Biotransformation of codeine to morphine in freely suspended cells and immobilized cultures of Spirulina platensis

Both freely suspended cells and immobilized cultures of Spirulina platensis, a blue-green alga, biotransformed exogenously fed codeine, an opium alkaloid, to morphine. The external addition of codeine to the culture medium did not affect the growth of S. platensis. Immobilization of Spirulina in a calcium alginate gel matrix was optimized by using 2% (w/v) sodium alginate and reducing the concentration of nutrients of Zarrouk's medium, which caused destabilization of the calcium alginate gel. The accumulation of morphine increased gradually and reached maxima of 330g 100ml^–1 culture at 105h in freely suspended and 351g 100ml^–1 at 96h in immobilized Spirulina cultures. Accumulation of morphine was detected only in the medium, whereas cells did not show accumulation. The immobilized Spirulina cultures showed marginally higher conversion of codeine to morphine over freely suspended cultures.     

Factors influencing secondary somatic embryogenesis inMalus x domestica Borkh. (cv ‘Gloster 69’)

A procedure for regeneration of apple plants through secondary somatic embryogenesis (SSE) was developed in apple Gloster 69. Primary somatic embryos were produced from cotyledon-derived cultures of immature zygotic embryos. These somatic embryos were multiplied by secondary somatic embryogenesis (SSE) on media with different Plant Growth Regulator (PGR) combinations. The highest SSE rate (55.5%) was obtained with a combination of NAA (5.3 M), BAP (0.9 M) and KIN (0.9 M) or with TDZ alone (10 M). In addition, effects of explant source, somatic embryo size, type and concentrations of carbohydrates and gelling agents on SSE were investigated. The optimum SSE (>73%) was obtained by the culture of large size somatic embryos or cotyledon-like structures on medium containing a combination of NAA/BAP/KIN or TDZ (10 M) alone, maltose (175 mM) and Phytagel (2.8 g/1).     

CO2 gas exchange of benthic microalgae during exposure to air: a technique for the rapid assessment of primary production

A method of measuring CO₂gas exchange (caused, for example, by microalgal photosynthesis on emersed tidal mudflats) using open flow IR gas analyzers is described. The analyzers are integrated in a conventional portable photosynthesis system (LI-6400, LI-COR, Nebraska, USA), which allows manipulation and automatic recording of environmental parameters at the field site. Special bottomless measuring chambers are placed directly on the surface sediment. Measurements are performed under natural light conditions and ambient CO₂concentrations, as well as under different CO₂concentrations in air, and various PAR radiation levels produced by a LED light source built into one of the measurement chambers. First results from tidal channel banks in a north Brazilian mangrove system at Bragança (Pará, Brazil) under controlled conditions show a marked response of CO₂assimilation to CO₂concentration and to irradiance. Photosynthesis at 100molmol^–1CO₂in air in one sample of a well-developed algal mat was saturated at 309mol photons m^–2s^–1, but increased with increasing ambient CO₂concentrations (350 and 1000mol mol^–1CO₂) in the measuring chamber. Net CO₂assimilation was 0.8mol CO₂m^–2s^–1at 100mol mol^–1CO₂, 5.9mol CO₂m^–2s^–1at 350mol mol^–1CO₂and 9.8mol CO₂m^–2s^–1at 1000mol mol^–1CO₂. Compensation irradiance decreased and apparent photon yield increased with ambient CO₂concentration. Measurements under natural conditions resulted in a quick response of CO₂exchange rates when light conditions changed. We recommend the measuring system for rapid estimations of benthic primary production and as a valuable field research tool in connection with certain ecophysiological aspects under changing environmental conditions.     

Production of glucosamine containing disaccharides of the Lewis-A and -X types employing glycosidases

Disaccharide derivatives of interest for inhibition studies and for synthesis of the blood group determinants Lewis-a and Lewis-x were obtained with glycosidases as catalysts. Thus, Fuc(1–4)(6-OBn)GlcNH₂SEt and Gal1–3(6-OBn)GlcNH₂-SEt were produced employing (6-OBn)GlcNH₂SEt as acceptor and -L-fucosidase and -D-galactosidase, respectively, as catalysts. The phthalimido derivative of lactosamine, Gal1-4GlcNPhthSEt, was prepared from lactose employing GlcNPhthSEt as the acceptor and a yeast -galactosidase as catalyst. The reactions were both regio- and stereospecific, which allowed straightforward production of pure products on a g scale and higher.     

