Biopanning of a phage library using a Western blotting membrane is difficult because of high background binding. We propose a reliable biopanning method, namely, immunogel-biopanning, which is performed using immunoplates coated with a molecular species fractionated from a crude sample by native polyacrylamide gel electrophoresis (PAGE). The efficacy of this method was determined in model experiments using a human interleukin-18 (IL-18)-specific single chain Fv (scFv) phage clone.
The bioavailability of chondrosine was evaluated by its direct measurement as found in the blood plasma following removal of plasma proteins by perchloric acid. The postcolumn HPLC determination of chondrosine was performed on an SCX column (6 mm i.d.×150 mm), 0.35 mol/l boric acid (pH 5.2 adjusted by 0.1 mol/l NaOH) as an eluent (0.9 ml/min), 0.5% 2-cyanoacetamide and 1.0 M NaOH as fluorogenic reagents (0.25ml/min each) with a fluorescence detector (ex. 331nm, em. 383nm). Two separate animal studies were conducted. In study 1, adult male ddY mice (n=6) received i.v. chondrosine (1.0mg/kg body weight) and the plasma samples were collected. In the second study, 6 adult male ddY mice received p.o. chondrosine (400mg/kg body weight) and the plasma samples were collected. Blood plasma samples were deproteinized by perchloric acid, analyzed and the bioavailability of chondrosine was determined. Twenty five to fifty microliters of blood plasma were required for the assay. Chondrosine was absorbed after oral administration with two phases having two maximum values, 7.8±5.4 and 4.0±1.9 at 15μg/ml and 120min, respectively; it disappeared from the blood flow very quickly after intravenous administration. This study provides the first report of the bioavailability of orally administered chondrosine in mice.
Advanced glycation endproducts (AGEs) are believed to be secondary factors in the selective neuronal injury associated with several neurodegenerative disorders. In this study, we investigated the protective effects of (−)-epigallocatechin gallate (EGCG), a major monomer of green tea polyphenols, against AGEs-induced damage in neuron cells. The results showed that EGCG treatment protected against glyceraldehyde-derived AGE-induced neurotoxicity, which is associated with an increase in intracellular reactive oxygen species, as well as against decreases in intracellular catalase (CAT) and superoxide dismutase (SOD) activities. EGCG treatment also decreased malondialdehyde and carbonyl levels, and AGEs formation. Treatment with 10 μM EGCG upregulated SOD and CAT levels, whereas glutathione peroxidase activity was reduced. Furthermore, 5 μM EGCG was found to down-regulate the mRNA level of the AGE receptor (RAGE) in neuronal cells up to 2.5 fold, as determined by real time PCR. The results demonstrated that EGCG may exhibit protective effects against AGEs-induced injury in neuronal cells through its antioxidative properties, as well as by interfering with AGEs-RAGE interaction mediated pathways, suggesting a beneficial role for this tea catechin against neurodegenerative diseases.
(1R,9S)-β-Hydrastine (BHS) decreases the basal intracellular Ca2+ concentration ([Ca2+]i) in PC12 cells.5) This study examined the effects of (1R,9S)-BHS on [Ca2+]i in PC12 cells. (1R,9S)-BHS at 10—100 μM in combination with a high extracellular K+ level (56 mM) inhibited the release of dopamine in a concentration-dependent manner with an IC50 value of 66.5 μM. BHS at 100 μM inhibited the sustained increase in [Ca2+]i induced by a high K+ level (56 mM), and had an inhibitory effect on the 2 μM nifedipine-induced blockage of the K+-stimulated sustained increase in [Ca2+]i. In addition, (1R,9S)-BHS at 100 μM prevented the rapid and sustained increase in [Ca2+]i elicited by 20 mM caffeine, but did not have an effect on the increase induced by 1 μM thapsigargin, in the presence of external Ca2+. These results suggest that the active sites of (1R,9S)-BHS are mainly L-type Ca2+ channels and caffeine-sensitive Ca2+-permeable channels in PC12 cells.
The human Rad51 protein (HsRad51) catalyzes homologous pairing and strand exchange between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) during recombinational repair of double-stranded DNA breaks. An HsRad51 mutation that results in the substitution of Gln for Arg150 (R150Q) was found in bilateral breast cancer patients; however, the consequences of this R150Q mutation have not been elucidated. To determine how this HsRad51(R150Q) mutation affects HsRad51 function, in the present study, we purified the HsRad51(R150Q) mutant. The purified HsRad51(R150Q) was completely proficient in the ATP-hydrolyzing activity. A gel filtration analysis revealed that HsRad51(R150Q) also retained the polymer formation ability. In contrast, the ssDNA- and dsDNA-binding abilities of HsRad51(R150Q) were clearly reduced, as compared to those of HsRad51. These differences in the DNA-binding properties between HsRad51(R150Q) and HsRad51 may be important to account for the tumorigenesis in breast cancer patients with the HsRad51(R150Q) mutation.
A DNA-damaging agent, bleomycin, arrests the cell cycle at the G2 phase of Jurkat cells, which are defective in the G1 checkpoint, while microtubule-disrupting colchicine arrests it at M phase. Fungal cyclopeptides, malformin A1 and malformin C, were found to abrogate bleomycin-induced G2 arrest (IC50; 0.48 μM and 0.9 nM, respectively), resulting in a drastic decrease in cells in G2 phase and increase in cells in subG1 phase. On the other hand, malformins showed little effect on the colchicine-induced M phase arrest in Jurkat cells (IC50; 2.7 μM and 24 nM, respectively). Malformin C (0.026 μM) also abrogated bleomycin-induced G2 arrest in colon cancer-derived HCT-116 cells. These data strongly suggest that malformin C disrupted the cell cycle at the G2 checkpoint of cancer cells, leading to sensitization of the cancer cells to the anti-cancer reagent.
Schizophyllan (SPG) is used to treat cervical cancer in combination with irradiation to enhance the immunological surveillance system. Dectin-1 is a cell surface receptor for 1,3-β-glucan. In this study, we prepared two anti-Dectin-1 monoclonal antibodies, 4B2 and SC30 having a KD of 7.04×10−8 M and 1.55×10−7 M, respectively, and evaluated the role of Dectin-1 in SPG-induced anti-tumor activity in mice. Expression of Dectin-1 on peritoneal macrophages and binding of SPG to the cells were decreased by administration of 4B2 and SC30. SPG-mediated anti-tumor activity was inhibited by 4B2 and SC30. 4B2 and SC30 inhibited the binding of SPG to splenocytes from mice. The binding of SPG-biotin to Dectin-1-transfected HEK293 cells was inhibited by 4B2, but not SC30. 4B2 and SC30 differ in their influence on Dectin-1 between primary cells and transduced cells, and Dectin-1 effects 1,3-β-glucan-mediated anti-tumor activity in mice by binding to SPG.
