S-1 and oxaliplatin versus tegafur-uracil and leucovorin as post-operative adjuvant chemotherapy in patients with high-risk stage III colon cancer: updated 5-year survival of the phase III ACTS-CC 02 trial.

BACKGROUND: The ACTS-CC 02 trial demonstrated that S-1 plus oxaliplatin (SOX) was not superior to tegafur-uracil and leucovorin (UFT/LV) in terms of disease-free survival (DFS) as adjuvant chemotherapy for high-risk stage III colon cancer (any T, N2, or positive nodes around the origin of the feeding arteries). We now report the final overall survival (OS) and subgroup analysis according to the pathological stage (TNM 7th edition) for treatment efficacy. PATIENTS AND METHODS: Patients who underwent curative resection for pathologically confirmed high-risk stage III colon cancer were randomly assigned to receive either UFT/LV (300 mg/m2 of UFT and 75 mg/day of LV on days 1-28, every 35 days, five cycles) or SOX (100 mg/m2 of oxaliplatin on day 1 and 80 mg/m2/day of S-1 on days 1-14, every 21 days, eight cycles). The primary endpoint was DFS and the patients' data were updated in February 2020. RESULTS: A total of 478 patients in the UFT/LV group and 477 patients in the SOX group were included in the final analysis. With a median follow-up time of 74.3 months, the 5-year DFS rate was 55.2% in the UFT/LV group and 58.1% in the SOX group [stratified hazard ratio (HR) 0.92; 95% confidence interval (CI) 0.76-1.11; P = 0.3973], and the 5-year OS rates were 78.3% and 79.1%, respectively (stratified HR 0.97; 95% CI 0.76-1.24; P = 0.8175). In the subgroup analysis, the 5-year OS rates in patients with T4N2b disease were 51.0% and 64.1% in the UFT/LV and SOX groups, respectively (HR 0.72; 95% CI 0.40-1.31). CONCLUSION: Our final analysis reconfirmed that SOX as adjuvant chemotherapy is not superior to UFT/LV in terms of DFS in patients with high-risk stage III colon cancer. The 5-year OS rate was similar in the UFT/LV and SOX groups.

Influence of food on the gastric motor effect of the Kampo medicine rikkunshito in rat.

BACKGROUND: Rikkunshito, one of the Kampo medicines, is widely prescribed as a remedy for various upper gastrointestinal syndromes. The effect of rikkunshito is related to endogenous ghrelin and its active ingredient atractylodin enhances ghrelin receptor signaling. Kampo medicines are traditionally administered before or between meals; however, no definitive benefit of the timing of administration has been proven yet. To clarify the influence of food on the pharmacological action of rikkunshito, we investigated the gastric motor activity and pharmacokinetic profiles of atractylodin after the administration of rikkunshito in fasted and fed rats. METHODS: Phase III-like contractions in the gastric antrum after an injection of ghrelin were measured using a strain gauge force transducer. Rikkunshito was administered to rats during fasting or after a nutrient test meal. Ghrelin was injected 30 minutes later and gastric motility was evaluated. Furthermore, after rikkunshito administration, the pharmacokinetic profiles of atractylodin in the plasma and brain of fasted and free-fed rats were assessed. KEY RESULTS: Rikkunshito administration potentiated ghrelin-induced phase III-like contractions under fasting conditions. This effect was attenuated in animals fed a test meal. Atractylodin was detected pharmacokinetically in the plasma and brain after rikkunshito administration in rats, and free-fed rats exhibited a decreased maximum concentration of plasma atractylodin and a delayed time to reach the maximum concentration. CONCLUSIONS & INFERENCES: We show that the pharmacological action of rikkunshito is influenced by food in rats. The efficacy of rikkunshito may be associated with decreased absorption of its active ingredient atractylodin when food is in the stomach.

Prebiotic properties of epilactose.

