Immunohistochemical localization of aldehyde and xanthine oxidase in rat tissues using polyclonal antibodies

Tissues from male Wistar rats, fixed with 4% paraformaldehyde and embedded in paraffin, were studied with immunoperoxidase techniques using polyclonal antibodies raised against aldehyde oxidase or xanthine oxidase purified from rat liver. Immunohistochemical studies demonstrated that aldehyde oxidase-bearing cells were strongly stained in renal tubules, esophageal, gastric, intestinal and bronchial epithelium as well as liver cytoplasm. Weak but positive immunoreactivity was observed on the pulmonary alveolar epithelial cells, gastric glands and intestinal goblet cells. In contrast, it was demonstrated that cells with xanthine oxidase were strongly stained in renal tubules, esophageal, gastric, and small and large intestinal and bronchial epithelia etc. Positive immunostaining was also found in adrenal gland, skeletal muscle, spleen and cerebral hippocampus. Immunoreactivity againt aldehyde oxidase was not found in adrenal gland, spleen, mesentery or aorta, while immunoreactivity against xanthine oxidase was not found in mesentery or aorta. Although the significance of this ubiquitous and similar localization of aldehyde and xanthine oxidase seems unclear at present, these results may provide a clue as to the full understanding of the pathophysiological role of these oxidases in tissues.     

The concept of biological control methods in aquaculture

Microbial techniques of biocontrol using the interaction ofmicroorganisms to repress the growth of deleterious bacteria andviruses were developed. The bacterial strain used in this work alsoimproved the growth of fishes and crustaceans. Using the conceptandprocedures of the biocontrol method described here, the aquacultureproduction became stable and evenincreased.     

Dopaminergic Receptors Linked to Adenylate Cyclase in Human Cerebromicrovascular Endothelium

Cultured endothelium derived from three fractions of human cerebral microvessels was used to characterize dopamine (DA) receptors linked to adenylate cyclase activity. DA or D1 agonist, (+/-)-SKF-82958 hydrobromide, stimulated endothelial cyclic AMP formation in a dose-dependent manner. The selective D1 antagonist, (+/-)SCH-23390, inhibited in a dose-dependent manner the production of cyclic AMP induced by DA. The affinity for the D1 receptor appeared to be greater in endothelium derived from large and small microvessels than from capillaries. Cholera toxin ADP-ribosylation of Gs proteins abolished the DA stimulatory effect on endothelial adenylate cyclase, whereas pertussis toxin ADP-ribosylation enhanced the DA-inducible formation, indicating the presence of both D1 and D2 receptors. Agonists of alpha 1-adrenergic receptors (phenylephrine, 6-fluoronorepinephrine) or serotonin (5-HT), which stimulated the production of cyclic AMP, had no additive effect on DA-stimulated cyclic AMP formation. Incubation of these agents with DA produced the same or lower levels of cyclic AMP as compared to that formed by DA alone. The effect of alpha 1-adrenergic agonists or 5-HT on DA production of cyclic AMP was partially prevented by the D2 antagonist, S(-)-sulpiride, or ketanserin (5-HT2 greater than alpha 1 greater than H1 antagonists), respectively. These findings represent the first demonstration of D1- (stimulatory) and D2- (inhibitory) receptors linked to adenylate cyclase in microvascular endothelium derived from human brain. The data also indicate that dopaminergic receptors can interact with either alpha 1-adrenergic or or 5-HT receptors in endothelium on the adenylate cyclase level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Putative inositol 1,4,5-trisphosphate binding proteins in rat brain cytosol.

In previous works, we synthesized a series of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) analogs, with a substituent on the second carbon of the inositol ring. Using these analogs, the Ins(1,4,5)P3 affinity media were also synthesized (Hirata, M., Watanabe, Y., Ishimatsu, T., Yanaga, F., Koga, T., and Ozaki, S. (1990) Biochem. Biophys. Res. Commun. 168, 379-386). When the cytosol fraction from the rat brain was applied to an Ins(1,4,5)P3 affinity column, an eluate with a 2 M NaCl solution was found to have remarkable Ins(1,4,5)P3-binding activity. The active fraction was further fractionated with gel filtration chromatography, and two proteins with an apparent molecular mass of 130 or 85 kDa were found to be Ins(1,4,5)P3-binding proteins but with no Ins(1,4,5)P3 metabolizing activities. Partial amino acid sequences determined after proteolysis and reversed-phase chromatography revealed that the protein with an apparent molecular mass of 85 kDa is the delta-isozyme of phospholipase C and that of 130 kDa has no sequence the same as the Ins(1,4,5)P3-recognizing proteins hitherto examined. Ins(1,4,5)P3 at concentrations greater than 1 microM strongly inhibited 85-kDa phospholipase C delta activity, without changing its dependence on the concentrations of free Ca2+ and H+. Among inositol phosphates examined, Ins(3,4,5,6)P4 inhibited the binding of [3H]Ins(1,4,5)P3 to the 130-kDa protein at much the same concentrations as seen with Ins(1,4,5)P3. This report seems to be the first evidence for the presence of soluble Ins(1,4,5)P3-binding proteins in the rat brain, one of which is the delta isozyme of phospholipase C.     

