Significant change in the structure of a ribozyme upon introduction of a phosphorothioate linkage at P9: NMR reveals a conformational fluctuation in the core region of a hammerhead ribozyme.

A modified hammerhead ribozyme (R32S) with a phosphorothioate linkage between G(8) and A(9), a site that is considered to play a crucial role in catalysis, was examined by high-resolution 1H and (31)P nuclear magnetic resonance (NMR) spectroscopy. Signals due to imino protons that corresponded to stems were observed, but the anticipated signals due to imino protons adjacent to the phosphorothioate linkage were not detected and the (31)P signal due to the phosphorothioate linkage was also absent irrespective of the presence or absence of the substrate. (31)P NMR is known to reflect backbone mobility, and thus the absence of signals indicated that the introduction of sulfur at P9 had increased the mobility of the backbone near the phosphorothioate linkage. The addition of metal ions did not regenerate the signals that had disappeared, a result that implied that the structure of the core region of the hammerhead ribozyme had fluctuated even in the presence of metal ions. Furthermore, kinetic analysis suggested that most of the R32S-substrate complexes generated in the absence of Mg(2+) ions were still in an inactive form and that Mg(2+) ions induced a further conformational change that converted such complexes to an activated state. Finally, according to available NMR studies, signals due to the imino protons of the central core region that includes the P9 metal binding site were broadened or not observed, suggesting that this catalytically important region might be intrinsically flexible. Our present analysis revealed a significant change in the structure of the ribozyme upon the introduction of the single phosphorothioate linkage at P9 that is in general considered to be a conservative modification.

Protective effect of fluvastatin sodium (XU-62-320), a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on oxidative modification of human low-density lipoprotein in vitro.

We investigated the protective effect of fluvastatin sodium on the oxidation of low-density lipoprotein (LDL) induced in vitro by copper ions. The extent of lipid peroxidation was assessed by monitoring the increase of UV absorbance at 234 nm, which is the peak absorbance of a conjugated diene. Fluvastatin sodium (1-30 microM) not only prolonged the lag time of oxidation in the initiation step, but also decreased the rate of oxidation in the propagation step, both concentration dependently. Fluvastatin sodium and alpha-tocopherol showed an additive effect when both compounds were added before oxidation. However, when the lag time was prolonged initially by alpha-tocopherol, and fluvastatin sodium and alpha-tocopherol, were further added into the reaction mixture at the end point of the lag phase, fluvastatin sodium still showed an antioxidative effect, whereas alpha-tocopherol showed a pro-oxidative effect. Therefore, the antioxidative property of fluvastatin sodium differs from that of alpha-tocopherol. In this experiment, as neither the double bond-reduced derivative of fluvastatin sodium nor pravastatin sodium showed any protective effect, we concluded that the antioxidative effect of fluvastatin sodium is not a common property of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, but may be derived from its unique chemical structure. Since the oxidative modification of LDL plays an important role in the genesis of atherosclerosis, fluvastatin sodium may help reduce the risk of atherosclerosis, not only by reducing plasma LDL levels but also by protecting LDL from oxidative modification.

Antioxidative property of T-0970, a new ureidophenol derivative.

We investigated the antioxidative property of T-0970, a newly synthesized ureidophenol derivative. The inhibitory effect of T-0970 on spontaneous lipid peroxidation in rat brain was 10 times greater than those of well-known antioxidants such as butylhydroxytoluene (BHT), probucol and alpha-tocopherol. T-0970 also showed dose-dependent free radical scavenging activities in vitro for both superoxide anions and hydroxyl radicals. The radical-scavenging potencies of T-0970 were about 10-30 times stronger than those of BHT. We evaluated the in vivo antioxidative ability of T-0970 in the animal model of acute oxidative tissue injury in rats. Intraperitoneal injection of ferric nitrilotriacetate (Fe/NTA) caused an acute and remarkable increase in the level of thiobarbituric acid-reactive substances (TBARS) in both plasma and the liver, and also resulted in a considerable elevation of the plasma levels of GOT and GPT indicative of hepatic injury. Both oral and intravenous administration of T-0970 dose-dependently depressed these diagnostic parameters. These results indicate that T-0970 may have a therapeutic potential in various diseases associated with oxidative tissue injury.