Previously, we have reported that the exposure of PC12 cells to the aluminum–maltolate complex (Al(maltol)3) results in decreased cell viability via the apoptotic cell death pathway. In this study, we have used several nitric oxide synthase (NOS) inhibitors and the NO generator diethylenetriamine NONOate (DETA NONOate) to examine whether or not intracellular nitric oxide (NO) generation is involved in the onset mechanism of Al(maltol)3-induced cell death. Cell viability was assessed by measuring lactate dehydrogenase (LDH) release and caspase-3 activity. Treatment of the cells with 150 μM Al(maltol)3 for 48 h resulted in intracellular NO generation. Exposure of the cells to DETA NONOate also induced a marked decrease in cell viability. Pre-treatment of the cells with a general NOS inhibitor or with a selective inducible NOS (iNOS) inhibitor effectively prevented Al(maltol)3-induced cell death. However, a neuronal NOS (nNOS) inhibitor did not exhibit any protective effect against Al(maltol)3-induced cell death. In addition, ascorbic acid markedly inhibited Al(maltol)3- and DETA NONOate-induced cell death. Based on these results, we discussed the involvement of intracellular NO generation in the onset mechanisms of Al(maltol)3-induced cell death.
Alpha-ketoglutarate is a key intermediate in the Krebs cycle, and a rate-limiting cofactor of prolyl-4-hydroxylase. It also has a potent effect on increasing the proline pool during collagen production, but the details underlying the boosting effect on collagen production by α-ketoglutarate remain as yet unreported. To investigate the effects of α-ketoglutarate on procollagen production and wrinkle formation, we conducted experiments in cultured human dermal fibroblasts and UVB-irradiated hairless mice. Based on ELISA measurements, α-ketoglutarate (10 μM) stimulated procollagen production in fibroblasts by 25.6±4.6% compared to vehicle (dH2O)-treated control cells. Also, we demonstrated that α-ketoglutarate increased activities of prolidase, which is known to play an important role in collagen metabolism, in fibroblasts and N-benzyloxycarbonyl-L-proline (Cbz-Pro), prolidase inhibitor, inhibited procollagen synthesis by α-ketoglutarate in fibroblasts. To determine the effect of topically applied α-ketoglutarate on wrinkle formation, α-ketoglutarate (1%) and vehicle (70% propylene glycol, 30% ethanol) were applied on the dorsal skin of UVB-induced hairless mice for twelve weeks. We found that α-ketoglutarate decreased wrinkle formation upon long-term topical application. These results suggest that α-ketoglutarate diminishes UVB-induced wrinkle formation by increasing collagen production, through a pathway that involves prolidase activation. Therefore, application of α-ketoglutarate may represent an effective anti-wrinkle agent for the cosmetic field.
Irinotecan, a DNA topoisomerase I inhibitor, is widely used in cancer chemotherapy. However, little is known of the mechanisms of its antitumor effects and the development of drug resistance in human hepatocellular carcinoma (HCC). In this study, we investigated the effects of short-term culture with SN-38, the active metabolite of irinotecan, on apoptosis in Huh7 cells. The cells were cultured with SN-38 for 24, 72, and 120 h, and apoptosis was determined using the terminal dUTP nick-end labeling (TUNEL) assay. The expressions of p53, apoptosis-related proteins, and P-glycoprotein (P-gp), a protein conferring the multidrug-resistant phenotype, were analyzed using Western blotting. Induced expression of P-gp was detected using fluorescence microscopy. SN-38 significantly induced apoptosis in Huh7 cells at 24 h. SN-38 also increased the expression of p53, Bax, and caspase-9 and decreased Bcl-xL expression in Huh7 cells. SN-38 decreased p53 expression and increased P-gp expression after 120 h, resulting in inhibition of apoptosis. This inhibition was reversed by the addition of verapamil to the culture medium during 120 h incubation. SN-38-induced P-gp expression was additionally enhanced by p53 decoy oligodeoxynucleotide. The changes in P-gp expression were directly moderated by p53 gene downregulation, suggesting that it plays a role in the mechanism of drug resistance. These results suggest that the accumulation of irinotecan in HCC leads to the development of drug resistance.
The present study investigated the segmental discrepancy of the rat distal colonic anion transport induced by luminal forskolin and the possible underlying mechanisms using short-circuit current recording technique and comparative quantity real-time PCR analysis. Forskolin-induced ISC in the segment next to lymph node (DC1) and the segment 4 cm away from lymph node (DC4) were 4.09±0.66 μA/cm2 and 18.84±3.18 μA/cm2 (n=13), respectively, which were blocked by luminal Cl− channel blocker, glybenclamide (1 mM) (n=5, p<0.01), as well as removal of extracellular Cl− and HCO3− in both DC1 and DC4 (n=5, p<0.001). Furthermore luminal pretreatment with K+ blockers, TEA (5 mM) and glybenclamide (100 μM) didn't affect forskolin and bumetanide-enhanced ISC. Reducing serosal Cl− concentration increased forskolin-induced ISC by 90% in DC1 but decreased forskolin-induced ISC in DC4 by 50%. Furthermore, pretreatment with serosal bumetanide, an inhibitor of Na+-K+-2Cl− cotransporter, enhanced forskolin-induced ISC by 87% in DC1, from 4.09±0.66 μA/cm2 to 7.65±0.53 μA/cm2 (n=6, p<0.01), but inhibited forskolin-induced ISC by 50% in DC4, from 29.19±4.51 μA/cm2 to 15.06±4.10 μA/cm2 (n=6, p<0.05). Pretreatment with luminal amiloride (10 μM), an inhibitor of ENaC, and serosal 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) (200 μM), an inhibitor of NBC, significantly inhibited the forskolin-induced ISC in DC1 (n=6, p<0.05), but not in DC4. Real-time PCR analysis did not show the significant differences between the two segments in the expression amounts of CFTR and NKCC mRNAs, however the expression of NBC mRNA in DC4 was significantly higher than that in DC1. In conclusion, the rat distal colon manifests a segmental discrepancy in anion transport stimulated by luminal forskolin. HCO3− might be predominantly involved in the forskolin-induced anion secretion in DC1, but Cl− might be the main component of the anion secretion in DC4.
