The capacity of gene delivery vehicles is considered to be a critical factor determining the success of gene therapy. To date, various types of gene delivery vehicle have been developed. Among them, recombinant adeno-virus (Ad) vectors have potential that has favored their worldwide use in vitro and in vivo. Conventional Ad vectors are composed of subgroup C Ad serotype 5 (Ad5), although it has been clarified that the drawbacks of Ad5 vectors are a high seroprevalence of Ad5 in adults and low transduction efficiencies in cells lacking the primary receptor for Ad5, coxsackievirus and adenovirus receptor. To overcome these problems, we developed a novel Ad vector fully composed of Ad serotype 35 (Ad35). Ad35 vectors show a wide tropism for human cells because Ad35 binds to human CD46, which is ubiquitously expressed on almost all human cells, as a primary receptor. In addition, anti-Ad5 antibodies do not inhibit Ad35 vector-mediated transduction and the seroprevalence of Ad35 in adults is lower than that of Ad5. This paper reviews our studies on the development and evaluation of Ad35 vectors. Ad vectors derived from other Ad serotypes different from Ad5, including Ad35, are expected to be gene delivery vehicles alternative to conventional Ad5 vectors.
Monosodium L-glutamate (MSG) elicits a unique taste termed umami and is widely used as a flavor enhancer in a variety of cuisines. Recent studies suggest the existence of L-glutamate (GLU) receptors and its transduction molecules in the gut mucosa as well as in the oral cavity. The vagal gastric afferent fibers respond specifically to the luminal stimulation of GLU in the stomach. GLU administration in the stomach also activates several brain areas (insular cortex, basal ganglia, limbic system, and hypothalamus). Ingestion of MSG enhanced secretion of digestive juices and insulin. Spontaneous ingestion of an MSG solution at the most preferred concentration (1% (w/v)) reduced weight gain, fat deposition, and plasma leptin levels without affecting food intake, naso-anal length (an index of somatic development), and lean mass in rats. These results suggest that umami signaling via gustatory and visceral pathways may play an important role in the process of digestion, absorption, metabolism, and other physiological functions via activation of the brain.
L-Glutamate and 5′-ribonucleotides such as guanosine-5′-monophosphate (GMP) and inosine-5′-monophosphate (IMP) elicit a unique taste called ‘umami’ that is distinct from the tastes of sweet, salty, sour, and bitter. For umami, like sweet and bitter compounds, taste signaling is initiated by binding of tastants to G-protein-coupled receptors (GPCR) in taste bud cells. To date, several GPCRs for umami compounds have been identified in taste cells, including the heterodimer T1R1/T1R3, and truncated type 1 and 4 metabotropic glutamate receptors missing most of the N-terminal extracellular domain (taste-mGluR4 and truncated-mGluR1). Apparently contradictory data in T1R3 knock-out (KO) mouse models have been reported. One study showed that behavioral preference and taste nerve responses to umami stimuli in T1R3-KO mice were totally abolished, suggesting that T1R1/T1R3 is a sole receptor for umami taste. The other reported reduced but not abolished responses to umami in T1R3-KO mice, suggesting existence of multiple receptors for umami taste. In this paper, we summarized the data from recent studies that further addressed this issue by using different experimental techniques. Some of the studies provided additional evidence for the existence of umami receptor systems mediated by mGluR1 and mGluR4 in addition to T1R1/T1R3. It is proposed that the signal mediated by the pathway involving T1R1/T1R3 may play a different role from that derived from the mGluRs. The former occurs mainly in the anterior tongue, and plays a major role in preference behavior, whereas the latter occurs mainly in the posterior tongue and contributes to behavioral discrimination between umami and other taste compounds.
Physiologic studies conducted in rats have demonstrated that afferent fibers of the gastric branch of the vagus nerve increase their firing rate with the intragastric administration of the amino acid glutamate, and the increased firing rate is blocked by the depletion of serotonin (5-HT), administration of the blocker for the serotonin type-3 receptor (SR3), or nitric oxide synthase (NOS). To understand glutamate signaling in the gastric mucosa at the cellular level, we have been studying rats as an animal model using anatomic and immunohistochemical procedures. Our results have indicated that 5-HT-immunoreactive (ir) cells are present in the superficial part of the gastric mucosal epithelium and in the base of the fundic glands, whereas immunoreactivity for SR3 is localized in the neck and its vicinity of the fundic glands. Further, NOS1/neuronal NOS-ir cells with a bipolar shape are located in the lamina propria where a dense network of neuronal cells is present. These results suggest that complex cellular events take place during intragastric glutamate signaling.
