Effect of carbon dioxide and ethylene on berberine production and cell browning in Thalictrum minus cell cultures

Cultured cells of Thalictrum minus L. (Ranunculaceae), transferred from culture flasks to a bubble column bioreactor, produced little berberine and turned dark brown, even when supplied with sufficient oxygen. This phenomenon was ascribed to the removal of CO₂ from the culture medium by bubbling air, and could be reproduced in flask cultures artificially deprived of CO₂. The induction of cell browning by exogenously administered ethylene suggested that CO₂ probably acts antagonistically against endogenously generated C₂H₄. The physiological damage caused by forced aeration could be prevented by adding 2 % CO₂ to the air in the bioreactor.     

An Analysis of Osterhout and Hill's Salt Bridge Experiments in Characeae Internode

Conduction of action potentials in Chara internodal cells wasblocked at a 5%-urethane treated region. The action potentialscould be propagated beyond this region when an electric bridgewas built across it with a low enough resistance that the actioncurrent across it could depolarize the membrane at the distaljunction of the bridge up to the threshold level. After recoveryof propagation, the configuration of the action current flowingthrough the bridge changed from monophasic to diphasic. Coursesof the monophasic action current and the depolarizing potentialof the resting membrane were in parallel with the course ofaction potential, although they were slightly out of phase witheach other. The magnitudes of the current and depolarizing potentialagreed well with those estimated using a simplified equivalentcircuit of the bridge arrangement or with those observed usingan electric model circuit. ¹Present address: Department of Physiology, Tohoku UniversitySchool of Dentistry, Seiryo-machi, Sendai 980, Japan. ²Present address: Biology Laboratory, Kyoritsu Women's University,Hachioji, Tokyo 193, Japan. (Received December 18, 1985; Accepted March 26, 1986)     

Glucosylation of esculetin by plant cell suspension cultures

Cell suspension cultures of Lithospermum erythrorhizon, Gardenia jasminoides and Nicotiana tabacum were capable of glucosylating esculetin to esculin (7-hydroxycoumarin-6-O-β-D-glucoside). Especially, a culture strain of Lithospermum erythrorhizon was superior in the esculetin glucosylating capability; 40 to 50% of esculetin administered to the culture medium at early stationary growth stage was converted into esculin within 24 h. The rate of glucosylation was also dependent on the growth stage and the medium composition especially growth hormones and sugar.     

Plant saponins can affect DNA recombination in cultured mammalian cells

Some plant extracts and products are known to affect mammalian cells, tissues and organisms as they contain a toxic substance or a metabolic stimulant. Our biochemical investigations revealed that some plant saponins can increase the cellular DNA repair activity and the general recombinase activity measured by in vitro assay (1). In the experiments described here, HeLa cells were cultured for several days with plant saponins or flavonoids and analyzed to measure i) recombination activity of the cell extract by induction of Tcr colonies from two mutant DNAs (mutants 1 and 2, which are both tetracycline sensitive) after transformation into E. coli recA-, and ii) repair synthesis of nuclear DNA followed by incorporation of 3H-thymidine. Saikosaponins a, b1, d, ginsenosides Rb1, Re, Rh and flavonoid baicalin caused a significant stimulation of intermolecular recombination. It is worth noting that none of the plant saponins and flavonoids had any inhibitory or toxic effect at concentrations less than 25 micrograms/ml in the culture media.     

DNA analysis by microfabricated capillary electrophoresis device.

The LIGA (Lithographie Galvanoformung Abformung) process using synchrotron radiation lithography is applied to the microfabrication of capillary array electrophoresis (CAE) device. Laser-induced fluorescence detection system for the CAE device has been constructed by the modification of laser confocal fluorescence microscopy. DNA molecules were detected during migrating in the microchannels filled with polymer separation matrices under electric field to optimize the separation conditions for DNA analysis. Based on this observation, we demonstrated that microfabricated CAE device is realized the fast separation of DNA.     

