Synthetic inositol trisphosphate analogs and their effects on phosphatase, kinase, and the release of Ca2+

A series of inositol 1,4,5-trisphosphate (IP3) analogs and positional isomers was examined to explore the structure-activity relationships among IP3 5-phosphatase, IP3 3-kinase, and the release of Ca2+. All analogs with additional groups on the 2nd position of IP3 inhibited the hydrolysis of [5-32P]IP3 catalyzed by erythrocyte ghosts, with a lower Ki value than seen with IP3. IP3 dehydroxylated at the 2nd position also had a lower Ki, while 2,4,5-IP3 or cyclic(1:2), 4,5-IP3 had higher Ki values. Among these compounds 2-deoxy-IP3 was as potent as IP3 in inhibiting the phosphorylation by [3H] IP3-3-kinase in rat brain cytosol. The other compounds, except for 2,4,5-IP3 inhibited the phosphorylation, however, 2-30 times higher concentrations were required. By lowering free Ca2+, the concentrations required for half-maximal inhibition were low, while those of IP3, 2-deoxy-IP3, and positional isomers remained unchanged. These compounds acted as full agonists in releasing Ca2+ from permeabilized macrophages, although 1.6-50-fold higher concentrations than IP3 were required. These compounds also inhibited the binding of [3H]IP3 to rat cerebellum and bovine adrenal cortex microsomes, but the potencies were 2.9-33 times less than that of IP3. Thus, the 2nd position of IP3 can be modified with only a slight loss of biological activity.     

Alternative splicing mechanism in a cytochrome P-450 (P-450PB-1) gene generates the two mRNAs coding for proteins of different functions

Two mRNAs for P-450PB-1 and P-450PB-1(ps) are about 2 kilobase pairs long and have identical sequences with each other except for one short region of high variability (Kimura, H., Yoshioka, H., Sogawa, K., Sakai, Y., and Fujii-Kuriyama, Y. (1988) J. Biol. Chem. 263, 701-707). To clarify the origin of the short replacement block between the two mRNAs, we isolated several genomic clones containing relevant gene sequences. Sequence analysis of these genomic clones revealed that the two short segments specific for the two mRNAs are tandemly arranged in a genomic sequence and form exonic sequences equipped with AG and GT sequences on their 5' and 3' ends, respectively, and the putative consensus sequences for the lariat formation. The two short sequences lie between the two exonic sequences coding for the common part of the two mRNAs. Taken together with the structure of the related P-450(M-1) gene (Morishima, N., Yoshioka, H., Higashi, Y., Sogawa, K., and Fujii-Kuriyama, Y. (1987) Biochemistry 26, 8279-8285), all these results clearly demonstrate that the two mRNAs are generated from a single gene by alternative splicing at the eighth exons. The synthesis of the two mRNAs is regulated temporally in livers of male and female rats and brains of the female animals. One of the two mRNAs codes for a monooxygenase of P-450PB-1, and the other (P-450PB-1(ps) mRNA) lacks the sequence coding for the heme-binding site conserved among all species of P-450 molecules, and, therefore, it cannot function as a monooxygenase. The immunoblot analysis using an antibody specific for the 15-mer peptide uniquely encoded by P-450PB-1(ps) mRNA shows that the P-450PB-1(ps) peptide is synthesized at least in rat livers of both sexes in temporally regulated manners and is bound to the microsomal membranes. The function of this peptide remains to be seen.     

Particle size of airborne mouse crude and defined allergens

Laboratory animal allergy is a serious occupational diseases of many workers and scientists engaged in animal experimentation. Control measures depend upon characterization of allergens including airborne particles. This study measured the particle size of crude mouse urine and pelt aeroallergens generated in mouse housing rooms and compared them with mouse serum albumin, a defined major allergen. Allergens were detected by specific immunological methods. Most crude and defined allergens (74.5-86.4%) concentrated on a filter with a retention size greater than 7 microns. In distrubed air, allergen concentration increased 1.4 (albumin) to 5 (crude) fold and the proportion of small particles increased from 1.4% in calm air to 4.5% in distrubed air. This information on the generation and size distribution of aeroallergens will be important in the development of effective counter measures.     

Retinoic acid ambivalently regulates the expression of MyoD1 in the myogenic cells in the limb buds of the early developmental stages.

The expression of MyoD1 in myogenic cells located in the muscle prospective region of the limb bud at stage 20-22 was highly sensitive to retinoic acid. Unlike RAR-beta, the expression of MyoD1 mRNA in the muscle precursor cells was significantly increased by retinoic acid at lower concentrations (0.1-10 nM), but inhibited by it at higher concentrations (0.1-1 microM). The ambivalent modulation of MyoD1 expression suggested that MyoD1 expression is regulated by not only the retinoic acid receptor and its response element, but also by other factors. Retinoic acid may be involved in the differentiation of the myogenic cells during early development.     

Purification and partial characterization of two types of growth-inhibitory protein latently present in rabbit serum.

Normal rabbit serum contained two kinds of growth-inhibitory protein, GI-I and GI-II, in latent forms. These latent inhibitors were activated by incubation at 37 degrees C for 12 h, and their activation was lowered by inhibitors for serine, cysteine and metalloproteinases. Both growth inhibitors were highly purified in active forms by successive column chromatographies. GI-I showed a major protein band with an Mr of 18,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while GI-II showed a major protein band with an Mr of 36,000. GI-I and GI-II half-inhibited the growth of rat tumorigenic cell line (RSV-BRL) at concentrations of 0.5 ng/ml and 10 ng/ml, excess concentrations. Of the 15 cell lines tested, GI-I specifically inhibited the growth of rodent and lagomorph cells, whereas GI-II nonspecifically inhibited the growth of all cell lines tested. Specificities for cell type and malignancy were not observed with either inhibitor. These growth inhibitors were stable to a reducing reagent and proteinase inhibitors, but labile to urea, acid, organic solvents, trypsin, plasmin and heating at 95 degrees C for 5 min. These properties suggested that both growth inhibitors might be distinct from known growth-inhibitory factors.     