The appropriate recognition of nutrients in gastrointestinal tract has an important role in the maintenance of healthy body functions. Perception of nutrients in gastrointestinal tract influences not only the regulation of gastrointestinal functions such as digestion and absorption, but also the subsequent formation of ingestive behaviors and food preferences. Glutamate is widely distributed amino acids, approximately 15—45% in the dietary protein as the total of free- and bound-forms. To note is that, glutamate is implicated in numerous physiological and metabolic functions in the body as a nutrient, in addition, free glutamate in the food is also known as the source of umami, one of the five basic taste. Taste is basically percepted on the tongue in the oral cavity via the specific taste sensors (receptors and/or transporters), however, the recent studies suggest the functional roles of taste sensors in the regulation of gastrointestinal tract. Interestingly, we recently found the presence of specific glutamate-sensing systems in the lumen of the stomach. In addition, we have been vigorously studying the physiological roles of dietary free glutamate in the body through experimental animals and human. In this topic, we review the beneficial roles of dietary free glutamate in the regulation of gastrointestinal functions and propose some application of the benefits for the clinical gastrointestinal disorders.
Vesicular glutamate transporter (VGLUT) is responsible for the active transport of L-glutamate into synaptic vesicles and, thus, plays an essential role in the glutamatergic chemical transmission in the central and peripheral nervous systems. Recent studies indicated that VGLUT is also expressed and localized in various secretory vesicles in non-neuronal peripheral organelles such as hormone-containing secretory granules in endocrine cells. L-Glutamate is stored in VGLUT-containing organelles, secreted upon stimulation, and then acts as a paracrine and/or autocrine modulator to regulate cellular functions. Thus, VGLUT is a key molecule for glutamate signaling and is the core of a novel signaling system.
Utilizing the human genome database, the recently developed G-protein-coupled receptor (GPCR) deorphanizing strategy successfully identified multiple receptors of free fatty acids (FFAs) and is proposed to play a critical role in a variety of physiologic homeostasis mechanisms. GPR40 and GPR120 are activated by medium- and long-chain FFAs, whereas GPR41 and GPR43 are activated by short-chain FFAs. GPR40, which is preferentially expressed in pancreatic β-cells, mediates insulin secretion. On the other hand, GPR120, which is abundantly expressed in the intestine, functions as a receptor for unsaturated long-chain FFAs and promotes the secretion of glucagon-like peptide-1 (GLP-1). In this review, we summarize the identification, structure, and pharmacology of the receptors and speculate on the respective physiologic roles that FFA receptor family members may play.
Recently, it has been clarified that glutamate (Glu) can stimulate the umami taste as well as the visceral sensation to help the gastric protein digestion. Our survey suggests the possibility that the amount of free Glu in hospital foods is lower than that in ordinary foods. In the present study, monosodium glutamate (MSG) was supplemented to meals for 11 elderly inpatients during 2 months, and the fortification effects on their nutritional status, general condition, and quality of life (QOL) were investigated. The degree of recognition was improved, and peripheral lymphocytes were increased, even when there was no change in nutritional intake or protein nutritional status. Based on these results, we concluded that appropriate utilization of Glu for nutritional care of the elderly people would be useful for improving QOL.
Bleomycin is well known as causative molecule for acute lung injury and interstitial pneumonia. The free radical production from bleomycin is thought to play an important role in the pathogenesis of acute lung injury and interstitial pneumonia. However, there was no direct evidence of free radical production in this model. Therefore, we examined in vivo radical production by mice treated with a bleomycin using electron spin resonance with the spin trap, α-(4-pyridyl-1-oxide)-N-tert-butylnitrone. Six hours after instillation of bleomycin, the lung exposed to bleomycin gave a lipid-derived free radical adduct, which would support evidence for in vitro lipid peroxidation resulting from bleomycin administration. In the treatment of deferoxamine, chelating agent for iron and other metals, to reduce the bleomycin induced free radical production, parallel to decrease the lipid-derived free radical production by deferoxamine, pathophysiological findings of lung injuries were improved by deferoxamine. In conclusion, this is a first paper of in vivo direct evidence of production of free radical from bleomycin-induced lung injury. It is suggested that this method may be used in many kinds of lung disease models, which have potentials of free radical production to cause lung damage.
A simple capillary electrophoretic method based on 1-phenyl-3-methyl-5-pyrazolon (PMP) derivatization has been developed for simultaneous separation and determination of nine aldoses (xylose, arabinose, glucose, rhamnose, fucose, galactose, mannose, glucuronic acid and galacturonic acid). The separation of PMP-labeled monosaccharide derivatives was carried out in uncoated capillary (48.5 cm×75 μm i.d.) and under the selected optimum conditions of pH 11.0, 200 mM borate buffer at applied voltage 15 kV and capillary temperature 20 °C, the nine PMP-monosaccharides could be perfectly separated from each other within 40 min. Furthermore, the developed method was firstly applied to determine the sugar composition in the polysaccharide isolated from Chinese Codonopsis pilosula. The results showed that C. pilosula polysaccharide was a typically acidic heteropolysaccharide and was composed of arabinose, glucose, rhamnose, galactose, mannose, glucuronic acid and galacturonic acid in the molar contents of 48.1, 103.5, 16.1, 48.5, 7.5, 4.2 and 119.1 μM, respectively. The assay results were satisfactory.
