Within the recent years novel lead optimisations for histamine H3 receptor antagonists made their way from bench to bedside. Structure–activity relationships, cross-affinities and side effects as well as pharmacokinetic profiling will be discussed on selected promising compound series. Due to diversity in potential therapeutic applications and in some cases a controversial debate, different indications will be highlighted with the potential and the problems of the test compounds, e.g. sleep-wake disorders, attention-deficient hyperactivity disorder, epilepsy, cognitive impairment, schizophrenia, obesity and neuropathic pain. First data published on clinical trials phase II (IIb) are presented showing proof of concept of H3 receptor antagonists in narcolepsy and photo-induced epilepsy.
Brownian dynamics simulations of a wild type and mutants of bovine pancreatic trypsin inhibitors were performed using the program brownian. The results of the simulations were consistent with experimentally determined stabilities of these proteins, and the computational times of the simulations were much less than those of molecular dynamics simulations. These results indicate that Brownian dynamics simulations can be useful for investigating structural features of proteins that have some mutants, such as drug-metabolizing enzymes.
Recently, an interesting debate arose about the nature (substrate versus inhibitor) of esculetin, a coumarin derivative, for mushroom polyphenol oxidase (PPO). The present study examined the behavior of PPOs preparations from fungal and plant origin towards esculetin as a substrate. Both enzymes were able to oxidize esculetin though at a slow rate. A higher sensitivity was reached when the assay was performed in the presence of 3-methyl-2-benzothiazolinone hydrazone (MBTH) even with a lower amount of PPO. These observations unambiguously confirmed that esculetin has to be considered a substrate for mushroom polyphenol oxidase. The oxidation of esculetin was also demonstrated for the first time by a fungal laccase. This should be taken into account because some mushroom PPO preparations could exert contaminant laccase activity. In addition, a PPO preparation from Ferula communis was demonstrated to use esculetin as a substrate. Umbelliferone, the monophenolic precursor of esculetin along the phenylpropanoid pathway, behaved as a competitive inhibitor for the monophenolase activity of mushroom PPO with a Ki value=0.014 mM. This is worth a mention because only a few couples of mono- and corresponding o-diphenol show such opposite behavior towards PPO. A possible role of PPO in the esculetin fate along biosynthesis pathway of coumarin derivatives is also discussed.
The cDNAs (Espals) encoding phenylalanine ammonia-lyase (PAL) were cloned from Ephedra sinica by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers and by 5′ and 3′-rapid amplification of cDNA ends (RACE). 2166 bp of the open reading frame (ORF) encoded 722 amino acids; sequence analyses of Espal clones suggested that at least four isoforms of EsPAL (EsPAL1, 2, 3, 4) existed, with nine amino acids substitution in their sequences. Phylogenetic analysis of EsPAL and PALs from other plant species revealed that EsPAL and Pinus PAL formed a gymnosperm-type PAL subfamily. The recombinant EsPAL1 to 4 functionally catalyzed a PAL reaction and their Km, Vmax, Kcat and Kcat/Km values did not show significant differences. Semi-quantitative RT-PCR analysis indicated that the expression of Espal genes in the roots was higher than in the plant's aerial parts. In addition, the activity of PAL in the roots was also higher than in the aerial parts. These results suggest that Espal genes are expressed in the whole plant but are dominant in the roots rather than in the aerial parts.
This study was designed to evaluate the effect of diphenyl diselenide in a classical model of alloxan-induced diabetes in rats. Oxidative stress is involved in alloxan toxic effects and we have hypothesized that diphenyl diselenide via its antioxidant properties could confer protection against alloxan pancreatic toxicity. Diabetes was induced by administration of alloxan (150 mg/kg, intravenously). Diphenyl diselenide (10 mg/kg, subcutaneously) was administered for 6 d before (prevention group) or for 6 d after (remediation group) diabetes induction. Diphenyl diselenide treatment reduced the blood glucose and fructosamine levels, which were increased in alloxan-treated rats. However, the delta-aminolevulinate dehydratase (δ-ALA-D) activity inhibited by alloxan was not restored by diphenyl diselenide. Moreover, diphenyl diselenide caused by itself an inhibition in hepatic and renal δ-ALA-D activity. Our findings suggest that the acute treatment with diphenyl diselenide reduces the hyperglycemia but does not improve δ-ALA-D activity decreased by alloxan. Although the dose of diphenyl diselenide used here for treating diabetic animals has been relatively high and produced toxic effects, the compound or analogous molecules might not be rejected as a promising anti-hyperglycemic agent.
