Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate

Adventitious roots of ginseng were treated with methyl jasmonate (MJ) up to 150m and cultured for 40days. Up to 100m MJ inhibited the root growth but increase ginsenoside accumulation. In a two-stage bioreactor culture, total ginsenosides, after elicitation with 100 m MJ peaked after 10days at 48mgg^–1 dry wt and then dropped sharply. Of the two groups of ginsenosides (Rb and Rg), higher amounts of Rb accumulated in the adventitious roots.Revisions requested; 2 July 2004; Revisions received 30 June 2004; 3 September 2004     

Fehlender Einfluß von Licht auf die Reversion der UV-induzierten spektralen Veränderung von Cytidylsäure

Zusammenfassung Das Lichtprodukt, welches nach Belichtung von UV-bestrahlter Cytidylsäure (CMP) in früheren Versuchen zu entstehen schien und über das von Park u. Kaplan berichtet worden war, konnte in neuen Versuchen des zweiten Autors nicht wiedergefunden werden. Nachbehandlung mit Licht wie auch Dunkelheit bei ca. 25°C ergaben offenbar die gleiche Substanz mit dem Absorptionsspektrum von normalem CMP.

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Summary The photoproduct after illumination of UV-irradiated Cytidylic acid (CMP) reported by Park and Kaplan earlier could not be reproduced by the 2^nd author. Both, postincubation with and without light at about 25°C led to the formation of obviously the same substance with the absorption spectrum of normal CMP.

     

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     

Influence of environmental reference points on learned orientation in fish (Phoxinus)

Summary Fish were trained to find food at the north position in a circular tank. The starting cue was the burning of a light 45° from north. When the tank was rotated 180° they sought food in the south position. Elimination tests proved the orientation marks to be minute marks on the wall and floor of the tank and not the sun.The unusual ability of a fish to thus appraise his immediate environment and to use inconspicuous landmarks for orientation aid in explaining the homing ability of lake and shore fishes.In two other units, each different, an artificial sun in the artificial sky was used as an orientation point by the fish only after moving all other marks continually during training. Long periods of frequent training were necessary, nevertheless, because these fish did eventually associate a 90° angle of the sun to a feeding position suggests the need for exploring further the fish's use of the sun in orientation.In honor of Karl von Frisch on his seventieth birthday.Fulbright Research Scholar, 1954/55, Zoologisches Institut, Universität München, Germany. Appreciation for laboratory space, equipment and helpful consultation is extended to Prof. K. v. Frisch, and to Karl Boellinger and F. Schutz for faithful aid in the testing and training.     

Studies of the action of ceramide-like substances (d- andl-PDMP) on sphingolipid glycosyltransferases and purified lactosylceramide synthase

We have studied the effects ofD-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and itsL-enantiomer on glycosphingolipids in cultured normal human kidney proximal tubular cells. We found thatD-PDMP exerted a concentration-dependent reduction in the metabolic labelling and cellular levels of glucosylceramide (GlcCer), lactosylceramide (LacCer), and the globo-series glycosphingolipids, GbOse₃Cer and GbOse₄Cer. It also directly inhibited the activity of UDP-glucose:ceramide 1 4-glucosyltransferase (GlcT-1) and UDP-galactose: GlcCer 1 4 galactosyltransferase (GalT-2). In contrast,L-PDMP had opposite effects on the metabolic labelling of GlcCer, LacCer, and GbOse₃Cer. The levels of GlcCer and LacCer were increased, while the labelling and level of GbOse₄Cer were strongly reduced. Purified GalT-2 from human kidney was inhibited byD-PDMP and stimulated byL-PDMP. It appears likely that the different glycosphingolipid glycosyltransferases possess similar binding sites for the ceramide moiety, which are blocked by binding toD-PDMP and, in the case of GbOse₄Cer synthase, byL-PDMP as well. The stimulatory effects ofL-PDMP on GlcCer and LacCer synthases may be the result of binding to a modulatory site on the glycosyltransferases; in intact cells, the enzyme-analog complex may afford protection against the normal catabolic inactivation of the enzymes.Abbreviations GalT-2 UDP-galactose:GlcCer -galactosyltransferase - GbOse₃Cer Gal1 4Gal1 GlcCer - GbOse₄Cer GalNAc1 3Gal1 4Gal1 GlcCer - GlcCer glucosylceramide - GlcT-1 UDP-glucose:ceramide -glucosyltransferase - GSLs glycosphingolipids - LacCer lactosylceramide - PDMP threo-1-phenyl-2-decanolyamino-3-morpholino-1-propanol     

