We previously showed that enzyme immunoassay (EIA) of β-methyldigoxin (MDx3) using anti-MDx3 3′-hemisuccinate–bovine serum albumin antiserum (Antiserum-I) was superior to that using commercial anti-digoxin antiserum (Antiserum-II) in terms of specificity and that pretreatment of human serum with phenyl boric acid (PBA) column was effective. In the present study, we examined the precision of EIA using Antiserum-I and the recovery of MDx3 after PBA column treatment in rat serum, and also investigated pharmacokinetic changes of MDx3 in rats. The intra- and inter-assay variations and recovery tests using Antiserum-I were good. The PBA column was effective in selectively separating MDx3 from rat serum containing MDx3 and its metabolites. The recovery tests using Antiserum-I with PBA column showed about 110% and the interference of metabolites of MDx3 was negligible. Serum concentration–time courses of MDx3 by EIA using Antiserum-I with PBA column and Antiserum-I were lower than that using Antiserum-II. The distribution volume at steady state and total body clearance values of MDx3 in these conditions were significantly higher than those using Antiserum-II. The usefulness of PBA column was ascertained, while effects of PBA column on these parameters were not significant. In addition, rapid absorption of MDx3 was observed by EIA using Antiserum-I with PBA column. These results suggest that EIA using Antiserum-I with PBA column for the pretreatment of serum samples should be a more useful and valuable system in therapeutic drug monitoring and pharmacokinetic studies of the unchanged type of MDx3 than Antiserum-II.
We transfected the α-chain of human FcεRI into rat basophilic leukemia cell line RBL-2H3, established several stable transfected cells, and screened them by β-hexosaminidase release induced by sensitization with human IgE and stimulation with anti-human IgE antibody. A cloned cell line RBL-hEIa-2B12 was the strongest responder among the transfected cell clones. The concentrations of cytosolic free Ca2+ concentration in the human IgE-sensitized cells increased after stimulation with anti-human IgE antibody. Thus, it is suggested that the α-chain of human FcεRI is associated with the β-chain and/or γ-chain of rat FcεRI, and that they form functional high affinity IgE receptor complexes. The total IgE concentrations of the sera from allergic patients were determined by using the β-hexosaminidase release assay, where the transfected cells were sensitized with diluted and heat-inactivated (at 56 °C for 30 min) serum and stimulated with anti-human IgE antibody. The IgE concentration obtained correlated with those measured by an enzyme immunoassay method. β-Hexosaminidase release induced by stimulation with 5 times diluted serum was sometimes less than the release induced by the same serum; diluted 25 times or 125 times, suggesting that these serum contained factors that blocked IgE binding to FcεRI or cross-linking by anti-human IgE antibody. The results suggested that our system will be useful for detecting FcεRIα-bindable IgE in human serum.
In plants gibberellins (GAs) are responsible for triggering stem or internodal elongation. To comprehend the molecular basis of internodal elongation in rice, a proteomics approach using differentially displayed proteins on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was carried out to identify the proteins expressed during the GA controlled leaf-sheath elongation response. Out of 352 protein spots detected on 2-D PAGE, 32 proteins showed modulation in the expression levels in GA3-treated leaf-sheath for 48 h as compared to control. These proteins were analyzed using protein sequencer and/or mass spectrometry in conjunction with the protein database to assign putative identities. The twin protein spots (LS079 and LS083), identified as calreticulin, showed different isoelectric points and expression level in GA3-treated leaf-sheath. The expression level of LS083 (pI 4.0) was down-regulated as compared to the up-regulation of LS079 (pI 4.3). In the presence of GA3 and growth inhibitor, uniconazole and abscisic acid, respectively, no elongation in leaf-sheath occurred and calreticulin did not shift from LS083 to LS079. Over-expression of calreticulin in rice inhibited the callus regeneration and seedling growth. These results suggest that calreticulin is an important component in the GA signaling pathway that regulates rice seedling leaf-sheath elongation.
