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In recent years, lifestyle-related diseases such as hypertension and diabetes have been on the rise. These conditions can cause serious conditions such as myocardial and cerebral infarctions. Therefore, proper control of blood pressure and blood glucose levels is important issues in preventive medicine. Traditional fermented foods have been shown to have various functions, and their effects on lifestyle-related diseases have attracted particular attention. In this study, we investigated the effects of fermented soybeans and rice bran (OE-1) and supplements containing OE-1 on blood glucose levels and weight changes. We identified an inhibitory effect on elevated blood glucose levels upon administration of OE-1, and this effect was thought to be due to digestive enzyme inhibition. These effects of foods containing OE-1 are expected to have a positive effect on the prevention and improvement of lifestyle-related diseases as health foods.

We developed a new inhalation exposure method to evaluate effects of synthetic cannabimimetics that are being distributed as new, unregulated drugs in the Tokyo area. We selected the commercial product "SOUTOU" containing AB-CHMINACA and 5F-AMB as the test drug and dried marshmallow (Althaea officinalis) leaves as the negative control. A half cigarette packed with dried marshmallow leaves or SOUTOU was ignited, then mainstream smoke from each was delivered to five mice in an exposure box. After the cigarettes were fully consumed, neurobehavioral observations and a catalepsy test were performed at 15, 30 and 60 min after exposure. The effluent air from the exposure box was poured into impingers containing acetonitrile (first impinger) and dimethyl sulfoxide (second impinger). The resulting solutions were analyzed to assess decomposition of the synthetic cannabimimetics. Mice exposed to SOUTOU smoke showed many excitement behaviors and some suppressive behaviors at 15, 30 and 60 min. These clearly included cannabimimetic specific pharmacological actions. Negative control mice also showed some suppressive behaviors at 15 min but these were attenuated at later times, nearly disappearing at 60 min. In addition, the behavioral effects observed in controls were less pronounced than those in SOUTOU exposed mice. The inhalation exposure method developed in our study would be effective for determining cannabinoid specific pharmacological effects of illegal drugs, as well as for assessing the presence of active compound(s) by comparing the test substance with a negative control.

A lysozyme-chitosan conjugate preparation (LYZOX), produced from egg white lysozyme and chitosan by Maillard reaction, is a commercial product developed as a cosmetic ingredient or food additive. Effects of LYZOX on in vitro growth of Candida albicans were examined. C. albicans cells were treated with LYZOX for 3 hrs, and then washed and cultured for an additional 16 hrs in modified RPMI1640 medium. Mycelial growth of C. albicans was clearly inhibited by more than 100 μg/ml of LYZOX in a concentration-dependent manner. On the other hand, corresponding concentration of chitosan or lysozyme or their mixture only scarcely showed clear inhibitory effect. Similarly, anti-Candida activity of the combination of LYZOX and decanoic acid, a middle-chain fatty acid, was also examined. Inhibitory activity of this combination against mycelial growth of C. albicans was very potent and appeared synergistic, since fractionated inhibitory concentration (FIC) index for 70% growth inhibition was calculated to be 0.20. Oral application of this combination improved the symptoms of Candida-infected-tongue in an experimental murine candidiasis model. On the basis of these results, the possible application of LYZOX as a new functional product with anti-candida activity was discussed.

Alcohol hangover is an unpleasant state caused by acetaldehyde, which is produced by partial oxidation of ethanol. Treating hangover is important from the viewpoint of preventing excessive drinking. In the present study, we attempted to produce mouse model of alcohol hangover by intraperitoneal pretreatment with cyanamide at 12.5 mg/kg followed by oral ethanol at 1.6 g/kg. The mice showed decrease of spontaneous locomotor activity and food intake. Thus, it is suggested that the hangover model was successively established by co-administration of cyanamide and ethanol. Solmack, a product based on herbal medicine for stomach anda pproved as medical drug, recovered decrease of spontaneous locomotor activity and tended to recover decrease of food intake in the.hangover model mice. Other refreshing drinks did not show such effects, though they contain herbal medicine ingredients to some extent. Our model in mice might be useful to detect effective treatment for alcohol hangover.

We studied the carcinogenic effects caused by repetitive irradiation at a low dose, which has received attention in recent years, and examined the experimental methods used to evaluate radiation-induced carcinogenesis. For this experiment, we selected a mouse with as few autochthonous cancers as possible. Skin cancer was selected as the target for analysis, because it is a rare cancer in mice. Beta-rays were selected as the radiation source. The advantage of using beta-rays is weaker penetration power into tissues, thus protecting organs, such as the digestive and hematogenous organs. The benefit of our experimental method is that only skin cancer requires monitoring, and it is possible to perform long-term experiments. The back skin of mice was exposed repetitively to beta-rays three times a week until the occurrence of cancer or death, and the dose per exposure ranged from 0.5 to 11.8 Gy. With the high-dose range (2.5-11.8 Gy), the latency period and carcinogenic rate were almost the same in each experimental group. When the dose was reduced to 1-1.5 Gy, the latency period increased, but the carcinogenic rate remained. When the dose was further reduced to 0.5 Gy, skin cancer never happened, even though we continued irradiation until death of the last mouse in this group. The lifespan of 0.5 Gy group mice was the same as that of the controls. We showed that the 0.5 Gy dose did not cause cancer, even in mice exposed repetitively throughout their life span, and thus refer to 0.5 Gy as the threshold-like dose.