The genome sequencing project revealed presence of two active chalcone synthase (CHS) homologues (At1g02050 and At4g34850) in the model plant Arabidopsis thaliana. We report herein the two genes encode closely related novel plant-specific type III polyketide synthases (PKSs) that produces long-chain alkyl α-pyrones. PKS-A (At1g02050) and PKS-B (At4g34850) share significantly low amino acid sequence identity (20—40%) with other type III PKSs, and the phylogenetic tree analysis revealed that they form a separate cluster located closely to those of bacterial type III PKSs. When expressed in Escherichia coli, both PKS-A and PKS-B accepted unusually long (up to the C20 chain-length) fatty acyl CoAs as a starter substrate, and carried out sequential condensations with malonyl-CoA to produce triketide and tetraketide α-pyrones. Interestingly, despite the low sequence identity, homology modeling revealed that the active-site architecture of PKS-A and PKS-B showed similarity to that of a bacterial type III PKS from Mycobacterium tuberculosis.
4,4′-Butylidenebis(6-t-butyl-m-cresol) (BBBC) can be eluted from disposable gloves made of nitrile-butadiene rubber and possibly also detected in food. We have reported that BBBC is an androgen and estrogen antagonist. In this report, BBBC (0.1, 1.0 mg/kg body weight (bw)/day) was subcutaneously administered to pregnant rats from gestation days 11 through 18 and the effects on male offspring (postnatal day (PND) 102) were examined. Body weight at lactation and brain weight at PND 102 were decreased in the 1.0 mg/kg bw BBBC-treated group. Altered levels and turnover of the monoamines dopamine (DA), serotonin (5-HT), and noradrenalin (NA) as well as their metabolites were detected. In the prefrontal cortex DA, 3,4-dihydroxyphenylacetic acid (DOPAC), 5-HT, 5-hydroxyindole-3-acetic acid (5-HIAA) levels were significantly increased, but homovanillic acid (HVA)/DA was decreased. In the striatum NA level, DOPAC/DA and HVA/DA were significantly increased, but 3-methoxy-4-hydrophenylglycol hemipiperazinium (MHPG) level and MHPG/NA were decreased. In hippocampus MHPG level was significantly decreased. In hypothalamus 5-HIAA level and 5-HIAA/5-HT were significantly increased. These results suggested that prenatal exposure to BBBC affects the central nervous system of male rat offspring, and BBBC may be an endocrine disrupting-chemical during the fetal periods.
The Gα subunits of the G12 family of heterotrimeric guanine nucleotide-binding proteins (G proteins), defined by Gα12 and Gα13, have many cellular functions in common, including stimulation of stress fiber formation and focal adhesion assembly via the small GTPase RhoA activation. We and others previously showed that Gα12 and Gα13 mediate neurite retraction in neuronal cell lines, but their roles in primary cultured neurons have not been adequately understood. Here, we found that expression of constitutively active mutants of Gα12 or Gα13 caused growth cone collapse dependent on Rho-kinase activity in hippocampal neurons. The stimulation of thrombin and lysophosphatidic acid (LPA) receptors, which have been thought to selectively couple to Gα12 and Gα13, respectively, caused growth cone collapse and suppressed axon branching dependent on Rho-kinase activity in hippocampal neurons. Thrombin- and LPA-induced growth cone collapse was suppressed by both single knockdown of Gα12 and Gα13 with short hairpin RNAs and this suppression was augmented by double knockdown of both Gα12 and Gα13. These results suggest that thrombin and LPA receptors couple to both Gα12 and Gα13 for growth cone collapse.
Post-translational modification with ISG15 (interferon-stimulated gene 15 kDa) (ISGylation) is mediated by a sequential reaction similar to ubiquitination, and various target proteins for ISGylation have been identified. We previously reported that ISGylation of the E2 ubiquitin-conjugating enzyme Ubc13 suppresses its E2 activity. Ubc13 forms a heterodimer with Uev1A, a ubiquitin-conjugating enzyme variant, and the Ubc13–Uev1A complex catalyzes the assembly of a Lys63-linked polyubiquitin chain, which plays a non-proteolytic role in the nuclear factor (NF)-κB pathway. In this study, we examined the effect of ISGylation on tumor necrosis factor receptor-associated factor (TRAF)-6/transforming growth factor β-activated kinase (TAK)-1-dependent NF-κB activation. We found that expression of the ISGylation system suppresses NF-κB activation via TRAF6 and TAK1 and that the level of polyubiquitinated TRAF6 is reduced by expression of the ISGylation system. Taken together, the results suggest that the NF-κB pathway is negatively regulated by ISGylation.
