Heterogeneity of the retinal G-protein transducin from frog rod photoreceptors. Biochemical identification and characterization of new subunits.

Transducin, a retinal G-protein, has been shown to exist as heterotrimers of alpha (39,000), beta (36,000), and gamma (approximately 7,000) subunits. Blue Sepharose CL-6B column chromatography of a transducin preparation extracted with a metal-free, low salt buffer containing GTP showed three distinct alpha and two distinct beta gamma activities in frog (Rana catesbeiana) rod outer segment. The binding of a hydrolysis-resistant GTP analog in these alpha fractions was proportional to the amount of the M(r) 39,000 protein. The first alpha was eluted in a complex with an inhibitory subunit of cGMP phosphodiesterase, but alpha subunits in the second and the third fractions were not complexed with any proteins. Two-dimensional gel electrophoresis and characterization with regard to the interaction with the inhibitory subunit of cGMP phosphodiesterase suggested that the first and the second alpha s were the same protein; however, the third alpha showed different characters as follows. We designated alpha in the first two fractions as alpha 1, and alpha in the third fraction as alpha 2. Nonlinear regression analysis for the binding of a hydrolysis-resistant GTP analog to both alpha subunits revealed a single class of GTP binding sites with an apparent stoichiometry of 1 mol of GTP/mol of alpha. Compared with alpha 1, alpha 2 required larger amounts of rhodopsin and beta gamma for the binding of a hydrolysis-resistant GTP analog. alpha 2 also showed less binding with the inhibitory subunit of cGMP phosphodiesterase. Both alpha 1 and alpha 2 complexed with beta gamma or beta delta (described below) were substrates for pertussis toxin-dependent ADP-ribosylation. The protein profiles of two beta gamma fractions revealed that the main fraction was composed of a beta gamma complex; however, the second active fraction was composed of beta complexed with delta (M(r) 12,000). Compared with beta gamma, beta delta stimulated GTP binding to alpha 1 at approximately 10-fold higher concentration. Two-dimensional gel electrophoresis revealed five beta and two gamma isoforms in beta gamma. Only one beta isoform was present in beta delta. The diversity of transducin subunits may reflect different signaling pathways in visual signal transduction.     

Convulsions in senescence-accelerated mice (SAM-R/1/Eis).

Senescence-accelerated mice (SAM) are one of the animal models used for studying senescence, which consist of several substrains such as SAM-R/1, R/2, P/1, P/2. SAM-R/1/Eis maintained in Eisai Tsukuba Research Laboratories, Ibaraki, Japan, was originally introduced as a substrain of a normal control SAM-R/1 from Kyoto University, Japan. We have noted signs of convulsions in SAM-R/1/Eis mice during routine animal care, particularly while changing cages. We identified the clinical signs and determined the concentrations of glucose and immunoreactive insulin in plasma of SAM-R/1/Eis mice. There were no differences in the male:female ratios of mice showing prodrome only, grand mal, or no-signs. The ages at which prodrome and grand mal were first noted peaked between 20 and 25 weeks. Concentrations of glucose and immunoreactive insulin in plasma did not indicate the mice were in insulin hypoglycemia, which is one cause of convulsions. AKR strain mice, some of which originated with the SAM strain are known to become convulsive by repeated "throwing" stimulations. Conversely, in SAM-R/1/Eis, throwing stimuli are not needed to cause convulsive signs. Thus it is likely that in SAM-R/1/Eis mice the signs are triggered by repeating mild environmental changes, such as changing cages. The results of this study show that SAM-R/1/Eis is neither a normal control strain, nor an original SAM-R/1 strain. But it is possible that SAM-R/1/Eis is another useful animal model for studying spontaneous convulsion.     

Evaluation of the micronucleus test using a Chinese hamster cell line as an alternative to the conventional in vitro chromosomal aberration test.

The in vitro micronucleus (MN) test was carried out simultaneously with the conventional chromosomal aberration (CA) test on 11 clastogenic chemicals or spindle poisons with different modes of action using a Chinese hamster cell line (CHL). The method of slide preparation for the MN test was the same as that for the conventional metaphase analysis, except that 1% acetic acid in methanol was used as the cell suspension medium for air-drying (to preserve the cytoplasm around the nucleus). All chemicals tested induced micronuclei reproducibly and dose-dependently in good agreement with the results of metaphase analysis (r = 0.99). Since the MN test methodology is simple and the observation of MN is less subjective than that of CA, we conclude that the in vitro MN test would be a good alternative to the conventional CA test for screening the genotoxicity of chemicals.     

Topochemical analysis of morphiceptin and dermorphin bioactivities.