The plasmid-borne qacA and qacB genes encode a multidrug efflux protein. The proteins encoded by qacA and qacB mediate efflux of cationic antiseptic agents such as quaternary ammonium compounds. In methicillin-resistant Staphylococcus aureus (MRSA), qacA and qacB are widely prevalent and decrease antiseptic susceptibility. However, it is difficult to find the plasmids encoding qacA or qacB in community-associated MRSA (C-MRSA) isolated from patients with impetigo. Most MRSA, the strains causative of impetigo, carry the plasmid-borne exfoliative toxin-producing gene etb. To find the reason for the paucity of qacA or qacB in MRSA isolated from patients with impetigo, we performed transfer experiments of the plasmid pTZ2162qacB encoding qacB. The pTZ2162qacB was transferred to S. aureus strain RN4220 by transduction, although no pTZ2162qacB was transferred by conjugation. Additionally, pTZ2162qacB was transduced to MRSA carrying etb, and was coexistence with the plasmid encoding etb. Our results showed that pTZ2162qacB was horizontally transferred by transduction and was compatible with the plasmid encoding etb. Consequently, there will be risk of the emergence of C-MRSA with decreased antiseptic susceptibility among patients with impetigo.
Pharmacological studies with an aqueous extract obtained from leaves of Capraria biflora showed potent cytotoxic, analgesic, antimicrobial and anti-inflammatory activities. It has been demonstrated that biflorin possesses an in vitro cytotoxic activity against tumor cells. The in vivo antitumor activity of biflorin was evaluated on two mouse models, sarcoma 180 and Ehrlich carcinoma. Biflorin was active against both tumors with a very similar profile. In addition, biflorin was also able to increase the response elicited by 5-FU in mice inoculated with both tumors. The results showed a decrease in Ki67 staining in tumor cells from treated-animals when compared with non-treated groups, which suggests an inhibition of tumor proliferation rate. Histopathological analysis from kidneys and liver showed that biflorin possessed weak and reversible toxic effects. It was also demonstrated that biflorin acts as an immunoadjuvant agent, rising the production of ovalbumin-specific antibodies and inducing a discreet increase of the white pulp and nest of megakaryocytic in spleen of treated mice, which can be related to its antitumor properties.
Soyo-san is a traditional oriental medicinal formula, a mixture of 9 crude drugs, and it has been clinically used for treating mild depressive disorders. The purpose of the study was to examine the effect of Soyo-san on repeated stress-induced alterations of learning and memory on a Morris water maze (MWM) task and also the anxiety-related behavior on the elevated pulse maze (EPM) in ovariectomized female rats. We assessed the changes in the reactivity of the cholinergic system by measuring the immunoreactive neurons of choline acetyltransferase (ChAT) and reactivity of acetylcholinesterase (AChE) in the hippocampus, and the serum levels of corticosterone were assessed after behavioral testing. The female rats were randomly divided into three groups: the nonoperated and nonstressed group (normal), the ovariectomized and stressed group (control), and the ovariectomized, stressed and Soyo-san treated group (SOY). The rats were exposed to immobilization stress (IMO) for 14 d (2 h/d), and Soyo-san (400 mg/kg, i.p.) was administered 30 min before IMO stress. Treatments with SOY caused significant reversals of the stress-induced deficits in learning and memory on a spatial memory task, and it also produced an anxiolytic-like effect on the EPM, and increased the ChAT and AChE reactivities (p<0.05, respectively). The serum level of corticosterone in the SOY group was significantly lower than that in the control group (p<0.05). These results suggest that Soyo-san might prove to be an effective antidepressant agent.
We investigated the protective effect of Cuscutae semen (CS) on acute liver injury induced by dimethylnitrosamine (DMN) in Sprague-Dawley rats. CS is an important traditional herbal medicine widely used as a tonic and aphrodisiac to nourish the liver and kidney and to treat impotence and seminal emission. Rats were given a single intraperitoneal injection of DMN (40 mg/kg), and were then treated with CS daily by oral gavage for 4 d. Immunohistochemical studies for alpha-smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) were performed, along with hydroxyproline and biological assay. Liver injury caused by DMN-injection was significantly inhibited in the CS-treated group compared to the silymarin-treated group. The results of blood biological assay were significantly protected by CS in serum total protein (T-protein), T-bilirubin (T-bili), D-bilirubin (D-bili), GOT, GPT, and ALP. The hydroxyproline content and amount of active α-SMA and PCNA were significantly decreased in the CS-treated group than in the silymarin-treated group. CS exhibited an in vivo hepatoprotective effect and anti-fibrogenic effects against DMN-induced acute liver injury and inhibited the formation of hydroxyproline, which suggests that CS may be useful in preventing fibrogenesis after liver injury.
Agaricus blazei is a medicinal mushroom that possesses antimetastatic, antitumor, antimutagenic, and immunostimulating effects. However, the molecular mechanisms involved in A. blazei-mediated apoptosis remain unclear. In the present study, to elucidate the role of the Bcl-2 in A. blazei-mediated apoptosis, U937 cells were transfected with either empty vector (U937/vec) or vector containing cDNA encoding full-length Bcl-2 (U937/Bcl-2). As compared with U937/vec, U937/Bcl-2 cells exhibited a 4-fold greater expression of Bcl-2. Treatment of U937/vec with 1.0—4.0 mg/ml of A. blazei extract (ABE) for 24 h resulted in a significant induction of morphologic features indicative of apoptosis. In contrast, U937/Bcl-2 exposed to the same ABE treatment only exhibited a slight induction of apoptotic features. ABE-induced apoptosis was accompanied by downregulation of antiapoptotic proteins such as X-linked inhibitor of apoptosis protein (XIAP), inhibitor of apoptosis protein (cIAP)-2 and Bcl-2, activation of caspase-3, and cleavage of poly(ADP-ribose)polymerase (PARP). Ectopic expression of Bcl-2 was associated with significantly induced expression of antiapoptotic proteins, such as cIAP-2 and Bcl-2, but not XIAP. Ectopic expression of Bcl-2 also reduced caspase-3 activation and PARP cleavage in ABE treated U937 cells. Furthermore, treatment with the caspase-3 inhibitor z-DEVD-fmk was sufficient to restore cell viability following ABE treatment. This increase in viability was ascribed to downregulation of caspase-3 and blockage of PARP and PLC-γ cleavage. ABE also triggered the downregulation of Akt, and combined treatment with LY294002 (an inhibitor of Akt) significantly decreased cell viability. The results indicated that major regulators of ABE-induced apoptosis in human leukemic U937 cells are Bcl-2 and caspase-3, which are associated with dephosphorylation of the Akt signal pathway.