Fluvastatin depresses the enhanced lipid peroxidation in vitamin E-deficient hamsters.

Fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, has recently been reported to have the antioxidative activity in vitro. However, it is still unclear whether chronic treatment with this drug actually leads to amelioration of the redox status in the body. In this study, we investigated the antioxidative effect of fluvastatin in vivo, using a vitamin E-deficient hamster model, an in vivo model of enhanced oxidative stress. After pre-treatment with a vitamin E-deficient diet for 2 months, fluvastatin, pravastatin or probucol was added to the diet for 1 month. Vitamin E deficiency caused a significant increase in the levels of plasma oxidative stress markers such as 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha) and hydroperoxides. Furthermore, there was a significant increase in the oxidizability of plasma lipids in the vitamin E-deficient animals, indicating that the oxidative stress was increased in the circulation. Fluvastatin markedly depressed the above oxidative stress markers in plasma, and significantly decreased the oxidizability of plasma lipids without affecting their levels. Probucol, a reference antioxidant, also showed a similar effect while pravastatin, another HMG-CoA reductase inhibitor, showed only a weak improvement. We suggest that the treatment with fluvastatin leads to a reduction of oxidative stress in vivo, which is mainly derived from its antioxidative property rather than its lipid-lowering activity.

Bone metabolic analysis in patients with primary hyperparathyroidism.

Surgical treatment for primary hyperparathyroidism (PHPT) improves not only the calcium and phosphate metabolism but also the bone metabolism. This study was conducted to analyze the bone metabolism after PHPT operations. Bone mineral density (BMD) was measured by dual-photon absorptiometry in 50 patients before and after operation. Osteocalcin (OC) and alkaline-phosphatase activity (Alp) in serum were measured before and after surgery as markers of bone formation, and urinary deoxypiridinorine (DPD) as an index of osteoclast activity. The 50 patients under study were 40 women (80%) and ten men (20%). Increases in BMD at the lumbar spine were remarkable at three months following operation. Slow but steady progress was made until six months, reaching a plateau thereafter. The increase in BMD of lumbar spine was approximately 10%. Urinary DPD was the most sensitive among the three bone metabolic markers. Although serum Alp and OC remained high after operation, urinary DPD was normalized earlier. The discrepancy of bone formation and resorption was shown after operation and this contributed to the increases in BMD in the first six months.

A discussion of anti-Aspergillus niger glucose oxidase monoclonal antibody reactivity to red blood cells of several species.

We observed that mouse spleen cells from rosettes with autologous red blood cells (RBCs) and that rosette-formation was suppressed by anti-Aspergillus niger glucose oxidase monoclonal antibody (mAb). In the present study, we investigated whether RBCs of species besides mice have the structure recognized by anti-A. niger glucose oxidase mAb by using rosette-formation and complement-mediated hemolysis. Lysates of monkey and human RBCs did not suppress rosette-formation whereas autologous (mouse), rat and sheep RBC lysates partially suppressed rosette-formation. Those lysates exerted their suppressive activity after they had been treated at 56 degrees C for 30 min. A. niger glucose oxidase also suppressed rosette-formation with or without treatment at 56 degrees C for 30 min. Alternatively, anti-A. niger glucose oxidase mAb lysed mouse, rat and sheep RBCs but not human RBCs with complement. These findings suggest that the cell surfaces of mouse, rat and sheep RBCs have a structure which can be recognized by anti-A. niger glucose oxidase mAb while the cell surfaces of monkey and human RBCs do not.

Decreased turnovers of glutathione and ascorbic acid in watanabe heritable hyperlipidemic rabbits.