We recently reported that cellobiose 2-epimerase from Ruminococcus albus effectively converted lactose to epilactose. In this study, we examined the biological effects of epilactose on intestinal microbiota, bile acid metabolism, and postadministrative plasma glucose by animal tests. Dietary supplementation with epilactose or fructooligosaccharide (4.5% each) increased cecal wall weight and cecal contents and decreased the pH of the cecal contents in Wistar-ST rats. The number of total anaerobes tended to be greater in rats fed epilactose and fructooligosaccharide than in those fed the control diet. Lactobacilli and bifidobacteria were more numerous in rats fed epilactose and fructooligosaccharide diets than in those fed the control diet. Analysis of clone libraries of 16S rRNA suggests that supplementation with epilactose did not induce the proliferation of harmful bacteria belonging to classes Clostridia or Bacteroidetes. Epilactose, as well as fructooligosaccharide, inhibited the conversion of primary bile acids to secondary bile acids, which are suggested to be promoters of colon cancer. In addition, oral administration of epilactose did not elevate the plasma glucose concentration in ddY mice. These results clearly indicate that epilactose is a promising prebiotic. We also showed that cellobiose 2-epimerase converted lactose in cow milk and a spray-dried ultrafiltrate of cheese whey to epilactose. Cellobiose 2-epimerase may increase the value of dairy products by changing lactose to epilactose possessing prebiotic properties.

Specificity of 17beta-oestradiol and benzo[a]pyrene oxidation by polymorphic human cytochrome P4501B1 variants substituted at residues 48, 119 and 432.

1. Eight human cytochrome P4501B1 (CYP1B1) allelic variants, namely Arg48 Ala119 Leu432, Arg48 Ala119 Val432 Gly48 Ala119 Leu432, Gly48 Ala119 Val432, Arg48 Ser119 Leu432, Arg48 Ser119 Val432, Gly48 Ser119 Leu432 and Gly48 Ser119 Va1432 (all with Asn453), were expressed in Escherichia coli together with human NADPH-P450 reductase and their catalytic specificities towards oxidation of 17beta-oestradiol and benzo[a]pyrene were determined. 2. All of the CYP1B1 variants expressed in bacterial membranes showed Fe2+.CO versus Fe2+ difference spectra with wavelength maxima at 446 nm and they reacted with antibodies raised against recombinant human CYP1B1 in immunoblots. The ratio of expression of the reductase to CYP1B1 in these eight preparations ranged from 0.2 to 0.5. 3. CYP1B1 Arg48 variants tended to have higher activities for 17beta-oestradiol 4-hydroxylation than Gly48 variants, although there were no significant variations in 17beta-oestradiol 2-hydroxylation activity in these eight CYP1B1 variants. Interestingly, ratios of formation of 17beta-oestradiol 4-hydroxylation to 2-hydroxylation by these CYP1B1 variants were higher in all of the Val432 forms than the corresponding Leu432 forms. 4. In contrast, Leu432 forms of CYP1B1 showed higher rates of oxidation of benzo[a]pyrene (to the 7,8-dihydoxy-7,8-dihydrodiol in the presence of epoxide hydrolase) than did the Val432 forms. 5. These results suggest that polymorphic human CYP1B1 variants may cause some altered catalytic specificity with 17beta-oestradiol and benzo[a]pyrene and may influence susceptibilities of individuals towards endogenous and exogenous carcinogens.

Physiological mechanism-based analysis of dose-dependent gastrointestinal absorption of L-carnitine in rats.