Change in membrane potential induced by streptolysin O,a pore-forming toxin: flow cytometric analysis using a voltage-sensitive fluorescent probe and rat thymic lymphocytes

Streptolysin O (SLO) is a bacterial pore-forming toxin that is employed to permeabilize cell membranes in some biological experiments. SLO forms various types of pores with different shapes, increasing membrane ion permeability and subsequently inducing changes in membrane potential. To characterize the pores formed by SLO, the changes in membrane potential induced by SLO in rat lymphocytes were considered using flow cytometry with a voltage-sensitive fluorescent probe, bis-(1,3-dibutylbarbituric acid)trimethine oxonol (Oxonol). SLO caused three types of membrane potential responses accessed with Oxonol. One type induces a great decrease in Oxonol fluorescence (large hyperpolarization) that may be elicited via the increase of Ca²⁺-dependent K⁺ permeability by SLO-induced influx of external Ca²⁺. A second type is an increase in Oxonol fluorescence (depolarization) that may be caused by a nonspecific increase in membrane cation permeability. The third type is a small decrease in Oxonol fluorescence (small hyperpolarization), probably via an increase in Cl^– permeability. That SLO transitionally changes membrane ion permeability may have implications in the pathology of pyogenic group streptococci infections in which SLO is thought to be one of the key virulence factors.     

Evaluation of ready-to-use liquid reagents for clinical chemistry in laboratory animals.

We evaluated the ready-to-use liquid reagents for clinical chemistry (6 tests), to assess their suitability for use in the toxicology laboratory setting. Hitachi 736 automated analyzer was used for the analyses. The evaluation included the following studies: Precision, Linearity, Effects of interference substances such as hemolytic hemoglobin, bilirubin, turbidity to the analytical values and correlation to the solid reagents, which are prepared each time they are needed. The precision and linearity data were within the reagents' specifications. Results of comparison of the liquid reagents and the solid reagents in analyzing plasma samples of rats, dogs and monkeys were generally good except for a bias in results for GOT and GPT, regardless of the animal species tested. It is concluded that these types of liquid reagents can be used in clinical pathology examinations in animal studies.     

Transcriptional regulator SpxA1a controls the resistance of the honey bee pathogen Melissococcus plutonius to the antimicrobial activity of royal jelly

Royal jelly (RJ), a brood food of honey bees, has strong antimicrobial activity. Melissococcus plutonius, the causative agent of European foulbrood of honey bees, exhibits resistance to this antimicrobial activity and infects larvae orally. Among three genetically distinct groups (CC3, CC12 and CC13) of M. plutonius, CC3 strains exhibit the strongest RJ resistance. In this study, to identify genes involved in RJ resistance, we generated an RJ-susceptible derivative from a highly RJ-resistant CC3 strain by UV mutagenesis. Genome sequence analysis of the derivative revealed the presence of a frameshift mutation in the putative regulator gene spxA1a. The deletion of spxA1a from a CC3 strain resulted in increased susceptibility to RJ and its antimicrobial component 10-hydroxy-2-decenoic acid. Moreover, the mutant became susceptible to low-pH and oxidative stress, which may be encountered in brood foods. Differentially expressed gene analysis using wild-type and spxA1a mutants revealed that 45 protein-coding genes were commonly upregulated in spxA1a-positive strains. Many upregulated genes were located in a prophage region, and some highly upregulated genes were annotated as universal/general stress proteins, oxidoreductase/reductase, chaperons and superoxide dismutase. These results suggest that SpxA1a is a key regulator to control the tolerance status of M. plutonius against stress in honey bee colonies.     

A convenient route for synthesis of 2-isopropyliden-5-methyl-4-hexen-1-yl butyrate, the sex pheromone of Planococcus kraunhiae (Hemiptera: Pseudococcidae), by use of β,γ to α,β double-bond migration in an unsaturated aldehyde

The Japanese mealybug Planococcus kraunhiae (Kuwana) is an important pest which spoils many kinds of fruit in Japan. Because conventional application of insecticides is often ineffective, alternative strategies are being investigated for management of this pest. Recent studies revealed that a pheromone-based technique which interferes with sexual communication, i.e. mating disruption, was promising. However, mating disruption usually requires a substantial amount of a pheromone. I therefore developed a new and convenient route for synthesis of the P. kraunhiae pheromone, 2-isopropyliden-5-methyl-4-hexen-1-yl butyrate (fujikonyl butyrate). First, a commercially available isomer of fujikonol, 2-isopropenyl-5-methyl-4-hexen-1-ol (lavandulol), was oxidized, furnishing the corresponding aldehyde (lavandulal). The β,γ double bond of lavandulal smoothly migrated to the α,β position in the presence of acids, and as a consequence, the corresponding aldehyde of fujikonol (fujikonal) was obtained. Fujikonal was then reduced to fujikonol, which was esterified with butyric acid to give the pheromone of P. kraunhiae. All the reactions were accomplished under very mild conditions (room temperature to 50 °C) with good yields. Moreover, only small amounts of by-products were generated. The synthetic pheromone obtained by this method can be used as a mating disruptant.