Secretory phospholipase A2 (sPLA2) is a growing family of structurally related, disulfide-rich, low molecular weight, lipolytic enzymes with a His-Asp catalytic dyad. sPLA2s are distributed in a wide variety of vertebrate and invertebrate animals, plants, bacteria, and viruses, and there are 10 catalytically active sPLA2 isozymes in mammals. Although the structural bases for mammalian sPLA2s have been well documented, their physiological functions are still subject to debate. Individual mammalian sPLA2s have distinct enzymatic properties and display distinct tissue expression patterns, suggesting that each enzyme acts on distinct phospholipid membrane moieties in vivo. In this article, we briefly review our latest understanding of the possible physiological functions of sPLA2s, in keeping with their diverse actions on mammalian and nonmammalian cell membranes.
The phospholipase A2 receptor (PLA2R) is a type I transmembrane glycoprotein related to the C-type animal lectin family such as the mannose receptor. PLA2R regulates a variety of biological responses elicited by secretory phospholipase A2s (sPLA2s). Group IB sPLA2 acts as an endogenous ligand to induce cell proliferation and lipid mediator production. Analysis of PLA2R-deficient mice suggested a potential role of the sPLA2-IB/PLA2R pathway in the production of proinflammatory cytokines during endotoxic shock. PLA2R is also involved in the clearance of sPLA2s, especially group X sPLA2, to protect their exaggerated reactions by potent enzymatic activities. In circulation, the soluble form of PLA2R is constitutively present as an endogenous inhibitor for mammalian sPLA2s.
Cytosolic phospholipase A2α (cPLA2α) preferentially hydrolyzes phospholipids containing arachidonic acid and plays a key role in the biosynthesis of eicosanoids. This review discusses the essential features of cPLA2α regulation and addresses new insights into the functional properties of this enzyme. Full activation of the enzyme requires Ca2+ binding to an N-terminal C2 domain and phosphorylation on serine residues. Ca2+ binding induces translocation of cPLA2α from the cytosol to the perinuclear membranes. Serine phosphorylation is mediated by mitogen-activated protein kinases (MAPKs), Ca2+/calmodulin-dependent protein kinase II, and MAPK-interacting kinase Mnk1. Interaction with proteins and lipids, which include vimentin, annexins, NADPH oxidase, phosphatidylcholine, phosphatidylinositol 4,5-bisphosphate (PIP2), and ceramide-1-phosphate, can also modulate the activity of cPLA2α. Recent evidence has established the physiological and pathological roles of cPLA2α using cPLA2α knockout mice. This enzyme has been implicated in fertility, striated muscle growth, renal concentration, postischemic brain injury, arthritis, inflammatory bone resorption, intestinal polyposis, pulmonary fibrosis, acute respiratory distress syndrome, and autoimmune encephalomyelitis. Now novel three paralogs, cPLA2β, cPLA2γ, and cPLA2δ, have been identified in humans. cPLA2γ is distinct from others in that it is farnesylated and lacks the C2 domain. Biological roles for these new enzymes have not yet been defined.
The catalytic activity of calcium-independent phospholipase A2 (iPLA2), which is classified as a group VI PLA2, is regulated by protein kinase C, calmodulin, and others such as reactive oxygen species. Numerous findings have shown that iPLA2 is involved in stimulus-induced arachidonic acid release and lysophospholipid generation, although the participation is dependent upon the cell type and stimulus. The catalytic action of iPLA2 is known to be responsible for phospholipid remodeling as a housekeeping function. However, it has been widely accepted that arachidonic acid and lysophospholipid generated by iPLA2 act as a signaling molecule in cellular functions. Those include eicosanoid production, glucose-induced insulin secretion, Fas-induced apoptosis, cellular proliferation, membrane traffic in fusion, contribution to myocardial ischemia, and others. In this review, the functional role of iPLA2 in cellular responses upon stimulation is the focus.
As detailed in previous reviews, phospholipase A2 (PLA2) enzymes belonging to the secretory PLA2 (sPLA2), cytosolic PLA2 (cPLA2), and Ca2+-independent PLA2 (iPLA2) families may play specific physiologic and pathologic roles. In the past two years, there have been considerable advances in the understanding of the regulatory functions of individual PLA2s. This short article focuses on the latest topics in the PLA2 field, which have offered new insights into this intriguing enzyme family. Specifically, I describe a novel cellular action and unexplored in vivo functions of sPLA2, expanding regulatory aspects of cPLA2, and unique functional roles of iPLA2 in apoptosis, Ca2+ homeostasis, and myocardial ischemia.
