Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38)

A novel neuropeptide with 38 residues (PACAP38) was isolated from ovine hypothalamic tissues using the pituitary adenylate cyclase activation in rat pituitary cell cultures as a parameter of the biological activity (Miyata et al, Biochem. Biophys. Res. Commun. 164, 567-574, 1989). From the side fractions obtained during the purification of PACAP38, a shorter form peptide with 27 residues corresponding to the N-terminal 27 amino acids of PACAP38 and amidated C-terminus was isolated and named as PACAP27. Synthetic PACAP27 showed a biological activity of adenylate cyclase stimulation comparable to PACAP38. Moreover PACAP27 which shows a considerable homology with vasoactive intestinal polypeptide (VIP) demonstrated a similar vasodepressor activity as VIP, but the adenylate cyclase stimulating activity was about 1000 times greater than VIP.     

The nucleotide sequence of the mouse immunoglobulin epsilon gene: comparison with the human epsilon gene sequence

We have determined the nucleotide sequence of the immunoglobulin epsilon gene cloned from newborn mouse DNA. The epsilon gene sequence allows prediction of the amino acid sequence of the constant region of the epsilon chain and comparison of it with sequences of the human epsilon and other mouse immunoglobulin genes. The epsilon gene was shown to be under the weakest selection pressure at the protein level among the immunoglobulin genes although the divergence at the synonymous position is similar. Our results suggest that the epsilon gene may be dispensable, which is in accord with the fact that IgE has only obscure roles in the immune defense system but has an undesirable role as a mediator of hypersensitivity. The sequence data suggest that the human and murine epsilon genes were derived from different ancestors duplicated a long time ago. The amino acid sequence of the epsilon chain is more homologous to those of the gamma chains than the other mouse heavy chains. Two membrane exons, separated by an 80-base intron, were identified 1.7 kb 3' to the CH4 domain of the epsilon gene and shown to conserve a hydrophobic portion similar to those of other heavy chain genes. RNA blot hybridization showed that the epsilon membrane exons are transcribed into two species of mRNA in an IgE hybridoma.     

Differences in liver homogenates from Donryu, Fischer, Sprague-Dawley and Wistar strains of rat in the drug-metabolizing enzyme assay and the salmonella/hepatic S9 activation test

Comparison studies for detecting differences between liver microsome and S9 preparations from 4 strains (Donryu, Fischer, Sprague-Dawley, Wistar) of young male rats were carried out with pretreatment of the animals by inducers such as PCBs and PB plus 5,6-BF. Each microsome fraction was assayed for the enzymic activity of metabolism of model substrates such as aniline, benzophetamine, BP, DMN and 7-ethoxycoumarin. The hepatic S9 sample was also compared, as regards its metabolizing ability to activate 9 pre-mutagens (2AA, AAF, o-AAT, BP, DAB, DMBA, DMN, m-PDA, quinoline) to directly acting mutagens in the Salmonella/hepatic S9 activation test by using TA98, TA100 and TA1537 strains with or without cytochrome P450 inhibitors (SKF-525A, metyrapone, 7,8-benzoflavone).In the enzymic assay with PCBs-induced microsomes, BP hydroxylation revealed a strain-specific difference: the microsomes from Fischer and Wistar rats were more effective for metabolizing BP than those from the other strains of rat. The effect of induction by PB plus 5,6-BF for Fischer rats showed relatively higher enzymic activity in the same induction group. Other microsomes prepared from rats with and without induction by PB plus 5,6-BF did not show a clear-cut strain dependency in the enzymic activities assayed.In the mutation experiments with hepatic S9 samples, the examination of DAB and quinoline revealed a marked strain difference when S9 samples prepared from PCBs-pretreated and PB-plus-5,6-BF-induced rats were used: the S9 sample from Fischer rats was available for activating the two pre-mutagens to directly acting mutagens. No marked difference in the metabolic activation of the remaining 7-pre-mutagens was observed on other S9 preparations.In examinations of mutagenicity activities with the use of three inhibitors, the two S9 preparations made with the two induction methods showed inhibition profiles closely similar to each other. However, there were minor differences in the profiles by these inhibitors.From these findings it was concluded that Fischer rat-liver S9 is useful for detecting mutagens in the metabolic activation test, when induction by PB plus 5,6-BF was used in the Ames Salmonella test.     

Solid phase fluoroimmunoassay for 11-deoxycortisol in serum using 21-amino-17-hydroxyprogesterone

Solid phase fluoroimmunoassay of serum 11-deoxycortisol (17,21-dihydroxy-4-pregnene-3,20-dione) was established using fluorescein isothiocyanate-labelled 11-deoxycortisol and anti-11-deoxycortisol antibody-conjugated polyacrylamide beads. 21-Amino-17-hydroxyprogesterone (21-amino-17-hydroxy-4-pregnene-3,20-dione) was synthesized as a useful derivative for preparing the fluorescent dye conjugate. Serum 11-deoxycortisol was measured with this assay system after extraction and purification by Sephadex LH-20 column chromatography. The minimal amount of 11-deoxycortisol detected was 40 pg/tube and the measurable range was from 0.04 to 5.0 microgram/dl. Intra- and inter-assay coefficients of variation were 8.3% (n=6) and 9.8% (n=5), respectively. 11-deoxycortisol values determined by the present assay correlated well with those determined by radioimmunoassay. The present assay is particularly suitable for estimating the conditions of the pituitary and adrenocortical functions.     