A simple and rapid high-performance liquid chromatographic method has been developed for determination in human plasma of isepamicin (ISP), an aminoglycoside antibiotic agent. After protein precipitation and clean-up procedure to remove lipophilic contaminants, ISP is derivatized pre-column with 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate for fluorescence detection. Chromatographic separations are achieved using C18 column and mobile phase consisting of 20 mM KH2PO4 containing 8 mM triethylamine (pH 7.0) and acetonitrile (78/22, v/v). Amikacin was used as an internal standard. The calibration curve was linear over a concentration range of 0.5—50 μg/ml. The limit of quantification was 0.5 μg/ml. The intra- and inter-day variabilities of ISP were both less than 17.5%. Both derivatives were stable for at least a week at ambient condition. This assay procedure should have useful application in therapeutic drug monitoring of ISP.
To optimize ginsenosides hydrolyzing β-glucosidase production from Aspergillus niger, response surface methodology was carried out in two stages. The Plackett–Burman design was achieved to screen the important variables that influence β-glucosidase production. Among 10 variables (wheat bran, soybean powder, CaCl2, ginsenosides, KH2PO4, MgSO4, polyethylene glycol (PEG), medium volume, inoculum size, and stirring speed), it was found that wheat bran, KH2PO4, and stirring speed had significant effect on β-glucosidase activity due to very low p-values (p<0.05). Subsequently, wheat bran, KH2PO4, and stirring speed were further optimized using central composite design. The optimal β-glucosidase production was predicted to be 4650.14 U/ml with the combination of factors (wheat bran, 34.51 g/l; KH2PO4, 1.78 g/l; stirring speed, 161.60 rpm/min). Finally, under optimal fermentation conditions, ginsenoside Rb1 was converted to Rd and F2 by A. niger within 10 min. Little compound K was detected at 30 min, and finally F2 was completely transformed to compound K within 8 h. The putative conversion pathway of Rb1 by A. niger was Rb1, Rd, F2, and compound K.
To determine the antioxidant role of glutathione (GSH) in human red blood cells (RBCs), we investigated the effect of disrupting GSH homeostasis on the oxidative modification of thiol-dependent enzymes by exposure to tert-butyl hydroperoxide (BHP). When hemolysate was incubated with BHP, significant decreases in enzyme activity were observed. However, the inactivation did not occur in intact RBC suspensions that were exposed to BHP. In this study, we used two independent treatments aimed at decreasing the level of reduced form of GSH, pre-incubation with a glutathione reductase inhibitor or glucose-free medium to examine the influences of preventing GSH-dependent antioxidant and reactivation activity on thiol-dependent enzyme. Pyruvate kinase (PK) activity clearly decreased along with depletion of GSH compared to other glycolytic enzyme activities by BHP exposure in RBCs. The addition of GSH, but not glucose, before BHP exposure completely prevented the inactivation of PK in hemolysate; however, partial reactivation of inactivated PK was observed by post-addition of both GSH and glutaredoxin at an early stage during BHP exposure. Moreover, hydroxyl radicals but not hydrogen peroxide inactivated PK. These results suggest that PK is highly susceptible to radicals and that GSH is essential to protect PK activity by not only directly scavenging radicals but also by systematically reactivating oxidized enzyme in human RBCs.
Bacterial membrane constituents, such as Ornithine-containing lipid (OL) and the lipid A portion of lipopolysaccharide, trigger various immune responses through recognition by Toll-like receptor (TLR) 4. Usually, these lipids are dissolved in a small amount of aqueous or organic solvent before being added to the culture medium for examination of their biological activities. Macrophages stimulated with OL or lipid A sonically dissolved in saline released both interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). In contrast, macrophages stimulated with OL or lipid A sonically dissolved in ethanol or dimethyl sulfoxide (DMSO) secreted much TNF-α, but very little IL-1β. These results, taken together, indicate that how an endotoxin is prepared affects its biological activities. In addition, electromicroscopic analysis revealed that sonication of air-dried OL or lipid A in DMSO produced larger particles than those produced in saline, suggesting that the process of preparing lipidic TLR4-ligands affects their physical state including particle size, and that the physical state might be an important determinant of biological activity.