The human adenovirus type 5 (Ad5) early-region 1A (E1A) proteins have been shown to have strong tumor-suppressive activities in human tumor cells and to enhance the sensitivity of a variety of malignant tumors to apoptosis induced by ionizing radiation and chemotherapeutic agents. However, the inherent limitations of E1A gene therapy prevent its application, such as the efficiency of expression, precision of targeting, and toxicity of vector. This prompted us to construct an E1A expression vector (pPIC9/E1A) and express the E1A protein in the methylotrophic yeast Pichia pastoris. The E1A protein was purified using two steps of ion-exchange column chromatography on HiTrap Q and HiTrap SP. The analysis indicated that the E1A protein/liposome inhibited S-180 tumor growth and also rendered the S-180 tumor strongly susceptible to the anticancer drug bleomycin in vivo. Furthermore, tunnel assay clearly revealed that the mechanism was induction of cellular apoptosis. Importantly, the E1A protein overcame the limitations of gene therapy. Thus the E1A protein may be a useful therapeutic agent for some malignant tumors.
Primary cultivated amnion epitherial cells prepared from amnion tissue of human fetal membrane (Amnion-cells) were stimulated with influenza virus-hemagglutinin (IV-HA), fractionated from a commercialized IV-HA vaccine by DEAE Sephacel column chromatography. From 72—96 h after stimulation, chromosomal DNA fragmentation and the appearance of in situ TUNEL stained-positive cells were revealed. Amnion-cell DNA fragmentation was inhibited in the presence of glycophorin A or C purified from the human erythrocyte membrane fraction, but not inhibited with free N-acetyl-neuraminic acid. RT-PCR and Western blotting analysis showed that anti-oxidative enzymes were altered with incubation period, accompanied by the expression of the cellular oxidative stress-related caspase cascade. Pre-stimulation of Amnion-cells with hemin, a heme oxygenase-1 inducer, significantly attenuated IV-HA induced DNA fragmentation. It is concluded that IV-HA induces apoptosis in Amnion-cells, and that this apoptotic induction may be facilitated by certain sialoglycoproteins on the cell surface, and is related to changes in the intracellular redox condition.
The Na+/H+ exchangers (NHEs) comprise a family of membrane proteins that catalyze the electroneutral exchange of Na+ and H+. Calcineurin homologous protein (CHP) acts as a crucial cofactor for NHE activity through direct interaction with the carboxyl-terminal tail region of NHEs. We have cloned a new rat CHP isoform (rCHP2) and characterized the binding property to NHEs and the tissue distribution. rCHP2 binds to the juxtamembrane region of plasma membrane-type NHE isoforms (NHE1—5) in vivo and in vitro as well as rCHP1 (original rat CHP). Interestingly, CHP2 is predominantly expressed in the small and large intestine although rCHP1 shows relatively ubiquitous expression at both the mRNA and protein levels. In situ hybridization experiments demonstrated the abundant expression of CHP2 in the epithelial cell layer of villi of the small intestine in contrast with the expression of CHP1 in both the epithelial layer and connective tissues. These results suggest that CHP2 functions in the absorptive epithelium for the intestine with NHE(s).
Calreticulin is an abundant endo/sarcoplasmic reticulum Ca2+-binding protein. To investigate whether calreticulin (CRO1) is involved in the cold-stress response in rice, a transgenic plant was constructed. The transcriptional level was decreased within 30 min and recovered within 2 h of a cold treatment. The calreticulin protein was shifted from a soluble fraction to an insoluble fraction by cold stress. Endogenous abscisic acid (ABA) is an important factor in cold response, and the synthesis of ABA was strongly induced in CRO1-sense transgenic rice, the same as in cold-sensitive rice. The phosphorylation of calreticulin increased after cold treatment. Over-expression of calreticulin enhanced the activities of 47 kDa Ca2+-dependent protein kinase (CDPK) that had been induced by cold treatment. The 47-kDa CDPK activity increases more in the cold sensitive variety IR36 and the sense transgenic rice than it does in other varieties. The synthesis of ABA, phosphorylation of calreticulin and 47-kDa CDPK activity induced in sense transgenic rice were the same as in cold-sensitive rice and the phosphorylation of antisense transgenic rice was similar to that of cold-tolerant rice. These results suggest that the calreticulin is involved in the signaling pathway leading to response to cold stress.
