Chromosomes of Temnocephala minor,an ectosymbiotic turbellarian on Australian crayfish found in Kagoshima Prefecture,with karyological notes on exotic turbellarians found in Japan

Records of exotic turbellarian species found in Japan are reviewed from taxonomic and karyological viewpoints. Temnocephala minor Haswell, 1888, an ectocommensal on a freshwater crayfish of Australia, was found from culture ponds of Cherax tenuimanus (introduced from W. Australia) in Kagoshima Prefecture. T. minor had the chromosome number of 2x = 18 (2sm + 2m + 2m + 2sm + 2m + 2m + 2m + 2sm + 2m). The following 3 species of exotic freshwater triclads were recorded from tanks and ponds used for tropical fish culture: Dugesia austroasiatica Kawakatsu, 1985 (2x = 16), Dugesia tigrina (Girard, 1850) (2x = 16) and Rhodax? sp. (3x = 24; 3x = 24 &; 3x + 1LB + 1SB = 25 + 1SB). The following 3 species of exotic terrestrial triclads were recorded: Bipalium nobile Kawakatsu et Makino, 1982 (2x = 10), Bipalium kewense Moseley, 1878 (2x = 18), and Platydemus manokwari de Beauchamp, 1962 (n = 6, 2x = 12). An extensive occurrence of P. manokwari in the Southwest Islands of Japan may be due to an unexpected introduction of the animal in very recent years.     

Over-production of Thermus protease in dense culture of Escherichia coli using two carbon sources

Escherichia coli JM109(DE3) harboring expression plasmid pkAQNÆC30, which carries the Thermus protease aqualysin I (AQI) gene, was cultivated with glucose as a sole carbon source. The final cell concentration was over 15 g dry weight/l and the amount of AQI produced reached approximately 130 kU/ml broth. Moreover, by using two carbon sources, glucose and glycerol, the production yield was increased to over 200 kU AQI/ml, while suppressing the formation of inhibitory acetic acid.     

DIVERSITY OF HALOGENATED SECONDARY METABOLITES IN THE RED ALGA LAURENCIA NIPPONICA (RHODOMELACEAE,CERAMIALES)1,2

Many morphologically similar, but chemically distinct, populations have been found in the marine red alga Laurencia nipponica Yamada (Rhodomelaceae, Ceramiales) growing in Japan. Each chemical type is characterized by a specific end-product of halogenated secondaly metabolite synthesis: chamigrane-type sesquiterpenoids such as prepacifenol and halochamigrene epoxide and C₁₅ bromoethers such as laurencin, laureatin, isoprelaurefucin, epilaurallene, and kumausallene. These seven types of secondary metabolite syntheses remained the same in the wild and under various culture conditions. Because bromoethers and terpenoids are probably synthesized by different metabolic pathways, it is virtually certain that different sets of enzymes participate in their synthesis. Prepacifenol- and laureatin-producing populations were selected as representatives of terpenoid and bromoether groups, respectively. F₁ tetrasporophytes derived from crosses between reciprocal, female and male gametophytes of prepacifenol- and laureatin-producing strains bore both types of metabolites, suggesting that the genes Producing these enzyme systems are encoded by nuclear genomes. The F₁ gametophytes resulting from the reciprocal crosses produced either prepacifenol or laureatin, and the four individuals derived from spore tetrads (a set of tetraspores derived from a single tetrasporangium) produced either prepacifenol or laureatin in a 1:1 ratio, indicating that genes participating in terpenoid synthsis and those participating in bromoether synthesis are on different loci of homologous chromosomes and are segregated at meiosis (tetrasporogenesis). One individual of this interpopulational F₁ gamtophyte produced both parental types of metabolite, perhaps indicating the occurrence of a recombination type. Natural hybrid individuals, including such recombination-type gametophytes, were found in a sympatric locality at which these two chemical types occur. F₁ tetrasporophytes derived from crosses between respective prepacifenol- and laureatin-producing strains and their F₁ gametohytes produced only parental-type metabolite-producing plants. These results indicate that the diverse chemical types can be referred to as races (chemical races).     

