Existence of a Novel Hemagglutinin Having No Protease Activity in Vibrio mimicus

The protease elaborated by Vibrio mimicus is known to possess hemagglutinating ability to chicken erythrocytes, the well-known HA/protease. A non-protease hemagglutinin (HA) with strong agglutinating ability towards rabbit erythrocytes was obtained from 32 hr culture supernatant of a pathogenic environmental strain of V. mimicus. This HA (V. mimicus HA: VMHA) appeared stable at relatively higher temperature and agglutinated the erythrocytes from rabbit, guinea pig and mouse but not the erythrocytes from chicken, bovine, horse and sheep. Simple sugars, metal ions and chelating agents failed to inhibit the activity of VMHA. The activity of VMHA was found to be sensitive to digestion by proteolytic enzymes including HA/protease. These results provide evidence for the existence of novel HA other than HA/protease in V. mimicus.     

Histochemical study on the maturation of human megakaryocytes using microfluorometry

Summary A microcytofluorometrical DNA measurement was basically studied and was applied to single megakaryocytes previously identified on a Wright-Giemsa stained smear. The smear was first photographed and the location of each megakaryocyte was recorded on a cell map. The smear was then bleached with 50% acid ethanol and absolute methanol, and re-stained with 4,6-diamidino-2-phenylindole (DAPI) reagent (pH 7.4) at 4° C. Nuclear blue fluorescence was observed and the intensity of this fluorescence was proportional to the amount of DNA with the coefficient of variation (CV) of 3.6% when stained for 30 min. After 30 min DAPI staining, the DNA measurement was microcytofluorometrically performed in single megakaryocytes which had been morphologically classified into 4 groups on the basis of cytoplasmic maturation, Bessis' classification, assessed on Wright-Giemsa-stained bone-marrow smears from normal human beings. The histograms of the cells did not show any difference in DNA ploidy distribution among the classes: that is, the DNA histograms disclosed ploidy distribution from 4 N to 64 N with the largest population of 16 N. These findings suggest that nuclear DNA synthesis is completed before platelet production starts. This method is useful for comparing the morphological features and DNA content of single megakaryocytes.     

Regulation of Histamine Turnover via Muscarinic and Nicotinic Receptors in the Brain

To clarify the regulation of central histaminergic (HAergic) activity by cholinergic receptors, the effects of drugs that stimulate the cholinergic system on brain histamine (HA) turnover were examined, in vivo, in mice and rats. The HA turnover was estimated from the accumulation of tele-methylhistamine (t-MH) during the 90-min period after administration of pargyline (65 mg/kg, i.p.). In the whole brain of mice, oxotremorine, at doses higher than 0.05 mg/kg, s.c., significantly inhibited the HA turnover, this effect being completely antagonized by atropine but not by methylatropine. A large dose of nicotine (10 mg/kg, s.c.) also significantly inhibited the HA turnover. This inhibitory effect was antagonized by mecamylamine but not by atropine or hexamethonium. A cholinesterase inhibitor, physostigmine, at doses higher than 0.1 mg/kg, s.c., significantly inhibited the HA turnover. This effect was antagonized by atropine but not at all by mecamylamine. None of these cholinergic antagonists used affected the steady-state t-MH level or HA turnover by themselves. In the rat brain, physostigmine (0.1 and 0.3 mg/kg, s.c.) also decreased the HA turnover. This inhibitory effect of physostigmine was especially marked in the striatum and cerebral cortex where muscarinic receptors are present in high density. Oxotremorine (0.2 mg/kg, s.c.) and nicotine (1 mg/kg, s.c.) also decreased the HA turnover in the rat brain. However, these effects showed no marked regional differences. These results suggest that the stimulation of central muscarinic receptors potently inhibits the HAergic activity in the brain and that strong stimulation of central nicotinic receptors can also induce a similar effect.     

Purification and characterization of extracellular soluble and membrane-bound insoluble alkaline phosphatases possessing phosphodiesterase activities in Bacillus subtilis.

