Shingo Tsuyama

Visualization of gaseous monoxide reception by soluble guanylate cyclase in the rat retina

Immunohistochemistry using novel monoclonal antibodies (mAbs) allowed us to uncover tissue activities of soluble guanylate cyclase (sGC) fine tuned by NO and CO. Upon NO and CO applications in vitro, purified sGC increased the affinity to mAb3221 by 100‐ and 10‐fold, respectively, but not to mAb28131. Immunohistochemistry for gas‐generating enzymes revealed that NO occurred in amacrine, bipolar, and Müllers glia cells (MGCs), whereas CO was derived mostly from heme oxygenase (HO)‐2 in MGCs. Basal sGC immunoreactivities in vivo to mAb3221 but not to mAb28131 were enhanced by injecting L‐arginine and attenuated by blocking NO synthases, suggesting the ability of the former mAb to sense NO. Comparison of mAb‐assisted immunohistochemistry suggested that sGC activities were enhanced by zinc protoporphyrin‐IX, an HO inhibitor, and repressed completely by blocking NO. However, suggested roles of CO played in situ varied among different retinal layers. In inner plexiform and inner nuclear layers located in the proximity of the cellular NO sources, CO serves as a simple inhibitor of local sGC, while playing roles in housekeeping sGC activation in external limiting membrane standing far from them. These results suggest that CO generated in MGCs is a diffusible gas mediator regulating sGC in both autocrine and paracrine manners.

ADP-ribosylation of nonmuscle actin with component I of C2 toxin

C2 toxin elaborated by Clostridium botulinum type C is composed of two dissimilar protein components, designated components I and II. Component I of the toxin caused ADP-ribosylation of a protein of Mr 45,000 in chicken tissue homogenates and also purified nonmuscle but not muscle actin. The endogenous ADP-ribosylation of intracellular actin with C2 toxin was correlated with the morphological change in intact culture cells caused by the toxin. These results indicate that the biological activity of the toxin involves a novel enzymatic activity of component I, which catalyzes the preferential ADP-ribosylation of nonmuscle actin of the target cells.

EphB2 and ephrin-B1 expressed in the adult kidney regulate the cytoarchitecture of medullary tubule cells through Rho family GTPases

Eph receptors and ephrin ligands are membrane-bound cell-cell communication molecules with well-defined functions in development, but their expression patterns and functions in many adult tissues are still largely unknown. We have detected substantial levels of the EphB2 and EphB6 receptors and the ephrin-B1 ligand in the adult mouse kidney by RT-PCR amplification. Immunolocalization experiments revealed that EphB2 is localized in the tubules of the inner and outer medulla and EphB6 is in the tubules of the outer medulla and cortex. By contrast, ephrin-B1 was detected in tubules throughout the whole nephron. Consistent with the overlapping expression of the EphB2 receptor and the ephrin-B1 ligand in the medulla, EphB2 is tyrosine-phosphorylated, and therefore activated, in the kidney. In the outer medulla, however, EphB2 signaling may be attenuated by the co-expressed kinase-inactive EphB6 receptor. Interestingly, we found that EphB signaling induces RhoA activation and Rac1 inactivation as well as cell retraction, enlargement of focal adhesions and prominent stress fibers in primary cultures of medullary tubule cells. These results suggest that EphB receptor signaling through Rho family GTPases regulates the cytoarchitecture and spatial organization of the tubule cells in the adult kidney medulla and, therefore, may affect the reabsorption ability of the kidney.

Mono‐ADP‐Ribosylation in Brain: Purification and Characterization of ADP‐Ribosyltransferases Affecting Actin from Rat Brain

Four ADP‐ribosyltransferases that acted on non‐muscle actin were purified more than 3,000‐fold from rat brain extract. The molecular weights of these brain ADP‐ribosyltransferases were 66,000 as estimated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and gel filtration on TSK gel G3000SW. The Km values for NAD were approximately 20 μM. These enzymes were not inhibited by thymidine or nicotinamide, but were inhibited by ADP and ADP‐ribose. Two soluble ADP‐ribosylation factors purified from rat brain had different effects on the four ADP‐ribosyltransferases during the ADP‐ribosylation of non‐muscle actin. These ADP‐ribosyltransferases modified not only actin but also the stimulatory guanine nucleotide‐binding protein of adenylate cyclase, Gs, and another guanine nucleotide‐binding protein in brain, Go. These findings suggest that the four brain ADP‐ribosyltransferases are concerned with nerve functions in the central nervous system.

Oscillation of ADP-ribosyl cyclase activity during the cell cycle and function of cyclic ADP-ribose in a unicellular organism, Euglena gracilis.

In Euglena gracilis, the activity of ADP‐ribosyl cyclase, which produces cyclic ADP‐ribose, oscillated during the cell cycle in a synchronous culture induced by a light‐dark cycle, and a marked increase in the activity was observed in the G2 phase. Similarly, the ADP‐ribosyl cyclase activity rose extremely immediately before cell division started, when synchronous cell division was induced by adding cobalamin (which is an essential growth factor and participates in DNA synthesis in this organism) to its deficient culture. Further, cADPR in these cells showed a maximum level immediately before cell division started. A dose‐dependent Ca2+ release was observed when microsomes were incubated with cADPR.