A gene with a high-nucleotide sequence homology to the edeine B1 amidinohydrolase gene of Bacillus brevis was identified in the database of the Bacillus subtilis genome. The gene was isolated, expressed in Escherichia coli, and the gene product was analyzed with regard to the characteristics of its enzyme activity. A 32-kDa protein encoded by the ywhG gene showed a 69.8% amino acid sequence-homology to the edeine B1 amidinohydrolase of B. brevis. Among various guanidino-compounds, edeine B1 and agmatine were both efficiently hydrolyzed by the protein encoded by the ywhG gene, although edeine B1 was a more potent substrate than agmatine in this assay system. These data indicate that the protein encoded by the ywhG gene is an agmatinase that is essential for polyamine biosynthesis in B. subtilis.
A fragment of chromosomal DNA from Enterococcus faecalis ATCC 29212 was cloned using Escherichia coli KAM32 host cells lacking major multidrug efflux pumps. E. coli KAM32 cells were sensitive to many antimicrobial agents, and the transformed cells harboring a recombinant plasmid became resistant to several structurally unrelated antimicrobial agents such as tetraphenylphosphonium chloride, 4′,6-diamidino-2-phenylindole (DAPI), Hoechst 33342, acriflavine, benzalkonium chloride, norfloxacin and ethidium bromide. This suggests that the cloned DNA fragment carries a gene(s) encoding a multidrug efflux pump. Determination of the nucleotide sequence of the cloned DNA revealed a gene designated as emeA. The transformed E. coli cells showed efflux activity of several antimicrobial agents such as DAPI, Hoechst 33342 and acriflavine. Efflux of DAPI via EmeA was strongly inhibited by reserpine.
The ameliorating effects of LiuWei Dihuang Wang (LDW) after single, one-week or two-week treatment of scopolamine (SCOP)-induced and p-chloroamphetamine (PCA)-induced amnesia by using the passive avoidance task and the facilitatory effects on two-way active avoidance performance in rats were studied. LDW (2 g/kg) after single treatment significantly prolonged the shortened step-through latency induced by SCOP and PCA. Then, SCOP- and PCA-induced amnesia was reversed by 1 and 0.1—1 g/kg LDW with one-week consecutive treatment respectively. For two-week consecutive treatment with LDW, the reversal from SCOP- and PCA-induced amnesia required only 0.01 g/kg. However, the rats treated with LDW only at 0.5, but not 0.01—0.1 g/kg, before or after each training session showed an increasing number of avoidances and a decreasing number of escapes on days 2—5 of learning. LDW at any dose alone did not influence the step-through latency in the training trial produced by non-shock rats, the motor activity and pentobarbital-induced hypnosis in rodents. These results suggest that LDW possesses the anti-amnestic and cognitive-enhancing activities related to the memory processes, and these activities were parallel to treatment duration and dependent on the learning models.
Injecting muscarinic receptor agonists into a specific area of the brainstem produces an antinociceptive response. The present study investigates whether direct injections of the cholinergic agonist, carbachol, into the rat nucleus reticularis gigantocellularis (NRGC)/nucleus reticularis gigantocellularis alpha (NRGCα) of the rostral ventrolateral medulla evokes antinociception, and then examines the interference action of cholinergic antagonists in rats. Microinjections of carbachol (0.75, 1.5, 3 μg/site) prolonged hot plate (HP) and tail flick (TF) responses to noxious heat stimuli in a dose-dependent manner. The level of carbachol-induced antinociception during the HP and TF tests reached a maximum at 5—15 min after carbachol administration in all groups. Thereafter, the peak level progressively decreased and reached the baseline by the end of the experiment. Antinociception induced by carbachol at 3 μg/site was attenuated by the prior administration of the muscarinic receptor antagonist, atropine (200, 500 ng/site). On the other hand, the nicotinic autonomic ganglion blocker, mecamylamine (1, 3 μg/site), did not affect subsequent carbachol-induced antinociception. These results suggest that the antinociceptive effects induced by a microinjection of carbachol depend on muscarinic, but not nicotinic, mechanisms within the rat NRGC/NRGCα.
Paeonia radix is the root of Paeonia japonica MIYABE, a perennial plant classified in the family Paeoniaceae. In the present study, the effects of Paeonia radix on performance in treadmill exercise, and 5-hydroxytryptamine (5-HT) synthesis and tryptophan hydroxylase (TPH) expression in the dorsal raphe were investigated. Time to exhaustion in treadmill exercise was increased and exercise-induced increases in 5-HT synthesis and TPH expression in the dorsal raphe were shown to be suppressed by Paeonia radix treatment; 5-HT synthesis and TPH expression were inhibited by Paeonia radix treatment under resting conditions as well. In sum, treatment with Paeonia radix, inhibiting 5-HT synthesis and TPH expression, may bring about reduced fatigue, both during exercise and the resting state.
