Glycine is a well-documented cytoprotective agent and protects mammalian intestine against ischemia-reperfusion injury, irradiation and experimentally induced colitis. The specific glycine transporter GLYT1 is found throughout the human intestine where it is responsible for some 30—50% of glycine uptake into intestinal epithelial cells across the basolateral membrane and appears to function to maintain glycine supply to enterocytes and colonocytes. This paper reviews current knowledge of GLYT1 and presents recent evidence supporting its essential role in glycine mediated cytoprotection in intestinal absorptive cells. Regulatory mechanisms involved in intestinal expression of GLYT1 are discussed and the potential of glycine for use as an anti-inflammatory, protective agent in the management of inflammatory bowel disease examined.
Gaseous molecules such as nitric oxide (NO), hydrogen sulfide (H2S), or carbon monoxide (CO) are involved in the regulation of colonic water and salt transport, which can be switched between absorption and secretion. Nitric oxide is produced from the amino acid L-arginine by different isoforms of the enzyme NO synthase, which are expressed both by enteric neurones and by the colonic epithelium. NO donors evoke a transepithelial Cl− secretion in vitro. Most actions of NO are mediated by a stimulation of guanosine 5′ cyclic monophosphate (cGMP) synthesis via activation of the soluble guanylate cyclase. In rat colon, NO possesses several main action sites: a stimulation of apical Cl− channels most probably not related to cGMP-dependent phosphorylation, and an increase in the cytosolic Ca2+ concentration, which stimulates a Ca2+-dependent K+ conductance in the basolateral membrane. Hydrogen sulfide, produced during the metabolism of the amino acid L-cysteine, also evokes a Cl− secretion, either by stimulation of secretomotor submucosal neurones as in guinea-pig colon or by activating Ca2+-dependent and ATP-sensitive K+ channels as in rat colon. The third gasotransmitter, CO, produced during the degradation of heme, evokes anion secretion carried by Cl− and HCO3−. This response is mainly caused by the activation of apical anion channels and a stimulation of Ca2+-dependent K+ channels via an increase of the cytosolic Ca2+ concentration. Consequently, gaseous molecules produced by enteric neurones, epithelial cells, as well—in the case of H2S—the microbial flora affect key transport enzymes involved in colonic ion transport.
A number of transport mechanisms in the colonic epithelium contribute to HCO3− movement across the apical and basolateral membranes, but this ion has been largely regarded as a by-product of the transport functions it is involved in, such as NaCl- or short chain fatty acid (SCFA) absorption. However, emerging data points to several specific roles of HCO3− for colonic epithelial physiology, including pH control in the colonic surface microenvironment, which is important for transport- and immune functions, as well as the secretion and the rheological properties of the mucus gel. Furthermore, recent studies have demonstrated that colonic HCO3− transporters are expressed in a highly segmental as well as species-specific manner. This review summarizes recently gathered information on the functional anatomy of the colon, the roles of HCO3− in the colonic epithelium, colonic mucosal integrity, and the expression and function of HCO3− transporting mechanisms in health and disease.
The importance of intracellular pH (pHi) in the regulation of diverse cellular activities ranging from cell proliferation and differentiation to cell cycle, migration and apoptosis has long been recognised. More recently, extracellular pH (pHo), in particular that of relatively inaccessible compartments or domains that occur between cells in tissues, has begun to be acknowledged as a relevant signal in cell regulation. This should not be surprising given the abundant reports highlighting the pHo-dependence of the activity of membrane proteins facing the extracellular space such as receptors, transporters, ion channels and enzymes. Changes in pH affect the ionisation state of proteins through the effect on their titratable groups. There are proteins, however, which respond to pH shifts with conformational changes that are crucial for catalysis or transport activity. In such cases protons act as signalling molecules capable of eliciting fast and localised responses. We provide examples of ion channels that appear fastidiously designed to respond to extracellular pH in a manner that suggests specific functions in transporting epithelia. We shall also present ideas as to how these channels participate in complex transepithelial transport processes and provide preliminary experiments illustrating a new way to gauge pHo in confined spaces of native epithelial tissue.