Semi-vioxanthin isolated from marine-derived fungus was assessed for immunoregulatory activity in mouse RAW264.7 macrophages. In the present study, the facilitative effects of semi-vioxanthin on tumor necrosis factor-α (TNF-α) and its mRNA expression and on expression of the co-stimulatory molecules, cluster of differentiation (CD) 80, CD86 and major histocompatibility complex class II (MHC II), as well as the molecular mechanism underlying the immunologic enhancement properties of semi-vioxanthin were studied. Our results clearly indicated that semi-vioxanthin treatment resulted in the degradation of IκBα, which led to the activation and nuclear translocation of the p65 subunit of nuclear factor-κB (NF-κB), as determined by immunoblotting, immunofluorescence and electrophoretic mobility shift assays (EMSA). Moreover, TNF-α production was prevented by NF-κB and mitogen-activated protein kinase (MAPK) inhibitors. Inhibition of NF-κB and extracellular signal regulated kinases (ERK1/2) activity by specific inhibitors blunted the effect of semi-vioxanthin on the up-regulation of CD80, CD86 and MHCII expression, but neither p38 MAPK nor c-Jun N-terminal kinase (JNK) inhibitor had this effect. Thus, we demonstrate that semi-vioxanthin regulates TNF-α production through NF-κB and MAPK signaling pathways. Activation of NF-κB and ERK1/2 were necessary for CD80, CD86 and MHCII expression induced by semi-vioxanthin. These data suggest that semi-vioxanthin has immunoregulatory effects.
In vitro interaction of fluconazole and baicalein (BE) was investigated against 30 fluconazole-resistant clinical isolates of Candida albicans. Synergistic activities were determined using the checkerboard microdilution assay based on the fractional inhibitory concentration indices. Organisms were also tested against the 2 drugs singly and in combination using time–kill methods. Both fluconazole and BE showed weak antifungal activity when tested alone. However, the combination of fluconazole and BE showed strong antifungal activity against most of the fluconazole-resistant isolates tested. The findings of time–kill curves confirmed the interaction. Yeast cells grown in the presence of BE exhibited a reduced extrusion of Rhodamine 6G, which indicates the inhibition of efflux pumps by BE. This novel synergism of fluconazole and BE that can overcome drug-resistance in yeast may prove useful in combined treatment of fungal infections.
To assess whether azelnidipine (AZN) exerts renoprotective effects, 20-week-old adult male stroke-prone spontaneously hypertensive rats (SHRsp) were treated with AZN 10 mg/kg/d (n=6), olmesartan (OLM) 3 mg/kg/d (n=4), hydralazine (HYD) 20 mg/kg/d (n=3), or water (control; n=5). Each test agent was administered by oral gavage for 12 weeks. Systolic blood pressure (SBP) was measured every 2 weeks and urinary protein excretion (UproV) every 3 weeks. At the age of 32 weeks, the rats were sacrificed and blood and kidneys collected for biochemical, histological, and immunohistochemical studies. All drug treatments significantly (p<0.05) reduced SBP, UproV, and blood biochemical parameters such as creatinine, total cholesterol, and blood urea nitrogen. Masson trichrome staining and immunohistochemical staining revealed significant (p<0.05) reductions of interstitial fibrosis, collagen type III, nicotinamide-adenine dinucleotide/nicotinamide-adenine dinucleotide phosphate oxidase, and p22phox and p47phox components expression in the AZN- and OLM-treated groups in comparison with rats treated with HYD and control animals. ED1, 4-hydroxy-2-nonenal (4-HNE), and heat shock protein (HSP)-47 expression was also reduced in the AZN- and OLM-treated groups versus in HYD and control animals. These results indicate that not only OLM but also AZN exerts renoprotective effects through inhibition of macrophage infiltration and antioxidant activity in SHRsp model of renal injury.