Type-1 diabetic patients experience hyperketonemia caused by an increase in fatty acid metabolism. Thus, the aim of this study was to measure the effect of ketone bodies as suppressors of oxidizing species produced by stimulated neutrophils. Both acetoacetate and 3-hydroxybutyrate have suppressive effect on the respiratory burst measured by luminol-enhanced chemiluminescence. Through measurements of hypochlorous acid production, using neutrophils or the myeloperoxidase/H2O2/Cl− system, it was found that acetoacetate but not 3-hydroxybutyrate is able to inhibit the generation of this antimicrobial oxidant. The superoxide anion scavenging properties were confirmed by ferricytochrome C reduction and lucigenin-enhanced chemiluminescence assays. However, ketone bodies did not alter the rate of oxygen uptake by stimulated neutrophils, measured with an oxygen electrode. A strong inhibition of the expression of the cytokine IL-8 by cultured neutrophils was also observed; this is discussed with reference to the antioxidant-like property of acetoacetate.
Orally ingested non-steroidal anti-inflammatory drugs (NSAIDs) and acid in gastric secretions are gastric irritants that co-exist at the surface of the gastric mucosa. Here, we examined the individual and combined effects of indomethacin, a typical NSAID, and hydrochloric acid on cell death in primary cultures of guinea pig gastric mucosal cells. Indomethacin alone (at concentrations less than 200 μM) did not induce apoptosis; however, hydrochloric acid-induced apoptosis was stimulated in the presence of indomethacin (50—200 μM). Isobologram analysis confirmed the presence of a cytotoxic synergy between indomethacin and hydrochloric acid. The synergistic response between the two gastric irritants was also observed for necrosis. Given that the IC50 value of indomethacin for inhibition of prostaglandin synthesis is about 5 nM, the synergistic response between indomethacin and hydrochloric acid appears to be independent of the inhibition of cyclooxygenase activity by indomethacin.
We recently suggested that the transferrin (Tf)–gallium-67 (67Ga) complex dissociated on the surface of the hepatocytes after partial hepatectomy and free 67Ga bound to heparan sulfate in the extracellular matrix. In the present study, we investigated whether the entering of indium-111 (111In) and iron-59 (59Fe) with high affinity to transferrin differed from the entering of 67Ga by the hepatocytes after partial hepatectomy. 111In was almost taken by the plasma and little taken by the red blood cell. On the other hand, the uptake of 59Fe by the red blood cell was higher than plasma. The uptake of 59Fe by the bone marrow was significantly higher than that of 111In. The uptake of 111In and 59Fe by the liver tissue was reached a maximum 2 d after partial hepatectomy but the uptake ratio of 111In was lower than that of 59Fe. We suspected that the uptake of 59Fe by the liver tissue was the highest because of the high binding affinity of Tf–59Fe to Tf-receptor. The entering of 111In and 59Fe into the hepatocytes was also reached a maximum 2 d after partial hepatectomy but the ratio of 59Fe was slightly lower than that of 111In. These results suggested that the binding affinity to Tf could have played a crucial role in the differences of the entering of 111In, 59Fe and 67Ga into the hepatocytes of partially hepatectomized rats.
On thyrotropin-releasing hormone (TRH) metabolism, pyroglutamyl aminopeptidase II (PAP-II), a zinc-dependent ectoenzyme primarily located in the central nervous system, is believed to play a predominant role. Recently we cloned pyroglutamyl aminopeptidase I (PAP-I) which is known for specifically removing a L-pyroglutamate (L-pGlu) residue from the amino terminus of proteins and peptides including TRH. To investigate possible contribution of PAP-I toward TRH metabolism, we conducted biochemical and immunohistochemical characterization using recombinant rat, mouse and human PAP-Is and an antibody raised against rat PAP-I. The Km values toward TRH by the recombinant PAP-Is were about 0.05 mM, being similar value to the reported value of recombinant PAP-II. The L-pGlu-cleaving activities toward TRH in rat brain homogenate were inhibited by a PAP-II specific inhibitor 1,10-phenanthroline, but not inhibited by the antibody against rat PAP-I. Immunohistochemical study in rats revealed heterogeneous distribution of PAP-I in the pituitary, the target tissue of TRH, but the distribution was cytosolic. Taken together, these results suggested that PAP-I might not be dominantly involved in the degradation of TRH in rats. Additionally, we found that PAP-I was localized in the renal proximal tubules. Further investigations are needed for elucidating the function of PAP-I in these restricted sites.
