Transformation and expression of a cloned δ-endotoxin gene in bacillus thuringiensis

Abstract A shuttle vector containing the replication region of a resident plasmid of B. thuringiensis , was used to determine the conditions allowing efficient transformation of B. thuringiensis by electroporation. Using this plasmid a δ-endotoxin gene was cloned and expressed both in Escherichia coli and B. thuringiensis . It was shown that this gene was poorly expressed in the wild type situation whereas after cloning in acrystalliferous strains of B. thuringiensis large amounts of crystal protein were obtained.     

Elicitor-induced metabolic changes in cell cultures of chickpea (Cicer arietinum L.) cultivars resistant and susceptible to Ascochyta rabiei

Cell-suspension cultures of two chickpea (Cicer arietinum L.) cultivars, resistant (ILC 3279) and susceptible (ILC 1929) to the fungus Ascochyta rabiei (Pass.) Lab., showed differential accumulation of the phytoalexins medicarpin and maackiain, and transient induction of related enzyme activities after application of an A. rabiei-derived elicitor. The chalcone-synthase (CHS) activity (EC 2.3.1.74) which is involved in the first part of phytoalexin biosynthesis exhibited a maximum 8–12 h after elicitation in the cells of both cultivars. Concomitant with the fivefold-higher phytoalexin accumulation, CHS activity increased twofold in the cells of the resistant cultivar. The maximum of the elicitor-induced CHS-mRNA activity was determined 4 h after onset of induction in the cultures of both cultivars, although in cells of cultivar ILC 3279 this mRNA activity was induced at a level twofold higher than that in cells of the susceptible race ILC 1929. Investigations of CHS isoenzymes by two-dimensional gel electrophoresis of immunoprecipitated in-vitro-translated protein indicated the presence of five proteins. In the cells of both cultivars only two of the isoenzymes were induced after elicitor treatment. Analysis of the total in-vitro-translated proteins by two-dimensional gel electrophoresis showed that the constitutively expressed patterns of mRNA activities in the cell cultures of the two cultivars were identical. After elicitation, considerably more translatable mRNAs were induced in the cells of cultivar ILC 3279. The few induced proteins, and their respective mRNA activities, which could be detected in the cells of the susceptible cultivar, all existed in the cells of the resistant cultivar, too. One highly induced protein (M_r 18 kDa) found in the cells of cultivar ILC 3279 reached its maximum mRNA activity 6 h after elicitor application. The amount of this protein was hardly increased in the cells of the susceptible cultivar. This protein appears to be excreted from the cells into the growth medium.Abbreviations CHS chalcone synthase - IEF isoelectric focussing - ILC international legume chickpea - PR-protein pathogenesis-related protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Financial support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. The authors thank Dr. K. Hahlbrock (Max-Planck-Institut für Züchtungsforschung, Köln, FRG) for provision of antisera and the International Centre for Agricultural Research in the Dry Areas (Aleppo, Syria) for plant material.     

Energy expenditure and locomotor activity in mice selected for food intake adjusted for body weight

Summary The aim of this study was to examine the differences in physical activity and their contribution to differences in energy utilization in mice, selected either high or low for food intake, adjusted for body weight, which show correlated responses in lean content and metabolic rate. Simultaneous measurements of fasting metabolic rate and activity were made in lines of mice selected at either: a young age, 4-to 6-week food intake corrected for 4-week body weight; or an older age, 8- to 10-week food intake corrected for mean weight at 8 and 10 weeks of age. Correlated response in metabolic rate was found to have been accompanied by changes in locomotor activity near the ages at selection in both sets of lines. Activity, however, accounted for only a small proportion of variation in fasting heat production, generally less than 5%, although a highly positive correlation (r=0.63) between the two traits was found. It was concluded that selection for food intake adjusted for body weight has led to correlated response in physical activity. In consequence, mice selected in the upward direction expend some of the excess energy intake rather than assimilating it as body mass and are, therefore, slightly leaner than their counterparts selected in the downward direction.     