The effect on bone tissue of β-ecdysterone, a type of ecdysteroid found in many plants, has not been previously investigated. In this study, we found that β-ecdysterone treatment significantly induced alkaline phosphatase (ALP) activity in mesenchymal stem cells in a dose-dependent manner. Real-time polymerase chain reaction (PCR) showed that Runx2, osteocalcin, and type I collagen expression also increased. ICI182780, a specific estrogen receptor antagonist, inhibited the upregulation of ALP activity. Moreover, β-ecdysterone promoted estrogen receptor (ER) reporter gene activity; however, ICI182780 reversed its effect, suggesting that β-ecdysterone has stimulatory effects on osteogenic differentiation via the ER. Furthermore, β-ecdysterone alleviated osteoporosis symptoms in a mouse model without obvious side effects. Therefore β-ecdysterone may be a promising candidate drug for the treatment of osteoporosis.
The effects of fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), were studied in normophagic and food-restriction-induced hyperphagic middle-aged Wistar rats. Normophagic intact Wistar rats were given fluvoxamine (100 mg/kg/d, per os (p.o.)) or vehicle for 10 d. Hyperphagic middle-aged Wistar rats were subjected to 10 d of food restriction; they were allowed to refeed for 10 d, with ad libitum food access and administered fluvoxamine (100 mg/kg/d, p.o.) or vehicle during the 10-d refeeding period. Fluvoxamine administration to normophagic middle-aged Wistar rats affected neither their weight nor food intake. However, administration to food-restricted rats showed inhibitory effects of weight gain and food intake during 10 d of refeeding. Fluvoxamine-treated rats showed significantly lower neuropeptide Y (NPY) immunostaining levels in the paraventricular nucleus (PVN) and dorsomedial hypothalamic nucleus (DMH) than untreated controls. Hypophagic and weight-inhibiting effects of fluvoxamine might be mediated via decreased NPY in PVN and DMH. These results suggest that the appetite-controlling effect of fluvoxamine might be responsive to the rats' appetite condition.
In this study, we established a drug screening system based on transcriptional regulation of vascular endothelial growth factor (VEGF) under hypoxia-inducible factor-1α control. We cloned the neomycin-resistance gene into the plasmid GL (pGL)3-promoter vector to generate the pGL3-promoter-neo vector. The 3 copies of the 47-bp fragment that contained the hypoxia response element of VEGF were synthesized and inserted in front of the minimal promoter of the pGL3-promoter-neo vector to generate p3HRE-luc-neo. The recombinant reporter gene vectors were transfected into EAhy926 cells, and stable cell lines were obtained. The positive cell line was selected for its ability to express luciferase in response to hypoxia. We demonstrated that CoCl2 significantly enhances luciferase activity in a concentration-dependent fashion. We then optimized the cell density and incubation time under hypoxia which were used to screen. The assay exhibited a low background and was an ideal model for high-throughput screening for human VEGF regulators.
The pharmacological profiles of antigen-induced immediate airway response (IAR) in rats are not fully understood. In this study, we established an ovalbumin (OVA)-induced IAR model using noninvasive measurement in rats, and evaluated the effects of commonly used and effective antiasthmatic drugs, i.e. ketotifen (antihistamine), pranlukast (anti-leukotriene C4/D4/E4 (LT)), seratrodast (anti-thromboxane A2 (TXA2)), salbutamol (β2-agonist), and prednisolone (steroid). The rat IAR model exhibited an optimal rapid airway response, and salbutamol inhalation completely suppressed the IAR. Ketotifen inhibited only the quick phase (QP; the reaction from 3 to 6 min after challenge), while pranlukast and seratrodast suppressed only the early phase (EP; the reaction from 6 to 30 min after challenge). Prednisolone inhibited both QP and EP. Further, continuous administration of compound 48/80, which depletes connective tissue mast cells (CTMC), partially inhibited QP but not EP. In conclusion, these findings suggest that the pharmacological profiles of noninvasive rat IAR are similar to those of asthmatic patients, and that rat IAR exhibits additional, immunological diverse characteristics, i.e. QP caused by the exocytosis of mediators in CTMCs and EP mediated by LT and TXA2, which are produced by mucosal mast cells (MMCs) and possibly by other types of cells. This is the first report about the comprehensive pharmacological profiles of rodent IAR model, and these analyses of rat IAR model may help expand our understanding of the diverse mechanisms underlying human asthmatic diseases.