The therapeutic value of an antirheumatic alkaloid, sinomenine (SIN), was investigated in the acute experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). SIN is a bioactive alkaloid derived from the Chinese medicinal plant, Sinomenium acutum REHDER & E. H. WILSON (Family Menispermaceae). Chinese doctors have utilized this plant to treat rheumatic and arthritic diseases for over one thousand years. Experiments in which EAE-induced Lewis rats exhibit an acute monophasic episode of disease demonstrated that SIN is effective in preventing clinical signs of disease. The therapeutic effect on disease activity was observed at preonset administration times and at various doses tested. Consistent with disease activity in vivo, SIN-treated animals have reduced cellular infiltration within the spinal cord along with decreased TNF-α and IFN-γ expression levels. SIN can significantly inhibit proliferation response of splenocytes induced by MBP68—82. TNF-α and IFN-γ, secreted by splenocytes induced by MBP68—82 are inhibited by SIN by dose-dependence manner. The mRNA levels of CC chemokines, RANTES, MIP-1α and MCP-1, are inhibited in SIN-treated EAE rats. The data in this proof of concept study support the premise that SIN may be a promising new therapeutic intervention in MS.
To evaluate the pharmacological characteristics of SA12590, a new oxime-derivative of the ethacrynic acid (ECA) derivative SA9000, we examined both its ocular hypotensive effects (in ocular normotensive cats and cynomolgus monkeys) and its potential corneal toxicity (in rats). A 50 μl topical administration of 3% SA12590 significantly reduced intraocular pressure (IOP) (by 3.5 mmHg) in anesthetized cats (p<0.05). Twenty-four hours after 3 drops (5-min intervals) of 20 μl 3% SA12590, IOP was reduced by 8 mmHg (p<0.05, n=4) in conscious monkeys without evidence of corneal toxicity. Three days' daily single 20 μl dosing with 3% SA12590 reduced IOP by 4 mmHg (p<0.01, n=3) at 72 h after the first administration in conscious monkeys. The toxicity of topically administered 20 μl 3% SA9000 or SA12590 (3 drops with 5-min intervals) on rat corneal epithelium was assessed using a photo-slit lamp. In this study, 3% SA12590, unlike 3% SA9000, exhibited no corneal toxicity. In a glutathione assay for sulfhydryl (SH) reactivity, SA12590, unlike SA9000, displayed no in vitro SH reactivity. Thus, oxime-modification may both improve efficacy towards IOP upon topical administration and improve the safety profile, probably by enhancing corneal penetration and minimizing SH reactivity-related toxicity. These findings indicate that SA12590 has potential as a new ocular hypotensive drug.
Chalcones belong to the flavonoid family from plant origin and some of them possess anti-inflammatory activity. Recently, several natural and synthetic chalcone derivatives were reported to inhibit inducible nitric oxide synthase (iNOS)-catalyzed NO production in cell cultures. In the present study, to find the optimal chemical structures and to elucidate their action mechanisms, 41 synthetic chalcones having the substituent(s) on A- and B-rings were prepared and their effects on iNOS-catalyzed NO production were evaluated using lipopolysaccharide (LPS)-treated RAW 264.7 cells. When simultaneously added with LPS, 2′-methoxy-3,4-dichlorochalcone (Ch15), 2′-hydroxy-6′-methoxychalcone (Ch29), 2′-hydroxy-3-bromo-6′-methoxychalcone (Ch31) and 2′-hydroxy-4′,6′-dimethoxychalcone (Ch35) among the tested compounds potently inhibited NO production (IC50s, 7.1—9.6 μM). The favorable chemical structures were found to be a methoxyl substitution in A-ring at an adjacent position (2′ or 6′) to carbonyl moiety with/without 2′-(or 6′-)hydroxyl group and 3-halogen substitution in B-ring. When the cellular action mechanisms of Ch15, Ch31 and Ch35 were further examined using Western blotting and electrophoretic mobility shift assay, it was revealed that Ch15 and Ch31 clearly down-regulated iNOS expression while Ch35 did not. Moreover, Ch15 and Ch31 were proved to suppress the nuclear transcription factor-κB activation. From the results, it is suggested that certain chalcone derivatives potently inhibit iNOS-catalyzed NO production by the different cellular mechanisms, iNOS down-regulation and/or iNOS inhibition, depending on their chemical structures. These chalcone derivatives may possibly be used as lead compounds for developing new anti-inflammatory agents.
Pharmacokinetics and pharmacodynamics of novel oral sustained-release granules based on swelling polymer incorporation layer system (SPILA granules) containing morphine hydrochloride was evaluated. SPILA granules were designed to release morphine faster in neutral environment than in acidic one to keep higher plasma levels over a protracted period, especially after 12 h post dose. SPILA granules were orally administered to beagle dogs to compare the pharmacokinetics with commercially available twice-a-day dosage form, MS Contin®. Tmax and AUC0—24 h values of SPILA granules were 6 h and 191 μg·h/ml, respectively. Tmax and AUC0—24 h values of MS Contin® were 2 h and 146 μg·h/ml, respectively. Relative bioavailability following SPILA granules administration to twice-a-day MS Contin® (30 mg) administration was 131%. In rats, analgesic effect was evaluated over 24 h. SPILA granules and aqueous solutions were administered to rats to compare the analgesic effect. AUC0—24 h value for SPILA granules was 8.88 μg·h/ml, which was a little lower than that for the aqueous solution (10.1 μg·h/ml), whereas the analgesic effect after SPILA granules once-a-day administration expressed as AURC (1701% Analgesia·h) was similar to that after the aqueous solution 4 times-a-day administration (1603% Analgesia·h). These results indicate that SPILA granules based on the pH-dependent release regulating polymer system can be a good candidate for an oral once-a-day sustained-release dosage form.
In the course of the investigation of effects of newly synthesized matrix metalloproteinase inhibitors (MMPIs), FYK-1388, FYK-1352 and F61-1008, which have strong and broad matrix metalloproteinase (MMP) inhibitory activity, on wound healing in streptozotocin (STZ)-induced diabetic rats, strong reducing effects on serum triacylglycerol (TG) have been found. Namely, when examined using breaking wound strength as an index, MMPIs did not significantly facilitate wound healing in STZ-induced diabetic rats. Unexpectedly, however, the treatment of STZ-induced diabetic rats with MMPIs markedly lowered the serum level of TG without changing the blood glucose level. Among these compounds tested, FYK-1388 was the most effective, and the compound reduced serum concentrations of TG and cholesterol and levels of very low-density lipoprotein (VLDL)-TG and low density lipoprotein (LDL)-cholesterol in a dose-dependent manner. FYK-1388 did not affect serum levels of free fatty acids, high-density lipoprotein (HDL)-cholesterol, aspartate aminotransferase and alanine aminotransferase, mass of body fat, liver weights, and hepatic contents of TG and cholesterol. Moreover, treatment of Zucker fa/fa rats with FYK-1388 lowered serum levels of TG and cholesterol without changing blood levels of glucose and insulin. Since the structures of these MMPIs markedly differ from those of the hypotriglyceridemic drugs that are used clinically, it seems plausible that these MMPIs could be used as a new type of hypotriglyceridemic drug.