Changes in cell wall polysaccharides in developing barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) coleoptiles

Cell wall polysaccharides in developing barley coleoptiles were examined using acetic acid–nitric acid extraction, alditol acetate and methylation analyses and enzymatic digestion. The coleoptile cell wall from imbibed grain was rich in pectic polysaccharides (30 mol%), arabinoxylan (25 mol%), cellulose (25 mol%) and xyloglucan (6 mol%), but contained only low levels of (13,14)--D-glucan (1 mol%). During 5 days of coleoptile growth, pectic polysaccharides decreased steadily to about 9 mol%, while (13,14)--D-glucan increased to 10 mol%. Following the cessation of growth of the coleoptiles at about 5 days, (13,14)--D-glucan content rapidly decreased to 1 mol%. The cellulose content of the walls remained at about 35–40 mol% throughout coleoptile growth. Similarly, arabinoxylan content remained essentially constant at 25–30 mol% during growth, although the ratio of substituted to unsubstituted 4-linked xylosyl units decreased from about 4:1 to 1:1. Xyloglucan content ranged from 6 mol% to 10 mol% and the oligosaccharide profile determined using a xyloglucan-specific endoglucanase and MALDI-TOF mass spectrometry indicated that the oligosaccharides XXGG and XXGGG were the principal components, with one and two acetyl groups, respectively, Thus, dramatic changes in wall composition were detected during the growth of barley coleoptiles, both with respect to the relative abundance of individual wall constituents and to the fine structure of the arabinoxylans.     

Inhibition of hexose transport in the human erythrocyte by 5, 5′-dithiobis(2-nitrobenzoic acid): Role of an exofacial carrier sulfhydryl group

Summary The sulfhydryl reagent 5, 5-dithiobis (2-nitrobenzoic acid) (DTNB) was used to study the functional role of an exofacial sulfhydryl group on the human erythrocyte hexose carrier. Above 1mm DTNB rapidly inhibited erythrocyte 3-O-methylglucose influx, but only to about half of control rates. Efflux was also inhibited, but to a lesser extent. Uptake inhibition was completely reversed by incubation and washing with 10mm cysteine, whereas it was only partially reduced by washing in buffer alone, suggesting both covalent and noncovalent interactions. The covalent thiol-reversible reaction of DTNB occurred on the exofacial carrier, since (i) penetration of DTNB into cells was minimal, (ii) blockade of potential uptake via the anion transporter did not affect DTNB-induced hexose transport inhibition, and (iii) DTNB protected from transport inhibition by the impermeant sulfhydryl reagent glutathione-maleimide-I. Maltose at 120mm accelerated the covalent transport inhibition induced by DTNB, whereas 6.5 m cytochalasin B had the opposite effect, indicating under the one-site carrier model that the reactive sulfhydryl is on the outward-facing carrier but not in the substrate-binding site. In contrast to glutathione-maleimide-I, however, DTNB did not restrict the ability of the carrier to reorient inwardly, since it did not affect equilibrium cytochalasin B binding. Thus, carrier conformation determines exposure of the exofacial carrier sulfydryl, but reaction of this group may not always lock the carrier in an outward-facing conformation.     

