Compartmental study of rat renal phospholipid metabolism

Phospholipid content and metabolism were studied in rat renal papillary, medullary and cortical slices. The highest concentration of phospholipids was found in cortex and the lowest in papilla samples (ratio cortex/medulla, 1.3; cortex/papilla, 3.7). The profile of the various phospholipids was different depending on the zone. The most important difference was the relative concentrations of sphingomyelin (CerPCho) and phosphatidylinositol (PtdIns) with ratios for PtdIns/CerPCho of 5.0, 3.3 and 2.5 in papilla, medulla, and cortex, respectively. In the three zones, PtdIns showed the highest specific activity for [2-¹⁴C]glycerol and [1-¹⁴C]arachidonic acid incorporation. By contrast, a higher amount of [1-¹⁴C]palmitic acid was incorporated into phosphatidylcholine than into any other phospholipid. The various radioactive precursors were only poorly incorporated into phosphatidylethanolamine. No radioactivity was associated with phosphatidylserine. The papilla possesses the most active phospholipid metabolism of all the pathways studied.     

Coordinate packaging of newly synthesized phosphatidylcholine and phosphatidylglycerol in lamellar bodies in alveolar type II cells

Methylamine, a weak base, inhibits packaging of newly synthesized phosphatidylcholine (PC) in lamellar bodies in 20–22 h cultured alveolar type II cells, suggesting a role for acidic pH of lamellar bodies. In this study, we tested if (i) the packaging of PC is similarly regulated in freshly isolated type II cells and (ii) methylamine also inhibits the packaging of other surfactant phospholipids, particularly, phosphatidylglycerol (PG). The latter would suggest coordinated packaging so as to maintain the phospholipid composition of lung surfactant. During the short-term metabolic labeling experiments in freshly isolated type II cells, methylamine treatment decreased the incorporation of radioactive precursors into PC, disaturated PC (DSPC), and PG of lamellar bodies but not of the microsomes, when compared with controls. The calculated packaging (the percentage of microsomal lipid packaged in lamellar bodies) of each phospholipid was similarly decreased (∼50%) in methylamine-treated cells, suggesting coordinated packaging of surfactant phospholipids in lamellar bodies. Equilibrium-labeling studies with freshly isolated type II cells (as is routinely done for studies on surfactant secretion) ± methylamine showed that in methylamine-treated cells, the secretion of PC and PG was decreased (possibly due to decreased packaging), but the phospholipid composition of released surfactant (measured by radioactivity distribution) was unchanged; and the PC content (measured by mass or radioactivity) of lamellar bodies was lower, but the PC composition (as percentage of total phospholipids) was unchanged when compared with control cells. We speculate that the newly synthesized surfactant phospholipids, PC, DSPC, and PG, are coordinately transported into lamellar bodies by a mechanism requiring the acidic pH, presumably, of lamellar bodies.     

微生物降解甲基叔丁基醚及其关键酶的研究进展

为了减少机动车排放的CO和O3等污染气体,提高汽油的辛烷值,甲基叔丁基醚（MTBE）作为燃料氧化剂而广泛应用于汽油中。与此同时,由于MTBE具有很高的水溶性和难降解性,使用过程中的泄露带来了严重的污染问题。其自身的化学特性也给去除环境中的MTBE增添了很多困难。生物降解是土壤地下水修复中公认的有效又可以节省成本的方法。国外已经报道MTBE能在好氧或厌氧的条件下被微生物通过直接代谢或共代谢的方式而降解,国内在此方面的研究刚刚开始,尤其在降解机理方面的研究还存在很大的空白。本文对能够降解MTBE的微生物进行了总结,并且重点阐述了在MTBE降解过程中起作用的关键酶。     

Altered phospholipid metabolism in sodium butyrate-induced differentiation of C6 glioma cells

