Pharmacological treatment for cerebral ischemia and cerebral vasospasm following subarachnoid hemorrhage (SAH) cannot attain sufficiently high concentrations of the drugs in the cerebrospinal fluid (CSF) without precipitating systemic side effects. We recently developed a liposomal drug delivery system for intrathecal application that can maintain effective concentrations of cerebral vasodilator, fasudil, in the CSF. A single intrathecal injection of liposomal fasudil could maintain a therapeutic drug concentration in the CSF over a period time due to their sustained-release property, significantly decreasing infarct size in a rat model of acute ischemia and reducing vasoconstriction of the rat and dog basilar artery in a model of SAH. In this review, we are introducing our new less-invasive intrathecal drug delivery system that provides an alternative and safe method to deliver therapeutic agents.
It is desirable to diagnose hepatocellular carcinoma (HCC) in the early stages during its development since its treatment is usually difficult. We previously proposed a new diagnostic method that made use of the total metallothionein (MT), zinc (Zn), and copper (Cu) concentrations in the liver of the HCC patients. We recently found that MT-1 is involved in the metabolism or detoxification of toxic metals, such as cadmium; on the other hand, MT-2 is responsible for the homeostasis of essential metals such as copper, in experimental models such as Long Evans Cinnamon (LEC) rats. In order to device a better diagnostic method than the one we proposed previously, in this study, we newly propose an improved method that includes the discriminative determination data regarding the MT isomers, namely, MT-1 and MT-2, in the liver of patients with or without HCC as compared with the total MT level. The total MT and Zn concentrations in the HCC patients were confirmed to be significantly lower than those in patients without hepatic disorders (Ctrl). In contrast, Cu concentrations of the HCC patients were higher than those of the Ctrl patients. In addition, in the juxta-tumor portion with HCC, MT-1 concentrations were significantly higher than those of MT-2. In contrast, the MT-1 concentrations in the tumor portion were significantly lower than that in the juxta-tumor portion. In addition, MT-1/MT-2 ratio in the tumor portion was significantly lower than that of the juxta-tumor portion. By using parameters such as concentrations of Cu, Zn, total MT, and MT isomers, we performed the multivariate discriminative analysis (MDA). The results suggest that the concentrations of MT isomers change depending on the progress of the tumor, and information on MT isomers and trace elements is very useful in determining the stage of the chronic hepatic disorder.
A high performance liquid chromatography (HPLC) method was developed for the first time to quantify simultaneously the six major active ingredients in Acanthopanax senticosus (RUPR. et MAXIM.) HARMS, namely protocatechuic acid, syringin, chlorogenic acid, caffeic acid, liriodendrin and isofraxidin. The analysis was performed by a reverse phase gradient elution with an aqueous mobile phase (containing 0.05% phosphoric acid) modified by acetonitrile and diode-array multiple-wavelength UV detector (DAD). Six regression equations showed good linear relationships between the peak area of each marker and concentration. The recoveries of the markers listed above were 92.3%, 93.9%, 90.3%, 93.1%, 94.3% and 90.7%, respectively. The relative standard deviation of intra-day and inter-day were less than 2.7% and 3.1%, respectively. This method was validated for specificity, accuracy, precision and limits of quantification. Medicinal materials of ten commercial brands were analyzed and found to contain different amounts of the six bioactive markers. The method developed can be used for the quality control of Acanthopanax senticosus (RUPR. et MAXIM.) HARMS.
Synthetic DNA templates were compared with authentic cDNA templates as standards for the real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The single-stranded DNA template used here targeted the multidrug resistant transporter P-glycoprotein/MDR1. The double-stranded DNA template, targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was synthesized using an exonuclease-free large fragment E. coli DNA polymerase I. The human colon carcinoma cell line Caco-2 and human duodenum biopsies were used to prepare the authentic cDNA templates. The standard lines were comparable for the synthetic DNA templates and authentic cDNA templates. Long-term cryopreservation at −80 °C resulted in the destabilization of the synthetic single-stranded DNA template compared with the authentic cDNA templates in the case of MDR1, whereas for GAPDH, the stability of the synthetic double-stranded DNA template was comparable with that of the authentic cDNA templates. Even for the synthetic DNA templates, repetitive freeze-thawing resulted in destabilization, especially at lower concentrations, and degradation products might have interfered with the RT-PCR's efficiency. The synthetic DNA templates are better than the authentic cDNA templates, but more than 5 cycles of repetitive freeze-thawing should be avoided.
The mouse GATA-4 gene is separated by six introns, and this gene organization is conserved in rodents and man. The transcriptional start site of the GATA-4 gene is essentially the same in rat heart, stomach and testis, and in cultured cells expressing GATA-4 such as TM3, TM4, I-10 and P19.CL6 cells. The 5′-upstream of the GATA-4 gene is also conserved in rodents and man. We examined its promoter activity by means of luciferase reporter gene assay using testis-derived TM3 and TM4 cells. The GC-boxes and E-box located in the several tens of base pairs upstream of the transcriptional start sites of the GATA-4 gene were found to be critical for its promoter activity in these cells, consistent with the mode of transcription characteristics of the TATA-less promoter. P19.CL6 cells differentiate into beating cardiomyocytes upon induction by DMSO, accompanied by stimulation of the transcription of heart-specific genes including GATA-4. Interestingly, they exhibit increased luciferase reporter gene activity upon induction by DMSO. Both proximal tandem GC-boxes and the E-box are also contributed to the reporter gene activity in P19.CL6 cells.
