Relative efficacies of 1,4-diazepines on GABA-stimulated chloride influx in rat brain vesicles

The effects of 1,4-diazepines with two annelated heterocycles [brotizolam (WE 941), ciclotizolam (WE 973) and WE 1008] on gamma-aminobutyric acid (GABA)-stimulated chloride influx into rat brain membrane vesicles were examined. Brotizolam enhanced GABA (30 microM)-stimulated 36Cl- influx (146.1% of control), while ciclotizolam and WE 1008 showed only a small enhancement (119.3% and 119.1%, respectively) of GABA-stimulated 36Cl- uptake. Brotizolam resulted in a left shift of the GABA dose response curve at lower concentrations of GABA (10 microM), while at higher concentrations of GABA (1 mM), brotizolam caused a reduction of the maximal response. The enhancement of GABA-stimulated 36Cl- uptake by brotizolam (0.1 microM) was antagonized by Ro 15-1788. At higher concentration of GABA (300 microM), brotizolam inhibited GABA-stimulated 36Cl- uptake in a dose dependent manner and Ro15-1788 failed to antagonize this effect. These results suggest that 1) brotizolam produces an enhancement of GABA (30 microM)-stimulated chloride influx through the benzodiazepine receptor. 2) brotizolam inhibition of GABA (300 microM)-stimulated chloride influx involves an additional mechanism, and 3) the sedative-hypnotic action of brotizolam may be related to its high efficacy at the benzodiazepine/GABA-gated chloride channel.     

Single subunit structure of the human thyrotropin receptor.

We have produced rabbit antibody against a synthetic peptide corresponding to N-terminal region of the extracellular domain of human thyrotropin receptor (hTSH-R) (N peptide, aminoacid residues 29-57). Western blot analysis revealed that N-peptide antibody recognized recombinant hTSH-R stably expressing in CHO-K1 cells as a mol. wt. about 104 kDa regardless in the presence or absence of disulfide-reducing agent. The band was not detected in untransfected CHO-K1 cells and no band was also stained by the antibody absorbed with N-peptide. In a reducing condition, the antibody also bound the rat receptor from FRTL5 cells as the same molecular size (104 kDa). These results clearly indicate that TSH-R is composed of a single subunit and that two subunit model for the TSH-R may reflect artifactual proteolytic cleavage of the receptor during membrane preparation.     

Effect of dietary tin deficiency on growth and mineral status in rats

To clarify the influence of dietary tin deficiency on growth and mineral status, the following two different synthetic diets were fed to male Wistar rats: group 1—a diet containing 1.99 μg tin/g; group 2—a diet containing 17 ng tin/g. The rats in group 2 showed poor growth, lowered response to sound, and alopecia, with decreased food efficiency compared with rats in group 1. The changes of mineral concentrations in tissues observed in group 2, compared with group 1, are summarized as follows: calcium concentration in lung increased; magnesium concentration in lung decreased; iron concentrations in spleen and kidney increased; iron concentration in femoral muscle decreased; zinc concentration in heart decreased; copper concentrations in heart and tibia decreased; manganese concentrations in femoral muscle and tibia decreased. These results suggest that tin may be essential for rat growth.     

Stimulatory effect of glycine betaine on l-lysine fermentation

Summary The growth rate, sugar consumption rate, and production rate of an l-lysine producing Brevibacterium lactofermentum mutant were stimulated by addition of exogenous glycine betaine. Glycine betaine stimulated the growth rate especially in media of inhibitory osmotic stress, and the stimulation was independent of any specific solute. Therefore growth stimulation by glycine betaine was considered to be an osmoprotective effect. A strong enhancement of the sugar consumption rate and the l-lysine production rate was observed even with resting cells under osmotic stress as well as in a fermentation with growing cells. These data indicated that the osmoprotective effects of glycine betaine on l-lysine production can be independent of protein synthesis. Offprint requests to: Yoshio Kawahara     

Effects of Mutations of Rad50, Rad51, Rad52, and Related Genes on Illegitimate Recombination in Saccharomyces Cerevisiae

To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rad51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.     

Resuscitation of Vibrio cholerae O1 strain TSI-4 from a viable but nonculturable state by heat shock

Abstract Vibrio cholerae strain TSI-4 was incubated in an M9 salt solution at 15 °C for more than 100 days. The plate counts showed no viable cells on day 30, but a broth culture from that day showed the growth of bacteria. However, after 35 days the bacteria entered the nonculturable state, based on the assessment of both the plate counts and broth culture. A portion of the culture was heated at 45 °C for 1 min in a water bath and subsequently plated onto a nutrient agar plate. More than 1000 colonies were recovered after this heat-shock treatment. The recovered cells showed the same chromosomal DNA pattern in the restriction map and the same outer membrane protein pattern in SDS-PAGE. Recovery of viable cells by heat-shock was achieved in cultures grown on M9 salt but not from cultures grown in phosphate-buffered saline. This suggests that the presence of NH₄Cl in the M9 salt solution may support the growth of the bacteria in a low nutrient medium, while also playing an important role in resuscitation.     