We evaluated the dose-dependent (saturable) gastrointestinal absorption of L-carnitine, a lipid-lowering agent, in rats by a physiological mechanism-based approach to clarify its absorption characteristics and to examine the in vitro (in situ)-in vivo correlation in intestinal transport. The intestinal absorption rate constant (ka), which was estimated by the analysis of gastrointestinal disposition, decreased markedly from 0.1061 to 0.0042 min(-1) when the dose was increased from 0.05 micromol rat(-1) (low dose) to 100 micromol rat(-1) (high dose). The dose-dependence in ka was attributable to the saturability of intestinal transport that, in the perfused intestine, was similar to the saturability in ka. At the high dose, the apparent absorption rate constant (k'a) of 0.0021 min(-1), which was estimated by the analysis of plasma concentrations after oral administration, was an order of magnitude smaller than the gastric emptying rate constant (kg) of 0.059 min(-1) and comparable with the ka of 0.0042 min(-1), suggesting that the gastrointestinal absorption of L-carnitine is absorption-limited in the intestine. At the low dose, where intestinal L-carnitine absorption was far more efficient, the k'a of 0.0172 min(-1) was smaller than the ka of 0.1061 min(-1) and closer to the kg of 0.072 min(-1), suggesting that apparent absorption was retarded by gastric emptying which is less efficient than intestinal absorption. This shift in the rate-determining process with an increase in dose explains the less marked dose dependence in k'a compared with ka. The bioavailability decreased from 100 to 42% with an increase in dose. This could be accounted for quantitatively by a reduction in the fraction absorbed (F(a,oral)) due to a reduction in ka, assuming first-order absorption during the transit time of T(si) through the small intestine (F(a,oral) = 1 - exp(-ka x T(si))). Thus, using L-carnitine as a model, this study has successfully demonstrated that the saturability in gastrointestinal absorption can be correlated with the intestinal transport in a quantitative and mechanism-based manner. This should be of help not only for developing more efficient oral L-carnitine delivery strategies, taking advantage of in vitro (in situ) information about the intestinal transport mechanism, but also for establishing a more generally applicable in vitro (in situ)-in vivo correlation in gastrointestinal absorption.

Fractional absorption of L-carnitine after oral administration in rats: evaluation of absorption site and dose dependency.

We evaluated the fractional absorption of L-carnitine, a gamma-amino acid essential cofactor for the transfer of long-chain fatty acids, in rats in vivo after oral administration to determine its absorption behavior. At both low (0.05 micromol/rat) and high (100 micromol/rat) doses, L-carnitine was recovered only from the region of the cecum and below at 10 h after administration. During a major shift in distribution from cecum at 10 h to feces at 24 h, there was no significant change in the total recovery at each dose, suggesting that L-carnitine absorption is negligible in the cecum and the large intestine (colon and rectum). However, the recovery of L-carnitine was incomplete and the fraction recovered was larger at the high dose than at the low dose. The fractions absorbed were estimated to be 96.7 and 33.0% for the low and high doses, respectively, as these were the fractions that disappeared from the gastrointestinal tract. These values were comparable with 100 and 42%, respectively, of bioavailability values by the pharmacokinetic analysis of plasma concentration data in our preceding study [Matsuda et al., Biopharmaceutics & Drug Disposition, in press]. These results suggest that L-carnitine is significantly absorbed only in the small intestine, without undergoing first-pass degradation, and in a dose-dependent manner presumably due to the involvement of saturable transport by L-carnitine carriers. Consistent with the suggestions in vivo, L-carnitine absorption in the closed intestinal loop in situ was concentration-dependent in the small intestine but not in the large intestine, and the apparent membrane permeability in the large intestine was smaller by an order of magnitude than that of passive transport in the small intestine. These findings support our preceding kinetic modeling strategy assuming the small intestine to be the sole absorption site, and should be of help in guiding studies on development of more efficient oral L-carnitine delivery strategies.

Isolation and identification of acetyl-CoA carboxylase inhibitors from green tea (Camellia sinensis).

An aqueous methanol extract from green tea showed potent acetyl-CoA carboxylase inhibitory activity. An active compound was isolated from the extract and identified as (-)-epigallocatechin gallate by instrumental analyses. The IC50 value of (-)-epigallocatechin gallate was 3.1 x 10(-4) M. Among tea catechins and related compounds, nearly equal activity was found in (-)-epigallocatechin gallate and (-)-epicatechin gallate, whereas (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, gallic acid and methyl gallate each had no inhibitory activity. These results indicate that the 3-O-gallate group of the catechin structure was necessary for this activity. (-)-Epigallocatechin gallate inhibited triglyceride accumulation in 3T3-L1 cells at a concentration of 1.0 x 10(-7) M or higher.