Epicutaneously administered chemical antigens like 2,4-dinitrofluorobenzene (DNFB), evoke an atopic dermatitis (AD)-like dermatitis reaction in NC/Nga mice under specific pathogen free (SPF) conditions. Astragalus membranaceus (AM), is a popular herbal medicine used to treat allergic diseases in East Asia. In the present study, we examined whether AM suppress AD-like skin lesions in NC/Nga mice treated with DNFB under SPF conditions. Oral administration of AM to DNFB-treated NC/Nga mice was found to inhibit ear thickness increases and the skin lesions induced by DNFB. Moreover, IFN-γ production by CD4+ T cells from the lymph nodes of DNFB-treated NC/Nga mice was significantly inhibited by AM treatment, although levels of IL-4 and total IgE in serum were not. Study findings suggest that AM may suppress the development of AD-like dermatitis in DNFB-treated NC/Nga mice by reducing IFN-γ production.
We determined the DNA sequences of the internal transcribed spacer 1 and 2 (ITS 1 and 2), the 5.8S rRNA gene and most of the 28S rRNA gene of Poria cocos for the first time, and conducted analysis of 20 samples including cultured mycelias and crude drug materials obtained from various localities and markets. Direct sequencing of the ITS 1 and 2 regions of the samples, except for four wild samples, showed that they had identical DNA sequences for ITS 1 and 2 with nucleotide lengths of 997 bps and 460 bps, respectively. By cloning, the four wild samples were found to have combined sequences of common ITS sequences with 1 or 2-base-pair insertions. Altogether both ITS 1 and 2 sequences were substantially longer than those of other fungal crude drugs such as Ganoderma lucidum and Polyporus umbellatus. Thus, Poria cocos could be distinguished from these crude drugs and fakes by comparing the nucleotide length of PCR products of ITS 1 and 2. Contrary to the basic homogeneity in ITS 1 and 2, three types (Group 1, 2, 3) of the 28S rRNA gene with distinctive differences in length and sequence were found. Furthermore, Group 1 could be divided into three subgroups depending on differences at nucleotide position 690. Products with different types of 28S rRNA gene were found in crude drugs from Yunnan and Anhui Provinces as well as the Korean Peninsula, suggesting that the locality of the crude drugs does not guarantee genetic uniformity. The result of DNA typing of Poria cocos may help discrimination of the quality of the crude drug by genotype.
The previous data indicated that the testosterone (Tes)-induced relaxation of thoracic aorta is greater in spontaneously hypertensive rats (SHR) than in normotensive rats (Wistar-Kyoto rats; WKY) and that there were differences between SHR and WKY in the functions of KATP, Kv, and KCa channels. The present study was carried out to ascertain the mechanisms of the Tes-induced relaxation. Indomethacin (30 μM) pretreatment suppressed the Tes-induced relaxation. Following noradrenalin (NA)-induced vasoconstriction, the relaxation induced by Tes was significantly attenuated by endothelium removal in SHR (not in WKY), but the dilatory effect of Tes following KCl-induced vasoconstriction was not attenuated by endothelium removal. After tetraethylammonium (KCa channel inhibitor) or iberiotoxin (large conductance, Ca2+ activated BK channel inhibitor) pretreatment, the Tes-induced relaxation was attenuated in SHR, but not in WKY. This attenuation in SHR was not observed after endothelium removal. The above results suggest that the relaxation induced by Tes following NA-induced vasoconstriction in SHR results from hyperpolarization due to BK channel opening.
Glucocorticoids such as prednisolone are used for their anti-inflammatory properties. But there is evidence to suggest that under certain conditions, glucocorticoids have pro-inflammatory effects, for example, enhancement of IL-1β production. To date, it has been reported that IL-1β production intensity was associated with single nucleotide polymorphisms at positions −1470, −511, and −31 in the promoter region and at position 3954 in exon 5 of the IL-1β gene. In the present study, it was examined whether these IL-1β genotypes were associated with the suppressive effect of prednisolone on IL-1β production in human peripheral blood mononuclear cells (PBMC) stimulated by lipopolysaccharide (LPS). A midrange concentration (10−6 M) of prednisolone suppressed the LPS-induced increase in IL-1β mRNA expression and protein release, while higher concentrations (10−5 M, 10−4 M) exhibited less suppression or had a synergistic stimulative effect on IL-1β production in certain subjects. Under treatment with 10−4 M prednisolone, the levels of IL-1β protein production stimulated by LPS in PBMC extracted from the subjects with the IL-1β TT−31, TC−31, and CC−31 genotypes were suppressed to 6.0±3.4%, 31.4±57.0%, and 87.7±84.8%, respectively, of the level in prednisolone-untreated control cells (TT−31vs. CC−31, p<0.05). Glucocorticoid-based anti-inflammatory therapy might be less effective in patients with the IL-1β TC−31 and CC−31 genotypes than those with the TT−31 genotype.
Hyperuricemia and gout appear to be rapidly increasing worldwide and frequently cause symptoms of metabolic syndrome. Dietary flavonoids have their potential beneficial effects on human health. In the present study, 15 flavonoids (quercetin, morin, myricetin, kaempferol, icariin, apigenin, luteolin, baicalin, silibinin, naringenin, formonoetin, genistein, puerarin, daidzin and naringin dihydrochalcone) were selected to investigate for their hypouricemic action in mice. Oral administration of quercetin, morin, myricetin, kaempferol, apigenin and puerarin at 50 and 100 mg/kg for 3 d was able to elicit hypouricemic actions in hyperuricemic mice induced by potassium oxonate. Luteolin, formonoetin and naringenin showed the significant effects only at 100 mg/kg. Quercetin, puerarin, myricetin, morin and kaempferol significantly reduced liver uric acid level in hyperuricemic animals. In addition, quercetin, morin, myricetin, kaempferol and puerarin exhibited significant inhibition on the liver xanthine oxidase (XOD) activities. It seems to be likely that these flavonoids reduce serum urate levels by mainly inhibiting XOD activity. However, the hypouricemic effect of apigenin observed seemed not to parallel with the changes in liver uric acid level and liver XOD activity, implying that apigenin might act via other mechanisms apart from inhibiting enzyme activity simply. Analysis of the chemical structure showed that a planar structure with the hydroxyl groups played a crucial role in hypouricemic activity of flavonoids. The exact mechanism of the hypouricemic action of flavonoids in vivo should be investigated in the future.