Serological Characterization of Trichosporon cutaneum and Related Species

Recent molecular biological, chemical, physiological and morphological studies indicate that Trichosporon cutaneum and related species should be reclassified. In this study, antigenic characteristics of the species were determined. The results of adsorption experiments revealed that there were at least three serological types: I, II and III. Specific factor sera I, II and III were prepared on the basis of adsorption experiments and isolates were serotyped by cell slide agglutination (CSA). Since the CSA test was difficult to read in some strains, the results of the CSA test were compared with the findings from an enzyme-linked immunosorbent assay (ELISA). For the ELISA, crude polysaccharide antigens prepared from the culture supernatant were used as the antigen. The types determined by ELISA correlated well with those determined by the CSA test. These data suggest that T. cutaneum and related species have at least three serological types, and that the typing can be done by either CSA or ELISA.     

Cloning and sequence analysis of the highly expressed melanin-synthesizing gene operon from Streptomyces castaneoglobisporus

 Streptomyces castaneoglobisporus HUT6202 overproduces a diffusible melanin-like pigment. An operon, designated mel, containing a gene that encodes tyrosinase, which is involved in the synthesis of melanin pigment, was cloned from the chromosomal DNA of the microorganism into the high-copy plasmid pAK114 and expressed in S. lividans. The tyrosinase activity of the transformed cells was at approximately a 110-fold higher level than that of the same host carrying the plasmid pIJ702, which has the same replication origin as pAK114 and carries the mel operon from S. antibioticus. The sequence analysis of the S. castaneoglobisporus mel operon revealed that an open- reading frame consisting of 378 base pairs(bp), designated ORF378, was found upstream of the tyrosinase gene (TYRC) consisting of 819 bp. In the present study, we constructed a chimeric mel operon consisting of ORF378 from S. castaneoglobisporus and the tyrosinase gene (TYRA) from S. antibioticus. The chimeric mel operon or the S. antibioticus mel operon, which consists of ORF438 and TYRA, expressed the tyrosinase activity in Escherichia coli intracellularly when located under the control of lacZ promoter, and the tyrosinase activity from the former was at a 30-fold higher level than that from the latter. This suggests that the gene contributing to the high expression of the tyrosinase activity in S. castaneoglobisporus is ORF378, rather than TYRC. Received: 12 June 1995/Received revision: 24 July 1995/Accepted: 7 September 1995     

Neurological disorder and excessive accumulation of calcium in brain of clinically vitamin A-deficient rats

Three groups of rats were fed two types of synthetic diets for 52 d. The—A group was allowed free access to a vitamin A-deficient diet and showed classical signs of vitamin A deficiency. The brain was the only organ in our experiment where no significant weight difference was present among the three groups. In the brain, calcium concentration was significantly higher in the—A group when compared with the PF (Pair-fed; allowed restricted amount of control diet) and +A groups (allowed free access to control diet). In the tibia, calcium and magnesium concentrations were significantly lower in the—A group when compared with other two groups. Excessive accumulation of calcium in brain and apparently similar unbalance in bone, mineral concentration were observed in central nervous system (CNS) degenerative diseases. Our results suggest that abnormal metabolism of calcium and magnesium in some tissues and excessive accumulation of calcium in brain may be responsible for the development of neurological disorders in vitamin A-deficient rats.     

Structure, function and expression of a murine homeobox protein AREC3, a homologue of Drosophila sine oculis gene product, and implication in development.

The cDNA clones encoding ARE(Na,K-ATPase alpha1 subunit gene regulatory element) binding protein AREC3 were isolated from myoblast C2C12 cells and mouse skeletal muscle cDNA library. At least four alternatively spliced forms of AREC3 cDNA were identified. Sequence analysis indicates that AREC3 has an extensive homology with the Drosophila sine oculis gene product required for development of the entire visual system [Cheyette et al.(1994) Neuron 12, 977-996]. The homologous region including a homeodomain is required for specific DNA binding to ARE. A transactivation domain was identified in the C-terminal part of the AREC3 by reporter gene assays using GAL4-AREC3 fusion protein constructs. Immunohistochemistry revealed that AREC3 localized to the nucleus and cytoplasm of myoblast C2C12 cells, and the production of AREC3 is augmented during muscle differentiation. Western blot analysis indicated that the 115 kDa form of AREC3 protein is increased in the cytoplasmic extract, and the 67kDa form is increased both in nuclear and cytoplasmic extracts of C2C12 cells during muscle differentiation.