Inhibition of Ca(2+) signalling by the sphingosine 1-phosphate receptor S1P(1)

The lysophospholipid, sphingosine 1-phosphate (S1P), regulates a multitude of cellular functions by activating specific G protein-coupled receptors (GPCRs) (S1P(1-5), plus three newly identified S1P receptors). The G(i)-coupled S1P(1) receptor inhibits adenylyl cyclase, stimulates mitogen-activated protein kinases (MAP kinases) and cell migration, and is required for blood vessel maturation. Here, we report that S1P(1) inhibits Ca(2+) signalling in a number of cell types. In HEK-293 cells, which endogenously express S1P(1-3), overexpression of S1P(1) reduced intracellular free Ca(2+) concentration ([Ca(2+)](i)) increases induced by various receptor agonists as well as thapsigargin. The inhibitory Ca(2+) signalling of S1P(1) was blocked by pertussis toxin (PTX) and the protein kinase C (PKC) inhibitor, G?6976, and imitated by phorbol ester and overexpression of classical PKC isoforms. Activation of S1P(1) stably expressed in RH7777 cells, which endogenously do not express S1P receptors, also inhibited Ca(2+) signalling, without mediating Ca(2+) mobilization on its own. It is concluded that the widely expressed S1P receptor S1P(1) inhibits Ca(2+) signalling, most likely via G(i) proteins and classical PKC isoforms. Co-expression of S1P(1) with S1P(3), but not S1P(2), reversed the inhibitory effect of S1P(1), furthermore suggesting a specific interplay of S1P receptor subtypes usually found within a single cell type.     

Inhibition of tumor growth by poly(ethylene glycol) derivatives of anti-ErbB2 antibodies

Poly(ethylene glycol) (PEG) modification of substances with antitumor activity was shown to enhance penetration into growing solid tumors and extend antitumor effects. Accordingly, PEG was introduced as a modifier to two types of monoclonal antibodies (N12 and L26) specific to the ErbB2 (HER2) oncoprotein. These antibodies suppress the growth of tumors overexpressing ErbB2 (e.g. N87 human tumor) and the effect of PEG on their antitumor activity was evaluated. Methoxy-PEG-maleimide conjugated to sulfhydryl groups at the hinge region of the antibodies impaired their antibody binding to N87 tumor cells and did not enhance the antitumor inhibitory activity in tumor-bearing mice. A branched N-hydroxysuccinimide-activated PEG (PEG2), conjugated through amino groups of the protein, was used for binding to the whole antibody (Ab) or to its monomeric Fab′ fragment. When tested against N87 cells in vitro, the binding activity and antitumor cytotoxic effects of Ab-PEG2 were mostly preserved. PEG2 modification did not seem to alter the tumor-inhibitory activity of the antibodies in vivo and the same pattern of tumor development was observed during the first few weeks following administration. However, the stimulating effects of PEG were observed at later stages of tumor growth since tumor development was either slowed down or completely arrested. Furthermore, a second tumor implanted into the same mice during this later stage was significantly or completely inhibited, as compared to results in mice injected with the unmodified antibody. The Fab′-PEG2 monomeric derivative was also shown to be effective in inhibiting the growth of a second tumor. The extended and prolonged enhancing effect of PEG on the antitumor activity of antibodies or Fab′ fragments directed against ErbB2 may be of importance in the treatment of ErbB2-overexpressing neoplasms. Received: 2 September 1999 / Accepted: 19 February 2000     

Effects of short-term waterlogging on the growth and yield of peas (Pisum sativum)

The effects of waterlogging the soil for up to 5 days on the growth of peas at different stages of development were investigated in glasshouse and outdoor experiments. During waterlogging the oxygen content of the soil declined to less than 2% within 2–3 days. Subsequently, leaf senescence was hastened, stem growth rate was slowed and yield was decreased. The effects on yield were mainly attributable to the development of fewer nodes and fewer pods. Waterlogging for 24 h or more just before flowering restricted growth and yield. The effects of waterlogging at earlier and later stages were less marked. Waterlogging at any period after flower buds were visible prevented further increase in root dry weight.     