AKR1C20, a member of the aldo-keto reductase (AKR) superfamily, found by mouse genomic analysis, exhibits the highest sequence identity (89%) with mouse liver 17β-hydroxysteroid dehydrogenase (HSD) type 5, but its function remains unknown. In this report, we have expressed the recombinant AKR1C20 from its cDNA, and examined its properties. The purified enzyme was a 36-kDa monomer, and showed both 17β-HSD and 3α-HSD activities in the presence of NADP(H) as the coenzymes. While the Km values for testosterone and 5α-dihydrotestosterone were high (>0.2 mM), those for 3α-hydroxy- and 3-keto-steroids were low (0.3—5 μM), resulting in high catalytic efficiency for the substrates. Although no significant dehydrogenase activity towards non-steroidal alcohols was observed, the enzyme highly reduced α-dicarbonyl compounds such as 16-ketoestrone, 9,10-phenanthrenequinone, acenaphthenequinone, 1-phenylisatin and camphorquinone. The pH optima of the dehydrogenase and reductase activities were 10.5 and 6.5—7.5, respectively. The enzyme was inhibited by sulfobromophthalein, hexestrol, indomethacin and flufenamic acid. The properties of AKR1C20 are distinct from those of previously known mouse 17β-HSD type 5 (AKR1C6), 3α-HSD (AKR1C14) and other members of the AKR1C subfamily. Thus, AKR1C20 is a novel 3α(17β)-HSD, which may also function as a reductase for xenobiotic α-dicarbonyl compounds.
Intersimple sequence repeats (ISSR) molecular fingerprinting markers have been employed to authenticate eight populations of Dendrobium officinale using 10 primers selected from 76 ISSR primers. A total of 127 DNA fragments were amplified, of which 115 were polymorphic (90.5% of all bands). Sixteen specific authentication markers have been found. To enhance the efficiency of authentication, ISSR fingerprinting codes have been constructed using six polymorphic bands for authenticating D. officinale populations. Eight wild populations of D. officinale have been authenticated accurately using ISSR.
FK614 is a structurally novel class of peroxisome proliferator-activated receptor γ (PPARγ) agonist, with the mechanism of its insulin-sensitizing action most likely due to activation of PPARγ. In this study, properties of FK614 for PPARγ binding, ability to induce conformational change, and coactivator recruitment were investigated. FK614, rosiglitazone, and pioglitazone competed specific binding of [3H]rosiglitazone to PPARγ with Ki values of 11 nM, 47 nM, and 1.3 μM, respectively. Limited trypsin digestion of PPARγ with FK614 or rosiglitazone produced distinct patterns of digested polypeptides, suggesting that FK614 directly binds to PPARγ but induces specific alterations in receptor conformation. FK614 induced interaction of PPARγ with nuclear receptor coactivator CBP but of lower magnitude than rosiglitazone and pioglitazone. The estimated Kd values of FK614-, rosiglitazone-, and pioglitazone-PPARγ complex to CBP peptide were 1.8, 0.64, and 0.72 μM, respectively, indicating FK614-PPARγ complex exhibits a lower affinity for CBP peptide compared to other agonist-PPARγ complexes. When tested the effect of FK614 on CBP recruitment induced by 9(S)-hydroxyoctadecadienoic acid, an endogenous ligand, FK614 negatively modulated PPARγ activation. The unique properties of FK614 may underlie the molecular basis of ligand-dependent transcriptional modulation mediated by PPARγ.
Meiosis activating sterol (MAS), the intermediate of cholesterol biosynthesis, is an important substance to stimulate oocytes maturation in FSH-induced signal transduction pathway. Lanosterol 14α-demethylase (CYP51) converts lanosterol to MAS. Although MAS is firstly isolated from bovine testis, the information about bovine CYP51 gene and its expression is little. In present studies, the cDNA cloning, genomic structure, chromosomal mapping, and expression patterns of bovine CYP51 were demonstrated. The cDNA coding bovine CYP51 contains a 1509 bp open reading frame and a 1119 bp 3′ untranslated region. And the bovine CYP51 genen includes 10 exons and spans about 17 kb. Screening the cattle RH5000 panel bovine CYP51 is mapped to chromosome 4 (0cR). The sequenced promoter region is TATA-less and contains several highly conserved regulatory elements, such as GC-box, cAMP-responsive elements (CRE), sterol regulatory element (SRE) which is important fragment for its transcription. No evidence of processed pseudogenes is found using long PCR and Southern blot. Northern blot analysis reveals that an approximately 2.7 kb mRNA is expressed in all the examined bovine tissues, while a 1.8 kb mRNA is found only in the mature bovine testis where the MAS is accumulated. Immunochemistry analysis shows that leydig cells express the highest level of the CYP51 protein in testis. Among different stages follicles it is localized primarily to the oocytes with the level varying slightly. Granulosa cells of primordial, primary and secondary follicles show background staining. While granulosa cells facing the antrum and cumulus granulosa cells of antral follicles show considerably heavier staining. The highest level is expressed in corpus lutea. These data indicate a stage- and cell type-specific expression of CYP51 protein in bovine oogenesis.
Pheromones affect gonadal functions and sexual behaviors. Information in regard to pheromones is received by the vomeronasal organ (VNO) and transmitted to the accessory olfactory bulb (AOB). We investigated the physiological role of the α1B and β3 subunits of the N (neuronal)-type voltage-dependent Ca2+ channel in the neurotransduction in the accessory olfactory (vomeronasal) system using α1B-deficient mice and β3-deficient mice. RT-PCR studies showed the existence of β1, β2, β3, β4, α1A, α1B, and α1C subunits of voltage-dependent Ca2+ channels in the mouse VNO. Immunohistochemical studies showed that the α1A, α1B, and α1C subunits of voltage-dependent Ca2+ channels exist in the sensory neurons and supporting cells of the mouse VNO. Exposure of the VNO to urine samples excreted from male mice induced lower Fos-immunoreactivity in the periglomerular (PG) cells of the AOBs in α1B-deficient female mice than in those of wild mice. The density of Fos-immunoreactive (Fos-ir) cells after exposure to female urine samples at the periglomerular cell layer of α1B-deficient male mice was lower than that of wild mice. Exposure of the VNO of β3-deficient female mice to male urine samples also induced low Fos-ir cells in the periglomerular cell layer of the AOB. These data suggest the importance of the α1B and β3 subunits of the N-type voltage-dependent Ca2+ channel for the pheromone signal transduction system.
