Interaction of volkensin with HeLa cells: binding,uptake, intracellular localization,degradation and exocytosis

Among two-chain ribosome-inactivating proteins (RIPs), volkensin is the most toxic to cells and animals, and is retrogradely axonally transported in the rat central nervous system, being an effective suicide transport agent. Here we studied the binding, endocytosis, intracellular routeing, degradation and exocytosis of this RIP. The interaction of volkensin with HeLa cells was compared to that of nigrin b, as an example of a type 2 RIP with low toxicity, and of ricin, as a reference toxin. Nigrin b and volkensin bound to cells with comparable affinity (approx. 10^-10 M) and had a similar number of binding sites (2 × 105/cell), two-log lower than that reported for ricin. The cellular uptake of volkensin was lower than that reported for nigrin b and ricin. Confocal microscopy showed the rapid localization of volkensin in the Golgi stacks with a perinuclear localization similar to that of ricin, while nigrin b was distributed between cytoplasmic dots and the Golgi compartment. Consistently, brefeldin A, which disrupts the Golgi apparatus, protected cells from the inhibition of protein synthesis by volkensin or ricin, whereas it was ineffective in the case of nigrin b. Of the cell-released RIPs, 57% of volkensin and only 5% of ricin were active, whilst exocytosed nigrin b was totally inactive. Despite the low binding to, and uptake by, cells, the high cytotoxicity of volkensin may depend on (i) routeing to the Golgi apparatus, (ii) the low level of degradation, (iii) rapid recycling and (iv) the high percentage of active toxin remaining after exocytosis.Received 21 April 2004; received after revision 26 May 2004; accepted 9 June 2004     

Enzymes of Sphingolipid metabolism in Drosophila melanogaster

Sphingolipids are important structural components of membranes that delimit the boundaries of cellular compartments, cells and organisms. They play an equally important role as second messengers, and transduce signals across or within the compartments they define to initiate physiological changes during development, differentiation and a host of other cellular events. For well over a century Drosophila melanogaster has served as a useful model organism to understand some of the fundamental tenets of development, differentiation and signaling in eukaryotic organisms. Directed approaches to study sphingolipid biology in Drosophila have been initiated only recently. Nevertheless, earlier phenotypic studies conducted on genes of unknown biochemical function have recently been recognized as mutants of enzymes of sphingolipid metabolism. Genome sequencing and annotation have aided the identification of homologs of recently discovered genes. Here we present an overview of studies on enzymes of the de novo sphingolipid biosynthetic pathway, known mutants and their phenotypic characterization in Drosophila.Received 14 June 2004; received after revision 15 August 2004; accepted 21 August 2004     

Cytochromes P450 and metabolism of xenobiotics

Cytochromes P450 (henceforth P450s) are involved in a variety of metabolic and biosynthetic processes. The number of known P450 enzymes exceeds 1000, while the endogenous substrates of most of them remain unknown. All P450 enzymes exhibit similarity in their structure and general mechanism of action; however, there are significant differences in the detailed function of individual enzymes as well as in the structures and properties of their active sites. This review discusses the properties of the most important P450 enzymes taking part in drug metabolism in humans. P450 3A4 is of paramount importance, because it is the most abundant P450 in the human liver and is known to metabolize the majority of drugs whose biotransformation is known. Genetically dependent variabilities of individual P450 activities and levels are described, documenting the importance of pharmacogenetics aimed at explaining differences in the response of the organism to various drugs. Received 7 November 2000; received after revision 9 January 2001; accepted 10 January 2001     

Uterine glucose metabolism in the prepubertal rat treated neonatally with androgen,estrogen, and antihormones

Summary Estrogen secretion during infancy may selectively enhance the phosphogluconate oxidative pathway in the rat uterus, for altered estrogen-stimulated glucose oxidation prepubertally is correlated (+0.91) with impaired ovarian development and not uterine estrogen receptor content.This work was supported in part by NSF Research Grant PCM-8409586.     