To determine the role of nitric oxide (NO) in rat liver transporter regulation, we investigated in precision-cut liver slices the effect of NO and peroxynitrite (ONOO−), a reaction product of NO with superoxide (O2−), on mRNA levels of 13 influx and efflux transporters. To inactivate Kupffer cells (KCs), liver slices were prepared from rats treated with gadolinium chloride (Gd). Transporter mRNA levels were determined after incubation of untreated (normal-slices) and Gd-pretreated slices (Gd-slices) for 18 h with Spermine NONOate (SpNO), an NO donor, and SIN-1 (3-(4-morpholinyl) sydnonimine hydrochloride, SIN), a ONOO− donor. SpNO and SIN varied all transporter mRNA levels examined, except organic anion transporting polypeptide 1b2 (Oatp1b2/Oatp4). SpNO in normal-slices and SIN in Gd- and normal-slices generally decreased influx and increased efflux transporter transcription. In contrast, these effects were not observed in Gd-slices treated with SpNO. SpNO and SIN in normal-slices commonly decreased organic anion transporter 2 (Oat2) and increased multidrug resistance-associated protein 2 (Mrp2) transcription, but differentially regulated bile salt export pump (Bsep) and multidrug resistance protein 2 (Mdr2) transcription, the up-regulation by SpNO and the down-regulation by SIN. In addition, the induction of tumor necrosis factor (TNF)-α and interleukin (IL)-1β was not observed after incubation with SpNO or SIN. These findings suggest that NO and ONOO− play a role in the regulation of rat transporter transcription in hepatocytes, which communicate with KCs, in a proinflammatory cytokine-independent manner.
Environmental pollutants including halogenated and polycyclic aromatic hydrocarbons activate the aryl hydrocarbon receptor (AhR) and thereby cause a wide range of pathological changes. Development of AhR antagonists will be useful for prevention and treatment of diseases related to AhR activation. Towards this end, we aimed in the present study at seeking for potential inhibitors of the AhR pathway in mycelial extracts using the dioxin responsive element-based sensing via secreted alkaline phosphatase (DRESSA). Through the screening of 13 mycelia, extracts prepared from Phellinus linteus, Cordyceps militaris and Hericium erinaceum inhibited activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin, benzo[a]pyrene or 3-methylcholanthrene. Subsequent studies revealed that only Phellinus linteus suppressed activation of AhR and AhR-dependent gene expression triggered by all of these agonists. Cigarette smoke is known to contain a number of halogenated and polycyclic aromatic hydrocarbons. We found that Phellinus linteus has the potential to block activation of AhR and AhR-dependent gene expression triggered by cigarette smoke. Furthermore, the inhibitory effect of Phellinus linteus on the AhR pathway was independent of; 1) depression of AhR or AhR nuclear translocator, and 2) induction of AhR repressor. We conclude that Phellinus linteus contains potent inhibitor(s) of AhR activation and may be useful for prevention of pathologies associated with aberrant activation of AhR.
Manganese (Mn) plays an important role in the etiology of several neurobehavioral disorders, but there is a lack of data regarding its specific effects on neurotransduction, especially dopaminergic neurotransduction. We investigated the relationship between motor deficits and alterations in the expression of tyrosine hydroxylase (TH) and dopamine D2-like receptors (DR), including the three dopaminergic subtypes, D2, D3, and D4, in low- and high-dose Mn-treated mice. After administration of Mn (intraperitoneal injections of 20 or 40 mg/kg MnCl2·4H2O once per day for 5 d), motor activity and expression of TH and DR were examined in the striatum of the mouse brain. Mn treatment resulted in significant decrease in coordination and/or impaired motor learning after 5 d of treatment and this effect remained until 10 d after the end of Mn treatment. The expression of dopamine D2-like receptor D2 (DRD2), but not TH, DRD3, or DRD4, in the striatum was dose-dependent, and statistically significant increases were seen at the mRNA and protein levels. These findings indicate that Mn-induced motor deficits may be modulated in part by the expression of DRD2 in the striatum. In addition, our results suggest that the disturbance of dopaminergic neurotransmission mediated by DRD2 may be involved in the pathogenesis of Mn neurotoxicity.
We evaluated the effects of antimicrobial drugs on four strains of Pseudomonas aeruginosa that are resistant to eight widely used antipseudomonal drugs (piperacillin, piperacillin-tazobactam, imipenem, meropenem, ceftazidime, aztreonam, amikacin, ciprofloxacin) and colistin. In the killing test, colistin (2 μg/ml) was the most effective, followed by aztreonam (48 μg/ml), piperacillin-tazobactam (192—4 μg/ml), piperacillin (192 μg/ml), and a three drug combination of azetreonam (16 μg/ml), ceftazidime (16 μg/ml), and amikacin (4 μg/ml). Six hours after drug addition, colistin (2 μg/ml), aztreonam (48 μg/ml), piperacillin-tazobactam (192—4 μg/ml), piperacillin (192 μg/ml), and the above three drug combination had bacteriostatic effects on all four strains. Colistin, three time breakpoint of aztreonam, piperacillin, or piperacillin-tazobactam, and the three drug combination of aztreonam, ceftazidime, and amikacin were effective in vitro.
