Improvement of biosynthesis and accumulation of bioactive compounds by elicitation in adventitious root cultures of Polygonum multiflorum

Applied Microbiology and Biotechnology - We examined the effects of abiotic (methyl jasmonate [MeJA] and salicylic acid [SA]) and biotic (yeast extract and chitosan) elicitors for improvement of...     

The influence of optimization target selection on the structure of arterial tree models generated by constrained constructive optimization

The computational method of constrained constructive optimization was used to generate complex arterial model trees by optimization with respect to a target function. Changing the target function also changes the tree structure obtained. For a parameterized family of target functions a series of trees was created, showing visually striking differences in structure that can also be quantified by appropriately chosen numerical indexes. Blood transport path length, pressure profile, and an index for relative segment orientation show clear dependencies on the optimization target, and the nature of changes can be explained on theoretical grounds. The main goal was to display, quantify, and explain the structural changes induced by different optimization target functions.     

Agrobacterium-mediated transformation and regeneration of fertile transgenic plants of chinese cabbage (brassica campestris ssp. pekinensis cv. ‘spring flavor’)

A procedure for the regeneration of fertile transgenic Chinese cabbage (Brassica campestris ssp. pekinensis cv. Spring Flavor) is presented in this report. The protocol is based on infection of cotyledon explants of 5-d-old seedlings with an Agrobacterium tumefaciens strain LBA4404 carrying a disarmed binary vector pTOK/BKS-1. The T-DNA region of this binary vector contains the nopaline synthase/neomycin phosphotransferase II (nptII) chimeric gene for kanamycin resistance and the cauliflower mosaic virus 35S/coat protein gene of tobacco mosaic virus L (TMV-L) chimeric gene. After co-cultivation for 48 h, the cotyledonary petioles were placed on shoot induction media containing 15 mg/L kanamycin sulfate. Shoot induction was continued for 3–4 weeks, then subcultured once and after 2 weeks the shoots were transferred to root induction medium. After 1 week 8 putatively transformed plantlets from 200 cotyledon explants were obtained and transferred to greenhouse. Six of them grew to maturity, produced normal flowers and set seeds. Polymerase chain reaction and Southern blot hybridization analyses confirmed the introduction of the T-DNA into the Chinese cabbage genome. Further, Western blot analysis using polyclonal TMV antiserum showed most of the regenerants (5 out of 6) expressed TMV coat protein gene. Stable inheritance of the inserted clone was investigated in the next generation.     

Diverse reactions catalyzed by naphthalene dioxygenase fromPseudomonas sp strain NCIB 9816

Naphthalene dioxygenase (NDO) fromPseudomonas sp strain NCIB 9816 is a multicomponent enzyme system which initiates naphthalene catabolism by catalyzing the addition of both atoms of molecular oxygen and two hydrogen atoms to the substrate to yield enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. NDO has a relaxed substrate specificity and catalyzes the dioxygenation of many related 2- and 3-ring aromatic and hydroaromatic (benzocyclic) compounds to their respectivecis-diols. Biotransformations with a diol-accumulating mutant, recombinant strains and purified enzyme components have established that in addition tocis-dihydroxylation, NDO also catalyzes a variety of other oxidations which include monohydroxylation, desaturation (dehydrogenation),O-andN-dealkylation and sulfoxidation reactions. In several cases, the absolute stereochemistry of the oxidation products formed by NDO are opposite to those formed by toluene dioxygenase (TDO). The reactions catalyzed by NDO and other microbial dioxygenases can yield specific hydroxylated compounds which can serve as chiral synthons in the preparation of a variety of compounds of interest to pharmaceutical and specialty chemical industries. We present here recent work documenting the diverse array of oxidation reactions catalyzed by NDO. The trends observed in the oxidation of a series of benzocyclic aromatic compounds are compared to those observed with TDO and provide the basis for prediction of regio- and stereospecificity in the oxidation of related substrates. Based on the types of reactions catalyzed and the biochemical characteristics of NDO, a mechanism for oxygen activation by NDO is proposed.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Histochemisches Enzymmuster der Mamma unter dem experimentellen Einfluß von Geschlechtshormonen

Zusammenfassung In histochemischen Untersuchungen an der Mamma von Ratten soll die Beeinflussung der Enzymaktivität durch Östrogene und Progesteron demonstriert werden. Die Untersuchungen wurden an 40 weiblichen juvenilen und kastrierten Albinoratten durchgeführt, von denen die 1. Gruppe Follikelhormon (Progynon, 5 /d), die 2. Gruppe Corpus luteum-Hormon (Proluton, 1 mg/d) erhielten. Neben Kontrolltieren gleichen Alters wurden die Brustdrüsen unbehandelter infantiler Tiere untersucht. Am 3., 5., 10. und 20. Tag nach Injektionsbeginn wurden die Tiere getötet und die Brustdrüsen histologisch sowie enzymhistochemisch durch den Nachweis von alkalischer Phosphatase (Azofarbstoff und Schwermetallsimultanmethode), Glukose-6-Phosphatase, Adenosintriphosphatase, 5-Nukleotidase, Glukose-6-Phosphat-Dehydrogenase, Succinodehydrogenase und Laktatdehydrogenase aufgearbeitet.Es wurde festgestellt, daß unter dem Einfluß der Behandlung mit Geschlechtshormonen ein Anstieg der Enzymaktivität einsetzt, wobei Östrogen eine dominierende Bedeutung hat. Diese zeigt sich vor allem in den ersten 5 Tagen, während danach eine gewisse Gleichförmigkeit in der Intensität dieser Reaktion eintritt. Die Ergebnisse lassen sich dem Wandel der Zellstruktur unter gleichartigen hormonalen Einflüssen zuordnen und sind damit Ausdruck hormonal induzierter Stoffwechselleistungen.

