Spectrophotometric determination of inorganic phosphate in the presence of thiol compounds

A spectrophotometric method for inorganic phosphate determination in the presence of thiol compounds is described. Thiol compounds, which interfere with the measurement of inorganic phosphate by a modification of the method of Gomori, are removed by carboxymethylation by iodoacetate prior to the formation and reduction of phosphomolybdate complex. A linear standard curve is obtained by this method, and the method is suitable for the assay of a phosphate-releasing enzyme when the measurement must be performed in the presence of thiol compounds.     

Light and electron microscopic studies on capillaries of the goat cardiac muscle with special reference to the topographical relationship between the vessels and Purkinje fibers

The capillaries of the cardiac muscle were investigated in the goat by means of light microscopy, transmission electron microscopy and scanning electron microscopy, and the following results were obtained. The capillaries of the working cardiac muscles were numerous, arranged mainly parallel to the long axis of muscle cells and formed dense elongated networks. On the contrary, those of the terminal Purkinje fibers were relatively few in number, oriented in various directions and formed loose and circularly meshed networks surrounding the fibers. Such findings were discussed in correlation with the physiology and functional morphology of various types of the cardiac muscle cells.     

Mutations in the hepatocyte nuclear factor-4alpha gene in Japanese with non-insulin-dependent diabetes: a nucleotide substitution in the polypyrimidine tract of intron 1b.

Mutations of the hepatocyte nuclear factor 4 alpha (HNF-4alpha) gene have been demonstrated in maturity-onset diabetes of the young (MODY) 1 families. To investigate the possibility that the HNF-4alpha gene contributes to the onset of non-insulin-dependent diabetes mellitus (NIDDM) in Japanese patients, we screened all exons and flanking introns of this gene for mutations in 100 patients with NIDDM diagnosed after 25 years of age. We identified two missense mutations: M49V in exon 1c and T1301 in exon 4; and two nucleotide substitutions in introns: cytosine to thymidine at -5 nt in intron 1b and adenine to thymidine at -21 nt in intron 5. We screened an additional 220 diabetic subjects for the polymorphism in intron 1b. The c/t substitution in intron 1b was associated with NIDDM. This substitution in the polypyrimidine tract, an important cis-acting element directing intron removal, is likely to influence pre-mRNA splicing of this gene. T1301 in exon 4 was observed in only two diabetic subjects. This mutation could influence the conformation of this peptide, resulting in changes in ligand binding domain function. M49V in exon 1c was found in both diabetic and non-diabetic subjects; isoforms HNF-4alpha 4, 5, and 6 with this mutation may impair glucose metabolism in tissue. In contrast to the primary cause of nonsense and missense mutations of the HNF-4alpha gene in MODY1, the nucleotide substitution in intron 1b may partially contribute to development of NIDDM in combination with other genetic and environmental factors.     

Immunohistochemical studies on peptide- and amine-containing endocrine cells and nerves in the gut and the rectal gland of the ratfish Chimaera monstrosa

Summary The occurrence and distribution of endocrine cells and nerves were immunohistochemically demonstrated in the gut and rectal gland of the ratfish Chimaera monstrosa (Holocephala). The epithelium of the gut mucosa revealed open-type endocrine cells exhibiting immunoreactivity for serotonin (5HT), gastrin/cholecystokinin (CCK), pancreatic polypeptide (PP)/FMRFamide, somatostatin, glucagon, substance P or gastrin-releasing peptide (GRP). The rectum contained a large number of closed-type endocrine cells in the basal layer of its stratified epithelium; the majority contained 5HT- and GRP-like immunoreactivity in the same cytoplasm, whereas others were immunoreactive for substance P. The rectal gland revealed closed-type endocrine cells located in the collecting duct epithelium. Most of these contained substance P-like immunoreactivity, although some reacted either to antibody against somatostatin or against 5HT. Four types of nerves were identified in the gut and the rectal gland. The nerve cells and fibers that were immunoreactive for vasoactive intestinal peptide (VIP) and GRP formed dense plexuses in the lamina propria, submucosa and muscular layer of the gut and rectal gland. A sparse network of gastrin- and 5HT-immunoreactive nerve fibers was found in the mucosa and the muscular layer of the gut. The present study demonstrated for the first time the occurrence of the closed-type endocrine cells in the mucosa of the rectum and rectal gland of the ratfish. These abundant cells presumably secrete 5HT and/or peptides in response to mechanical stimuli in the gut and the rectal gland. The peptide-containing nerves may be involved in the regulation of secretion by the rectal gland.     

