Here we describe recent applications and technical advancements of functional multineuron calcium imaging (fMCI), which monitors the firing activity of more than a thousand neurons through their somatic Ca2+ signals. fMCI is used for analysis of various neural circuits under normal and pathological conditions. In vitro fMCI is made more sophisticated by using multipoint illumination and scanning technology with spinning-disk and low-laser-intensity imaging, electron-multiplying charge-coupled device cameras, etc. In vivo fMCI is still developing. We review optical technologies for fast scanning imaging, deep tissue imaging, and recording from moving animals.
Landiolol is an ultra-short-acting β1-adrenergic receptor blocking agent that is used for both perioperative and postoperative patients with tachycardia during general anesthesia. Validated HPLC-UV methods that quantitatively determine landiolol and its major metabolite (M-1) in human blood were reported for clinical research of landiolol. These analytes were recovered from the same blood sample using a multi-step extraction process and determined with two different HPLC conditions. These methods were validated over concentration ranges of 0.05 to 10 μg/ml for landiolol and 0.1 to 20 μg/ml for M-1 and were found to have acceptable accuracy, precision, linearity, and selectivity. These methods are useful to the characterize of blood pharmacokinetics of landiolol and M-1 for clinical research.
TBR31-2 is one of the bone marrow stromal cell lines. Differentiation toward osteogenic cells and calcification was observed when TBR31-2 cells were cultured for 4 weeks. Bone morphogenetic protein-2 (BMP-2) stimulated alkaline phosphatase (ALP) activity in a dose- and time-dependent manner. On the other hand, troglitazone increased oil droplet accumulation in a dose-dependent manner. In the presence of BMP-2, an increase of expression in osteogenic cell differentiation marker genes and a decrease of expression in adipocyte differentiation marker genes were observed with the exception of the induced expression of peroxisome proliferator-activated receptor γ (PPARγ), however, troglitazone, a ligand of PPARγ treatment exhibited the opposite tendency. Interestingly, treatment with both BMP-2 and troglitazone resulted in a decrease of ALP activity and an increase of oil droplet accumulation. Reverse tanscription-polymerase chain reaction (RT-PCR) analysis also indicated that osteogenic differentiation markers decreased and that adipocyte differentiation markers increased. Thus, when the cells were cultured with BMP-2, osteogenic differentiation was enhanced while the expression of PPARγ was maintained, and the addition of troglitazone caused a significant number of differentiated cells into adipocytes. These findings indicate that BMP-2 enhanced osteogenic differentiation and the expression of adipogenic transcription factor (PPARγ) followed by osteogenic differentiation without activation of PPARγ by its ligand.
We investigated the effects of extracellular ATP on TBR31-2 cells established from the bone marrow of transgenic mice harboring the temperature-sensitive simian virus (SV) 40 T-antigen gene. These cells showed the capacity to differentiate toward osteoblasts and could be enhanced by bone morphogenetic protein (BMP)-2, an inducer of osteoblasts. The intracellular calcium ion level ([Ca2+]i) in differentiating TBR31-2 cells was measured by fluorescence confocal microscopic imaging using the Ca2+-sensitive probe, Calcium Green 1/AM. P2 receptor agonists, such as ATP (1 μM), uridine 5′-triphosphate (1 μM), and ADP (1 μM), significantly increased the [Ca2+]i of TBR31-2 cells in 2-d and 5-d cultures, but a potent P2X receptor agonist, α,β-methylene ATP (10 μM), did not increase [Ca2+]i. The increase in [Ca2+]i induced by ATP in the 2-d culture tended to be higher than in the 5-d culture. The increase in [Ca2+]i of both cultures was inhibited by pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid, a P2 receptor antagonist. However, in an external Ca2+-free condition ATP-induced increase in [Ca2+]i was unchanged at either stage. U73122, phospholipase C inhibitor and Thapsigargin, a calcium-pump inhibitor, significantly inhibited the increase in [Ca2+]i at both stages. Reverse transcription-polymerase chain reaction analysis showed that the expression of P2Y receptor mRNA was higher in the 2-d culture than in the 5-d culture. These results indicate that ATP induces the increase in [Ca2+]i from the calcium store through activating P2Y receptors in TBR31-2 cells and that the 2-d culture can respond to ATP more than the 5-d culture due to the higher expression of P2Y receptors. This suggests that the physiological role of ATP in osteoblasts is altered during differentiation.