Reaction center light harvesting B875 complexes from Rhodocyclus gelatinosus: characterization and identification of quinones

Reaction center-B875 pigment-protein complexes were purified from Rhodocyclus gelatinosus. The proteic components consist of 7–8 polypeptides among which some were identified by their apparent molecular weights: the light harvesting B875 polypeptides and of 8 and 6 kDa, reaction center L (23 kDa), M (28 kDa) and H (34 kDa), cytochrome c (43 kDa). Four c-type hemes were found per reaction center. Flash-induced absorbance changes showed the presence of both Q_A and Q_B in the complex. Charge recombination times were determined to be: 1.16±0.2 (n=30) for P⁺Q_AQ_B ^- and 7–10 ms for P⁺Q_A ^- in presence of herbicides. From quinone analysis on one hand and kinetics of charge recombination on the other hand, we proposed that in the reaction center of Rhodocyclus gelatinosus Q_A is menaquinone 8 and Q_B is ubiquinone 8.     

Structure-activity relationships on hyperglycemia by representatives of the adipokinetic/hyperglycemic hormone family in Blaberus discoidalis cockroaches

Summary Several members of the adipokinetic/hyperglycemic neurohormone family from several different invertebrate species have been prepared by solid-phase peptide synthesis and assayed by a modified in vivo hyperglycemic bioassay in Blaberus discoidalis cockroaches. The hypertrehalosemic hormone (HrTH) is the endogenous hypertrehalosemic factor for B. discoidalis and was the most potent peptide in the assay. The more divergent the sequence of a family member from Blaberus HrTH, the less potent was the bioanalog. Manduca adipokinetic hormone is the most divergent peptide of the family and was totally inactive in the bioassay. Locusta adipokinetic hormone I had reduced maximum activity in the assay, which suggests that Ser⁵ is an important residue for the transduction of the hyperglycemic response. The direct relation between bioanalog similarity to Blaberus HrTH sequence and potency suggests that the hormone and target cell receptor for HrTH have evolved to maintain an optimal fit.Abbreviations AKH adipokinetic hormone - HrGH hyperglycemic hormone - HrTH hypertrehalosemic hormone - RPCH red pigmentconcentrating hormone - CAH cardioacceleratory hormone. Hormone abbreviations are according to the convention of Gäde and Raina (1989) except that the genus names are not abbreviated     

固氮酶铁钼辅因子FeMo-co在水与非水体系内重组活力与催化活力的比较研究

 本文提出一种测定FeMo-co催化活力的反应体系,用此反应体系,在测定FeMo-co催化活力的过程中,FeMo-co与变种UW45抽提液的重组活性始终保持不变。讨论了水含量、还原剂对FeMo-co催化活力和重组活力的影响。     

Chinese hamster ovary cells continuously secrete a cysteine endopeptidase

Summary The protease activity in serum-free conditioned medium of chinese hamster ovary (CHO) cells was measured using peptidyl (or aminoacyl)-4-methylcoumaryl-7-amides (MCAs) as the substrates. Aminopeptidase increased in level as amounts of nonviable cells increased during cultivation in serum-free medium, indicating that the activity seems to be originated from intracellular proteases. The activity toward Boc-Leu-Arg-Arg-MCA, which was strongly inhibited by p-chloromercuribenzonate and N-ethylmaleimide, was the strongest among those toward peptidyl-MCAs in the conditioned medium within 48 h-cultivation in serum-free medium. In contrast to the case of aminopeptidase activity, the endopeptidase activity decreased in level after 48 h-cultivation although amounts of nonviable cells increases. Thus, CHO cells continuously secrete the cysteine proteases. This work was supported by the management of the Research Association for Biotechnology as a part of the R&D of Basic Technology for Future Industries sponsored by NEDO (New Energy and Industrial Technology Development Organization).     

Radical Cations in Aromatic Hydrocarbon Carcinogenesis

Most carcinogens, including polycyclic aromatic hydrocarbons (PAH), require metabolic activation to produce the ultimate electrophilic species that bind covalently with cellular macromolecules to trigger the cancer process. Metabolic activation of PAH can be understood in terms of two main pathways: one-electron oxidation to yield reactive intermediate radical cations and monooxygenation to produce bay-region diol epoxides. The reason we have postulated that one-electron oxidation plays an important role in the activation of PAH derives from certain common characteristics of the radical cation chemistry of the most potent carcinogenic PAH. Two main features common to these PAH are: 1) a relatively low ionization potential, which allows easy metabolic removal of one electron, and 2) charge localization in the PAH radical cation that renders this intermediate specifically and efficiently reactive toward nucleophiles. Equally important, cytochrome P-450 and mammalian peroxidases catalyze one-electron oxidation. This mechanism plays a role in the binding of PAH to DNA. Chemical, biochemical and biological evidence will be presented supporting the important role of one-electron oxidation in the activation of PAH leading to initiation of cancer.