A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C₁₈ cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar G_M3-ganglioside is simple and rapid relative to previously described methods. Recovery of G_M3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (G_M3 synthase; ST-1), the transfer of [¹⁴C] sialic acid from CMP-[¹⁴C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations G_M3 G_M3-ganglioside - II³NeuAc-LacCer NeuAc2-3Gal1-4Glc1-1Cer - G_D1a G_D1a-ganglioside, IV³NeuAc, II³NeuAc-GgOse₄Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer - G_D3 G_D3-ganglioside, II³(NeuAc)₂LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer - GgOse₄Cer asialo-G_M1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer - FucG_MI fucosyl-G_MI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer - ST-1 G_M3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase - LacCer lactosylceramide, Gal1-4Glc1-1Cer - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride     

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

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

A Cytogenetic Study of the Leaf Beetle Genus Cyrtonus (Coleoptera, Chrysomelidae)

The chromosomes of ten species of Cyrtonus and the genome sizes of six are surveyed. Among the total of 15 chromosomally studied species, 11 have 2n=28 chromosomes and a 13+Xy_p male meioformula, three have 2n=40 and 19+Xy_p and one 2n=46 and 22+Xy_p. All but one species with 28 chromosomes show only metacentric or submetacentric chromosomes, whereas the species with 40 and 46 chromosomes display some telocentrics or subtelocentrics, that are probably derived from the former by centric fissions. However, since the number of major chromosome arms is strikingly higher in these latter species (NF=70 and 78) than in the 28-chromosome species (mostly NF=56), other chromosomal rearrangements such as pericentric inversions or heterochromatin accretions could also be involved. The genome sizes display a narrow range, from 1C=0.6–1.22pg, and they are not significantly correlated with the chromosome numbers. Some possible factors implied in the rough chromosomal evolution of Cyrtonus are discussed in relation to a few other genera of the subfamily Chrysomelinae.     

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 Distance in Housekeeping Genes Between <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> and <Emphasis Type="Italic">Plasmodium reichenowi</Emphasis> and Within <Emphasis Type="Italic">P. falciparum</Emphasis>

The time to the most recent common ancestor of the extant populations of Plasmodium falciparum is controversial. The controversy primarily stems from the limited availability of sequences from Plasmodium reichenowi, a chimpanzee malaria parasite closely related to P. falciparum. Since the rate of nucleotide substitution differs in different loci and DNA regions, the estimation of genetic distance between P. falciparum and P. reichenowi should be performed using orthologous sequences that are evolving neutrally. Here, we obtained full-length sequences of two housekeeping genes, sarcoplasmic and endoplasmic reticulum Ca²⁺-ATPase (serca) and lactate dehydrogenase (ldh), from 11 isolates of P. falciparum and 1 isolate of P. reichenowi and estimate the interspecific genetic distance (divergence) between the two species and intraspecific genetic distance (polymorphism) within P. falciparum. Interspecific distance and intraspecific distance at synonymous sites of interspecies-conserved regions of serca and ldh were 0.0672±0.0088 and 0.0011±0.0007, respectively, using the Nei and Gojobori method. Based on the ratio of interspecific distance to intraspecific distance, the time to the most recent common ancestor of P. falciparum was estimated to be (8.30±5.40) × 10⁴ and (11.62±7.56) × 10⁴ years ago, assuming the divergence time of the two parasite species to be 5 and 7 million years ago, respectively.This article contains an online supplementary table.Reviewing Editor: Dr. Martin Kreitman     

The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update

Mitochondrial dysfunction has been shown to participate in the induction of apoptosis and has even been suggested to be central to the apoptotic pathway. Indeed, opening of the mitochondrial permeability transition pore has been demonstrated to induce depolarization of the transmembrane potential (_m), release of apoptogenic factors and loss of oxidative phosphorylation. In some apoptotic systems, loss of _m may be an early event in the apoptotic process. However, there are emerging data suggesting that, depending on the model of apoptosis, the loss of _m may not be an early requirement for apoptosis, but on the contrary may be a consequence of the apoptotic-signaling pathway. Furthermore, to add to these conflicting data, loss of _m has been demonstrated to not be required for cytochrome c release, whereas release of apoptosis inducing factor AIF is dependent upon disruption of _m early in the apoptotic pathway. Together, the existing literature suggests that depending on the cell system under investigation and the apoptotic stimuli used, dissipation of _m may or may not be an early event in the apoptotic pathway. Discrepancies in this area of apoptosis research may be attributed to the fluorochromes used to detect _m. Differential degrees of sensitivity of these fluorochromes exist, and there are also important factors that contribute to their ability to accurately discriminate changes in _m.     