Über Konstitution und Erbgang eines neuen Delphinidinglycosids „Floridorin“ aus der Garteniris-Sorte cv. ‘Floridor’ (Cayeux 1929)

Zusammenfassung Bei der diploiden hohen Garten-Iris cv. Floridor (Cayeux 1929) wurde als bisher einziger Sorte ein neues Delphinidinglycosid Floridorin aufgefunden. Seine chemische Konstitution wurde als Delphinidin-3-Glukose-Rhamnose-p-Cumarsäure aufgeklärt. Es fand sich zusammen mit dem schon von uns aufgeklärten Anthozyan Tulipanin (Delphinidin-3-Glucose-Rhamnose). Das Hauptanthozyan der anderen Garten-Iris ist das von uns neuerdings nachuntersuchte Violanin. Die Untersuchungen wurden mit bereits von uns angegebenen neueren Methoden ausgeführt, wie der stufenweisen Hydrolyse und dem oxidativen Abbau. Das neue Floridorin zeigte bei den diploiden Garten-Iris einen monohybriden rezessiven Erbgang gegen Violanin. Die Blüten der Sorten, die Floridorin enthalten, sind schon mit dem Auge an einem charakteristischen taubenblauen Farbton zu erkennen.

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On the constitution and inheritance of a new delphinidine glycoside Floridorin from the cultivated iris variety cv. Floridor (Cayeux 1929)Studies on anthocyanins LI

Summary The diploid tall bearded garden Iris cv. Floridor (Cayeux 1929) proved to be thus far the only variety with a different anthocyanine, called Floridorin. Its chemical structure has been found to be delphinidine-3-glucose-rhamnose-p-coumaric-acid. It occurs together with tulipanine already analyzed by us as delphinidine-3-glucose-rhamnose. The main anthocyanine of thePogoniris garden varieties proved to be violanin the structure of which has been studied by us lately. The investigations were carried out by some newer methods, such as partial hydrolysis and oxidative degradation already published by us. The new Floridorin gives a monohybrid recessive Mendelian ratio with other diploid varieties ofPogoniris colored by violanine. The varieties colored by Floridorin show a characteristic greyish blue coloration which can be recognized with the naked eye.

     

Mapping of β-glucan content and β-glucanase activity loci in barley grain and malt

Genetic study of -glucan content and -glucanase activity has been facilitated by recent developments in quantitative trait loci (QTL) analysis. QTL for barley and malt -glucan content and for green and finished malt -glucanase activity were mapped using a 123-point molecular marker linkage map from the cross of Steptoe/Morex. Three QTL for barley -glucan, 6 QTL for malt -glucan, 3 QTL for -glucanase in green malt and 5 QTL for -glucanase in finished malt were detected by interval mapping procedures. The QTL with the largest effects on barley -glucan, malt glucan, green malt -glucanase and finished malt glucanase were identified on chromosomes 2,1,4 and 7, respectively. A genome map-based approach allows for dissection of relationships among barley and malt glucan content, green and finished malt -glucanase activity, and other malting quality parameters.     

Quantitative measurement of cell-bubble attachment in bubble columns using foam flotation techniques

A simple and convenient system for quantitatively measuring the number of adsorbed animal cells per unit of bubble surface area (, unit: cells/cm2) was developed. The system was successfully applied to recombinant Chinese hamster ovary (r-CHO) suspension cultures to investigate the dynamic cell-bubble attachment in a bubble column. In serum-free medium, values increased with bubble rising height (H) and cell concentration (C) and then became constant (about 1750 cells/cm2) when H and C were sufficiently high. In medium containing protective additives, the trends of values with H were similar to that in serum-free medium. Compared with serum-free medium, polyvinyl-pyrrolidone (PVP) increased the values to 1941 cell/cm2 whereas other tested additives decreased the values of in some different degree.     

Glycosylphosphatidylinositol (GPI) hydrolysis by Transforming Growth Factor-β1 (TGF-β1) as a potential early step in the inhibition of epithelial cell proliferation

Glycosylphosphatidylinositol (GPI) was previously identified in rabbit articular chondrocytes as being a precursor of inositolphosphate glycan (IPG), released upon (Transforming Growth Factor-) (TGF-) exposure, and capable of mimicking the proliferative effects of the growth factor. Here, using mink lung epithelial cells (CCL 64), which are known to be growth-inhibited by TGF-, we studied the potential role of GPI-derived molecules in the antiproliferative effect of TGF-1. We first identified an endogenous pool of GPI material and three different anionic forms of IPG in epithelial cells pre-labeled with [3H] glucosamine. Shortly (8 min) after TGF-1 addition, the cells responded by a rapid and transient hydrolysis of GPI, accompanied by the release of the most anionic form of IPG. This TGF--released IPG, after partial purification, was shown to decrease the proliferation of CCL 64 cells. Moreover, anti-IPG antibodies reduced the effects of TGF- and blocked the effects of partially purified IPG. These data strongly suggest that GPI hydrolysis may be an early step of the TGF- signalling pathway involved in growth inhibition of epithelial cells.     