We evaluated the microbial contamination of 17 types of vegetable and 10 types of fruit after 30-s washing with tap water with and without subsequent disinfection by 10-min immersion in 0.01% (100 ppm) sodium hypochlorite. The mean microbial contamination level of 9 types of leafy vegetable was 2.8×105 colony-forming units (CFU)/g after washing with water and 3.4×104 CFU/g after washing followed by disinfection. The mean microbial contamination level of 8 types of nonleafy vegetable was 3.4×104 CFU/g after washing with water and 1.0×104 CFU/g after washing followed by disinfection. The mean microbial contamination level of 10 types of unpeeled fleshy fruit was 9.3×103 CFU/g after washing with water and 1.3×103 CFU/g after washing followed by disinfection. The contaminants in vegetables and unpeeled fruit were similar after washing and after washing followed by disinfection, including Pseudomonas fluorescens and Pseudomonas aeruginosa. The contamination did not markedly decrease even after disinfection with sodium hypochlorite. However, the flesh of each type of peeled fruit showed no or only low levels of contamination (≤10 CFU/g), probably caused by the transfer of microorganisms from the skin of fruit via fruit knives.
Piscidin, a 22-residue cationic peptide isolated from the mast cells of hybrid striped bass, has potent antimicrobial activities. In the present study, we investigated the fungicidal activity and mode of action of piscidins. Fungicidal and hemolytic assays were examined in order to assess their potency and toxicity, respectively. The results showed that fungicidal and hemolytic activities were higher for piscidin 1 (P1) than piscidin 3 (P3). Additionally, the abilities to permeabilize the model phospholipids membranes were also higher for P1 than P3, which were consistent with the biological activities of P1 and P3. These results suggest that the biological action of the peptides may be carried out through the lipid membrane. To understand the fungicidal properties of P1, we focused on a membrane-active mechanism of the peptide by in vivo testing against Candida albicans as the model organism. Flow cytometric analysis by using bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] and protoplast regeneration experiments showed that P1 caused fungal membrane damage. Furthermore, fluorescence analysis, using 1,6-diphenyl-1,3,5-hexatriene, revealed that these peptides created pores in fungal membranes. Thus, the present study demonstrated that piscidins exert their fungicidal effects by disrupting fungal membrane through pore formation.
Tacrolimus is an agent used in clinical immunosuppressive drug therapies. A wide spectrum of adverse effects has been reported in association with this immunosuppressor, including neurotoxic effect. The upper limit of therapeutic blood concentrations of tacrolimus has been described as 30 ng/ml in immunosuppressed patients. We investigated the effect of this therapeutic dose of tacrolimus on the expression and activity of the multidrug resistance protein 1 (MDR1 or Pgp, P-glycoprotein) and ATP-binding cassette transporters A5 (ABCA5) in human brain microvascular endothelial cells (HBMEC), derived from Blood-Brain Barrier (BBB) endothelium, these being the most predominantly expressed transcripts in these cells. The expression and activity of MDR1 transporter decreased with 30 ng/ml tacrolimus. The cell viability was not changed with the therapeutic dose used. By contrast, ABCA5 transcripts, of unknown role as yet, increased their expression at this concentration. We propose that the secondary cytotoxic effects of this immunosuppressor on CSN, besides the functional blockade related to multidrug resistance proteins, such as MDR1, and probably ABCA5, could be linked to variations in the expression levels of these proteins at the BBB.
Several methods are used to evaluate gastric motility in rodents, but they all have technical limitations. Recent technical developments enable a convenient method to evaluate gastric motility. The 13C-acetic acid breath test in rodents is a non-invasive and repeatable method that can be used without physical restraints. The present study aimed to validate the 13C-acetic acid breath test by measuring the effects of loperamide, morphine, mosapride, and itopride on gastric emptying in mice. Loperamide (1—10 mg/kg) and morphine (1.25—10 mg/kg) slowed gastric emptying and decreased the maximum concentration (Cmax) and area under the curve (AUC90 min) value in a dose-dependent manner. Mosapride (0.2—5 mg/kg) accelerated gastric emptying and increased Cmax value. Mosapride (20 mg/kg) did not accelerate gastric emptying on the 13C-breath test. Itopride (30 mg/kg, per os) significantly accelerated gastric emptying compared with the vehicle group. In a comparison with the conventional phenol red test, there was a correlation between the Cmax value of breath test and gastric emptying (%) of phenol red tests in treatment with loperamide or mosapride. These results indicate that the 13C-acetic acid breath test is an accurate, noninvasive, and simple method for monitoring gastric emptying in mice. This method is useful to assess the effect of drugs and gut function pharmacologically.