The mammalian thioredoxin reductase (TrxR) is a selenocysteine-containing flavoprotein that regulates the thioredoxin system, one of the major systems that maintain the intracellular redox balance. We previously reported that cytosolic TrxR (TrxR1), one of three mammalian TrxR isozymes, was induced by treatment with cadmium. In the present study, to study the role of cadmium-induced TrxR1 in cellular defense, we silenced the expression of TrxR1 in HeLa cells by using small interfering RNA and examined the effect of TrxR1 silencing on the sensitivity of the cells toward cadmium. We found that the gene silencing of TrxR1 had a dual effect on cadmium-induced cell death, depending on the concentration of cadmium. The TrxR1 silencing increased the sensitivity toward a low dose (less than 10 μM) of cadmium but decreased the sensitivity toward a high dose of cadmium. These results suggested that TrxR1 might play an important role in the cellular defense system against cadmium in two ways. TrxR1 might rescue the cells from a low dose of cadmium-induced moderate injury, while it might promote the death of cells severely injured by a high dose of cadmium.
Vesicular glutamate transporter (VGLUT) plays an essential role in L-glutamate signaling in neurons and some peripheral tissues through vesicular storage of L-glutamate in secretory vesicles. To investigate the topology of VGLUT in membranes, we prepared site-directed antibodies against the amino-terminal (anti-N), 1st putative loop (anti-L), and carboxyl terminal (anti-C) regions. None of the antibodies reacted with VGLUT2 expressed in COS cells because they could not gain access to the antigen. However, both the anti-N and anti-C antibodies recognized VGLUT2 when the cells were permeabilized with digitonin, while the anti-L antibodies did not. Immunological reactivity to anti-L-antibodies appeared when the cells were permeabilized with Triton X-100. These results suggest that both the amino-terminal and carboxyl-terminal regions of VGLUT2 in membranes face the cytoplasm while the 1st loop faces the lumen.
An extract from ginger (root of Zingiber officinale) reduced the minimum inhibitory concentrations (MICs) of aminoglycosides in vancomycin-resistant enterococci (VRE). The effective compound was isolated and identified as -gingerol. In the presence of -gingerol at 1/10 concentration of its own MIC, the MIC of arbekacin was lowered by 1/32 to 1/16. -Gingerol also reduced the MICs of other aminoglycosides, and of bacitracin and polymixin B, but not of other antimicrobial agents tested. Because -gingerol reduced the MICs of several aminoglycosides both in strains possessing or lacking aminoglycoside-modification enzymes, it seems that the effect of -gingerol is not related to these enzymes, which mainly confer bacterial resistance against aminoglycosides. It seemed that a detergent-like effect of -gingerol potentiated the antimicrobial activity of the aminoglycosides. In fact, some detergents such as sodium dodecyl sulfate (SDS) and Triton X-100 reduced the MICs of aminoglycosides, bacitracin and polymixin B in VRE. Since the intrinsic resistance to aminoglycosides in enterococci is due to low level of entry of the drugs into the cells, increase in the membrane permeability caused by -gingerol will enhance the influx of aminoglycosides into enterococcal cells.
The growth rate of Candida albicans at the lag growth phase was not influenced by sodium sulfite, but that at the log growth phase was remarkably inhibited. C. albicans was able to grow in aerobic conditions both with and without glucose. Under anaerobic conditions, the growth of C. albicans was completely inhibited in glucose-free RPMI1640 medium. Ethanol production was increased at the lag growth phase, but decreased at the log growth phase. When oligomycin was added to the aerobic growth condition, the growth at log growth phase was remarkably inhibited. The expression of SDH2 mRNA was low in the lag growth phase and high in the log growth phase. We concluded that C. albicans obtained ATP by fermentation at the lag growth phase, and needed oxygen to generate ATP by oxidative phosphorylation at the log growth phase.
We cloned a gene responsible for multidrug resistance from the chromosomal DNA of Klebsiella pneumoniae MGH78578 that showed multidrug resistance. We designated the gene kmrA. The deduced amino acid sequence of KmrA was similar to that of SmvA that is responsible for methyl viologen-resistance in Salmonella enterica sv. Typhi and Typhimurium. Introduction of the cloned kmrA gene into drug-hypersensitive Escherichia coli KAM32 cells made them resistant to acriflavine, 4′,6-diamidino-2-phenylindole (DAPI), Hoechst 33342, tetraphenylphosphonium chloride (TPPCl), methyl viologen and ethidium bromide. We observed elevated energy-dependent efflux of ethidium in E. coli cells carrying the kmrA gene compared with control cells. We also cloned the smvA gene from S. enterica sv. Typhimurium LT2 and investigated the resistance pattern for several drugs. The pattern was similar between KmrA from K. pneumoniae and SmvA from S. enterica.
There are more than 30 genes for putative multidrug efflux pumps in the chromosome of Staphylococcus aureus. Only a few of these have been analyzed so far. Here we cloned a new gene, SA1972, using a PCR method, from the chromosome of S. aureus N315. We found that the product SA1972 could lead to elevated resistance against several antimicrobial agents such as norfloxacin, acriflavine and ethidium bromide. We designated the gene as sdrM. We observed elevated energy-dependent efflux of acriflavine in S. aureus cells introduced with the sdrM gene. We conclude that SdrM is a multidrug efflux pump belonging to the major facilitator (MF) superfamily.