Effect of oleic acid on mitochondrial oxidative phosphorylation in rat brain slices

We tested the effect of oleic acid on oxidative phosphorylation and free fatty acid composition in rat brain slices simultaneously to investigate the relationship between the change in respiratory control ratio and the uptake of oleic acid in the brain mitochondria. The uncoupling of mitochondria was observed when the ratio of oleic acid to stearic acid in the free fatty acid fraction was nearly doubled, but was not recovered even by the addition of fatty acid-free bovine serum albumin. The data suggest that the intactness of oxidative phosphorylation of brain mitochondria is maintained by the precise control of the free fatty acid composition in the mitochondrial membranes.     

Possible involvement of calcium ions in the hatching of Schistosoma mansoni eggs in water

The possibility of involvement of calcium ions in the hatching of Schistosoma mansoni eggs in water is described. The hatching of S. mansoni eggs under low osmotic pressure was partially inhibited by EGTA (5 mM), lanthanum chloride (1-5 mM), and ruthenium red (0.1-1 mM). The reagents used in these experiments were not toxic to the eggs however, because miracidia hatched normally when the reagents were removed.     

Efficient production of wheat-barley hybrids and preferential elimination of barley chromosomes

Summary Intergeneric hybridization between four common wheat cultivars, Triticum aestivum L. cultivars Chinese Spring, Norin 12, Norin 61, and Shinchunaga, and cultivated barley, Hordeum vulgare L. cultivars Betzes, Nyugoruden, Harunanijou, and Kinai 5 were carried out in a greenhouse under 15 – 20 °C and long-day (15 h) photoperiod conditions. Two days prior to pollination, a 100 mg/1 2,4-D solution was injected into wheat stems. Among wheat cultivars, Norin 12, Norin 61, and Shinchunaga showed higher crossabilities than that of Chinese Spring, suggesting the presence of crossability gene(s) other than the kr system of Chinese Spring. Variation was also found among the barley cultivars as male parents. Betzes barley showed the highest crossability with wheat. Thus, the cross Norin 12×Betzes showed the highest crossability (8.25%), followed by Norin 61 ×Betzes (6.04%), Shinchunaga×Betzes (5.00%), and Shinchunaga×Kinai 5 (5.00%). The embryos were rescued by culture at 15–20 days after pollination. Seventyfour plants were obtained from 82 embryos. The morphology of the hybrid plants resembled that of wheat parents. Among 60 seedlings observed, 28 had 28 chromosomes, 8 had 21, 23 had aneuploid numbers of chromosomes (22–27), and 1 had 29 chromosomes. About half of the aneuploid hybrids showed mosaicism for chromosome number. By analyzing five isozyme markers of barley chromosomes, the chromosome constitutions of the aneuploid hybrids were determined. Barley chromosomes 1 and 5 were found to be preferentially eliminated in the hybrids, while chromosomes 2 and 4 were eliminated infrequently. The conditions and genetic factors for high crossability and the tendency of barley chromosome elimination are discussed.     

A Zn2+-dependent and histone H1-specific protease in sea urchin sperm

Protease activity was extracted from sea urchin sperm with 1% Triton X-100 and partially purified by DEAE-cellulose and Sephadex G-100 chromatography. The enzyme preferentially degraded histone H1, while showing only a weak activity toward other histones. Heat-denatured casein and bovine serum albumin were not digested by this enzyme under the present experimental conditions. This protease hydrolyzed only Boc-Val-Leu-Lys-MCA among various peptidyl-MCAs. The optimal pH ranged from 7 to 11. Its molecular weight was about 41,000. Among various known inhibitors of proteases, only omicron-phenanthroline effectively inhibited the activity. The enzyme was stimulated by Zn2+ or Co2+. It was inactivated by omicron-phenanthroline but could be reactivated by the addition of Zn2+ or Co2+. Therefore, this protease seems to be a metalloprotease dependent on Zn2+ or Co2+. The insensitivity of this enzyme to phosphoramidon and its very restricted substrate specificity suggest that this enzyme is very different from other metalloproteases described hitherto.     