Angelicae Decursivae Radix (‘Jeonho’ in Korean) is prescribed as the root of Angelica decursiva (=Peucedanum decursivum) and Peucedanum praeruptorum in Korean pharmacopoeia. However, Anthricus sylvestris has been usually distributed on the market because it is identical to the Korean plant name ‘Jeonho’. Furthermore, due to the morphological similarity of the aerial parts and herbal medicines, the correct identification of these roots is difficult. Therefore, to develop a reliable method for discriminating among A. decursiva (=P. decursivum), P. praeruptorum and A. sylvestris, we applied the tools of molecular genetics, such as the analysis of ribosomal DNA internal transcribed spacer (rDNA-ITS) and random amplified polymorphic DNA (RAPD). In the comparison of rDNA-ITS sequences, we found a specific primer region for the identification of A. sylvestris among three varieties of the herb that produced a 273 bp strand of DNA specific to A. sylvestris. As the result of RAPD analysis, we developed one sequence characterized amplified region (SCAR) marker for A. decursiva and P. praeruptorum that amplified a 363 bp DNA fragment specific to both A. decursiva and P. praeruptorum and two markers for P. praeruptorum that amplified 145 bp and 305 bp DNA fragments specific to P. praeruptorum. Furthermore, we established the SCAR markers for the simultaneous discrimination of the three species by applying a multiplex-polymerase chain reaction (PCR) with a combination of primers. This method of discrimination would be useful in preventing the distribution of adulterates because it can identify each herb and distinguish it from inauthentic substitutions.
Aldehyde oxidase (AO) is a homodimer with a subunit molecular mass of approximately 150 kDa. Each subunit consists of about 20 kDa 2Fe-2S cluster domain storing reducing equivalents, about 40 kDa flavine adenine dinucleotide (FAD) domain and about 85 kDa molybdenum cofactor (MoCo) domain containing a substrate binding site. In order to clarify the properties of each domain, especially substrate binding domain, chimeric cDNAs were constructed by mutual exchange of 2Fe-2S/FAD and MoCo domains between monkey and rat. Chimeric monkey/rat AO was referred to one with monkey type 2Fe-2S/FAD domains and a rat type MoCo domain. Rat/monkey AO was vice versa. AO-catalyzed 2-oxidation activities of (S)-RS-8359 were measured using the expressed enzyme in Escherichia coli. Substrate inhibition was seen in rat AO and chimeric monkey/rat AO, but not in monkey AO and chimeric rat/monkey AO, suggesting that the phenomenon might be dependent on the natures of MoCo domain of rat. A biphasic Eadie–Hofstee profile was observed in monkey AO and chimeric rat/monkey AO, but not rat AO and chimeric monkey/rat AO, indicating that the biphasic profile might be related to the properties of MoCo domain of monkey. Two-fold greater Vmax values were observed in monkey AO than in chimeric rat/monkey AO, and in chimeric monkey/rat AO than in rat AO, suggesting that monkey has the more effective electron transfer system than rat. Thus, the use of chimeric enzymes revealed that 2Fe-2S/FAD and MoCo domains affect the velocity and the quantitative profiles of AO-catalyzed (S)-RS-8359 2-oxidation, respectively.
Recently, we showed that a biosurfactatnt 4-O-[(4′,6′-di-O-acethyl-2′,3′-di-O-alkanoyl)-β-D-mannopyranosyl] meso-erythritol A (MEL-A) greatly increased the efficiency of gene transfection mediated by cationic liposomes in vitro. We then studied whether the high transfection efficiency of these liposomes is maintained in vivo for tumor cells in the mouse abdominal cavity. When a complex of the liposomes and plasmid DNA was injected intraperitoneally into C57BL/6J mice bearing B16/BL6 tumors, we found that the biosurfactant significantly increased liposome-mediated gene transfection to the mouse tumor cells. The transfection efficiency of the plasmids into the solid tumors by the cationic liposomes of cholesteryl-3β-carboxyamidoethylene-N-hydroxyethylamine (OH-Chol) with MEL-A increased by about 100-fold compared with that by the cationic liposomes of DC-Chol (commercially available) without MEL-A. The results suggest that nonviral vectors with MEL-A are very useful for gene transfection in vivo.
