Development of a novel tocopheryl ester for suppression of lipid accumulation without cytotoxicity by optimization of dicarboxylic ester moiety

Tocopheryl succinate (Tsuc) is a succinic acid ester of the well-known antioxidant α-tocopherol (T). Tsuc exhibits various biological activities, including tumor growth suppression via activation of cell signaling and prevention of lipid accumulation in mouse adipocyte 3T3-L1 cells. The latter findings suggest that Tsuc may be a drug candidate for the treatment of obesity. However, Tsuc was found to induce apoptosis of normal cells (in addition to cancer cells), demonstrating the need to reduce the cytotoxicity of Tsuc without losing the suppression effect on lipid accumulation. Based on our previous findings, we focused on the ester structure of Tsuc for controlling cytotoxicity. Herein, we examined the cytotoxicity and lipid accumulation suppression effect of various T ester derivatives. We found that the terminal carboxylic group is necessary for suppression of lipid accumulation. We synthesized tocopheryl glutarate (Tglu) and tocopheryl adipate (Tadi) by elongation of carbon atoms 1 and 2 of the dicarboxylic moiety, respectively. Tglu and Tadi did not show any cytotoxicity, and both esters suppressed lipid accumulation, although their suppression activities were weaker than that of Tsuc. Tadi showed a more potent lipid accumulation inhibitory effect than Tglu. Although Tadi inhibited lipogenesis and promoted lipolysis, lipolysis was induced at lower concentrations than inhibition of lipogenesis, suggesting that Tadi mainly affects lipolysis. Taken together, we succeeded in the reduction of cytotoxicity, without loss of the suppression effect on lipid accumulation, by elongation of the dicarboxylic moiety of Tsuc. Tadi may be a promising candidate as an anti-obesity drug.     

Evidence for specificity of cultivable bacteria associated with arbuscular mycorrhizal fungal spores

Bacteria associated with arbuscular mycorrhizal (AM) fungal spores may play functional roles in interactions between AM fungi, plant hosts and defence against plant pathogens. To study AM fungal spore-associated bacteria (AMB) with regard to diversity, source effects (AM fungal species, plant host) and antagonistic properties, we isolated AMB from surface-decontaminated spores of Glomus intraradices and Glomus mosseae extracted from field rhizospheres of Festuca ovina and Leucanthemum vulgare. Analysis of 385 AMB was carried out by fatty acid methyl ester (FAME) profile analysis, and some also identified using 16S rRNA gene sequence analysis. The AMB were tested for capacity to inhibit growth in vitro of Rhizoctonia solani and production of fluorescent siderophores. Half of the AMB isolates could be identified to species (similarity index 0.6) within 16 genera and 36 species. AMB were most abundant in the genera Arthrobacter and Pseudomonas and in a cluster of unidentified isolates related to Stenotrophomonas. The AMB composition was affected by AM fungal species and to some extent by plant species. The occurrence of antagonistic isolates depended on AM fungal species, but not plant host, and originated from G. intraradices spores. AM fungal spores appear to host certain sets of AMB, of which some can contribute to resistance by AM fungi against plant pathogens.     

Susceptibility of the summer fruit tortrix moth, Adoxophyes orana (Lepidoptera: Tortricidae), to chlorpyrifos and strategies for insecticidal control in orchards

The susceptibility of populations of the summer fruit tortrix moth, Adoxophyes orana, from apple orchards in Kent, England, to chlorpyrifos was determined in 1992, 1994 and 1995, by topically dosing larvae feeding on leaves or adults adhered to pheromone trap sticky bases. LD₅₀ values (range 6.3 to 23.2 ng chlorpyrifos per individual) for first or second generation males, second generation females or for first generation fourth instar larvae collected in the field were significantly greater (2–3 fold) for populations from orchards with a long history of treatment with broad-spectrum organophosphorous and other insecticides than for those from untreated orchards (range 1.6 to 8.1 ng chlorpyrifos per individual), though there was considerable variation between sites and years. Twelve replicated orchard experiments between 1993 and 1995 were used to examine a range of different strategies for insecticidal control. Applications of chlorpyrifos against overwintered larvae feeding in trusses in spring gave, at best, a 75% reduction in larval numbers, insufficient to prevent damage by the subsequent generation in summer. However, two applications of fenoxycarb (one immediately pre-and one immediately post-blossom of apple cv. Cox) completely prevented successful subsequent development of the pest, and a single (post-blossom) spray nearly so. The timing of application of chlorpyrifos against first generation larvae hatching from eggs in June was shown to be critical and currently used methods of spray-timing were unreliable. The effective persistence of chlorpyrifos was short (< 7 days). Though good control could be achieved with a single spray, a better strategy was to apply a series of sprays at 7-day intervals to cover the egg hatch period. Sprays of Bacillus thuringiensis at the same timing intervals gave, maximally, an 80% reduction in larval numbers, but usually less: improved control was not achieved by applying a programme of sprays compared to a single spray, though a novel, more potent, formulation was significantly more active. Triazophos was also effective as an egg hatch spray. Sprays of chlorpyrifos or tebufenozide between late September and mid-October 1995 against second or third instar larvae migrating to overwintering sites before diapause, failed to reduce significantly the numbers of subsequent overwintering larvae or the numbers that emerged to feed on blossom trusses in spring.     