Manganese superoxide dismutase (MnSOD) is an antioxidative enzyme that scavenges superoxide radicals and is localized in the mitochondrial matrix. MnSOD is induced by a variety of stimuli through nuclear factor (NF)-κB and AP-1 activation. We investigated the expression of MnSOD in HeLa cells exposed to various agents interfering with endoplasmic reticulum (ER) functions. All agents caused an increase in the mRNA and protein levels of MnSOD. Although ER stress-responsive genes often are up-regulated by ATF6, IRE1 and XBP1, which are ER stress-related transcription factors/transducers, the overexpression of neither molecule affected the levels of MnSOD mRNA and protein. Furthermore, we showed that ER stress reagents induced NF-κB and AP-1 activation that were inhibited by a dominant-negative IRE1 mutant. We finally demonstrated that ER stress-induced MnSOD expression was reduced by the IRE1 mutant. These results suggest that the MnSOD expression is controlled by ER stress through IRE1-mediated NF-κB and AP-1 activation.
Biotransformation of sinesetin (1) by larvae of Spodoptera litura was investigated. Compound 1 was converted to a new flavonoid, (+)-5,7,3′,4′-tetramethoxyflavone-6-O-β-D-glucoside (4), and two known flavones, 4′-hydroxy-5,6,7,3′-tetramethoxyflavone (2) and 6-hydroxy-5,7,3′,4′-tetramethoxyflavone (3). These structures were established by IR, HR-EI-MS, HR-FAB-MS, 1D NMR, and 2D NMR spectral studies. The results indicate that the metabolic reaction of compound 1 by larvae of S. litura proceeded along two pathways; the main pathway is demethylation at the C-6 position followed by glucosylation, and the minor pathway is demethylation at the C-4′ position.
UDP-N-acetylglucosaminyl transferase (OGT) resides in both cytosolic and nuclear compartments and catalyzes O-linked glycosylation of various proteins. In the current study, we have extracted protein from nuclear DNA (chromatin protein) using 0.2% NP-40 detergent. Addition of chromatin protein to either cytosolic or nuclear preparations (containing abundant OGT) resulted in a dose-dependent loss of OGT activity. Since chromatin-mediated loss of OGT activity could be restored by immunopurification of OGT, we conclude that loss of enzyme activity is not due to direct inactivation of OGT. Addition of UDP-galactose (to saturate potential UDP binding proteins) effectively restored OGT activity in cytosol containing chromatin protein. This indicates that chromatin protein inhibits OGT activity by binding UDP-GlcNAc. These studies suggest that nuclear substrate availability may comprise one of the in vivo mechanisms regulating OGT activity and O-linked glycosylation of nuclear proteins. This is potentially significant, since most transcription factors are O-linked glycosylated and such post-translational modifications can alter gene expression.
Traditionally, Corydalis tuber has been used for the control of pain including headache, stomachache, and neuralgia. In the present study, modulation of the Corydalis tuber on glycine-activated ion current in the acutely dissociated periaqueductal gray (PAG) neurons was studied by a nystatin-perforated patch-clamp technique. High concentrations of Corydalis tuber elicited ion current, which was suppressed by strychnine application, while low concentrations of Corydalis tuber reduced glycine-induced ion current in the PAG neurons. Inhibitory action of Corydalis tuber on glycine-activated ion current was partially abolished by treatment with naltrexone, a non-selective opioid antagonist. Application of N-methylmalemide (NEM), a sulfhydryl alkylating agent, also partially abolished the inhibitory action of Corydalis tuber on glycine-activated ion current in the PAG neurons. These results suggest that the inhibitory effect of Corydalis tuber on glycine-activated ion current in the PAG neurons is one of the analgesic mechanisms of the Corydalis tuber.
Oxidative stress plays an important role in chronic complications of diabetes. In the present study the antioxidant effect of oral administration of ethanolic extract of Eugenia jambolana seed kernel on tissue antioxidant enzymes and lipid peroxidation in liver and kidney of streptozotocin-induced diabetic rats was evaluated. Administration of seed kernel to diabetic rats significantly decreased the levels of blood glucose, glycosylated hemoglobin and increased body weight gain, plasma insulin and hemoglobin. The diabetic rats showed the low activities of superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione content in liver and kidney, which were restored to near normal levels by treatment with the seed kernel extract. The increased levels of lipid peroxidation and hydroperoxides in diabetic rats were reverted back to near normal levels after the treatment with seed kernel extract. Diabetic rats treated with seed kernel extract restored almost normal architecture of liver and kidney and were confirmed by histopathological examination. The present study reveals the efficacy of Eugenia jambolana seed kernel in the amelioration of diabetes, which may be attributed to its hypoglycemic property along with its antioxidant potential. The antioxidant effect of Eugenia jambolana seed kernel was also compared with glibenclamide, a standard hypoglycemic drug.
