Lipoprotein lipases and stress hormones: studies with glucocorticoids and cholera toxin

The intravenous injection of cholera toxin in rats 17 h prior to experimentation results in increased levels of insulin and corticosterone in the blood. This is accompanied by a rise in lipoprotein lipase activity in muscle and a decrease in adipose tissue. Pre- and postheparin blood levels of the enzyme are increased, representing the higher overall muscle activity. Hepatic lipase is decreased by cholera toxin treatment. These enzyme changes are accompanied by increased levels of non-esterified fatty acids, ketone bodies and unesterified cholesterol in the blood, whereas triacylglycerol levels are lowered. The lipoprotein triacylglycerol secretion is not affected by cholera toxin, suggesting increased triacylglycerol removal from the blood. On the other hand the unesterified cholesterol removal may be decreased due to the decreased hepatic lipase activity. Administration of excess glucocorticoid 2 days prior to blood and tissue sampling also resulted in a rise in lipoprotein lipase, a decrease in hepatic lipase activity and an increase of non-esterified fatty acids. In contrast to the effect of cholera toxin, the triacylglycerol levels were increased. Adrenalectomy, whether by inhibition of 11-beta-steroid hydroxylase or by surgical intervention, did not abolish the choleratoxin effects. It is concluded that corticosterone increase is not essential to the cholera toxin effects. Corticosterone itself probably causes an increase of cyclic AMP and/or Ca2+ levels, as is discussed.     

昆虫延长滞育的研究

持续时间达1年以上的滞育,称为延长滞育,延长滞育的诱导、维持、解除均不同于简单滞育(滞育期短于1年)。本论文系统阐述了昆虫延长滞育的类型、滞育诱导及解除的环境因子、延长滞育物候学和生物学特性,延长滞育的遗传学及延长滞育的生态学意义。延长滞育是昆虫生活史的重要组成部分,是一种普遍现象。     

昆虫滞育研究进展

首先对昆虫滞育的若干名词概念作出解释,按照滞育过程的阶段特点划分为3个时期:(1)滞育前期(包括滞育诱导和滞育准备);(2)滞育期(包括滞育进入、滞育维持和滞育解除);(3)滞育后期。然后重点介绍这3个阶段近期分子和细胞水平上的重要工作,对一些科学问题进行讨论。     

Eco-physiological phases of insect diapause

Insect diapause is a dynamic process consisting of several successive phases. The conception and naming of the phases is unsettled and, sometimes, ambiguous in the literature. In this paper, the ontogeny of diapause was reviewed and the most often used terms and the best substantiated phases were highlighted, explained and re-defined. The aim was to propose relatively simple and generally applicable terminological system. The phases of diapause induction, preparation, initiation, maintenance, termination and post-diapause quiescence were distinguished. The specific progression through diapause phases in each species, population (genotype), or even individual, is based on (thus far largely unknown) physiological processes, the actual expression of which is significantly modified by diverse environmental factors. Thus, such phases are eco-physiological in their nature.     

Photoperiodic determination of insect development and diapause

Summary The Dual System Theory of photoperiodic determination was found to be consistent with experimental data on diapause induction in response to skeleton photoperiods. Symmetrical and asymmetrical skeleton photoperiods of both diel and nondiel durations were investigated. The theoretical model was shown to predict accurately the incidence of diapause among larvae of the European corn borer,Ostrinia nubilalis, that had been reared under the different photoperiodic regimes.Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, and by a research grant (GM 07557) from the National Institutes of Health.     

Photoperiodic determination of insect development and diapause

Summary The Photoperiodic Determination Model proposed earlier (I, II) is further elaborated, and its applicability to nondiel photoperiods tested. Model-generated predictions of diapause incidences were in good agreement with observed incidences among larvae of the European corn borer,Ostrinia nubilalis, reared under photoperiods from 15 to 50 hrs duration with scotophases of from 9 to 18 hrs.Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, and by a research grant (GM-07557) from the National Institutes of Health.     