To study the anti-infectious effect of a vascular allograft, the antimicrobial activity of tryptophan metabolites mediated by indoleamine 2,3-dioxygenase was determined. The growth of methicillin-resistant Staphylococcus aureus (MRSA) over 10 h in extracts from post-transplantation vascular allograft was significantly slower than that of extracts from non-transplantation vascular allograft regardless of the presence of tryptophan (p<0.05). When the antimicrobial activity of the tryptophan metabolites in the L-tryptophan–L-kynurenine pathway was examined, 3-hydroxy-DL-kynurenine and α-picolinic acid had strong antibacterial activity against MRSA, S. epidermidis, Escherichia coli, and multidrug-resistant Pseudomonas aeruginosa, although antimicrobial activities of anthranilic acid, 3-hydroxyanthranilic acid, and quinolinic acid against them were low. The results showed that, of the tested tryptophan metabolites, 3-hydroxy-DL-kynurenine and α-picolinic acid contributed to the anti-infectious effects of the allograft by inhibiting of the growth of microorganisms.
Candida albicans (C. albicans) is known as an opportunistic pathogen that changes from a yeast form to a hyphae form in response to various outside environmental signals. The addition of propranolol inhibited hyphae formation of C. albicans. Propranolol inhibited the expression of agglutinin like sequence 3 (ALS3) and ALS8mRNA, which are regulated by the cAMP-EFG1 pathway in C. albicans. Propranolol did not affect the expression of CST20, HST7 or CPH1mRNA, which are components of the mitogen-activated protein (MAP) kinase cascade in C. albicans. The expression of CYR1mRNA, which encodes adenylate cyclase of C. albicans, was not affected by propranolol. These findings indicated that the interruption of hyphae formation by propranolol is caused by inhibition of the cAMP-EFG1 pathway, but not effects on the MAP kinase cascade.
It has been reported that Janus tyrosine kinase (JAK)-dependent signaling pathways play a critical role in the pathogenesis of numerous malignancies and immune reactions, and inhibition of JAK has been implicated in cell growth inhibition. The role which JAK has on osteoclast differentiation and anti-bone resorptive activity is not well understood. In this study, we investigated the effects of a pan-JAK inhibitor, pyridone 6, on osteoclast differentiation and bone-resorption in vitro and ex vivo. Pyridone 6 inhibited osteoclast differentiation in mouse bone marrow macrophage (BMM) cultures stimulated by the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) and co-cultures of bone marrow cells and osteoblasts. Pyridone 6 suppressed the expression of c-Fos and nuclear factor of activated T cells (NFAT) c1 in BMMs. It also inhibited the bone resorptive activity of mature osteoclasts that was accompanied by disruption of actin rings. Pyridone 6 also suppressed I-κB degradation and extracellular signal-regulated kinase (ERK) in mature osteoclasts, suggesting that these are the key molecules that pyridone 6 targets in the inhibition of osteoclast function. These results demonstrate inhibition of JAK may be useful for the treatment of bone-resorptive diseases, such as osteoporosis.
The present study was performed to investigate the effects of histamine H1-antagonists on the sleep–awake state in rats placed on a grid suspended over water in comparison with rats placed on sawdust. When rats were placed on the grid suspended over water, significant increases in the awake time and decreases in non-rapid eye movement (non-REM) sleep time were observed compared with in rats on sawdust, even when measured hourly for 6 h. Diphenhydramine, chlorpheniramine and promethazine caused a significant decrease in the awake time and increase in non-REM sleep time in rats placed on the grid suspended over water for 1—2 h and/or 2—3 h after administration. On the other hand, in rats placed on sawdust, no significant differences were observed in the awake time and non-REM sleep time with diphenhydramine and chlorpheniramine compared with the control. Different from these two drugs, promethazine caused a significant decrease in the awake time and increase in non-REM sleep time 1—2 h and 2—3 h after administration even when rats were placed on sawdust at a relatively high dose. These results clearly indicate that histamine H1-antagonists had potent effects on decreasing the awake time and increasing non-REM sleep time under the conditions of an activated histaminergic system.