A membrane-bound insoluble alkaline phosphatase (APase) and an extracellular soluble APase were purified, respectively, from a membrane preparation of Bacillus subtilis 6160-BC6, which carries a mutation to produce APase constitutively, and from a culture fluid of a mutant strain. RAN 1, isolated from strain 6160-BC6, which produces an extracellular soluble APase. The two preparations were homogeneous, as judged by sodium dodecyl sulfate discontinuous gel electrophoresis and by gel electrophoreses in the presence of 8 M urea at pH 9.3 and 4.3. RAN 1 APase was crystallized. Both preparations possessed phosphatase and phosphodiesterase activities, and their pH optima were both at 9.5. They were competitively inhibited by phosphate or arsenate and were activated by the addition of Ca2+ but not by Zn2+. The APase and alkaline phosphodiesterase activities seemed to be contained in the same protein molecule. The molecular weight of 6160-BC6 APase was estimated to be 46,000 +/- 1,000, and that of RAN 1 APase was estimated to be 45,000 +/- 1,000. The largest difference between the 6160-BC6 and RAN 1 APase's was in solubility in low-ionic-strength solutions. Present results suggest that each enzyme is composed of a single polypeptide chain and that 6160-BC6 APase aggregates in solutions of low ionic strength.     

Effect of furosemide on urinary excretion of prostaglandin E in normal volunteers and pateints with essential hypertension

Urinary excretion of prostaglandin E was measured radioimmunologically in 19 healthy persons ( 15 men and 4 women ) and in 16 patients ( 10 men and 6 women ) with essential hypertension before and after the administration of furosemide. The excretion rates were increased from 26.3±3.0 to 64.5±11.3 ng/hr in the former and from 11.9±2.7 to 26.9±85 ng/hr in the latter. There was a significant difference between them, healthy subjects showing a greater increase than patients with essential hypertension.There was an obvious sexual difference in urinary excretion of prostaglandin. In men, greater increase in the excretion rates was found than in the women. Greater increases were also obtained in healthy men than in hypertensive men and in healthy women than in hypertensive women. The present results suggest that furosemide enhances urinary excretion of prostaglandin E by mechanisms which entails either an increase in prostaglandin synthesis or a decrease in renal metabolism.     

Mutation of Bacillus subtilis causing hyperproduction of alpha-amylase and protease, and its synergistic effect.

Mutants that had a genetic lesion increasing the production of alpha-amylase and protease simultaneously were isolated from a transformable strain of Bacillus subtilis Marburg by N-methyl-N'-nitro-N-nitrosoguanidine treatment. These mutants produced two to three times more alpha-amylase and five to 16 times more protease than their parent and were tentatively referred to as AP mutants. As this mutation seems to have occurred at a single gene of the bacterial chromosome and was not located near the alpha-amylase structural gene, the gene was designated as "pap." When pap- and amyR2 (an alpha amylase regulator gene) or pap- and ProH coexisted in the same cell, synergistic effects of the two genetic characters were observed on the alpha-amylase and protease production, respectively. Upon introduction of the pap mutation, the following phenotypic changes were observed in addition to changes in alpha-amylase and protease productivity. (i) Mutants lost the character of competence for the transformation. (ii) When cells were cultured at 30 C for 30 h, mutant cells became filament owing to the formation of chains of cells. (iii) Autolysis of cells was decreased in the mutants. When pap- was transferred to the wild strain by deoxyribonucleic acid-mediated transformation, the transformants showed all these phenotypic alterations simultaneously.     