We examined the changes in phospholipid metabolisms in sodium butyrate-treated C6 glioma cells. Treatment of 2.5 mM sodium butyrate for 24 h induced an increase in the activity of glutamine synthetase, suggesting that these cells were under differentiation. Similar treatment was associated with (i) increased arachidonic acid incorporation into phosphatidylcholine, and (ii) decreased arachidonic acid incorporation into phosphatidylinositol and (iii) phosphatidylethanolamine. These effects were subsequently investigated by examining the acylation process, de novo biosynthesis, and the agonist-stimulated phosphoinositides hydrolysis in these cells. Our results indicated that sodium butyrate stimulated the acylation of arachidonic acid into lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylinositol. The glycerol incorporation into these lipids was not affected, but the inositol incorporation into these lipids was not affected, but the inositol incorporation into total chloroform extracts and Pl and phosphatidylinositol 4-phosphate was decreased in the sodium butyrate-treated cells. Moreover, the accumulation of the rapid histamine-stimulated phosphoinositide metabolites, i.e., inositol monophosphate, inositol diphosphate, and inositol triphosphate (IP₃) was decreased in these cells. To elucidate whether the decreased inositol phosphates were due to a decrease in the phosphoinositides hydrolysis, we measured the transient IP₃ production directly by a receptor-binding assay. Our results indicated that histamine-stimulated transient IP₃ formations were decreased. Taken together, these results indicated that multiple changes by multiple mechanisms of phospholipid metabolisms were found in sodium butyrate-treated C6 glioma cells. The decreased IP₃ formation and its subsequent action, i.e., Ca²⁺ mobilization, may play an early but pivotal role by which sodium butyrate induces C6 glioma cell differentiation.     

Effect of trifluoperazine on certain arterial wall lipid-metabolizing enzymes inducing atherosclerosis in rhesus monkeys

The effect of trifluoperazine (TFP) was investigated on arterial wall lipid-metabolizing enzymes like acyl-CoA:cholesterol acyltransferase (ACAT) and cholesterol ester hydrolase (CEH) in rhesus monkeys. The activity was determined in aortic wall homogenates obtained from rhesus monkeys fed an atherogenic diet coupled with intramuscular injections of adrenaline and TFP. Although TFP had no significant effect on serum cholesterol and triglycerides, it decreased significantly the formation of atherosclerotic lesions by decreasing the esterification of cholesterol, by inhibiting ACAT and enhancing its utilization by activating CEH. Hence, the preventive effect of TFP on the development of atherosclerosis in rhesus monkeys is mediated through its ability to influence the activities of arterial wall lipid-metabolizing enzymes like ACAT and CEH.     

High doses of atorvastatin and simvastatin induce key enzymes involved in VLDL production

Treatments with high doses of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors may induce the expression of sterol regulatory element binding protein (SREBP)-target genes, causing different effects from those attributed to the reduction of hepatic cholesterol content. The aim of this study was to investigate the effects of high doses of statins on the key enzymes involved in VLDL production in normolipidemic rats. To examine whether the effects caused by statin treatment are a consequence of HMG-CoA reductase inhibition, we tested the effect of atorvastation on these enzymes in mevalonatefed rats. Atorvastatin and simvastatin enhanced not only HMG-CoA reductase but also the expression of the SREBP-2 gene itself. As a result of the overexpression of SREBP-2 caused by the statin treatment, genes regulated basically by SREBP-1, as FA synthase and acetyl-coenzyme A carboxylase, were also induced and their mRNA levels increased. DAG acyltransferase and microsomal IG transfer protein mRNA levels as well as phosphatidate phosphohydrolase activity were increased by both statins. Simvastatin raised liver cholesterol content, ACAT mRNA levles, and CTP:phosphocholine cytidylyltransferase activity, whereas it reduced liver DAG and phospholipid content. Mevalonate feeding reversed all changes induced by the atorvastatin treatment. These results show that treatment with high doses of statins induces key enzymes controlling rat liver lipid synthesis and VLDL assembly, probably as a result of SREBP-2 overexpression. Despite the induction of the key enzymes involved in VLDL production, both statins markedly reduced plasma TG levels, suggesting that different mechanisms may be involved in the hypotriglyceridemic effect of statins at high or low doses.     

