Shozo Takamura

Augmentation of various immune reactivities of tumor-bearing hosts with an extract of Cordyceps sinensis

In order to enhance general reactivity of immune system in the tumor-bearing host, we employed extract ofCordyceps sinensis (CSE) as a biological response modifier.Cordyceps sinensis is an interesting material produced by a kind of mushroom parasitic to larval moths and was used to hasten recovery from exhaustion in ancient China.In this experiment, C57BL/6 mice implanted subcutaneously with syngeneic EL-4 lymphoma cells were employed as the host. Oral administration of the extract leads to a reduction of tumor size and prolongation of the host survival time. As judged by plaque-forming cells against T-dependent (sheep erythrocytes) and T-independent (bacterial lipopolysaccharide) antigens, CSE showed to augment the antibody responses. As for the activities of peritoneal macrophages, chemotaxis was dramatically depressed within a few days after EL-4 transplantation up to the end of life, but treatment with CSE at −14, −7, −4, +4, +7 and +10 days after the tumor transplantation augmented the activity about four times stronger than that of control. Phagocytic activity of macrophages was also decreased in tumor-bearing mice treated with cyclophosphamide (100 mg/kg) 3 and 5 days after tumor transplantation. But administration of CSE restored the activity to more than the normal level. The overall efficacy of CSE was tested with protective activity against systemic infection bySalmonella enteritides. The tumor-bearing mice receiving this medicine lived significantly longer than any other groups without CSE.

Comparison of gentamicin nephrotoxicity between rats and mice

Toxic effects of gentamicin administration (10-80 mg/kg body weight, subcutaneously (s.c.), once daily for 7 days) on several enzyme activities of kidney and duodenal mucosa together with other parameters were compared between male rats and mice. In Wistar rat kidney, tubular brush border Mg(2+)-dependent, HCO3(-)-stimulated ATPase (Mg(2+)-HCO3(-)-ATPase) activity was inhibited by 40-80 mg/kg gentamicin in an almost dose-dependent manner with no changes in microsomal Mg(2+)-Na(+)-K(+)-ATPase activity. Cytosol carbonic anhydrase (CA) activity was inhibited only by 80 mg/kg gentamicin. In rat duodenal mucosa, Mg(2+)-HCO3(-)-ATPase and CA activities were unchanged by any dose of gentamicin. Rat serum urea nitrogen (UN), GOT and GPT concentrations and urinary N-acetyl-beta-D-glucosaminidase (NAG) activity were significantly increased by 80 mg/kg gentamicin. In ddY mice, however, almost all parameters described above were unaffected by gentamicin except for the urinary NAG activity which was increased only by 80 mg/kg gentamicin. The concentration of gentamicin in cytosol of rat whole kidney was approximately 3.4-fold higher compared with that in mouse kidney after 80 mg/kg treatment. In light microscopic analysis, 80 mg/kg gentamicin produced necrosis in the greater part of rat kidney proximal tubuli with no pathological findings in mouse kidney. In conclusion, Mg(2+)-HCO3(-)-ATPase activity in brush border membrane of rat proximal tubuli was selectively damaged in gentamicin nephrotoxicity, indicating that the rats are the suitable model for studies of gentamicin nephrotoxicity in humans.

Characterization of a streptococcal antitumor glycoprotein (SAGP).

An acidic antitumor glycoprotein (SAGP) was purified from a crude extract of Streptococcus pyogenes, Su strain. Intraperitoneal injection with SAGP (20 mg protein/kg/day for 4 consecutive days) prolonged the life span of mice inoculated i.p. with Ehrlich ascite carcinoma cells and methylcholanthrene-induced fibrosarcoma cells (Meth A) up to 244% and 169% of that of the control mice, respectively. These in vivo antitumor effects were reduced in immunosuppressed mice. The effector spleen cells from the Meth A-inoculated and SAGP-injected mice showed a considerable cytostatic activity on Meth A cells in vitro, and immunosuppression studies suggested that carrageenan-sensitive and/or asialo-GM1 positive spleen cells are responsible for the in vivo antitumor effect of SAGP. SAGP inhibited the cell growth of cultured cell lines including transformed hamster embryonic lung cells, murine leukemia L 1210, Meth A and human promyelocytic leukemia HL60 cells. The IC50s for the cell growth of these cells were all below 0.1 microg protein/ml. SAGP inhibited the incorporation of nucleic acid precursors into Meth A cells. It seems that sulfhydryl groups of the SAGP molecule are essential for the expression of the antitumor action of SAGP. The cell growth-inhibitory activity of SAGP was diminished in Meth A cells preincubated with pertussis toxin (IAP), whereas it was augmented in the cells preincubated with cholera toxin (CTX), suggesting the involvement of toxin-sensitive GTP (G)-proteins in the SAGP-action. IAP and CTX-catalyzed ADP ribosylation assays confirmed that SAGP augmented the activity of IAP-sensitive G-protein. In addition, this augmentation was detected neither in Meth A cells incubated with heat-inactivated SAGP nor in SAGP-insensitive L929 cells. SAGP induced apoptosis in Meth A and HL60 cells as assessed by DNA fragmentation. A single dose injection of SAGP (100 mg protein/kg, i.v., s.c., or i.p.) into mice produced no toxic signs except occasional pain responses observed for one week after the injection. Thus, SAGP is a low toxic substance that shows in vivo antitumor activity by modulating immune responses of the host, and also exhibits in vitro cell-growth inhibition through IAP-sensitive G-protein.