The aim of the present study was to investigate the protective effects of (−)-epigallocatechin-3-gallate (EGCG), the main polyphenolic constituent of green tea, in aging mice induced by D-galactose (D-gal). The aging mice model was induced by subcutaneous (s.c.) injection of D-gal (150 mg/kg) once daily for 6 weeks. EGCG (2 mg/kg or 6 mg/kg) was administered intragastrically (i.g.) once daily for 4 weeks after 2-week D-gal injection. The water maze test was used to evaluate the learning and memory function of mice. The activities of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) and the contents of malondialdehyde (MDA) in the hippocampus were measured using different biochemical kits to estimate the changes in the antioxidative ability of mice. TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining method was used to detect neuronal apoptosis, and the activation and expression of proapoptotic protein caspase-3 in the hippocampus were observed and analyzed using immunohistochemical staining and the Western blot method to evaluate apoptosis in the brain. The results indicated that subcutaneous injection of D-gal induced learning and memory impairment in mice, decreased T-SOD and GSH-Px activities, increased MDA contents in the hippocampus, and increased the cell apoptosis index and cleaved caspase-3 protein expression in the hippocampus. Oral administration of EGCG (2 mg/kg or 6 mg/kg) for 4 weeks significantly improved the cognitive deficits in mice and elevated T-SOD and GSH-Px activities, decreased MDA contents in the hippocampus, and reduced the cell apoptosis index and expression of cleaved caspase-3 in the mouse hippocampus. The results suggest that EGCG has potent neuroprotective effects on aging mice induced by D-gal through antioxidative and antiapoptotic mechanisms, indicating that EGCG is worthy of further study in aging.
Gentamicin (GM) is an antibiotic widely used in treating severe gram-negative infections. However, its clinical use is limited by its nephrotoxicity. Several lines of evidence indicate that free radicals are important mediators of gentamicin nephrotoxicity. Therefore, the aim of this work was to investigate the possible protective effect of the flavonoid quercetin, an antioxidant, on gentamicin-induced nephrotoxicity. For this purpose, rats were divided into four groups. First group served as a control and injected with the normal saline, second group was injected with quercetin (50 mg/kg/d, per os) for 7 d, third group was injected with gentamicin (80 mg/kg/d, intraperitoneally) for 7 d and the fourth group of animals was injected with quercetin plus gentamicin simultaneously for 7 d. Total protein levels were estimated in 24-h urine samples to assess kidney dysfunction. The rats were sacrificed on the seventh day and kidneys were collected for histopathological studies. Blood urea nitrogen (BUN) and creatinine levels were measured in the blood. Moreover, glutathione (GSH), lipid peroxide (TBARS) levels, superoxide dismutase (SOD) and catalase (CAT) activities were determined in renal tissues. GM-treated rats showed early kidney dysfunction as urinary total protein, BUN and serum creatinine levels were significantly increased. The significant decrease in GSH levels, SOD, CAT activities and increase in TBARS levels, indicated that GM-induced nephrotoxicity was mediated through oxidative stress reactions. Histopathological examination of GM-treated rats revealed degenerative changes in glomeruli and tubules. On the other hand, simultaneous administration of quercetin plus gentamicin protected kidney tissues against nephrotoxic effects of gentamicin as evidenced from amelioration of histopathological changes and normalization of kidney biochemical parameters.
Calycosin-7-O-β-D-glucopyranoside (CCGR) is the main isoflavonoid compound isolated from Astragalus membranaceus var. Mongholicu (BGE.) HSIAO, a Chinese herb medicine traditionally used to treat viral myocarditis. In this study, its antiviral activities against coxsackie virus B3 (CVB3) causing myocarditis were investigated. In vitro assay showed that CCGR displayed a low cytotoxicity and effectively inhibited CVB3-mediated cytopathic effects on Vero cells with an IC50 value of 25 μg/ml. In an acute myocarditis murine model, treatment with 24 mg/kg CCGR for 14 d significantly improved the survival rate of mice infected with CVB3, alleviated pathological damages of cardiac muscles in the myocarditis mice, reduced the virus titers in the heart, decreased heart indexes and improved left ventricular function. These results showed that CCGR exerted significant antiviral activities against CVB3 both in vitro and in vivo, and identified CCGR as one of active ingredients in Astragalus membranaceus for the treatment of viral myocarditis.