Gastric proton pump (H+, K+-ATPase) secretes H+ of acid (HCl) via the luminal membrane of parietal cells. For the HCl secretion, Cl−- and K+-transporting proteins are required. Recent our studies have demonstrated that K+–Cl− cotransporters (KCC3a and KCC4) are expressed in gastric parietal cells. KCC3a is associated with Na+, K+-ATPase in the basolateral membrane, and KCC4 is associated with H+, K+-ATPase in the apical canalicular membrane. This paper summarizes the functional association between KCCs and P-type ATPases and the contribution of these complexes to acid secretion in gastric parietal cells.
The parietal cell of the gastric gland is a highly differentiated cell responsible for the gastric hydrochloric acid secretion into the lumen of the stomach. In response to stimulation of acid secretion, the parietal cells undergo well-characterized morphological transformations to recruit H+/K+-ATPase from the cytoplasmic tubulovesicles to the apical canalicular membrane. Besides H+ extrusion via H+/K+-ATPase, Cl− efflux and K+ recycling across the apical canalicular membrane are necessary via chloride and potassium channels/transporters, respectively. In the last decade, a number of molecular candidates for the Cl− efflux and K+ recycling have been identified in the apical canalicular membrane of the parietal cell. This review focuses on the functional transformation of gastric parietal cells and intracellular trafficking of ion channels/transporters expressed in the apical canalicular membrane associated with gastric acid secretion.
This work investigates the anti-inflammatory mechanism of saikosaponin A (SA), a major component of Bupleurum falcatum LINNE. SA significantly inhibited phorbol myristate acetate (PMA) plus A23187-induced the production and expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in human mast cell (HMC)-1 cells. SA suppressed PMA plus A23187-induced phosphorylation of extracellular signal-regulated kinase and p38. When HMC-1 cells were treated with SA, translocation of nuclear factor (NF)-κB/Rel A into nucleus and degradation of inhibitor of NF-κB (IκB) in cytoplasm were inhibited. SA decreased PMA plus A23187-induced cystein-aspartic acid protease (caspase)-1 activity. IL-1β production was also inhibited by SA. Finally, SA significantly decreased the number of nasal rubs and serum TNF-α level in the ovalbumin-sensitized allergic rhinitis mouse model. The underlying mechanism involves, at least in part, inactivation of caspase-1, which provides new evidence for therapeutic application of SA to target inflammatory processes.
The active type of coagulation factor X (factor Xa) activates various cell-types through protease-activated receptor 2 (PAR2). We previously reported that a factor Xa inhibitor could suppress Thy-1 nephritis. Considering that fibrin deposition is observed in diabetic nephropathy as well as in glomerulonephritis, this study examined the roles of the coagulation pathway and factor Xa in the development of diabetic nephropathy using type 2 diabetic model mice. Diabetic (db/db) and normoglycemic (m+/m+) mice were immunohistochemically evaluated for their expression/deposition of PAR2, transforming growth factor (TGF)-β, fibrin, extracellular matrix (ECM) proteins, and CD31 at week 20. Significantly greater numbers of PAR2-positive cells and larger amounts of fibronectin, and collagen IV depositions were observed in the glomeruli of db/db mice than those in m+/m+ mice. Next, expression of PAR2 versus deposition of collagen IV and fibronectin was compared between week 20 and week 30, and the number of PAR2-positive cells in the glomeruli decreased in contrast with the increased accumulation of ECM proteins. In an intervention study, fondaparinux, a factor Xa inhibitor, was subcutaneously administered for ten weeks from week 10 to 20. Fondaparinux treatment significantly suppressed urinary protein, glomerular hypertrophy, fibrin deposition, expression of connective tissue growth factor, and ECM proteins deposition together with CD31-positive capillaries. These results suggest that coagulation pathway and glomerular PAR2 expression are upregulated in the early phase of diabetes, together with the increase of profibrotic cytokines expression, ECM proteins deposition and CD-31-positive vessels. Factor Xa inhibition may ameliorate glomerular neoangiogenesis and ECM accumulation in diabetic nephropathy.