Synthesis and metabolism of vertebrate-type steroids by tissues of insects: A critical evaluation

Summary This review covers the synthesis and the metabolism of vertebrate-type steroids (progesterone, testosterone, estradiol, corticosteroids) by insect tissues and discusses the significance of the reactions for insect physiology. Biosynthesis of vertebrate-type steroids from cholesterol hitherto has been demonstrated in only two insect species, i.e. the water beetleAcilius sulcatus (Coleoptera) and the tobacco hornwormManduca sexta (Lepidoptera). InAcilius, steroid synthesis is associated with exosecretion (chemical defense). Nothing, however, is known about a physiological role of the C₂₁ steroid conjugate present in ovaries and eggs ofManduca. No synthesis of vertebrate-type steroids was observed in any other insect investigated to date. Most metabolic conversions of steroids by insects concerned oxidoreduction of oxygen groups (hydroxysteroid dehydrogenase activity) and (polar and apolar) conjugate formation. All important enzymatic steps involved in synthesis and catabolism, as known from studies with tissues of vertebrates, were not, or hardly observed. The conclusion is drawn that typical vertebrate-type (C₂₁, C₁₉ and C₁₈) steroids probably do not act as physiologically active substances in insects.     

A comparison of cycloartenol and lanosterol biosynthesis and metabolism by the Oomycetes

Summary Representative members in each of the four orders of Oomycetes (Phytophthora cactorum, Peronosporales;Lagenidium callinectes, L. giganteum, Lagenidiales;Saprolegnia ferax, Saprolegniales;Apodachylella completa, Leptomitales) have been examined for their ability to synthesize and polycyclize squalene-oxide (SO) to a tetracyclic product and to differentiate between cycloartenol and lanosterol metabolism to sterols.P. cactorum andL. giganteum failed to synthesize or metabolize SO, cycloartenol or lanosterol. While the other three fungi synthesized sterols via SO and lanosterol, a minor metabolism of added cycloartenol to the 4,4-desmethyl-14-methylcyclosteroid dehydropollinastanol was observed.Acknowledgment.Saprolegnia ferax-ATCC 3605 (1),Lagenidium callinectes ATCC 24973 (2),Apodachlyella completa (3) were obtained from Dr J. Aronson, Arizona State Univ. (3),Lagenidium gigateum was obtained from a drainage ditch in California (4), andPhytophthora cactorum was obtained from the U.C. Berkeley Fungal Collection (5). The fungi were cultured as previously described: Cultures 1,2,3: Berg, L. B., Ph. D. dissertation, Univ. of MD., College Park (1983); culture 4: Kerwin, J.L., and Washino, R.K., Exp. Mycol.7 (1983) 109; culture 5: Nes, W.D., and Stafford, A.E., Lipids19 (1984) 544. Preliminary observations involving the labeled substrates were presented by Le, P.H., Nes, W.D., and Parish, E.J. J. Am. Oil Chem. Soc.62 (1985) 655(A). Please address all correspondence to W.D. Nes, Plant Physiology and Chemistry Research Unit, ARS-US Dept of Agriculture, Berkeley, CA 94710, USA.     

Influence of phenobarbital and TCDD on the hepatic metabolism of TCDD in the dog

Summary The influence of phenobarbital and TCDD pretreatment on the formation and biliary excretion of TCDD-metabolites following single doses of³H-TCDD was investigated. Without pretreatment, 24.5% of the absorbed³H-TCDD dose was excreted in the bile within 110 h. Phenobarbital did not influence this rate, whereas a single dose of 10 g of unlabeled TCDD/kg b.wt nine days earlier resulted in a doubling of the amount of radioactive material eliminated in the bile (47.4%).     

Demonstration of vitamin D3 metabolism inmytilus edulis

Summary Radiolabeled vitamin D₃ was converted into several polar metabolites upon incubation with tissue homogenates from the common musselMytilus edulis. Chromatographic analysis indicated that the metabolites have chromatographic mobilities different from those of known standards. The results suggest that vitamin D₃ is metabolized in mussels via pathways that differ from the vertebrate systems.Acknowledgments. The authors thank Dr Pirjo Rantamäki for the mussels and F. Hoffmann-La Roche & Co. for supplying the vitamin D₃ metabolite standards. This work was supported by a grant from the Magnus Ehrnrooth Foundation to T. K.     