Effects of mitomycin C on the expression of proliferating cell nuclear antigen after filtering surgery in rabbits.

BACKGROUND: The aim of the study was to evaluate the effects of mitomycin C on the cell replication activity in wound healing following experimental filtration surgery. METHODS: Trabeculectomy with or without application of mitomycin C was performed on albino rabbit eyes. At 1, 4, 7, 14, and 28 days after surgery, the expression of proliferating cell nuclear antigen, a marker of cell proliferation, in the filtering site was examined immunohistochemically using a streptavidin-biotin complex method. RESULTS: In control eyes that underwent trabeculectomy but did not receive mitomycin C, the number of immunoreactive cells increased 4-7 days after operation and decreased markedly at around 14 days. The filtering site was obstructed histologically at 4-7 days after operation. In the mitomycin C-treated eyes, immunoreactive cells appeared 4 days after surgery but disappeared by 7 days at the surgical site. The number of immunoreactive cells in the treated eyes was much lower than that in control eyes. CONCLUSION: The cell replication activity was markedly inhibited by administration of mitomycin C. The filtering site remained open for 28 days after surgery, whereas it was completely obstructed within 7 days in control eyes. Immunocytochemistry for proliferating cell nuclear antigen, as used in this study, is a simple and reliable method for detection of cell replication activity.

Isolation and identification of alpha-glucosidase inhibitors from tochu-cha (Eucommia ulmoides).

alpha-Glucosidase inhibitory activity was found in aqueous methanol extracts of tochu-cha, dried leaves of Eucommia ulmoides (Eucommiaceae). Five active principles against yeast enzyme were isolated and characterized. Among them, quercetin (1, Ki: 8.5 x 10(-6) M) was considered to contribute mostly to the activity of the tochu leaves. In regard to an animal alpha-glucosidase, rat intestinal sucrase activity was also inhibited by 1.

Dose-dependent gastrointestinal absorption of 5-fluorouracil in rats in vivo.

Dose-dependent gastrointestinal absorption of 5-fluorouracil (5-FU) was kinetically evaluated in rats in vivo by analyzing gastrointestinal disposition after oral administration, where a linear model assuming first-order gastric emptying followed by first-order intestinal absorption was fitted to remaining fraction versus time profiles for the stomach and small intestine to estimate the rate constants of gastric emptying (kg) and intestinal absorption (Ka). With an increase in dose from 1.5 nmol/rat (low dow) to 15 mumol/rat (high dose), the Ka decreased from 5.95 to 0.55 min-1, suggesting the involvement of carrier-mediated transport. This study is the first to demonstrate the dose-dependent gastrointestinal absorption of 5-FU in vivo, though it has long been suggested in situ and in vitro. Meanwhile, at both the low and high doses, the Kg values, which were unaffected by dose (0.069 and 0.082 min-1, respectively, for the low and high doses), were smaller than the Ka values by an order of magnitude or more and the recovery of 5-FU was negligible, compared with that of inulin (a nonabsorbable marker), in the most distal segment of ileum. These results suggest that, regardless of dose, 5-FU is highly absorbable in a gastric emptying-limited manner. Thus, well-publicized bioavailability problems (low and erratic) of 5-FU may be attributable to extensive and variable first-pass metabolism rather than poor and variable gastrointestinal absorption.

[Usefulness of the 24-hour delayed film of upper gastrointestinal series for the diagnosis of sigmoidovesical fistula: report of 3 cases].

Recently, sigmoidovesical fistula is not a rare disease as a result of the change of our food style and increase in the age ratio. However, in general, the preoperative image diagnosis is difficult. We have experienced three cases of sigmoidovesical fistula, examined by barium enema, cystography, upper gastrointestinal series, cystoscopy, colonic fiberscopy and computed tomography. The fistulas were identified preoperatively by the 24-hour delayed film of upper gastrointestinal series. By this method, the regions of sigmoidovesical fistula were identified, and the patients were operated. We concluded that the 24-hour delayed film in upper gastrointestinal series might be useful to diagnose the sigmoidovesical fistula.