Tubeimoside I (TBMS I), an extract from Chinese herbal medicine Bolbostemma paniculatum (MAXIM.) FRANQUET (Cucurbitaceae) has been shown as a potent anti-tumor agent for a variety of human cancers, but yet to be evaluated for hepatoma that is highly prevalent in Eastern Asian countries including China. Here, we examined in vitro the cytotoxic effects of TBMS I on human hepatoma (HepG2) and normal liver (L-02) cell lines. We also investigated TBMS I-induced molecular events related to apoptosis in HepG2 cells. The results show that TBMS I inhibited the proliferation of both HepG2 and L-02 cells in a dose- and time-dependent manner, but HepG2 cells appeared more sensitive to the agent. When exposed to TBMS I for 24, 48 and 72 h, IC50 for HepG2 cells versus L-02 cells were 15.5 vs. 23.1, 11.7 vs. 16.2, 9.2 vs. 13.1 (μM, p<0.01), respectively. TBMS I induced cell shrinkage, nuclear condensation and fragmentation, cell cycle arrest at the G2/M phase, mitochondrial membrane disruption, release of cytochrome c from the mitochondria, activation of caspase 3 and 9, and shifting Bax/Bcl-2 ratio from being anti-apoptotic to pro-apoptotic, all indicative of initiation and progression of apoptosis involving mitochondrial dysfunction. Taken together, these results indicate for the first time that TBMS I potently inhibited growth in HepG2 cells by mediating a cascade of apoptosis signaling pathways. Considering its sensitivity of HepG2 cells, preferential distribution in the liver and natural product origin, TBMS I therefore may have a great potential as a chemotheraputic drug candidate for hepatoma.
We designed to study the role of mitochondria in astaxanthin-induced apoptosis in hepatocellular carcinoma cells. Effect of astaxanthin on cell proliferation was studied by using methyl thiazolyl tetrazolium (MTT) in three tumor cell lines (CBRH-7919, SHZ-88 and Lewis) and normal human hepatocyte HL-7702 cell. Cell apoptosis rate, changes of mitochondrial morphology, mitochondrial transmembrane potential and electron transport chain were evaluated respectively. Expressions of B cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2 associated X protein (Bax) were detected by Western blot. Results as following, astaxanthin had little effect on HL-7702 cell, however its inhibition was most pronounced in CBRH-7919 cell line with an IC50 of 39 μM. This dose of astaxanthin and CBRH-7919 cell line were chosen for further studies. Astaxanthin could induce cell apoptosis and mitochondrial membrane damage. The mitochondrial transmembrane potential and function of electron transport chain were decreased. The expression of Bcl-2 protein was down-regulated but that of Bax protein was up-regulated. In conclusion, astaxanthin showed anticancer effect by inducing cell apoptosis through the regulation of mitochondrial-dependent manner.
Translocation of two Rac/Rop guanosine 5′-triphosphate-binding proteins from Scoparia dulcis, Sdrac-1 and Sdrac-2, was examined employing transformed belladonna which overproduces these proteins as glutathione-S-transferase-tagged forms. The transferase activities of the fused proteins in microsomal fraction of belladonna markedly increased by the incubation with methyl jasmonate either in Sdrac-1 or Sdrac-2 transformant, while low and constant activities were observed in the untreated control. Recombinant Sdrac-2 protein was found to bind to prenyl chain in the presence of cell extracts prepared from methyl jasmonate-treated S. dulcis, however, Sdrac-1 was palmitoylated by the addition of the cell extracts. These results suggest that both Sdrac-1 and Sdrac-2 translocate to plant membranes by the stimulation with methyl jasmonate, however, targeting of these proteins is triggered by the independent modification mechanisms, palmitoylation for Sdrac-1 and prenylation for Sdrac-2.