The persistence of latent human immunodeficiency virus type 1 (HIV-1)-infected cellular reservoirs, despite prolonged treatment with highly active antiretroviral therapy (HAART), represents a major hurdle to virus eradication. In this study, we evaluated the effect of Japanese herbal medicine on the induction of HIV-1 replication in latently infected monocytic cell line, U1, in order to eradicate virus efficiently. We found that Mao-to was able to induce HIV-1 replication either alone or in combination with tumor necrosis factor-alpha (TNF-α). Among the four components of Mao-to, only Ephedrae herba had strong effects in inducing HIV-1 replication. Analysis by Western blotting revealed that Ephedrae herba induced the nuclear translocation of nuclear factor-kappa B (NF-κB). Reporter assay data also showed that Ephedrae herba and, slightly, Mao-to activated the NF-κB promoter, indicating that these herbal agents may induce HIV-1 replication through NF-κB activation. These findings suggest that Mao-to and its component, Ephedrea herba, may be good candidates to augment HAART by inducing the expression of latent HIV-1 with the ultimate goal of eliminating persistent viral reservoirs in individuals infected with HIV-1.
The aim of this study was to investigate the effect of grapefruit juice intake on the antinociception of morphine in rats. The antinociception of morphine (30 mg/kg, per os (p.o.)) was significantly enhanced by the oral administration of grapefruit juice (2 ml/rat). Further, the effect of grapefruit juice was examined in morphine-tolerant rats. The repeated administration of morphine (100 mg/kg p.o.) for 5 d caused a marked decrease in the antinociception, indicating the development of morphine-tolerance. In the morphine-tolerant rats, oral administration of grapefruit juice potentiated significantly the antinociceptive effect of morphine. To examine the pharmacokinetics of morphine after the repeated treatment with morphine for 5 d, microdialysis probes were implanted into the jugular vein and spinal intrathecal space in rats. The morphine concentrations in the blood and intrathecal cerebrospinal fluid (CSF) were gradually decreased by the repeated treatment with morphine. The grapefruit juice treatment significantly increased the blood concentration of morphine in morphine-tolerant rats. These results suggest that oral administration of grapefruit juice enhances the morphine antinociception by increasing the intestinal absorption of this agent.
The photodynamic effect of the glycoconjugated photosensitizer library containing 16 glycoconjugated 5,10,15,20-tetraphenylporphyrins and 8 glycoconjugated 5,10,15,20-tetraphenylchlorins were examined in HeLa cells, and analyzed by two approaches, namely, physiological properties (cellular uptake and reactive oxygen species (ROS)) and structural features of glycoconjugated photosensitizers. All glycoconjugated photosensitizers showed no cytotoxicity in the dark at a concentration of 5 μM. The photocytotoxicity profiles poorly related to the amount of cellular uptake of the photosensitizers. Photocytotoxicities of the glycoconjugated photosensitizers were inhibited by the ROS inhibitor, sodium azide. The result clearly suggests that singlet oxygen is a dominant species in all cases. The glycoconjugated photosensitizers examined have three structural features, namely, (1) the kind of sugar moieties, (2) the kind of light-absorbing moiety and (3) the substitution position of the sugar moiety. In regard to the sugar moieties, the photosensitizers bearing D-xylose tend to show higher photocytotoxicity than other photosensitizers, while those bearing D-arabinose tend to show lower photocytotoxicity. The photocytotoxicity with respect to the light-absorbing moiety tends to increase in the order of zinc porphyrin<porphyrin<chlorin. The optical density at the QI band fairly explained the results. As an effect of the substitution position of the sugar moieties, the photosensitizers bearing sugar moieties at the meta-position of phenyl group showed remarkably high activity compared with para-substituted ones, and the difference could not be explained by the optical density. Confocal laser scanning microscopy revealed that the meta-substituted photosensitizers are not readily deactivated from the excited state in the cellular microenvironment, and this may explain their potent photocytotoxicity.
Tanshen has long been widely used as a traditional Chinese medicine. Tanshinone IIA (Tan IIA) is the most abundant lipophilic constituent of Tanshen which has antitumor activity but the mechanism is poorly understood. Some preliminary reports hypothesized that it is a DNA intercalator and that the furano-o-quinone moiety could produce free radicals responsible for its cytotoxicity. Here the interaction of Tan IIA with DNA was explored in detail using fluorescence, viscosimetry, and molecular modeling. Tan IIA was found to bind with DNA in the minor groove rather than act as an intercalator. Furthermore, the results of immunofluorescence showed that Tan IIA does not produce free radicals in vivo to damage DNA. The former hypothesis was thus negated. The furan oxygen plays the key role in the antitumor ability of Tan IIA because it is involved in the groove-binding, but not in the production of free radicals. The molecular basis illustrated here could be responsible for all the findings in the structure-relationship studies of tanshinone cytotoxicity.
