Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

Genetic diversity and population structure of the gizzard shad,Konosirus punctatus (Clupeidae,Pisces), in Korean waters based on mitochondrial DNA control region sequences

In this study, we clarified the population structure of the gizzard shad, Konosirus punctatus, in Korean waters. We analyzed 896 base pairs of the mitochondrial DNA control region in 182 individuals, which were sampled from eight localities between the East Sea and the Yellow Sea. The haplotype diversity (h) was very high (0.9662–1.0000) but the nucleotide diversity (π) was very low (0.0061–0.0434). A neighbor-joining tree showed that the population clustered into two reciprocal monophyletic groups, lineages A and B. Lineage A is distributed on all coasts of Korea, from the Yellow Sea to the East Sea, declining to the east, whereas lineage B is distributed in the East Sea and Korea Strait, disappearing completely from middle Korea Strait to the west. Analysis of molecular variance showed strong structuring (F _ST = 0.856; P < 0.0001) between the two lineages. Neutrality tests and mismatch distribution analyses showed that a recent rapid expansion event occurred only in lineage A. Our results suggest that the management unit of the Korean gizzard shad may be divided in two, lineage A and lineage B.     

Differentiation of joints in transplants developing on chorioallantois

The wing of the chick embryos (the 17th-21st stages of development according to Hamburger--Hamilton) were transplanted on the chorioallantois of the chick embryo-recipients, incubated for 8.5-9.5 days. Differentiation of the joints was studied in serial histological sections and in translucent preparations of the skeleton stained with alcian blue. The transplants for the investigation were taken on the 1st-11th days after transplantation. In the transplants all three segments of the wing always developed. The development of the external form of the extremity, chondrogenesis and osteogenesis of the skeletal anlages were about 24 h late. Histological changes, specific for the early period of the articular interzone and cleft formation corresponded to the control embryos data, but were one day younger. In future the changes did not progress, and passed into regression, demonstrating as fusion of the articular surfaces. In the transplants blood vessels formed networks of irregular form that surrounded the articular zones. Some branches run from them into mesenchyme, situating around the joint. According to the literature data, these vessels are connected with formation of the articular cleft and in the control embryos blood vessels of the articular capsule develop from them. In the transplants they are dilated, twisted (especially in the ulnar joint area) and do not penetrate into the developing prechondral and then into the cartilage bridges of the fusing articular surfaces. Numerous blood accumulations, as well as extravasates are often seen near the deformed anlages of bones. Thus, disturbance of blood supply in the transplants and lack of innervation in them, discussed in the literature, result in fusion of the articular surfaces.     

Citric acid production by Aspergillus niger immobilized on polyurethane foam

Summary Citric acid was produced using Aspergillus niger immobilized on polyurethane foam in a bubble column reactor. Most of the adsorbed cells remained on the support and, as a result, high oxygen tension was maintained during the reactor operation. However, uncontrolled growth of the pellets made continuous reactor operation difficult. The citric acid productivity obtained from 15 vol.% foam particles containing immobilized cells was 0.135 g/l per hour. This productivity of immobilized cells was almost the same as that of free cells. The oxygen level dropped to half saturation in 5 days in the immobilized cell culture in contrast to 2 days in the free cell culture.     

Ultrastructure of poplar cortical cells during the transition from growing to wintering stages and vice versa

Summary Electron microscopic studies revealed that major cytological changes in the cortical cells of poplar (Populus euramericana cv. gelrica) began to occur in early September in conjunction with the metabolic transition from the growing to the wintering stage. During this transition, the cells became temporarily rich in endoplasmic reticulum, polysomes and vesicles. As the conspicuous formation of organelles progressed, the large vacuoles became smaller and filled with osmiophilic materials. Undefined organelles (protein-lipid bodies) also increased in number. From late October until March, organelles involved in protein synthesis were sparsely distributed in the cells, indicating that the number of these organelles is probably linked to the seasonal cycle of protein synthesis. In early February, after release from dormancy, fusion of vacuoles proceeded in the cells. The inclusion of organelles and a gradual decrease in the amount of osmiophilic materials in the vacuoles occurred at this stage. Subsequently, the structure of the cells continued to undergo changes to accommodate growth, which occurred in early May.     