This work presents behavioral effects of (O-methyl)-N-2,6-dihydroxybenzoyl-tyramine (riparin III) isolated from the unripe fruit of Aniba riparia (NEES) MEZ (Lauraceae) in animal models of open field, rota rod, elevated plus maze and hole board tests in mice. Riparin III (ripIII) was administered orally, in male mice, at single doses of 25 and 50 mg/kg. The results showed that ripIII, at both doses, had no effects on the spontaneous motor activity in the rota rod test nor in the number of squares crossed in the open field test. However, riparin III decreased the number of grooming and rearing. In the plus maze test, ripIII, at both doses increased the following parameters: percentage of entries in the open arms (PEOA), time of permanence in the open arms (TPOA) and percentage of time of permanence in the open arms (PTOA) and at the dose of 50 mg/kg, increased the number of entries in the open arms (NEOA). Similarly, ripIII, at both doses, showed an increase in the number of head dips into the holes of the hole board test. These results show that riparin III presents anxiolytic effects in the plus maze and hole board tests which are not influenced by the locomotor activity in the open field test.
The anti-diabetic and hypolipidemic effects of aqueous-extract from the flower of Inula japonica (IJ) and its two fractions (IJR and IJP) were investigated in alloxan-induced diabetic mice. An oral glucose tolerance test (OGTT) of IJ was also performed in normal and diabetic mice. The results showed that IJ (1000 mg/kg), IJR (500 mg/kg) and IJP (250 mg/kg) significantly reduced blood glucose levels in diabetic mice by oral administration (p<0.01). IJ and IJP markedly decreased serum triglyceride concentrations (p<0.05) in diabetic mice. Their hypoglycemic activities were better than gliclazide (40 mg/kg) and compared with metformin (250 mg/kg). IJ raised plasma insulin levels in alloxan-induced diabetic mice. IJ, IJR and IJP significantly decreased the consumption of water and food in diabetic mice. OGTT showed that IJ slightly lowered blood glucose levels in normal mice, but significantly decreased blood glucose in diabetic mice between 60—150 min after a glucose load (p<0.05). The data indicated that IJ has both anti-diabetic and hypolipidemic effects.
The anticancer activity of dichloromethane extract of guduchi [Tinospora cordifolia (WILLD.) MIERS ex HOOK. F. & THOMS. Family: Menispermaceae (TCE)] in the mice transplanted with Ehrlich ascites carcinoma (EAC) was investigated. The EAC mice receiving 25, 30, 40, 50 and 100 mg/kg, TCE showed a dose dependent elevation in tumor-free survival and a highest number of survivors were observed at 50 mg/kg TCE, which was considered as an optimum dose for its neoplastic action. The average survival time (AST) and median survival time (MST) for this dose were approximately 56 and 55 d, respectively when compared with 19 d of non-drug treated controls. Administration of 50 mg/kg TCE resulted in 100% long-term survivors (up to 90 d). An attempt was also made to evaluate the effectiveness of TCE in the various stages of tumor development, where 50 mg/kg TCE was administered intraperitoneally after 1, 3, 6, 9, 12 or 15 d of tumor inoculation and these days have been arbitrarily designated as stage I, II, III, IV or V, respectively for reasons of clarity. The greatest anticancer activity was recorded for stage I, II and III where number of long term survivors (LTS) was approximately 33, 25 and 17%, respectively. However, treatment of mice at stage IV and V did not increase LTS, despite an increase in AST and MST. The EAC mice receiving 50 mg/kg TCE showed a time dependent depletion in the glutathione (GSH) activity up to 12 h post-treatment and marginal elevation thereafter. This depletion in GSH was accompanied by a drastic elevation in lipid peroxidation (LPx) and a maximum elevation in LPx was observed at 6 h that declined gradually thereafter. TCE exerted cytotoxic effect on tumor cells by reducing the GSH concentration and increase in LPx simultaneously.
We have clarified that Eriobotrya japonica seed extract has strong antioxidative activity, and is effective for the prevention and treatment of various diseases, such as hepatopathy and nephropathy. In this study, to investigate the influences of components of Eriobotrya japonica seed extract on its antioxidative activity, extracts were prepared using various solvents (n-hexane (Hex), ethyl acetate (EtOAc), n-butanol (n-BuOH), methanol (MeOH) and H2O) and the antioxidative activity of the solvent fractions and components was evaluated based on the scavenging of various radicals (DPPH and O2−) measured by the ESR method and the inhibition of Fe3+-ADP induced NADPH dependent lipid peroxidation in rat liver microsomes. The radical scavenging activities and inhibitory activities on lipid peroxidation differed among the solvent fractions and components. In the n-BuOH, MeOH and H2O fractions, radical scavenging activity and inhibitory activity on lipid peroxidation were high. In addition, these fractions contained abundant polyphenols, and the radical scavenging activity increased with the polyphenol content. In the low-polar Hex and EtOAc fractions, the radical scavenging activity was low, but the lipid peroxidation inhibition activity was high. These fractions contained β-sitosterol, and the inhibitory activity on lipid peroxidation was high. Based on these findings, the antioxidative activity of Eriobotrya japonica seed extract may be derived from many components involved in a complex mechanism, resulting in high activity.
The pineal gland and its main hormone, melatonin (MLT), are involved in a variety of physiological processes. MLT is a member of the indolamine family and has significant antioxidative activity. Acetaminophen (AA) is the most widely used medication in the world, both by prescription and over the counter. In large doses, AA is hepatotoxic causing oxidative stress and lipid peroxidation. Therefore, antioxidants have been used to protect against the toxicity of AA. Here, we examined in vitro and in vivo the protective effects of MLT against AA-induced toxicity in mice. MLT (100 μM) had a significant protective effect on the AA (7 mM)-induced loss of cell viability in mouse primary cultured hepatocytes as determined using the 3H-thymidine incorporation assay and MTT assay. The AA-induced generation of reactive oxygen species (ROS) peaked at 6 h and was followed by an increase in lipid peroxidation at 12 h in hepatocytes. MLT (0.1, 1, 10 or 100 μM) dose-dependently attenuated the increase in both production of ROS and lipid peroxidation by AA. Similarly, in vivo, AA (400, 600 or 800 mg/kg, intraperitonealy)-induced mortality and hepatotoxicity were significantly decreased by MLT (10 mg/kg, subcutaneously). Pretreatment with MLT had a greater protective effect on the hepatotoxicity of AA than post-treatment. However, MLT had no protective effect on the antipyretic effect or antinociception caused by AA. These results suggest that MLT is potentially useful for preventing AA-induced toxicity, but not the antipyretic effect or antinociception caused by AA.