Indole-3-acetaldehyde oxidase of pea seedlings

Indole-3-acetaldehyde oxidase was partially purified from the epicotyl of Pisum satiyum seedlings by column chromatography using CM-Sephadex and Sephadex G-150. The enzyme was only active in the presence of molecular oxygen. The activity was maximal at pH 8.0, and the Km value for indole-3-acetaldehyde was 1.4 × 10⁻³ M . The enzyme was inhibited strongly by p -hydroxymercuribenzoate, cyanide and hydroxylamine, suggesting that it contains sulfhydryl group(s) and a metal component such as iron.     

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 10. IN VIVO AND IN VITRO SYNTHESIS OF alpha-AMYLASE IN RICE SEED SCUTELLUM

Scutellar tissues were dissected from germinating rice seeds and the incorporation of [³⁵S]methionine into the α-amylase molecule was examined by in vivo and in vitro assay systems. Immunoprecipitation with monospecific anti-α-amylase immunoglobulin G raised against the purified enzyme preparation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography were used to identify α-amylase and its possible precursor molecule. Using freshly prepared scutellar tissues, it was demonstrated that α-amylase is synthesized de novo in the scutellar epithelium and secreted into endosperm. The synthesis of α-amylase directed by the polyadenylic acid-containing ribonucleic acid isolated from the scutellar tissues was also established using the translation system of either wheat germ extract or reticulocyte lysate. The immunoprecipitable product obtained in the in vitro translation system was smaller in molecular weight than that synthesized in vivo on the basis of mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results are discussed in relation to the processing of the nascent polypeptide precursor of the enzyme molecule and the introduction of the oligosaccharide chain to the cleaved polypeptide to make up the mature form of α-amylase.     

Lysis of growing fissin-yeast cells induced by aculeacin A,a new antifungal antibiotic

Cells of Schizosaccharomyces pombe grown in the presence of aculeacin A, a peptide antibiotic, were lysed resulting the death of cells. Under high osmolarity, the cellular lysis induced by aculeacin A was considerably reduced. The use of synchronous-culture systems distinguished cell elongation from cell division revealed that the sites of aculeacin A-induced lysis on the fission yeast were the end(s) and the cell plate region, corresponded to the regions of the cell wall synthesis. Aculeacin A-resistant survivors exhibited morphological alterations which were swollen at one or both ends of the cell and appeared drumstick or dumbbel like; the wall of the bulge region was observed to be stained with a fluorescent brightener, as well as that of the cell plate region. These effects of aculeacin A are discussed as compared with effects of 2-deoxy-D-glucose.     

The preferential codon usages in variable and constant regions of immunoglobulin genes are quite distinct from each other.

The pattern of codon utilization in the variable and constant regions of immunoglobulin genes are compared. It is shown that, in these regions, codon utilizations are quite distinct from one another: For most degenerate codons, there is a selective bias that prefers C and/or G ending codons to U and/or A ending codons in the constant region compared with the bias in the variable region. This would strongly suggest that, in immunoglobulin genes, the bias in code word usage is determined by other factors than those concerning with the translational mechanism such as tRNA availability and codon-anticodon interaction. A possibility is also suggested that this differance of code word usage between them is due to the existence of secondary structure in the constant region but not in the variable region.     

Endocrine function in a case of beta-adrenergic hyperdynamic circulatory state.

Endocrine functions were investigated in a case of "beta-adrenergic hyperdynamic circulatory state". This state was diagnosed by (1) typical symptoms of cardiac awareness, (2) physical findings (increments of pulse rate and blood pressure by changing positions or walking), (3) increase in cardiac output (5.25 l/min leads to 14.03 l/min) and decrease in circulatory time (10.8 sec leads to 5.5 sec) by isoproterenol infusion (0.02 mug/min/kg body weight), (4) rapid loss of symptoms and above findings by propranolol treatment (30 mg per os daily) and reappearance by discontinuing medication. The mechanism of insulin response to glucose has been a controversy as to whether the secretion is transmitted by beta-receptor or independent glucose receptor. And in this physiologic beta-adrenergic state, it was found that insulin responses in IVGTT and OGTT were within normal limit. When beta-adrenergic condition was corrected by propranolol treatment, insulin responses were shown lowered, though in the normal range. This could be reproduced by discontinuing medication. Insulin, glucagon and growth hormone secretions caused by arginine were also found normal, but during the period the patient was on propranolol therapy, all responses were decreased, within the normal range. These results do not positively support the idea that glucose receptor is linked to beta-receptor. They do not either agree with the contention that secretions of insulin, glucagon and growth hormone induced by arginine are mediated through beta-receptors.     

Leaf structure and function in evergreen trees and shrubs of Japanese warm temperate rain forest I. The structure of the lamina

Information has been obtained as a part of a wider study of leaf structure, water relations and mineral status, which is to include work on a considerable variety of evergreen forests. Fifteen structural features have been investigated in well-lit leaves of 60 dicotyledonous species of Japanese Warm Temperate Rain Forest, this sample giving an 81% cover of the relevant species (Table 1). The salient features of the average leaf are summarised on p. 200. The leaves of shrubs and small trees have been compared with those of tall and medium trees; some of the differences were unexpected. For example, the leaves of the lower growing species tended to be thicker and to have thicker outer walls. The leaves of the whole Japanese sample were compared with those of 40 species in Tropical Lowland Rain Forest in New Britain. Although the average leaf area of the Japanese species was less than one tenth of that of the species from New Britain, the thickness and internal structure of the leaf and the size and density of the stomata were quite similar in the two sets of leaves (Table 2). The Japanese leaves were somewhat more xeromorphic in that they had thicker outer walls in the upper and lower epidermis.