Inhibition of thymidylate synthase by glyceraldehyde 3-phosphate

1. A number of common metabolites which had carbonyl and/or phosphate groups were tested for their ability to alter the activity of thymidylate synthase from Lactobacillus casei. Glyceraldehyde 3-phosphate was found to be an effective inhibitor of thymidylate synthase. 2. Glyceraldehyde 3-phosphate reversibly inhibited thymidylate synthase with a K1 of 12-13 microM; the inhibition was competitive with dUMP and noncompetitive with 5,10-methylenetetrahydrofolate which is consistent with an ordered addition of substrates.     

Inhibition of ribulose 1,5-bisphosphate carboxylase/oxygenase by 2-carboxyarabinitol-1-phosphate

In some plants, 2-carboxy-d-arabinitol 1-phosphate (CA 1P) is tightly bound to catalytic sites of ribulose, 1,5-bisphosphate carboxylase/oxygenase (rubisco). This inhibitor's tight binding property results from its close resemblance to the transition state intermediate of the carboxylase reaction. Amounts of CA 1P present in leaves varies with light level, giving CA 1P characteristics of a diurnal modulator of rubisco activity. Recently, a specific phosphatase was found that degrades CA 1P, providing a mechanism to account for its disappearance in the light. The route of synthesis of CA 1P is not known, but could involve the branched chain sugar, hamamelose. There appear to be two means for diurnal regulation of the number of catalytic sites on rubisco: carbamylation mediated by the enzyme, rubisco activase, and binding of CA 1P. While strong evidence exists for the involvement of rubisco activase in rubisco regulation, the significance of CA 1P in rubisco regulation is enigmatic, given the lack of general occurrence of CA 1P in plant species. Alternatively, CA 1P may have a role in preventing the binding of metabolites to rubisco during the night and the noncatalytic binding of ribulose bisphosphate in the light.     

Inhibition of glycerol-3-phosphate acyltransferase by analogs of glycerol-3-phosphate.

Analogs of glycerol-3-phosphate were tested as substrates or inhibitors of the glycerol-3-phosphate acyltransferases of mitochondria and microsomes. (rac)-3,4-Dihydroxybutyl-1-phosphonate, (rac)-glyceraldehyde 3-phosphate, (rac)-3-hydroxy-4-oxobutyl-1-phosphonate, (1S,3S)-1,3,4-trihydroxybutyl-1-phosphonate, and (1R,3S)-1,3,4 trihydroxybutyl-1-phosphonate were competitive inhibitors of both mitochondrial and microsomal sn-glycerol-3-phosphate acyltransferase activity. An isosteric analog of dihydroxyacetone phosphate, 4-hydroxy-3-oxobutyl-1-phosphonate, was a much stronger competitive inhibitor of the microsomal than the mitochondrial enzyme. Phenethyl alcohol was a noncompetitive inhibitor of both the microsomal and the mitochondrial acyltransferases. The product of the mitochondrial acyltransferase reaction with (rac)-3,4-dihydroxybutyl-1- phosphonate was almost exclusively (rac)-4-palmitoyloxy-3-hydroxybutyl-1-phosphonate. The microsomal acylation reaction generated both the monoacyl product and (S)-3,4-dipalmitoyloxybutyl-1-phosphonate. The apparent Km for (S)-3,4-dihydroxybutyl-1-phosphonate was 2.50 and 1.38 mM for the mitochondrial and microsomal enzymes, respectively.     

Soil compaction and the growth of vining peas

The effects of differing soil conditions at two sites on the growth of vining peas are described. The work was carried out over several seasons having contrasting rainfall at the time of crop emergence. Topsoil compaction induced by tractor wheelings reduced plant population and yield of vining peas by up to 70%. On the compacted plots peas were unable to compensate for the low population which was demonstrated by the inclusion of a non-compacted treatment thinned to the same population as the compacted plots. The severity of compaction was greatest when soil conditions were dry at emergence and least severe when wet. It is suggested that compaction resulted in increased ethylene production in the root and shoot tissues and this may be a causal agent of the observed growth modifications. Recultivation of a compacted seed bed prior to sowing resulted in better establishment and higher yields than with the topsoil compacted but final yields were variable when compared to a ploughed control. Double digging did not increase yield above a ploughed control. In one year a 25% yield reduction was attributed to this treatment. The reasons for the reduction in yield are complex but it is suggested that the causal agent was modfied soil and plant water relations resulting in altered crop physiology. Tramlining the crop reduced yield in one year but when the frequency of wheeling damage is considered they would be unlikely to affect productivity.     