The therapeutic anti-diabetic effect of SMK001, a poly herbal formula was evaluated in the streptozotocin (STZ; 60 mg/kg, single intraperitoneal injection) induced diabetic rats. For therapeutic study, test articles were orally dosed once a day from 21 d after STZ-dosing at 100, 200 and 500 mg/kg/5 ml dosage levels for 4 weeks. The body weight changes, blood and urine glucose level changes were monitored with changes on the pancreas weight, and after sacrifice, the histopathological changes of pancreas and the changes of insulin- and glucagon-producing cells were also observed by immunohistochemistry. The results were compared to that of glibenclamide 5 mg/kg-dosing group. Significantly (p<0.01 or p<0.05) decrease of body weight, blood and urine glucose levels were detected in STZ-induced diabetic animals with disruption and disappearance of pancreatic islets. In addition, significantly (p<0.01) increase of glucagon- and decrease of insulin-producing cells were detected in STZ induced diabetic rats. However, these diabetic changes were significantly (p<0.01 or p<0.05) and dose dependently decreased in SMK001-dosing groups, and SMK001 100 mg/kg showed more favorable effects compared to that of glibenclamide 5 mg/kg. Based on these results, it is considered that SMK001 has favorable effect to inhibit the changes on the blood and urine glucose levels, body weight and the histopathological changes of pancreas in STZ induce diabetes.
We previously demonstrated that a serotonin (5-HT) precursor 5-hydroxytryptophan (5-HTP) increases serum leptin levels in mice. It was reported that administration of 5-HTP elicits hypophagia in rodents and humans. In the present study, we examined involvement of leptin in 5-HTP-elicited decreases in the milk intake of fasted mice. Serum leptin levels increased with increases in milk intake in mice, while 5-HTP strongly decreased milk intake in fasted mice compared to that in the control group. Serum leptin levels in fasted mice treated with 5-HTP were similar to those control mice after milk intake. As leptin is a powerful anorectic signal, 5-HTP-induced anorexia may be mediated by facilitation of leptin secretion.
We investigated whether P-glycoprotein (P-gp) ATPase activity of Caco-2 cell membranes could be estimated by measuring consumption of ATP using luciferin–luciferase reaction, and whether the results would be useful for assessment of the interactions between P-gp and drugs. The vanadate-sensitive ATPase activity of Caco-2 cell membranes was measured rapidly with high sensitivity using luciferin–luciferase reaction. Cyclosporin A, verapamil, digoxin and quinidine stimulated the ATPase activity concentration-dependently with Km values of 5.3, 0.9, 1.2 and 4.1 μM, respectively. These values except for digoxin were comparable with previous reports. The ATPase activity and P-gp mRNA expression in Caco-2 cells were induced by all-trans-retinoic acid, digoxin and levothyroxine, but not dexamethasone or rifampicin. This method was useful to assess interactions with P-gp and drugs, and was used to elucidate the mechanisms of interaction of levothyroxine and digoxin.
A promising approach to reduce the occurrence of cancer is its treatment, specifically by chemical intervention through minor dietary constituents. Epidemiological studies suggest that specific pharmacologically active agents present in the diet might reduce cancer. A remarkable surge of interest in chemoprevention research has, thus, lead to the identification of many phytochemicals of dietary origin as effective potential chemotherapeutic agents. In the present investigation, attempt has been made to study the potency of Kalpaamruthaa (KA), a herbal preparation, against cancer. The changes in level of glycoprotein components, marker enzymes and lysosomal enzymes were carried out in 7,12-dimethylbenz(a)anthracene (DMBA) induced Sprague–Dawley rats. The changes in the body weights and volume were also determined. KA was administered at the dosage level of 100, 200, 300, 400 and 500 mg/kg body weight (BW) in olive oil orally for 14 d, after the induction period is completed (90 d). On administration of KA, the levels of the above enzymes and the changes in the body weights and volume were significantly normalized in a dose dependant manner. The present study shows that KA is effective at the dosage level of 300 mg/kg body weight in mammary carcinoma bearing rats.
Bis(indole) alkaloids, of the topsentin class (1—4) and hamacanthin class (5—9), isolated from the marine sponge Spongosorites sp. were investigated using several biological assays. In the evaluation of antimicrobial activity against various strains of bacteria and fungi, compounds of the hamacanthin class exhibited more potent antibacterial activity than those of the topsentin class. Deoxytopsentin (1) and hamacanthin A (5) also exhibited significant antibacterial activity against methicillin-resistant Staphylococcus aureus, with MIC values of less than 12.5 μg/ml. In the antifungal activity test, hamacanthins, especially hamacanthin A (5), showed potent inhibitory activity against medically important pathogenic fungi. In contrast, all of the topsentins (1—4) were inactive against fungal growth. These compounds (1—9) also exhibited moderate cytotoxicity against cancer cell lines at concentrations between 1.1 and >20 μg/ml.
The aim of the present study is to investigate difference in sensitivity to glibenclamide, a sulfonylurea oral antidiabetic agent, among Wistar rats, Spontaneously Hypertensive rats (SHR/Izm) and Wistar-Kyoto rats (WKY/Izm). We examined the effect of glibenclamide on blood levels of glucose and insulin in these rat strains. Under anesthesia with pentobarbital sodium (50 mg/kg, i.p.), blood samples were collected before and 5—120 min after administration of glibenclamide (10 mg/kg, i.p.). Blood levels of glucose and insulin in each sample were measured by glucose oxidase method and radioimmunoassay, respectively. In 8 week-old rats of all strains tested, blood levels of glucose were decreased by glibenclamide. In 12—20-week-old rats, although blood levels of glucose in Wistar and SHR/Izm were decreased after glibenclamide administration, those of WKY/Izm were not decreased. In rats of this age, time-course and extent of increases in blood insulin levels observed after administration of glibenclamide in WKY/Izm was almost the same as that of SHR/Izm, however, smaller than that of Wistar. Both insulin secretions induced via inactivation of ATP-sensitive K+ channel and sensitivity of pancreatic β-cells to insulin seems to be decreased in WKY/Izm after 12 weeks of age. This phenomenon may explain the mechanism of glucose intolerance previously reported in WKY/Izm.