Downregulation of p‐COUMAROYL ESTER 3‐HYDROXYLASE in rice leads to altered cell wall structures and improves biomass saccharification

p‐Coumaroyl ester 3‐hydroxylase (C3′H) is a key enzyme involved in the biosynthesis of lignin, a phenylpropanoid polymer that is the major constituent of secondary cell walls in vascular plants. Although the crucial role of C3′H in lignification and its manipulation to upgrade lignocellulose have been investigated in eudicots, limited information is available in monocotyledonous grass species, despite their potential as biomass feedstocks. Here we address the pronounced impacts of C3′H deficiency on the structure and properties of grass cell walls. C3′H‐knockdown lines generated via RNA interference (RNAi)‐mediated gene silencing, with about 0.5% of the residual expression levels, reached maturity and set seeds. In contrast, C3′H‐knockout rice mutants generated via CRISPR/Cas9‐mediated mutagenesis were severely dwarfed and sterile. Cell wall analysis of the mature C3′H‐knockdown RNAi lines revealed that their lignins were largely enriched in p‐hydroxyphenyl (H) units while being substantially reduced in the normally dominant guaiacyl (G) and syringyl (S) units. Interestingly, however, the enrichment of H units was limited to within the non‐acylated lignin units, with grass‐specific γ‐p‐coumaroylated lignin units remaining apparently unchanged. Suppression of C3′H also resulted in relative augmentation in tricin residues in lignin as well as a substantial reduction in wall cross‐linking ferulates. Collectively, our data demonstrate that C3′H expression is an important determinant not only of lignin content and composition but also of the degree of cell wall cross‐linking. We also demonstrated that C3′H‐suppressed rice displays enhanced biomass saccharification.     

Oxidation of Arg-410 promotes the elimination of human serum albumin

The effect of the oxidation of amino acid residues on albumin on its in vivo elimination was investigated using mutants and oxidized HSAs. The single-residue mutants (H146A, K199A, W214A, R218H, R410A, Y411A) and oxidized HSAs were produced by the recombinant DNA techniques and incubation with a metal ion-catalyzed oxidation (MCO) system for 12, 24, 48 or 72 h. Pharmacokinetics were evaluated in mice after labeling with 111In. Structural and functional properties were examined by several spectroscopic techniques. Time-dependent increase in carbonyl group content resulted in increase in the liver clearance of oxidized HSAs. Slight decreases in alpha-helical content as the result of oxidation was induced by the increases in accessible hydrophobic areas and the net negative charge on the HSA molecule. No significant change in the pharmacokinetics and structural properties was observed for the W214A, R218H and Y411A mutants, but the properties for the H146A, K199A and R410A mutants were affected (extent of effect: R410A > K199A > H146A). The liver clearance of these proteins is closely correlated to hydrophobicity (r = 0.929, P < 0.01) and the net charge of the proteins (r=0.930, P < 0.01). The rate of elimination of HSA is closely related to the hydrophobicity and net charge of the molecule. Further, the R410A mutants had a short half-life and structure similar to oxidized HSA after oxidation. Therefore, the modification of Arg-410 via oxidative stress may promote the elimination of HSA.     

The budget of dissolved trace metals in Lake Biwa,Japan

A comprehensive study on the dynamics of dissolved elements (Mg, Al, Si, P, Ca, V, Cr, Mn, Fe, Ni, Zn, As, Sr, Y, W, and U) in Lake Biwa was carried out using a clean technique. Lake water samples (n = 523) were collected from six stations in the North Basin and three stations in the South Basin. River water samples (n = 178) were collected from 14 major rivers flowing into the North Basin. Rainwater samples (n = 89) were collected at Otsu. The river water was enriched with Mn, Al, Fe, P, and Zn and the rainwater was enriched with Zn, Al, Fe, and Mn compared to North Basin water during winter mixing. The residence times of dissolved species were estimated on the basis of input through the rivers and rain. The residence times for Ca, Mg, and Sr were about 8 years, the same as that for water. Mn, Al, Fe, and Zn showed the shortest residence times (0.05–0.19 year). A budget calculation suggested that more than 60% of the input of dissolved Si, P, V, Cr, Mn, Fe, Ni, and Zn was scavenged and retained in the lake sediments and/or discharged as suspended particles.