The effects of five levels of population density on various organs, the neuroendocrine system, skin function, skin blood perfusion, and blood parameters were studied in the hairless mouse. Skin barrier recovery was evaluated by measuring transepidermal water loss after tape stripping. Blood perfusion was measured by means of a laser Doppler imaging technique. The effect of a parasympathetic nerve stimulator, carpronium chloride, on skin function in the crowded animal model was also examined. A 7 d crowding (10, 15, 20 mice/cage) significantly increased the levels of corticosterone, catecholamines (norepinephrine, epinephrine and dopamine), glucose and serum lactate dehydrogenase activity in circulating blood, induced atrophy of kidney, ovary and thymus and hypertrophy of adrenal glands, and decreased body weight gain in comparison with the control (5 mice/cage). Crowding also increased epidermal thickness and epidermal proliferative activity, and decreased corneocyte size, rate of barrier recovery and skin blood perfusion. Most of these changes became more marked with increasing population density and/or longer exposure to a crowded environment. Isolation (1 mouse/cage) increased the level of norepinephrine and rate of skin blood perfusion, and significantly delayed barrier recovery. Repeated topical applications of carpronium chloride for 7 d improved the changes in skin blood perfusion, barrier recovery, kidney and ovary, and epidermal morphology induced by crowding. The crowded animal model could be useful for quantifying objectively the influence of crowded environment-induced stress on cutaneous function and blood perfusion.
In the present study, a series of 2-substituted-pyridines were synthesized and characterized by IR, 1H-NMR and Elemental Analysis. The compounds were assayed against seizures induced by maximal electro shock (MES) and pentylenetetrazole (scMet). Neurologic deficit was evaluated by the rotarod test. The decrease in the elevated motor activity by introceptive chemical stimuli (amphetamine antagonistic activity) was studied at the dose level of 25 and 50 mg/kg, antihistaminic and cardiac activity were also studied. All the compounds exhibited significant anticonvulsant activity. Compounds 2-(2′-piperazino-ethanoxy)pyridine, 2-(3′-morpholino-2′-hydroxypropyloxy)pyridine, 2-(3′-piperidino-2′-hydroxypropyloxy)pyridine and 2-(3′-piperazino-2′-hydroxypropyloxy)pyridine were most active of the series against MES-induced seizures. Compounds 2-(2′-piperazino-ethanoxy)pyridine, 2-(2′-phenylamino-ethanoxy)pyridine, 2-(3′-imidazolo-2′-hydroxypropyloxy)pyridine, 2-(3′-methylamino-2′-hydroxypropyloxy)pyridine and 2-(3′-piperidino-2′-hydroxypropyloxy)pyridine exhibited significant decrease in the elevated motor activity at the dose of 50 mg/kg. Remarkable sympathetic blocking activity was observed with 2-(3′-piperazino-2′-hydroxypropyloxy)pyridine, 2-(3′-piperidino-2′-hydroxypropyloxy)pyridine and 2-(3′-imidazolo-2′-hydroxypropyloxy)pyridine only. Compounds 2-(2′-morpholino-ethanoxy)pyridine, 2-(2′-piperidino-ethanoxy)pyridine, 2-(2′-piperazino-ethanoxy)pyridine, 2-(2′-imidazolo-ethanoxy)pyridine, 2-(2′-diphenylamino-ethanoxy)pyridine, 2-(2′-diethanolamino-ethanoxy)pyridine, 2-(2′-phenylamino-ethanoxy)pyridine and 2-(2′-(4″-hydroxy)phenylamino-ethanoxy)pyridine exhibited significant blocking of histamine induced contraction on guinea pig ileum.