Change in the rate of organelle movement during progression of the cell cycle inAdiantum protonemata

Summary The rate of organelle movement during progression of the cell cycle in single-celled protonemata of the fernAdiantum capillus-veneris is determined microscopically with a time-lapse video system. Under red light organelle movement is very slow (1.8 m/min) in early G₁ in the apical 100-m region. The rate of organelle movement becomes higher in proportion to distance from the nuclear region, reaching a plateau in the neighborhood of 300 m from the tip. Organelle movement during the progression of G₁ and S phases in the dark does not show a significant difference from that in early G₁ under red light. In M phase, however, organelle movement in the nuclear region slows down a few minutes after nucleolar disappearance and then stops until the beginning of cell plate formation. Organelle movement in the basal region of the protonema slows down, but does not stop, shortly after movement in the nuclear region has ceased. This indicates that a message is sent from the nuclear region to the basal region.     

The Synthesis and Antiviral Activity of Glycyrrhizic Acid Conjugates with α-D-Glucosamine and Some Glycosylamines

Glycyrrhizic acid and its 30-methyl ester were conjugated with 2-amino-1,3,4,6-tetra-O-acetyl-2-deoxy--D-glucopyranose, 2,3,4,6-tetra-O-acetyl--D-glucopyranosyl amine, 2,3,4-tri-O-acetyl--L-arabinopyranosyl amine, 2-acetamido-2-deoxy--D-glucopyranosyl amine, and -D-galactopyranosyl amine using N,N-dicyclohexylcarbodiimide and its mixtures with N-hydroxybenzotriazole. Structures of the conjugates were confirmed by IR, UV, ¹H, and ¹³C NMR spectroscopy. The glycoconjugate with the residues of 2-acetamido-2-deoxy--D-glucopyranosyl amine in the carbohydrate part of its molecule exhibited antiviral activity (ID₅₀ 4 g/ml) toward the herpes simplex type 1 virus (HSV-1) in the VERO cell culture. Two compounds demonstrated anti-HIV-1 activity (50–70% inhibition of p24) in a culture of MT-4 cells at concentrations of 0.5–20 g/ml.     

The genetics of adult-plant blackleg (Leptosphaeria maculans) resistance from Brassica juncea in B. napus

The genetic control of adult-plant blackleg (Leptosphaeria maculans) resistance in a Brassica napus line (579NO48-109-DG-1589), designated R13 possessing Brassica juncea-like resistance (JR), was elucidated by the analysis of segregation ratios in F₂ and F₃ populations from a cross between R13 and the highly blackleg-susceptible B. napus cultivar Tower. The F₂ segregration ratios were bimodal, demonstrating that blackleg resistance in R13 was controlled by major genes. Analysis of the segregation ratios for 13 F₃ families indicated that blackleg resistance in these families was controlled by three nuclear genes, which exhibited a complex interaction. Randomly sampled plants of F₃ progeny all had the normal diploid somatic chromosome number for B. napus. The similarities between the action of the three genes found in this study with those controlling blackleg resistance in B. juncea is discussed.     

The Effects of Hypoxia on Three Sympatric Shark Species: Physiological and Behavioral Responses

Behavioral and physiological responses to hypoxia were examined in three sympatric species of sharks: bonnethead shark Sphyrna tiburo, blacknose shark, Carcharhinus acronotus, and Florida smoothhound shark, Mustelus norrisi, using closed system respirometry. Sharks were exposed to normoxic and three levels of hypoxic conditions. Under normoxic conditions (5.5–6.4mg l^–1), shark routine swimming speed averaged 25.5 and 31.0cm s^–1 for obligate ram-ventilating S. tiburo and C. acronotus respectively, and 25.0cm s^–1 for buccal-ventilating M. norrisi. Routine oxygen consumption averaged about 234.6 mg O₂kg^–1h^–1 for S. tiburo, 437.2mg O₂kg^–1h^–1 for C. acronotus, and 161.4mg O₂ kg^–1 h^–1 for M. norrisi. For ram-ventilating sharks, mouth gape averaged 1.0cm whereas M. norrisi gillbeats averaged 56.0 beats min^–1. Swimming speeds, mouth gape, and oxygen consumption rate of S. tiburo and C. acronotus increased to a maximum of 37–39cm s^–1, 2.5–3.0cm and 496 and 599mg O₂ kg^–1 h^–1 under hypoxic conditions (2.5–3.4mg l^–1), respectively. M. norrisi decreased swimming speeds to 16cm s^–1 and oxygen consumption rate remained similar. Results support the hypothesis that obligate ram-ventilating sharks respond to hypoxia by increasing swimming speed and mouth gape while buccal-ventilating smoothhound sharks reduce activity.     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.     

