Electroconvulsive seizure (ECS) therapy is a clinically proven treatment for depression and is often effective even in patients resistant to chemical antidepressants. However, the molecular mechanisms underlying the therapeutic efficacy of ECS are not fully understood. Here, I review studies that show molecular, cellular, and behavioral changes by ECS treatment, and discuss the functions of ECS to underlie the action of antidepressant effects. In hippocampus, these changes cover gene induction, increased adult neurogenesis, and electrophysiological reactivity. Especially, the role of vascular endothelial growth factor (VEGF) in neurogenesis is discussed. Among other gene expression changes in hippocampus, a role of cyclooxygenase (COX)-2, an inducible type of the rate-limiting enzyme of prostanoid synthesis, is focused. ECS-induced changes in other brain regions such as prefrontal cortex and hypothalamus, and ECS-induced behavioral changes are also reviewed. Understanding the molecular, cellular, and behavioral changes by ECS will provide a new view to find potential targets for novel antidepressant design that are highlighted by these findings.
During the past decade formulation of vesicles as a tool to improve drug delivery, has created a lot of interest amongst the scientist working in the area of drug delivery systems. Vesicular system such as liposomes, niosomes, transferosomes, pharmacosomes and ethosomes provide an alternative to improve the drug delivery. Niosomes play an important role owing to their nonionic properties, in such drug delivery system. Design and development of novel drug delivery system (NDDS) has two prerequisites. First, it should deliver the drug in accordance with a predetermined rate and second it should release therapeutically effective amount of drug at the site of action. Conventional dosage forms are unable to meet these requisites. Niosomes are essentially non-ionic surfactant based multilamellar or unilamellar vesicles in which an aqueous solution of solute is entirely enclosed by a membrane resulting from the organization of surfactant macromolecules as bilayer. Niosomes are formed on hydration of non-ionic surfactant film which eventually hydrates imbibing or encapsulating the hydrating aqueous solution. The main aim of development of niosomes is to control the release of drug in a sustained way, modification of distribution profile of drug and for targeting the drug to the specific body site. This paper deals with composition, characterization/evaluation, merits, demerits and applications of niosomes.
Reactive oxygen species, including hydrogen peroxide (H2O2), are known to induce β-cell apoptosis. The present study investigated the role of Ca2+ in H2O2-induced apoptosis of the β-cell line INS-1. Annexin V assay with flow cytometry and DNA ladder assay demonstrated that treatment of INS-1 cells with 100 μM H2O2 for 18 h significantly increased apoptotic cells. A comparable level of apoptosis was also observed after 18 h when the cells were treated with 100 μM H2O2 only for initial 30 min. The H2O2-induced apoptosis was abolished by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA/AM), a chelator of intracellular Ca2+, by 2-aminoethoxydiphenylborate (2-APB), a blocker of inositol 1,4,5-trisphosphate (IP3) receptors and cation channels, and by xestospongin D, a blocker of IP3 receptors, and was partially blocked by SKF-96365, a non-selective cation channel blocker. However, nicardipine, an L-type voltage-dependent Ca2+ channel blocker, or N-(p-amylcinnamoyl)anthranilic acid (ACA), a TRPM2 blocker, had little effect on the apoptosis. The inhibitory effect of BAPTA/AM or 2-APB on the H2O2-induced apoptosis was largely attenuated when the drug was added 30 min or 1 h after start of the treatment with H2O2. These results suggest that the initial intracellular Ca2+ elevation induced by H2O2, which is mediated via IP3 receptors and store-operated cation channels, plays an obligatory role in the induction of β-cell apoptosis.
Cornuside, a secoiridoid glucoside compound, was isolated from the fruit of Cornus officinalis SIEB. et ZUCC. Cornuside has been reported to possess immunomodulatory and anti-inflammatory activities. However, the effects and mechanism of action of cornuside in inflammation have not been fully characterized. The present study was therefore designed to examine whether cornuside suppresses inflammatory response in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Cornuside significantly inhibited the LPS-induced production of nitric oxide, prostaglandin E2, tumor necrosis factor-alpha, interleukin-6 (IL-6), and IL-1beta. The mRNA and protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were also decreased by cornuside. Furthermore, cornuside significantly attenuated the LPS-stimulated phosphorylation and degradation of inhibitory kappa B-alpha and the subsequent translocation of the p65 subunit of nuclear factor-kappa B (NF-κB) to the nucleus. Cornuside also reduced the phosphorylations of extracellular-signal-related kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK1/2). These results suggest that the anti-inflammatory property of cornuside is related to the downregulations of iNOS and COX-2 due to NF-κB inhibition as well as the negative regulation of ERK1/2, p38, and JNK1/2 phosphorylations in RAW 264.7 cells.
Bufalin, a traditional Chinese medicine, has been reported as a protective factor in many tumors. We therefore investigated the effect of bufalin on platelet-derived growth factor (PDGF)-BB-induced proliferation of cultured rat mesangial cells. The effect of bufalin on cell proliferation and its underlying mechanisms were investigated in cultured rat mesangial cells (MCs) by the methylthiazoletetrazolium (MTT) assay, flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and cyclin-dependent kinases (CDK)2 and CDK4 kinase assays. Bufalin inhibited 20 ng/ml PDGF-BB-induced MC proliferation in a dose-dependent manner. Similar results were observed in different concentrations of bufalin, which blocked PDGF-BB-induced progression through G0/G1 to S phase of the cell cycle. Furthermore, bufalin not only inhibited upregulation of cyclin D1 and CDK4, but also downregulation of p21 in both mRNA and protein levels. Although bufalin did not affect p27 and CDK2 mRNA expression, it reversed downregulation of p27 and upregulation of CDK2 in protein level. Activity of CDK2 and CDK4 was also inhibited by bufalin. However, both bufalin and PDGF-BB did not affect cyclin E mRNA or protein expression. These results suggest that bufalin could inhibit MC proliferation by modulating cell cycle progress, indicating that bufalin could be a potential therapeutic agent for the prevention of mesangial proliferative glomerulonephritis.