Hypoxia inducible factor 1 alpha (HIF-1α) is frequently over-expressed in the numerous types of cancer and plays an important role in angiogenesis. In the present study, the inhibitory mechanism of rhapontigenin isolated from Vitis coignetiae was investigated on HIF-1α stability and angiogenesis in human prostate cancer PC-3 cells. Rhapontigenin significantly suppressed HIF-1α accumulation at protein level but not at mRNA level in PC-3 cells under hypoxia. Also, rhapontigenin suppressed hypoxia-induced HIF-1α activation in various cancer cells, such as colorectal adenocarcinoma (SW620), breast adenocarcinoma (MCF-7), fibrosarcoma (HT-1080) and prostate carcinoma (LNCaP). Interestingly, rhapontigenin had more potency in inhibition of hypoxia-induced HIF-1α expression than that of resveratrol, a known HIF-1α inhibitor. In addition, rhapontigenin promoted hypoxia-induced HIF-1α degradation and cycloheximide (CHX) blocked protein synthesis. A prolyl hydroxylase (PHD) inhibitor dimethyloxalylglycine (DMOG) is usually utilized to examine whether prolyl hydroxylation is involved in inhibition of HIF-1α accumulation. Here, DMOG recovered HIF-1α accumulation inhibited by rhapontigenin. Immunoprecipitation assay also revealed that rhapotigenin enhanced the binding of hydroxylated HIF-1α to von Hippel-Lindau (VHL) tumor suppressor protein. Furthermore, rhapontigenin reduced vascular endothelial growth factor (VEGF) secretion in hypoxic PC-3 cells as well as suppressed tube formation in human umbilical vein endothelial cells (HUVECs) treated by the conditioned media of hypoxic PC-3 cells. However, anti-angiogenic effect of rhapontigenin in hypoxic PC-3 cells was reversed by DMOG. Taken together, these findings suggest that rhapontigenin inhibits HIF-1α accumulation and angiogenesis in PC-3 prostate cancer cells.
Alterations by perfluorinated fatty acids (PFCAs) with a chain length of 6—9 carbons in the fatty acid profile of hepatic lipids of mice were investigated. The characteristic changes caused by all the PFCAs examined were increases in the contents and proportions of oleic acid (18 : 1), palmitoleic acid (16 : 1) and 8,11,14-eicosatrienoic acid (20 : 3) in hepatic lipids. Hepatic contents of palmitic acid were also increased by the treatments with the PFCAs. These effects were almost dependent on the hepatic concentrations of PFCA molecules regardless of their carbon chain length. Perfluorooctanoic acid elevated the expressions of mRNA encoding acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, stearoyl-CoA desaturase (SCD) (SCD1 and 2), chain elongase (ELOVL5), Δ6 desaturase (Fads2), 1-acylglycerophosphocholine acyltransferase (LPCAT) (LPCAT3). The four PFCAs examined induced microsomal SCD and LPCAT in hepatic concentration-dependent manners regardless of carbon chain length. One linear regression line was confirmed between LPCAT activity and hepatic concentration of PFCA at wide range of the concentration, whereas the induction of SCD was saturable at relatively low concentration of PFCAs. These results suggest (i) that PFCAs with a chain length of 6—9 carbons change the fatty acid profile of hepatic lipids by increasing contents and proportions of 16 : 1, 18 : 1 and 20 : 3, (ii) that these alterations in fatty acid profile are caused by up-regulation of SCD, de novo fatty acid synthesis, chain elongase and Δ6 desaturase and (iii) that the mechanism underlying SCD induction is, in part, mediated through peroxisome proliferator-activated receptor α.