A daily eatable candy that has possible protective activity against oral candidiasis was experimentally produced. The candy was made from reduced-maltose as main constituent and from several natural products, such as oligonol (depolymerized polyphenols derived from lychee), cinnamon (cassia), citral, and capric acid, which are known to have anti-Candida activity in vitro and in vivo. The candy effectively inhibited the mycelial growth of C. albicans, even when it was diluted 1,000 times with culture media. We assessed the protective activity of the candy against murine candidiasis. When 50μl of candy dissolved and diluted 4 times with water was administered 3 times into the oral cavity of Candida infected mice, the score of lesions on the Candida-infected tongues improved on day 2. These findings suggest that this candy has potential as food that provides protective activity against oral candidiasis.

Numerous rodent models of depression have been reported, most requiring a long experimental period and significant effort. We explored a new potential mouse model for depression by investigating whether exposure to a 15-day chronic stress paradigm could induce depression-like behavior in ICR mice. Animals in the stress-exposed groups were subjected to 3 h of restraint while immersed in a 28°C water bath daily for 15 consecutive days. Immobility time in the forced swim test was increased in the chronic stress-exposed mice compared with the controls. Serum corticosterone levels were also much higher in the stressed mice than in the control mice. Hippocampal cell survival (BrdU-positive cells) and neurotrophic factor (NGF, TrkA) mRNA levels were significantly decreased in the chronic stress-exposed mice compared with controls. Administration of the anti-depressant drugs clomipramine (20 mg/kg/d) or imipramine (30 mg/kg/d) did not change the immobility time in the forced swim test, but treatment with lithium (100 mg/kg/d) did result in slight improvement. These results suggest that this 15-day chronic stress paradigm can induce depression-like behavior and neurological changes, in a short time and with minimal effort, facilitating the assessment of treatments for depression.

As an adverse event, it has been reported that anticoagulation activity of warfarin was enhanced by simultaneous intakes of glucosamine and chondroitin sulfate. However, it is unclear whether these is a causative relation. Therefore, in the present study, we evaluated whether glucosamine and chondroitin sulfate enhanced the anticoagulant action of warfarin in mice in vivo, focusing on hepatic cytochrome P450 (CYPs)-mediated mechanisms. Mice were fed a diet containing various doses of glucosamine or chondroitin sulfate (0, 0.3, 1% (w/w)) for 2 weeks, and given warfarin by gavage on the last 2 days of the treatment regimen. Doses of glucosamine and chondroitin sulfate were 443 mg/kg and 464 mg/kg in the 0.3% diet groups, and 1523 mg/kg and 1546 mg/kg in the 1% diet groups. We found that 1% glucosamine significantly shortened prothrombin time and thrombotest Owen in animals given warfarin. However, the two ingredients did not induce or inhibit hepatic CYPs, including (S)-warfarin hydroxylase. These findings suggest that glucosamine and chondroitin sulfate do not affect the anticoagulation activity of warfarin through hepatic CYP mediated-mechanisms.

The effect of S-PT84, a heat-killed preparation of Lactobacillus pentosus on growth of Candida albicans was examined in vitro and in vivo. The mycelial growth was effectively inhibited by S-PT84 and seemed to bind to the hyphae. We assessed the potential of S-PT84 for treatment of oral and gastric candidiasis using a murine model. When 2 mg of S-PT84 was administered three times into the oral cavity of orally Candida infected mice, the score of lesions on the tongue was improved on day 2. When 50 μl and 200 μl of S-PT84 (10 mg/ml) were administered three times into the oral cavity (0.5 mg × 3) and the stomach (2 mg × 3) of the same mouse model, the number of viable Candida cells in the stomach was reduced significantly on day 2. These findings suggest the possibility that S-PT84 has potential as a food ingredient supporting anti-Candida treatment, especially for Candida infection in the gastrointestinal tract.

Herb-drug interactions are mainly mediated by hepatic cytochrome P450 (CYP) enzymes. Here, we examined the effect of three herbs (valerian, salacia and black cohosh) on CYP activity in vivo in mice and in liver microsomes in vitro. Extracts which showed activity in the preliminary tests were then fed to mice at various doses (0, 0.5, 1.5 and 4.5%). Valerian did not show any effect on hepatic CYPs. Black cohosh increased the liver weight, total CYP content and CYP activities (2B and 3A) in a dose-dependent manner (up to 4.5%). Salacia inhibited CYP1A2 activity in liver microsomes in vitro. Also, salacia at the dietary dose of 4.5% suppressed body weight gain, decreased hepatic total CYP content and increased CYP activities (1A1, 2B and 2C). These findings suggest that black cohosh and salacia at high dose affect the activity of hepatic CYPs, and therefore may interact with drugs that are metabolized by CYP.

We have established spontaneously immortalized Schwann cell lines from adult ICR mice [IMS32] and Fischer344 rats [IFRS1]. IMS32 cells display distinct Schwann cell phenotypes such as a spindle-shaped morphology and the expression of glial cell markers (e.g. S100, glial fibrillary acidic protein (GFAP), p75 low-affinity neurotrophin receptor (p75(NTR))) and neurotrophic factors. In addition, conditioned medium obtained from IMS32 cells enhances neurite elongation of PC12 cells and mouse dorsal root ganglion (DRG) neurons. IMS32 cells have been utilized to investigate the action mechanisms of various molecules that accelerate peripheral nerve regeneration (e.g. ciliary neurotrophic factor, sonic hedgehog, galectin-1). Like IMS32 cells, IFRS1 cells retain the characteristic features of mature Schwann cells as described above. Furthermore, IFRS1 cells have been shown to myelinate neurites in coculture with adult rat DRG neurons and PC12 cells. Our current investigation with IFRS1 cells focuses on the molecular mechanisms of myelination-inducible factors, such as soluble neuregulin-1 type III and exendin-4. These Schwann cell lines can be valuable tools for exploring neuron-Schwann cell interactions, pathobiology of axonal degeneration and regeneration in the peripheral nervous system, and novel therapeutic approaches against neurological disorders in patients with relevant diseases.