Curcumin is a natural polyphenolic compound abundant in the rhizome of the perennial herb turmeric, Curcuma longa. It is commonly used as a dietary spice and coloring agent in cooking, and is used anecdotally as an herb in traditional Indian and Chinese medicine. It has been reported that curcumin has the potential to protect against cardiac inflammation through suppression of GATA-4 and nuclear factor-κB (NF-κB); however, no study to date has addressed the effect of curcumin on experimental autoimmune myocarditis (EAM) in rats. In this study, 8-week-old male Lewis rats were immunized with cardiac myosin to induce EAM. They were then divided randomly into a treatment or vehicle group and orally administrated curcumin (50 mg/kg/d) or 1% gum arabic, respectively, for 3 weeks after myosin injection. We performed hemodynamic, echocardiographic, hematoxylin and eosin staining, mast cell staining and Western blotting studies to evaluate the protective effect of curcumin in the acute phase of EAM. Cardiac functional parameters measured by hemodynamic and echocardiographic studies were significantly improved by curcumin treatment. Furthermore, curcumin reduced the heart weight-to-body weight ratio, area of inflammatory lesions and the myocardial protein level of NF-κB, interleukin (IL)-1β, tumor necrosis factor (TNF)-α and GATA-4. Our results indicate that curcumin has the potential to protect against cardiac inflammation through suppression of IL-1β, TNF-α, GATA-4 and NF-κB expresses, and may provide a novel therapeutic strategy for autoimmune myocarditis.
We investigated the effects of the α1-adrenergic agonist phenylephrine on platelet-derived growth factor (PDGF)-stimulated extracellular signal-regulated kinase (ERK) in primary cultures of adult rat hepatocytes. Hepatocytes were isolated and cultured with PDGF (10 ng/ml) and/or α-adrenergic agonist. Phosphorylated ERK isoforms (ERK1 and ERK2) were detected by Western blotting analysis using anti-phospho mitogen-activated protein kinase (MAPK) antibody. PDGF stimulated phosphorylation of ERK2 (42 kDa MAPK) by 2.0-fold within 3—5 min. The PDGF-induced ERK activation was abolished by AG1296 (10−7 M) or LY294002 (10−7 M) treatment. MAPK kinase inhibitor, PD98059 (10−6 M), completely inhibited the PDGF-induced increase in ERK activity. In addition, PDGF-induced mammalian target of rapamycin activity was completely inhibited by AG1296, LY294002, PD98059, or rapamycin treatment. Phenylephrine alone showed no effects on ERKs, but significantly increased phosphorylation of ERK2 induced by PDGF. Moreover, a synthetic analog of diacylglycerol (DG), phorbol 12-myristate 13 acetate (TPA; 10−7 M), potentiated PDGF-induced ERK2 phosphorylation, while ionomycin had no effect (10−6 M). The effects of phenylephrine and TPA were antagonized by the phospholipase C (PLC) inhibitor U73122 (10−7 M), and the protein kinase C (PKC) inhibitor GF109203X (10−7 M), respectively. Accordingly, PDGF-induced DNA synthesis and proliferation in the presence or absence of phenylephrine or TPA were completely inhibited by AG1296, LY294002, PD98059, or rapamycin treatment. These results suggest that activation of PLC/PKC by phenylephrine represent an indirect positive regulatory mechanism for stimulating ERK induced by 10 ng/ml PDGF.
Ferrous ferric chloride (FFC) is a distinct form of aqueous iron composed of a complex of ferrous chloride and ferric chloride that participates in both oxidation and reduction reactions, and stimulates the proliferation and differentiation of mammalian keratinocytes, melanocytes, and fibroblasts. However, it is not known whether FFC can stimulate their proliferation and differentiation without being added into culture media or painted on the skin. This study aims to clarify whether FFC can stimulate their proliferation and differentiation from a distance without being added to culture media. In this study, FFC-containing skin lotions were painted under the culture dishes (1 mm away from cells) or on the top of the covers of 1 to 5 polystyrene culture dishes (1 to 5 cm away) and tested for their proliferation- and differentiation-stimulating effects. FFC lotions stimulated the proliferation and differentiation of human keratinocytes, melanocytes, and fibroblasts from a distance of 1 mm to 1 cm. However, FFC lotions failed to stimulate the proliferation and differentiation of melanocytes from distances of 2 to 5 cm. Results using Teflon covers were similar to those of polystyrene covers. Moreover, the effects of FFC lotions painted on the top of the Teflon covers were completely lost by lead disks. These results suggest that FFC can stimulate the proliferation and differentiation of skin cells from a distance of 1 cm without being added into culture media through physical factors rather than chemical factors.
Human parainfluenza virus type 1 (hPIV1) generally does not show visible plaques in common cell lines, including Lewis lung carcinoma-monkey kidney (LLC-MK2) cells, by plaque formation assays for human parainfluenza virus type 3 (hPIV3) and Sendai virus. In several conditions of the plaque formation assay, complete elimination of serum proteins in the overlay medium was necessary for visualization of hPIV1-induced plaque formation in LLC-MK2 cells. We developed a plaque formation assay for hPIV1 isolation and titration in LLC-MK2 cells using an initial overlay medium of bovine serum albumin-free Eagle's minimum essential medium containing agarose and acetylated trypsin for 4—6 d followed by a second overlay staining medium containing agarose and neutral red. The assay allowed both laboratory and clinical hPIV1 strains to form large plaques. The plaque reduction assay was also performed with rabbit anti-hPIV1 antibody as a general evaluation model of viral inhibitors to decrease both the plaque number and size. The results indicate that the plaque formation assay is useful for hPIV1 isolation, titration, evaluation of antiviral reagents and epidemiologic research.