Toxoplasmosis is a parasitic disease caused by Toxoplasma gondii, with very few therapeutic treatment options. Typically, the choices for treatment are pyrimethamine and sulfadiazine, however their utility is limited because of drug toxicity and serious side effects. For these reasons, new drugs with lower toxicity are urgently needed. In this study, the compound oleuropein isolated from Fraxinus rhynchophylla showed anti-T. gondii effects in vitro and in vivo. In Madin–Darby bovine kidney cells, the selectivity of oleuropein was 8.9, which was higher than sulfadiazine and pyrimethamine (3.8 and 2.5, respectively). In infected mice, the inhibition ratio of T. gondii in the peritoneal cavity was 55.4% compared to the negative control group after treatment with 300 mg/kg oleuropein. In addition, inhibitory effects on granuloma, apoptosis, necrosis and cyst-formation were shown in sections of spleen and liver. Oleuropein is therefore a potentially useful anti-T. gondii candidate for clinical application.
To develop a clindamycin-loaded wound dressing, cross-linked hydrogel films were prepared using freeze–thawing method with various mixtures of polyvinyl alcohol (PVA) and sodium alginate (SA). The physicochemical properties such as swelling ratio, tensile strength and elongation of hydrogels were evaluated. The drug release from this clindamycin-loaded hydrogel, in vitro protein adsorption test and in vivo wound healing observations in rats were then performed. Increased SA concentration decreased the gelation %, maximum strength and break elongation, but it resulted into an increment in the swelling ability, elasticity and thermal stability of hydrogel film. However, SA had insignificant effect on the release of clindamycin. This hydrogel improved the healing rate of artificial wounds in rats. Thus, a clindamycin-loaded wound dressing with PVA and SA hydrogel should be a candidate for wound care.
We have prepared lipid emulsions of approximately 200 nm in diameter with soybean oil (SO) and a series of Pluronics with various numbers of oxyethylene units and about 60 oxypropylene units (SO/Pluronics), and studied the pharmacokinetics of menatetrenone incorporated into SO/Pluronics in rats. Emulsions of approximately 200 nm in diameter were obtained when SO contents were 2.5% and 20% (w/w) for 2.4% (w/w) PL101 and Pluronics that more than 30% was made up by oxyethylene units, respectively. The half-life of menatetrenone in plasma when oxyethylene units made up less than 30% of the Pluronic (SO/PL101 and SO/PP103) was similar to that for SO/egg yolk phosphatides (SO/EYP), but longer than that when oxyethylene units composed more than 40% of the Pluronic (SO/PP104 and SO/PF108, by 3- and 10-fold, respectively). Pretreatment with dextran sulfate 500000, an inhibitor of emulsion uptake by the reticuloendothelial system (RES), resulted in a higher plasma concentration and a lower liver uptake of menatetrenone as SO/PL101 at 10 min and SO/PP103 at 60 min, indicating that both SO/PL101 and SO/PP103 were taken up by the RES, although SO/PP103 required some time to be recognized by the RES. These findings suggested that larger numbers of oxyethylene units of Pluronics with 60 oxypropylene units were required for the longer plasma circulation of SO/Pluronics due to evasion of the RES.
To conserve both form and function in the oral area, effective and selective drugs against oral cancer will be required. We focused on MUC1, a transmembrane glycoprotein, that is considered tumor-associated antigens (TAAs) for cancer therapy. Recently, studies were done to evaluate the patterns of MUC1 expression in oral squamous cell carcinomas (OSCCs) and it was found that higher MUC1 expression correlates with tumor invasion and metastasis. Using oral squamous cell carcinoma cell lines, we demonstrate here that tumor-specific targeting of MUC1 with the specific monoclonal antibody C595 has functional consequences with regard to complement deposition on MUC1-expressing oral cancer cell lines. Anti-MUC1 monoclonal antibody (mAb) also induced complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) to OSCC cells, and these effects were strongly correlated with MUC1 expression. Thus, these results indicate that anti-MUC1 mAb could provide a useful tool against OSCCs, and may provide insight into the development of low side-effect targeting therapy for this malignant disease.