In the present study, a new series of 2,6-diaryl-3-methyl-4-piperidones was synthesized by Mannich reaction (condensation) of ethyl–methyl ketone, substituted aromatic aldehydes and ammonium acetate. Oximes and thiosemicarbazone derivatives of 2,6-diaryl-3-methyl-4-piperidones were synthesized by reaction with hydroxylamine hydrochloride and thiosemicarbazide respectively. The chemical structures were confirmed by means of IR, 1H-, 13C-NMR and mass spectral data. The compounds were screened for acute toxicity, analgesic, local anaesthetic and antifungal activity. 2-(4-Methylphenyl)-3-methyl-6-(4-chlorophenyl)-piperidin-4-one 2 exhibited the highest analgesic and local anaesthetic activity. The oximes and thiosemicarbazones were completely devoid of analgesic and local anaesthetic activity. 2-(4-Methylphenyl)-3-methyl-6-(4-hydroxyphenyl)-piperidin-4-oxime 21 and 2-(4-methoxyphenyl)-3-methyl-6-(4-chlorophenyl)-piperidin-4-oxime 17 exhibited potent antifungal activity against Aspergillus niger. Antifungal activity against Candida albicans was observed only with 2-(4-dimethylaminophenyl)-3-methyl-6-(4-chlorophenyl)-piperidin-4-oxime 20. 2,6-Diaryl-3-methyl-4-piperidones did not exhibit antifungal property.
Wood creosote pills (P4) containing wood creosote and four herbal drugs, Gambir, Phellodendri Cortex, Glycyrrhizae Radix, and Citri Unshiu Pericarpium (CUP), have been used to treat food poisoning and diarrhea through self-medication in Japan. The mean dissolution time (MDT) of guaiacol, one of the active constituents of wood creosote, from P4 (138.3±3.3 min) was significantly longer than that (42.6±4.3 min) from pills (P0) containing only wood creosote. The MDT of the variant pills prepared from P4 without CUP (54.3±12.5 min) was found to be significantly shorter than that of P4. These findings suggest that CUP plays an important role in sustaining the dissolution of guaiacol from P4. The long MDT of guaiacol is considered one of the most important factors affecting the duration of efficacy after oral administration of wood creosote pills. The present findings are considered proof that CUP has been prescribed in traditional as well as new formulations of wood creosote pills.
Four minor components, along with the major cyanogenic glycosides, amygdalin and prunasin, were isolated from Prunus persica seeds (Persicae Semen; Tounin), and characterized as mandelic acid glycosides (β-gentiobioside and β-D-glucoside) and benzyl alcohol glycosides (β-gentiobioside and β-D-glucoside). The anti-tumor promoting activity of these compounds was examined in both in vitro and in vivo assays. All of the compounds significantly inhibited the Epstein-Barr virus early antigen activation induced by tumor promoter. In addition, they produced a delay of two-stage carcinogenesis on mouse skin that was comparable in potency to (−)-epigallocatechin gallate from green tea. Structure–activity relationships indicated that a substituent at the benzylic position with glycosidic linkage affected the in vitro and in vivo activities with an order of enhancing potency, CN<COOH<H.
To elucidate drug interaction between human immunodeficiency virus (HIV) protease inhibitors (PIs), the effect of indinavir (IDV) on the intestinal exsorption of other HIV PIs, amprenavir (APV), saquinavir (SQV) and nelfinavir (NFV) was investigated in rats using an in situ single perfusion method. IDV in the intestinal perfusate inhibited the exsorption of rhodamine 123 (Rho123), a known P-glycoprotein (P-gp) substrate, from blood into intestinal lumen in a concentration-dependent manner, and the inhibitory potency of 10 μM IDV in the perfusate was close to that of 10 μM cyclosporin A (CsA) in the perfusate. Ten μM of IDV in the intestinal perfusate also decreased significantly the exsorption clearance of Rho123 after intravenous administration. The IDV concentration in this system was not likely to cause hepatic interaction between HIV PIs, because the plasma IDV concentration was far below its inhibition constants for other HIV PIs in the liver microsomes. Thus, 10 μM of IDV was chosen to investigate the effect of this inhibition on the exsorption of APV, SQV and NFV. IDV in the intestinal perfusate markedly increased the exsorbed amounts of SQV and NFV but not APV after intravenous administrations. Their exsorption clearances, however, showed only a slight increasing tendency or remained unchanged. These findings suggest that in addition to P-gp inhibition, other factors such as CYP3A inhibition might be important in the drug interaction of IDV with APV, SQV and NFV after intravenous administration in rat small intestine. The results obtained in this study will provide useful information to discuss the interactions among PIs when a double protease therapy is used for in HIV-infected patients.