Worldwide, lung cancer is the most common form of cancer and often has a poor prognosis. Establishment of effective therapies for lung cancer is a major concern in clinical cancer research. We compared the cytotoxic effects of chemotherapeutic agents including cisplatin, 5-fluorouracil, vinorelbine and cladribine, on a human lung cancer cell line, A549, and its derivative transfected with the DNase γ gene. We observed selective cytotoxicity of cladribine on the DNase γ-expressing sub-cell line of A549. Cladribine induces selective apoptosis in DNase γ-expressing A549 cells, which depends on activation of caspases. These results suggest that a combination therapy that includes cladribine along with the introduction of DNase γ has potential as a new therapeutic strategy for lung cancer.
Our previous studies have demonstrated that lipid peroxidation in the lenses of hereditary cataract model rats (Ihara cataract rat (ICR)/f rats) caused a dysfunction in Ca2+ regulation. In the present study, we investigated the effect of in vitro hydrogen peroxide (H2O2) stimulation on lipid peroxide (LPO) and the activities of sarco-/endoplasmic reticulum and plasma membrane Ca2+-ATPase (SERCA and PMCA) in the ICR/f rat lenses. An increase in LPO level and decreases in the SERCA and PMCA activities were observed with increasing H2O2 concentration, and pretreatment with diethyldithiocarbamate, a potent radical scavenger, attenuated these changes in normal and ICR/f rat lenses. The glutathione levels, glutathione peroxidase and glutathione reductase activities are significantly lower in ICR/f rat lenses than in normal rat lenses. Furthermore, we presented as two kinetic parameters such as DP (defense point) and Ks (reactive constant) analyzed from above various biological responses vs. H2O2 concentration–profile curves using a one-exponential equation. The DPs for LPO, SERCA and PMCA in ICR/f rat lenses is lower than in normal rat lenses. In contrast to the results in DP, the Ks for LPO, SERCA and PMCA in ICR/f rat lenses is higher than in normal rat lenses. In addition, the closed relationship of was observed between DP and Ks for LPO, SERCA and PMCA. These results show that the resistance to H2O2 in the ICR/f rat lenses is lower than that of normal rats. The DP and Ks values can provide an useful information for resistances to various stimuli in cells and tissues.
Intestinal microbiotas of human subjects and effect of antibiotic treatment on them have been reported with cultivation independent methods. However, Japanese fecal microbiotas have not been studied enough. We have constructed a clone library method to obtain results within 3 d. In this study, intestinal microbiotas of 29 healthy Japanese adults, whose fecal samples were collected twice at 5 month intervals from each subject, were analyzed with our clone library method, and using those data as a benchmark effect of antibiotic treatment on intestinal microbiotas was evaluated. The fifty-eight fecal microbiotas were assessed based on percentages at genus level, and the variability was analyzed with a principal component analysis (PCA). PCA showed that the microbiotas divided into three groups depending on the large eigenvectors (genera Ruminococcus, Bacteroides, and Prevotella), and the dual samples from the twenty-two individuals have belonged to the same PCA group. It suggests that almost Japanese adults have own stable intestinal microbiota. The genera Ruminococcus and Bacteroides were present in almost subjects, while the genus Prevotella was found only in nine subjects (approximately 30%) which was preserved with 5 months intervals. Next, the microbiotas before and after antibiotic treatment were evaluated comparing with the 58 healthy adult microbiotas. The results showed that beta-lactams influenced profoundly on intestinal microbiotas and the effect of macrolides depended on the cases. It suggests that our clone library method could show overview of intestinal microbiota and would give us useful information about the effect of antibiotic treatment for daily clinical diagnosis.
To find anti-inflammatory compounds from the tuberous roots of Dioscorea batatas, we isolated 6-hydroxy-2,4,7-trimethoxyphenanthrene (batatasin I) from the dichloromethane (CH2Cl2) fraction of this plant. Batatasin I inhibited both the generation of prostaglandin D2 (PGD2), leukotriene C4 (LTC4) and degranulation reaction in mouse bone marrow-derived mast cells (BMMCs). This compound inhibited cyclooxygenase-2 (COX-2) dependent PGD2 generation in a dose dependent manner, with IC50 values of 1.78 μM. Western blotting probed with specific anti-COX-2 antibodies showed that the decrease in the quantity of the PGD2 generation was accompanied by a decrease in the COX-2 protein level. In addition, this compound also inhibited the production of 5-lipoxygenase (5-LOX) dependent LTC4 in a dose dependent manner (IC50, 1.56 μM). Batatasin I also inhibited the mast cell degranulation reaction (IC50, 6.7 μM) in BMMCs. This result indicates that batatasin I could be developed as an anti-inflammatory agent through further investigation.
The generation of nitric oxide (NO) via inducible NO synthase (iNOS) and reactive oxygen species plays a key role in cytokine-mediated pancreatic β-cell damage. Oxidative stress due to reactive oxygen species activates the nuclear factor-κB (NF-κB) transcription factor, which regulates iNOS expression. In this regard, suppression of the NF-κB pathway is a novel strategy for protecting β-cells from damage. This study was performed to explore the effects of kazinol U, a prenylated flavan from Broussonetia kazinoki, on the NF-κB activation pathway in interleukin-1β (IL-1β)- and interferon-γ (IFN-γ)-treated β-cells. The cytotoxic effects of cytokines were completely abolished when RINm5F cells or islets were pretreated with kazinol U. Kazinol U inhibited the nuclear translocation and DNA binding of NF-κB subunits, which correlated with the inhibitory effects on IκB kinase (IKK) phosphorylation and IκBα degradation. In addition, kazinol U suppressed NO and hydrogen peroxide production and apoptotic cell death by cytokines in RINm5F cells. The protective effects of kazinol U were further demonstrated by normal insulin secretion of cytokine-treated islets in response to glucose. Taken together, these results suggest that using kazinol U to block the NF-κB pathway in pancreatic β-cells reduces cell damage. Therefore, kazinol U may have therapeutic value in delaying pancreatic β-cell destruction in type 1 diabetes.