Effect of phospholipid n‐3 polyunsaturated fatty acids on rat lipid metabolism

It has been demonstrated that the amount and type of dietary fat are factors involved in the risk of arteriosclerosis and coronary or cerebral artery disease through lipid metabolism. In this study, we investigated the effects of phospholipids (PLs) containing n‐3PUFAs on lipid metabolism in rats. PLs containing n‐3PUFAs were prepared from squid (Todarodes pacificus) mantle muscle. Groups of male Wistar rats were fed AIN93G diet containing soybean oil (SO, 7%), fish oil (1.2%) + SO (5.8%), soybean PLs (1.8%) + SO (5.2%), or PLs containing n‐3PUFAs (1.8%) + SO (5.2%). The following indicators were assayed as indexes of lipid metabolism: TAG and cholesterol in serum and liver, fecal cholesterol, bile‐acid excretion, and liver mRNA expression levels of genes encoding proteins involved in cholesterol homeostasis. Serum and liver TAG contents decreased significantly in the group fed PLs containing n‐3PUFAs as compared to other groups, accompanied by a significant decline in the expression level of sterol regulatory element binding protein‐1c. The decrease in cholesterol content in the group fed PLs containing n‐3PUFAs was due to the increase in fecal cholesterol excretion and the increase of mRNA expression levels of ATP‐binding cassette (ABC) G5 and ABCG8 in liver. Practical applications : PLs containing n‐3PUFAs decreased serum and liver TAG contents compared with that induced by soybean PLs. Further, PLs containing n‐3PUFAs can induce a reduction in serum and liver cholesterol concentrations as well as the triglyceride‐reducing effect of conventional n‐3PUFAs containing TAG. In other words, dietary n‐3PUFAs contained in PLs can prevent life‐style diseases such as hyperlipidemia, arteriosclerosis and coronary, or cerebral artery disease more effectively than TAG containing n‐3PUFAs. Therefore, it is expected that the risk of lifestyle diseases would be decreased if PL containing n‐3PUFAs can be supplied routinely. In this study, PLs containing n‐3PUFAs were prepared from squid mantle muscle. On an industrial scale, such PLs can be produced from various unused resources and waste materials of fisheries. We conclude that highly functional foods could be developed based on the findings of this study, and would be available for health promotion worldwide.     

Synthesis of libraries of 16beta-aminopropyl estradiol derivatives for targeting two key steroidogenic enzymes

Two libraries, each consisting of 48 16beta-aminopropyl estradiol derivatives, phenols and sulfamates, respectively, were synthesized by solid-phase parallel chemistry through a seven-step reaction sequence. Following the attachment of a C18-steroid sulfamate precursor on a trityl chloride resin, diversity elements were first introduced on the 16beta-aminopropyl chain of the steroid by acylation reactions with eight Fmoc-amino acids. After deprotection, the free amine function of the resulting compounds was reacted with six carboxylic acids for the introduction of a second diversity level. The two variants employed for the cleavage of compounds from the solid support, acidic and nucleophilic, allowed the corresponding libraries of sulfamate and phenol derivatives in yields of 8-50 % and 13-58 % to be obtained with an average HPLC purity of 94 % and 91 %, respectively. Potent steroid sulfatase inhibitors and interesting SAR results were generated from the screening of the sulfamate library. Furthermore, moderate inhibitors of type 1 17beta-HSD resulted from the partial screening of phenol library. Thus, these two categories of compounds were synthesized to rapidly identify potential inhibitors of steroid biosynthesis for the hormonal therapy of estrogen-dependent diseases, and also to demonstrate the versatility and efficiency of the recently developed sulfamate linker.     

初始生物素含量波动时谷氨酸发酵关键酶系的酶活变化模式

谷氨酸发酵培养基中的生物素初始含量会随玉米浆来源、批次的不同出现波动,进而影响谷氨酸发酵性能和稳定性。本文研究分析了谷氨酸发酵中,初始生物素含量正常/不当,特别是初始含量不当、采取补救措施条件下的丙酮酸、异柠檬酸和α-酮戊二酸代谢节点处关键酶的活性变化。生物素匮乏时,催化主代谢途径的关键酶活性均被弱化,特别是α-酮戊二酸脱氢酶完全失活,能量代谢主要靠乙醛酸循环维持。当发现生物素匮乏并补加生物素后,中心代谢途径关键酶--丙酮酸脱氢酶的活性恢复到正常水平,TCA重新成为主要供能途径。生物素过量时,丙酮酸羧化酶和谷氨酸脱氢酶活性基本不变,而其他关键酶均被激活。当发现生物素过量并添加吐温40后,丙酮酸脱氢酶和异柠檬酸脱氢酶依旧保持很高活性,而α-酮戊二酸脱氢酶和异柠檬酸裂解酶的活性则下降到正常水平。采用上述补救措施可以挽救因初始生物素匮乏或过量所引起的错误发酵,终酸浓度均可恢复到对照（生物素亚适量）水平（75～80 g·L^-1）。另外,对生物素初始含量和吐温添加进行复合式调控,终酸浓度可进一步提高,达到87 g·L^-1,比对照提高8.8%。     

Correlation of omega-3 levels in serum phospholipid from 2053 human blood samples with key fatty acid ratios

Background  

This research was conducted to explore the relationships between the levels of omega-3 fatty acids in serum phospholipid and key fatty acid ratios including potential cut-offs for risk factor assessment with respect to coronary heart disease and fatal ischemic heart disease.     