The combined effect of terpinen-4-ol, the main component of tea tree oil, and capric acid against mycelial growth of Candida albicans and murine oral candidiasis was evaluated in vitro and in vivo. Mycelial growth of C. albicans was estimated by the Cristal violet method. Combination of these compounds revealed a potent synergistic inhibition of growth. Therapeutic efficacy of the combination was evaluated microbiologically in murine oral candidiasis, and its application of the compounds clearly demonstrated therapeutic activity. Based on these results, the combined agent of terpinen-4-ol and capric acid was discussed as a possible candidate for oral candidiasis therapy.

Selective serotonin reuptake inhibitors (SSRIs) are known to reduce post-myocardial infarction (MI)-induced morbidity and mortality. However, the molecular mechanism underlying SSRI-induced cardioprotection remains unclear. Here, we investigated the role of sigma-1 receptor (Sig-1R) stimulation with fluvoxamine on myocardial hypertrophy and cardioprotection. Male ICR mice were subjected to transverse aortic constriction (TAC) in the cardiac aortic arch. To confirm the cardioprotective role of Sig-1R stimulation by fluvoxamine, we treated mice with fluvoxamine (0.5 or 1 mg/kg) orally once a day for 4 weeks after onset of aortic banding. Interestingly, in untreated mice, Sig-1R expression in the left ventricle (LV) markedly decreased over 4 weeks with increased hypertrophy. By contrast, fluvoxamine administration significantly attenuated TAC-induced myocardial hypertrophy concomitant with recovery of Sig-1R expression in LV. Fluvoxamine also attenuated hypertrophy-induced impaired LV fractional shortening. The fluvoxamine cardioprotective effect was nullified by treatment with a Sig-1R antagonist, NE-100 (1 mg/kg). Importantly, another SSRI with very low affinity for Sig-1R, paroxetine, did not exhibit antihypertrophic effects in TAC mice and in cultured cardiomyocyte treated with angiotensin II. Fluvoxamine treatment significantly restored TAC-induced impaired Akt and eNOS phosphorylation in LV. Our findings suggest that fluvoxamine protects heart against TAC-induced cardiac dysfunction via upregulation of Sig-1R and stimulation of Sig-1R-mediated Akt-eNOS signaling in mice. This is the first report of a potential role of Sig-1R stimulation by fluvoxamine in preventing cardiac hypertrophy and myocardial injury in TAC mice.

To evaluate the effect of the thiocarbamate antifungal agent liranaftate on inflammation and itchiness, footpad edema by phorbol 12-myristate 13-acetate (PMA) and the paw-licking accompanying by perceptual stimuli by compound 48/80 were examined. The effect of liranaftate application to mouse footpad on paw-licking time by compound 48/80 was observed. Topical administration of 4% liranaftate 1 hr before compound 48/80 did not suppress the paw-licking time, while pyrilamine, an anti-histamine agent, suppressed it significantly. As liranaftate was reported to suppress the ear inflammation induced by PMA, the effect of this agent on the footpad edema by PMA was examined. Liranaftate application significantly suppressed the increase in footpad swelling 24 hr after application of PMA, as true with ear inflammation. In this condition, we measured the paw-licking time by compound 48/80, but the suppression of time was not observed by the agent with or without the suppression of footpad inflammation. From these observations, we conclude that liranaftate treatment suppresses late phase inflammatory reaction in feet, perhaps accompanied by cytokine production, though it may not relieve acute stimuli and itchiness through an anti-histamine effect directly.

The protective effects of notoginseng against hepatic damage were investigated in mice. To prepare a model animal of hepatitis, a mixture of lipopolysaccharide and galactosamine (LPS/GAlN) was administered intraperitoneally, leading to the impairment of hepatic function. Extracts of notoginseng or its components (ginsenoside Rb1 and ginsenoside Rg1) were orally administered 2 h before LPS/GalN injection. Eight hours after LPS/GalN injection, blood and liver tissue samples were collected. The levels of serum aspartate amino transferase (AST), alanine aminotransferase (ALT), tumor necrosis factor (TNF)-α, and interferon (IFN)-γ were measured using commercial assay kits. Histologic changes in the tissue samples were also observed after hematoxylin-eosin staining. LPS/GalN administration increased the serum levels of AST and ALT, and histologic changes were noted, indicating hepatic cell damage. Prior to the increase in ALT, the serum levels of TNF-α and IFN-γ were elevated after LPS/GalN injection. Pretreatment of the mice with either notoginseng extract or gensenoside Rb1 and Rg1 attenuated the LPS/GalN-induced hepatic damage markedly. The protective effects of the components against hepatic damage appeared to be less potent than those of the crude extract and prescription of notoginseng. Notoginseng may be clinically useful in patients with hepatitis.

Fucoxanthin-containing oil extracted from microalga, Chaetoseros sp., was subjected to genotoxicity studies, the bacterial reverse mutation test and the micronucleus test in mice. The number of revertant colonies in fucoxanthin oil-treated plates of all strains tested was less than twice the number of colonies in the negative control, regardless of the presence of the metabolic activator in the bacterial reverse mutation test. In the micronucleus test, 500, 1,000, 2,000 mg/kg body weight of fucoxanthin oil was administered orally to mice. There was no significant increase in micronucleus frequency in bone marrow cells. These results suggest that fucoxanthin oil does not exhibit genotoxicity.