In this study, luteolin-7-O-glucoside (L7G), an herbal medicine isolated from Ailanthus altissima, inhibited 5-lipoxygenase (5-LOX)-dependent leukotriene C4 (LTC4) production in bone marrow-derived mast cells (BMMCs) in a concentration-dependent manner with an IC50 of 3.0 μM. To determine the action mechanism of L7G, we performed immunoblotting for cytosolic phospholipase A2 (cPLA2) and mitogen-activated protein kinases (MAPKs) following c-kit ligand (KL)-induced activation of BMMCs with or without L7G. Inhibition of LTC4 production by L7G was accompanied by a decrease in cPLA2 phosphorylation, which occurred via the extracellular signal-regulated protein kinase-1/2 (ERK1/2) and p38 and c-Jun N-terminal kinase (JNK) pathways. In addition, L7G also attenuated mast cell degranulation in a dose-dependent manner (IC50, 22.8 μM) through inhibition of phospholipase Cγ1 (PLCγ1) phosphorylation. Our results suggest that the anti-asthmatic activity of L7G may be mediated in part via the inhibition of LTC4 generation and mast cell degranulation.
Reprogramming differentiated cells toward stem cells may have long-term applications in stem-cell research and regenerative medicine. Here we report on the dedifferentiation of human epidermal keratinocytes into their precursor cells in vitro with basic fibroblast growth factor (bFGF) but not external gene intervention. After incubation of human terminally differentiating keratinocytes, some of the surviving keratinocytes reverted from a differentiated to a dedifferentiated state, as evidenced by re-expression of biological markers of native keratinocyte stem cells (nKSCs), including β1-integrin, CK19 and CK14. Moreover, these dedifferentiation-derived KSCs (dKSCs) showed an ability for high colony formation correlated with cell cycle analysis showing a marked accumulation in S phases, acquired a similar regional distribution of both α6-integrin and CD71 expression at the ultrastructural level, and had a increased proliferative capacity by releasing telomerase from nucleolar sites to nucleoplasmic distribution. However, on comparing dKSCs with nKSCs, 2 points seem noteworthy: (1) the proportion of transit amplifying cells in dKSCs treated with bFGF is much higher than that in nKSCs and (2) regional differences exist in the subcellular localization of telomerase in nKSCs and dKSCs. Most nKSCs showed a prominent nucleolar concentration of human telomerase reverse transcriptase expression, whereas most dKSCs showed a more diffuse intranuclear distribution of telomerase or even signal depletion at nucleoli relative to the general nucleoplasm. These results indicate that bFGF could induce the terminally differentiating epidermal keratinocytes to convert into their precursor cells, which offers a new approach for generating residual healthy stem cells for wound repair and regeneration.
Guinea pigs and rats are both common animal models for hyperlipidemia studies. However, many recent studies have suggested that rats do not develop hypertriglyceridemia in response to cholesterol feeding. In the present work, the differences in triglyceride metabolism between guinea pigs and rats were investigated. Feeding a high-fat diet containing 0.1% cholesterol and 10% lard for 4 weeks led to a significant increase in plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG) and free fatty acid (FFA) in guinea pigs but not in rats. By contrast, hepatic TG levels in rats were greatly increased in response to the high-fat diet, while it remained unchanged in guinea pigs. Furthermore, the hepatic acyl CoA:diacylglycerol acyltransferase (DGAT) activity and microsomal triglyceride transfer protein (MTTP) mRNA levels in guinea pigs fed a high-fat diet were significantly higher than those in the control group, which implies an increased very-low-density lipoprotein (VLDL)-TG secretion rate in guinea pigs in response to a high-fat diet. Hepatic carnitine palmitoyltransferase-1 (CPT-1) activity and peroxisome proliferator-activated receptor-α (PPARα) mRNA levels were upregulated in guinea pigs, but not rats, fed a high-fat diet. These findings may explain the differences in plasma and hepatic TG concentrations between guinea pigs and rats. These results suggest that there are differences in triglyceride metabolism between the two species when fed high-fat diets.
Inflammation takes responsibility for the seawater aspiration-induced lung injury. Tanshinone IIA (TIIA) can protect lipopolysaccharide-induced lung injury in mice through the inhibition of inflammation, but it is not reported whether TIIA have a protective effect on lung injury induced by seawater aspiration. Macrophage migration inhibitory factor (MIF) plays an important role in acute lung injury. In this study, we observed the effect of TIIA on the seawater aspiration-induced lung injury and the role of MIF in it. Seawater was aspirated into trachea of rats to make the lung injury model. TIIA was administered to investigate its beneficial effect on seawater-induced acute lung injury. The results showed that seawater aspiration led to hyoxemia, pulmonary edema, neutrophil infiltration, and lung histopathologic changes, with the elevated MIF expression in the lung tissues and plasma. However, these changes were attenuated by TIIA. In macrophage cells we also demonstrated that TIIA could inhibit MIF expression, nuclear factor κB (NF-κB) activity and release of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) induced by seawater. Besides, pretreatment with (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid (ISO-1), the MIF antagonist, elevated NF-κB and cytokines induced by seawater were also reduced markedly. Furthermore, rMIF treatment alone increased the phosphorylation level of NF-κB and release of cytokines, which was almost abolished by TIIA. Taken together, our results suggested that TIIA exert a protective effect on the seawater aspiration-induced lung injury partly through downregulation of MIF and the subsequent NF-κB activity, as well as expression of IL-6 and TNF-α.
