A Clostridium perfringens Vector for the Selection of Promoters

A promoter selection vector for Clostridium perfringens genes was constructed from a C. perfringens-Escherichia coli shuttle vector, pJIR418. The plasmid carries a promoterless chloramphenicol acetyltransferase gene (catP), derived from pIP401, downstream of the multiple cloning sites of pUC18. When a promoter region of the phospholipase C gene was inserted into one of the cloning sites, derivatives of C. perfringens strain 13 carrying the resultant plasmid acquired resistance to chloramphenicol. This plasmid should be a useful reporter system for C. perfringens genes.     

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.     

Charged-particle mutagenesis. 1. Cytotoxic and mutagenic effects of high-LET charged iron particles on human skin fibroblasts.

Cytotoxic and mutagenic effects of high-LET charged iron (56Fe) particles were measured quantitatively using primary cultures of human skin fibroblasts. Argon and lanthanum particles and gamma rays were used in comparative studies. The span of LETs selected was from 150 keV/microns (330 MeV/u) to 920 keV/microns (600 MeV/u). Mutations were scored at the hypoxanthine guanine phosphoribosyl transferase (HPRT) locus using 6-thio-guanine (6-TG) for selection. Exposure to these high-LET charged particles resulted in exponential survival curves. Mutation induction, however, was fitted by the linear model. The relative biological effectiveness (RBE) for cell killing ranged from 3.7 to 1.3, while that for mutation induction ranged from 5.7 to 0.5. Both the RBE for cell killing and the RBE for mutagenesis decreased with increasing LET over the range of 1.50 to 920 keV/microns. The inactivation cross section (sigma i) and the action cross section for mutation induction (sigma m) ranged from 32.9 to 92.0 microns2 and 1.45 to 5.56 X 10(-3) microns2; the maximum values were obtained by 56Fe with an LET of 200 keV/microns. The mutagenicity (sigma m/sigma i) ranged from 2.05 to 7.99 X 10(-5) with an inverse relationship to LET.     

Nonspecific lipid transfer protein in castor bean cotyledon cells: subcellular localization and a possible role in lipid metabolism.

The subcellular localization and several biochemical activities of nonspecific lipid transfer protein (nsLTP) were investigated. A section of a castor bean cotyledon cell was labeled with anti-nsLTP serum followed by protein A-gold. Gold particles were more abundant in the glyoxysome matrix and the vessel cell wall than in other areas. Cell fractionation analysis of 6-day-old castor bean cotyledons by sucrose density gradient centrifugation demonstrated that 13% of nsLTP was distributed in the glyoxysomal fraction, identified on the basis of catalase as a marker, and 87% in the soluble fraction near the top of the gradient. The location of castor bean nsLTP in glyoxysomes was further confirmed by in vitro import experiments. The synthesized precursor of nsLTP (pro-nsLTP-C) was incorporated into intact castor bean glyoxysomes and processed to the mature form after import into the glyoxysomes, but it was not imported into canine pancreatic microsomes. Castor bean nsLTP-A was found to possess the ability to bind oleic acid and oleoyl-CoA by means of a method involving Lipidex 1000. The dissociation constants (Kd) for oleic acid and oleoyl-CoA binding to nsLTP-A were 4.8 and 5.0 microM, respectively. The saturated binding capacities (Bmax) for oleic acid and oleoyl-CoA per mol of nsLTP-A were 1.1 and 1.2 mol, respectively. When acyl-CoA oxidase activity was assayed in the glyoxysomal fraction, marked enhancement of the activity was observed in the presence of nsLTP. These results suggest the possibility that nsLTP regulates fatty acid beta-oxidation through the enhancement of acyl-CoA oxidase activity in glyoxysomes. The occurrence of castor bean nsLTP in the vessel wall was discussed.     

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.