Stabile production of a thrombin resistant pro-urokinase derivative (PRO-UKS1) by Namalwa KJM-1 cells adapted to serum-free medium

Pro-UKS1 was designed as a thrombin-resistant derivative of pro-urokinase (pro-UK) by introducing a glycosylation site using site-directed mutagenesis. An expression plasmid for pro-UKS1, pMo1UKS1SEd1-5, was constructed and introduced into Namalwa KJM-1 cells (Hosoiet al., 1988), and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKS1 producer (resistant to 500 nM of MTX), clone 41-8, was selected and further characterized. Clone 41-8 was cultured in serum-free ITPSGF medium (Hosoiet al., 1988). Under the conventional conditions, the concentration of pro-UKS1 reached 26 g ml^–1. Addition of glucose and tri-iodothyronine (T₃) improved productivity, and the maximal productivity of pro-UKS1 was 67 g ml^–1 day^–1. In this conditioned medium, content of pro-UKS1 was above 80% of total proteins.Abbreviations BSA bovine serum albumin - dhfr dihydrofolate reductase - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - kb kilobase pairs - kDa kilodaltons - MTX Methotrexate - PBS phosphate buffered saline - pro-UK pro-urokinase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - T₃ tri-iodothyronine - Tween-PBS phosphate buffered saline containing 0.05% Tween 80     

Crystalline hemoprotein from Pseudomonas that catalyzes oxidation of side chain of tryptophan and other indole derivatives.

A new enzyme which catalyzes the oxidation of the side chain of tryptophan and other indole derivatives, has been purified to apparent homogeneity from Pseudomonas and crystallized. The overall purification was about 25-fold with a yield of 4.5%. The purified enzyme was apparently homogeneous as judged by polyacrylamide gel electrophoresis. The molecular weight estimated by gel filtration was approximately 280,000 and sedimentation coefficient (S20,w) was 11 by sucrose density gradient ultracentrifugation. The absorption spectra indicated that the enzyme was a hemoprotein. The purified enzyme was shown to catalyze the reaction in which 1 mol each of NH3 and CO2 was formed at the expense of 1 mol each of L-tryptophan and molecular oxygen. Neither peroxidase nor catalase activity was detected in the purified enzyme and no formation of H2O2 was observed during the enzyme reaction. The product(s) of the reaction was unstable but was converted to and was identified as its stable quinoxaline derivative, 2-(3-indolyl)quinoxaline, in the presence of o-phenylenediamine. These results indicate that the product of the reaction was 3-indolylglycoaldehyde or 3-indolylglyoxal. A variety of other indole derivatives such as D-tryptophan, 5-hydroxyl-L-tryptophan, tryptamine, serotonin, melatonin, N-acetyl-L-tryptophan, N-acetyl-L-tryptophanamide, 3-indoleacetamide, 3-indolelactic acid, 3-indolepropionic acid, 3-indoleethanol, and skatole were also substrates.     

Clinical analysis on steroids. XII. Occurrence of D-homoannulation during the hot acid hydrolysis of 5β-pregnane-3α,20α-diol disulfate

Two D-homosteroids were isolated from the hydrolyzate of 5β-pregnane -3α,20α-diol disulfate (II) when it was refluxed in 3N hydrochloric acid. The structures of these steroids have been elucidated as 17α-methyl-D-homo-5β-androstane-3α, 17aβ-diol (VI) and 17α-methyl-17aγb-chloro-D-homo-5β-androstan-3α-ol (VIII) by instrumental analyses. The former was identical with a synthetic specimen derived from 5β-pregnane-3α,20β-diol di-sulfate (IV) by uranediol rearrangement. The main hydrolyzates obtained were 17α-ethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol (V) and 5β-pregnane-3α, 20α-diol (III).