The RNA interference (RNAi) effect is an alternative technology to antisense DNA as an experimental method of down-regulating a specific target protein. Optimal gene therapy for tumors must deliver synthetic small interfering RNA (siRNA) to tumor cells with high efficiency and minimal toxicity. Previously, we reported that cationic nanoparticles composed of cholesteryl-3β-carboxyamidoethylene-N-hydroxyethylamine and Tween 80 (NP-OH) could deliver plasmid DNA with high transfection efficiency. In this study, to apply NP-OH for siRNA transfection, we optimized the charge ratio (+/−) of NP-OH/siRNA and nanoplex-forming solution, and evaluated the transfection efficiency into PC-3 cells. Positively charged nanoplex prepared in the presence of sodium chloride exhibited efficient siRNA transfer. The distribution of siRNA after transfection was strongly detected both in the cytoplasm and nucleus. Furthermore, we demonstrated that NP-OH nanoplex of bcl-2 siRNA significantly inhibited tumor growth compared with control siRNA, and the efficacy was comparable to commercial products. The results of the experiments showed that NP-OH nanoparticles have potential as a viable vector candidate for synthetic siRNA delivery.
The purpose of the present study was to evaluate the effect of kainic acid (KA)-induced acute seizures on the pharmacokinetic profiles of antiepileptic drug, carbamazepine (CBZ) in mice. Experimental acute seizure in mice was induced by intraperitoneal injection of KA (30 mg/kg), and mice were provided for experiments after 48 h of KA treatment. The portal plasma concentrations of CBZ and its metabolite carbamazepine-10,11-epoxide (CBZ-epo) had trends to decrease as compared to the control mice, whereas the brain CBZ and CBZ-epo concentrations was actually lower in KA treated mice. On the other hand, the exsorption of CBZ from blood to the intestinal lumen via P-glycoprotein (P-gp) in KA treated-mice was significantly increased in parallel with that of Rhodamine-123 (Rho123), a P-gp substrate. Western blotting analysis for intestinal and cerebral P-gp showed that the P-gp expression was induced in the KA-treated mice. The apparent brain-to-plasma concentration ratio (Kp) of CBZ in the KA-treated mice showed significant decrease but that of CBZ-epo did not. Moreover, in the KA-treated mice, the percentage of protein binding was significantly increased, and found to be an inverse proportion in the relationship between the Kp and protein binding of CBZ. In conclusion, the mechanism responsible for a decreased brain CBZ concentration in the KA-induced seizure mice is based on the up-regulation of P-gp function in tissues and plasma protein binding of CBZ.
The effect of a thin film of a zwitterionic random copolymer composed of carboxybetaine [1-carboxy-N,N-dimethyl-N-(2′-methacryloyloxyethyl)methanaminium inner salt] (CMB) and n-butyl methacrylate (BMA), poly(CMB-r-BMA) (CMB, 30 mol%), on the healing of a full-thickness excisional and incisional wound in hairless rats was examined. The poly(CMB-r-BMA) film significantly enhanced wound closure and complete healing of a full-thickness excisional wound compared with the effect of the poly(n-butyl methacrylate) (PBMA) and the poly(ethylene terephthalate) (PET) films. However, the poly(CMB-r-BMA) film did not enhance healing of a full-thickness incisional wound in hairless rats. The amount of proteins adsorbed and that of neutrophiles adhered onto the poly(CMB-r-BMA) film were significantly smaller than those onto the PBMA and PET films. The results suggested that various cells and growth factors in the wound exudate are utilized effectively by covering an excisional wound with the poly(CMB-r-BMA) film, resulting in acceleration of healing. In addition, the poly(CMB-r-BMA) film significantly enhanced healing of a full-thickness excisional wound in hairless rats compared with the effect of Tegaderm® as wound dressings. The poly(CMB-r-BMA) film has potential as a new wound dressing.