The editorial committee has noticed that this publication as a Note in Biological and Pharmaceutical Bulletin contains data
sets identical to those presented in papers, already published in other journals, from the same laboratory with the same corresponding
author. Due to a violation of the editorial policies of the journal, this Note has been retracted by the committee.
The Editorial Committee of the Pharmaceutical Society of Japan (April 15, 2014)
Acori graminei Rhizoma is one of the best-known traditional herbal medicines frequently used for the treatment of cardiovascular symptoms in Asian countries. The anti-ischemic effect of Acori graminei Rhizoma on ischemia-induced isolated rat heart was investigated through analysis of changes in perfusion pressure, aortic flow, coronary flow, and cardiac output. The subjects in this study were divided into two groups, an ischemia-induced group without any treatment (I), and an ischemia-induced group with Acori graminei Rhizoma treatment (I+AGR). There were no significant differences in perfusion pressure, aortic flow, coronary flow, or cardiac output between the two groups before ischemia was induced. The supply of oxygen and buffer was stopped for 10 min to induce ischemia in isolated rat hearts, and Acori graminei Rhizoma was administered while inducing ischemia. The data showed that Acori graminei Rhizoma treatment significantly prevented decreases in perfusion pressure, aortic flow, coronary flow, and cardiac output under an ischemic condition. In addition, hemodynamics (except heart rate) of the AGR-treated group was significantly recovered 60 min after reperfusion compared to the control group, (systolic aortic pressure: 85.5% vs. 62.5%, aortic flow volume: 68.1% vs. 49.4%, coronary flow volume: 86.8% vs. 60.1%, and cardiac output: 73.1% vs. 54.1%, p<0.01). These results suggest that Acori graminei Rhizoma has distinct anti-ischemic effects.
The oral anti-inflammatory activity of 4,4′-dihydroxy-α-truxillic acid (1) was compared with that of two other nonsteroidal anti-inflammatory drugs, loxoprofen sodium (LOX) and diclofenac sodium (DIC). The activity of 1 against the inflammatory pain response induced by formalin was comparable to that of LOX, but weaker than that of DIC. In the monosodium urate (MSU)-induced acute inflammatory model, 1 showed stronger anti-inflammatory activity than both LOX and DIC. The ED50 value for 1 was 4.5 μmol/kg, while the values for LOX and DIC were 65 and 25 μmol/kg, respectively. Otherwise, the oral single-dose toxicity of 1 was investigated in both sexes of Sprague–Dawley rats administered once at a dose of 2000 mg/kg. 1 showed no death, clinical signs, changes in body weight or pathological findings related to the treatment. In addition, no mutagenicity was observed in the reverse mutation assay. Furthermore, 1 did not show any ulcerogenic activity at doses ranging from 30 to 300 mg/kg in rat. Thus, 1 might be considered to be an effective anti-inflammatory agent with no deleterious adverse effect.
The discovery of drugs for the treatment of allergic disease is an important subject in human health. Stimulation of mast cells starts the process of degranulation resulting in releasing of mediators, such as histamine. In this report, we investigated the effect of aqueous extract of Dracocephalum argunense FISCH. (Labiatae) (DAAE) on the mast cell-mediated allergic response and studied its possible mechanisms of action, focusing on the histamine release and pro-inflammatory cytokine secretion in mast cells. DAAE inhibited compound 48/80-induced systemic reactions and serum histamine release in mice. In addition, DAAE attenuated IgE-mediated skin allergic reaction. DAAE dose-dependently reduced IgE-induced histamine release from mast cells. The level of cAMP was transiently increased by treatment of DAAE. DAAE specifically blocked the phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore A23187-induced p38 mitogen-activated protein kinase (MAPK) activation. DAAE decreased the secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-6 in mast cells. Our findings provide evidence that DAAE inhibits mast cell derived allergic reactions, and involvement of cAMP for histamine release and p38 MAPK for pro-inflammatory cytokine secretion in these effects.
Our recent study demonstrated that the dimeric structure of α-truxillic acid derivatives played an important role in the expression of their anti-inflammatory activities. In the present report, to investigate the correlation between the structure and anti-inflammatory activity, α-truxillic acid (1) and its derivatives (2—6), β-truxinic acid (7) and its derivatives (8—10) were prepared, and their activities were evaluated in the formalin test. All compounds showed only weak or no activities against the neurogenic pain response, but demonstrated significant activities against the inflammatory pain response induced by formalin. The highest anti-inflammatory activities were observed for α-truxillic acid (1) and its derivative 4,4′-dihydroxy-α-truxillic acid (2). In addition, α-truxillic acid (1) and its derivative, α-truxillic acid bis(p-nitrophenyl)ester (5), showed higher anti-inflammatory activities than β-truxinic acid (7) and the corresponding derivative (10). Furthermore, free carboxylic acids (1, 2) showed higher activities than their dimethyl esters (3, 4) and bis(p-nitrophenyl)ester (5). These results confirmed that the α-formation of dimeric structure and the free carboxylic acid were also important for the expression of anti-inflammatory activities. Otherwise, 4,4′-dichloro-β-truxinic acid (8) had higher activity than its parent compound 7; furthermore, 1,3-dibenzoyl-2,4-di(4-chlorophenyl)cyclobutane (6) also showed strong anti-inflammatory activity. These results suggested that substituents in the phenyl groups were also important for the expression of anti-inflammatory activity. In order to gain information about their activity intensity, the anti-inflammatory activities of 2 and 4,4′-dichlorolated derivatives (6, 8) were compared with that of indomethacin (a nonsteroidal anti-inflammatory drug) in the formalin test. As a result, compounds 2, 6 and 8 showed stronger anti-inflammatory activities than indomethacin. These results suggested that α-truxillic acid and β-truxinic acid derivatives might be developed into a new type of anti-inflammatory drug.