The effects of a newly synthesized compound, 6-acetoamido-1-acetyl-1-indazole (TAS-3-124), on autoimmune diseases were studied. We used animal models of collagen-induced arthritis (CIA) in mice and experimental autoimmune encephalomyelitis (EAE) in rats to evaluate the efficacy of TAS-3-124. TAS-3-124 at doses of 100 and 300 mg/kg p.o. inhibited the development of CIA, decreasing the swelling of fore- and hind-limbs and bone destruction in knee joints. This agent also suppressed the delayed type hypersensitivity reaction (DTH) against type II collagen. These effects were confirmed by histopathological examination and measurement of the expression of mRNA of proinflammatory cytokines in the knee joint. In addition, TAS-3-124 at a dose of 300 mg/kg inhibited the development of EAE and the DTH to myelin basic protein (MBP) in rats. Moreover, TAS-3-124 inhibited the production of proinflammatory cytokines including interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-6 but not T cell derived cytokines in mice. These demonstrate the efficacy of TAS-3-124 against experimental autoimmune disease, probably due to the suppression of the production of proinflammatory cytokines in the pathological lesion.
Cerebrocortical cell cultures were prepared from 1-d-old rats. On post-culture day 6, 5-hydroxytryptamine (5-HT) was added to the medium and cells were exposed for another 3 d. 5-HT elicited cytotoxicity in a dose-dependent manner, and the survival rate of neuronal cells was decreased to 64.9±5.0% at 0.1 mM concentration. Chromatin staining with Hoechst 33258 and electron microscopy revealed that the 5-HT-induced neuronal death was entirely due to necrosis. Pretreatments with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antisense oligonucleotide and several classical apoptotic inhibitors did not exhibit neuroprotection in this paradigm. Northern blot analysis showed that the enhancement of GAPDH mRNA levels was undetected during cell death. The present results demonstrate that GAPDH overexpression is not involved in the 5-HT-induced necrotic death pathway.
We reported previously that chronic hypoperfusion induced by permanent occlusion of the bilateral common carotid arteries (2VO) in rats caused progressive cognitive deficits and neuronal damage in the hippocampus and the white matter. These changes are similar to those observed in human dementia. Reverse transcription-polymerase chain reaction (RT-PCR) differential display was carried out to identify mRNAs encoding the intrinsic factors involved in permanent ischemia from the 2VO rat brain. Over 20 clones which showed different expression levels in 2VO and sham-operated rats were isolated. One of these, named vof-16, was markedly enhanced the expression by 2VO. The whole sequence of vof-16 mRNA was 2098 nt. The distribution of vof-16 transcripts was examined by RT-PCR and in situ hybridization. The results revealed that vof-16 was abundant in the hippocampus, the tenia tecta, the piriform cortex and the area around the aorta. The expression levels of vof-16 in 2VO and sham-operated rat hippocampus were determined by a quantitative PCR method. The expression was abundant in the hippocampus of rats with cognitive impairment induced by 2VO. In contrast, the expression levels of vof-16 were lower in the 2VO rats with no impairment and in sham-operated rats. These results suggest that the expression levels of vof-16 may be related to the cognitive impairment induced by chronic ischemia after 2VO.
Dosiinpartner (DSP) is a newly developed dietary functional food to help control weight. The aim of this study was to evaluate whether DSP combined with a high-fat (HF) diet could influence body weight, fat accumulation, and plasma glucose levels. Mice were fed for 8 weeks with normal diet, HF diet, and HF+10% or 20% DSP diet. Body weight was recorded at 1 week, and plasma levels of total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and glucose were analyzed at the end of the study. Weight increases in the 10% or 20% DSP group were significantly less than in the HF diet group (p<0.05). Plasma total cholesterol and LDL cholesterol levels decreased by 48.3% and 26.8% in the 10% DSP group and by 42.9% and 34.9% in the 20% DSP group, respectively. However, the HDL cholesterol level was unchanged. Glucose levels also decreased by 80.6% in the 10% DSP group but was almost the same in the HF and 20% DSP groups. Our findings indicate that DSP may be beneficial in the regulation of high-fat diet-induced overweight and other complications such as circulatory disorders and diabetes mellitus.