We defined the staining applicability of Histofine MOUSESTAIN, Mouse MAX-PO and Rat MAX-PO for fixed frozen and fresh frozen tissue sections besides paraffin embedded tissue sections. Two different types of cool air dried tissue sections, fresh frozen tissues and fixed frozen tissues of nine-week old Slc: ICR mouse and nine-week old Slc: SD rat, were prepared for immunohistochemistry (IHC) staining in accordance with conventional method. Additional two steps of 1 and 2 to our recommended procedure for paraffin embedded tissue sections were examined as follows: STEP 1: Adjust reaction time of polymer; STEP 2: Add 0.2% of glutaraldehyde (GA) solution before the STEP 1, when background staining is still observed by STEP 1. The steps 1 and 2 resulted in elimination of background staining of most Histofine series. Therefore, polymer detection systems, Histofine series, are applicable for IHC staining of two different types of frozen tissue sections as well as paraffin embedded tissue sections.

We assessed the potential of oligonol, a low molecular polyphenol formulation prepared from lychee fruits, for treatment of oral candidiasis using a murine model. Oligonol at concentration of more than 313 microg/ml inhibited the mycelial growth of Candida albicans in vitro. When 50 microl of oligonol (20 mg/ml ) was administered three times into the oral cavity of orally Candida -infected mice, the number of viable Candida cells in the oral cavity was reduced significantly and the score of lesions on the tongue recovered on day 2. These findings suggest that oligonol could have potential as a food component supporting anti- Candida treatment.

To evaluate the anti-inflammatory activity of the thiocarbamate antifungal agent liranaftate, the edema and the neutrophil accumulation detected by the activity of neutrophil marker enzyme, myeloperoxidase (MPO), were examined following application of liranaftate to mouse ears with inflammation induced by phorbol 12-myristate 13-acetate (PMA). Topical 20 microl administration of liranaftate in a dose-range between 1-4% suppressed the increase in ear thickness 6 hr after PMA application dose-dependently. Similarly, it decreased the weight increase of an ear section after 24 hr dose-dependently. More than 1% of liranaftate also suppressed augmentation of MPO activity of the ear section. This and histological observation indicate that liranaftate treatment suppressed neutrophil accumulation in PMA-applied ear lesion. From these results, we discussed that liranaftate might suppress inflammatory symptoms caused by trychophytosis in a clinical condition.

Pharmacokinetics of tebipenem pivoxil (TBPM-PI), a novel oral carbapenem antibiotic, were known in various laboratory animal. (1) In mouse, rat, dog and monkey, TBPM-PI were absorbed quickly, and the bioavailability was (71.4, 59.1, 34.8 and 44.9%, respectively. (2) TBPM-PI was quickly converted to tebipenem (TBPM), an active form of TBPM-PI. Through blood circulation, TBPM was distributed into the kidney at a high concentration and eliminated quickly. There was no other tissue than the kidney, in which TBPM was highly distributed and remained for a long time. In addition, low penetration to the central nervous system was confirmed. The penetration ratio of TBPM to ELF, that is the ratio of ELF concentration to plasma concentration of TBPM, was 21.8 +/- 14.7%. (3) Serum protein bindings of TBPM in the range of 0.1-100 microg/ml were 90.4-98.3% for mouse, 78.5-90.0% for rat, 15.7-18.7% for dog, 35.3-39.3% for monkey and 59.7-73.9% for human. (4) In vitro metabolism was investigated in plasma, liver S9 fractions and small intestinal S9 fractions derived from infant and adult animals. TBPM-PI was transformed into TBPM quickly in any matrices. It was confirmed that absorbed TBPM-PI was quickly transformed into TBPM or LJC 11,562 (opened ring TBPM) in the plasma after oral administration of 14C-TBPM-PI to infant or adult rat and monkey. TBPM-PI and opened ring TBPM-PI was not detected in plasma and urine samples. In rat and monkey, the oral absorption, distribution, metabolite and excretion of TBPM-PI were not so much different between infant and adult animals. (5) Liver metabolic enzyme system was little affected by 7-days repeated administration of 1-100 mg/kg TBPM-PI. IC50 values of TBPM-PI and TBPM for human CYP isoforms were estimated to be 100 microg/ml or higher. (6) After single oral administration of 10 mg/kg 14C-TBPM-PI to rat, 36.9-42.7% and 58.3-62.2% of radioactivity was excreted to urine and feces, respectively, by 120 hours after administration. The majority of dosage was excreted out of body by 48 hours after administration. After single intravenous administration of 10 mg/kg 14C-TBPM, 87.4% and 11.4% of radioactivity was excreted in urine and bile, respectively, by 24 hours after administration. The majority of dosage was excreted out of body by 4 hours after administration.

We investigated physiological and behavioral characteristics of socially stressed animals in a resident-intruder paradigm. ICR male mice (resident, n = 14) were exposed individually to a novel male conspecific (intruder, n = 14) in their homecage for 30 min. Along with behavioral analyses, the skin temperatures of both the resident and the intruder were measured simultaneously using a multipoint radiation thermometer. There were no significant differences between the resident and intruder in the amount of locomotion, flight and aggressive behaviors. The mean skin temperature of the residents during the interaction was higher than before the interaction. In addition, the skin temperatures of the intruders were consistently higher than the residents. The results suggest that social stress causes elevation in skin temperature as well as stress-induced hyperthermia in core temperature. Moreover, infrared radiation thermometers may provide an alternative means of measuring physiological parameters of two (or more) subjects simultaneously in the study of animal social behavior.

We have identified the vaspin gene(serpina12), which is up-regulated in visceral white adipose tissues (WATs) of Otsuka Long-Evans Tokushima fatty (OLETF) rat, an animal model of abdominal obesity and type 2 diabetes. Vaspin mRNA was barely detectable at 6 weeks of age, but was abundantly and exclusively expressed in visceral WATs at 30 weeks of age, when OLETF rats reach their peak body weight. However, vaspin mRNA decreased with worsening of diabetes and body weight loss. Vaspin mRNA increased with administration of thiazolidinediones, i.e. pioglitazone. Administration of recombinant vaspin into high fat high sucrose (HFHS) chow-induced obese ICR mice improved glucose tolerance and insulin sensitivity. Vaspin may be the compensatory molecule in the pathogenesis of metabolic syndrome and vaspin recombinant protein or vaspin-mimicking agents such as vaspin analogues, antibodies or small molecule agents would link to drug discovery and development.