Oral bioavailability is one of the important criteria for development of a drug-lead candidate. In this study, the absorptive characteristics and the efflux mechanism of a mercaptoacetamide-based histone deacetyalse (HDAC) inhibitor, coded as W2, were investigated using Caco-2 cells. The transport of W2 was asymmetric as indicated by 1.85 fold higher basolateral to apical (BL to AP) than apical to basolateral (AP to BL) flux. Such asymmetry was associated with multidrug resistance associated protein 1 (MRP1) and P-glycoprotein (P-gp), as evidenced by specific inhibition of these proteins. In the presence of verapamil and cyclosporin A, potent inhibitors of P-gp, the apparent permeability ratio (Papp BL to AP/Papp AP to BL) of W2 was decreased from 1.85 to 0.73 and 1.03, respectively, and the absorption from apical to basolateral side was enhanced from 13.3±0.2×10−6 cm/s to 17.3±0.12×10−6 cm/s and 19±0.3×10−6 cm/s, respectively. Upon addition of quinidine, a mixed P-gp and MRP1 inhibitor, the permeation of W2 from the apical side was significantly increased (Papp 17.1±0.32×10−6 cm/s) while the efflux was inhibited (Papp 21.3±0.19×10−6 cm/s). Furthermore, the influence of the MRP1 inhibitors, indomethacin and N-benzyl-indomethacin (NBI) was evaluated. NBI treatment attenuated the basolateral to apical flux of W2 (Papp 20.3±0.1×10−6 cm/s), whereas this effect was completely abrogated by indomethacin (Papp 11±0.4×10−6 cm/s). The results suggest that P-gp and MRP1 transporters are capable of mediating the efflux of W2 and might play a significant role in its oral absorption.
In this study we elucidated the effects of berberine, a major alkaloid component contained in medicinal herbs, such as Phellodendri Cortex and Coptidis Rhizoma, on ischemic neuronal damage in mouse organotypic hippocampal slice cultures (OHSCs) caused by oxygen and glucose deprivation (OGD) and N-methyl-D-aspartate (NMDA) -type glutamate receptor stimulation. Hippocampal slices obtained from 7-d-old ICR mice were cultured for 10 d before the experiments. Ischemia-related damage was induced by OGD (5, 15, 45 min) or NMDA (10 μM) treatment, and was evaluated by measuring propidium iodide (PI) uptake. Levels of apoptotic marker proteins, B-cell lymphoma 2 (Bcl-2) and phosphorylated-Bcl-2 (p-Bcl-2), in the OHSCs were measured as indices of biochemical neuronal cell damage by Western blotting. Berberine (5, 25 μM) or the NMDA antagonist MK-801 (25 μM) was added to the medium 30 min before OGD or NMDA treatment. OGD time-dependently increased PI uptake of the OHSCs. Both berberine (5, 25 μM) and MK-801 (25 μM) significantly inhibited PI uptake at 24 h after 45-min OGD treatment and PI uptake in OHSCs exposed to NMDA for 24 h. OGD treatment also significantly increased the level of p-Bcl-2 but not that of Bcl-2 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in OHSCs. Berberine (5—25 μM) significantly suppressed the OGD-induced increase of p-Bcl-2 level in OHSCs when tissue was exposed to the alkaloid prior to OGD or simultaneously with OGD. These findings suggest that berberine has protective effects against ischemic damage in mouse OHSCs and that the effects are at least partly mediated by suppression of Bcl-2 phosphorylation.
2,4,2′,4′-Tetrahydroxy-3-(3-methyl-2-butenyl)-chalcone (TMBC), a naturally occurring compound from Morus nigra, modulated melanogenesis by inhibiting tyrosinase. TMBC inhibited the L-dopa oxidase activity of mushroom tyrosinase with an IC50 value of 0.95±0.04 μM, which was more potent than kojic acid (IC50=24.88±1.13 μM), a well-known tyrosinase inhibitor. The kinetic studies of tyrosinase inhibition revealed that TMBC acts as a competitive inhibitor of mushroom tyrosinase with L-dopa as the substrate. Furthermore, TMBC effectively inhibited both cellular tyrosinase activity and melanin biosynthesis in B16 melanoma cells without significant cytotoxicity. The inhibitory effect of TMBC on melanogenesis was attributed to the direct inhibition of tyrosinase activity, rather than the suppression of tyrosinase gene expression. These results indicated that TMBC may be a new promising pigmentation-altering agent for cosmetic or therapeutic applications.
Total ethanol extract and saponins from Chinese herb Radix Astragali (Huangqi) have been previously shown to possess anti-hepatitis B virus (HBV) activities in vitro. To identify the active ingredients, we isolated a triterpenoid saponin that was determined to be astragaloside IV. In the human HBV-transfected liver cell line HepG2 2.2.15, astragaloside IV effectively suppressed secretion of HBV antigens with inhibition rates of 23.6% for the secretion of Hepatitis B surface antigen (HBsAg) and 22.9% for that of Hepatitis B e antigen (HBeAg) at 100 μg/ml after 9 d of treatment. The inhibitory activity of astragaloside IV on secretion of HBV antigens is more potent than that of 3TC without significant cytotoxicity. In duck hepatitis B virus (DHBV)-infected ducklings, astragaloside IV caused 64.0% inhibition at 120 mg/kg, 49.6% inhibition at 40 mg/kg, and 41.7% inhibition at 10 mg/kg to serum DHBVs after 10 d of treatment, and also reduced serum DHBV DNA levels. Together, our results demonstrate that astragaloside IV possesses potent anti-HBV activity.