Oxidative stress has been postulated to play important roles in the pathogenesis of various diseases such as atherosclerosis in hyperlipidemic subjects. Although the possible role of oxidation of low-density lipoprotein (LDL) in the etiology of atherosclerosis has been studied extensively, the turnover of endogenous antioxidants, which is an important protection system against oxidative stress, remains to be elucidated. The aim of our study was to determine the change of the turnover of endogenous antioxidants such as glutathione and ascorbic acid in case of hyperlipidemia, using Japanese white rabbits (JW) and Watanabe heritable hyperlipidemic rabbits (WHHL). The levels of total glutathione and low molecular weight thiols in the liver, kidney, and other organs in both strains of rabbits were similar. However, a kinetic analysis using L-buthionine-(S,R)-sulfoximine revealed that the rate of glutathione turnover in the liver and kidney of WHHL was about 50%) lower than that of JW. Furthermore, intravenously administered ascorbic acid disappeared more slowly in WHHL than in JW. These results indicate that the turnovers of both glutathione and ascorbic acid in WHHL are depressed in comparison with that in JW. These changes would be closely related to the increased oxidizability of lipids in the circulation of hyperlipidemic subjects.

[Antitumor effect of UFT against differentiated thyroid cancer].

We have determined the levels of 5-FU, tegafur and uracil in the thyroid cancer and normal thyroid tissue in the patients with differentiated carcinoma who were administered UFT 600 mg/day p.o. preoperatively for six days. 5-FU and uracil levels in the thyroid cancer tissue were significantly higher than in normal thyroid tissue. However, tegafur level did not show significant differences in any tissues. Two cases with differentiated carcinoma, which resulted in PR after prolonged administration of UFT were presented. These findings suggest that oral administration of UFT for a long term is a useful treatment for advanced differentiated thyroid cancer.

[Concentrations of 5-fluorouracil (5-FU) in serum and tissues at venous injection of tegafur or 5-FU--clinical study on colorectal cancer].

Tissue and serum concentrations of 5-fluorouracil (5-FU) after daily slow venous injection of tegafur or 5-FU for 5 days were measured. The serum concentration of 5-FU elevated to the highest level 6 hours after the venous injection (mean: 30 ng/ml). Compared with the tegafur injection, the serum concentration of 5-FU elevated to a higher peak level 6 hours after the 5-FU injection (mean: 827 ng/ml). The serum concentration of 5-FU after the tegafur injection tended to be maintained longer than after the 5-FU injection. When tegafur was injected, the concentration of 5-FU in cancer tissue or lymphnodes was significantly higher than in normal tissue. On the other hand, no significant difference was detected among the concentrations of 5-FU in the above three tissues when 5-FU was injected. Moreover, a significantly higher concentration of 5-FU in lymphnodes was caused by the tegafur injection compared to the 5-FU injection.

[Efficacy of the treatment of gastric cancer as neo-adjuvant chemotherapy of 48 hour continuous intravenous infusion of 5-fluorouracil (5-FU) with leucovorin (LV)].

A 51-year-old female with inoperable gastric cancer and with infiltration of pancreatic tail diagnosed by abdominal CT was treated with leucovorin (LV) and 5-fluorouracil (5-FU). The regimen was: LV 30 mg/body/24 hr prior to 5-FU 1,000 mg/m2/day for 48 hrs. This treatment was repeated 6 times. After treatment, the size of tumor decreased so that the patient was able to be operated (Total gastrectomy with partial distal pancreatico-splenectomy). During the treatment, patient showed no side effect except for slight nausea. This neo-adjuvant chemotherapy might be a recommendable treatment of advanced gastric cancer.

[Efficacy of a 48-hour infusion of 5-fluorouracil in patients with stage IV gastric cancer after palliative gastrectomy].

Efficacy of a 48-hour infusion of 5-FU in patients with stage IV gastric cancer was investigated. A one-cycle regimen consisted of a 48-hour infusion of 5-FU (30 mg/kg/24 hours) every week for 6 weeks. Fourteen patients with stage IV gastric cancer who underwent absolutely non-curative gastrectomy, received 5-FU infusion postoperatively. Ten of the 14 received more than 2 cycles. Survival rates in these patients (group I) were evaluated by Kaplan-Meier method and compared with those of matched pair controls (group II) who received other anticancer agents. Side effects were generally mild. Nausea during infusion was observed in only 3 cases. The 50% survival period was 393 days in group I and 135 days in group II, respectively. The survival curve in group I was significantly higher than in group II (p = 0.05). From these results, this treatment is considered to be very effective.

[Result of surgical treatment to benign thyroid tumor].