Abuse of volatile organic solvents among youth remains a major social problem. Organic solvents are cheap and relatively easy to obtain, so they carry the risk of becoming a "gateway drug" for users. The effect of repeated inhalation of toluene on subsequent responses to other drugs of abuse is unclear. In the present study, we investigated the effect of toluene inhalation on methamphetamine-induced behavioral change using a newly developed sealed inhalation shuttlebox. The influence of the cyclic AMP response element binding (CREB) protein expression following toluene inhalation was also examined. Mice were exposed to toluene or air once daily for five days. Methamphetamine produced significant hyperlocomotion in air-exposed mice. This stimulatory effect of methamphetamine was significantly enhanced following repeated inhalation of toluene. Furthermore, repeated toluene inhalation increased the levels of CREB proteins in the limbic forebrain. The present study demonstrated that adaptation of the adenylate cyclase system following repeated toluene inhalation might be involved in the expression of behavioral sensitization to subsequent methamphetamine administration. Inhalant abuse could thus be associated with the risk of other substances of abuse.

It has been clinically reported that chronic exposure to diphenylarsinic acid (DPAA) induced prominent cerebellar symptoms in apartment building residents in Kamisu, Japan. The aim of the present study was then to investigate the effect of chronic treatment with DPAA on the central motor impairment in mice. In the present study, we found that chronic in vivo exposure to a high dose of DPAA induced motor impairment in adult mice. This impairment was reversed by withdrawal following chronic DPAA treatment. The [35S]GTPgammaS binding assay showed the down-regulation of the dopamine receptor function in the striatum in adult mice treated with DPAA. We also found that neonatal exposure to a low dose of DPAA induced motor learning impairment in mice. Furthermore, treatment with an extremely low dose of DPAA caused the activation of caspase-3, the increase in glial fibrillary acidic protein-like immunoreactivity (IR) and the reduction in levels of myelin-associated glycoprotein-IR in mouse cerebellum neuron/glia co-cultures. In addition, we found that neonatal exposure to a low dose of DPAA induced anxiogenic behavior in a plus maze in mice. Taken together, these results suggest that chronic treatment with DPAA may induce motor impairment in adult mice. Moreover, neonatal exposure to DPAA leads to the irreversible motor impairment associated with abnormalities in the cerebellum.

The aim of the present study was to investigate the role of protein kinases within the spinal cord in the development of a neuropathic pain-like state induced by partial sciatic nerve ligation in mice. Thermal hyperalgesia induced by nerve ligation in mice was markedly suppressed by either repeated intrathecal (i.t.) pre-treatment or post-treatment with the selective protein kinase (PKC) inhibitor RO-32-0432 and the selective Rho kinase inhibitor Y-27632. In contrast, sciatic nerve ligation-induced thermal hyperalgesia was not observed by repeated i.t. pre-treatment with the selective PKA inhibitor KT5720. Interestingly, thermal hyperalgesia induced by nerve ligation in mice was significantly suppressed by repeated i.t. post-treatment with fasudil, which possesses the inhibitory effect of several protein kinases including PKC and Rho kinase. Collectively, these findings suggest that a long-lasting activation of PKC and RhoA/Rho kinase pathways in the spinal cord may be responsible for the development of thermal hyperalgesia induced by nerve ligation in mice. The present data raise the fascinating possibility that i.t. or epidural administration with fasudil may be useful for the treatment of neuropathic pain.

Adult bone marrow consists of two different compartments, a vascular compartment of sinusoid and a hematopoietic compartment consisting of stromal cells and hematopoietic cells. In the hematopoietic compartment, stromal cells play an important role in the formation of the microenvironment for hematopoiesis. To clarify the relationship between hematopoietic cells and stromal cells, particularly reticular cells and macrophages, we examined the femur bone marrow of ICR mouse fetuses and neonates using F4/80 immunostaining and three-dimensional reconstruction under light and electron microscopy. In the fetal femurs, the marrow cavity formed early from 15 days of gestation, and it showed a marked increase in volume thereafter. On the basis of the appearance of hematopoietic cells, marrow development could be classified into two stages, a pre-hematopoietic stage from 15 days of gestation to two days of age, and a beginning stage of hematopoiesis thereafter. The pre-hematopoietic bone marrow contains not only stromal reticular cells but also macrophages, and both types of stromal cells were strongly positive to F4/80 monoclonal antibody. These F4/80-positive reticular cells had a triangular cell profile with long and slender cytoplasmic processes. Reticular cells often contained large lysosomes of not only dying neutrophils but also erythroblast nuclei. A few erythroblasts accumulated around the processes, and the number of erythroblasts around reticular cells increased with bone marrow development. On the other hand, macrophages were located either close to sinusoids or in sinusoid lumen, and a close relationship to hematopoietic cells was hardly noticeable. At the beginning stage of hematopoiesis, F4/80-positive reticular cells extended their long and slender cytoplasmic processes, and the number and length of the processes appeared markedly increased. The three-dimensional cell surface of the F4/80-positive reticular cells became very complex. Numerous erythroblasts accumulated around the processes, and erythroblastic islands could gradually be recognized after four days of age. In the erythroblastic islands, central reticular cells were F4/80-positive and contained numerous large phagosomes originating from the expelled nuclei of erythroblasts. Although macrophages contained large phagosomes, the relationship between macrophages and hematopoietic cells could not clearly be elucidated even at the beginning stage of hematopoiesis. At the onset of bone marrow hematopoiesis, the hematopoietic compartment contained two kinds of F4/80-positive phagocytes, i.e., reticular cells and macrophages. In marrow erythroblastic islands, not macrophages but F4/80-positive reticular cells were located at the center of each island.

