apart from the earlier reported carbohydrate chains, also small amounts of trisialo tri- and tri-antennary oligosaccharides were found. The primary structures of the latter carbohydrate chains have been determined by 500-MHz¹H-NMR spectroscopy to beAbbreviations hCG human chorionic gonadotropin - hCG- -subunit - hCG- -subunit - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F (E.C. 3.5.1.52) - endo-F endo--N-acetylglucosaminidase-F (E.C. 3.2.1.96) - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - CBB coomassie brilliant blue R 250 - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - Man mannose - Gal galactose - Fuc fucose     

Genetic transformation of alfalfa somatic embryos and their clonal propagation through repetitive somatic embryogenesis

Genetically transformed alfalfa (Medicago sativa L., cv. Zajearska 83) plantlets were obtained by inoculating somatic embryos with Agrobacterium tumefaciens strains A281/pGA472 and LBA4404/pBI121. Single somatic embryos, 5–7 mm long, were released from a repetitively embryogenic culture, wounded, and cocultivated with the bacteria. The agar-solidified culture medium contained mineral salts, vitamins, 40 g l^–1 sucrose, 1 g l^–1 yeast extract and 0.05 mg l^–1 BA. Five clones, transformed with A281/pGA472, and 4 clones transformed with LBA4404/pBI121, were selected for proliferation by repetitive somatic embryogenesis, on media containing 100 mg l^–1 of kanamycin. The transformation of kanamycin-resistant clones was confirmed by assaying the activity of neomycin phosphotransferase II and/or -glucuronidase enzymes, and by the Southern blot analysis. It is suggested that the transformation/regeneration system based on somatic embryogenesis may be suitable for establishing transgenic alfalfa lines. The relatively low frequency of embryo transformation is compensated for by abundant proliferation in secondary somatic embryogenesis.Abbreviations BA 6-benzyladenine - GUS -glucuronidase - Km kanamycin - NPTII neomycin phosphotransferase II - X-gluc 5-bromo-4-chloro-3-indolyl--glucuronic acid - BM basal medium     

Evidence for non-proteinaceous inhibitor(s) of β-glucuronidase in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>L.) leaf and root tissues

The GUS gene of E. coli, encoding -glucuronidase, has been widely used as a reporter gene in plant transformation. However, -glucuronidase activity in transgenic wheat leaf or root tissue is rarely observed or reported. To address this question, we investigated three wheat lines transformed with the GUS reporter gene. We found all three lines expressed GUS mRNA as well as -glucuronidase protein in their leaf and root tissues as detected by RNA gel blot, ELISA, and immunoblot analyses. However, -glucuronidase enzyme activity was only detected in pollen grains from the transgenic plants. Fluorometric and histochemical assays performed in the presence of wheat tissue extracts indicated that wheat leaf and root tissues contain inhibitor(s) of -glucuronidase activity, but pollen grains contain much lower concentrations. Further characterizations indicated that the inhibitor(s) is of low molecular weight (<10 kDa) and is non-proteinaceous.     

Oxygen free radicals induced release of lysosomal enzymes in vitro

Summary The effect of oxygen free radicals, generated by xanthine and xanthine oxidase, was studied on the release of lysosomal hydrolase from rat liver lysosomes in vitro. A lysosomal enriched subcellular fraction was prepared, using differential centrifugation technique, from the homogenate of rat liver. The biochemical purity of the lysosomal fraction was established by using the markers of different cellular organelles. Oxygen free radicals were generated in vitro by the addition of xanthine and xanthine oxidase. The release of lysosomal hydrolase (-glucuronidase) from the lysosomal fraction was measured. There was a 3 to 4 fold increase in the release of -glucuronidase activity in the presence of xanthine and xanthine oxidase when compared to that in the absence of xanthine and xanthine oxidase. In the presence of superoxide dismutase (SOD), a scavenger of oxygen free radicals, the xanthine and xanthine oxidase system was unable to induce the release of -glucuronidase activity from the lysosomes. Sonication (2 bursts for 15 sec each) and Lubrol (2 mg/10 mg lysosomal protein) treatment, which are known to cause membrane disruption, also induced the release of -glucuronidase from lysosomal fraction. This release of -glucuronidase by sonication and lubrol treatment was not prevented by SOD. These data indicate that lysosomal disruption is a consequence of oxygen free radicals, generated by xanthine and xanthine oxidase.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EGTA Ethylene Glycol Bis-(-aminoethyl ether)N,N,-N,N-tetracetic acid - Tris Tris (hydroxymethyl) aminomethane - SOD Superoxide Dismutase     

Molecular screening and fetal diagnosis of β-thalassemia in the Italian population