The anti-rhinitis properties of Pleurotus pulmonarius were investigated in BALB/c mice. A single administration of Pleurotus Pulmonarius caused no significant effect on antigen-induced nasal rubbing and sneezing at a dose of 500 mg/kg, but a significant inhibition was observed after 2 weeks of repeated treatment at this dose, and at a dose of 200 mg/kg, it also caused a significant inhibition after repeated administration for 4 weeks. Pleurotus pulmonarius showed no significant inhibitory effect on the production of IgE. In addition, Pleurotus pulmonarius caused no inhibition of histamine-induced nasal rubbing and sneezing at a dose of 500 mg/kg, but in vitro study, it inhibited histamine release from rat mast cells induced by compound 48/80 at the soluble supernatant solution of 30 and 100 μg/ml of Pleurotus pulmonarius suspended in PBS. These results demonstrated that Pleurotus pulmonarius may be effective in the relief of symptoms of allergic rhinitis through inhibition of histamine release.
In our previous study, a novel phenylbutenoid dimer (±)-trans-3-(3,4-dimethoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene (PSC), isolated from Zingiber cassumunar ROXB. (Zingiberaceae), inhibited proliferation of various human cancer cells with the IC50 values ranging 10 to 30 μM. Prompted by these anti-proliferative effects, we performed additional studies in A549 human lung cancer cells in order to investigate the mechanism of action. PSC arrested cell cycle progression at the G0/G1 phase in a concentration- and time-dependent manner. PSC dose-dependently induced cyclin-dependent kinase (CDK) inhibitor p21 expression, whereas the expression of cyclin D1, cyclin A, CDK4, CDK2, and proliferating cell nuclear antigen (PCNA) were decreased by treatment with PSC. These results suggest that one of the anti-proliferative mechanisms of PSC is to suppress cell cycle progression by increasing p21 expression and down-regulating cyclins and CDKs. This study characterizes additional biological activity of this novel phenylbutenoid dimer and expands its therapeutic potential for cancer as a chemotherapeutic agent derived from natural products.
Dipropofol has a strong antibacterial activity against Gram-positive bacteria. However, it lacked the solubility in water and this property was supposed to limit its efficacy. We tried to improve the solubility and found a new solubilization method of dipropofol in water by the addition of a monosaccharide or ascorbic acid.
Aqueous and hydro-ethanolic extracts of Bauhinia microstachya leaves (AEBM and HEBM) were investigated for their phenolic content and phytochemical profile (by spectrophotometry and HPLC), and for their antioxidant activities and free radical scavenging potential in different in vitro systems (TRAP, TEAC, TBARS, nitric oxide, superoxide and hydroxyl radical). HEBM presented a 27.4% higher content of phenolics when compared to AEBM and a distinct phytochemical profile was observed. Our work suggests that both extracts have potent antioxidant activities and that their antioxidant capacity and efficiency vary according to the radical-generating system. In general, HEBM was more effective than AEBM in avoiding ROS-generating damage and in scavenging the various radicals formed. Nevertheless, when results were normalized to total phenolic content, a different profile of antioxidant activities and free radical scavenging potential was observed, particularly against oxidative lipid damage and superoxide radical. B. microstachya extracts may be considered an interesting source of natural antioxidants as well as other phenolic-rich plants.
For the accurate identification of medicinal licorice species, nucleotide sequences of four types of DNA regions were researched for 205 specimens, including three species used as licorice: Glycyrrhiza uralensis, Glycyrrhiza glabra, and Glycyrrhiza inflata. The four DNA regions were the internal transcribed spacer (ITS) on nuclear ribosomal DNA, the rbcL gene, the matK gene, and the trnH–psbA intergenic region on chloroplast DNA (cpDNA). Ten genotypes were consequently recognized as combinations of the sequence data obtained from the four DNA regions. Species-specific genotypes were defined from the frequency of the appearance of species in each genotype and from the phylogenetic relationships of the 10 genotypes. This revealed the possibility of identifying licorice species based on the 10 genotypes. Next, comparison of species identifications by each DNA region suggested that efficient identification of licorice species is possible using the genetic information obtained from the ITS and trnH–psbA intergenic region. Additionally, concerning the phylogenetic relationships of the Glycyrrhiza species used as licorice, it is suggested from the genetic information of the four types of DNA regions that G. glabra is more closely related to G. inflata than to G. uralensis. In the G. uralensis examined, four genotypes were recognized as intra specific variations. The appearance frequency of each genotype in G. uralensis differed according to the area in China. G. uralensis may have expanded its distribution areas from western to eastern China because many licorices with the phylogenetic ancestral genotype were observed in western areas, while many with the derivative genotype were observed in eastern areas.
The antioxidant activities of plant extracts from Eysenhardtia platycarpa, E. punctata, and E. subcoriacea (Fabaceae) species, used in Mexican traditional medicine for the treatment of diabetes complications, were analyzed in a rat pancreas homogenate model. Methanolic extracts of E. platycarpa, E. punctata, and E. subcoriacea protected the pancreatic homogenate from 2,2-azo-bis(2-amidinopropane)dihydrochloride (AAPH)-induced damage. The inhibitory effect was dose-dependent at concentrations of 10—1000 ppm and correlated with 1,1 diphenyl-2-picrylhydrazyl (DDPH) radical scavenger capacity. 3-O-Acetyl-11α,12α-epoxy-oleanan-28,13β-olide (1, EC50=21.2±2.2 μM), (+)-catechin (2, EC50=7.4±1.1 μM), and (+)-catechin 3-O-β-D-galactopyranoside (3, EC50=11.5±1.5 μM), natural constituents isolated from the branches of E. platycarpa, displayed significant antioxidant activity. Compounds 1 and 2 significantly increased (p<0.001) the pancreatic glutathione (GSH) concentration alone and in combination with AAPH treatment. Compound 1 was obtained from oleanolic acid by relay synthesis via acetylation, bromo-lactonization, dehydrobromination, and oxidation, and its antioxidant effect was evaluated on streptozotocin (STZ)-induced diabetic rats. On its own, 1 at a dose of 100 mg/kg b. wt. (i.p.) for 5 d significantly increased the activities of glutathione peroxidase (GSHPx) and catalase (CAT). Simultaneous treatment of 1 (100 mg/kg b. wt.) and STZ significantly reduced the pancreatic thiobarbituric acid reactive substances (TBARS) concentration together with a significant increase in the activities of GSHPx and CAT, preventing hyperglycemia induced by STZ after 5 d of treatment. The present study demonstrates the antioxidant and antihyperglycemic activities of compound 1 isolated from Eysenhardtia species used in traditional medicine.