Surgical results of 81 cases with various benign thyroid tumors were reviewed. All had undergone mainly hemi-lobectomy including isthmectomy and had been followed up for more than 2 years. Sixty-eight (84%) have been well without any complaint. However, 5 of 15 cases with adenomatous goiter (AG) again developed nodule(s) in the remnant thyroid and 1 of 37 cases with adenoma developed cancer in the remnant. In order to prevent recurrence in the cases with AG, total thyroidectomy may be required with the meticulous selection of the case. For the cases with AG which undergone hemilobectomy or lumpectomy, TSH suppression treatment may be also required after surgery. It is of caution that the nodule developed in the thyroid after surgery may be not always the same nodule as primary one.

In vivo anti-tumor activities of gelatin.

AIM: As reported previously, porcine skin gelatin exerted direct anti-tumor effect in vitro and induced anti-tumor peritoneal macrophages in vitro. The present study investigated whether or not the gelatin exerted anti-tumor effect in vivo. METHODS: In vitro anti-tumor activities of peritoneal macrophages and the gelatin were evaluated with tritium thymidine uptake by target tumor cells. In vivo anti-tumor activity was evaluated with the survival of tumor-bearing animals and the size of the tumor. RESULTS: Intraperitoneal daily administration of 12.5 mg of the gelatin prolonged the survival of mice which had been intraperitoneally inoculated with MH134 (hepatic cell carcinoma cell line) or Colon 26 (colon carcinoma cell line) tumor cells, and there were no tumors in case of MH134 cells inoculation. Intraperitoneal daily administration of 12.5 mg of the gelatin did not affect growth of subcutaneous MH134 tumor. The gelatin administered subcutaneously did not affect the survival of mice with intraperitoneal MH134 tumor. On the other hand, bovine skin gelatin administered subcutaneously achieved statistically significant prolongation of the survival. The contact of MH134 cells with porcine skin gelatin for 5 min was required for the gelatin to exert its anti-tumor activity in vitro. Porcine skin gelatin of 12.5 mg injected intraperitoneally was detected as protein in the peritoneal cavity 5 min after the injection. Peritoneal macrophages elicited by intraperitoneal injection with porcine skin gelatin suppressed tritium thymidine uptake by MH134 cells more strongly than those elicited by thioglycollate injection. CONCLUSION: Porcine skin gelatin administered intraperitoneally prolonged the survival of tumor-bearing mice via activation of peritoneal macrophages and involvement of direct anti-tumor activity of porcine skin gelatin. Key Words: porcine skin, gelatin, dissemination.

Serum factors that suppress cytotoxic effect of methotrexate.

AIM: To study the phenomenon that human erythroid leukemia K-562 cells are more sensitive to cytotoxic effect of antimetabolites when cultured in a serum-free medium than in a conventional medium containing fetal calf serum (FCS). METHODS: Cytotoxic effects of methotrexate, azaserine and 5-fluorouracil were estimated by accessing the lactate dehydrogenase (LDH) activity of viable tumor cells. Proteins of FCS were separated using two-dimensional electrophoresis followed by mass spectrometry analysis. RESULTS: Addition of 10% FCS attenuated anti-tumor activity of methotrexate and azaserine against K-562 cells compared with serum-free medium. Such an activity of FCS was different for each serum lot. Comparison of the proteins in active serum lot with those in not active one using two-dimensional electrophoresis showed that in the active serum there were proteins 150 kDa, which were absent in the not active serum lot. Mass spectrometry indicated that all those proteins had the amino acid sequence of albumin. Sera of one healthy volunteer and two patients with thyroid cancer also attenuated the activity of the agent. CONCLUSION: Several lots of FCS and human serum demonstrated the ability to attenuate the cytotoxic effect of methotrexate in vitro, possibly due to the formation of albumin dimers/MTX complexes.

Pharmacologically active components of Todopon Puok (Fagraea racemosa), a medicinal plant from Borneo.