To examine whether cisplatin affects the multidrug transporter MDR1/P-glycoprotein in the kidneys, the effects of cisplatin on cell sensitivity to an anticancer drug, MDR1 function and expression were examined by assessing the growth inhibition by the MDR1 substrate paclitaxel, the uptake and efflux of the MDR1 substrate Rhodamine123 and the level of MDR1 mRNA, respectively. Porcine kidney epithelial LLC-PK1 cells were used, as they have a structure and function similar to those of renal proximal tubular cells and physiologically express low levels of MDR1. The growth inhibitory curve of LLC-PK1 cells by paclitaxel was shifted to a higher concentration range by pretreatment with 1 μM cisplatin for 48 h. The uptake and efflux of Rhodamine123 were significantly reduced and enhanced, respectively, by pretreatment with 1 μM cisplatin for 48 h. This enhanced efflux was suppressed by the representative MDR1 substrate/inhibitor ciclosporin. The expression of MDR1 mRNA was increased by the existence of cisplatin for 48 h. These observations taken together suggested that the transient exposure to cisplatin could cause the up-regulation of MDR1 in LLC-PK1 cells.
The usefulness of kinetic analysis of pharmacological response data was discussed in investigating the ductus arteriosus dilating effect (DADE) of lipo-PGE1 (a lipid emulsion preparation of prostaglandin E1 for injection) preparations. Lipo-PGE1 was administered intravenously via the umbilical vein by a bolus injection or an infusion in newborn rats 60 min after the delivery. The DADE data were expressed as the inner diameter ratio between the ductus arteriosus and the main pulmonary artery, and were analyzed by a pharmacological response kinetic (PRK) model consisting of an Emax model and a simple pharmacokinetic model as the pharmacodynamic- and the pharmacokinetic-component, respectively. The latter component includes the release process of free-PGE1 from the lipid phase of lipo-PGE1, followed by distribution to the effect compartment. The Emax value was estimated by the maximal DADE observed 10 min after the bolus administration of each dose, and the value was fixed in the PRK analysis. The regression curves given by simultaneous non-linear least squares regression analysis were satisfactorily fitted to the observed DADE data at all doses. Prediction of the DADE of lipo-PGE1 in an infusion study was satisfactorily done using the estimated parameters in the i.v.-study. These findings indicate that PRK modeling based on the intensities of the observed pharmacological response-time data is a meaningful tool in some targeting-type drugs, for which pharmacokinetic analysis itself is meaningless or acquisition of pharmacokinetic data is technically impossible, in predicting the time courses of the drug's pharmacological response in different dosage regimens.
Propofol (2,6-diisopropylphenol), widely used an intravenous anesthetic, is rapidly metabolized to its glucuronide in the in vivo studies. Kinetic parameters for the glucuronidation of propofol and its analogs, such as 2,5-diisopropylphenol, 2-tert-butyl-6-methylphenol, 2-tert-butyl-5-methylphenol, 2,6-dimethylphenol and 2,5-dimethylphenol, were determined in vitro using human and rat liver microsomes. 2,5-Dimethylphenol and 2-tert-butyl-6-methylphenol exhibited the highest and lowest glucuronidation rates, respectively. Substitutes at the 2,6-positions gave lower glucuronidation rates than those at the 2,5-positions in both the human and rat microsomes. 2,5-Diisopropylphenol was glucuronidated at a lower rate in human than propofol. The affinity of uridine 5′-diphosphate (UDP)-glucuronosyltransferase for disubstituted phenols, such as propofol, 2,5-diisopropylphenol, 2,5-dimethylphenol, and 2-tert-butyl-6-methylphenol, gave higher Km values in human liver microsomes than in rat ones, and lower Vmax values showed similar relationship, expect for Vmax in propofol. The alkyl group at the 6 position showed a higher Km for glucuronidation by a steric hindrance in the human and rat microsomes. Our results propose that the glucuronidation of propofol and its analogs may not be explained by only a steric hindrance.