Detection of the Nicotiana rustica chloroplast genome coding for the large subunit of Fraction I protein in a somatic hybrid in which only the N. tabacum chloroplast genome appeared to have been expressed

Nine plants were produced from anthers of a somatic hybrid which had been obtained by fusion of Nicotiana tabacum L. and N. rustica L. protoplants. As determined by electrofocusing, the Fraction I protein of the original somatic hybrid had largesubunit polypeptides exclusively of the N. tabacum type. Two of the plants regenerated from anthers contained Fraction-I-protein large subunits exclusively of the N. rustica type. Since each plant was regenerated from a single cell, the somatic hybrid must have had cells containing both the N. tabacum and N. rustica chloroplast genome although the latter was not expressed. Possibilities to account for this non-expression of a chloroplast genome in the somatic hybrid are discussed.     

A synthesis of 3′,4-́dideoxykanamycin B

3′,4′-Dideoxykanamycin B, the kanamycin B derivative that is active against resistant bacteria, was prepared from kanamycin B viaN-tosylation, 3′,4′-O-sulphonylation, 3′,4′-unsaturation, and hydrogenation. The unsaturated intermediate was obtained from the 3′,4′-di-O-sulphonyl derivatives by the action of sodium iodide in N,N-dimethylformamide; if zinc dust was added in this reaction, aziridine derivatives were formed, Removal of the tosyl group was successfully performed by using sodium in ammonia-ethylamine.     

Histamine release from rat mast cells by Vibrio vulnificus protease

Abstract Vibrio vulnificus protease (VVP) stimulated histamine release from isolated mast cells in a dose- and temperature-dependent manner within a range of 0.2–4.0 μ g/0.5 ml. Histamine release was accompanied by degranulation, and no leakage of lactate dehydrogenase from cells was observed, indicating that the histamine release was not due to cytolysis but to exocytosis. This release, completed within 30 s at 37°C, suggested that the mechanism of action of VVP on mast cells is different from that of other proteases, such as trypsin or α-chymotrypsin, which release histamine from the cells slowly.     

Diacylglycerol kinase alpha regulates globular adiponectin-induced reactive oxygen species

It has previously been reported that the globular form of adiponectin (gAd), mature adipocyte-derived cytokine, induced generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. This study investigated whether diacylglycerol kinases (DGKs), enzymes functioning in sub-cellular signalling pathways, had a role on gAd-induced ROS generation in RAW 264 cells. Administration of R59022, a specific inhibitor for DGK, reduced gAd-induced ROS generation and NO release. RAW 264 cell expressed DGKα mRNA. Depression of DGKα mRNA by RNA interference significantly reduced the ROS generation in response to gAd treatment. Interestingly, transfection with the DGKα-specific small interfering RNA attenuated the expression level of Nox1 mRNA in gAd-treated RAW 264 cells. In addition, the DGKα knockdown with siRNA suppressed gAd-induced NO release.     

Antioxidative enzymes in seedlings of Nelumbo nucifera germinated under water

Dry seeds of anoxia-tolerant lotus ( Nelumbo nucifera Gaertn= Nelumbium speciosum Willd.) have green shoots with plastids containing chlorophyll, so photosynthesis starts even in seedlings germinated under water, namely hypoxia. Here we investigated antioxidative enzyme changes in N. nucifera seedlings responding to oxygen deficiency. The activity of superoxide dismutase (SOD; EC 1.15.1.1), dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2) were lower in seedlings germinated under water (submerged condition) in darkness (SD seedlings) than those found in seedlings germinated in air and darkness (AD seedlings). In contrast, ascorbate peroxidase (APX; EC 1.11.1.11) activity was higher in SD seedlings and the activity of catalase (EC 1.11.1.6) and monodehydroascorbate reductase (MDAR; EC 1.6.5.4) in SD seedlings was nearly the same as in AD seedlings. When SD seedlings were exposed to air, the activity of SOD, DHAR and GR increased, while the activity of catalase and MDAR decreased. Seven electrophoretically distinct SOD isozymes were detectable in N. nucifera . The levels of plastidic Cu,Zn-SODs and Fe-SOD in SD seedlings were comparable with those found in AD seedlings, which may reflect the maintenance of green plastids in SD seedlings as well as in AD seedlings. These results were substantially different from those previously found in rice seedlings germinated under water.