Ultrastructural localisation of TGF-beta exposure in dentine by chemical treatment

Transforming growth factor- (TGF-) sequestered in dentine matrix has an important role in dental tissue repair after injury and its exposure at sites of injury may stimulate tertiary dentinogenesis. This study aimed to investigate the expression of TGF- isoforms in mature human dentine matrix and the ability of chemical treatments to expose TGF- on the cut surface of dentine using gold immunolabelling and subsequent scanning electron microscopy examination. TGF-1 was the only isoform that could be detected in human dentine and the nature of the chemical treatment of the tissue influenced its detection. EDTA treatment provided good exposure of TGF-1 on the dentine surface, whilst citric acid and sodium hypochlorite treatments revealed lesser amounts of this isoform. Only minimal staining for TGF-1 was observed in samples treated with phosphate-buffered saline. TGF-2 and -3 could not be detected in the specimens with any of the treatments. This study suggests that TGF-1 is the only TGF- isoform expressed by human odontoblasts to be sequestered in dentine implying that differences in isoform–extracellular matrix interactions may exist. Information on chemical treatment of tissue specimens for immunostaining may provide a useful basis for selection of tissue preparation techniques for clinical restorative treatment procedures to facilitate TGF- mediated reparative processes at sites of dental injury.     

Molecular cloning and expression of multiple cellulase genes of Ruminococcus flavefaciens strain 186 in Escherichia coli

Summary Cellulase genes of the ruminant micro-organism Ruminococcus flavefaciens strain 186 have been cloned and expressed in Escherichia coli using the bacteriophage vector NM1149. Twenty-six clones showed expression of endo--1,4-d-glucanases and were divided into four groups according to their insert sizes of approximately 2, 3, 4 or 9 kilobases (kb). Two of the clones with 4 kb inserts also showed exo--1,4-d glucanase activity while two clones with 9 kb inserts showed -glucosidase activity. One of the clones with 9 kb inserts (M903) showed the activities of all three cellulase activities. In addition, two of the 4 kb-insert clones and one 9 kb-insert clone degraded Avicel (PH101).     

Male swarms at landmarks and scramble competition polygyny inPolistes gallicus (Hymenoptera: Vespidae)

At the end of summer, males of Polistes gallicusfly in swarms around vertical landmarks and land in clusters on their favorite perches, where they drag their legs and abdomen. Here males occasionally crowd around a perched female; they make no effort to defend an exclusive mating territory but instead attempt to copulate by displacing rivals from the female. In this work we describe this spatial-nuptial system, which entails site fidelity without territoriality, unisexual swarms, common patrol routes, collective sexual approaches, and scramble competition polygyny. Mating success is evaluated in relation to the familiarity with flight paths (routine patrollers versus newcomers), to the type of sexual approach (single males versus in- group males), and, in the laboratory, to the individual activity level.     

Isolation and characterization of β-glucosidases from Aspergillus nidulans mutant USDB 1183

Two extracellular -glucosidases (cellobiase, EC 3.2.1.21), I and II, from Aspergillus nidulans USDB 1183 were purified to homogeneity with molecular weights of 240,000 and 78,000, respectively. Both hydrolysed laminaribiose, -gentiobiose, cellobiose, p-nitrophenyl--L-glucoside, phenyl--L-glucoside, o-nitrophenyl--L-glucoside, salicin and methyl--L-glucoside but not -linked disaccharides. Both were competitively inhibited by glucose and non-competitively (mixed) inhibited by glucono-1,5-lactone. -Glucosidase I was more susceptible to inhibition by Ag⁺ and less inhibited by Fe²⁺ and Fe³⁺ than -glucosidase II.     