Oxidative stress-induced cell damage has been implicated in a variety of neurodegenerative disorders. In the current study, we investigated the protective role of ginsenoside Rd against the cytotoxicity induced by exposure to hydrogen peroxide (H2O2) and the underlying mechanism in the PC12 cell line. The protective effects of ginsenoside Rd (1, 10 μM) on H2O2-induced cytotoxicity may be ascribed to its antioxidative properties by reducing the intracellular reactive oxygen species level; decreasing malondialdehyde production, a common index of lipid peroxidation; and enhancing the antioxidant enzymatic activities of superoxide dismutase and glutathione peroxidase. Additionally, ginsenoside Rd could stabilize the mitochondrial membrane potential after H2O2 exposure. These findings suggested that ginsenoside Rd may be considered a potential antioxidant agent and should encourage further research in neurodegenerative diseases to explore the potential neuroprotective effects of ginsenoside Rd.
Epileptic patients are at a significantly higher risk for impairments of cognitive function and behavioral abnormalities. In this study, we investigated the effect of repeated electroconvulsive shock (ECS)-induced seizures on the spontaneous alternation behavior in a Y-maze test and the locomotor activity in an open-field test, and examined the effects of anti-epileptic drugs in rats. ECS was administered for seven consecutive days and the Y-maze and open-field tests were performed 24 h after the last ECS administration. The repeated electroconvulsive seizures significantly impaired the spontaneous alternation and increased the locomotor activity. Moreover, these behavioral changes induced by the seven administrations of ECS persisted for at least 28 d. The inhibition of the ECS-induced seizures through the daily pretreatment of the phenytoin (120 mg/kg, intraperitoneally (i.p.)), phenobarbital (60 mg/kg, i.p.) and valproate (400 mg/kg, i.p.) abolished the locomotor hyperactivity in the open-field test. The impaired spontaneous alternation behavior in the Y-maze test was also significantly suppressed by the treatment with phenytoin and valproate. However, phenobarbital injection produced no significant ameliorating effect in the Y-maze test. These results suggest that the inhibition of ECS-induced seizures through phenytoin and valproate injections suppress the development of impairment of spontaneous alternation and the locomotor hyperactivity.
During our isolation of biologically active substances from the spores of Ganoderma lucidum (Reishi Houshi, G. lucidum) guided by the inhibitory activity on HL-60 cell proliferation, NMR spectroscopic and mass spectrometric data indicate the substance contains a mixture of several long chain fatty acids. Hence, in this study, we have examined the inhibitory effects of an ethanolic extract of the spores of G. lucidum as the spore extract, on the proliferation of various human cancer cell lines by comparison with several authentic long chain fatty acids. Of the fatty acids we examined nonadecanoic acid (C19:0) showed the highest inhibitory activity for HL-60 cell proliferation with IC50 values of 68±7 μM followed by heptadecanoic acid (C17:0, 120±23 μM), octa- (C18:0, 127±4 μM) and hexadecanoic acids (C16:0, 132±25 μM), respectively. The corresponding unsaturated fatty acids containing one double bond such as cis-10-nonadecenoic acid (C19:1), cis-9-octadecenoic acid (C18:1), cis-10-heptadecenoic acid (C17:1) and cis-9-hexadecenoic acid (C16:1) were less effective. The ethanolic extract of spores of G. lucidum were shown by annexin-V FITC/PI double staining to induce apoptosis of HL-60 cells in a similar way to cis-10-nonadecenoic acid (C19:1). These unsaturated fatty acids probably inhibit tumor necrosis factor production induced by lipopolysaccharide in a mouse macrophage preparation. Our results suggest the spores of G. lucidum contain 19-carbon fatty acids as one of the components for characteristics of its physiological effects.
Peroxisome proliferator-activated receptor gamma (PPARγ) agonists of the thiazolidinedione class are widely used for the treatment of type 2 diabetes subjects due to their ability to improve insulin resistance. Troglitazone and ciglitazone belong to the PPARγ agonists of thiazolidinediones. We report here that troglitazone but not ciglitazone increased IL-1β induced cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression in vascular smooth muscle cell (VSMC) from Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Potentiated expression of COX-2 and iNOS by troglitazone was inhibited by MG-132, a specific inhibitor of inhibitory factor κB (IκB) activation. Troglitazone treatment of these cells also resulted in a dose-dependent increase in IL-1β induced IκBα phosphorylation. These data suggest that troglitazone is capable of increasing IL-1β induced COX-2 and iNOS expression through an IκBα dependent mechanism in VSMC from WKY and SHR.