The concomitant use of low concentration beta-lactam antibiotics antagonizes the activity of vancomycin against some strains of MRSA. These strains, called beta-lactam antibiotic-induced vancomycin resistant MRSA (BIVR), have been increasing for a number of years. We previously reported that the combination of VCM and ceftizoxime displayed this antagonism not only in vitro, but also in vivo, in a systemic infection caused by BIVR in mice. In the present study, we validated the antagonism in combinations of VCM with other beta-lactams, i.e., flomoxef (FMOX), ampicillin (AMPC), azthreonam (AZT) and imipenem/cilastatin (IPM/CS), in systemic infections and pneumoniain in mice. The survival rate of the mice with systemic infections caused by BIVR treated with combinations of FMOX, AMPC, AZT, and IPM/CS with VCM were significantly lower than with VCM monotherapy, and the number of residual viable cells in the kidneys of mice treated with combinations of FMOX and IPM/CS with VCM were significantly higher than with VCM monotherapy The number of residual viable cells in the lungs of mice with pneumonia caused by BIVR treated with the combination of IPM/CS and VCM was significantly higher than with VCM monotherapy. On the other hand, the survival rate with combination therapy consistings IPM/CS plus teicoplanin (T EIC) was significantly higher, and the number of residual viable cells in the kidney was significantly lower, than with TEIC monotherapy alone. In the mice with pneumonia, the number of residual viable cells in the lung after combination therapy with IPM/CS and TEIC was significantly lower than with TEIC monotherapy. Combination therapy with beta-lactams plus VCM showed antagonistic in models of systemic infection and pulmonary infection caused by BIVR, whereas combination therapy consisting of a beta-lactam plus TEIC had a synergistic effect in the same models, even though VCM and TEIC are member of the same glycopeptide antibiotic class.

Diphtheria-tetanus-acellular pertussis vaccine (DTaP) developed in Japan is now widely used worldwide. DTaP is safer than the diphtheria-tetanus-whole-cell pertussis vaccine (DTwP) and has fewer severe side effects, but local reactions such as redness, swelling, and induration are still reported. The pathophysiological mechanism of these reactions is controversial. To clarify the cause of local reactions, we conducted studies using the mouse model. After administering either one or two abdominal subcutaneous DTaP inoculations, we observed changes in histopathology at the injection site at 24h, 48h, and 7 days. The control group, inoculated with physiologic saline, showed no significant changes either pathologically or with the naked eye. All mice after DTaP vaccination showed indurations at the injection site. Pathologically, we watched leukocyte invasion into or around the site, especially neutrophils and eosinophils. After the first vaccination, the extent of the invasion was strong 24h and 7 days later. At 24h following the second vaccination, a dramatic leukocyte invasion seen persisted at 7days. At 7 days after the first vaccination, peripheral fibrosis had begun, and when a second vaccination was administered, it began even earlier at the second site. These histopathological changes show that local reactions are caused by both inflammatory and allergic responses. Because this mouse study resulted in the same pattern of reactions observed in humans, this method will be useful for studies focusing on local reactions.

Tissue plasminogen activator (tPA) is a serine protease that catalyzes the conversion of plasminogen (plg) to plasmin. The tPA-plasmin system plays a role in synaptic plasticity and remodeling. In this review, we focused on the role of tPA-plasmin system in the rewarding effect of morphine. A single morphine treatment induced tPA mRNA and protein expression in a naloxone-sensitive manner, which was associated with an increase in the enzyme activity in the nucleus accumbens (NAc). The acute effect of morphine in inducing tPA expression was diminished after repeated administration. No differences were observed in the morphine-induced antinociceptive effect between wild-type and tPA knockout (tPA-/-) mice. Morphine-induced conditioned place preference and hyperlocomotion were significantly reduced in tPA-/- and pLg-/- mice, being accompanied by a loss of morphine-induced dopamine release in the NAc. Microinjection of either exogenous tPA or plasmin into the NAc significantly potentiated morphine-induced dopamine release in the NAc of ICR mice. In contrast, plasminogen activator inhibitor-1 (PAI-1) dose-dependently reduced morphine-induced dopamine release. Furthermore, the defect of morphine-induced dopamine release and hyperlocomotion in tPA-/- mice was reversed by microinjections of either exogenous tPA or plasmin into the NAc. Our findings demonstrate a novel function of the tPA-plasmin system in regulating dopamine release in the NAc, which is involved in the morphine reward.

In the process of investigating the hypolipidemic effects of Spirulina platensis, we found that the aqueous extract of S. platensis may inhibit the intestinal absorption of dietary fat by inhibiting pancreatic lipase activity. The aqueous extract of S. platensis (500 m/kg) reduced the elevation of rat plasma triacylglycerol levels after oral administration of the lipid emulsion 2 h after administration. To clarify the hypolipidemic effects of S. platensis, the active component was isolated and designated 1'-O-(palmitonyl)-2'-O-(caprylonyl) glyceryl-beta-alpha-D-galactopyranoside (glycolipid H-b2). Glycolipid H-b2 was found to inhibit pancreatic lipase activity in a dose-dependent manner. The fractions containing glycolipid H-b2 (250 mg/kg) reduced the elevation of rat plasma triacylglycerol levels after oral administration of the lipid emulsion 2 h after administration. Furthermore, we examined the effects of phycocyanin isolated from S. platensis on pancreatic lipase activity. Phycocyanin inhibited the pancreatic lipase activity in a dose-dependent manner. These results suggest that the inhibitory effects of S. platensis on postprandial triacylglycerolemia may be due in part to the inhibition of pancreatic lipase activity by glycolipid H-b2 and phycocyanin.

