Mass propagation of shoots of Stevia rebaudiana using a large scale bioreactor

A procedure for the mass propagation of multiple shoots of Stevia rebaudiana is described. Isolated shoot primordia were used as the inoculum to obtain clusters of shoot primordia. Such clusters were grown in a 500 liter bioreactor to obtain shoots. A total of 64.6 Kg of shoots were propagated from 460 g of the inoculated shoot primordia. These shoots were easily acclimatized in soil.     

Physical Characterization of the Chromosomal DNA of the Yeast Saccharomyces exiguus

The number of chromosomes in the yeast Saccharomyces exiguuswas determined to be thirteen by two-dimensional pulsed-fieldgel electrophoresis. The thirteen chromosomes ranged in DNAsize from 520 to 2,600 kbp, with a total length of approximately14 Mbp. Numbers I to XIII were assigned to the chromosomes indecreasing order of DNA length. Southern hybridization analysisusing total DNAs from S. exiguus and S. cerevisiae as probesshowed that there was no significant homology between the chromosomalDNAs of the two species, except in the case of the chromosomalDNA that included rDNA. When rDNA and genes LEU2, TRP1, URA3and HO of S. cerevisiae were used as hybridization probes, itwas apparent that S. exiguus had DNA sequences homologous tothe rDNA and to the LEU2 and HO genes. In S. exiguus, rDNA-likeand LEU2-like DNAs were located on chromosomes I and IX, respectively,and HO-like DNA was located on chromosome VI or VII. (Received May 17, 1993; Accepted July 15, 1993)     

Some properties and occurrence of cytochrome c-552 in the aerobic photosynthetic bacterium Roseobacter denitrificans

Characteristics and occurrence of cytochrome c-552 from an aerobic photosynthetic bacterium, Roseobacter denitrificans, were described.Relative molecular mass of the cytrochrome was 13.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 15,000 by gel filtration. This cytochrome was a acidic protein having a pI of 5.6 and E_m was +215 mV at pH 7.0. Absorption peaks were at 278, 408 and 524 nm in the oxidized form and 416, 523 and 552 nm in the reduced form.Amino acid composition and N-terminal amino acid sequence of cytochrome c-552 determined for 24 residues had low similarities to those of cytochrome c-551 of this bacterium, which is homologous to cytochrome c ₂, although the physico-chemical properties of these two cytochromes were similar to each other.Cytochrome c-552 was maximally synthesized in the light under aerobic conditions but not in the dark. The synthesis also occurred in the presence of alternative acceptors such as trimethylamine N-oxide (TMAO) and nitrate under anaerobic conditions. Our results suggest that cytochrome c-552 is involved in TMAO respiration and denitrification in R. denitrificans, although the effect of light remains to be solved.Abbreviations E_m Midpoint redox potential - PAGE Polyacrylamide ge electrophoresis - SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis - TMAO Trimethylamine N-oxide     

Interleukin-5 induces tumor suppression by peritoneal exudate cells in mice

The antitumor activity of peritoneal exudate cells (PEC) induced by murine interleukin-5 (mIL-5) was examined using Meth-A sarcoma cells transplanted into the peritoneal cavity of mice. Although in vitro treatment of Meth-A sarcoma cells with mIL-5 did not result in inhibition of their growth, treatment of mice intraperitoneally with mIL-5 (1 g/day) from day –5 to +5 (tumor cells were inoculated on day 0) led to a significant increase in survival or even rejection of tumor cells. This antitumor effect depended on the dose of mIL-5. Interestingly, there was identical therapeutic activity when the protocol of days –10 to –1 was used as opposed to –5 to +5. In addition, post-treatment with mIL-5 from day +1 to +10 was ineffective. This suggests that the therapeutic activity of IL-5 is largely prophylactic. Under the former condition, the number of PEC was found to increase over 50-fold when compared to levels in control mice. Moreover, the antitumor effect of mIL-5 was completely abolished by subcutaneous injection of anti-mIL-5 monoclonal antibodies. The treatment of mice injected intraperitoneally with human IL-2 also resulted in an increase in survival. Winn assay experiments using PEC recovered from mIL-5-treated mice (1g/day, from day –10 to –1) revealed that these PEC could mediate antitumor activity against Meth-A sarcoma cells. Furthermore, when the cured mice were re-injected with Meth-A sarcoma cells or syngeneic MOPC 104E cells, they could reject Meth-A sarcoma cells but not MOPC 104E cells, indicating that immune memory had been generated. These results suggest that IL-5 augumented the PEC tumoricidal activity but we have no indication that the tumoricidal activity was mediated through a mIL-5-dependent mechanism.     