Adipocytokines secreted from adipocytes have been extensively analyzed due to their role as key factors in various complications of obesity, including arterial sclerosis, liver steatosis, insulin resistance, and diabetes. Several in vivo and in vitro studies have suggested that adipocyte maturation is related to fluctuations in adipocytokine secretion. However, the relationship between adipocyte maturation and adipocytokine levels has not been fully elucidated. Therefore, we sought to clarify the link between adipocytokine gene expression and adipocyte maturation through systematic analysis. We quantified mRNA for six adipocytokine genes: adiponectin, resistin, leptin, plasminogen activator inhibitor 1 (PAI-1), heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), and visfatin, in adipose tissue, in primary cultured adipocytes obtained from an obese Zucker rat, and in the preadipocyte cell line 3T3-L1. Moreover, to elucidate the role of adipocytokines in adipocyte maturation, adipocytokine expression levels were analyzed during maturation. Although fluctuations in adipocytokine gene expression were heterogeneous, gene expression was highly similar during maturation of primary cultured adipocytes from obese and non-obese rats, suggesting that the maturation process is independent from processes that lead to obesity. Moreover, the expression patterns of adiponectin, resistin and leptin mRNA in 3T3-L1 cells were highly similar to those in primary cultured adipocytes, indicating that these adipocytokines could be common maturation markers for primary cultured adipocytes obtained from obese and non-obese rats, and for preadipocyte cell lines.
For the purpose of investigating the effects of S100B on the development of Parkinsion's disease (PD), a high-performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry (HPLC/MS-ESI-TOF) metabonomic approach was established to study the mesencephalon profiling of brain-specific human S100B transgenic mice. In order to obtain more full-scale chemical information of metabolites, two kinds of separation mechanism, including reversed-phase (RP) column chromatography and hydrophilic interaction liquid chromatography (HILIC) column, were combined to use. Acquired data were subjected to principal component analysis (PCA) to investigate the effects of S100B protein on mice mesencephalon metabolite profiles. Potential biomarkers were screened by using Mass Hunter Prossional Profiller (MPP) and were identified by the accurate mass. Twelve metabolites in mesencephalon of S100B transgenic mice were identified as potential biomarkers, among which, glutamic acid (Glu) detected by RP/MS in negative ionization mode, gamma-aminobutyric acid (GABA) and tryptophan (Trp) detected by HILIC/MS in positive ionization mode, phenylalanine (Phe) and histidine (His) detected by HILIC/MS in negative ionization mode, related to metabolic pathway of neurotransmitters in mice central nervous system. The analytical technique used in this paper was able to detect biochemical changes in mesencephalon of S100B transgenic mice, which may be helpful to understand the action mechanism of S100B protein in the development of PD.
A simple, specific, sensitive LC/MS/MS method for the quantitation of tenofovir (TFV) in monkey plasma was developed and validated. After the addition of adefovir as an internal standard (IS), methanol was used to produce a protein-free extract. Isocratic chromatographic separation was performed on a reverse-phase Discovery C18 column (4.6×250 mm, 5 μm). The mobile phase consisted of methanol–water–formic acid (20 : 80 : 0.5, v/v/v). Detection of TFV and the IS was achieved with electrospray ionization (ESI)-MS/MS in the positive ion mode using 288/176 and 274/162 transitions, respectively. The analytical range was set at 0.005—1.250 μg/ml using a 200 μl plasma sample. The intra- and inter-day precision values were less than 11.4%, and accuracy ranged from 0.4 to 2.9% in all quality control samples. The method was fully validated for its sensitivity, selectivity, accuracy and precision, matrix effect, recovery, and stability. Due to the high polarity of TFV, the major challenge was to circumvent ion suppression when quantitating the plasma concentration of TFV using the LC/MS/MS method. To avoid ion suppression, sufficient chromatographic separation was the most effective means for the present purposes. Moreover, it was found that the reconstitution solvents of the dried residue had a significant impact on LC peak shapes. The validated method was successfully applied to a bioequivalence study in 6 monkeys after the oral administration of two ester prodrugs of TFV (equivalent to TFV 20 mg/kg). The method permits laboratory scientists with access to the appropriate instrumentation to perform rapid TFV determination.