Inhibition of sweet potato glucose 6-phosphate dehydrogenase by NADPH2 and ATP

NADPH₂ and ATP competitively inhibit sweet potato glucose 6-phosphatedehydrogenase with NADP and glucose 6-phosphate (G6P), respectively.At pH 8.0, a Lineweaver-Burk plot of the reciprocal rate againstreciprocal G6P concentration was concave downwards in the presenceand absence of ATP, whereas a double reciprocal plot followedthe Michaelis-Menten relationship at pH 7.0, irrespective ofthe presence of ATP. Many of the other metabolic intermediatestested had no effects on the enzyme reaction. ¹ This paper constitutes Part 96 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. ² Present address: Institute of Applied Microbiology, Universityof Tokyo Bunkyo-ku, Tokyo 113. (Received October 20, 1971; )     

Induction of tumor-inhibitory macrophages with a novel synthetic immunomodulator, 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738)

3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738) has been investigated for its immunomodulatory effect on murine macrophages. Incubation of macrophages harvested from the peritoneal cavities of normal mice with the compound for 48 to 72 hr rendered these cells inhibitory to the growth of tumor cells in vitro. Activation of tumor-inhibitory macrophages occurred over a range of concentrations (0.025 to 0.1 micrograms/ml) producing no direct inhibitory effects on tumor cells. Treatment of effector cells with carrageenan abrogated the effect, whereas treatment with anti-Thy-1.2 antibody and C did not, suggesting that the primary effectors were macrophages rather than T lymphocytes. These activated macrophages also manifested in vitro tumor cytolysis. In vivo studies indicated that peritoneal macrophages from mice treated with single oral doses of 100 to 400 mg/kg of the compound were also inhibitory to tumor cell growth in vitro. Effector macrophages became demonstrable in mice as early as 1 day after drug administration, reached peak activity at day 12, and disappeared by day 31, indicating a rapid onset but long-persisting effect. The tumor cytostatic activity of these macrophages was augmented by endotoxin at the dose of endotoxin that, in itself, had no effect. The addition of protease inhibitors, N-alpha-p-tosyl-L-lysine chloromethyl ketone and aprotinin, to cultures markedly diminished the cytostatic effect, suggesting that the release of neutral protease(s) could account for the inhibitory effects of the macrophages. On the other hand, hydrogen peroxide and arginase seemed excluded as the mechanism of action because the effect was not sensitive to treatment with catalase and exogenous arginine. The present findings indicate that CL 246,738 is an orally active immunopotentiator capable of inducing tumor-inhibitory macrophages both in vitro and in vivo.     

The mechanism of action of 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738) in the potentiation of natural killer cells

An effort has been made to determine the mechanism by which the immunomodulator 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738) enhances the cytotoxic activity of natural killer (NK) cells. Orally administered CL 246,738 produced augmentation of NK cell activity in mice in a dose-related fashion over a dose range of 10 to 160 mg/kg, with a peak stimulation occurring at 40 mg/kg. The stimulatory effect was short-lived and only persisted for 3 days after a single oral dose of the drug. However, it could be boosted by a subsequent treatment. With anti-asialo GM-1 (anti-ASGM-1) antibody used as an NK cell marker, it was determined that the compound increased the number of ASGM-1-positive cells in mice, as indicated by radioimmunoassay and immunofluorescence staining. NK cells of beige mice were also activated by CL 246,738. Furthermore, the compound at concentrations of 0.02 to 0.2 microgram/ml induced NK cell activity in vitro, with a minimum 3-day incubation being required for optimal activation. This effect was dependent on the presence of macrophages and was inhibited by anti-IFN-alpha + beta but not anti-IFN-beta antibody. Taken together, it is postulated that the compound functions by stimulating macrophages to release IFN-alpha, which subsequently activates NK cells. As an effective stimulator of IFN and NK cells, CL 246,738 may prove clinically useful in the immunotherapy of certain types of malignancy.     