Characterization of two different Ca2+ uptake and IP3-sensitive Ca2+ release mechanisms in microsomal Ca2+ pools of rat pancreatic acinar cells

We have examined the effect of the Ca²⁺ (Mg²⁺)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent ⁴⁵Ca²⁺ uptake into IP₃-sensitive Ca²⁺ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40–50% of total Ca²⁺ uptake was inhibited by TG up to 10 nm (apparent K_i4.2 nm, Ca²⁺ pool I). An additional increase of inhibition up to 85–90% of total Ca²⁺ uptake could be achieved at 15 to 20 nm of TG (apparent K_i12.1 nm, Ca²⁺ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent K_i10 m). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca²⁺ uptake. About 30–40% of total Ca²⁺ uptake was inhibited by 100 m of vanadate (apparent K_i18 m, Ca²⁺ pool II). The remaining 60–70% could be inhibited either by vanadate at concentrations up to 1 mm (apparent K_i300 m) or by TG up to 10 nm (Ca²⁺ pool I). The amount of IP₃-induced Ca²⁺ release was constant at 25% over a wide range of Ca²⁺ filling. About 10–20% remained unreleasable by IP₃. Reduction of IP₃ releasable Ca²⁺ in the presence of inhibitors showed similar dose-response curves as Ca²⁺ uptake (apparent K_i 3.0 nm for IP₃-induced Ca²⁺ release as compared to 4.2 nm for Ca²⁺ uptake at TG up to 10 nm) indicating that the highly TG-sensitive Ca²⁺ pump fills the IP₃-sensitive Ca²⁺ pool I. At TG concentrations >10 nm which blocked Ca²⁺ pool II the apparent K_i values were 11.3 and 12.1 nm, respectively. For inhibition by vanadate up to 100 m the apparent K_i values were 18 m for Ca²⁺ uptake and 7 m for Ca²⁺ release (Ca²⁺ pool II). At vanadate concentrations up to 1 mm the apparent K_i values were 300 and 200 m, respectively (Ca²⁺ pool I). Both Ca²⁺ pools I and II also showed different sensitivities to IP₃. Dose-response curves for IP₃ in the absence of inhibitors (control) showed an apparent K_m value for IP₃ at 0.6 m. In the presence of TG (inhibition of Ca²⁺ pool I) the curve was shifted to the left with an apparent K_m for IP₃ at 0.08 m. In the presence of vanadate (inhibition of Ca²⁺ pool II), the apparent K_m for IP₃ was 2.1 m. These data allow the conclusion that there are at least three different Ca²⁺ uptake mechanisms present in pancreatic acinar cells: TG- and IP₃ insensitive but highly vanadate-sensitive Ca²⁺ uptake occurs into membrane vesicles derived from plasma membranes. Two Ca²⁺ pools with different TG-, vanadate- and IP₃-sensitivities are most likely located in the endoplasmic reticulum at different cell sites, which could have functional implications for hormonal stimulation of pancreatic acinar cells.This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 246. The authors wish to thank Dr. KlausDieter Preuß for valuable discussions and Mrs. Gabriele Mörschbächer for excellent secretarial help.     

Methylobacterium rhodesianum MB 126 possesses two acetoacetyl-CoA reductases

Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Methylobacterium rhodesianum MB 126, an NADPH-linked d(-)--hydroxybutyryl-CoA forming reductase (enzyme A) and an NADH-and NADPH-linked l(+)--hydroxybutyryl-CoA forming reductase (enzyme B). Enzyme A and B give apparent K _m values of 15 M and 30 M for AcAc-CoA, 18 M for NADPH and 30 M for NADH, respectively. They are inhibited by AcAc-CoA at concentrations higher than 25 M and 50 M, respectively. The contribution of the two reductases to poly--hydroxybutyrate synthesis is discussed.     

Organ-specific crystalline structures of ferritin cores inβ-thalassemia/hemoglobin E

Summary The cores of ferritins isolated from different organs of human subjects with-thalassemia/hemoglobin E (-thal/HbE) disease have different size distributions and crystallinities depending on the source organ. These patients have not been treated by hypertransfusion regimen or iron chelation therapy.-Thal/HbE spleens and livers yield ferritin cores which are less crystalline than those isolated from normal spleens and livers, reflecting the more rapid deposition of iron in the diseased state. Ferritins isolated from the hearts and pancreases of-thal/HbE subjects were found to have larger, more crystalline cores than those from the-thal/HbE livers and spleens, possibly as a consequence of the role of the heart and pancreas as long-term iron deposition sites in this iron overload pathology.     