Summary This paper reports our experience of molecular screening and fetal diagnosis of -thalassemia in 457 at risk couples of Italian descent. Molecular screening was carried out by dot blot analysis on amplified DNA with oligonucleotide probes complementary to the eight most common mutations in Italians [39 (CT); 6 (-A); ⁺-87 (CG); ⁺ IVSI nt 110 (GA); IVSI nt 1 (GA); ⁺ IVSI nt 6 (TC); IVSII nt 1 (GA); ⁺ IVSII nt 745 (CG)]. By using this approach, we have been able to define the mutation in 92.8% of cases. The rest (all but four) were defined by direct sequencing and this led to the detection of nine rare mutations [76 (-C); ⁺ IVSI nt 5 (GA); ⁺ IVSI nt 5 (GC); ⁺ IVSI -1 (cod 30) (GC); ⁺-87 (CT), -290 bp del.; ⁺-101 (CT)], and to the characterization of a novel mutation consisting of the deletion of the G at the invariant AG of the IVSII splice acceptor site of the -globin gene ( IVSII nt 850-1 bp). In the remaining four cases, the -globin gene showed entirely normal sequences and the -globin gene cluster was intact, as indicated by Southern blot analysis. Fetal diagnosis was carried out by dot blot analysis with the oligonucleotide probes defined in the parents. The procedure is simple and reliable, and the results can be obtained within 1 week of sampling. No misdiagnosis has so far occurred. The results indicate that fetal diagnosis of -thalassemia by DNA analysis may be obtained in practically all cases (even in a population showing marked heterogeneity of -thalassemia) by the combination of dot blot analysis for detecting common mutations, and direct sequencing for defining those that are uncommon.     

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.     

Detection and partial characterization of two antigenically distinct β-amylases in developing kernels of wheat

A -amylase (EC 3.2.1.2) was identified in the outer pericarp (P) of developing seeds of wheat (Triticum aestivum L.) and compared with the well known -amylase which is synthesized during seed development in the starchy endosperm (E). The enzyme P already exists in the tissues before anthesis and vanishes at the time when E starts to accumulate. The isoelectric-focusing patterns of P and E are very similar. The relative molecular weight (M_r) of P is slightly higher than that of E (66 and 64.5 kDa, respectively). Both P and E exhibit common epitopes in addition to epitopes specific for each of them. The two enzymes were identified in small amounts in the green tissues of the developing seeds (inner pericarp and testa). No antigenic difference was detected between P and the -amylases of roots and leaves.Abbreviations P pericarp -amylase - E endosperm -amylase - IS1 anti--amylase immune serum - IS2 anti- and anti- amylase immune serum - IS3 anti- amylase immune serum - IEF isoelectric focusing - IgG immunoglobulin G The authors thank Dr. P. Ziegler (Universität Bayreuth, FRG) for stimulating discussion and for useful suggestions during the writing of the text. The authors thank Miss C. Mayer for her skillful technical assistance.     

Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting gene transfer efficiency during early transformation steps

The factors influencing transfer of an intron — containing -glucuronidase gene to apple leaf explants were studied during early steps of an Agrobacterium tumefaciens-mediated transformation procedure. The gene transfer process was evaluated by counting the number of -glucuronidase expressing leaf zones immediately after cocultivation, as well as by counting the number of -glucuronidase expressing calli developing on the explants after 6 weeks of postcultivation in the presence of 50 mg/l kanamycin. Of three different tested disarmed A. tumefaciens strains, EHA101(pEHA101) was the most effective for apple transformation. Cocultivation of leaf explants with A. tumefaciens on a medium with a high cytokinin level was more conducive to gene transfer than cocultivation on media with high auxin concentrations. Precultivation of leaf explants, prior to cocultivation, slightly increased the number of -glucuronidase expressing zones measured immediately after cocultivation, but it drastically decreased the number of transformed calli appearing on the explants 6 weeks after infection. Other factors examined were: Agrobacterium cell density during infection, bacterial growth phase, nature of the carbon source, explant age, and explant genotype.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - CaMV35S 35S RNA of cauliflower mosaic virus - EDTA ethylenediaminetetraacetate - FeNaEDTA ethylenediaminetetraacetate ferric-sodium salt - GusA -glucuronidase - gusA ß-glucuronidase gene of Escherichia coli - gusA-intron ß-glucuronidase gene containing an intron in the coding region - IBA indole butyric acid - 2iP N6-2-isopentenyl adenine - NAA naphthaleneacetic acid - nptII neomycinphosphotransferase II gene - X-Gluc 5-bromo-4-chloro-3-indolyl ß-D-glucuronide     