Selective inhibition of MAO-A in serotonergic synaptosomes by two amphetamine metabolites, p-hydroxyamphetamine and p-hydroxynorephedrine

The present study was carried out mainly to clarify whether the two amphetamine metabolites, p-hydroxyamphetamine (P-OHA) and p-hydroxynorephedrine (p-OHN) are taken up by mouse brain 5-hydroxytryptamine (5-HT) nerve terminals to inhibit type A monoamine oxidase (MAO-A) and then potentiate the abnormal behavior, head-twitch. Of the two metabolites, only intracerebroventricular p-OHA, at 80 μg/mouse, sufficient to cause a head-twitch response (HTR), appreciably inhibited MAO-A activity without affecting MAO-B activity in homogenates of the mouse striatum, hypothalamus and the rest of the forebrain; and p-OHN did not inhibit either type of MAO at the dose tested. Estimation of intra- and extrasynaptosomal MAO-A activity showed that both metabolites significantly inhibited only the intrasynaptosomal deamination of 5-HT by MAO-A with p-OHA being more potent. Taken together with our previous findings, these present results clearly indicate that p-OHA may accumulate in the 5-HT nerve terminals through the uptake system, and concomitantly inhibit MAO-A activity. These actions of p-OHA may increase intraneuronal 5-HT levels and then potentiate 5-HT release to cause interaction with the post-synaptic 5-HT receptors.     

Mapping of the entomocidal fragment of Spodoptera -specific Bacillus thuringiensis toxin CryIC

 Insecticidal CryI protoxins of Bacillus thuringiensis are activated by proteolysis in the midgut of insects. A conservation of proteolytic cleavage sites in the CryI proteins facilitates the expression of active toxins in transgenic plants to obtain protection from various insects. However, the engineering of CryIC toxins has, thus far, failed to yield applicable resistance to armyworms of Spodoptera species representing common insect pests worldwide. To improve the production of recombinant CryIC toxins, we established a CryIC consensus sequence by comparative analysis of three cryIC genes and tested the stability and protease sensitivity of truncated CryIC toxins in Escherichia coli and in vitro. In contrast to previous data, the boundaries of trypsin-resistant CryIC core toxin were mapped to amino acid residues I28 and R627. Proteolysis of the truncated CryIC proteins showed that Spodoptera midgut proteases may further shorten the C-terminus of CryIC toxin to residue A615. However, C-terminal truncation of CryIC to residue L614, and a mutation causing amino acid replacement I610T, abolished the insecticidal activity of CryIC toxin to S. littoralis larvae, as well as its resistance to trypsin and Spodoptera midgut proteases. Because no CryIC toxin carrying a proteolytically processed N-terminus could be stably expressed in bacteria, our data indicate that, in contrast to other CryI poteins, an entomocidal fragment located between amino acid positions 1 and 627 is required for stable production of recombinant CryIC toxins. Received: 15 April 1996/Accepted: 10 July 1996     

Isolation of a root-specific cDNA encoding a ns-LTP-like protein from the roots of bean (Phaseolus vulgaris L.) seedlings

A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome.     

Biodegradation of the mixtures of 4-chlorophenol and phenol by Comamonas testosteroni CPW301

A 4-chlorophenol (4-CP)-degrading bacterium, strain CPW301, was isolated from soil and identified as Comamonas testosteroni. This strain dechlorinated and degraded 4-CP via a meta-cleavage pathway. CPW301 could also utilize phenol as a carbon and energy source without the accumulation of any metabolites via the same meta-cleavage pathway. When phenol was added as a additional substrate, CPW301 could degrade 4-CP and phenol simultaneously. The addition of phenol greatly accelerated the degradation of 4-CP due to the increased cell mass. The simultaneous degradation of the 4-CP and phenol is useful not only for enhanced cell growth but also for the bioremediation of both compounds, which are normally present in hazardous waste sites as a mixture.