Correlation of omega-3 levels in serum phospholipid from 2053 human blood samples with key fatty acid ratios

Background

The incidence of primary osteoporosis is higher in Japan than in USA and European countries. Recently, the importance of preventive medicine has been gradually recognized in the field of orthopaedic surgery with a concept that peak bone mass should be increased in childhood as much as possible for the prevention of osteoporosis. Under such background, we have developed a new bean snack with an aim to improve bone volume loss. In this study, we examined the effects of a newly developed snack on bone volume and density in osteoporosis model mice.

Methods

Orchiectomy (ORX) and ovariectomy (OVX) were performed for C57BL/6J mice of twelve-week-old (Jackson Laboratory, Bar Harbar, ME, USA) were used in this experiment. We prepared and given three types of powder diet e.g.: normal calcium diet (NCD, Ca: 0.9%, Clea Japan Co., Tokyo, Japan), low calcium diet (LCD, Ca: 0.63%, Clea Japan Co.,) and special diet (SCD, Ca: 0.9%). Eighteen weeks after surgery, all the animals were sacrified and prepared for histomorphometric analysis to quantify bone density and bone mineral content.

Results

As a result of histomorphometric examination, SCD was revealed to enhance bone volume irrespective of age and sex. The bone density was increased significantly in osteoporosis model mice fed the newly developmental snack as compared with the control mice. The bone mineral content was also enhanced significantly. These phenomena were revealed in both sexes.

Conclusion

It is shown that the newly developed bean snack is highly effective for the improvement of bone volume loss irrespective of sex. We demonstrated that newly developmental snack supplements may be a useful preventive measure for Japanese whose bone mineral density values are less than the ideal condition.     

Effect of carbamate pesticides on the induction of hepatic enzymes of the rat and on the microsomal phospholipid changes

The influence of two carbamine pesticides i.e., manebe and carbaryl upon the hepatic microsomal enzymes induction in the rat was studied. Both substances, when administered by themselves, affect only slightly liver weight, P 450 cytochrome rates and bilirubin glucuronosyltransferase, in the microsome fraction of the hepatic homogenate. It seems, however, that carbaryl is involved in producing a slight induction, whereas manebe acts inversely. Yet, manebe changes largely the induction effects of phenobarbital when associated with the latter. In the animal treated simultaneously with manebe and phenobarbital, the increase in the rate of hepatic microsomal P 450 cytochrome as well as the variations in the distribution of fatty acids in phospholipids, are significantly lower than in the animal solely treated with phenobarbital.     

电压扰动对EAD代谢通量中微生物与关键酶活性的影响

为探究电压扰动对EAD代谢通量中微生物与关键酶活性的影响,实验采用单室EAD反应器,以电解电压为扰动变量,利用CNA构建代谢模型,对扰动前后微生物群落、酶活水平及通量变化进行讨论。结果表明,电压扰动后,产甲烷途径偏向于氢营养型产甲烷,约占38.5%,且0.6 V扰动时表现出最佳的甲烷通量,为0.5222 g。EAD体系中的H₂的主要来源是阴极和NADH,电解电压的扰动会影响H₂的产生,进而会影响产甲烷通量。在0.6 V扰动后,生物膜中Trichloromonas的相对丰度高达 40.2%,分别是1.0 V和1.4 V的1.52倍和1.13倍,同时CoI、PTA、AK与CoF₄₂₀均表现出最佳的酶活水平,关键酶活水平和Trichloromonas的相对丰度与产甲烷代谢通量表现出较好的一致性,而阳极生物膜中微生物多样性也是影响EAD产甲烷通量的重要因素。     

电压扰动对EAD代谢通量中微生物与关键酶活性的影响

为探究电压扰动对EAD代谢通量中微生物与关键酶活性的影响,实验采用单室EAD反应器,以电解电压为扰动变量,利用CNA构建代谢模型,对扰动前后微生物群落、酶活水平及通量变化进行讨论。结果表明,电压扰动后,产甲烷途径偏向于氢营养型产甲烷,约占38.5%,且0.6 V扰动时表现出最佳的甲烷通量,为0.5222 g。EAD体系中的H₂的主要来源是阴极和NADH,电解电压的扰动会影响H₂的产生,进而会影响产甲烷通量。在0.6 V扰动后,生物膜中Trichloromonas的相对丰度高达 40.2%,分别是1.0 V和1.4 V的1.52倍和1.13倍,同时CoI、PTA、AK与CoF₄₂₀均表现出最佳的酶活水平,关键酶活水平和Trichloromonas的相对丰度与产甲烷代谢通量表现出较好的一致性,而阳极生物膜中微生物多样性也是影响EAD产甲烷通量的重要因素。     