Adriamycin (ADM)-encapsulated thermosensitive liposomes (ts-lip-ADM) and common liposomes (lip-ADM) were developed and evaluated. The encapsulation efficiency of the two liposomes were above 99%, and the average sizes of liposomes were about 120 nm. Temperature-dependent drug release from loaded liposomes in vitro was investigated: more than 90% of loaded ADM was released from ts-lip-ADM within 30 min at 42°C, while less than 3% was released from lip-ADM at 42°C beyond 120 min. An in vitro model of blood brain barrier (BBB) was established and evaluated by permeability and transendothelial electrical resistance (TEER). The model was employed to study the permeability of liposomes in vitro. The permeability of ts-lip-ADM could be increased significantly after the temperature was raised to 42°C, which was about 10—16, 22—38, 38—45, 50—105 fold to that of ts-lip-ADM (37°C), lip-ADM (42°C), lip-ADM (37°C) and free ADM, respectively. C6 glioma-bearing mice model was developed and used to evaluate body distribution and anti-tumor efficacy in vivo. Mice were IV injected at a drug dose of 10 mg/kg. After administration the heads of mice were heated in water bath at 42°C for 30 min. The maximum brain concentration of ts-lip-ADM was 6.4, 3.7 fold compared with that of ADM solution and lip-ADM, respectively. The survival time of mice administered ts-lip-ADM (44 d) was remarkably longer than that of other three groups. This study indicates that ADM-encapsulated thermosensitive liposomes combined hyperthermia could enhance ADM delivery across BBB and prolong survival time of glioma-bearing mice.
Due to the predominance of multiple-antibiotic-resistant Klebsiella pneumoniae strains, the search for new approaches for the prevention of K. pneumoniae infections is now under intensive investigation. The objective of the present study was to investigate the effects of high mobility group nucleosomal binding domain 2 (HMGN2) protein, which acts on the bladder epithelial cells T 24, on the invasion of K. pneumoniae 03183 and explore its possible mechanisms. Pretreatment with HMGN2 significantly reduced K. pneumoniae 03183 uptake by T 24 cells. In T 24 cells, there were no detectable cytotoxic effects of HMGN2 at any concentration between 32 and 256 μg/ml after 2 h incubation. HMGN2 exhibited no appreciable antibacterial activity against K. pneumoniae 03183. Fluorescence microscopy and flow cytometry analysis revealed that HMGN2 blocked K. pneumoniae 03183-induced actin polymerization. K. pneumoniae 03183-induced phosphorylation of extracellular signal-regulated kinase (ERK) and cofilin were prevented by pretreatment with HMGN2. These results indicated that pretreatment with HMGN2 inhibited cofilin phosphorylation and then induced actin disruption which may block ERK phosphorylation. These changes led to inhibition of K. pneumoniae 03183 invasion of T 24 bladder epithelial cells.
Senescence of endothelial cells has been proposed to play an important role in endothelial dysfunction and atherogenesis. In the present study we aimed to investigate whether ginsenoside Rb1, a major constituent of ginseng, protects endothelial cells from H2O2-induced endothelial senescence. While H2O2 induced premature senescent-like phenotype of human umbilical vein endothelial cells (HUVECs), as judged by increased senescence-associated β-galactosidase (SA-β-gal) activity, enlarged, flattened cell morphology and sustained growth arrest, our results demonstrated that Rb1 protected endothelial cells from oxidative stress induced senescence. Mechanistically, we found that Rb1 could markedly increase intracellular superoxide dismutase (Cu/Zn SOD/SOD1) activity and decrease the malondialdehyde (MDA) level in H2O2-treated HUVECs, and suppress the generation of intracellular reactive oxygen species (ROS). Consistent with these findings, Rb1 could effectively restore the protein expression of Cu/Zn SOD, which was down-regulated in H2O2 treated cells. Taken together, our data demonstrate that Rb1 exhibits antioxidant effects and antagonizes H2O2-induced cellular senescence.
Formation of α-synuclein aggregates is a key step in Parkinson's disease pathogenesis although the etiology remains elusive. α-Synuclein is accumulated in degenerating neurons, leading to the production of filamentous inclusions such as Lewy bodies. However, the in vitro overexpression of α-synuclein alone failed to induce inclusion bodies consisting of phosphorylated α-synuclein. The seeded aggregates-initiated polymerization of α-synuclein and tau has been reported elsewhere. What molecule is an initiator of filamentous inclusions remains to be defined. Here, we report that leucine-rich repeat kinase 2 (LRRK2)-cotransfection together with α-synuclein enhance the aggregate formation, phosphorylation, release to extracellular media of α-synuclein, and the cell-to-cell transmission into neighboring cells in human neuroblastoma SH-SY5Y cells. In cells transfected with α-synuclein alone, the proteins were distributed in the cytosol and did not form inclusions. On the other hand, the inclusions and phosphorylation of α-synuclein were formed in cells cotransfected with α-synuclein and LRRK2 G2019S mutant together. LRRK2 G2019S-cotransfected PC12 cells also induced the aggregates. Furthermore, the cell-to-cell transmission of α-synuclein and the cell toxicity were also enhanced by either LRRK2 wild type or G2019S mutant, whereas the cell viability was not decreased in cells transfected with α-synuclein alone. These results suggest that overexpression of LRRK2, especially G2019S mutant, whose functions remain unclear, initiate the aggregate formation, release and transmission of α-synuclein, resulting in the propagation of α-synuclein to neighboring cells and reduction of cell viability.