The present study was undertaken to assess whether A-3922, a dihydrobenzofuran derivative that possesses antioxidative effects, had any preventive effect on the onset and/or progression of diabetic cataract. Male Wistar rats were received a bolus intravenous injection of streptozotocin (65 mg/kg) and were given 5% glucose in drinking water for 10 weeks. The diabetic rats were divided into two groups and treated with 30 mg/kg/d A-3922 or vehicle during the experimental period. The opacities of eye lenses were observed by using both our original device and a slit lamp microscope. The lens opacities were initially detected as early as the 2nd week and the cataracts were developed in similar fashion in both A-3922-treated and untreated diabetic rats until 7th week, suggesting that A-3922 did not show any appreciable effect on the onset of diabetic cataract. In the later period (8th week or later), however, progression of cataract was retarded and significant reductions in both the total cataract score and the degree of opacity were apparently observed on 10th week of A-3922-treated diabetic rats. These results suggest that A-3922 can delay the progression but not the onset of diabetic cataract, and it has a possibility to be a candidate for drugs of cataract associated with diabetes.
Accumulating evidence suggests that nitric oxide (NO) and prostaglandin E2 (PGE2) are involved in the pathogenesis of various chronic inflammatory diseases and cancer. During the course of a screening program to identify natural anti-inflammatory substances, we isolated the compound 2-amino-3H-phenoxazin-3-one (APO) from an extract of the edible brown mushroom Agaricus bisporus IMBACH. APO inhibited NO production by mouse peritoneal macrophages in response to the pro-inflammatory stimuli lipopolysaccharide (LPS) and interferon (IFN)-γ (LPS/IFN-γ) at low concentrations (IC50=1.5 μM) through reduced inducible NO synthase protein expression. PGE2 production by LPS/IFN-γ-stimulated macrophages was inhibited by APO at much lower concentrations (IC50=0.27 μM) than those required for the inhibition of NO production. Mechanistic analysis showed that APO inhibited both cyclooxygenase (COX)-1 and COX-2 enzyme activities with almost equal selectivity. Secretion of NO and the pro-inflammatory cytokine IL-6 by IFN-γ-activated RAW264.7 cells, a murine macrophage-like cell line, was also dose-dependently reduced by APO. Furthermore, APO increased the secretion of the anti-inflammatory cytokine IL-4 by antigen-stimulated T cells and promoted the polarization of CD4+ Th cells toward the anti-inflammatory Th2 phenotype at equimolar concentrations that inhibited NO production. Our results suggested that APO induced polarization toward the Th2 subset, at least in part through the down-regulation of IL-12 production. Thus, APO appears to have potent anti-inflammatory and immunoregulatory properties that may provide a promising therapeutic strategy for the treatment of T cell-mediated inflammatory autoimmune diseases as well as for bacteria-induced chronic-inflammatory diseases.
Lavandulyl flavanones of Sophora flavescens roots are anti-malarial, anti-inflammatory, and cytotoxic. Here, we examined whether four lavandulyl flavanones, (2S)-2′-methoxykurarinone (1), sophoraflavanone G (2), leachianone A (3), and (−)-kurarinone (4), isolated from S. flavescens could protect HT22 immortalized hippocampal cells against glutamate-induced oxidative stress. Compounds 1 and 2 induced the expression of heme oxygenase (HO)-1 and increased HO activity dose- and time-dependently. These two compounds also suppressed glutamate-induced reactive oxygen species generation in HT22 cells, whereas compounds 3 and 4 were not protective. These two lavandulyl flavanones (compounds 1, 2) may protect against glutamate-induced neurotoxicity via HO-1 induction.
To characterize active principles for prevention and treatment of diabetic complications, the isolation of protein glycation inhibitors from the fruiting body of Phellinus linteus was conducted in vitro using the model systems of hemoglobin-δ-gluconolactone (early stage), bovine serum albumin-methylglyoxal (middle stage), and Nα-acetyl-glycyl-lysine methyl ester-D-ribose (last stage) assays. Nine compounds were isolated from the active ethylacetate fraction of the fruiting body and identified as protocatechuic acid (1), protocatechualdehyde (2), caffeic acid (3), ellagic acid (4), hispidin (5), davallialactone (6), hypholomine B (7), interfungins A (8), and inoscavin A (9) by spectroscopic analyses. At the early stage of protein glycation, compounds 6, 8, and 9 exhibited inhibitory activity on hemoglobin A1C formation. For the middle stage, compounds 2, 6, and 9 showed a significant inhibitory effect on methylglyoxal-medicated protein modification and their IC50 values were 144.28, 213.15, and 158.66 μM, respectively. At the last stage of glycation, compound 8 was found to be a potent inhibitor of the cross-linking of proteins, which was more effective than that of aminoguanidine, a well-known inhibitor for advanced glycation end products. Consequently, compound 8 showed the most potent inhibitory effects at each stage of protein glycation. This mechanism may help to provide a protective effect against hyperglycemia-mediated protein damage.