We examined the cytotoxic effect of maytanprine isolated from the methanol extract of Maytenus diversifolia on human leukemia K562 cells using a flow cytometer and compared its cytotoxicity with that of maytansine, a potent cytotoxic maytansinoid. Maytanprine at concentrations of 0.03 nM or more (up to 1 nM) attenuated cell growth with decreasing cell viability and increased the population of shrunken cells in a concentration-dependent manner. Complete inhibition of growth by maytanprine was observed at concentrations of 0.3 nM or more. The compound at 0.03 nM markedly decreased the population at G0G1 phase in the cell cycle, but only slightly decreased that in the G2M phase, suggesting the possibility that it inhibits or delays cell division, and increased the population of cells with hypodiploidal DNA (apoptotic cells). The potency of maytanprine in inhibiting cell growth was greater than that of maytansine, although the inhibitory action of maytanprine was similar to that of maytansine. The results suggest that maytanprine exerts a potent inhibitory action on the growth of human leukemia K562 cells. M. diversifolia is one natural source of maytanprine, which is more cytotoxic than maytansine.
A basic peptide with mass weight of 7.597 kDa was isolated and purified from the Naja atra venom by using the combination of ion exchange chromatography and reverse phase high performance liquid chromatography. N-terminal protein sequence determination revealed that this peptide was a weak neurotoxin. Neurotoxicity and cytotoxicity assay were performed. It was noticed that although the analysis of protein sequence did not show it was much more basic, this neurotoxin was eluted out after a cardiotoxin-like basic protein (CLBP). It was also found that, despite of low neurotoxicity, when applied to two non-neural cell lines including K562 cells and K1735-M2 cells, this weak neurotoxin exhibits synergic effects with cardiotoxins, which is firstly reported. It was presumed that the synergic effect might be due to the presence of their common characteristic tertiary structure, three-finger structure. This fact might bring us some new sights about the functions of the un-lethal components in the complex venom system and may help us to understand how the venom really works as an integrative system.
Neuroprotective effects of estrogen and estrogen-like chemicals on neurodegenerative diseases, especially Parkinson's disease, have been well established. In the present study, we compared the effects of Bak Foong Pill (BFP), a well-known gynaecological tonic in China, and 17β-estradiol, on dopamine transporter (DAT) and tyrosine hydroxylase (TH) gene expression patterns in ovariectomized, 1-methyl-4-phenyl-1,2,3,6-tetrahyrdropyridine (MPTP)-induced Parkinson's disease (PD) model mice, using multiplex reverse transcription-polymerase chain reaction (RT-PCR). MPTP, a specific dopaminergic neurotoxin, significantly decreased DAT and TH mRNA levels in the striatum, midbrain and cerebellum, but not the cortex, of C57BL/6 mice. However, MPTP-challenge with BFP pretreatment demonstrated reduced neurotoxicity, with DAT and TH mRNA levels either not affected by MPTP or affected to a significantly lesser extent in the midbrain and striatum as compared to the MPTP treated controls. 17β-estradiol treatment prevented MPTP-induced reduction of DAT expression in striatum and midbrain, but failed to alter TH expression. These results suggest that BFP is able to protect dopaminergic neurons against MPTP-induced neuronal damage in a mechanism that is different from the protective effect of estrogen.
PM-F2-OB is one of the most well-known traditional herbal medicines that are frequently used for the treatment of obesity in Korea. The anti-obesity effect of PM-F2-OB on rats fed a high-fat diet was investigated through analyses of changes in body weight, kidney fat weight, and blood biochemicals including cholesterol, free fatty acid, BUN, creatinine, HDL, LDL, phospholipids, SGOT, SGPT, total lipids, and triglycerides. The subjects in this study were divided into four groups: a normal group with a standard diet (N); a PM-F2-OB treatment group fed a standard diet (N+PM-F2-OB); a control group fed a high-fat diet (C); and a PM-F2-OB treatment group fed a high-fat diet (C+PM-F2-OB). There were no significant differences in body weight change between the N and N+PM-F2-OB treatments. Also, there was no significant difference in the amount of food intake between the C and C+PM-F2-OB treatments. These results suggest that PM-F2-OB has no significant toxicity and does not induce a dislike for that diet due to its smell or taste. Rats were administered a high-fat diet (20% (w/w)) for six weeks to induce obesity. The study shows that PM-F2-OB significantly prevented increases in body weight, cholesterol, LDL and total lipids that resulted from the high-fat diet. PM-F2-OB also decreased kidney fat weight and free fatty acid, phospholipid, and triglyceride concentrations induced by the high-fat diet to level equals or below the normal diet group. It was concluded from the results that PM-F2-OB has a distinct anti-obesity effect.