To elucidate the topochemical requirements for bioactivities of morphiceptin (Tyr-Pro-Phe-Pro-NH2) and dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), we have designed and synthesized two diastereomers Tyr-(L and D)-(NMe)Ala-Phe-D-Pro-NH2. Both the analogs display high activities in the guinea pig ileum assay. The only difference in the composition of these two diastereomers arises from the chirality at residue 2. The high mu-receptor activities are attributed to structures where the Tyr1-L-(NMe)Ala2 amide bond assumes a cis configuration while the Tyr1-D-(NMe)Ala2 amide bond assumes a trans configuration. Accessible space studied for the second residues of these molecules confirms the fact that the L-(NMe)Ala2 analog belongs to the morphiceptin family of opioids while the D-(NMe)Ala2 analog belongs to the dermorphin class of opioids. The similarity in the spacial array of the analogs explains their high mu-receptor activities and indicates that they are likely recognized at the same opioid receptor.     

Hernandulcin in hairy root cultures of Lippia dulcis

The hairy root culture of Lippia dulcis Trev., Verbenaceae, was established by transformation with Agrobacterium rhizogenes A4. The transformed roots grew well in Murashige and Skoog medium containing 2% sucrose. The roots turned light green when they were cultured under 16 h/day light. The green hairy roots produced the sweet sesquiterpene hernandulcin (ca. 0.25 mg/g dry wt) together with 20 other mono- and sesquiterpenes, while no terpenes were detected in the nontransformed root cultures. The growth and hernandulcin production in the hairy root cultures were influenced by the addition of auxins to the medium. The addition of a low concentration of chitosan (0.2 – 10.0 mg / l) enhanced the production of hernandulcin 5-fold.Abbreviations Cht chitosan - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog(1962)     

Determination of eperisone in human plasma by gas chromatography—mass spectrometry

A gas chromatographic—mass spectrometric method was developed to determine eperisone hydrochloride, 4′-ethyl-2-methyl-3-piperidinopropiophenone hydrochloride, in human plasma over the concentration range 0.2–40 ng/ml. Excellent sensitivity was achieved by selection of a favorable fragment ion, m/z 98, of eperisone and reduction of heat decomposition of eperisone by using a splitless injector and a shortened capillary column. The method described here allows the determination of plasma concentrations as low as 0.2 ng/ml, the concentration attained 6 h after a single oral administration of 50 mg. At eperisone hydrochloride concentrations higher than 0.5 ng/ml, the mean inter-day variation of accuracy of the assay was less than 12%.     

Fitness and its components in Drosophila melanogaster

Fitness and its components in Drosophila melanogaster were estimated under the homozygous condition. All of these parameters, except for egg-to-adult viability and female fecundity, were measured under interspecific competition with D. hydei. The following results were obtained: (1) Significant genetic variation was detected in fitness, productivity, adult viability, total egg-to-adult viability and female fecundity. (2) Significant correlation was obtained between fitness and productivity (rp = 0.792 for phenotypic correlation, rg = 0.945 for genotypic correlation), between fitness and total egg-to-adult viability (rp = 0.355, rg = 0.395), and between fitness and female fecundity (rp = 0.451, rg = 0.511). (3) There was no significant correlation between total egg-to-adult viability and fecundity (rp = -0.046). The biological implications of the interrelationship between fitness and its components are discussed.     

An epidemic of echovirus 18 in 1988 in Japan--high association with clinical manifestation of exanthem. A report of the National Epidemiological Surveillance of Infectious Agents in Japan

Laboratory reports of isolation of echovirus serotype 18 (E18) slightly increased in the summer of 1987 followed by a sharp increase with a peak in July, 1988. A total of 1,094 isolations were reported during these two years from 39 laboratories participating in the National Epidemiological Surveillance of Infectious Agents in Japan. When compared with the previous E18 outbreaks, a higher proportion of children at two years of age or under (58.3%) and a much higher incidence of exanthem (46.4%) were remarkable. Meningitis-associated isolations were reported in 30.6%, less than half of the percentage of the previous epidemic.     

Kinetic analysis of the acid-alkaline conversion of horseradish peroxidases.

The nature of the acid-alkaline conversion of horseradish peroxidases was studied by measuring four rate constants in reactions, E + H+ (k1) in equilibrium (k2) EH+ and E + H2O (k3) in equilibrium (k4) EH+ + OH-, where EH+ and E denote the acid and alkaline forms of the enzymes. The values of k1, (k2 + k3), and k4 were obtained by measuring the relaxation rates of the acid leads to alkaline and alkaline leads to acid conversions by means of th pH jump method with a stopped-flow apparatus. The value of k3 could also be obtained by measuring the rate of reactions between hydrogen peroxide and peroxidases at alkaline pH. The measurements were conducted with four peroxidases having different pKa values: peroxidase A )pKa = 9.3), peroxidase C (pKa = 11.1), diacetyldeuteroperoxidase A (pKa = 7.7), and diacetyldeuteroperoxidase C (pKa = 9.1). The value of k1 was about 10(10) M-1 s-1 in the reaction of the four enzymes while k4 was quite different between the enzymes. The pKa was determined by k3 and k4 for the natural peroxidases and by k1 and k2 for the diacetyldeuteroperoxidases. The mechanism of the acid-alkaline conversion was discussed in comparison with that of metmyoglobin.