Oral administration of a methanolic extract of Piper nigrum leaf (PN-ext, 50, 200 and 500 mg/kg) showed a potent dose-dependent inhibition of dinitrofluorobenzene (DNFB)-induced cutaneous reaction at 1 h [immediate phase response (IPR)] after and 24 h [late phase response (LPR)] after DNFB challenge in mice which were passively sensitized with anti-dinitrophenyl (DNP) IgE antibody. Ear swelling inhibitory effect of PN-ext (50, 200 and 500 mg/kg, per os (p.o.)) on very late phase response (vLPR) in the model mice was significant but weaker than that on IPR. Oral administration of PN-ext (50, 200 and 500 mg/kg for 7 d) inhibited picryl chloride (PC)-induced ear swelling in PC sensitized mice. PN-ext exhibited in vitro inhibitory effect on compound 48/80-induced histamine release from rat peritoneal mast cells. Two lignans of PN-ext, (−)-cubebin (1) and (−)-3,4-dimethoxy-3,4-desmethylenedioxycubebin (2), were identified as major active principles having histamine release inhibitory activity.
Defining a quantitative and reliable relationship between in vitro drug release and in vivo absorption is highly desired for rational development, optimization, and evaluation of controlled-release dosage forms and manufacturing process. During the development of once daily extended-release (ER) tablet of glipizide, a predictive in vitro drug release method was designed and statistically evaluated using three formulations with varying release rates. In order to establish internally and externally validated level A in vitro–in vivo correlation (IVIVC), a total of three different ER formulations of glipizide were used to evaluate a linear IVIVC model based on the in vitro test method. For internal validation, a single-dose four-way cross over study (n=6) was performed using fast-, moderate-, and slow-releasing ER formulations and an immediate-release (IR) of glipizide as reference. In vitro release rate data were obtained for each formulation using the United States Pharmacopeia (USP) apparatus II, paddle stirrer at 50 and 100 rev. min−1 in 0.1 m hydrochloric acid (HCl) and pH 6.8 phosphate buffer. The f2 metric (similarity factor) was used to analyze the dissolution data. The formulations were compared using area under the plasma concentration–time curve, AUC0—∞, time to reach peak plasma concentration, Tmax, and peak plasma concentration, Cmax, while correlation was determined between in vitro release and in vivo absorption. A linear correlation model was developed using percent absorbed data versus percent dissolved from the three formulations. Predicted glipizide concentrations were obtained by convolution of the in vivo absorption rates. Prediction errors were estimated for Cmax and AUC0—∞ to determine the validity of the correlation. Apparatus II, pH 6.8 at 100 rev. min−1 was found to be the most discriminating dissolution method. Linear regression analysis of the mean percentage of dose absorbed versus the mean percentage of in vitro release resulted in a significant correlation (r2≥0.9) for the three formulations.
Lichens and their secondary metabolites have attracted the interest of many researchers. Some species have been shown to contain substances with remarkable biologic activity, as antimicrobial, mainly against Gram positive bacteria; antineoplasic acting on solid and ascetic tumors, or in culture cells; antiviral; hypotensive; and spasmolytic effects. The aim of this study was to isolate and characterize atranorin, one of the major constituents which presents in Cladina kalbii (DES ABB.) AHTI., and analyze its antinociceptive effect. The antinociceptive activity was verified in acetic acid-induced writhing test and formalin test with mice. In this work it was observed that atranorin was effective in significant reducing (p<0.05) abdominal writhing at doses of 200 and 400 mg/kg (p.o.) by 52.6 and 61.3%, respectively, when compared to control group (vehicle). The formalin test showed in 200 and 400 mg/kg (p.o.) that atranorin injection was able to inhibit the inflammatory processes (second phase) dose dependently.
Sildenafil is widely administered for the treatment of erectile dysfunction. Recently, sensorineural hearing loss following the ingestion of sildenafil was reported in one male patient. We examined hearing in mice that were administered high doses of sildenafil for up to 105 d. To assess hearing impairment, we evaluated auditory brainstem responses, auditory middle latency responses, and otoacoustic emissions. At high doses, sildenafil increased the hearing threshold shift of auditory brainstem responses. High-dose sildenafil treatment also resulted in delayed latency of both auditory brainstem responses and auditory middle responses. Otoacoustic emissions differed between control and high-dose sildenafil groups with long-term treatment. Collectively, these data demonstrate that high-dose and long-term sildenafil administration can induce hearing impairment in mice.