Liriope platyphylla (LP) has been used as a tonic, antitussive, and expectorant in Korea for many years. In this study, we found that the buthanol fraction of Liriope platyphylla (BLP)-conditioned media of C6 and primary astrocyte induced the neurite outgrowth of PC12 cells, and that the effect was reversed by addition of nerve growth factor (NGF)-antibody and GF109203X, an inhibitor of protein kinase (PKC). Furthermore, we demonstrated that BLP increased the expression and secretion of NGF. GF109203X also decreased NGF expression in C6 cells. Taken together, our results suggest that astroglial NGF enhanced by BLP in a PKC-dependent pathway contributed to the induction of neurite outgrowth of PC12 cells.
Co-culture conditions for Duboisia myoporoides–D. leichhardtii hybrid hairy root induction were investigated using leaf explants and Agrobacterium rhizogenes ATCC 15834. The bacteria density and duration of co-culture greatly affected the induction rate; the highest rate of 50% was obtained when the leaf explants were co-cultured for 2 d with 106 bacteria. One hairy root clone that showed the fastest root growth was selected and used for comparison study with adventitious roots cultured with 0.5 mg/l indole-3-acetic acid (IAA). The hairy roots cultured in Murashige and Skoog (MS) liquid medium grew well and yielded much more tropane alkaloids (35 mg/l scopolamine and 17 mg/l hyoscyamine) than adventitious roots cultured in 0.5 mg/l IAA after 6 weeks of culture at 25 °C in the dark. The hairy and adventitious roots (2.5 cm) grown in liquid media were divided into 5 parts (each 0.5 cm) along the root axis. Distribution of scopolamine and IAA was then determined by enzyme-linked immunosorbent assay (ELISA). Inverse relationship between contents of scopolamine and IAA was observed in the hairy roots; increase of scopolamine and decrease of IAA were proportional to the distance from the root meristem. In contrast, the contents of scopolamine and IAA were relatively constant in the adventitious roots. In shoot regeneration experiments, the hairy and adventitious root segments (1 cm) were placed onto 1/2 MS solid medium containing various concentrations of IAA and BA cultured at 25 °C under 16 h light. In adventitious roots, the shoots regenerated on media containing 6-benzyladenine (BA) (0.5 to 5 mg/l), and 100% regeneration was observed in medium with 0.1 mg/l IAA and 2 mg/l BA. On the other hand, shoot regeneration was only observed in 33% of hairy roots cultured on medium containing 5 mg/l BA.
Anogeissus latifolia is widely used in the Indian indigenous system of medicine and is reported to contain leucocyanidins and tannoid principles like ellagic acid and its derivatives. In view of its wide use and its chemical composition, this study was aimed at examining the antioxidant activity of the extract of A. latifolia. The extract was studied for total antioxidant capacity, hydrogen-donating ability, nitric oxide, superoxide scavenging activity, hydrogen peroxide decomposition activity along with lipid peroxidation. Integral antioxidative capacity was determined by chemiluminescence assay. The extract was also studied for lipid peroxidation assay by thiobarbituric acid-reactive substances (TBARS) method using rat liver homogenate. The results indicate that A. latifolia extract has potent antioxidant activity. Also to ascertain the possible reason for the potent activity, percentage of gallic acid was estimated and was found to be 0.95%, which could be one of the reasons for potent antioxidant activity exhibited by the plant.