We found a novel octapeptide (H-YRNWFGRW-NH2) mimicking sialyl Lewis X (sLeX) carbohydrate from a chemical peptide library with anti-sLeX monoclonal antibody (MAb) 2H5. The peptide libraries were constructed by Fmoc-based solid-phase methodology using the mix-split method. The octapeptide sequence was determined by the iterative deconvolution method using anti-sLeX MAb 2H5. To define the important residues for interaction with anti-sLeX MAb 2H5, alanine-scanning analogues of H-YRNWFGRW-NH2 were synthesized. Substitution of Tyr1, Trp4, Arg7 and Trp8 to Ala resulted in a marked drop in affinity. This result indicates that aromatic and cationic amino residues have a key role in interacting with anti-sLeX MAb 2H5. The binding property of the octapeptide was evaluated with anti-sLeX MAb 2H5 and human E-selectin. The octapeptide showed high inhibitory potency (IC50=17.8 nM) for sLeX and competitively inhibited the binding of anti-sLeX MAb 2H5 in a dose-dependent manner. The octapeptide had high affinity (Kd=0.168 μM) for E-selectin and this binding was inhibited by sLeX. These results suggest that octapeptide binds to anti-sLeX MAb 2H5 or E-selectin at the sLeX binding site and sterically interferes with the recognition of anti-sLeX MAb 2H5 or E-selectin with sLeX. This peptide may be a useful lead compound for an anti-inflammatory agent targeting selectin.
Tumor necrosis factor α (TNF-α), which is a primary cytokine responsible for inflammatory responses in skin, induces the synthesis of matrix metalloproteinase-9 (MMP-9), which causes skin aging. The protective effects of 3-deoxysappanchalcone against TNF-α-induced damage was investigated using human skin keratinocytes. The results showed that 3-deoxysappanchalcone inhibited MMP-9 expression at the protein and mRNA level, by blocking the activation of activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB). Taken together, the inhibitory activity of 3-deoxysappanchalcone on MMP-9 expression and production in TNF-α-treated cells was found to be mediated by the suppression of AP-1 and NF-κB activation.
We examined the feasibility of using submicron-sized liposomes (ssLips) for retinal delivery of hydrophilic compounds, which would also have a wide range of applications. To evaluate the uptake into conjunctival cell line and the intraocular behavior of hydrophilic compound-containing ssLips after eyedrop application, fluorometric investigation was carried out by using a hydrophilic fluorescence probe, 5(6)-carboxyfluorescein (CF). A relatively high amount of CF (>50%) could be incorporated into an internal phase of ssLips by a calcium acetate gradient method. CF being entrapped within the liposomes markedly enhanced both the uptake of CF into conjunctival cells and CF-oriented emission in the retina in mice after eyedrop application, while the free CF did not clear delivery efficiency in both in vitro and in vivo study. In addition, the cellular uptake and luminescence intensity in the retina were higher when a ssLip formulation composed of L-α-distearoyl phosphatidylcholine was applied than when a ssLip formulation composed of egg phosphatidylcholine was applied. Consequently, ssLips of appropriate composition were considered to have good potential to carry hydrophilic compounds into the retina.
As part of an ongoing search for immunomodulatory components aimed at the anti-complementary effect, ginsenosides isolated from processed ginseng were found to have inhibitory activity on complement activation through classical pathways. Activity-guided fractionation was used to isolate four ginsenosides, namely ginsenoside Rg6, F4, Rk3, and Rh4. Ginsenoside Rk3 and Rh4 had a 3 fold higher inhibition activity than rosmarinic acid which was used as a positive control while ginsenoside Rg6 and F4 showed only mild effects similar to that of the positive control. The results suggest that the activity of the corresponding ginsenosides may be increased by the glycosyl moiety at the C6 position rather than the double bond conformation at C20, and ginsenoside Rk3 and Rh4 could have a role in treating inflammatory diseases.