Affinity Profiles of Novel δ-Receptor Selective Benzofuran Derivatives of Non-Peptide Opioids

Highly selective heterocyclic opioid ligands with potent -antagonist activity have been developed on the basis of the message-address concept. Using this strategy, benzofuran derivatives corresponding to the non-selective opioid antagonist, naloxone, and to the -opioid receptor selective agonists, oxymorphone and oxycodone, were synthesized. In vitro opioid receptor binding profiles and agonist/antagonist character of these compounds were determined in rat brain membrane preparations with highly selective radioligands. All three benzofuran derivatives displayed high affinities for the -opioid receptor, much less potency toward the -binding site, and were the least effective at the -site. The results indicated that the addition of the bezofuran moiety to these fused ring opioids confers -receptor selectivity. The Na⁺ indices suggested a partial agonist character for oxymorphone- and oxycodone-benzofuran, and an antagonist character for naloxone-benzofuran. These compounds were capable of irreversible inhibition of opioid binding sites in a dosedependent.     

The production of copper,zinc and molybdenum deficiencies in crop plants grown in sand culture with special reference to some effects of water supply and seed reserves

Summary (1) A method is described for the production of acute deficiencies of copper, zinc or molybdenum in large scale sand cultures in which an adequate and satisfactory supply of water was obtained by treating rain water with ion-exchange resins. Results compared favourably with the use of glass distilled water.(2) Visual symptoms of copper, zinc and molybdenum deficiencies in large seeded leguminous crops, and in other species are summarised.(3) Seed reserves played an important part in the incidence of copper and molybdenum deficiencies and to a lesser extent of zinc deficiency in pea, runner bean, dwarf bean and broad bean.(4) The deficient seed was obtained by harvesting seed from plants grown in the respective deficiency sand cultures, and was compared with seed obtained similarly from complete nutrient sand cultures or commercially from field-grown plants.(5) Seed reserves of molybdenum were usually sufficient for a complete generation but reserves of copper and zinc were not adequate for the whole copper or zinc requirements of the plants.(6) Analytical data showed a marked decrease in the copper or molybdenum content of deficient seed, and a less striking decrease in zinc.(7) Foliage of deficient plants showed decreased content of copper and molybdenum and often of zinc compared with plants given the complete nutrient treatment, but use of deficient seed only decreased yields without further appreciably decreasing the mineral content of the foliage.     

Free Ia Eα chain expression in the E α + :E β − : recombinant strain A.TFR5

Molecular and biochemical techniques have been used to explore the reasons behind low E chain expression in the E ⁺ E ^– I-region recombinant strain, A.TFR5. A.TFR5 (A ^f E ^k, ap5), a recombinant between A.CA (A ^f E ^f) and A.TL (A ^k E ^k), carries the E ^k subregion. Previous results have shown that it expresses the E chain, but at reduced levels relative to E ⁺ E ⁺ strains. No E chains were detected, which is consistent with the A.TFR5E gene being derived from the A.CA parent, which carries the null E ^f allele. In this paper, the defect in E-chain expression is explored. Restriction fragment length polymorphism analysis has localized the recombination event in A.TFR5 approximately 30 kb upstream of E, in the region of the large intervening sequence of E. Northern blot analysis of total RNA from A.TFR5 shows normal amounts of the E message, but no E message. Two-dimensional gel analysis of 15 min pulse-labeled A.TFR5, A.CA, and A.TL E immunoprecipitates shows decreased levels of the intracellular E chain in A.TFR5 relative to A.TL. However, analysis of total cell extracts shows normal levels of this protein. A glycoprotein fraction isolated from total cell extracts of 5 h labeled cells contains normal amounts of intracellular E, but decreased amounts of the mature cell-surface protein. These data suggest that in the absence of E, the E chain (1) takes on an altered conformation that is not as efficiently recognized by alloantibodies, and (2) is found in normal levels as the partially glycosylated intracellular precursor, but is not processed and/or transported efficiently to the cell surface.     