Mice exposed to various stresses, especially restrained-stress, revealed the anxiogenic effect detected by the light-dark test. Under this condition, a remarkable decrease in [35S]GTPgammaS binding to membranes from the prefrontal cortex, amygdala and hypothalamus of restrained-stress mice stimulated by the selective 5-HT1A receptor agonist 5-carboxamidotriptamine (5-CT) was clearly observed, whereas a significant increase in [35S]GTPgammaS binding stimulated by the 5-HT1A receptor agonist was clearly observed in the dorsal raphe nuclei (DRN) of restrained-stress mice. The immunohistochemical study showed a drastic reduction in phosphorylated-CREB-like immunoreactivity in the DRN of restrained-stress mice. Furthermore, we found a drastic reduction in myelin-associated glycoprotein (MAG)-like immunoreactivity (MAG-IR) in the DRN, amygdala and hypothalamus, indicating the direct suppression of synaptic transmission in these regions. It has been accepted that GSK3beta in the Wnt signal pathway plays an important role in various neuronal functions including apoptosis, clustering of synapsin I and early growth and axonal remodeling. In the present study, the increase in protein levels of GSK3beta and phosphorylated-GSK3beta to cytosol fractions of the amygdala was noted in restrained-stress mice. Taken together, these results suggest that restrained stress may directly affect the 5-HT1A receptor-regulated synaptic transmission in the brain, leading to the expression of the anxiogenic effect in mice. It is well known that various stresses induce intracellular oxidative stress. The present study was then undertaken to investigate the effect of the stimulation of 5-HT1A receptors on oxidative stress. Treatment with H2O2 caused the activation of caspase-3-positive cells and the reduction in levels of MAG-IR in the limbic neuron/glia cocultures as compared to medium alone. The stimulation of 5-HT1A receptor by 5-CT produced a dramatic protection against H2O2-triggered activation of caspase-3 and reduction in levels of MAG-IR. These results suggest that 5-HT1A receptors were involved in the modulation of anxiety and the understanding of molecular mechanisms of 5-HT1A receptor-related cascades may pave the way for new therapeutic strategies for affective disorders.

Background: Vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase-activating peptide (PACAP), are members of a VIP/secretin/glucagon family. These peptides were demonstrated to possess the neuroprotective properties. However, these peptides are not suited to be developed as a medicine for brain ischemia because of their susceptibilities to endopeptidases., Methods: We examined the effects of IK 312548 (IK), VIP derivative, and Ac-PACAP, PACAP derivative, on the 10 min two-vessel occlusion (2 VO) model in C 57 BL/6 N mice lacking a part of the posterior communicating artery, and the 30 min middle cerebral artery occlusion (MCAO) model in ICR mice. A 10 ml x kg(-1) dose of each derivative (final concentration; 1 fmol x kg(-1) and 100 pmol x kg(-1)) was injected intraperitoneally (i.p.) to each animal just after the preparation of brain ischemia., Results: In 2 VO experiments, the number of neuronal cells in hippocampus was significantly reduced. However IK and Ac-PACAP treatments inhibited such reductions of neuronal cells in a dose-dependent manner. Particularly, between 1 pmol x kg(-1) and 100 pmol x kg(-) IK, and also between 10 fmol x kg(-1) and 1 pmol x kg(-1) Ac-PACAP significantly protected neuronal cell loss. In MCAO experiments, more than 60% of hemisphere was damaged. By treatment of IK (1-100 pmol x kg(-1)) and Ac-PACAP (1 fmol-1 pmol x kg(-1)), the range of brain damage decreased in a dose-dependent manner., Conclusions: Ac-PACAP and IK after the brain ischemia could pass the blood-brain barrier and protect brain cell damage.

Zingiber officinale Roscoe has been used as a folk medicine in China. An aqueous extract of Z. officinale Roscoe inhibited the hydrolysis of triolein emulsified with phosphatidylcholine by pancreatic lipase in vitro and it reduced the elevation of rat plasma triacylglycerol levels 1 and 2 h after oral administration of a lipid emulsion containing corn oil. These results suggest that the aqueous extract of Z. officinale Roscoe might inhibit the intestinal absorption of dietary fat by inhibiting its hydrolysis. Therefore we investigated the antiobesity effects of the aqueous extract of Z. officinale Roscoe by feeding a high-fat diet to mice for 8 weeks. Body weights at 2-8 weeks and final parametrial adipose tissue weights were significantly lower in mice fed the high-fat diet containing 3% aqueous extract of Z. officinale Roscoe than in the controls fed the high-fat diet. Feeding a high-fat diet containing 1% aqueous extract of Z. officinale Roscoe also significantly reduced final parametrial adipose tissue weights that were elevated in mice fed the high-fat diet alone. Our data suggest that the antiobesity effect of aqueous extract of Z. officinale Roscoe in mice fed a high-fat diet may be due in part to the inhibition of intestinal absorption of dietary fat by the active compounds of Z. officinale Roscoe.