Hyaluronic acid (HA) is a naturally-occurring ligand that can be useful for targeting liver endothelial cells. We describe herein the development of a new HA-lipid conjugate for the efficient delivery of liposomes to liver endothelial cells. When free HA coated cationic liposomes were injected into mice, their accumulation in the liver was significantly decreased depending on the content of free HA, while accumulation in the lung was not significantly changed. When cationic liposomes modified with HA-stearylamine (HA-SA conjugate) were injected in mice, liver accumulation was increased depending on the amount of HA-SA conjugate and accumulation in the lung was drastically reduced, compared to non-modified liposomes. Confocal imaging analyses showed that HA-SA modified liposomes were accumulated to a greater extent along with blood vessels than non-modified liposomes, suggesitng that HA-SA modified liposomes are distributed in endothelial cells in the liver. Collectively, these findings indicate that an HA-SA conjugate is a useful material that can be used to modify liposomes and for delivering bioactive liposomal cargoes to liver endothelial cells.
Cannabinoids elicit biological responses through two types of specific receptors, CB1 and CB2. Immune cells including naïve B-lymphocytes are known to selectively express peripheral cannabinoid receptors, CB2. Although the immunosuppressive effects of cannabinoids have become widely known, the mechanisms underlying their effects are not well understood. In this study, we demonstrated that splenic lymphocytes migrated toward a synthetic cannabinoid receptor agonist, WIN55,212-2. There is an optimal concentration range for induction of lymphocyte migration and a high dose fails to induce cell migration. Furthermore, a high dose of WIN55,212-2 significantly inhibited CXCL12-induced chemotaxis of lymphocytes. The inhibitory effect was transient and reversible. The inhibition was also observed when purified B-lymphocytes were used for CXCL12-induced chemotaxis. These results provide novel information regarding the cellular mechanisms underlying the effects of cannabinoids on the immune system.
Peroxisome proliferator-activated receptor γ (PPARγ; NR1C3) is known as a key regulator of adipocytogenesis and the molecular target of thiazolidinediones (TZDs), also known as antidiabetic agents. Despite the clinical benefits of TZDs, their use is often associated with adverse effects including peripheral edema, congestive heart failure, and weight gain. Here we report the identification and characterization of a non-thiazolidinedione PPARγ partial agonist, Cerco-A, which is a derivative of the natural product, (−)-cercosporamide. Cerco-A was found to be a binder of the PPARγ ligand-binding domain in a ligand competitive binding assay and showed a unique cofactor recruitment profile compared to rosiglitazone. A crystal structure analysis revealed that Cerco-A binds to PPARγ without direct hydrogen bonding to helix12. In PPARγ transcriptional activation assay and an adipocyte differentiation assay, Cerco-A was a potent partial agonist of PPARγ. After a 14-day oral administration, once per day of Cerco-A in Zucker diabetic fatty (ZDF) rats, an apparent decrease of plasma glucose and triglyceride was observed, as with pioglitazone. To evaluate drug safety, Cerco-A was administered for 13 days orally in non-diabetic Zucker fatty (ZF) rats. Each of the hemodilution parameters (hematocrit, red blood cells number, and hemoglobin), which are considered as undesirable effects of TZDs, was improved significantly compared to pioglitazone. While Cerco-A showed body weight gain, as with pioglitazone, Cerco-A had significantly lower effects on heart and white adipose tissues weight gain. The results suggest that Cerco-A offers beneficial effects on glycemic control with attenuated undesirable side effects.
Transient receptor potential vanilloid 1 (TRPV1) is primarily expressed in central and peripheral terminals of non-myelinated primary afferent neurons. We previously showed that AS1928370, a novel TRPV1 antagonist that can prevent ligand-induced activation but not proton-induced activation, ameliorates neuropathic pain in rats without hyperthermic effect. In this study, we investigated its analgesic profile in mice. AS1928370 showed good oral bioavailability and high penetration into the brain and spinal cord in mice. The mean plasma-to-brain and plasma-to-spinal cord ratios were 4.3 and 3.5, respectively. Pretreatment with AS1928370 significantly suppressed both capsaicin-induced acute pain and withdrawal response in hot plate test at 10—30 mg/kg per os (p.o.). At lower oral doses (0.3—1.0 mg/kg), AS1928370 improved mechanical allodynia in mice undergoing spinal nerve ligation. Intrathecal administration of AS1928370 (30 μg/body) also significantly suppressed mechanical allodynia. In addition, AS1928370 showed no effect on locomotor activity up to 30 mg/kg p.o. These results suggest that spinal TRPV1 has an important role in the transmission of neuropathic pain and that the central nervous system (CNS) penetrant TRPV1 receptor antagonist AS1928370 is a promising candidate for treating neuropathic pain.
Proanthocyanidin is a phenolic compound present in plants, that has antioxidant, antinociceptive, anti-emetic, and neuroprotective properties. We investigated the actions of proanthocyanidin from grape seeds on 5-hydroxytryptamine (5-HT)3 receptors in NCB-20 neuroblastoma cells using a whole-cell voltage clamp technique. Co-treatment of proanthocyanidin (0.3—100 μg/ml) and 3 μM 5-HT (near EC50) produced a slight inhibition of 5-HT-induced inward peak current (I5-HT) in NCB-20 cells, but pretreatment with proanthocyanidin for 30 s before application of 5-HT induced a much larger inhibition of I5-HT in an irreversible, concentration- and time-dependent manner (IC50=6.5±0.4 μg/ml, Hill coefficient=2.5±0.1). Proanthocyanidin also produced a concentration-dependent inhibition of currents induced by 30 μM 5-HT, near-maximal concentration (IC50=22.1±0.4 μg/ml, Hill coefficient=2.4±0.1). High concentrations (≧30 μg/ml) of proanthocyanidin caused a concentration-dependent inhibition of the activation and desensitization of currents induced by 30 μM 5-HT. Further studies showed that pretreatment of 20 μg/ml proanthocyanidin caused not only a rightward shift of the dose–response curve for 5-HT (EC50 shift from 2.7±0.4 to 6.2±0.5 μM), but also a decreased Emax (inhibition by 37.5±1.3%). The proanthocyanidin-induced inhibition of 5-HT3 receptors did not show a significant difference within the testing holding potential ranges (−50—+30 mV). These results suggest that proanthocyanidin inhibits 5-HT3 receptor function in NCB-20 cells in a noncompetitive mode, and that this inhibitory effect of proanthocyanidin probably contributes to the pharmacological actions of proanthocyanidin.