Methanothrix soehngenii gen. nov. sp. nov., a new acetotrophic non-hydrogen-oxidizing methane bacterium

A new genus of methanogenic bacteria is described, which was isolated from a mesophilic sewage digester. It is most probably the filamentous bacterium, earlier referred to asMethanobacterium soehngenii, fat rod or acetate organism. The single non-motile, non-sporeforming cells are rod-shaped (0.8×2 m) and are normally combined end to end in long filaments, surrounded by a sheath-like structure. The filaments form characteristic bundles.Methanothrix soehngenii decarboxylates acetate, yielding methane and carbon dioxide. Other methanogenic substrates (H₂–CO₂, formate, methanol, methylamines) are not used for growth or methane formation. Formate is split into hydrogen and carbon dioxide. The temperature optimum for growth and methane formation is 37°C and the optimal pH range is 7.4–7.8. Sulfide and ammonia serve as sulfur and nitrogen source respectively. Oxygen completely inhibits growth and methane formation, but the bacteria do not loose their viability when exposed to high oxygen concentrations. 100 mg/l vancomycin showed no inhibition of growth and methanogenesis. No growth and methane formation was observed in the presence of: 2-bromoethanesulfonic acid, viologen dyes, chloroform, and KCN. The bacterium has a growth yield on acetate of 1.1–1.4 g biomass per mol acetate. The apparent K _S of the acetate conversion system to methane and carbon dioxide is 0.7 mmol/l. The DNA base composition is 51.9 mol% guanine plus cytosine. The nameMethanothrix is proposed for this new genus of filamentous methane bacterium. The type species,Methanothrix soehngenii sp. nov., is named in honor of N. L. Söhngen.     

Association of gangliosides to fibroblasts in culture: A study performed with GM1 [14C]-labelled at the sialic acid acetyl group

The preparation of a G_M1-ganglioside (GM1) [¹⁴C]-labelled in the sialic acid residue is reported. This can be obtained by re-N-acetylation in the presence of [1-¹⁴C]-acetic anhydride, of a GM1 derivative de-N-acetylated specifically on the sialic acid residue by alkaline hydrolysis of GM1 with tetramethylammonium hydroxide. The radiolabelled GM1 is utilized to investigate the binding properties and the mode of interaction of GM1 with cultured fibroblasts. Three different forms of association (one serum-removable, one trypsin-removable and one trypsin-stable) have been recognized to occur in a way that depended on cell culture conditions (presence or absence of fetal calf serum), ganglioside concentration (from, 5×10^–9 M to 10^–4 M) and incubation time (up to 24 h). Some metabolic modifications of GM1 during the period of high cell viability were also investigated.Abbreviations GM1 G_M1-ganglioside, II³NeuAc-GgOse₄Cer - FCS fetal calf serum - EMEM Eaglés Minimum Essential Medium with Earlés salts - PBS Dulbecco phosphate buffered saline without calcium and magnesium     

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     

Ganglioside lactones:1H-NMR determination of the inner ester position of GD1b-ganglioside lactone naturally occurring in human brain or produced by chemical synthesis

The complete definition of the chemical structure of G_D1b-ganglioside (G_D1b) lactone isolated from human brain has been given by means of spectrometric and spectroscopic analyses. G_D1h lactone contains a single ester linkage involving the external sialic acid carboxyl group and the C-9 hydroxyl group of the internal sialic acid unit. A synthetic lactone of G_D1b prepared treating G_D1b with glacial acetic acid characterized in the same way showed an identical chemical structure.Abbreviations: Ganglioside nomenclature is according to Svennerholm [16] and the IUPAC-IUB Recommendations [17] G_M1 G_M1-ganglioside, II³NeuAc-GgOse₄Cer, Gal1-3GalNac1-4[NeuAc2-3]Gal1-4Glc1-1Cer - G_D1b G_D1b-ganglioside, II³(NeuAc)₂GgOse₄Cer, Gal1-3GalNAc1-4[NeuAc2-8NeuAc2-3]Gal1-4Glc1-1Cer - G_D1b lactone G_D1b-L, Gal1-3GalNAc1-4[NeuAc(1-9)2-8NeuAc2-3]Gal1-4Glc1-1Cer - Cer ceramide - FAB-MS fast atom bombardment-mass spectrometry - ¹H-NMR proteon nuclear magnetic resonance - 1D-NMR one dimensional NMR - 2D-COSY two dimensional correlated spectroscopy - DMSO-d₆ deuterated dimethylsulfoxide     