Antitumor action of an acidic glycoprotein (SAGP) fromStreptococcus pyogenes in mice

We have shown that an acidic glycoprotein (SAGP) isolated from a cell-free extract ofStreptococcus pyogenes (Su strain) prolonged the life-span of Ehrlich ascites carcinoma (EAC)bearing mice. The present study shows that the life-span prolonging effect of SAGP in EAC-bearing mice was reduced by whole body X-irradiation before EAC inoculation. SAGP (500μg protein/mouse/day X 4, i.p.) also showed a life-span prolonging effect (T/C (%) = 169) on Meth A fibrosarcoma (Meth A)-bearing mice, but the effect of SAGP was abrogated by an i.p. pretreatment of the host with carrageenan, an antimacrophage agent. The spleen cells from the Meth A-inoculated and SAGP-treated mice were found to have a considerable cytostatic activity by a3H-thymidine incorporation assay. But the activity disappeared in the presence of carrageenan. These results suggest that thein vivo antitumor effects of SAGP are mediated through its immunomodulating action.

Sexual difference and organ specificity of the effect of estradiol on carbonic anhydrase and Mg2+ -HCO3− -ATPase activities isolated from duodenal mucosa and kidney cortex of male and female rats: preliminary study with crude enzyme samples

Effects of the s.c. administration of various doses of estradiol propionate (E.P.; 25-500 micrograms/kg) on the activities of carbonic anhydrase (CA), Mg(2+)-dependent ATPase and Mg(2+)-dependent, HCO3(-)-stimulated ATPase (Mg(2+)-HCO3(-)-ATPase) in rat duodenal mucosa and kidney cortex, and on body weight, organ weight and serum concentrations of testosterone and estradiol-17 beta, were examined in adult male, female, testectomized and ovariectomized rats. In normal male rats, activities of cytosol CA and brush border Mg(2+)-HCO3(-)-ATPase in the kidney were increased in a dose-dependent manner and reached 1.6- and 2-fold of controls, respectively, after consecutive administration (daily for 7 days) of 500 micrograms E.P. with no changes in either enzyme activities in duodenal mucosa. The positive correlations (P less than 0.01) were observed by linear regression analysis between serum concentration of estradiol-17 beta and kidney cytosol CA or kidney brush border Mg(2+)-HCO3(-)-ATPase activities. In normal female rats, activities of cytosol CA and brush border Mg(2+)-HCO3(-)-ATPase in the duodenal mucosa, and brush border Mg(2+)-HCO3(-)-ATPase activity in the kidney were increased by E.P. administration (100 and 500 micrograms/kg, daily for 7 days), however, kidney cytosol CA activity did not change by any dosage. Behavior of a part of both enzymes to E.P. in testectomized rats was altered almost in the same way to that observed in normal female rats and vice versa in ovariectomized rats. Body weight was decreased, in general, by consecutive administration of E.P. in a dose-dependent manner, and kidney weight was increased by E.P. in both male and female rats.

Protective effects of 2,3-dimercaptopropane-1-sulfonate on mercuric chloride-induced acute inhibition of enzymes from rat duodenal mucosa and kidney cortex

Acute inhibitory effects of mercuric chloride on the activities of several enzymes from rat duodenal mucosa and kidney cortex and the protection provided by 2,3-dimercaptopropane-1-sulfonate (DMPS) were examined. Activities of carbonic anhydrase in homogenate, brush border and cytosol and of Mg(2+)-dependent, HCO3-stimulated ATPase in homogenate and brush border of duodenal mucosa and kidney cortex and activities of kidney microsomal Mg(2+)-dependent, Na(+)-K(+)-stimulated ATPase were all decreased following administration of HgCl2 (1-3 mg Hg/kg body weight s.c. once daily for 3 days). Decreases occurred generally in a dose-dependent manner and went back to near normal or normal levels by the combined administration of 20 and 30 mg DMPS/kg per day for 3 days. The concentration of serum urea nitrogen was increased about 8 times by the administration of 2 mg Hg/kg and restored to normal by the concomitant administration of 30 mg DMPS/kg. After the administration of 2 mg Hg/kg per day for 3 days, about half of the renal cortical proximal tubuli were necrotic. Administration of 30 mg DMPS/kg restored these changes to almost normal. The results suggest that DMPS is useful in mitigating acute Hg poisoning and that part of the protective effect of DMPS on enzyme damage induced by Hg poisoning may be due to its chelating action.