Greek classicism in living structure? Some deductive pathways in animal morphology

Classical temples in ancient Greece show two deterministic illusionistic principles of architecture, which govern their functional design: geometric proportionalism and a set of illusion-strengthening rules in the proportionalism's stochastic margin. Animal morphology, in its mechanistic-deductive revival, applies just one architectural principle, which is not always satisfactory. Whether a Greek Classical situation occurs in the architecture of living structure is to be investigated by extreme testing with deductive methods.Three deductive methods for explanation of living structure in animal morphology are proposed: the parts, the compromise, and the transformation deduction. The methods are based upon the systems concept for an organism, the flow chart for a functionalistic picture, and the network chart for a structuralistic picture, whereas the optimal design serves as the architectural principle for living structure. These methods show clearly the high explanatory power of deductive methods in morphology, but they also make one open end most explicit: neutral issues do exist.Full explanation of living structure asks for three entries: functional design within architectural and transformational constraints. The transformational constraint brings necessarily in a stochastic component: an at random variation being a sort of free management space. This variation must be a variation from the deterministic principle of the optimal design, since any transformation requires space for plasticity in structure and action, and flexibility in role fulfilling. Nevertheless, finally the question comes up whether for animal structure a similar situation exists as in Greek Classical temples. This means that the at random variation, that is found when the optimal design is used to explain structure, comprises apart from a stochastic part also real deviations being yet another deterministic part. This deterministic part could be a set of rules that governs actualization in the free management space.     

Immunogold-cytochemical labelling of β-glucosidase in the white-rot fungus Coriolus versicolor

Summary Immunogold cytochemical labelling of hyphal sections of Coriolus versicolor showed that -glucosidase was localised in the extracellular mucilage, cell wall layers and cell interior in hyphae grown on glucose-rich malt extract medium whereas in hyphae grown with carboxymethylcellulose (CMC) as sole carbon source, most labelling was in the cell wall layers and cell interior. Little mucilage was visible around hyphae from these cultures. Hyphae from beechwood cultures showed gold labelling of -glucosidase in mucilage and fungal cell walls with some intracellular labelling. Biochemical studies of enzyme activity showed that similar amounts of enzyme were detected in the growth medium when cultures were grown on CMC medium, in agitated liquid cultures or in stationary cultures. In agitated cultures grown on glucose-rich malt extract, the activity of -glucosidase in the medium was 100 times less than that detected in stationary cultures on the same medium. However activity in the hyphae of stationary CMC-grown cultures was similar to that in hyphae from stationary glucose-rich cultures. These data confirm the patterns of gold labelling observed in hyphae from stationary cultures on glucose-rich malt extract when -glucosidase was immobilised in the extracellular mucilage layer around the hyphae. In this paper we propose that a primary function of the extracellular mucilage produced by hyphae of C. versicolor in vivo is to serve as a matrix for immobilisation of -glucosidase. Its substrate, cellobiose, which is released as a result of endo-and exoglucanase hydrolysis of cellulose, is absorbed and retained by the gel filtration properties of the mucilage, so encountering the immobilised -glucosidase. Glucose produced by this reaction is retained within the mucilage matrix around the hyphae before intracellular absorption.Offprint requests to: C. S. Evans     

Untersuchungen zur Regulation der Bacteriochlorophyll-Synthese bei Rhodospirillum rubrum

Zusammenfassung Wenn Rhodospirillum rubrum aus aeroben Dunkelkulturen in ein synthetisches Medium übertragen und anaerob im Licht bebrütet wird, beginnt die Bacteriochlorophyllbildung bereits nach 1 Std, das Wachstum erst nach 5–8 Std Bebrütung. — Puromycin (10 g/ml) und Chloramphenicol (20 g/ml) hemmen in diesen Kulturen Protein- und Pigmentsynthese vollständig. Eine Hemmung wird auch beobachtet, wenn die Antibiotica erst mehrere Stunden nach Beginn der Lichtbebrütung zugesetzt werden. Synthese von Thylakoidprotein und Bacteriochlorophyll scheinen regulatorisch gekoppelt zu sein.Actinomycin C (D) (40 g/ml) und Mitomycin (1 g/ml) hemmen die Bacteriochlorophyllbildung enbenfalls. Die Thylakoidbildung ist vom Vorhandensein einer funktionsfähigen DNS und RNS abhängig.Die spezifische Aktivität der -Aminolaevulinsäuresynthetase nimmt in anaeroben Lichtkulturen gegenüber aeroben Dunkelkulturen um etwa das Vierfache zu. Sie wird durch die verwendeten antibiotica nicht beeinflußt. Die Biosynthese des Enzyms wird durch Mitomycin und Puromycin, nicht aber durch Actinomycin gehemmt.