Topical microemulsion of capsaicin without surfactant was developed in this study. In these systems, the oil phase was benzyl alcohol, and the cosurfactant was propylene glycol and ethanol. The drop-size of the systems was measured by dynamic light scattering method in order to distinguish true solution from microemulsion. The transdermal performance of the microemulsions was evaluated in vitro by Franz diffusion cells fitted with rat skins. The results showed the drop-size of the microemulsions without surfactant was smaller than that of the systems with Tween 80 and the permeation rate of capsaicin decreased as the content of Tween 80 increased. In the system composed of water, benzyl alcohol and propylene glycol, the permeation rate increased with the enhancement of benzyl alcohol and water. But water content had little effect on the permeation rate in the microemulsions with ethanol as cosurfactant.
An efficient protocol for the in vitro germination and propagation of Larrea divaricata CAV. (Jarilla) was established. To determine the effect of different growth regulators on the growth rates and phenol production, apical-node microshoots from in vitro germinated plantlets were incubated on the following media: 1) full-strength MS (Murashige and Skoog) salt medium with different ratios of α-naphthaleneacetic acid (NAA) and N6-benzyladenine (BA); 2) after pre-treatment with indolebutyric acid (IBA), transfer to MS medium of different inorganic salt strengths; and 3) full-strength MS salt medium with different ratios of sucrose and IBA. Successful microshoot rooting percentages were achieved by the second and third strategies, the highest being 87.5—100%. The maximum principal shoot length and node number obtained by the second strategy corresponded to the plantlets previously induced with 50 μM IBA, and grown on half- or full-strength MS salt media (7.03±0.93 and 9.86±1.07 cm, respectively) while in the third strategy the most efficient micropropagation medium was full-strength MS salt medium supplemented with 7.5 μM IBA: 3% (w/v) sucrose (7.05±1.08 and 7.0±1.51 cm, respectively). The phenol concentration was determined by analytical HPLC. The highest content of nordihydroguiaretic acid (NDGA) accumulated in microplants of L. divaricata cultivated on half-strength MS salt medium (35.90±3.82 mg/g DW). Reducing the MS medium salt concentration by half, in the absence of IBA, it resulted in a higher NDGA production. NDGA production was not sensitive to the variation of IBA concentration. The medium supplemented with 5% (w/v) sucrose and 2.5 μM IBA induced not only a higher NDGA production but also a higher quercetin production.
Treatment of dermal wounds with macromolecular agents such as natural polymers is one of the research areas of the biomaterial science. Fucoidan is a sulphated polysaccharide which is commonly obtained from seaweeds. The great number of studies on the different pharmacological properties of fucoidan is present, but there is limited information about using of fucoidan in the treatment of dermal burns. The aim of this study was to prepare fucoidan-chitosan hydrogels and to investigate their treatment efficiency on dermal burns. Hydrogels were prepared by swelling the polymers in acidic solution and their swelling, mechanical (hardness, cohesiveness and adhesiveness) and bioadhesive properties were investigated. The viscosity and water absorption capacity of formulations increased with increase in the polymer concentration. In contrast to the cohesiveness results, the adhesiveness of hydrogels increased with the polymer concentration. The bioadhesion was changed between 0.012—0.142 mJ·cm−2 and enhanced with addition of fucoidan into gel formulations. It was formed dermal burns on seven adult male New Zealand white rabbits and the optimum gel formulation applied on the wounds. Control and treatment group biopsy samples were taken on days 7, 14 and 21 and each burn wound site was evaluated histopathologically. No edema was seen in tested groups except control after 3 d treatment. After 7 d treatment, fibroplasia and scar were fixed on wounds treated with fucoidan-chitosan gel and fucoidan solution. The best regeneration on dermal papillary formation and the fastest closure of the wounds were observed in fucoidan-chitosan hydrogels after 14 d treatment.
In the present study we extracted three isomeric acetogenins (Ace) from the seeds of Annona cherimolia MILL. (Annonaceae) and determined their genotoxic and cytotoxic potential in mice. Our results showed a significant increase in the rate of micronucleated polychromatic erythrocytes induced by Ace with respect to the value for the control group; the effect was less pronounced than that observed with daunorubicin (Dau). To evaluate cytotoxicity, we determined the proportion of polychromatic erythrocytes with respect to the number of normochromatic erythrocytes, and we found an inhibitory effect induced by Ace that was quite similar to that observed with Dau. Besides, by means of the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide test, we also determined the cytotoxicity of Ace in both a normal fibroblast mouse cell line and in a line derived from human colon cancer. In this assay, the strongest decrease in viability corresponded to the malignant cell line. Our results established for the first time the genotoxic capacity of these compounds in vivo, and confirmed their cytotoxic potential in cultured cells.