The lignans of (+)-pinoresinol, (+)-epipinoresinol, (+)-lariciresinol and (+)-isolariciresinol together with phenols such as syringaldehyde and 7,8-dihydro-7-oxy-coniferyl alcohol were isolated from Todopon Puok (Fagraea racemosa Jack ex Wall), a medicinal plant from Borneo, using a bioassay of the relaxation effect on norepinephrine (NE)-induced contraction in rat aortic strips. The plant extract also exhibited analgesic properties in the acetic acid-induced writhing and tail pressure tests in mice, with the activity being concentrated in the lignan fraction. (+)-Pinoresinol showed analgesic effect on writhing symptoms in mice which were dose-dependent, and produced local anesthesia in guinea pigs.

Precise frequency-difference measurement between the 1.66-mum transitions of methane.

We determined 66 frequency differences (FD's) between rovibrational lines of methane at the 1.66-mum region. Following the technique developed by Nakagawa et al.[Opt.Lett.20, 410 (1995)], we measured the FD's as the optical beat frequency between two external-cavity diode lasers locked at 1-MHz-wide saturated absorption dips of the methane lines. Even though the methane lines often overlap in Doppler-limited resolution, the spectrometer that we use resolves them and determines their FD's with better than 40-kHz precision. This fact demonstrates that the methane lines are promising candidates for frequency reference and that this technique has great potential for high-resolution molecular spectroscopy.

Superoxide anion scavenging properties of fluvastatin and its metabolites.

We investigated the in vitro superoxide anion scavenging activities of fluvastatin and its metabolites. Fluvastatin showed dose-dependent superoxide anion scavenging activity in the NADH/phenazine methosulphate (PMS)/nitroblue tetrazolium (NBT) system, and the effect was as potent as the reference antioxidant, trolox, which is a water-soluble alpha-tocopherol derivative. The superoxide anion scavenging activities of the major metabolites of fluvastatin (M2, M3, M4, M7) were also determined in this system. All of these metabolites showed the activity. In particular, M2 and M3, which possess a phenolic hydroxyl group at the 5 or 6-position of the indole moiety, respectively, showed 3 times stronger activities than that of fluvastatin. Further, we also determined the effects of fluvastatin, M2 and M3 on phorbol myristate acetate (PMA)-induced superoxide anion generation in human peripheral blood polymorphonuclear leukocytes (PMN). The compounds tested also showed a depressing effect on the amount of superoxide anion in this system. We suggest that fluvastatin and its metabolites have the potential to protect cells or lipids from oxidative modification mediated by superoxide anion.

Inhibitory effects of fluvastatin and its metabolites on hydrogen peroxide-induced oxidative destruction of hemin and low-density lipoprotein.

Some 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which are used as hypolipidemic drugs, have been reported to have the potential to reduce the oxidizability of plasma low-density lipoprotein (LDL) when they are administered in vivo. Their in vivo mechanism is believed to be closely related to their hypolipidemic action based on the HMG-CoA reductase inhibitory activity. We hypothesized that some type of HMG-CoA reductase inhibitor has additional mechanism inhibiting LDL oxidation in vivo due not to its hypolipidemic action but to its direct antioxidative effect based on its unique chemical structure. We directly compared in vitro the antioxidative effects of well-known HMG-CoA reductase inhibitors (fluvastatin, pravastatin, simvastatin, cerivastatin and atorvastatin) on the hydrogen peroxide-induced oxidative destruction of hemin and LDL. Fluvastatin but not the others showed the inhibitory effect on this system. Its effect was dose-dependent and almost as strong as the natural antioxidants, alpha-tocopherol and ascorbic acid. Further, M2, which is a hydroxylated metabolite of fluvastatin, showed stronger antioxidative activity than did fluvastatin. We suggest that among these HMG-CoA reductase inhibitors, fluvastatin especially has an ability to retard the LDL oxidation which is based on not only its hypolipidemic action but also its direct antioxidative effect.

The effectiveness of administering a minimal dose of octreotide long-term prior to surgery for insulinoma: report of a case.

We report herein the case of an 80-year-old woman with insulinoma who was regarded as an unsuitable candidate for immediate surgery due to her advanced age and obesity, for whom octreotide, a long-acting analogue of somatostatin, was used to improve her hypoglycemia and hyperinsulinemia without hyperalimentation. Administering a minimal dose of octreotide for a long period resulted in the improvement of leg edema, weight control, and cardiopulmonary function, and resection of the pancreatic tumor was safely carried out without any complications.