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Histochemical enzymepattern of the mammary gland during the experimental influence of sex hormones

Summary The influence of estrogene und progesterone on the activity of enzymes is to be demonstrated by histochemical examinations of the mammary gland of rats. The examinations were made with 40 female juvenile and castrated albino rats of which the 1st group was given follicle-hormone (progynon, 5 /d), the 2nd group corpus-luteum-hormone (proluton, 1 mg/d). Besides control animals of the same age the mammary glands of untreated infantile animals were examined. On the 3rd, 5th, 10th, and 20th day after the beginning of the injections the animals were killed and the mammary glands were treated histologically as well as enzymehistochemically by examining their reaction to alcaline phosphatase, glucose-6-phosphatase, adenosine triphosphatase, 5-nucleotidase, glucose-6-phosphatase-dehydrogenase, succinic dehydrogenase, and lactic dehydrogenase.We found that under the influence of sex hormones the activity of enzymes increased and that estrogene had a dominating importance. This importance could be demonstrated during the first five days, while later these reactions showed a certain balance in their intensity. The results correspond to the change of the cellstructure under similar hormonal influences and thus are results of hormonally induced changes of metabolism.

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Herrn Prof. Dr. Karl-Heinz Bässler, Physiolog.-chem. Institut der Universität Mainz, sei für sachkundige Beratung in biochemischen Fragen vielmals gedankt.     

The effects of 1-naphthaleneacetic acid and N6-benzyladenine on the growth of Cymbidium forrestii rhizomes in vitro

An Asiatic orchid, Cymbidium forrestii, was clonally propagated using seed-derived rhizomes as explants. The rhizomes were cultured and proliferated on Murashige and Skoog medium supplemented with various growth substances. Auxins stimulated rhizome growth by increasing branching and fresh weight of the explant, with 1-naphthaleneacetic acid (NAA) being the most effective auxin. All auxins tested suppressed normal shoot formation. The apical meristem of the rhizome reacted to exogenously applied auxin by reducing the cytoplasmic zone of the apical meristem and causing meristem derivatives to rapidly differentiate into vacuolated parenchyma cells. Leaf formation and development was retarded in the presence of auxin. Cytokinins generally reduced rhizome growth and the number of branches, but benzyladenine (BA) can induce shoot formation in vitro. BA induced the cytoplasmic zone of the apical meristem to enlarge and enhanced leaf development. A 5% (w/v) sucrose concentration was most effective in shoot induction when combined with 5 mg1^-1 BA. Activated charcoal promoted rhizome growth; however, shoot formation was inhibited.     

Micropropagation of Raphanus sativus L. var. longipinnatus (Japanese radish) cv. Gungjung

Shoot cultures were established from seedling shoot tips of Raphanus sativus var. longipinnatus Bailey cv. Gungjung, (Japanese radish) cultured on a Murashige-Skoog medium supplemented with ca. 4.5–135 M kinetin or N⁶-benzyladenine. The latter cytokinin supported overall better growth, and 22.2 M was adopted for maintenance of established cultures. The nitrate: ammonium levels in the medium proved optimal for growth and shoot proliferation and both these parameters were significantly increased by addition of adenine sulfate or sodium phosphate. Rooting of excised shoots was achieved on auxin containing medium. Indole-3-butyric acid (ca. 5 or 10 M) also enhanced shoot growth. Plants were easily established in soil, appeared morphologically normal, and flowered.     

Antibody-antigen complex formation with immobilized immunoglobulins.

We have investigated the complex formation between an immobilized monoclonal antibody and antigens that differ in size about 50-fold. As a model system, we used an iodinated progesterone derivative and a progesterone-horseradish peroxidase conjugate as tracer and a monoclonal antibody as binding protein. The antibody was immobilized by four different methods: physical adsorption, chemical binding, and binding via protein G in the absence or presence of a protective protein (gelatin). These investigations have shown that the performance of competitive immunoassays is determined by a combination of factors: (a) the relative size of the analyte and the tracer, (b) the antibody density on the solid matrix, (c) the method of immobilization of the antibody, and (d) the binding constants between antibody-analyte and antibody-tracer. All of these interactions have to be considered in designing an optimal immunoassay. The smaller antigen can form a 3- to 35-fold higher maximal complex density than the larger antigen. Dose-response curves are less affected by the size of the tracer than by the binding constant with the antibody. A large enzyme tracer with a relatively low binding constant can, therefore, provide a more sensitive assay. On the other hand, the increase in complex density achieved with a smaller tracer yields a higher signal that in turn can provide a better signal-to-noise ratio in highly sensitive competitive solid-phase immunoassays. We have suggested a model for antibody immobilization that accounts for the interdependence of tracer size, complex formation, and antibody density. The methods described can be used to design and optimize immunoassays of predefined performance characteristics. The results are particularly useful for converting radioimmunoassays to enzyme immunoassays.