The original plant of Citrus hassaku Hort. Tanaka was found at the Jyoudo Temple in Inno-shima, Hiroshima Prefecture, Japan by Ekei Shounin in the Edo Era. Citrus hassaku has been called "jagada" when it was first discovered. Since this fruit was seasoned for eating around "hassaku" (August 1st of the lunar calendar), it was given the scientific name as "C. hassaku." Today, the fresh raw fruits of C. Hassaku are cultivated as a seasonal food, and the most famous producing district of C. hassaku fruit is Wakayama Prefecture, representing about 60% of the Japanese output. The immature fruits of C. hassaku and its three main flavanone glycosides (naringin, neohesperidin and narirutin) was found to shown inhibitory activities on a compound 48/80 induced histamine released from mast cells, and anti-allergic effects on dinitrofluorobenzen (DNFB)-induced triphasic skin reactions in mice. The contents of the three flavanone glycosides in the immature fruits were higher than those in mature fruits. The application of extracts from the immature fruits of C. hassaku to skin care for patients with atopitic dermatitis has resulted in improving effects for 76% of the patients. Similar efficacy was obtained for patients pollinosis.

Combined effects of arbekacin (ABK) with biapenem (BIPM) were examined on both in vitro and in vivo model of a mixture of MRSA and Pseudomonas aeruginosa. As a result, significant effect in vitro was observed in combined use of ABK (1/2 MIC) with BIPM (1/4 and 1/2 MIC) against MRSA as compared with ABK or BIPM alone. Against P. aeruginosa combined effect was also observed, showing reduction of viable cells to the limitation of detection within 2 hours. Moreover, with respect to the protective effect on mixed systemic infection of MRSA and P. aeruginosa, the combined treatment with ABK and BIPM showed more excellent efficacy as compared with the single use of each drug.

Background and Objectives: Intravenous myocardial contrast echocardiography is used clinically, but use in small animals has not been evaluated. The conditions for myocardial opacification and the feasibility of myocardial contrast echocardiography were examined in mice., Methods: Closed chest mice were examined. The left ventricular short-axis view at the mid papillary muscle level was recorded before and after NC100100 injection using a SONOS 5500 (PHILIPS). Real time and intermittent triggering (every 5 beats) myocardial contrast echocardiography was performed with a mechanical index of low (0.3), middle (0.7), and high (1.5). Open chest mice were examined after left anterior descending coronary artery ligation. Myocardial contrast echocardiography was performed using intermittent triggering imaging (every 5 beats) with high mechanical index (1.5). The ratio of the non-perfused area to the whole left ventricular wall area was compared with that of the non-stained area by Evans Blue., Results: The left ventricular myocardium was opacified in any setting. Good opacification was acquired at middle mechanical index (0.7) in real time myocardial contrast echocardiography and at high mechanical index (1.5) in intermittent myocardial contrast echocardiography. The opacified and non-opacified myocardium were clearly identified in all mice with coronary ligation. The non-opacified area ratio showed a good correlation with the non-stained area ratio (y = 0.93x + 0.51, r = 0.94, p < 0.05)., Conclusions: Myocardial contrast echocardiography can be used to assess myocardial perfusion and determine the ischemic area accurately in situ in the mouse.

In the developing tooth, 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) causes hypoplasia and hypomineralization of enamel and dentine. The present study was undertaken to clarify the effects of HEBP on the formation of dental tissues of tooth germs in an organ culture system. Mandibular first molars from 17.5-day-old mouse embryos were cultured with or without 250 microM HEBP in culture medium. Cultured tooth germs were analyzed by histological examination and by immunohistochemical localization using anti-amelogenin antibody. In cultured tooth germs treated with HEBP before the commencement of calcification in dentine, calcification of dentine matrix was inhibited completely and enamel formation was not observed. Ameloblasts were directly adjacent to dentine matrix. However, immunohistochemical data indicated that these ameloblasts secreted amelogenin. In the experiments of adding HEBP to cultured tooth germs on culture day 13, calcified dentine and enamel had formed before the administration of HEBP, but the dentine matrix newly formed after the administration of HEBP had not calcified. It was confirmed by immunohistochemical observations that enamel matrix-like material had penetrated into uncalcified dentine matrix and accumulated in dental papilla of tooth germs. However, no enamel matrix-like material was observed in calcified dentine and predentine underneath the calcified dentine by immunohistochemical staining. From these results, it might be concluded that ameloblasts secreted enamel matrix in the presence of HEBP and diffused through uncalcified dentine matrix into dental papilla. These findings suggests the calcification of dentine might be essential for the physical barrier to accumulate the enamel matrix and form a distinct layer of enamel as enamel.

6-Acyldecahydro [1,6]naphthyridines were synthesized as derivatives of matrine-type and allomatrine-type alkaloids, and the structure-activity relations were examined by the acetic acid-induced abdominal contraction test. All synthesized derivatives produced the antinociception in mice. The antinociceptive potencies of 15a-c and 16a-c were lower than those of 17a-c, 18a-c, 19a-c and 20a-c. Furthermore, those of the matrine-type derivatives 17b and 17c are greater than other derivatives. These findings suggest that less hindered tertiary amine and highly lipophilic acyl group are better functional groups for the greater antinociceptive potencies. Furthermore, these findings suggest that A or B ring of 1 and 2 are not essential for the antinociceptive effect.

The prophylactic effects of a Basidiomycetes preparation, AHCC, against lethal Candida albicans infection were investigated in non treated or immunosuppressed mice. In the cyclophosphamide-or 5-fluorouracil (5-FU)-treated leukopenic mice and nontreated mice, the intraperitoneal administration of AHCC prior to C. albicans infection clearly prolonged the survival periods of the infected mice. In doxorubicin-treated mice, AHCC was less but significantly effective. On the other hand, in prednisolone-treated mice, AHCC was not effective. Oral administration of AHCC also protected the 5-FU-treated mice from lethal Candida infection, as indicated by prolongation of the survival periods and inhibition of Candida growth in the kidneys of these mice and by the increase in a number of neutrophils in their peripheral blood. These results suggested that AHCC may display a protective role against opportunistic fungal infection in leukopenic hosts.