Discoidin domain receptors belong to the cell surface receptor tyrosine kinase family and recognize collagens for their activating ligands. They have been implicated for cell growth and migration and their elevated expressions were observed in various human cancers. When we expressed human Discoidin domain receptor 2 (DDR2) in insect cells, the protein was targeted properly into the cell membrane and this could enforce the cells to adhere on culture plate coated with type I collagen. By taking advantage of this, we established a novel insect cell based screening protocol to identify chemicals which inhibit the interaction between DDR2 and collagen. We screened a drug-compound library to select an anti-cancer drug, actinomycin D, as the inhibitory compound. Actinomycin D prevented the activation of DDR2 by type I collagen in human embryonic kidney 293 cells with an IC50 value of 9 μM, while it did not interfere with the activation of other receptor tyrosine kinases by their ligands. In conclusion we identified a new biological function of actinomycin D and the insect cell based method provides a useful protocol for screening inhibitors against the association of DDR2 with collagen.
In the present study, we investigated the effects of 3-oxoolean-12-en-27-oic acid (3-OA) isolated from the underground parts of Aceriphyllum rossii (Saxifragaceae) on the viability and apoptosis of HL-60 human promyelocytic leukemia cells, and the mechanisms underlying its action. 3-OA-treated HL-60 cells and HeLa human cervix adenocarcinoma cells displayed several apoptotic features, such as, DNA fragmentation, DNA laddering by agarose gel electrophoresis, and hypodiploid DNA contents by flow cytometry, and 3-OA also caused the activations of caspase-8, -9 and -3. Pretreatment with z-VAD-fmk (a broad-caspase inhibitor) almost completely suppressed 3-OA-induced DNA ladder formation and hypodiploid DNA contents, thereby implicating the caspase cascade in the apoptotic process. In addition, z-IETD-fmk (a caspase-8 inhibitor) and z-DEVD-fmk (a caspase-3 inhibitor) also completely neutralized the apoptotic effect of 3-OA in HL-60 cells. Furthermore, 3-OA increased Fas-related protein contents and the mRNA expressions of Fas ligand (FasL), Fas, and Fas-associated death domain (FADD). Preincubation with anti-Fas or anti-FasL blocking antibodies completely prevented 3-OA-induced apoptosis. Taken together, these results suggest that 3-oxoolean-12-en-27-oic acid induces apoptosis by activating caspase-8 via FasL-stimulated death receptor signaling.
Herba Cistanches is a common traditional Chinese medicine that has been used to reinforce the vital function of kidney and induce laxation for more than two thousands years. Four Cistanche species were found as Herba Cistanches in China herbal markets, including C. deserticola, C. tubulosa, C. salsa and C. sinensis. Phenylethanoid glycosides, particularly echinacoside and acteoside, are considered as active ingredients in Cistanche species. The contents of these compounds showed variation in different species and geographical sources. Standard chemical fingerprints were generated from each of four Cistanche species, which could be identification markers. In genetic analysis of Cistanche species, ninety-four inter-simple sequence repeat (ISSR) primers were used for polymerase chain reaction (PCR) amplification, and of which eight primers were found to be sufficient to distinguish different Cistanche species. As a result, the chemical fingerprint combined with the genetic fingerprint for distinction of Cistanche species could serve as markers for quality control of Herba Cistanches.
The methanolic extract from the seeds of Psoralea corylifolia was found to inhibit production of nitric oxide (NO) in lipopolysaccharide-activated mouse peritoneal macrophages. Among the isolated compounds, bavachinin (IC50=26 μM), isobavachalcone (17 μM), neobavaisoflavone (ca. 29 μM), corylifol A (ca. 21 μM), and psoralidin (ca. 23 μM) significantly inhibited the accumulation of nitrite (NO2−) as a marker of production of NO. Bakuchiol, which is mainly contained in the extract, also showed weak activity at 10 μM, but cytotoxic effects were observed more than 30 μM.