Therapeutic effects of hybrid liposomes (L-α-dimyristoylphosphatidylcholine (DMPC)/docosahexaenoic acid (DHA)) composed of 50 mol% DMPC and 50 mol% DHA on the metastasis of human colon carcinoma (HCT116) cells were examined in vivo. DMPC/DHA having a hydrodynamic diameter less than 100 nm were preserved for one month. Remarkably high therapeutic effects were obtained in the hepatic metastasis mouse models of HCT116 cells after the intravenous injection of DMPC/DHA. The histological analysis indicated the induction of apoptosis was observed in the liver section of the hepatic metastasis mouse models treated with DMPC/DHA in vivo. Furthermore, prolonged survival was obtained in the hepatic metastasis mouse models after the treatment with DMPC/DHA. Therapeutic effects of DMPC/DHA without any drugs on the hepatic metastasis were revealed on the basis of histological and biochemical analyses for the first time in vivo.
The unripe fruits of Rubus coreanus (Rosaceae) are used in traditional Chinese medicine to relieve kidney dysfunction. In the present study, we evaluated the protective effects of the triterpenoid glycoside niga-ichigoside F1 (NIF1) and of its aglycone 23-hydroxytormentic acid (23-HTA) isolated from the unripe fruits of Rubus coreanus (Rosaceae) against cisplatin-induced cytotoxicity in renal epithelial LLC-PK1 cells. Pretreating LLC-PK1 cells with 23-HTA or NIF1 was found to prevent cisplatin-induced cytotoxicity and apoptosis. In addition, 23-HTA or NIF1 pretreatment significantly improved the changes associated with cisplatin toxicity by increasing levels of glutathione (GSH) and decreasing levels of malondialdehyde (MDA) and reactive oxygen species (ROS). The activity of antioxidant enzymes including catalase (CAT) and superoxide dismutase (SOD) was significantly lower in cisplatin-treated LL-PK1 cells, and 23-HTA or NIF1 treatment notably increased the these enzyme activity and protein and mRNA levels of CAT and manganese SOD (MnSOD). Moreover, cisplatin caused a significant decrease in nuclear levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and pretreatment with 23-HTA or NIF1 significantly suppressed the cisplatin-induced translocation of Nrf2 in LLC-PK1 cells. Taken together, these results suggest that 23-HTA ameliorates cisplatin-induced toxicity via modulation of antioxidant enzymes through activation of Nrf2 in LLC-PK1 cells.
Adipocyte differentiation has been a target in anti-obesity strategies and is known to be closely related to lipid metabolism. Ceramide, a major sphingolipid metabolite, has been implicated in differentiation. In this study, we investigated whether ceramide biosynthesis is related to adipogenesis in 3T3-L1 cells. Preadipocytes can be differentiated synchronously by a mixture of adipogenic inducers including 3-isobutyl-1-methylxanthine, dexamethasone and insulin. The number of lipid droplets and the triglyceride content, which are differentiation biomarkers, gradually increased during adipogenesis. Interestingly, ceramide and sphingosine contents in the differentiated cells were decreased compared to those in preadipocytes. When the preadipocytes were treated with an 3-isobutyl-1-methylxanthine- or dexamethasone- or insulin-deficient mixture of inducers, the cellular ceramide levels were significantly increased compared with those in cells treated with the complete set of inducers. When preadipocytes were treated with 0, 0.1 or 1 μg/ml insulin along with 3-isobutyl-1-methylxanthine and dexamethasone, the ceramide levels were decreased and the triglyceride content was increased in a concentration-dependent manner. When the cells were treated with epigallocatechin gallate, an adipocyte differentiation inhibitor, during adipogenesis, the ceramide levels of adipocytes were increased and the fat content was decreased. In conclusion, our findings demonstrate that cellular ceramide levels are inversely correlated with adipocyte differentiation.