Kuma-zasa is Japanese folk medicine derived from plants of genus Sasa, family Bambusaceae. Although the plants of origin of Kuma-zasa were reported to be Sasa palmata, S. senanensis, S. yahikoensis, and S. kurilensis, authentication of those plants was difficult because of similarity in morphology. Several methods for the classification of genus Sasa are available, but none involve a genetic approach. Here, we performed the genetic profiling of genus Sasa, including the four species used medicinally. Thirteen sequences were observed in chloroplast DNA intron between rbcL and ORF106 and partial ORF106 regions of 34 specimens of 16 Sasa species and one specimen of Phyllostachys pubescens. We observed differences in alignment in this region among the specimens. The analyzed lengths varied from 759 to 821 bp depending on the specimen. There were nine base substitutions, eight successive thymines or adenines, and one to three repeat units of 31 bp. Moreover, we could not find species-specific alignment: different alignments were observed in specimens of the same species, while the same alignment was observed in specimens of different species. In the phylogenetic tree reconstructed by maximum parsimony analysis, medicinally used species did not form a cluster, although most of them were positioned close to each other. The genetic profiling of Sasa species would be of use in determining the botanical origin of the herbal medicine derived from the leaves of Sasa plants.
In the present study, we investigated the effect of saucernetin-7 (a biologically active compound isolated from the underground parts of Saururus chinensi) on the induction of apoptosis and the putative pathways of its action in HL-60 human promyelocytic leukemia cells. Saucernetin-7-treated HL-60 cells displayed several features of apoptosis, including DNA fragmentation, DNA laddering by agarose gel electrophoresis, and externalization of annexin-V targeted phosphatidylserine (PS) residues. z-VAD-fmk (a broad-caspase inhibitor) almost completely suppressed saucernetin-7-induced DNA ladder formation, thereby implicating the caspase cascade in the apoptotic process. We also observed that saucernetin-7 caused the activations of caspase-3, -8 and -9, and that it induced Bid cleavage, the mitochondrial translocation of Bax from the cytosol, and cytochrome c release from mitochondria, but it had no effect on Bcl-2 and Bcl-xL levels. Taken together, the present study demonstrates that saucernetin-7 is a potent inducer of apoptosis and that its activity is facilitated by caspase-8 activation, Bid cleavage, Bax translocation to mitochondria, release of cytochrome c into cytoplasm, and subsequently caspase-3 activation, which offers a potential mechanism for the apoptosis-inducing activity of saucernetin-7.
To evaluate active principles for diabetic complications from the black bamboo leaves, Phyllostachys nigra, eight compounds were isolated and tested for their effects on rat lens aldose reductase and advanced glycation endproducts. As a result, luteolin 6-C-(6″-O-trans-caffeoylglucoside) was found to show a strong aldose reductase and advanced glycation endproducts inhibition. This compound showed antioxidative activity measured in Photochem® apparatus. It is concluded, therefore, that luteolin 6-C-(6″-O-trans-caffeoylglucoside) (6), a flavone of this plant, have antioxidative as well as aldose reductase and advanced glycation endproducts inhibitory effects. As a result, this compound could be offered as a leading compound for further study as a new natural products drug for diabetic complications.
The acidic polysaccharide nostoflan was previously isolated as an antiviral component from the terrestrial alga Nostoc flagelliforme. In the present study, we examined the target for its anti-herpes simplex virus type 1 action. In time-of-addition experiments, the most sensitive stage of viral replication to nostoflan was found to be early events, including the virus binding and/or penetration processes. In order to determine what extent nostoflan may be involved in these processes, virus binding and penetration assays were separately performed. The results indicated that the inhibition of virus binding to but not penetration into host cells was responsible for the antiherpetic effect induced by nostoflan. Our study suggests that nostoflan may be a potential antiherpes agent.
Iron-deficiency anemia not only causes insufficient oxygen delivery to the body of the mother, but creates serious conditions in the fetus, such as insufficient oxygen delivery and poor development, and its treatment has become an important issue. To elucidate the mechanism of the anemia-ameliorating action of Tokishakuyakusan, we investigated the effect of administration of Tokishakuyakusan on anemia-related parameters and the serum transferrin and erythropoietin levels, erythrocyte morphology, and bone marrow cells in pregnant rats and in pregnant rats with iron-deficiency anemia. The results showed that Tokishakuyakusan significantly improved the low erythrocyte count, hemoglobin, and hematocrit values of the pregnant rats in the iron-deficient state, increased the proportion of normal erythrocytes in terms of erythrocyte morphology, increased the proportion of erythroblasts among bone marrow cells, and also significantly increased the erythropoietin and transferrin levels in the blood. These findings suggested that Tokishakuyakusan promotes erythrocyte differentiation in iron-deficiency anemia, and that it possesses anemia-ameliorating efficacy.
In this study, we attempted to enhance disulfiram (DSF) solubility using a 2-hydroxypropyl-β-cyclodextrin (HPβCD) and hydroxypropylmethylcellulose (HPMC). We also investigated the effect of an HPβCD solution containing DSF and HPMC (DSF eye drops) on cataract development in ICR/f rat. The solubility of DSF increased with increasing HPβCD concentration, and the solubility of DSF in HPβCD solution containing 0.1% HPMC was approximately 20% greater than that of DSF in HPβCD solution without HPMC. In in vivo transcorneal penetration experiments using rabbits, only diethyldithiocarbamate (DDC) was detected (DSF was not detected) in the aqueous humor. This DSF-DDC conversion in the cornea was inhibited by treatment with a sulfhydryl (SH) inhibitor, p-mercuribenzoate and N-ethylmaleimide, in in vitro transcorneal penetration experiments using rabbit corneas. On the other hand, the instillation of 0.25% and 0.5% DSF eye drops delayed cataract development in ICR/f rats, a recessive-type hereditary cataractous strain. The present study demonstrates that DSF in HPβCD solution with HPMC is converted to DDC by the catalysis of proteins containing SH residues in the cornea, and this DDC may cause the delay in cataract development in ICR/f rats.
Tulobuterol permeation through skin from various transdermal delivery systems has been compared for the bioequivalence among devices marketed. Both the permeation profiles across the hairless mouse skin and the release profiles from the devices were measured under well-controlled in vitro conditions. The release rate of the drug from the devices was appreciably higher than the penetration rate across the intact skin, indicating the skin-controlled delivery systems. However the deviation between the release rate and the permeation rate differs depending upon the system design. The brand patch showed the least difference between the release and permeation profiles among the brand and three generic devices examined. From the in vitro permeation profiles for both intact and stripped skins, the diffusion coefficient and the partition coefficient were evaluated on the basis of bi-layer skin model. The effect of the stratum corneum thickness was then simulated by SKIN-CAD®. The simulated profile has suggested that the clinical performance for transdermal tulobuterol delivery is influenced not only by the thickness of the stratum corneum but by the device design as well. This is particularly the case for the stratum corneum, thinner than about 10 μm or damaged skin with the decreased barrier capacity. For the stratum corneum thicker than 20 μm, on the other hand, the clinical performance may not be significantly influenced by the device designs investigated in this study.