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Summary If dark-aerobically grown Rhodospirillum rubrum is transferred to anaerobic conditions in the light, the synthesis of photosynthetic pigments starts after 1 or 2 hours. The growth of the culture begins in the synthetic medium not before 5 hours incubation. In these cultures Puromycin (10 g/ml) and Chloramphenicol (20 g/ml) inhibit synthesis of protein and bacteriochlorophyll both. An inhibition is also observed when the antibiotics are added some hours after the beginning of anaerobic light incubation.The synthesis of chromatophore protein and bacteriochlorophyll are likely connected by gen-regulation.Actinomycin C (D) (40 g/ml) and Mitomycin C (1 g/ml) inhibit the bacteriochlorophyll synthesis likewise. The effect of actinomycin is increased by preincubation with the antibiotic in the dark. Mitomycin C stops synthesis of bacteriochlorophyll and protein even if it added after preincubation in the light.The level of -aminolevulinic acid-synthetase increased fourfold in anaerobic light-cultures compared to dark-aerobically grown cells. The activity of the enzyme is not influenced by the antibiotics. But the rate of biosynthesis is inhibited by Puromycin and Mitomycin, but not by Actinomycin.

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Abkürzungen im Text ALS -Aminolaevulinsäure - B-Chlorophyll Bacteriochlorophyll - DNS Desoxyribonucleinsäure - RNS Ribonucleinsäure     

Immobilization of digitoxin 12β-hydroxylase,a cytochrome P-450-dependent enzyme from cell cultures of Digitalis lanata EHRH

Attempts were made to immobilize digitoxin 12-hydroxylase, a membrane-bound, cytochrome P-450-dependent monooxygenase from cell cultures of Digitalis lanata. The optimum procedure was the entrapment of microsomes in 2% alginate by crosslinking the polysaccharide chains with CaCl₂. After the immobilization of the enzyme about 70% of its activity was retained. The kinetic data such as the pH optimum and the optimum substrate concentrations were identical for the immobilized enzyme and freely suspended microsomes. Using -methyldigitoxin as a substrate enzyme activity could be observed for more than 20 h. A continuous flow system for immobilized digitoxin 12-hydroxylase is described.Abbreviations -mdg -methyldigoxin - -mdt -methyldigitoxin     

Fixed-bed dialysis culture of a transfectoma cell line producing chimeric Fab-fragments with “nutrient-split”-feeding strategy

The very shear sensitive transfectoma cell line, FAB 10, producing a chimeric Fab-antibody fragment specific for the human carcinoembryonic antigen (CEA), was cultivated in fixed bed reactors where the cells were immobilized in macroporous carriers. As the cell line had a very low productivity, cultures with process integrated product enrichment were performed in a membrane dialysis bioreactor with integrated fixed bed, where low molecular weight metabolites are removed over the membrane and high molecular weight products are enriched. In a new nutrient-split feeding strategy for dialysis cultures concentrated medium was supplied directly to the fixed bed unit, whereas a buffer solution was used as dialysis fluid. With the dialysis technique up to 5800 g Fab l–1 could be obtained, more than 10 times higher compared to fixed bed cultures without dialysis or batch cultures with suspended cells. The nutrient-split-feeding strategy reduced the amount of medium required per mg of antibody significantly. © Rapid Science Ltd. 1998     

β-carotene from the abscisic acid producing strain of Cercospora rosicola

-carotene was isolated from an extract of Cercospora rosicola mycelia and identified on the basis of chromatographic properties and UV-visible spectroscopic evidence. The accumulation of -carotene was inhibited 71 and 52% by 10 M amounts of decylimidazole and fluridone, resepectively. Abscisic acid accumulation was inhibited 63% by decylimidazole and enhanced about 10% by fluridone. Several other inhibitors of carotenoid synthesis failed to inhibit ABA biosynthesis.     