The inhibitory effects of tea polyphenols (flavan-3-ol derivatives) on Cu2+ mediated oxidative modification of low density lipoprotein.

Tea polyphenols (flavan-3-ol derivatives) suppressed the oxidative modification of low density lipoprotein (LDL) which is assumed to be an important step in the pathogenesis of atherosclerosis lesions. Inhibitory experiments on the oxidative impairment of porcine serum LDL by flavan-3-ols were carried out by incubating them at 37 degrees C in the presence of 5 microM Cu2+. The oxidation of LDL was monitored either by an absorption increase at 234 nm due to the conjugated diene formation, or the formation of hydroperoxides and thiobarbituric acid reactive substances (TBARS). It was found that the oxidation was strongly inhibited by various flavan-3-ols, and a lag time over 100 min appeared, depending on the types of flavan-3-ols used. The activities based on the prolongation of the lag time were in the order of (-)-epigallocatechin (EGC) < (+)-catechin (C) < (-)-epicatechin (EC) < (-)-epicatechingallate (ECG) < (-)-epigallocatechingallate (EGCG). IC50 of flavan-3-ols on Cu2+ mediated hydroperoxides and TBARS formation of LDL were 0.90, 0.95 microM for ECG and 2.38, 2.74 microM for EGC, respectively. It was found that the Cu2+ mediated cholesterol ester degradation in LDL was almost completely inhibited by 5.0 microM C or EGCG. Cu2+ mediated apolipoprotein B-100 fragmentation was also inhibited (up to 60%) in the presence of C or EGCG.

Effects of the herbal medicine "Sai-rei-to" on in vitro interferon-gamma production of peripheral blood mononuclear cells.

The herbal medicine "Sai-rei-to" has been used in the treatment of swellings and edemas for about 3000 years in China. Recently, this drug has been prescribed as an adjuvant in the treatment of rheumatoid arthritis (RA) among Japan's western medicine doctors. It is thought to possess regulatory effects on the immune system, although its mode of action is not yet fully described. In the present in vitro study, we at first induced interferon-gamma (IFN-gamma) in the cultures of peripheral blood mononuclear cells of healthy volunteers by adding pokeweed mitogen (PWM), phytohemagglutinin (PHA), recombinant interleukin-2 (IL-2), or control medium. We then added "Sai-rei-to" to these cultures and investigated the effects of this drug on IFN-gamma production levels. The results demonstrated that "Sai-rei-to" had 2 different effects: (i) it inhibited the IFN-gamma production in cultures with PWM or PHA (which induced large volumes of IFN-gamma), and (ii) it increased IFN-gamma production in the cultures with IL-2 (IL-2 induced only small volumes of IFN-gamma). These findings indicate that "Sai-rei-to" possesses regulatory effects on the IFN-gamma production. IFN-gamma is an important cytokine in the immune system, and it has also attracted attention as a factor related to the pathogeneses of RA. Therefore, concomitant administration of such a medicine which can appropriately control IFN-gamma production in vivo could be beneficial for RA patients from the immunological viewpoint. Clinical experience in the past has shown that "Sai-rei-to" has a very low incidence of side effects, and can be administered orally for long periods. We expect that concomitant administration of "Sai-rei-to" with current therapy could be clinically useful in the management of RA patients.

Neuronal activity in visual, auditory and polysensory areas in the monkey temporal cortex during visual fixation task.

The activity of 252 neurons in the inferotemporal visual area TEO, the superior temporal auditory area (AA), and the superior temporal polysensory area (STP) during the performance of a visual spot-fixation task and two variations, blink and tone tests, was examined in two behaving monkeys. A considerable number of not only TEO cells (45%) but also AA (29%) and STP (34%) cells were activated during the spot-fixation task, but unresponsive to the blanking of the spot during the fixation stage in the blink test. In addition, it was found that the activity of a third of the TEO, AA and STP cells which fired during the task-start stage in the spot-fixation task was modulated by cross-interaction between spot and tone simultaneously presented in the tone test: among these, the spot-induced activity of all TEO cells was enhanced by the tone, whereas the spot-induced activity of all AA and STP cells was suppressed by the tone. These findings are discussed in relation to the process of attending selectively to a fixation-spot.