The pharmacokinetics and metabolism of an α,β-blocker, amosulalol hydrochloride, were investigated in mice. After intravenous administration (10 mg/kg), the plasma concentration of the unchanged drug declined biphasically, with a terminal half-life of 1.1 h. The maximum plasma concentrations were reached at 0.25 h after oral administration, and then declined with apparent half-lives of 0.8—1.3 h. The systemic bioavailability of a 10-mg/kg dose was 38.7%. The area under the plasma concentration curve increased more than proportionally to the dose, which suggests metabolic saturation. After oral and intravenous administrations of 14C-labelled amosulalol hydrochloride, 64.7% and 81.0% of the radioactivity were recovered, respectively, in the urine within 48 h. HPLC-UV and LC/MS analyses demonstrated that the major urinary metabolite was the glucuronide of M-2 (desmethyl metabolite at the o-methoxyphenoxy group) followed by M-5, the M-3 glucuronide, and the M-4 glucuronide, in that order. In the bile sample, amosulalol carbamoyl glucuronide was found as a new metabolite of this drug.
This study was conducted to investigate the inhibitory effects of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) on cytochrome P450 3A (CYP3A). The inhibition of 6β-hydroxy testosterone formation from testosterone in rat liver microsomes was used as an index of CYP3A activity. In the present study, among the three types of fatty acids, the rank order of inhibitory effects of fatty acids was SFAs<MUFAs<PUFAs. Among the four PUFAs examined (linoleic acid, γ-linolenic acid, retinoic acid, and docosahexaenoic acid; DHA), the highest inhibitory effect of CYP3A-catalyzed testosterone metabolism was observed with DHA, which inhibited testosterone metabolism by 94%. The percentage inhibition (I) of fatty acids on testosterone 6β-hydroxylation was predicted from the number of double bonds and the carbon chain length of the fatty acids. The correlation between the predicted and observed I values yielded the r2 values of 0.759 and 0.907 using the linear and non-linear equations, respectively. Moreover, the goodness-of-fit assessed using the mean absolute error (MAE) was 17.6 with the linear model and 11.3 with the non-linear model. These observations suggested that fatty acids showed the variable inhibitory effects on CYP3A-mediated testosterone oxidation and that the number of double bonds, as well as the carbon chain lengths of fatty acids, may be important in the inhibition of CYP3A.
Fate of perfluorooctanoic acid (PFOA) after an intravenous injection to male rats at the dose of 0.041 mg/kg body weight was compared with that at the dose of 16.56 mg/kg body weight. In the liver, 52% and 27% of PFOA dosed was recovered 2 h after an intravenous injection at the low and the high doses, respectively. By contrast, larger proportion of PFOA dosed was distributed to serum, other tissues and carcass at the high dose compared with the low dose. Subcellular distribution of PFOA was determined in the liver. At the dose of 0.041 mg/kg, 45%, 34%, 18% and 3% were distributed to 8000 g pellet, 18000g pellet, 105000g pellet and 105000g supernatant fraction, respectively; 28%, 17%, 13% and 43% of PFOA were distributed to these fractions, respectively, at the dose of 16.56mg/kg. The higher the concentration of hepatic PFOA was, the more the PFOA was distributed to 105000g supernatant fraction. Biliary excretion index increased as PFOA concentration raised in the liver. These results suggest that PFOA is preferentially taken-up by the liver, and distributed to membrane fractions, especially 18000g pellet, and hardly excreted into bile when exposed at very low dose.
Long-term treatment with clomipramine (CMI), a tricyclic antidepressant, induces food craving and body weight gain in patients. The present study investigated the effects of chronic treatment with CMI on total food intake, macronutrient selection, and body weight gain in rats. Male Wistar rats were maintained on a dietary self-selection regime with separate sources of protein, fat and carbohydrate. Animals received i.p. injections of CMI (0, 3, 10, 30 mg/kg) during 27 consecutive days. Food consumption and body weight were recorded daily and results were calculated as average of three consecutive days, namely during pre-treatment (3 d before pharmacological treatment), treatment (7th—9th; 16th—18th and 25th—27th days), and post-treatment (28th—33rd days). Results showed that CMI (30 mg/kg) significantly decreased energy intake during all treatment period, an effect that was related to a decrease in both carbohydrate-rich diet intake and body weight gain. At dose of 3 mg/kg CMI increased the total energy intake in the 16th—18th days, suggesting an apparent biphasic effect of chronic treatment with CMI on caloric intake. Chronic administration with CMI (27 d) did not alter protein-rich or fat-rich diet consumption. The main result of this study indicated that chronic treatment with CMI decreases rather than increase food consumption and body weight gain in rats exposed to a macronutrient self-selection procedure.
SKG mice are a recently established experimental model for rheumatoid arthritis (RA). Although they spontaneously develop chronic autoimmune arthritis under conventional conditions, SKG mice failed to develop chronic arthritis in a strictly controlled specific pathogen-free (SPF) environment. Beta-glucan (BG) from Laminaria digitata, laminarin (LAM), induced arthritis under SPF conditions, thus BG would be a pathogenic factor for arthritis in SKG mice. Therefore, we prepared BG from Candida albicans, a pathogenic fungus and investigated whether BG from C. albicans induced arthritis in SKG mice under SPF conditions. SKG mice were injected intraperitoneally with particulate BG (oxidative-Candida albicans (OX-CA)), soluble BG (Candida soluble beta-glucan (CSBG)) from C. albicans and LAM as a positive control. In addition, schizophyllan (SPG) from Schizophyllum commune or Mycobacterium whole cells were injected into SKG mice to induce arthritis. Mice injected with OX-CA, CSBG and SPG had more severe arthritis than with LAM, and whole Mycobacterium cells. IL-6 concentration in sera from SKG mice injected with OX-CA or CSBG was high, whereas not detected in sera from mice treated with LAM. In histological analysis, infiltration of inflammatory cells was observed in SKG mice injected with BG. These results suggest that fungal infection may be a factor to induce and exacerbate autoimmune diseases such as RA.
We investigated the characteristics of binding of tamsulosin to α1-acid glycoprotein (AGP) genetic variants. The binding of tamsulosin to each of the human AGP variants was determined by ultrafiltration, and the binding characteristics for each variant were compared using binding parameters and inhibition of the binding by disopyramide and warfarin. The affinities of tamsulosin binding to a F1/S variant mixture and total AGP variants were relatively high (dissociation constants 1.6 μM). On the other hand, the dissociation constant for variant A was 14.9±2.53 μM. The binding of tamsulosin was competitively inhibited by warfarin but not by disopyramide. Tamsulosin appears to be a suitable compound for studying the characteristics of drug binding to human AGP F1/S variants under clinical conditions.