Molecular analysis of methane monooxygenase from Methylococcus capsulatus (Bath)

Methane monooxygenase (MMO) is the enzyme responsible for the conversion of methane to methanol in methanotrophic bacteria. In addition, this enzyme complex oxidizes a wide range of aliphatic and aromatic compounds in a number of potentially useful biotransformations. In this study, we have used biochemical data obtained from purification and characterization of the soluble MMO from Methylococcus capsulatus (Bath), to identify structural genes encoding this enzyme by oligonucleotide probing. The genes encoding the and subunits of MMO were found to be chromosomally located and were linked in this organism. We report here on the analysis of a recombinant plasmid containing 12 kilobases of Methylococcus DNA and provide the first evidence for the localization and linkage of genes encoding the methane monooxygenase enzyme complex. DNA sequence analysis suggests that the primary structures of the and subunit of MMO are completely novel and the complete sequence of these genes is presented.     

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

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

Establishment of fast-growing callus and root cultures of Cephalotaxus harringtonia

Summary Callus cultures were established from Cephalotaxus harringtonia (Japanese plumyew) stem expiants cultured on Murashige and Skoog medium supplemented with 4.5 M 2,4-dichlorophenoxyacetic acid and 0.05 M 6-furfurylaminopurine. The inclusion of 4.9 M 6-(,-dimethylallylamino) purine as the sole hormone significantly increased the growth rate of the callus. Organogenesis giving rise to both shoots and roots occurred upon transfer of the callus onto a hormonefree medium. Vitrification was common on all regenerated shoots cultured on Gelrite-containing medium. Regenerated roots were excised and established in McCown's woody plant medium. Doubling the phosphate and nitrate levels in the medium increased the growth of these root cultures.Abbreviations MS Murashige and Skoog basal medium - B5 Gamborg's B5 basal salt medium - WP McCown's woody plant basal salt medium - 2,4-D 2,4-dichlorophenoxyacetic acid - Kinetin 6-furfurylamino-purine - 2iP 6-(,-dimethylallylamino) purine - IBA indole-3-butyric acid     

Enhancement of photorespiration in immobilized <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> cells

Immobilization of Chlamydomonas reinhardtii in alginate increases its photorespiration rate. In the immobilized cells, the photorespiratory enzyme, phosphoglycolate phosphatase, was 75% higher than in freely suspended cells. Thus, the immobilized cells produced glycolate at twice the rate than in freely suspended cells when treated with aminooxyacetate (a transaminase inhibitor). With immobilized cells in a batch reactor, 270mol glycolatemg^–1 Chl was produced after 12h.Revisions requested 27 October 2004; Revisions received 13 December 2004     

Somatic embryogenesis and plant regeneration in wild cotton (Gossypium klotzschianum)

A simple and efficient method for high frequency somatic embryogenesis and plant regeneration from hypocotyl-derived cultures and suspension cultures of Gossypium klotzschianum Anderss, a wild, diploid species of cotton is described here. Embryogenic cultures were induced from hypocotyl sections on MSB medium with 0.9 M 2,4-D and 2.32 M kinetin. MSB medium containing 0.045 M 2,4-D, 0.93 M kinetin, 2.46 M IBA promoted embryogenic culture proliferation and embryo development. Suspension cultures with 0.23 M 2,4-D and 0.93 M kinetin also produced many embryos. Somatic embryos cultured on MSB medium with PGRs produced secondary embryos, and embryos developed into normal plantlets on PGR-free MSB medium. Regenerated plantlets were transferred onto the quarter-strength MSB medium with 0.5% active charcoal to avoid recallusing. Hypocotyls were better than cotyledons for culture induction and plant regeneration. 2,4-D and kinetin were essential for culture induction and maintenance.     

Interaction of aluminium ions with some amino acids present in human blood

Summary. The interaction of aluminium with some amino acids present in human blood was studied combining ion-chromatography (IC), atomic absorption spectrometry (AAS) and ultrafiltration (UF) techniques. An IC system for simultaneous determination of ornithine, lysine, glutamic acid, aspartic acid and tyrosine was developed. By adding aluminium to standard solutions of the amino acids and keeping the pH at 6 and 7 it was possible to verify that aluminium caused a reduction on the amino acid chromatographic signals. Similar experiment, carried out for copper showed the same behaviour (with different percentage of signal reductions) and validated the results for aluminium, considering that the interaction Cu-amino acid is well-established. The AAS analysis of sample fractions (500l) after the IC separation showed that aluminium (as copper as well) is not present in the fractions in which the amino acid peaks appear in the chromatogram. These approaches carried out with serum samples after UF showed that part of the free fraction of serum aluminium is distributed, besides other ligands, among these amino acids. It was found that in serum the affinity for aluminium followed the sequence Lys>Orn>Tyr>GluAsp.