Monounsaturated fatty acyl-coenzyme A is predictive of atherosclerosis in human apoB-100 transgenic, LDLr-/- mice

ACAT2, the enzyme responsible for the formation of cholesteryl esters incorporated into apolipoprotein B-containing lipoproteins by the small intestine and liver, forms predominantly cholesteryl oleate from acyl-CoA and free cholesterol. The accumulation of cholesteryl oleate in plasma lipoproteins has been found to be predictive of atherosclerosis. Accordingly, a method was developed in which fatty acyl-CoA subspecies could be extracted from mouse liver and quantified. Analyses were performed on liver tissue from mice fed one of four diets enriched with one particular type of dietary fatty acid: saturated, monounsaturated, n-3 polyunsaturated, or n-6 polyunsaturated. We found that the hepatic fatty acyl-CoA pools reflected the fatty acid composition of the diet fed. The highest percentage of fatty acyl-CoAs across all diet groups was in monoacyl-CoAs, and values were 36% and 46% for the n-3 and n-6 polyunsaturated diet groups and 55% and 62% in the saturated and monounsaturated diet groups, respectively. The percentage of hepatic acyl-CoA as oleoyl-CoA was also highly correlated to liver cholesteryl ester, plasma cholesterol, LDL molecular weight, and atherosclerosis extent. These data suggest that replacing monounsaturated with polyunsaturated fat can benefit coronary heart disease by reducing the availability of oleoyl-CoA in the substrate pool of hepatic ACAT2, thereby reducing cholesteryl oleate secretion and accumulation in plasma lipoproteins.     

Effects of Octadecanoylsucrose Derivatives on the Production of Inulinase by Apergillus niger SL-09

Summary The effect of the addition of octadecanoylsucrose esters to the growth medium on the production of inulinase by Aspergillus niger SL-09 was studied in batch culture using shake flasks. The activities of inulinase in vitro and in vivo formed by Aspergillus niger SL-09 was enhanced dramatically by the addition of sucrose ester S-770 to the medium, and it was confirmed that sucrose ester acted as a very efficient inducer for inulinase production. As a result, with the addition of 6 g sucrose ester l⁻¹ at the beginning of the culture, the enzyme activities were enhanced near 7-fold higher than that obtained in the basal medium.     

Inhibition by Cyclic AMP of Phorbol Ester-Potentiated Norepinephrine Release from Guinea Pig Brain Cortical Synaptosomes

The involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) and cyclic AMP-dependent protein kinase in the K+-evoked release of norepinephrine (NE) was studied using guinea pig brain cortical synaptosomes preloaded with [3H]NE. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, enhanced the K+-evoked release of [3H]NE, in a concentration-dependent manner, but with no effect on the spontaneous outflow and uptake of [3H]NE in the synaptosomes. The apparent affinity of the evoked release for added calcium but not the maximally evoked release was increased by TPA (10(-7) M). Inhibitors of PKC, polymyxin B, and a more potent inhibitor, staurosporine, counteracted the TPA-induced potentiation of the evoked release. Both forskolin and dibutyryl cyclic AMP (DBcAMP) enhanced the evoked release, but reduced the TPA-potentiated NE release. A novel inhibitor of cyclic AMP-dependent protein kinase, KT5720, blocked both the forskolin-induced increase in the evoked release and its inhibition of TPA-induced potentiation in the evoked release, thereby suggesting that forskolin or DBcAMP counteracts the Ca2+-dependent release of NE by activating cyclic AMP-dependent protein kinase. These results suggest that the activation of PKC potentiates the evoked release of NE and that the activation of cyclic AMP-dependent protein kinase acts negatively on the PKC-activated exocytotic neurotransmitter release process in brain synaptosomes of the guinea pig.     