Formation of biofilm in pathogenic bacteria defends them from antibiotics and the immune system of a host's life. Hence, investigation of the molecular mechanisms of biofilm formation and search for new substances counteracting this formation are becoming an attractive research area. In the course of our search for new inhibitors of biofilm formation in Mycobacterium species, we rediscovered a cyclic trihydroxamate siderophore, desferrioxamine E, from the culture of the marine-derived Actinomycete MS67. Desferrioxamine E inhibited biofilm formation of Mycobacterium smegmatis and M. bovis BACILLE de CALMETTE et GUÉRIN (BCG) with minimum inhibitory concentration (MIC) value of 10 μM, while no anti-microbial activity was observed up to 160 μM. Desferrioxamine E was also able to restore the anti-microbial activity of isoniazid against M. smegmatis by inhibiting biofilm formation. Mechanistic analysis of desferrioxamine E suggested that such inhibition might come from the depletion of iron in the medium, which is essential for biofilm formation in Mycobacterium species.
In our previous study, we reported that urechistachykinin I (U I) and II (U II) exerted antimicrobial effects. To find out how the tachykinin consensus sequence of the urechistachykinin peptide family affects its antimicrobial activity, analogues substituting the amino acid residues phenylalanine (Phe-6; Anal 1), glycine (Gly-8; Anal 2), and arginine (Arg-10; Anal 3) of U II to alanine (Ala) were designed. Subsequently, the antimicrobial activity was shown on the order of Anal 3>U II=Anal 2>Anal 1, and this activity pattern was correlated with membrane studies such as propidium iodide (PI) influx and fluorescein isothiocyanate dextran (FD) leakage assay. These results suggest that the antimicrobial activity is related to the hydrophobicity values of the peptides. In regards to the activity of U II, it is determined that the hydrophobic Phe-6 plays a more critical role than Gly-8 or Arg-10.
The present study reveals the pharmacological effect of chlorogenic acid, a major phenolic compound in plants, food, and coffee, on diabetic cataracts. Chlorogenic acid examined the inhibitory effects upon rat lens aldose reductase (AR) activity and xylose-induced rat lens opacity. The effect of chlorogenic acid on high glucose-induced cytotoxicity in lens epithelial cells was also examined. Chlorogenic acid showed potential inhibitory activity against rat lens AR, with an IC50 value of 0.95 μM. The xylose-induced opacity of lenses was significantly improved after treatment with chlorogenic acid in a dose-dependent manner. Chlorogenic acid prevented high glucose-induced cytotoxicity in human lens epithelial (HLE-B3) cells in a dose-dependent manner. These results suggest that chlorogenic acid may provide a potential therapeutic approach for prevention of diabetic complications, such as cataracts.
LEC-1 is the first tandem repeat-type galectin isolated from an animal system; this galectin has two carbohydrate recognition domains in a single polypeptide chain. Because its two lectin domains have different sugar-binding profiles, these domains are thought to interact with different carbohydrate ligands. In our previous study, we showed that a mutant of LEC-1 in which a cysteine residue was introduced at a unique position in the N-terminal lectin domain (Nh) can be cross-linked with a model glycoprotein ligand, bovine asialofetuin, by using a bifunctional photoactivatable cross-linking reagent, benzophenone-4-maleimide. In the present work, we applied the same procedure to the C-terminal lectin domain (Ch) of LEC-1. Cross-linked products were formed in the cases of two mutants in which a cysteine residue was introduced at Lys177 and Ser268, respectively. This method is very useful for capturing and assigning endogenous ligand glycoconjugates with relatively low affinities to each carbohydrate recognition domain of the whole tandem repeat-type galectin molecule.
FTY720 (Fingolimod) is known to have a significant therapeutic effect on experimental autoimmune encephalomyelitis (EAE). Here, we used an EAE mouse model, which had been established by immunizing C57BL/6J mice with a partial peptide of myelin oligodendrocyte glycoprotein (MOG35—55), to examine the relapse of EAE upon discontinuation of treatment with FTY720 alone or in combination with MOG35—55. Relapse was confirmed to occur in all animals (n=6) within one week after discontinuation of FTY720, with increase in the number of lymphocytes infiltrating the spinal cord and demyelination. However, in the case of combination therapy with FTY720 and MOG35—55, relapse following discontinuation of treatment was completely suppressed. The autoantigenic peptide might serve to suppress the clonal selection of relapse-associated autoantigen-specific T cells.