Inhibition of PTPs by H(2)O(2) regulates the activation of distinct MAPK pathways

It has been shown that endogenous production of reactive oxygen species (ROS) during T cell activation regulates signaling events including MAPK activation. Protein tyrosine phosphatases (PTPs) have been regarded as targets of ROS which modify the catalytic cysteine residues of the enzymes. We have analyzed the interplay between the inhibition of PTPs and the activation of MAPK by H(2)O(2). Stimulation of Jurkat T cells with H(2)O(2) induces the phosphorylation of ERK, p38, and JNK members of MAPK family. H(2)O(2) stimulation of T cells was found to inhibit the PTP activity of CD45, SHP-1, and HePTP. Transfection of cells with wtSHP-1 decreased H(2)O(2)-induced ERK and JNK phosphorylation without affecting p38 phosphorylation. Transfection with wtHePTP inhibited H(2)O(2)-induced ERK and p38 phosphorylation without inhibiting JNK phosphorylation. The Src-family kinase inhibitor, PP2, inhibited the H(2)O(2)-induced phosphorylation of ERK, p38, and JNK. The phospholipase C (PLC) inhibitor, U73122, or the protein kinase C (PKC) inhibitor, Ro-31-8425, blocked H(2)O(2)-induced ERK phosphorylation, whereas the same treatment did not inhibit p38 or JNK phosphorylation. Taken together, these results suggest that inhibition of PTPs by H(2)O(2) contributes to the induction of distinct MAPK activation profiles via differential signaling pathways.     

Inhibition of dinoflagellate growth by extracts of barley straw (Hordeum vulgare)

Growth of dinoflagellates representing three orders, the Gymnodiniales,Peridiniales, and the Prorocentrales was examined following treatment withbarley straw extract. Selected dinoflagellate taxa showed growth responsessimilar to those reported for freshwater algae including: inhibition(Gyrodinium galatheanum, Gymnodiniumsanguineum, Heterocapsa triquetra andH. pygmaea); stimulation (Gyrodinium instriatum,Prorocentrum minimum and P. micans); and no effect(Gyrodinium estuariale, G. uncatenum,Ceratium furca, Peridinium sp.).Although barley straw extracts do not appear to have value as a universalmanagement tool for dinoflagellates, they may have potential in management ofspecific taxa and possibly taxonomic groups.     

Inhibition of tumor growth in vitro by the extract of fagopyrum cymosum (fago-c)

Fagopyrum cymosum (Trev.) Meisn has long been used in China to treat various ailments of the lung, including lung tumors. This study investigated whether Fagopyrum cymosum extract (Fago-c) has effects on other organs. Human cancer cells derived from 10 different organs were employed, and their growths as affected by Fago-c were investigated. It was found that the growth of cancer cells from lung, liver, colon, leukocytes and bone is inhibited by Fago-c. However, cancer cells derived from prostate, cervix, ovary and brain are not sensitive to Fago-c, and the extract stimulates the growth of cancer cells from breast (MCF-7). Synergistic inhibition effect of Fago-c and daunomycin was observed in human lung cancer cells (H460). Cellular proteins from H460 cells treated with Fago-c were analyzed by 2D-gel electrophoresis. A protein (M.W./pI = 20K/5.9) was induced. The Fago-c extract was analyzed by High Performance Liquid Chromatography (HPLC). Four major and twenty minor components were identified. These studies indicate that the effect of Fago-c in inhibiting the growth of cell lines derived from certain organs.     

磷酸三(1-氯-2-丙基)酯降解菌筛选及其降解特性

【背景】磷酸三(1-氯-2-丙基)酯[tris-(1-chloro-2-propyl)phosphate,TCIPP]作为全球广泛关注的新兴有机污染物,具有环境赋存含量高、不易生物降解等特点,亟须开发TCIPP的高效去除技术。【目的】获得具有较高TCIPP降解效率并可用于TCIPP污染修复的新菌株。【方法】利用梯度提高无机盐培养基中TCIPP浓度的方法,从TCIPP污染土壤中筛选出1株能够降解液体中高浓度TCIPP (100 mg/L)的菌株,根据16S rRNA基因序列分析对其进行鉴定,并首次对其降解液体中TCIPP的特性进行研究。【结果】所筛选的TCIPP降解菌株DT-6为苍白杆菌(Ochrobactrum sp.),它能够利用TCIPP作为唯一碳源和能源;当TCIPP初始浓度为50 mg/L、培养时间为7 d时DT-6的生物量最大,对TCIPP的降解率也达到最高,为34.6%;蔗糖的加入能够显著促进DT-6的生长,但却抑制了其对TCIPP的降解。【结论】本研究报道了一株TCIPP高效降解菌Ochrobactrum sp. DT-6,能够为环境中TCIPP污染的生物修复提供新的种质...