We have assessed the release of histamine from mast cells by smooth muscle contraction. 0.3 μg/ml compound 48/80 showed no effect on concentration–response relationship of histamine in rabbit aorta. Compound 48/80 induced release of histamine from rat mast cells. When aorta was stimulated by compound 48/80 in the presence of mast cells, contraction was evoked in concentration-dependent manner. This mast cell-dependent contraction was completely blocked by H1 receptor antagonist, 1 μM diphenhydramine. When mast cells was treated with compound 48/80 inhibitor benzalkonium chloride, mast cell-dependent contraction was inhibited, although benzalkonium chloride itself showed no effect on concentration–response relationship of histamine in rabbit aorta. At high concentration of 10 μg/ml, benzalkonium chloride itself evoked histamine release from mast cells and indeed inhibitory effect of 10 μg/ml benzalkonium chloride on mast cell-dependent contraction was lower than that of 3 μg/ml. We have applied this bioassay to search anti-allergic ingredient from a total methanolic extract of Corydalis tuber (Corydalis turtschaninovii BESSER forma yanhusuo Y. H. CHOU et C. C. HSU). Successively, we have isolated five fractions. The fractions I—IV are identified to be corybulbine (1), tetrahydropalmatine (2), corydaline (3) and yuanhunine (4), respectively. Main component of fraction V is the mixture of 3 and canadine (5). Fractions II and V significantly inhibited mast cell-dependent contraction in rabbit aorta as well as inhibited histamine release from rat mast cells. Furthermore, fractions I, III and V inhibited histamine-induced contraction in rabbit aorta at non-competitive manner. From these results, combination of rat mast cells and rabbit aorta is good bioassay to search the anti-allergic ingredient, and we have obtained effective fractions from Corydalis tuber using this assay.
The EtOH extract from the rhizomes of Astilbe koreana (Saxifragaceae) exhibited potent antioxidant activity in our recent study. Since the oxidative stress is known to be involved in the inflammatory response after ultraviolet B (UVB) exposure, the ability of the A. koreana extract to inhibit UVB-induced prostaglandin E2 (PGE2) and nitric oxide (NO) production was examined. UVB irradiation (35 mJ/cm2) increased PGE2 and NO production, which were significantly decreased by pre-administration of the A. koreana extract in a dose-dependent manner. The A. koreana extract also preserved cellular antioxidant capacity after UVB irradiation, which was determined by glutathione (GSH) content. UVB irradiation enhanced the formation of ROS in the keratinocytes, which was determined using a 2,7′-dichlorofluorescein diacetate (DCFH-DA), a redox sensitive dye. The levels of intracellular reactive oxygen species (ROS) were also significantly reduced by pre-treatment of the A. koreana extract in a dose-dependent manner measured 9 h after UVB irradiation. The results suggest that the A. koreana extract may have a protective effect on the UVB-injured keratinocytes by inhibiting PGE2 and NO production, possibly through the inhibition of intracellular ROS accumulation.
Nine lignans and two butanolides were isolated from the stem bark of Machilus thunbergii and their structures were identified as machilin A (1), licarin B (2), zuonin B (3), macelignan (4), secoisolancifolide (5), isolancifolide (6), oleiferin C (7), meso-dihydroguaiaretic acid (8), licarin A (9), machilin F (10), and nectandrin B (11) by spectroscopic means. These compounds were assessed for their abilities to activate a caspase-3 activity in human promyeloid leukemic HL-60 cells. The intracellular caspase-3 activity of macelignan (4), oleiferin C (7), meso-dihydroguaiaretic acid (8), and licarin A (9) increased approximately 3.04, 6.16, 2.10, and 3.10-fold at 100 μM over that of untreated control. In addition, compounds 4, 7, 8, and 9 induced internucleosomal DNA fragmentation in HL-60 cells.
A homology-based cloning strategy yielded four cDNA clones encoding the open reading frame of carrot calmodulin, designated cam-4 and cam-8 from an oligogalacturonide elicitor-treated cell culture and cam-11 and cam-16 from cells exposed to ethylene, respectively. Reverse-transcription polymerase chain reaction analyses revealed that the expression of the cam-4 gene increased transiently when carrot seedlings were treated with oligogalacturonides, although, the cells incubated with fungal mycelial walls or ethylene did not show a significant change in the expression level. In contrast, marked and transient expression was observed for either cam-11 or cam-16 only when carrot cells were treated with ethylene. These results suggest that two classes of stimuli which are capable of triggering phytoalexin production in carrot cells, oligogalacturonides and ethylene, evoke the activation of the Ca2+ cascade in the cells by expressing distinct calmodulin genes to initiate the biosynthesis of the compound.