Feeding dried purple laver (nori) to vitamin B12-deficient rats significantly improves vitamin B12 status.

To clarify the bioavailability of vitamin B12 in lyophylized purple laver (nori; Porphyra yezoensis), total vitamin B12 and vitamin B12 analogue contents in the laver were determined, and the effects of feeding the laver to vitamin B12-deficient rats were investigated. The amount of total vitamin B12 in the dried purple laver was estimated to be 54.5 and 58.6 (se 5.3 and 7.5 respectively) microg/100 g dry weight by Lactobacillus bioassay and chemiluminescent assay with hog intrinsic factor respectively. The purple laver contained five types of biologically active vitamin B12 compounds (cyano-, hydroxo-, sulfito-, adenosyl- and methylcobalamin), in which the vitamin B12 coezymes (adenosyl- and methylcobalamin) comprised about 60 % of the total vitamin B12. When 9-week-old vitamin B12-deficient rats, which excreted substantial amounts of methylmalonic acid (71.7(se 20.2) micromol/d) in urine, were fed the diet supplemented with dried purple laver (10 microg/kg diet) for 20 d, urinary methylmalonic acid excretion (as an index of vitamin B12 deficiency) became undetectable and hepatic vitamin B12 (especially adenosylcobalamin) levels were significantly increased. These results indicate that vitamin B12 in dried purple laver is bioavailable to rats.

Accumulation of Trehalose as a Compatible Solute under Osmotic Stress in Euglena gracilis Z

ABSTRACT. The photosynthetic protozoon Euglena gracilis, accumulated a large amount of trehalose in the cells under salt or osmotic stresses. Radioactivity of [14C] paramylon, a β‐1,3‐polyglucan which was stored in the cells of E. gracilis. was degraded rapidly and this radioactivity was almost stoichiometrically incorporated into trehalose. The interconversion of trehalose from paramylon by salt or osmotic stresses was dependent on the concentrations or osmotic pressures, suggesting that E. gracilis accumulate trehalose as an osmoprotectant. After the removal of salt or osmotic stresses, trehalose was gradually degraded, however, it was not converted into paramylon.

Aberrant Utilization of Nitric Oxide and Regulation of Soluble Guanylate Cyclase in Rat Diabetic Retinopathy

Although nitric oxide (NO) was shown not only to exert biological activities through activation of soluble guanylate cyclase (sGC), but also to cause oxidative stress, mechanisms for switching these pathways are unknown. This study aimed to examine aberrant utilization of NO under disease conditions such as diabetes mellitus. Diabetes was induced in male Wistar rats by injecting streptozotocin (STZ; 50 mg/kg body weight, i.p.). Retina was perfusion-fixed for immunohistochemistry to detect the gas-mediated activation of sGC by anti-sGC antibodies that are function-sensitive [monoclonal antibody (MoAb) 3221] and -insensitive (MoAb28131). Regional lipid peroxidation was also examined by an anti-acrolein MoAb. At 6 weeks after STZ injection, inducible NO synthase induction became evident, coinciding with the overproduction of nitrotyrosine, followed by that of acrolein. Despite such NO overproduction, sGC did not exhibit any notable activation. When STZ-treated animals were posttreated with a derivative of superoxide dismutase that stays in circulation without undergoing renal ultrafiltration, immunoreactivities to MoAb3221 but not to MoAb28131 increased markedly in diabetic retina, suggesting that superoxide cancels free NO for local sGC activation. These results provide evidence of aberrant utilization of NO and suggest that superoxide plays a role in interfering with NO-mediated sGC activation for phototransducing events in this neural tissue.

Inositol 1,4,5-Trisphosphate and Cyclic ADP-Ribose Mobilize Ca2+ in a Protist, Euglena gracilis

Inositol 1,4,5-trisphosphate (InsP3) and cyclic ADP-ribose (cADPR) released Ca2+ from microsome fraction prepared from Euglena gracilis in dose-dependent manners. Caffeine, which also induced Ca2+ release from the microsomes, caused desensitization of the Ca2+ response to cADPR, although the Ca2+ response to InsP3 was not affected by caffeine. Further, ruthenium red inhibited the Ca2+ release induced by cADPR, but not by InsP3. These results suggest that cADPR functions as an endogenous messenger to activate a caffeine-sensitive, Ca(2+)-release mechanism, whereas InsP3 induces Ca2+ release by a distinct mechanism in E. gracilis.

Slit3 regulates cell motility through Rac/Cdc42 activation in lipopolysaccharide-stimulated macrophages.

Three slit genes, slit1 to slit3, have been cloned to date. Slit1 and slit2 act as chemorepellent factors for axon guidance. Slit3 is involved in the formation of the diaphragm and kidney during embryogenesis. However, its molecular function remains unclear. We found that slit3 expression was induced by lipopolysaccharide (LPS)‐stimulation in macrophages and that it was localized in the mitochondria and along the plasma membrane. Silencing of slit3 expression by RNA interference reduced cell motility and Rac/Cdc42 activation. These results suggest that slit3 functions as an intracellular signaling molecule for cell motility as part of the LPS‐induced signaling cascade.