An envelope-shaped film culture vessel for plant cell suspension cultures and metabolite production without agitation

An envelope-shaped film culture vessel (named Culture Bag) made of fluorocarbon polymer film, which is much more permeable to oxygen, nitrogen and carbon dioxide than other films, was found to be suitable to grow plant cells in liquid medium without agitation. Proliferous BY-2 tobacco cells showed almost the same growth in a Culture Bag of 12.5 m-thick film as that in a shake flask; the growth was lower in a Culture Bag of a thicker film. Lithospermum erythrorhizon cells produced almost the same amount of red naphthoquinone pigments (shikonin derivatives) in a Culture Bag of 12.5 m-thick film as those in a shake flask although the productivity was suppressed as the film thickness increased. L. erythrorhizon cells in a Culture Bag produced much less abnormal stress metabolites (orange-colored benzoquinone derivatives) than those in a shake flask, suggesting that culturing cells in the Culture Bag was less stressful due to its stationary liquid environment.     

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).     

Augmentation of antitumor resistance by a strain of unicellular green algae,Chlorella vulgaris

Summary Growth of Meth-A tumor in CDF1 mice was inhibited significantly by injection of a hot water extract of a strain of Chlorella vulgaris (CE) into the tumor or into the subcutaneous tissue near the tumor. The augmentation of resistance by CE may require the participation of T cells and macrophages, since it was abolished or reduced in athymic nude mice or mice treated with carrageenan, a macrophage blocker. Mice treated with CE exhibited antigen-specific augmented resistance against rechallenge with tumor. Moreover, the antitumor effect of CE was comparable with that of Corynebacterium parvum, but its mechanism of effect might be different.     

Comparative reproductive patterns in culture of differentGigartina subgenusMastocarpus andPetrocelis populations from northern Japan

Samples of theGigartina pacifica-ochotensis complex were collected at 21 localities around Hokkaido and northern Honshu. The carpospore and blade tip cultures showed 3 reproductive patterns. (1) 237 (86.8%) of the 273 cultured isolates derived from single plants have a direct type of life history. (2) 29 (10.6%) isolates exhibited a heteromorphic type with the alternation of foliose gametophytes and crustose tetrasporophytes. (3) 7 (2.6%) isolates showed a mixed pattern in which carposporelings developed intoPetrocelis-like crusts, basal discs with uprightGigartina blades, or chimera-like discs with compositePetrocelis-Gigartina anatomy. CulturedGigartina blades derived from bothG. pacifica andG. ochotensis were similar in morphology. In 18 cultures from 5 localitiesPetrocelis tetraspores developed into dioeciousGigartina gametophytes. A single tetrasporeling grew into aGigartina plant that reproduced directly. In hybridization experiments with 8 male and 14 female isolates from 4 localities on Hokkaido 85 (78.0%) of 109 were positive. On the basis of these and earlier studies it is concluded that a single species is present in northern Japan:G. pacifica Kjellm. has priority overG. ochotensis (Rupr.) Rupr. ex Yendo.     

Properties of membrane-bound α-glucosidases: Possible sugar receptor protein of the blowfly, Phormia regina

Previously reported PII-type α-glucosidase located in the precipitate of the labellar homogenate of the blowfly Phormia regina was solubilized by sodium deoxycholate (DOC) and further separated into three isozymes with different molecular weight: PII-M (mol. wt 9 × 10⁴). PII-D (mol. wt 2 × 10⁵) and PII-T (mol. wt 8 × 10⁵) by molecular sieve chromatography on Biogel P-300 or Ultragel AcA-34. These three isozymes had almost the same K_m's and relative values of V_m's for several substrates, suggesting that they had the same common active site.PII-D and PII-T are more strongly embedded in the membrane than PII-M, because the proportion of PII-D and PII-T was much increased when the remaining glucosidase in the precipitate after the first solubilization was reextracted by DOC. A large peak of α-glucosidase isozyme P-IV which preferentially hydrolyze sucrose eluted just after P-II (soluble P-II) when the supernatant fraction of the labellar homogenate was chromatographed on DEAE-Sephadex A-50. P-IV was scarcely present in the precipitate fraction.Soluble P-II had the same mol. wt as PII-M and had similar properties to PII-M except for the ratio of V_m's.A large proportion of PII-D was contained in the well washed labellar integuments, a preparation rich in labellar chemosensilla. It suggests that most of the insoluble α-glucosidase contained in the dendrite in labellar chemosensilla is PII-D. PII-D (and PII-T) are possible sites of the pyranose receptor molecule because their properties and localization agree well with those of the receptor.