The MeOH extracts of the ground part and the root of Boenninghausenia japonica NAKAI showed inhibitory activity against tumor cell growth. Fractionation of the extracts has resulted in isolation of 1,3-dihydroxy-4-(2′-hydroxy-3′-hydroxymethyl-3′,4′-epoxy-butyl)-N-methylacridone, 1,3-dihydroxy-4-[(Z)-3′-hydroxy-3′-methyl-buten-1′-yl]-N-methylacridone, 3-(1′,1′-dimethylallyl)-7-hydroxy-8-methoxy-2H-1-benzopyran-2-one, casegravol, cis-casegravol, and edgeworin in addition to 9 compounds reported from B. japonica and B. albiflora. The isolates from this plant and some related compounds were tested for antiproliferative activity against human gastric adenocarcinoma (MK-1), human uterus carcinoma (HeLa), and murine melanoma (B16F10) cells.
3,4,5-Trimethoxycinnamic acid (TMCA) is one of the constituents in Onji (roots of Polygala tenuifolia WILLD), an herbal medicine used for sedative in Japanese traditional Kampo medicine. Our previous study revealed that oral administration of this compound prolongs sleeping time induced by hexobarbital in mice to exhibit sedative action. In the present study, we investigate the effects of TMCA on the stress induced with repeated cold exposure or intracerebroventricular injection of corticotrophin-releasing hormone (CRH). Both types of stress significantly reduced the sleeping time induced with pentobarbital in rat, which was significantly prolonged by intraperitoneal injection of TMCA. The intracerebroventricular injection of CRH significantly augmented the content of norepinephrine (NE) in locus coeruleus (LC) of rats, which was significantly suppressed by the intracerebroventricular injection of TMCA. These findings suggest that TMCA would exhibit sedative effects by suppressing NE content in LC.
We have previously found that carboxymethylpullulan (CMPul) conjugated with sialyl Lewis X (Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc-, 2-3SLex) preferentially accumulates in the lymph nodes and spleen. In the present study, we investigated the structural requirements of the 2-3SLex moiety for this accumulation using rats. Radiolabeled CMPul conjugates with various degrees of substitution (d.s.) of the 2-3SLex moiety were intravenously administered to rats, and their tissue distributions were monitored by radioactivity. When the d.s. was more than 0.5, preferential accumulation in the lymph nodes as well as the spleen was observed. However, when the d.s. was 0.025, little effect of the 2-3SLex moiety was noted. Changes in the carbohydrate structure of 2-3SLex, i.e., a change to α2-6-linked sialic acid (Neu5Acα2-6Galβ1-4(Fucα1-3)GlcNAc-, 2-6SLex) or an elimination of the fucose (Neu5Acα2-3Galβ1-4GlcNAc-, sialyl N-acethyllactosamine (SLN)), also made the 2-3SLex moiety ineffective. Furthermore, Microautoradiography analyses revealed that 2-3SLex-CMPul was incorporated by particular subsets of macrophages in these tissues, and that CMPul and SLN-CMPul were also located in the same cells to a lesser extent. 2-3SLex-CMPul may be able to serve as a novel drug delivery carrier to target drugs to the peripheral lymphoid tissues.
A human fungal pathogen, Candida albicans, varies from the yeast form to the hyphal form due to various external signals. This morphogenetic transformation has been implicated in the development of pathogenicity. In this report, we show that calmodulin inhibitors (TFP and W-7) and an adenylatecyclase inhibitor (MDL-12-330A) suppressed the hyphae formation of C. albicans. Furthermore, the expression of hyphae-specific mRNAs located downstream from the RAS1-cAMP pathway was inhibited by these inhibitors. Suppression of hyphae formation by TFP or W-7 was not inhibited by the addition of cAMP, and these inhibitors did not affect the amount of cAMP in C. albicans. These results suggest that the Ca2+/calmodulin pathway contributes to hyphae formation and is related to the RAS1-cAMP pathway.
The opening of the adenosine triphosphate (ATP)–sensitive potassium (KATP) channel has been proposed as a therapeutic approach for ischemia. Here we examined the opening effect of KR-31378 on the KATP channel using patch clamp recording in neuroblastoma 2a (N2a) cells and investigated the neuroprotective effect of KR-31378 in organotypic hippocampal slice cultures exposed to oxygen/glucose deprivation. The treatment with KR-31378 (10 μM) to N2a cells seemed to induce KATP channel opening in a dose dependent manner. The opening effect of KR-31378 was more significant than that of other known KATP channel openers. Pretreatment with KR-31378 (10 μM) showed a neuroprotective effect in both CA1 and CA3 regions and its effect was attenuated by glibenclamide in a dose dependent manner in both areas. This remarkable neuroprotective effect of KR-31378 seemed to be mediated by the opening of the KATP channel. These results suggest that KR-31378 could be a possible neuroprotective agent against cerebral ischemia.