Studies on glucosyltransferase and endogenous glucosyl acceptor in Bacillus cereus AHU 1030 membranes

A glucosyltransferase, extracted from the membranes of Bacillus cereus AHU 1030 with Tris-HCl buffer containing 0.1% Triton X-100 at pH 9.5, was separated from an endogenous glucosyl acceptor by chromatography on DEAE-Sepharose CL-6B subsequent to chromatography on Sepharose 6B. Structural analysis data showed that the glucosyl acceptor was a glycerol phosphate polymer linked to beta-gentiobiosyl diglyceride. The enzyme catalyzed the transfer of glucosyl residues from UDP-glucose to C-2 of the glycerol residues of repeating units of the acceptor. On the other hand, a lipoteichoic acid which contained 0.3 D-alanine residue per phosphorus was isolated from the cells by phenol treatment at pH 4.6. Except for the presence of D-alanine, this lipoteichoic acid had the same structure as the glucosyl acceptor. The rate of glucosylation observed with the D-alanine-containing lipoteichoic acid as the substrate was less than 40% of that observed with the D-alanine-free lipoteichoic acid, indicating that the ester-linked D-alanine in the lipoteichoic acid interferes with the action of the glucosyltransferase. The enzyme also catalyzed glucosylation of poly(glycerol phosphate) which was synthesized in the reaction of a separate enzyme fraction with CDP-glycerol. Thus, it is likely that the glucosyltransferase functions in the synthesis of cell wall teichoic acid.     

Human liver microsomal cytochrome P-450 mephenytoin 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabolism. Purification and characterization of two similar forms involved in the reaction

Two forms of cytochrome P-450 (P-450), designated P-450MP-1 and P-450MP-2, were purified to electrophoretic homogeneity from human liver microsomes on the basis of mephenytoin 4-hydroxylase activity. Purified P-450MP-1 and P-450MP-2 contained 12-17 nmol of P-450/mg of protein and had apparent monomeric molecular weights of 48,000 and 50,000, respectively. P-450MP-1 and P-450MP-2 were found to be very similar proteins as judged by chromatographic behavior on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE- and CM-cellulose columns, spectral properties, amino acid composition, peptide mapping, double immunodiffusion analysis, immunoinhibition, and N-terminal amino acid sequences. In vitro translation of liver RNA yielded polypeptides migrating with P-450MP-1 or P-450MP-2, depending upon which form was in each sample, indicating that the two P-450s are translated from different mRNAs. When reconsituted with NADPH-cytochrome-P-450 reductase and L-alpha-dilauroyl-sn-glyceryo-3-phosphocholine, P-450MP-1 and P-450MP-2 gave apparently higher turnover numbers for mephenytoin 4-hydroxylation than did the P-450 in the microsomes. The addition of purified rat or human cytochrome b5 to the reconstituted system caused a significant increase in the hydroxylation activity; the maximum stimulation was obtained when the molar ratio of cytochrome b5 to P-450 was 3-fold. Rabbit anti-human cytochrome b5 inhibited NADH-cytochrome-c reductase and S-mephenytoin 4-hydroxylase activities in human liver microsomes. In the presence of cytochrome b5, the Km value for S-mephenytoin was 1.25 mM with all five purified cytochrome P-450s preparations, and Vmax values were 0.8-1.25 nmol of 4-hydroxy product formed per min/nmol of P-450. P-450MP is a relatively selective P-450 form that metabolizes substituted hydantoins well. Reactions catalyzed by purified P-450MP-1 and P-450MP-2 preparations and inhibited by anti-P-450MP in human liver microsomes include S-mephenytoin 4-hydroxylation, S-nirvanol 4-hydroxylation, S-mephenytoin N-demethylation, and diphenylhydantoin 4-hydroxylation. Thus, at least two very similar forms of human P-450 are involved in S-mephenytoin 4-hydroxylation, an activity which shows genetic polymorphism.