Incapacity of 5-hydroxytryptophan to act against the depression of tumor-resisting power of host animal which is induced by whole body x-irradiation]

The effect of L-5-hydroxytryptophan (L-5-HTP) against the depression of tumor-resisting power in host animal, which is induced by a whole body x-irradiation of 500 R, was studied. Host animal and tumor cell used were LAF, hybrid mouse and mastocytoma cell, respectively. The i.p. injected dose of L-5-HTP was 6.25 mg per mouse, which is most effective for protecting mice from radiation-induced hematopoietic death. In the experimental conditions, the agent was not effective to prevent the radiation-induced depression of tumor-resisting power of the animal.     

Rat liver cholesterol 7 alpha-hydroxylase. Pretranslational regulation for circadian rhythm.

A complete cDNA encoding cholesterol 7 alpha-monooxygenase (EC 1.14.13.17) which had been isolated from rat liver cDNA libraries by using specific antibodies to the enzyme (Noshiro, M., Nishimoto, M., and Okuda, K. (1989) FEBS Lett. 257, 97-100) was totally sequenced. The cDNA contained a 1,509-base pair open reading frame encoding 503 amino acid residues (Mr = 56,880) and an unusually long 3'-untranslated region rich in AT sequence in the total length of 3,545 base pairs. The predicted amino acid sequence displays less than 30% similarity to other sequenced cytochrome P-450s indicating that the 7 alpha-hydroxylase constitutes a novel family of cytochrome P-450. The AT-rich region often contained ATTTA motifs, 5'-AAT-3' or 5'-TAA-3' trinucleotides which were reported to be involved in rapidly degrading mRNA. Employing the specific antibodies and the cDNA as probes, a diurnal variation of the levels of the three factors, i.e. enzyme protein, mRNA, and enzyme activity, was studied on rat livers prepared at various times of the day. In normal animals, all three factors exhibited maximum level at 10:00 p.m. and minimum at 10:00 a.m. No significant sexual difference was observed. Cholestyramine feeding increased all three factors at 10:00 a.m. close to the maximum levels of the normal rats, but did not show a significant increase at 10:00 p.m. On the contrary, starvation markedly decreased all three factors either at 10:00 a.m. or at 10:00 p.m., while maintaining still the diurnal variation. A good correlation of the levels of mRNA to the enzyme activities and the protein levels demonstrates that pretranslational regulation is most likely a mechanism for the circadian rhythm of 7 alpha-hydroxylase. The marked diurnal fluctuation of the amount of protein and the level of mRNA also indicates their rapid turnover. The short half-life of mRNA could be correlated with the structure of the 3'-untranslated region of the mRNA characteristic of rapidly degrading mRNA, i.e. abundance of motif, AUUUA, and existence of 5'-AAU-3' or 5'-UAA-3' trinucleotides in single-stranded regions of the secondary structure.     

Mitotic checkpoints.

Entry into mitosis is triggered by activation of maturation promoting factor and a complex of p34cdc2 kinase and cyclin B. Activation induces nuclear lamina breakdown, chromosome condensation and mitotic spindle assembly. Exit from mitosis is initiated by the degradation of cyclin B and the subsequent inactivation of maturation-promoting factor. A more thorough understanding of the checkpoints for initiation of and exit from mitosis has evolved during the past few years.     

Aspartic acid residues at positions 190 and 192 of rat DNA polymerase beta are involved in primer binding

The sequence Gly-Asp-Met-Asp, spanning positions 189-192 of rat DNA polymerase beta, is similar to the sequence motif Gly-Asp-Thr-Asp that is highly conserved in a number of replicative DNA polymerases from eukaryotic cells, viruses, and phages. The role of this sequence in the catalytic function of rat DNA polymerase beta was investigated by individually changing each amino acid in this region by site-directed mutagenesis. The mutant enzymes DE190 and DE192, in which aspartic acid residues at positions 190 and 192, respectively, were replaced by glutamic acid, showed about 0.1% activity of the wild-type enzyme. On the other hand, the replacement of Gly-189 by alanine or Met-191 by isoleucine or threonine only slightly affected the enzyme activity. A gel mobility shift assay showed that DNA complexes with enzyme DE190 and especially with DE192 were less stable than the corresponding complex with the wild-type enzyme. Kinetic analysis with these mutant enzymes indicate that their Km's for primer DNA were about 10-fold higher than that of the wild type, while Km's for deoxyribonucleoside triphosphate were not changed. Since neither DE190 nor DE192 had any significant alteration in secondary structure, our results suggest that both Asp-190 and Asp-192 are located in the active site and are involved in the interaction of DNA polymerase beta with primer.     

Chromosomes of G2 arrested cells are easily analyzed by use of the 'tsBN2' mutation

The tsBN2 cell line, a temperature sensitive (ts) mutant of the BHK21/13 cell line (Nishimoto, T. et al. Somatic Cell Genet. 4, 323-340, 1978) has a ts defect in its regulatory mechanism for the initiation of chromosome condensation, the so-called, premature chromosome condensation (PCC) being induced at a nonpermissive temperature (Nishimoto, T. et al. Cell 15, 475-483, 1978, Nishimoto, T. et al. J. Cell Physiol. 109, 299-308, 1981). Using the 'tsBN2' mutation, we analyzed chromosomes of tsBN2 cells arrested in the G2 phase with neocarzinostatin (NCS) and found considerable aberrations, such as gaps, breaks and double minutes-like fragmentation, in addition to a typical G2-chromosome (a long filamental chromosome made up of two chromatids). Our results provide evidences that the tsBN2 cell line can be used for examinations of the chromosomes of cells arrested in the G2 phase.     

Design,synthesis and biological evaluation of novel 7-azaspiro[3.5]nonane derivatives as GPR119 agonists

The design and synthesis of a novel class of 7-azaspiro[3.5]nonane GPR119 agonists are described. In this series, optimization of the right piperidine N-capping group (R²) and the left aryl group (R³) led to the identification of compound 54g as a potent GPR119 agonist. Compound 54g showed a desirable PK profile in Sprague-Dawley (SD) rats and a favorable glucose lowering effect in diabetic rats.     

Enhancement of muscarinic receptor-coupled phosphatidyl inositol hydrolysis in diabetic bladder

We previously have shown an increase in muscarinic receptor density in streptozotocin (STZ)-induced diabetic and sucrosefed diuretic rat detrusor that correlates with an increase in the contractile response to muscarinic agonist (J Pharmacol Exp Ther 248: 81, 1989; Diabetes 40: 265, 1991). To investigate the signal transduction pathway involved in this altered functional response, we examined muscarinic receptor-coupled phosphatidylinositol metabolism in STZ-diabetic, sucrose-fed diuretic and age-matched control rat bladders. [³H]myo-inositol uptake was similar in all groups, but incorporation of myo-inositol into phosphatidylinositol (PI) was significantly increased in the diabetic bladder compared to the sucrose-fed and control rat bladders. Carbachol-induced increase in inositol phosphate (IPs) production was higher in the diabetic bladder than in bladders from control and sucrose-fed animals although the EC₅₀ values were similar for all groups. Enhanced inositol phosphate production after muscarinic agonist stimulation may be due not only to the upregulation of muscarinic receptors but also to the increased incorporation of myo-inositol into PI in the STZ-induced diabetic bladder.