胶凝面板堆石坝优化设计

采用非线性数学规划法对胶凝面板堆石坝进行断面优化设计,建立了以大坝的关键几何尺寸为设计变量,以造价为目标函数,以坝体稳定、应力、应力水平及几何尺寸为约束条件的优化设计数学模型,并采用罚函数法求解此优化模型。与原设计方案比较,优化设计方案的堆石方量少、造价低。与普通面板堆石坝相比,胶凝面板堆石坝的优化方案坝坡可以更陡,堆石方量更少、造价更低,且满足应力、稳定的约束条件,得到的断面安全可靠、经济合理。     

The effect of castration,testosterone and estradiol on14C-serotonin metabolism by organ cultures of male rat pineal glands

Summary The pineal gland of the male rat does not appear to rely on prior conversion of testosterone to estradiol for the stimulant effect of testosterone on pineal melatonin and 5-methoxytryptophol synthesis.     

具有双控制输入通道高阶不确定非线性系统控制设计

本文研究了一类高阶不确定非线性系统的全局渐近稳定控制设计问题.不同于现有结果,本文所研究的高阶非线性系统具有两个控制输入通道,即控制输入直接作用于两个一维子系统.这导致已有的控制设计方法不能或难以用来解决该类控制设计问题,因此,寻求新的控制设计方法,用以解决多控制输入通道高阶非线性系统的控制设计问题,是一个很有意义的研究课题.本文首先引入一个有效的反馈变换,从而在非线性系统满足适当假设条件下,成功地将该类系统的控制设计问题转化为规范的单控制输入通道情况下的控制设计问题.进而借助于已有结果,特别是增加幂次积分方法,给出所研究非线性系统的全局渐近稳定控制器的设计方法.尽管本文所研究的系统是具有多控制输入通道高阶非线性系统的特殊情况,所得结果具有一定的局限性,但对更一般情况具有借鉴和指导作用.最后,仿真算例验证了本文理论结果的有效性.     

我国高校科技创新到创业的推进机制研究

我国高校每年取得的科技成果在六千项至八千项之间,但是真正实现成果转化与产业化的还不到十分之一.应该转变观念,积极促成高校创新到创业的转化;科技创新与创业由一个部门归口管理,整合科技资源,探索产、学、研一体化的科研体制和运行机制;建立专利服务机构,提供科技成果转化孵化器,发展科技中介,从而健全学校科技成果产业化的链条,推进我国高校创新到创业的转化。     

Early effects of copper accumulation on methionine metabolism

Wilson's disease is characterized by longterm hepatic accumulation of copper leading to liver disease with reduction of S-adenosylmethionine synthesis. However, the initial changes in this pathway remain unknown and constitute the objective of the present study. Using the Long Evans Cinnamon rat model, early alterations were detected in the mRNA and protein levels, as well as in the activities of several enzymes of the methionine cycle. Notably, the main change was a redox-mediated 80% decrease in the mRNA levels of the methionine adenosyltransferase regulatory subunit as compared to the control group. Moreover, changes in S-adenosylmethionine, S-adenosylhomocysteine, methionine and glutathione levels were also observed. In addition, in vitro experiments show that copper affects the activity and folding of methionine adenosyltransferase catalytic subunits. Taken together, these observations indicate that early copper accumulation alters methionine metabolism with a pattern distinct from that described previously for other liver diseases.     

Cyclophosphamide-impaired regulation of hepatic heme metabolism

Summary Im male rats hepatic cytochromes b₅ and P-450 were reduced at different times after treatment with cyclophosphamide (CP) (200 mg/kg i.p. for 3 days). In contrast, microsomal heme did not change until 48 h after the last dose of CP, leading to accumulation of heme in a non-cytochromal form. Parallel to the above changes the heme metabolism showed derangement: -aminolaevulinate synthase, the rate-limiting enzyme in heme synthesis, was depressed and heme oxygenase, the enzyme which catalyzes the oxidative degradation of heme, was increased.     