Studies of phospholipid metabolism, proliferation, and secretion of stably transfected insulinoma cells that overexpress group VIA phospholipase A2

A cytosolic 84 kDa Group VIA phospholipase A₂ (iPLA₂β) that does not require Ca²⁺ for catalysis was cloned from Chinese hamster ovary (CHO) cells, murine P388D1 cells, pancreatic islet β-cells, and other sources. Proposed iPLA₂β functions include participation in phosphatidylcholine (PC) homeostasis by degrading excess PC generated in CHO cells that overexpress CTP:phosphocholine cytidylyltransferase (CT), which catalyzes the rate-limiting step in PC biosynthesis; participation in biosynthesis of arachidonate-containing PC species in P388D1 cells by generating lysophosphatidylcholine (IPC) acceptors for arachidonate incorporation; and participation in signaling events in insulin secretion from islet β-cells. To further examine iPLA₂β functions in β-cells, we prepared stably transfected INS-1 insulinoma cell lines that overexpress iPLA₂β activity eightfold compared to parental INS-1 cells or to INS-1 cells transfected with an empty retroviral vector that did not contain iPIA₂β cDNA. The iPLA₂β-overexpressing cells exhibit a twofold increase in CT activity compared to parental cells but little change in rates of [³H] choline incorporation into or disappearance from PC. Electrospray ionization (ESI) tandem mass spectrometric measurements indicate that iPLA₂β-overexpressing cells have 1.5-fold higher LPC levels than parental INS-1 cells but do not exhibit increased rates of [³H]arachidonate incorporation into phospholipids, and incorporation is unaffected by a bromoenol lactone (BEL) suicide substrate inhibitor of iPLA₂β. The rate of appearance of arachidonate-containing phosphatidylethanolamine species visualized by ESI mass spectrometry is also similar in iPLA₂β-overexpressing and parental INS-1 cells incubated with supplemental arachidonic acid, and this process is unaffected by BEL. Compared to parental INS-1 cells, iPLA₂β-overexpressing cells proliferate more rapidly and exhibit amplified insulin secretory responses to a protein kinase C-activating phorbol ester, glucose, and a cAMP analog. These findings suggest that iPLA₂β plays a signaling role in β-cells that differs from housekeeping functions in PC biosynthesis and degradation in P388D1 and CHO cells.     

Effect of n−3 fatty acids on the key enzymes involved in cholesterol and triglyceride turnover in rat liver

The effect of long-chain n−3 fatty acids on hepatic key enzymes of cholesterol metabolism and triglyceride biosynthesis was investigated in two rat models. In the first model, rats were intravenously infused for two weeks with a fat emulsion containing 20% of triglycerides in which either n−6 or n−3 fatty acids predominated. The treatment with n−3 fatty acids led to a reduction primarily of serum cholesterol (45%), but also of serum triglycerides (18%). HMG-CoA reductase activity and cholesterol 7α-hydroxylase activity were reduced by 45% and 36%, respectively. There were no significant effects on diacylglycerol acyltransferase (DGAT) or phosphatidate phosphohydrolase (PAP) activities. In the second model, rats were fed a diet enriched with sucrose, coconut oil and either sunflower oil (n−6 fatty acids) or fish oil (long-chain n−3 fatty acid ethyl esters). The treatment with n−3 fatty acids decreased serum triglycerides (41%) and, to a lesser extent, serum cholesterol (17%). Neither glycerol 3-phosphate acyltransferase (GPAT) or DGAT were affected by n−3 fatty acids. In contrast, PAP activity was reduced by 26%. HMG-CoA reductase was not significantly affected, whereas cholesterol 7α-hydroxylase activity was reduced by 36%. The results indicate that part of the TG-lowering effect of long-chain n−3 fatty acids may be mediated by inhibition of the soluble phosphatidate phosphohydrolase. The effect on serum cholesterol may be partly due to inhibition of HMG-CoA reductase.     

Effect of n-3 polyunsaturated fatty acid on gene expression of the critical enzymes involved in homocysteine metabolism

Background  

Previous studies showed that plasma n-3 polyunsaturated fatty acid (PUFA) was negatively associated with plasma homocysteine (Hcy).