[Extracorporeal shock wave lithotripsy monotherapy in the treatment of staghorn calculi].

From October, 1987 to September, 1989, 53 staghorn calculi of 51 patients underwent extracorporeal shock wave lithotripsy (ESWL) monotherapy by using Dornier HM3 lithotriptor. All patients were treated with double J stenting preoperatively. Mean number of shock waves was 6092 and mean number of sessions was 2.1. In 52 out of 53 kidneys (98%), the stones were disintegrated completely. Complete removal of the stone were observed in 29 kidneys (55%) 3 months after the last ESWL treatment. Complications consisted of fever attack (more than 38 degrees C) (26 patients), ileus (2), subcapsular hematoma (2) and gastrointestinal hemorrhage (1). They could be conservatively treated except one case with percutaneous nephrostomy. Supplementary procedures for the stone street were necessary in 23 patients. They consisted of ESWL (16 patients) and transuretheral lithotripsy (7). The indication of this procedure for the treatment of staghorn calculi was also discussed.

An immunomodulating protein, Ling Zhi-8 (LZ-8) prevents insulitis in non-obese diabetic mice.

Ling Zhi-8 (LZ-8), a novel and recently discovered immunomodulatory protein having in vivo immuno-suppressive activity, was tested for in vivo effect against Type 1 (insulin-dependent) diabetes mellitus in the nonobese diabetic mouse, the disease having immunologically mediated aetiology in this animal. LZ-8 had mitogenic activity in vitro towards spleen cells of the non-obese diabetic mice as previously shown towards those of DBA/2 mice. Intraperitoneal administration of LZ-8 twice weekly into the mice (10.3-12.6 mg/kg body weight) from 4 weeks of age prevented insulitis and an almost normal number of insulin producing cells were observed. Extreme insulitis and reduction of the number of insulin producing cells were observed in the pancreata of the untreated non-obese diabetic mouse. No cumulative incidence of diabetes mellitus was observed in the LZ-8 treated group, while cumulative incidences of 70% and 60% were observed in an untreated group followed up to 42 weeks of age when the incidence of diabetes was defined as a plasma glucose level of greater than 11 mmol/l and as a urine glucose level of greater than 2+, respectively. T cell subset population analysis was performed to further investigate the action of LZ-8 on the non-obese diabetic mouse which revealed that LZ-8 treatment increased in L3T4'/Lyt-2+ ratio.

A novel diagnostic method of Pneumocystis carinii. In situ hybridization of ribosomal ribonucleic acid with biotinylated oligonucleotide probes.

The reactivity and specificity of the in situ hybridization of ribosomal RNA in the diagnosis of Pneumocystis carinii were investigated. Three complementary oligonucleotide probes, 22 and 25 nucleotides long, corresponding to the species specific regions of 5S and 18S ribosomal RNA of Pneumocystis carinii were synthesized and labeled with biotinylated dUPT at the 3' termini. In situ hybridization was performed on formalin-fixed paraffin-embedded human lung tissues using the mixture of these probes and detected with the avidin-biotin peroxidase complex method. The reactions were positive in all 12 cases of Pneumocystis carinii pneumonia, but in none of the infections with other pathogenic agents, including virus (6 cases), mycobacteria (4 cases), protozoa (4 cases) and fungi (8 cases). The reactivity and specificity of this method was comparable with that of immunohistochemistry using a monoclonal anti-human Pneumocystis carinii antibody. Because the specificity of in situ hybridization is based on nucleotide sequences of ribosomal RNAs, that are constant among species, contrary to morphology of protista or expression of antigens, it should complement conventional staining and immunohistochemical methods, and provide a useful tool for the diagnosis of Pneumocystis carinii.

Effects of vitamin E administration on platelet function in diabetes mellitus.