The antidiabetic effects of corosolic acid (CA) were investigated in KK-Ay mice, an animal model of type 2 diabetes. CA (2 mg/kg body weight) reduced the blood glucose levels of KK-Ay mice 4 h after a single oral dose. CA (2 mg/kg) reduced the blood glucose levels in KK-Ay mice 2 weeks after a single oral dose and also significantly lowered plasma insulin levels were in KK-Ay mice under similar conditions. CA-treated KK-Ay mouse blood glucose significantly decreased in an insulin tolerance test. These results support the hypothesis that CA improves glucose metabolism by reducing insulin resistance. Therefore CA may be useful for the treatment of type 2 diabetes.
Fruits and vegetables contain numerous antioxidants such as carotenoids, vitamins, and phenolic phytochemicals. Recent studies have demonstrated that antioxidants may reduce the risk for diabetes or its complications. In this study, we investigated the effects of the chronic administration of Satsuma mandarin fruit on an antioxidant defense system in streptozotocin (STZ)-induced diabetic rat liver. After a ten-week administration of Satsuma mandarin, antioxidant enzymes and glutathione levels in the liver were evaluated. The superoxide dismutase (SOD), catalase, and glutathione-peroxidase (GPx) activities, and glutathione level in the STZ-induced diabetic rats liver decreased significantly compared with those in the age-matched normal rats. The glutathione-reductase (GR) activities did not differ significantly between these two groups. In contrast, the SOD, GR, and glutathione levels in the Satsuma mandarin (1% or 3%) diet-fed STZ-diabetic rat livers were significantly higher than those in the normal diet-fed STZ-diabetic rat livers. In addition, although the serum alanine aminotransferase and γ-glutamyl-aminotransferase concentrations of normal diet-fed STZ-diabetic rats were significantly higher than those of the age-matched normal rats, these increments of serum liver enzymes were diminished by the chronic administration of Satsuma mandarin. These results suggest that Satsuma mandarin may act as a suppressor against liver cell damage and inhibit the progression of liver dysfunction induced by chronic hyperglycemia.
Insecticidal and acaricidal activities of two geometrical isomers, (E)- and (Z)-butylidenephthalide isolated from Angelica acutiloba, against larvae and adults of fruit fly (Drosophila melanogaster), cat fleas (Ctenocephalides felis) and house dust mites (Dermatophagoides farinae and Tyrophagus putrescentiae) were investigated and compared with that of positive controls. (E)- and (Z)-Butylidenephthalide exhibited 50% lethal concentration (LC50) values of 2.07 and 0.94 μmol/ml of diet concentration against larvae of D. melanogaster, respectively. This indicated that two isomers of butylidenephthalide have geometrical stereoselectivity for larvicidal effect. Even though both (E)- and (Z)-butylidenephthalide also showed potent adulticidal and acaricidal activity against adults of D. melanogaster and two mites, there was no significant difference between two isomers. Insecticidal activity of both (E)- and (Z)-butylidenephthalide toward adults of C. felis was not detected even at the maximum concentration of 200 μg/cm2.
In this paper, we investigated the inhibitory mechanism of the production of nitric oxide (NO) by polyanions and liposomes composed of phosphatidylserine (PS-liposomes) focusing on cytokine production and mitogen activated protein kinase (MAP kinase) activation. NO production by macrophages was inhibited by treatment with oxidized lipoprotein (OxLDL), maleylated bovine serum albumin (mBSA), and heparin. No inhibitory effect was exhibited by poly-cytidilic acid (PolyC). To clarify the mechanism of the inhibitory effect of polyanions on NO production, we evaluated the productions of transforming growth factor-β (TGF-β) and interleukin (IL)-10 which are known to be anti-inflammatory cytokines. TGF-β was produced when macrophages were treated with OxLDL as was the case with PS-liposomes. No increase in TGF-β production was observed for mBSA, heparin, and PolyC. On the other hand, significant production of IL-10 was observed using mBSA. Extracellular signal-regulated kinase (ERK), a member of the MAP kinase superfamily, was activated when macrophages were treated with OxLDL as well as PS-liposomes. In the case of mBSA, the activation of ERK and c-Jun N-terminal kinase (JNK) was observed. No activation of p38 MAP kinase was observed using any of the polyanions. Although heparin had an inhibitory effect on NO production by macrophages, no activation of MAP kinase or production of TGF-β and IL-10 was observed. The inhibitory effect of these ligands on NO production may be regulated via different signaling pathways.
We evaluated the microbial contamination of nebulization solutions in medication cups from a total of 76 ultrasonic nebulizers in use in 10 hospitals. In addition, an interview survey was given to nurses to evaluate the disinfection methods of these ultrasonic nebulizers. Of a total of 76 nebulization solution samples, 11 (14.5%) were contaminated with 10—102 colony-forming units (CFU)/ml and 9 (11.8%) with 103—105 CFU/ml. The major contaminants were glucose non-fermentative bacilli such as Burkholderia cepacia, CDC gr.IV C-2, and Sphingomonas paucimobilis. Comparison of microbial contamination between the frequencies of disinfection showed a significantly lower number of contaminated samples when the cups were disinfected once daily than when disinfected once at intervals of 2—7 d (p=0.00037). In addition, comparison between the presence and absence of preservatives contained in the nebulization solution showed a significantly lower number of contaminated samples in the presence, rather than in the absence, of preservatives (p=0.00001). These results show that disinfection of ultrasonic nebulizers at 24-h intervals is desirable. In particular, when nebulization solutions not containing preservatives are used, disinfection at 24-h intervals is indispensable.