In this study, two terpenes with the same functional group; limonene oxide and pinene oxide were used at 5% w/v concentration in 50% v/v ethanol and 100% v/v propylene glycol (PG) to enhance the in vitro permeation of haloperidol (HP) through the human epidermis (or stratum corneum, SC). The enhancement mechanism of terpenes from both solvents was elucidated with HP-SC binding studies, Fourier transform infrared spectroscopy and differential scanning calorimetry. The enhancement activity of these terpenes was higher in 50% v/v ethanol than in 100% v/v PG. These terpenes in 50% v/v ethanol were predicted to provide the required therapeutic plasma concentration and daily-permeated amounts of the drug. Limonene oxide showed higher enhancement in both solvents, which was attributed to its less bulky structure. The terpenes in both solvents did not increase the partition of HP. Instrumental studies showed that these terpenes in 50% v/v ethanol extracted the SC lipids, disrupted the bilayer packing and partially fluidised the lipids. Limonene oxide in 100% v/v PG possibly disrupted the lipid bilayer, whilst leaving the overall bilayer structure intact and pinene oxide in the same vehicle fluidised the lipids within the ordered environment. This study showed that the mode of interactions of terpenes with SC were different in two solvent systems.
Several nutrients and drugs, which are known to be absorbed by specific carrier-mediated transport systems in the small intestine, had their transport investigated in the rat colon, by measuring uptake into everted sacs, to find if carrier-mediated transport systems may also be present in the colon. Among those transported by Na+-dependent carriers in the small intestine, D-glucose and taurocholate were found to be transported in an Na+-dependent manner in the colon, while 5-fluorouracil and ascorbate were not. It was also found that the colonic transports of D-glucose and taurocholate were saturable. These results suggest the presence in the colon of Na+-dependent carrier-mediated transport systems for D-glucose and taurocholate, but not for 5-fluorouracil and ascorbate. For nicotinate and methotrexate, which are transported by H+-dependent carriers in the small intestine, their transport was elevated at a lower pH (5.0) than the pH 7.4 in the colon, but not saturable. Therefore, the elevated transport of these acidic compounds may be explained by an increase in passive flux due to an increase in the fraction of the unionized and/or neutral forms, without postulating the presence of H+-dependent carrier-mediated transport systems in the colon. The transport activity of the suggested colonic transport systems for D-glucose and taurocholate was much lower than those of their respective counterparts in the small intestine. However, it may be possible to use them for oral drug delivery via the colon. Their physiological roles would also be of interest.
In this paper, the effects of H2O2, a typical reactive oxygen species (ROS), on cell proliferation or death were examined using the human cervical carcinoma cell line HeLa and its MDR1-overexpressing subline, Hvr100-6, which was established by stepwise exposure to vinblastine. It was confirmed that the growth of HeLa cells was enhanced by H2O2 at relatively low concentrations in a concentration-dependent manner, and the growth enhancement was suppressed by antioxidants. Doxorubicin and daunorubicin also enhanced the growth of HeLa cells at concentrations lower than IC50 values, and the antioxidants suppressed this effect, being consistent with the fact that both anticancer drugs generate ROS. The growth enhancement by H2O2 or doxorubicin and daunorubicin was not observed in Hvr100-6 cells. In addition, it was suggested that antioxidants had no effect on MDR1 mRNA expression in HeLa and Hvr100-6 cells, and thereby hardly reverse multidrug resistance in tumor cells.
We report that the synthetic peptide Prp106—126 (KTNMKHMAGAAAAGAVVGGLG-COOH) and the reversed peptide Prp126—106 (GLGGVVAGAAAAGAMHKMNTK-COOH) of human prion (hPrp) can express the decarboxylase activity for oxaloacetate in the presence of trifluoroethanol, similar to that of Oxaldie 1 (LAKLLKALAKLLKK-CONH2) reported previously. The degree of the relative activity of Prp106—126 and Prp126—106 to Oxaldie 1 is 0.47 and 0.21, respectively. Based on this experimental result, we applied the informational system method (ISM) developed by Veljkovic et al. to the amino acid sequence of Prp106—126 and Prp126—106 to extract a common factor. The same spectra were obtained, indicating that the same periodicity may be conserved on their sequences, as a necessary factor for expressing the same biological activity, irrespective of the orientation of the primary sequence.