The pharmacokinetics of vancomycin was retrospectively examined based on trough concentrations obtained during routine therapeutic drug monitoring to examine possible pharmacokinetic differences between adult Japanese cancer and non-cancer patients with various degrees of renal function. A total of 231 data points from 65 cancer patients and 41 non-cancer patients were collected, and patients' background, vancomycin dose, and vancomycin clearance estimated by an empirical Bayesian method were summarized. Regarding the patients' characteristics and clinical laboratory test data, no clear differences were found between the two groups. The relationship between vancomycin clearance and creatinine clearance were similar between the groups, suggesting little effect of malignancy on vancomycin clearance. After the sub-group comparisons regarding fluid retention and cancer type, no clear differences were found in the vancomycin clearance versus creatinine clearance relationship. We conclude that the initial dose of vancomycin should not necessarily be adjusted for cancer patients. For individualized vancomycin-based therapy, dose adjustment at the appropriate time is important according to information from routine therapeutic drug monitoring and clinical laboratory tests, and to observations of the efficacy, nephrotoxicity, and other conditions in each patient.
Sulfotransferase 1A3 (SULT1A3) is a phase II detoxifying enzyme of xenobiotics predominantly expressed in the intestinal epithelium. Recent increase in the use of herbal extracts as dietary supplements may lead to an increase in the possibility of dietary supplement–drug interactions. The purpose of the present study was to investigate the effects of 18 herbal extracts on SULT1A3 activity and the possibility of interaction between medicinal drugs and herbal extracts. We examined the inhibitory potencies of 18 herbal extracts on the sulfation of dopamine, a typical substrate of SULT1A3, and ritodrine, a β2 stimulant, by human recombinant SULT1A3. The sulfation of dopamine was inhibited by extracts of banaba, green tea, Rafuma, grape seed, peanut seed coat, gingko biloba leaf, St. John's wort, gymnema and milkthistle. The IC50 values of these herbal extracts were lower than the putative gastrointestinal concentration when the recommended dose was ingested. On the other hand, chlorella extract and rutin showed no inhibitory effects and wheat, mulberry and siberian ginseng had IC50 values exceedingly higher than the putative gastrointestinal concentration. The inhibitory profiles of herbal extracts for the sulfation of ritodrine were comparable to those for the sulfation of dopamine. In conclusion, the extracts of herbs such as banaba and green tea potently inhibited SULT1A3 activity. These extracts may increase the bioavailability of drugs whose bioavailabilities were limited by the function of SULT1A3 on the intestinal epithelium.
Periodontitis is initiated by accumulation of microbial plaque and activation of gingival inflammation through overexpression of matrix metalloproteinases (MMPs), leading to tissue destruction. Natural MMP inhibitors may be developed as therapeutic agents against periodontitis. In this study, panduratin A, a natural bioactive compound isolated from Kaempferia pandurata ROXB., was used to test its in vitro inhibitory activity against MMP-9 secretion from Porphyromonas gingivalis supernatant-induced human oral epidermoid carcinoma KB cells. Gelatin zymography, Western blot and RT-PCR analyses were performed to evaluate MMP-9 expression. The gelatin zymograms revealed that the main gelatinase secreted by P. gingivalis supernatant-induced KB cells migrated at 92 kDa, representing MMP-9. MMP-9 protein and mRNA levels were significantly decreased after panduratin A treatment (p<0.05). In contrast, panduratin A had no effect on tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 mRNA. Panduratin A also suppressed urokinase type plasminogen activator (uPA) mRNA expression. These results suggest that panduratin A could potentially prevent periodontal inflammation by decreasing the levels of MMP-9 protein and mRNA.