The spontaneously hypertensive rat (SHR)/NDmcr-cp (SHR-cp), which is a metabolic syndrome model rat, was reported to show hypercholesteremia, as compared with lean littermates. The serum total cholesterol level in SHR-cp at 18 weeks of age is higher than that of normotensive Wistar Kyoto rat (WKY), but that in SHR-cp at 10 weeks of age is the same. The objective of this study is to clarify whether there are differences in the system regulating serum cholesterol levels between SHR-cp and WKY at 10 weeks of age. Total serum cholesterol levels, and cholesterol levels of high density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein (VLDL) were similar in the two strains. However, the cholesterol levels in the liver of SHR-cp were lower than those of WKY. Next, mRNA levels of receptors (scavenger receptor class B type 1 [SRB1], LDL receptor [LDLR]) involved in uptake from serum to liver or enzymes of cholesterol catabolism (CYP7A1 and CYP8B1) and biosynthesis (mevalonate pyrophosphate decarboxylases [MPD]) in liver were compared between SHR-cp and WKY. High levels of MPD and LDLR and low levels of SRB1 were shown in SHR-cp, as compared with WKY. CYP7A1 and CYP8B1 levels were similar between SHR-cp and WKY. These results suggest that the serum cholesterol level in SHR-cp by the balance or regulation between the rise in cholesterol uptake and reduction in cholesterol biosynthesis in the liver is the same as that in WKY.
Fibroblast growth factor 21 (FGF21) is an effective metabolic regulator of glucose and lipid homeostasis in the context of insulin resistance, glucose intolerance and dyslipidemia in diabetic rodents and monkeys, and peroxisome proliferator-activated receptor α (PPARα) directly induces FGF21 expression in the rodent liver. Recent findings suggest that the effects and regulation of FGF21 qualitatively differ between rodents and humans. Here, we examined the effects of PPARα and PPARγ agonists on FGF21 mRNA expression in the mouse liver and in cultured hepatocytes. Intraperitoneal injection of both bezafibrate and pioglitazone induced FGF21 mRNA expression in the mouse liver. Rosiglitazone and pioglitazone as well as bezafibrate significantly induced FGF21 mRNA expression in cultured mouse hepatocytes. On the other hand, both rosiglitazone and pioglitazone significantly induced, whereas bezafibrate did not affect FGF21 mRNA expression in the human liver carcinoma cell line HepG2. Bezafibrate significantly induced pyruvate dehydrogenase kinase 4 mRNA expression, suggesting that HepG2 cells are sensitive to bezafibrate like the mouse liver. Our findings suggest that PPARγ-activating antidiabetic drugs such as rosiglitazone and pioglitazone induce FGF21 expression in mouse and human hepatocytes, and that PPARγ rather than PPARα might play an important role in human FGF21 production.
Protease-activated receptor-2 (PAR-2) is activated by serine proteases, such as trypsin and mast cell tryptase. Previous studies have demonstrated that both trypsin and PAR-2 activating peptide contract isolated rat urinary bladder preparations, however, the mechanisms are not fully understood. In the present study, we examined the role of bradykinin in contractions induced by trypsin and the PAR-2 agonist 2-furoyl-LIGRL-NH2 in urinary bladders isolated from control or cyclophosphamide (CYP)-induced cystitis rats. The contractile effects of trypsin were significantly greater in the preparations obtained from CYP-treated rats than in those from controls. The bradykinin B2 receptor antagonist Hoe 140 did not affect trypsin-induced contractions in control rat bladders, whereas it significantly reduced the contractile effects of trypsin on bladders from CYP-treated rats. On the other hand, Hoe 140 failed to affect contractions induced by the PAR-2 agonist 2-furoyl-LIGRL-NH2. These results suggest that the actions of trypsin on urinary bladders in cystitis rats are partly exerted through stimulation of bradykinin B2 receptor in a PAR-2-independent manner. This mechanism seems to be involved in the enhancement of trypsin-induced bladder contractions observed after induction of cystitis with CYP in rats.
A precise and convenient high-performance liquid chromatography (HPLC) method has been estab-lished to assay nilotinib in human plasma. Chromatographic separation of nilotinib was performed on a LiChrosphere®100 RP-18(e) column (250 mm×4.0 mm, 5 μm) using a mixture of acetonitrile and 0.01 M phosphate buffer (pH 3.0) (42 : 58, v/v) under isocratic conditions at a flow rate of 1.0 ml/min with ultraviolet (UV) detection at 266 nm. The calibration curve showed linearity at concentrations between 250 ng/ml and 5000 ng/ml (r2>0.999). The mean±S.D. absolute recovery of nilotinib from plasma was 99.2±3.3%. The coefficients of variation of both intra- and inter-day precision were below 9.1%. These results indicate that this new HPLC-based quantification may be useful for therapeutic drug monitoring of nilotinib to help manage treatment in patients with chronic myeloid leukemia in clinical practice.