Candida albicans is a medically important fungus which induces a disseminated candidasis and candidemia in immunocompromised hosts, and releases a polysaccharide fraction into the blood. We recently found that C. albicans released a water-soluble polysaccharide fraction (CAWS) into synthetic medium and demonstrated that CAWS was mainly composed of a complex of mannan and β-glucan. In the murine system, CAWS showed a lethality resembling anaphylactic shock when administered i.v., and induced coronary arteritis similar to Kawasaki Disease (KD) when given i.p. In the present study, we examined the biological activity of CAWS in the cell culture and found the following: i) CAWS slightly induced production of IFN-γ and IL-6 by splenocytes at lower dose (ca. 10 μg/ml), but at a higher dose strongly inhibited the proliferation of splenocytes induced by a B cell mitogen, lipopolysaccharide (LPS) and a T cell mitogen, concanavalin A. ii) The viability of these splenocytes monitored by propidium iodide staining was significantly reduced. iii) The addition of CAWS to a culture of monophage RAW264.7 cells significantly reduced cellular growth rate dose dependently. iv) The LPS-mediated synthesis of cytokines by RAW264.7 cells was significantly inhibited by CAWS. v) CAWS induced an aggregation of platelets in human platelet-rich plasma, and vi) CAWS inhibited the production of thrombomodulin by human umbilical endothelial cells and acted synergistically with TNF-α. Thus, CAWS strongly inhibited the cellular functions of leukocytes in vitro, partly through direct cytotoxicity. The enhanced production in injured cells of the vascular endothelium would be related to the local inflammatory response in the coronary artery.
The effects of Bak Foong Pill (BFP, also known as Bai Feng Wan), a preparation of crude drugs in wide clinical use for treatment of gynecological disorders, on blood coagulation and platelet aggregation were investigated. The anticoagulant effect of BFP was evaluated by using thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (APTT) assays. Results showed that BFP 70% ethanol extract (BFP-E-ext) significantly prolonged the TT in a dose-dependent manner with values of 17.6, 38.3, and 50.4 s at concentrations of 4.0, 6.0, and 12.0 mg/ml, respectively. Whereas, the BFP-E-ext did not show significant prolonging effect in PT and APTT assays. The results suggest that the anticoagulant effect of BFP is mediated by directly blocking thrombin, the key enzyme in the blood coagulation cascade. BFP-E-ext significantly inhibited platelet aggregation induced by collagen and adenosine diphosphate (ADP) with inhibition percentages of 74 and 52% at a concentration of 6.0 mg/ml, respectively, whereas, it exhibited a weak inhibitory activity on platelet aggregation induced by archidonic acid (AA). Comparing to BFP-E-ext, the effects of BFP aqueous extract (BFP-W-ext) on both anticoagulant and antiplatelet activities were significantly less potent. Moreover, the effects of the 26 ingredients of BFP on blood coagulation and platelet aggregation were separately evaluated with 19 ingredient herbs exhibiting anticoagulant effect and 10 exhibiting antiplatelet effect. The anticoagulant and antiplatelet effects of BFP were collectively demonstrated by in vivo assays showing prolonged bleeding times after BFP treatment for two weeks. The results of the present studies may provide explanations for beneficial effects of BFP on the circulation and indicate its potential use for cardiovascular diseases.
The protective effect of Baicalin, a flavonoid isolated from the root of Scutellaria baicalensis G., on H2O2-induced DNA single strand break (SSB) was examined in NIH3T3 mouse fibroblasts by Comet assay (single cell gel electrophoresis technique). When the cells were pulse-chased with H2O2 (0.1—0.5 mM) for 15 min in fetal bovine serum (FBS)-free Dulbecco's Modified Eagle's Medium (DMEM), SSB occurred in the DNA as reported elsewhere in dose-dependent manner. Baicalin (50, 100 μM) which was incubated with the cells for 24 h before the H2O2 chase did not give rise to significant protection against the SSB formation. However, when the time required to cause a change in the DNA damage histogram obtained by the Comet assay was precisely examined after the H2O2 chase, it was found that the H2O2 induced SSB was more promptly repaired in the cells pretreated with Baicalin prior to the H2O2 chase, compared to untreated control cells. At the same time, the cell viability examined by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide) (MTT) assay after the H2O2 abuse was moderately recovered in the Baicalin increased by the Baicalin treatment. It was thus concluded that Baicalin that was known as an antioxidant flavonoid in vitro also functions as a biological response modifier, improving the cellular repair potential of oxidatively damaged DNA.