改良法建立妊娠期高血压疾病大鼠模型

目的寻找理想的妊娠期高血压疾病动物模型。方法60只雌性Wistar大鼠分为六组,分别为正常妊娠组（A组）、左旋亚硝酸精氨酸甲酯（L-NAME）腹腔注射组（B组）、生理盐水注射组（C组）、假手术组（D组）、双侧子宫动脉结扎术组（E组）以及L-NAME腹腔注射联合腹主动脉缩窄术组（F组）,对大鼠血压、尿蛋白值以及胎鼠、胎盘、胎鼠头的重量进行监测并观察肾脏、胎盘的病理改变。结果F组孕鼠于孕13d即出现了蛋白尿、血压升高,较B组及E组出现时间早（P〈0.05）,其胎鼠体重、胎头重量下降较各组更明显（P〈0.05）,肾小球小动脉管壁增厚、管腔狭窄;胎盘出现血管间膜增厚、纤维蛋白沉积等妊娠高血压疾病的典型病理改变。结论L-NAME腹腔注射联合腹主动脉缩窄术是更为理想的建立妊娠高血压疾病动物模型的方法。     

Modulation of N-type calcium channel activity by G-proteins and protein kinase C

N-type voltage-gated calcium channel activity in rat superior cervical ganglion neurons is modulated by a variety of pathways. Activation of heterotrimeric G-proteins reduces whole-cell current amplitude, whereas phosphorylation by protein kinase C leads to an increase in current amplitude. It has been proposed that these two distinct pathways converge on the channel's pore-forming alpha(1B) subunit, such that the actions of one pathway can preclude those of the other. In this study, we have characterized further the actions of PKC on whole-cell barium currents in neonatal rat superior cervical ganglion neurons. We first examined whether the effects of G-protein-mediated inhibition and phosphorylation by PKC are mutually exclusive. G-proteins were activated by including 0.4 mM GTP or 0.1 mM GTP-gamma-S in the pipette, and PKC was activated by bath application of 500 nM phorbol 12-myristate 13-acetate (PMA). We found that activated PKC was unable to reverse GTP-gamma-S-induced inhibition unless prepulses were applied, indicating that reversal of inhibition by phosphorylation appears to occur only after dissociation of the G-protein from the channel. Once inhibition was relieved, activation of PKC was sufficient to prevent reinhibition of current by G-proteins, indicating that under phosphorylating conditions, channels are resistant to G-protein-mediated modulation. We then examined what effect, if any, phosphorylation by PKC has on N-type barium currents beyond antagonizing G-protein-mediated inhibition. We found that, although G-protein activation significantly affected peak current amplitude, fast inactivation, holding-potential-dependent inactivation, and voltage-dependent activation, when G-protein activation was minimized by dialysis of the cytoplasm with 0.1 mM GDP-beta-S, these parameters were not affected by bath application of PMA. These results indicate that, under our recording conditions, phosphorylation by PKC has no effect on whole-cell N-type currents, other than preventing inhibition by G-proteins.     

静注氟比洛芬酯预防肺叶手术中全麻患者苏醒期躁动的临床研究

目的:探讨肺叶手术中超前应用氟比洛芬酯对减轻全身麻醉苏醒期躁动发生率的影响。方法:60例肺叶切除术患者随机分为A组和B组,每组30例,两组均常规诱导全麻气管插管,术中瑞芬、丙泊酚、维库溴铵泵住维持麻醉,A组于术前给予氟比洛芬酯1 MG/KG。比较两组躁动得分、苏醒后(10 min、30 min、1 h、2 h、4 h、24 h)六个时间点疼痛评分及不良反应发生率。结果:两组出血时间、拔管时间、麻醉时间差异均无统计学意义(均P0.05);A组患者的躁动得分显著低于B组,差异有统计学意义(P0.05);两组的VAS评分差异有统计学意义(P0.05),不同时间点的VAS评分差异有统计学意义(P0.05),分组与时间之间存在交互作用(P0.05);A组患者的不良反应发生率低于B组,差异有统计学意义(P0.05)。结论:肺叶手术中超前应用氟比洛芬酯具有良好的镇痛效果,可以降低全身麻醉苏醒期躁动的发生率。     

Chemiluminescent immunoassay (CLIA) for salmon growth hormone (GH)

A highly sensitive and specific chemiluminescent immunoassay (CLIA) was developed for quantification of growth hormone (GH) in salmonid species. The CLIA for salmon GH was performed using the sandwich method with anti-GH IgG as the first antibody and chemiluminescent acridinium ester-labelled specific anti-GH F(ab′)₂ as the second antibody. The measurable range of salmon GH in the CLIA was 39–1250 pg/mL using a short assay (1 day) protocol and 3.9–125 pg/mL in a longer (2-day) assay. The dilution curve in the CLIA of serum from masu salmon (Oncorhynchus masou) was parallel to the standard curve of recombinant chum salmon (Oncorhynchus keta) GH. Seasonal changes of serum GH levels were measured in 1 year-old masu salmon cultivated in a pond from March to November. Their serum GH levels increased during smoltification from March to April, achieved a maximum level of 21 ng/mL in August, and then declined gradually to 11 ng/mL in October. © 1997 John Wiley & Sons, Ltd.