It is well known that nonsteroidal anti-inflammatory drugs (NSAIDs) have significant side effects, such as gastroenteropathy, and that rheumatoid arthritis patients taking NSAIDs are more susceptible to NSAIDs-induced gastric lesions as compared with patients with other diseases. We demonstrate the preventive effect of the co-administration of bittern water (BW, nigari-sui in Japanese), which enables the effective intake of Mg2+, on the ulcerogenic response to indomethacin in adjuvant-induced arthritis (AA) rats. Four kinds of BW with different Mg2+ contents; ranging from 10—200 mg/l Mg2+ (BW-10, 25, 50, 200) were used in this study. Arthritis was induced by the injection of 50 μl of a suspension of 10 mg/ml heat-killed butyricum (Mycobacterium butyricum) in Bayol F oil into the plantar region of the right hind foot and tail of rats. Oral administration of indomethacin (40 mg/kg) caused hemorrhagic lesions in the gastric mucosa of AA rats at 14 d after adjuvant injection, and the lesion score of AA rats administered indomethacin was significantly higher than that of normal rats administered indomethacin. The expression of the mRNA for inducible nitric oxide synthase (iNOS) mRNA expression and the production of nitric oxide (NO) in the gastric mucosa of AA rats were also increased by the administration of indomethacin. The co-administration of BWs decreased the ulcerogenic response to indomethacin in AA rats. In addition, the administration of BW attenuated the increase in iNOS mRNA expression and NO production in AA rats receiving indomethacin. The oral administration of Mg2+ to AA rats had a potent preventive effect on the ulcerogenic response to indomethacin in AA rats, probably due to an inhibition in the rise in iNOS and NO levels in the gastric mucosa.
Breast cancer is one of the most frequent female cancers in the Western world. Perturbation of estrogen levels by hormone replacement therapy or pregnancy is associated with a variety of diseases, including breast cancer. Estrogen supplementation is required to establish appropriate animal models for estrogen-related diseases. In this report, we demonstrated that supplementation with high doses of 17β-estradiol results in deaths in estrogen-dependent MCF-7 tumor xenograft model. Renal damage and bladder stone formation was implicated as a major cause of death. The mortality rate was significantly reduced when mice received a low dose of 17β-estradiol. We also confirmed that low dose of 17β-estradiol supplementation can support the growth of tumors in MCF-7 tumor xenograft model. These results suggest that low dose estrogen supplementation may be more appropriate in estrogen-dependent tumor xenograft models.
The improvement of diabetic complications such as lipid lowering and anti-oxidative potential of Hydrangea Dulcis Folium (HDF) was studied in streptozotocin-induced diabetic rats. Male Sprague-Dawley rats were divided into 3 groups after induction of streptozotocin (STZ)-diabetes: normal control; diabetic control; diabetic-HDF supplement (hot water extract 40 g/kg diet); and fed experimental diets for 3weeks. Serum glucose and insulin concentrations, serum lipid profile, intraperitoneal glucose tolerance test, and liver cytosolic antioxidant enzyme levels were measured. The HDF supplement significantly decreased serum glucose concentration, increased insulin level, and improved glucose homeostasis in diabetic control rats. The total cholesterol and triglyceride concentrations in the serum and liver were markedly reduced by HDF treatment in STZ-diabetic rats. Moreover, low density lipoprotein (LDL)-, VLDL-, and high density lipoprotein (HDL)-cholesterol levels were ameliorated in HDF supplemented diabetic rats. Decreased fecal excretions of cholesterol, triglyceride, and bile acid in diabetic rats were significantly increased by HDF consumption. HDF supplement reversed the effects of the oxidative stress system of liver in diabetic rats. Lipid peroxidation of diabetic rats, assessed by thiobarbituric acid reactive substance (TBARS), as well as superoxide dismutase (SOD) activities were significantly increased, and glutathione contents were decreased in diabetic rats. HDF supplement reverted these parameters to near normal value. Our data suggest that HDF supplement could be used to improve the glucose and lipid metabolism as well as to reduce the imbalance between the generation of reactive oxygen species (ROS) and the scavenging enzyme activity in preventing diabetic complications.
Synthetic retinoid Am80 is a potent modulator of the immune system. Am80 is effective in various experimentally induced autoimmune disorders. The purpose of this study is to confirm its effect on non-obese diabetic (NOD) mice, which spontaneously develop autoimmune type 1 diabetes. Am80 was orally administered in feed to 6 NOD mice per group at a dose of 0 (control), 0.1 (low) or 1 (high) mg/kg/d for 19 weeks. During the experiment period, the high urine glucose levels were observed in 33% mice of the control and low Am80 groups, whereas any mouse in the high Am80 group did not show abnormal urine glucose level. Histological examination showed that the average score of insulitis severity in the low Am80 group was similar to that in the control; however in the high Am80 group, the score was significantly reduced compared to that in the control group. Similarly, the severity of lymphocyte infiltration in the submandibular glands showed a tendency to decrease in the high Am80 group, but not in the low Am80 group, compared to the control. These data strongly suggest that the development of type 1 diabetes in NOD mice can be inhibited by oral administration of Am80.