Clotrimazole (CLO) is a local imidazolic antifungal agent. A major problem associated with the successful formulation of effective dosage forms containing CLO is its poor aqueous solubility, which presents a hindrance for the local availability of CLO and limits the effective antifungal therapy. In the present study, the effects of various concentrations of poly(amidoamine) (PAMAM) dendrimers generation 2 (G2) and generation 3 (G3) with amine (PAMAM-NH2) or hydroxyl surface groups (PAMAM-OH) on aqueous solubility and antifungal activity of CLO were studied. The obtained results showed that all tested PAMAM dendrimers improved the solubility of CLO and the more potent were PAMAM-NH2 dendrimers. The increase in solubility of CLO was highest at dendrimer concentration of 10 mg/ml. Microbiology studies indicated that only PAMAM-NH2 dendrimers significantly increased the antifungal activity of CLO (a 4—32-fold increase in the antifungal activity compared to pure CLO) and the most potent was dendrimer PAMAM-NH2 G2. These observations indicate that PAMAM dendrimers might be considered as potential carriers of CLO and provide further impetus to evaluate these polymers for use in basic drug delivery studies and to design semisolid dosage forms based on dendrimers with antimicrobial drugs, like CLO.
Galβ1-4Fuc disaccharide unit was recently reported to be the endogenous structure recognized by the galectin LEC-6 isolated from the nematode Caenorhabditis elegans. LEC-1, which is another major galectin from this organism, is a tandem repeat-type galectin that contains two carbohydrate recognition domains, the N-terminal lectin domain (LEC-1Nh) and the C-terminal lectin domain (LEC-1Ch), and was also found to have an affinity for the Galβ1-4Fuc disaccharide unit. In the present study, we compared the binding strengths of LEC-1, LEC-1Nh, and LEC-1Ch to Galβ1-4Fuc, Galβ1-3Fuc, and Galβ1-4GlcNAc units as well as to LEC-6-ligand N-glycans by using frontal affinity chromatography (FAC) analysis. The two lectin domains of LEC-1 exhibited the highest affinity for Galβ1-4Fuc, though sugar-binding properties differed somewhat between LEC-1Nh and LEC-1Ch. Furthermore, these two domains had significantly lower affinities for the LEC-6-binding glycans. These results suggest that the endogenous recognition unit of LEC-1 is likely to be Galβ1-4Fuc, and that the endogenous ligands for LEC-1 are different from those for LEC-6.
To study the endogenous counterpart of LEC-6, a major galectin in Caenorhabditis elegans, the proteomic analysis of glycoproteins captured by an immobilized LEC-6 column was performed using the nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique. A protein recovered in a significant amount was determined to be either F57F4.3 or F57F4.4, although the method used could not determine which protein was the actual counterpart. Because the knockdown of the F57F4.3/4 genes in C. elegans is reported to cause growth retardation, we performed a double knockdown of the lec-6 and F57F4.3/4 genes. Although the RNA-mediated interference (RNAi) of lec-6 led to no obvious phenotype, the RNAi of both the lec-6 and F57F4.3/4 genes led to a significant reduction in growth rate when compared to the RNAi of F57F4.3/4 alone. Furthermore, to clarify which protein, F57F4.3 or F57F4.4, was responsible for the retarded growth, the levels of the F57F4.3/4 proteins expressed in a C. elegans wild type and a mutant lacking part of the F57F4.3 gene were compared. The levels of protein expressed by the wild type and the mutant were not significantly different, suggesting that the F57F4.3 protein contributes very little to growth retardation and that the major glycoprotein that interacts with LEC-6 is the F57F4.4 protein. These results suggest that binding with LEC-6 supports the function of F57F4.4 and that their cooperative functioning regulates the growth of C. elegans.
Kaempferia parviflora (KP), a Zingiberaceae plant, is used as a folk medicine in Thailand for the treatment of various symptoms, including general pains, colic gastrointestinal disorders, and male impotence. In this study, the inhibitory activities of KP against xanthine oxidase (XOD) were investigated. The extract of KP rhizomes showed more potent inhibitory activity (38% at 500 μg/ml) than those of the other Zingiberaceae plants tested. Ten methoxyflavones were isolated from the KP extract as the major chemical components and their chemical structures were elucidated by X-ray crystallography. The structurally confirmed methoxyflavones were subjected to the XOD inhibitory test. Among them, 3,5,7,4′,5′-pentamethoxyflavone and 3′,4′,5,7-tetramethoxyflavone showed inhibitory activities (IC50 of 0.9 and >4 mM, respectively) and their modes of inhibition are clarified as competitive/non-competitive mixed type. To the best of our knowledge, this is the first report to present the inhibitory activities of KP, 3,5,7,4′,5′-pentamethoxyflavone and 3′,4′,5,7-tetramethoxyflavone against XOD.
Epidermis is one of the well-known estrogen target tissues. Information regarding estrogen metabolism in epidermis is still very limited compared to that of estrogen action. In the breast cancer tissue, 17β-estradiol (E2) is inactivated by sulfation and the expression level of estrogen sulfotransferase (SULT1E1) is inversely correlated with its malignancy. However, there is little datum about inactivation of estradiol in skin. In order to detect and measure E2 and its metabolites simultaneously, we established an assay method with radio HPLC. A majority of [3H] labeled E2 was converted to E2 sulfate in normal human epidermal keratinocyte (NHEK) cells. The estimated activity of sulfotransferase toward E2 at 20 nM was 0.11±0.01 (pmol/min/mg protein). Significant induction of estrogen sulfotransferase activity was observed in calcium-differentiated NHEK cells (0.58±0.07 (pmol/min/mg protein)). The gene expression of SULT1E1 was fifteen-fold higher in differentiated keratinocyte than in proliferating keratinocyte, whereas that of steroid sulfatase was reduced. These results suggest that E2 inactivation is primarily mediated by SULT1E1 in keratinocyte and E2 action is likely suppressed in epidermal differentiation.