Characterization and gene organization of Taiwan banded krait (Bungarus multicinctus) gamma-bungarotoxin

-Bungarotoxin was isolated from Bungarus multicinctus (Taiwan banded krait) venom using a combination of chromatography on a SP-Sephadex C-25 column and a reverse-phase high-performance liquid chromatography column. Circular dichroism (CD) measurement revealed that its secondary structure was dominant with -sheet structure as is that of snake venom -neurotoxins and cardiotoxins. -Bungarotoxin exhibits activity on inhibiting the binding of [³H]quinuclidinyl benzilate to the M2 muscarinic acetylcholine receptor subtype, and competes weakly with radioiodinated -bungarotoxin for binding to the Torpedo nicotinic acetylcholine receptor. Moreover, the toxin inhibits collagen-induced platelet aggregation, with an IC₅₀ of approximately 200 nM. The genomic DNA encoding the -bungarotoxin precursor is amplified by polymerase chain reaction (PCR). The gene is organized with three exons separated by two introns, and shares virtually identical overall organization with those reported for -neurotoxin and cardiotoxin genes, including similar intron insertions. The intron sequences of these genes share sequence identity up to 85%, but the exon sequences are highly variable. These observations suggest that -bungarotoxin, -neurotoxins, and cardiotoxins originate from a common ancestor, and the evolution of these genes shows a tendency to diversify the functions of snake venom proteins.     

Stereoselective aminoacylation of a dinucleoside monophosphate by the imidazolides ofDl-alanine and N-(tert-butoxycarbonyl)-Dl-alanine

Summary The aminoacylation of diinosine monophosphate (IpI) was studied. When the acylating agent was the imidazolide of N-(tert-butoxycarbonyl)-Dl-alanine, a 40% enantiomeric excess of thel isomer was incorporated at the internal 2 site and the positions of equilibrium for the 23 migration reaction differed for theD andl enantiomers. The reactivity of the nucleoside hydroxyl groups decreased in the order 2(3)>internal 2>5, and the extent of reaction was affected by the concentration of the imidazole buffer (pH 7.1). In contrast, reaction of IpI with the imidazolide of unprotectedDl-alanine led to an excess of theD isomer at the internal 2 site, while reaction with the N-carboxy anhydride ofDl-alanine proceeded without detectable stereoselection. The relevance of these results to the evolution of optical activity and the origin of genetically directed protein synthesis is discussed.     

A comparison and evaluation of some phytosociological techniques

Summary In three areas of vegetation (dune, mountain heath and salt marsh) the following phytosociological techniques have been tested and compared, using the same data: the Braun-Blanquet method; association and inverse analysis ofWilliams &Lambert; cluster analysis (agglomerative classification) based on different coefficients of similarity; and ordination (principal components analysis performed on matrices of different coefficients).The Braun-Blanquet method is considered to combine several advantages of the other methods and to be most economical in terms of efficiency (ratio of time input to information emerging).

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Zusammenfassung Auf drei verschiedenen Vegetationsflächen (Bergheide, Küstendünen und Salzwiesen) sind die folgenden pflanzensoziologischen Methoden geprüft und verglichen worden: die Methode von Braun-Blanquet; association analysis vonWilliams &Lambert (1959, 1961); Ordination (principal components analysis); und cluster analysis (Sokal &Sneath, 1963). Die letzteren beiden wurden mit verschiedenen Ähnlichkeitskoeffizienten geprüft.Auf Grund solchen Erfahrungen, zeigte sich die Braun-Blanquetische Methode leistungsfähiger als die anderen Methoden (d.h. optimale Einsicht in der Vegetation pro Arbeitsstunde). Sie vereinigte viele Vorteile der anderen Methoden.