Ultrastructural localization of corticotropin, β-lipotropin,and αand β-endorphin in the porcine anterior pituitary

Summary The peroxidase-antiperoxidase immunocytochemical technique was used to identify the ACTH/endorphin cells in the porcine pituitary at the ultrastructural level and to determine the precise subcellular localization of the pro-ACTH/endorphin fragments. The cells display different aspects: 1) large, regular shapes with numerous and large secretory granules; 2) small, irregular and angular shapes with small granules aligned along the periphery of the cell; and 3) intermediate forms. The presence of and -endorphin not only in the same cells but also in the same secretory granules that contain ACTH and -LPH clearly indicates that both the precursor or its fragments and the abovementioned peptides are stored in the same granules and released simultaneously by the corticotropic cells. The presence of FSH in some corticotropic cells is also discussed.Abbreviations used in this Article ACTH corticotropin - -MSH -melanotropin (ACTH I–I3) - CLIP corticotropin-like intermediate lobe peptide (ACTH 18–39) - -LPH -lipotropin - -MSH -melanotropin (-LPH 41–58); -endorphin (-LPH 61–91); -endorphin (-LPH 61–76)     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

Phytotoxic activity of selected water-soluble metabolites of Fusarium against Lemna minor L. (Duckweed)

Phytotoxicity and inhibitory effects of the fusarial toxins fumonisin B₁ (FB₁) [m.p. 103–105 °C], fusaric acid [m.p. 106–107 °C], butenolide (4-acetamido-4-hydroxy-2-butenoic acid lactone) [116–117 °C], 9, 10-dihydroxyfusaric acid [m.p. 150–155 ° C], and moniliformin on chlorophyll synthesis in the aquatic macrophyte Lemna minor (duckweed) were examined. FB₁ proved to be most active, reducing the growth of L. minor fronds and their ability to synthesize chlorophyll by 53% and 59%, respectively, at 0.7 g/ml. The growth rate of L. minor was reduced 59% by 6.7 g/ml fusaric acid, 62% by 66.7 g/ml butenolide, and 22% by 66.7 g/ml 9,10-dihydroxyfusaric acid. Moniliformin was the least phytotoxic to L. minor, with only a 16% suppression of growth rate and a 54% reduction in chlorophyll at 66.7 g/ml.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Changes in the content and constituents of the cuticular wax of Picea abies (L.) Karst. in relation to needle ageing and tree decline in five European forest areas

Cuticular wax composition of healthy and and declining mature Norway spruce trees [Picea abies (L.) Karst.] was investigated in five European forest areas. The amount of extracted wax and the content of alkanes and secondary alcohols were analysed as a function of the factors sample area (five areas, detailed below), needle age (current year to 2 years) and decline class (Class O to Class 2). Using a GC-MS, alkanes from C₂₀ to C₃₁ and the following alcohols were quantified: 10-nonacosanol, 5,10-nonacosandiol, 4,10-nonacosandiol and the triterpenol 24-methylenecycloartanol. According to our results, the total wax content as well as the alkane and alcohol content of waxes show a large variation corresponding to sample area and needle age. Ageing caused a highly significant increase in alkane content and a highly significant decrease in total wax and alcohol content. The decline class significantly influenced only the content of the long chain alkane C₃₁ (increase), the secondary alcohol 10-nonacosanol (decrease), and the triterpenic alcohol (decrease). Total wax weight was not influenced by tree damage. Thus, according to our results, needle ageing and progressive tree damage are correlated to different changes in the examined parameters.     

Nucleotide sequence of the maize chloroplast rpo B/C1/C2 operon: Comparison between the derived protein primary structures from various organisms with respect to functional domains

Summary The genes (rpo B/C₁/C₂) coding for the , , subnits of maize (Zea mays) chloroplast RNA polymerase have been located on the plastome and their nucleotide sequences established. The operon is part of a large inversion with respect to the tobacco and spinach chloroplast genomes and is flanked by the genes trnC and rps2. Notable features of the nucleotide sequence are the loss of an intron in rpoC₁, and an insertion of approximately 450 by in rpOC₂ compared to the dicotyledons tobacco, spinach and liver-wort. The derived amino acid sequence of this additional monocotyledon specific sequence is characterized by acidic heptameric repeat units containing stretches of glutamic acid, tyrosines and leucines with regular spacing. Other structural motifs, such as a nucleotide binding domain in the subunit and a zinc finger in the subunit, are compared at the amino acid level throughout the RNA polymerase subunits with the enzymes from other organisms in order to identify functionally important conserved regions.The sequence data presented in this paper will appear in the EMBL/Gen Bank/DDBJ Nucleotide Databases under the accession number X17318