Immunohistochemical demonstration of urotensins I and II in the caudal neurosecretory system of the Japanese charr,Salvelinus leucomaenis,retained in sea water

This paper is concerned with part of the role and function of the caudal neurosecretory system of the charr,Salvelinus leucomaenis, studied by immunohistochemistry. In order to elucidate the different histologic changes, we examined the immunoreactivities of urotenisn I (UI) and urotensin II (UII) in 3 experimental groups: the feral (river) fish, the fresh-water aquarium-, and sea water aquarium-retained fish. Coexistence of UI and UII was demonstrated in most of the smaller and larger neurons distributed in and near the urophyseal system of all 3 groups. However, some of the larger neurons were immunoreactive only to a single hormone, UI or UII. Merely a few neurons indicated no reactivity for either UI or UII. No such clearcut differences were encountered immunohistochemically in the 3 groups. Neuronal and urophysial immuno-reactivity to UI of feral and fresh-water-retained fish was slightly stronger than that of sea water-retained fish. Moreover, in sea water-retained fish, the intensity of immunoreactivity for UI was variable, and the number of neurons positive for UII only was somewhat larger than that in feral and fresh-water-retained fish. A series of UII-positive cerebrospinal fluid (CSF)-contacting neurons were seen in the ependymal and subependymal layers ventral to the central canal of the spinal cord in every group. These CSF-contacting neurons might constitute another neurosecretory system aside from the ordinary caudal neurosecretory system equipped with urophysis. In contrast to the hypothalamohypophysial neurosecretory system, the caudal neurosecretory system did not show any significant changes among the 3 groups. This suggests that urotensins I and II have no essential role in osmoregulation of the charr.     

Protein Kinases Are Involved in Prolonged Acetylcholine Release from Rat Hippocampus Induced by Thyrotropin-Releasing Hormone Analogue NS-3

Abstract: The effects of various protein kinase inhibitors on acetylcholine release from the rat hippocampus induced by the local application of NS-3 (montirelin hydrate, CG-3703), a thyrotropin-releasing hormone analogue, into the medial septum-diagonal band were examined using in vivo microdialysis. Perfusion of NS-3 (1 µ M ) into the medial septum-diagonal band for 20 min produced a pronounced and prolonged increase in the hippocampal acetylcholine efflux. Pretreatment of the medial septum-diagonal band with either K-252a, a nonselective protein kinase inhibitor, or selective protein kinase A inhibitor H-89 almost completely blocked the acetylcholine efflux evoked by NS-3, and selective protein kinase C inhibitor calphostin C inhibited the action of NS-3. On the other hand, NS-3 (0.1–10 µ M ) or TRH (1–100 µ M ) increased the cyclic AMP efflux from the medial septum-diagonal band in a concentration-dependent manner, as measured by microdialysis. These findings suggest that protein kinases A and C in the neurons of the medial septum-diagonal band are involved in the mechanism of the prolonged stimulation of acetylcholine release from the hippocampus induced by thyrotropin-releasing hormone and its analogue, NS-3.     

Effects of Nerve Growth Factor and Phorbol Derivative on Reactivation of Herpes Simplex Virus Type 1 in Cultured Cells of Latently Infected Adult Mouse Trigeminal Ganglia

Reactivation of herpes simplex virus type 1 (HSV-1) occurred rapidly in cells of latently infected adult mouse trigeminal ganglia which were cultured in serum-free medium in the presence of sufficient nerve growth factor (NGF). However, HSV-1 reactivation was delayed significantly in ganglionic cultures in the absence of exogenous NGF or in cultures treated with 2-aminopurine in the presence of NGF. The delayed viral reactivation in ganglionic cultures without NGF was accelerated by treatment with phorbol myristate acetate or dibutyryl cyclic AMP. Culture conditions which affected HSV-1 reactivation did not affect replication of HSV-1 in normal ganglionic cultures.     

Organization and expression inPseudomonas putida of the gene cluster involved in cephalosporin biosynthesis fromLysobacter lactamgenus YK90

TheLysobacter lactamgenus YK90pcbAB gene encoding -(l--aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase is located immediately upstream of thepcbC gene in the same orientation in the gene cluster involved in cephalosporin biosynthesis. ThepcbAB gene encodes a large polypeptide composed of 3722 amino acid residues with a molecular mass of 411 593 Da. The predicted amino acid sequence has a high degree of similarity with those of known ACV synthetases from fungi and actinomycetes. Within thepcbAB amino acid sequence, three conserved and repeated domains of about 600 amino acids were identified. The domains also share a high degree of similarity with non-ribosomal peptide synthetases such as gramicidin synthatase 2 ofBacillus brevis. ThepcbAB gene was expressed under the control of thelac promoter inPseudomonas putida. Expression of the gene cluster involved in cephalosporin biosynthesis inP. putida led to the accumulation of -lactam antibiotics. Deletion analysis of an open-reading frame located between thecefE andcefD genes from the gene cluster revealed that it encoded deacetylcephalosporin C synthetase (cefF). From the results presented here and those of previous studies, the genes involved in cephalosporin biosynthesis inL. lactamgenus appear to be clustered in the orderpcb AB-pcbC- cefE-cefF-cefD-bla in the same orientation within a 17-kb region of DNA.     

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.