A decrease in the vitamin E content of human diabetic platelets is closely associated with the accelerated platelet aggregation and platelet prostaglandin metabolism seen in patients with diabetes mellitus. We investigated the effect of vitamin E supplementation on these abnormalities of physiological function and prostaglandin metabolism in 14 non-insulin dependent diabetics with proliferative retinopathy. ADP-induced platelet aggregation was inhibited in vitro by the addition of vitamin E in a dose-dependent manner. However, in lower concentrations considered to be physiological doses in vivo, significantly greater inhibition was observed in diabetic platelets than in the control platelets. Next, alpha-tocopheryl nicotinate was administered to diabetics at a daily dose of 600 mg. The platelet vitamin E content was restored to control levels in 13 of the 14 patients after 2-4 weeks of daily administration. The ADP-induced platelet aggregation rate, platelet thromboxane B2 (TXB2, a stable metabolite of TXA2, a vasoconstrictor production, and plasma TXB2 level were low in all 14 diabetics. In contrast, plasma 6-keto-PGF 1 alpha (a stable metabolite of PGI2, a vasodilator) was significantly increased and therefore the 6-keto-PGF 1 alpha/TXB2 ratio in plasma was restored to within normal limits. These results indicate that vitamin E may improve platelet function and prostaglandin metabolism in diabetes mellitus and may be able to provide further beneficial effects in relation to the development of diabetic vascular complications.

Isolation and characterization of a new immunomodulatory protein, ling zhi-8 (LZ-8), from Ganoderma lucidium.

A novel protein with mitogenic activity in vitro and immunomodulating activity in vivo has been isolated from the mycelial extract of an Oriental medicinal fungus, ling zhi (Ganoderma lucidium). This protein was named ling zhi-8 (LZ-8) and its biochemical and immunological properties are described. LZ-8 was purified by two chromatographic systems, gel filtration and followed by ion-exchange, using an in vitro bioassay measuring blast-formation stimulatory activity toward mouse spleen lymphocytes to monitor purification. Analysis by several types of electrophoresis revealed a single band, with the molecular weight differing slightly depending on the system employed. Under reduced conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the method of Laemmli, U.K. ((1970) Nature 227, 680-685) indicated an apparent Mr = 17,100, while under nonreduced conditions an apparent Mr = 17,500 was found; and, using Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a value of apparent Mr = 13,100 was obtained. LZ-8 has an isoelectric point of 4.4, and sugar analysis indicated a low carbohydrate content (1.3%). Half-cysteine, histidine, and methionine were not detected from the analysis of amino acid composition after further purification of LZ-8 by reversed-phase high performance liquid chromatography. LZ-8 was capable of hemagglutinating sheep red blood cells, but no such activity was observed toward human red blood cells (A, B, AB, and O types). In vivo, LZ-8 prevents the production of systemic anaphylaxis reaction in mice if it has been administered repeatedly, and reduction of antibody production is the suggested mechanism. The mechanisms of hemagglutination of sheep red blood cells and of blast-formation stimulation of mouse spleen cells are also discussed.

Distinctive effect of ginseng saponins on development of morphine tolerance in guinea-pig ileum and mouse vas deferens.

Studies on the effect of ginseng saponins on the development of tolerance to morphine have been carried out using isolated preparations of guinea-pig ileum (GPI) and mouse vas deferens (MVD). Incubation of GPI preparation with morphine resulted in the development of tolerance to the inhibitory effect of morphine on the electrically evoked contractions. Ginseng total saponins and one of the constituents, protopanaxatriol saponin, suppressed the development of morphine tolerance in a concentration dependent manner in GPI preparation, though another constituent, protopanaxadiol saponin, did not affect the tolerance development substantially. In the MVD preparation, the development of tolerance to the morphine effect was observed as well, but none of the ginseng saponins affected it. It has been well established that electrically evoked contractions of GPI and MVD are mediated by acetylcholine and norepinephrine, respectively, and presumably their release is regulated presynaptically by opioid receptors. The fact that ginseng saponins suppressed the development of morphine tolerance only in the GPI preparation suggest that the inhibitory effect is mediated through and effect on the cholinergic system, without the involvement of direct action on opioid receptors.