Solvent Green 3 (SG), a model poorly water-soluble compound, was orally administered to rats with soybean oil emulsion or the Self-microemulsifying drug delivery system (SMEDDS) composed of Gelucire44/14. The bioavailability of SG after oral administration with SMEDDS was 1.7-fold higher than that with soybean oil emulsion. The intestinal absorption of lipid-based formulations themselves was evaluated by the in situ closed loop method. The effect of lipase and bile salt on their absorption was also evaluated. SMEDDS itself was rapidly absorbed in the intestine even in the absence of lipase and bile salt, and the absorption was increased by the addition of lipase and bile salt. On the other hand, no soybean oil emulsion was absorbed in the absence of lipase and bile salt. However, mixed micelle prepared from emulsion by incubating soybean oil emulsion with lipase and bile salt was rapidly absorbed through the intestine. Without lipase and bile salt, SG was not absorbed after administration with soybean oil emulsion. Therefore, we concluded that the degradation of soybean oil emulsion was needed for SG to be absorbed through the intestine. Furthermore, we investigated the intestinal absorption of SG after oral administration to rats whose chylomicron synthesis were inhibited by pretreatment with colchicine. Colchicine completely inhibited the intestinal absorption of SG after administration with each lipid-based formulation, suggesting that SG was absorbed from the intestine via a lymphatic route. Absorption of the dosage formulation should be paid attention when poorly water-soluble drugs are orally administered with lipid-based formulation.
The skin permeability and stability of formoterol fumarate (FF) in matrix patches containing l-menthol as an enhancer and N-methyl-2-pyrrolidone (NMP) as the solvent were investigated. Using a total of 28 matrix patches having a similar composition, containing ethylene–vinyl acetate (EVA) as the forming polymer and hydrogenated rosin glycerol ester (Ester Gum H) as the adhesive, the skin permeation of FF was found to increase with increasing l-menthol and NMP contents. FF in the matrix patches containing NMP in the range of 4.8—7.2% was stable, but stability decreased at higher values. With a standard matrix patch containing FF, the Cmax and AUC0—24 values were found to be 1.93 ng/ml after 4 h percutaneous application to rats and 25.6 ng·h/ml. The bioavailability after percutaneous exposure was equivalent to 15.2% of the AUC0—24 after intravenous administration. Percutaneous application proved efficacious with regard to control of simulated asthma at dose levels lower than those with which side effects occurred. Thus an optimized matrix patch containing FF was prepared with potential advantages for control of asthma.
This study was performed to evaluate variability in the pharmacokinetics of bepridil in 38 Japanese patients with arrhythmias, and to investigate the effects of aprindine as well as CYP2D6 and CYP3A5 polymorphisms on the oral clearance of bepridil. We determined the polymorphic alleles of CYP2D6 and CYP3A5 in each subject. The plasma concentration of bepridil at steady-state following repetitive oral administration was measured with an HPLC-based method, and the oral clearance was estimated using the nonlinear mixed effects model (NONMEM) program. Mean oral clearance was significantly greater in the patients with the CYP2D6*10 allele than in those without it. On the other hand, no significant effect of the CYP3A5 polymorphism was observed on the pharmacokinetics of bepridil. In addition, aprindine seemed to reduce the oral clearance of bepridil in the patients with the CYP2D6*10 allele.
To investigate the mechanism responsible for the intestinal absorption of a lipophilic organic cation, quinidine, we performed a pharmacokinetic analysis of transcellular transport across Caco-2 cell monolayers grown on a porous membrane. Basolateral-to-apical transport of the drug was almost constant in the concentration range of 100 nM—100 μM. Transcellular transport was greater in the apical-to-basolateral direction than in the opposite direction. Apical-to-basolateral transport was greater at a concentration of 100 μM than 100 nM. The calculated influx clearance value of the apical membrane was much greater than the other influx/efflux clearance values of cell membranes, and was 5.6-fold the influx clearance value of the basolateral membrane at the drug concentration of 100 μM. We also investigated the uptake of quinidine at the apical membrane of Caco-2 cells grown on plastic dishes. The uptake was markedly increased by alkalization of the apical medium at 37 °C, and was decreased at low temperature (4 °C). In addition, it was inhibited by diphenhydramine and levofloxacin, but not by carvedilol, rifamycin SV, or L-carnitine. These findings indicated that the influx at the apical membrane was the direction-determining step in the transcellular transport of quinidine across Caco-2 cell monolayers, and that some specific transport system was involved in this influx.
To clarify the mechanisms of gatifloxacin (GFLX)-induced hypoglycemia and hyperglycemia, the effect of GFLX on serum glucose levels was investigated in normal and diabetic rats. Rats received an intravenous injection of GFLX and their arterial blood was sampled periodically. Diabetic rats were produced by the intraperitoneal injection of streptozotocin and nicotinamide. In normal rats, the serum glucose concentration was decreased by GFLX at 25 and 50 mg/kg, while it was elevated 0.25 h after the injection of 100 mg/kg of GFLX. Serum immunoreactive insulin (IRI) levels increased as the dose of GFLX increased. The serum epinephrine concentration rose rapidly after the injection of GFLX at 50 and 100 mg/kg. In diabetic rats, the serum glucose concentration was actually increased by GFLX at 50 mg/kg. The baseline concentration of IRI was lower and the degree of the elevation caused by GFLX was smaller in diabetic rats. Both diabetic and control rats showed an increase in the serum epinephrine concentration after the injection of 50 mg/kg of GFLX. In conclusion, GFLX-induced secretion of insulin and epinephrine would contribute to the abnormalities in glucose homeostasis. The response of serum glucose to GFLX may differ between diabetic and normal rats due to the alteration of insulin secretion.