The regulation of trehalose metabolism in insects

Trehalose is a non-reducing disaccharide comprising two glucose molecules. It is present in high concentration as the main haemolymph (blood) sugar in insects. The synthesis of trehalose in the fat body (an organ analogous in function to a combination of liver and adipose tissue in vertebrates) is stimulated by neuropeptides (hypertrehalosaemic hormones), released from the corpora cardiaca, a neurohaemal organ associated with the brain. The peptides cause a decrease in the content of fructose 2,6-bisphosphate in fat body cells. Fructose 2,6-bisphosphate, acting synergistically with AMP, is a potent activator of the glycolytic enzyme 6-phosphofructokinase-1 and a strong inhibitor of the gluconeogenic enzyme fructose 1,6-bisphosphatase. This indicates that fructose 2,6-bisphosphate is a key metabolic signal in the regulation of trehalose synthesis in insects. Trehalose is hydrolysed by trehalase (E.C. 3.2.1.28). The activity of this enzyme is regulated in flight muscle, but the mechanism by which this is achieved is unknown. Trehalase from locust, flight muscle is a glycoprotein bound to membranes of the microsomal fraction. The enzyme can be activated by detergents in vitro and by short flight intervals in vivo, which indicates that changes in the membrane environment modulate trehalase activity under physiological conditions.     

Neuroendocrine control of carbohydrate metabolism in the freshwater bivalve molluscLamellidens marginalis

Summary Blood sugars and foot muscle glycogen were measured in the mussel,L. marginalis after ablation of the cerebral ganglia, and in mussels injected with cerebral ganglionic extract 3 h after ablation. There is a rise in the blood sugar and decrease in foot muscle glycogen 3 h after operation, but no change in sham-operated controls. The effect of ablation is reversed by injecting brain extract into ablated mussels. No such effect could be seen in the controls. The results are suggestive of the presence, in the cerebral ganglia, of a hypoglycaemic factor similar to insulin.     

The metabolism and function of sphingolipids and glycosphingolipids

Sphingolipids and glycosphingolipids are emerging as major players in many facets of cell physiology and pathophysiology. We now present an overview of sphingolipid biochemistry and physiology, followed by a brief presentation of recent advances in translational research related to sphingolipids. In discussing sphingolipid biochemistry, we focus on the structure of sphingolipids, and their biosynthetic pathways – the recent identification of most of the enzymes in this pathway has led to significant advances and better characterization of a number of the biosynthetic steps, and the relationship between them. We then discuss some roles of sphingolipids in cell physiology, particularly those of ceramide and sphingosine-1-phosphate, and mention current views about how these lipids act in signal transduction pathways. We end with a discussion of sphingolipids and glycosphingolipids in the etiology and pathology of a number of diseases, such as cancer, immunity, cystic fibrosis, emphysema, diabetes, and sepsis, areas in which sphingolipids are beginning to take a central position, even though many of the details remain to be elucidated. Received 13 February 2007; received after revision 19 April 2007; accepted 26 April 2007     

Regulation of carbohydrate metabolism by the optic tentacles of the garden snailCryptozona ligulata (Gastropoda-Stylommatophora)

Summary The presence of a hyperglycemic factor in the optic tentacles of the snailC. ligulata is reported here. A preliminary characterization based on crude extracts indicates the factor to be water-soluble, heat labile and to be an albumin. The ablation of optic tentacles and injection of optic tentacle extract into operated and normal snails caused a rise in blood sugar, total carbohydrate and glycogen in the foot muscle and mantle and a decrease in hepatopancreatic glycogen. The ablation also caused a fall in blood free amino acids and a rise in the tissues, which was reversed in the blood and foot muscle by injection of the extract. Possible conversion of amino acids to total carbohydrates and glycogen by gluconeogenesis is suggested.