Transient expression of the β-glucuronidase gene introduced into barley coleoptile cells by microinjection

A -glucuronidase gene was introduced directly into barley (Hordeum vulgare L. cv. Kobinkatagi) coleoptile cells by microinjection and transient expression of the gene was examined. Inner epidermis tissue of coleoptiles was excised and injected with plasmid DNA, pBI221, carrying cauliflower mosaic virus 35S promoter, -glucuronidase gene, and a nopaline synthase polyadenylation region. Histochemical assay for -glucuronidase production showed positive enzyme activity only in coleoptile cells injected with plasmid DNA. Expression of the -glucuronidase gene was examined chronologically using honogenates of injected coleoptile tissues. Glucuronidase activity first appeared after 6 hr, reached the maximum level 24 hr after injection, and decreased afterwards. These results suggest that microinjection of coleoptile tissues may be a useful approach for the genetic engineering of Gramineae plants in which protoplast regeneration is difficult.     

d-Xylose as inducer of the xylan-degrading enzyme system in the yeast Pullularia pullulans

Summary The specificity of induction of wooddegrading enzymes from Pullularia pullulans was investigated using series of mono-, di- and (14)--trisaccharides or glycanes. A strain of P. pullulans (1740), unable to grow on Avicel or carboxymethyl-cellulose (CMC), uses xylan and steamexploded wood as carbon sources. This strain, thus grown, was evaluated for various enzyme activities. d-Xylose was the nutritional inducer of -xylosidase and -xylanase. d-Glucuronic acid induced activity on CMC and -glucosidase activity was observed regardless of carbon source used. (14)--Xylobiose was not an inducer of -xylanase production, but high levels of this enzyme were obtained with either structural isomers (12) or (13)-. Since synthesis of this enzyme was stimulated by increasing xylose concentration yp to 40 g/l, it is suggested that xylose enters the cells by passive transport and is unable to induce a permease system.Affiliated to the Scientific, Technological and Medical University of Grenoble     

Enzymic activities and galactomannan mobilisation in germinating seeds of fenugreek (Trigonella fœnum-graecum L. Leguminosae)

Summary The activities of -galactosidase, -mannosidase and -mannosidase were determined in extracts from the endosperm and from the embryo of fenugreek seeds at different stages of germination. Endosperm homogenates contained little or no activity of the above enzymes in the early stages of germination, before the reserve galactomannan began to be mobilised. The onset of galactomannan breakdown coincided with the appearance of -galactosidase and -mannosidase activities, which increased throughout the period of galactomannan degradation and then remained constant. A similar rise in -galactosidase and -mannosidase activities occurred during galactomannan breakdown in dry-isolated endosperms incubated under germination conditions. The increase could be suppressed by metabolic inhibitors which also inhibit galactomannan breakdown. Embryo homogenates contained high -galactosidase, high -mannosidase and some -mannosidase activity at all stages of germination.No oligomannosyl -1,4 phosphorylase activity could be detected either in the endosperm or in the embryo.It is concluded that the galactomannan of fenugreek is broken down by a series of hydrolases secreted by the aleurone layer of the endosperm. They include -galactosidase, -mannosidase and probably also endo--mannanase.This is part four in a series of papers dealing with galactomannan metabolism. Part three: Planta (Berl.) 106, 44–60 (1972).     

Primary structure of the marmoset (Saguinus fuscicollis) hemoglobin. I. Use of tryptic maleylated peptides in the solubilization and sequence elucidation of the α- and β-chains

The primary structure of adult marmoset hemoglobin has been determined. The - and -chains of HbA were separated on a CM23 column in 8 M urea using a sodium phosphate gradient. Tryptic digests of the - and -chains were fractionated on a Dowex 50W-X2 column using a pH and pyridine acetate gradient. Large peptide fragments were obtained by the cyanogen bromide cleavage of the - and -chains, as well as by tryptic digestion of the maleylated - and -chains. The sequence was derived from the amino acid compositions and sequences of the individual tryptic peptide, automated sequence determination of intact - and -chains, as well as automated sequence determination of cyanogen bromide fragments and tryptic maleylated peptides derived from the - and -chains. The complete structure of marmoset adult hemoglobin is closely homologous to that of other primate hemoglobins. The sequence of the marmoset -chain differs from the -chain of human HbA at positions 8, 19, 23, 68, and 116. The -chain from marmoset HbA differs from the -chain of human HbA at positions 5, 13, 21, 50, 87, and 125.This work was supported in part by funds from a Physicians' Medical Education and Research Foundation Grant of the University of Tennessee Memorial Research Hospital and by NIH General Research Support Grant FR-5541 to the institution.