Photoperiodic determination of insect development and diapause

Summary The Dual System Theory of photoperiodic time measurement is shown to provide a satisfactory basis for the interpretation of: (1) photoperiodic determination of diapause; (2) effects of different photoperiodic regimes on circadian rhythms of adult eclosion; and (3) the phase response curve. A fundamental unity of photoperiodic time measurement in a wide variety of organisms is strongly suggested by the Dual System Theory.Research supported by the College of Agricultural and Life Sciences of the University of Wisconsin, and by a research grant (GM 07557) from the National Institutes of Health     

Photoperiodic determination of insect development and diapause

Summary The hypothesis advanced by James W. Truman to explain the photoperiodically regulated circadian rhythm of adult eclosion in a Saturniid moth has been modified and extended to form the basis of a proposed Developmental Determination Model. This model postulates the synthesis of a hypothetical substance (S) and the kinetics of its metabolism in the presence or absence of light energy. The relationship of scotophase duration to the temporal occurrence of postulated determination response thresholds was estimated in the terms of the hypothetical model. The temporal position of a postulated Determination Gate was also approximated in terms of the model system. The characteristics of the proposed model were designed to be consistent with the observed effects of different 24-hour photoperiods on the incidence of diapause among larvae of the European corn borer,Ostrinia nubilalis. The proposed Determination Model was shown to be capable of generating all four types of photoperiodic diapause reponse curves reported in existing literature.Research supported by the College of Agriculture and Life Sciences, University of Wisconsin, and by a research grant (GM 07557) from the National Institutes of Health.     

Quantification of gangliotetraose gangliosides with cholera toxin

A procedure is described for assay of GM1 and other gangliotetraose-type gangliosides at the picomole level. The gangliosides are absorbed onto polystyrene microwells and treated with neuraminidase and then with cholera toxin B subunits conjugated to horseradish peroxidase. Color is developed and quantified spectrophotometrically. Omission of neuraminidase gives a measure of GM1 alone. Linearity was obtained between 0.5 and 3 pmol. This procedure was applied to ganglioside mixtures isolated fron neuro-2A neuroblastoma and PC12 pheochromocytoma cells. For the latter, an additional step involving reaction with fucosidase increased the yield of GM1 due to the presence of fucosylated gangliosides. Application of the same reagents as a TLC overlay procedure to the gangliosides from neuro-2A cells revealed the presence of GD1a, GD1b, and GT1b in addition to GM1, thus confirming the presence of a family of gangliotetraose gangliosides.     

cAMP imaging of cells treated with pertussis toxin, cholera toxin, and anthrax edema toxin

The enzymatic activity of the three most studied bacterial toxins that increase the cytosolic cAMP level: pertussis toxin (PT), cholera toxin (CT), and anthrax edema toxin (ET), was imaged by fluorescence videomicroscopy. Three different cell lines were transfected with a fluorescence resonance energy transfer biosensor based on the PKA regulatory and catalytic subunits fused to CFP and YFP, respectively. Real-time imaging of cells expressing this cAMP biosensor provided time and space resolved pictures of the toxins action. The time course of the PT-induced cAMP increase suggests that its active subunit enters the cytosol more rapidly than that deduced by biochemical experiments. ET generated cAMP concentration gradients decreasing from the nucleus to the cell periphery. On the contrary, CT, which acts on the plasma membrane adenylate cyclase, did not. The potential of imaging methods in studying the mode of entry and the intracellular action of bacterial toxins is discussed.     

Interaction of cholera toxin B-subunit with human T-lymphocytes

In this work, ¹²⁵I-labeled cholera toxin B-subunit (CT-B) (specific activity 98 Ci/mmol) was prepared, and its high-affinity binding to human blood T-lymphocytes (K _d = 3.3 nM) was determined. The binding of the ¹²⁵I-labeled CT-B was inhibited by unlabeled interferon-α₂ (IFN-α₂), thymosin-α1 (TM-α₁), and by the synthetic peptide LKEKK, which corresponds to sequences 16-20 of human TM-α₁ and 131-135 of IFN-α₂ (K _i 0.8, 1.2, and 1.6 nM, respectively), but was not inhibited by the unlabeled synthetic peptide KKEKL with inverted sequence (K _i > 1 μM). In the concentration range of 10-1000 nM, both CT-B and peptide LKEKK dose-dependently increased the activity of soluble guanylate cyclase (sGC) but did not affect the activity of membrane-bound guanylate cyclase. The KKEKL peptide tested in parallel did not affect sGC activity. Thus, the CT-B and peptide LKEKK binding to a common receptor on the surface of T-lymphocytes leads to an increase in sGC activity.     

Chloroquine inhibition of cholera toxin

Cholera toxin (CT) stimulated adenylate cyclase and a phospholipase which elevated cellular levels of 3',5'-cyclic adenosine monophosphate (cAMP) and arachidonic acid (AA). The AA was quickly converted to prostaglandins (PGs) via the cyclo-oxygenase pathway. Chloroquine exerted minimal inhibition of cAMP levels in CT-treated cells, although CT-induced release of [3H]AA and PGs was blocked completely when the drug was added in concentrations as low as 0.1 mM (50 micrograms/ml). Inhibition of [3H]AA release was complete when chloroquine was added before or within 30 min after CT. The capacity of chloroquine to inhibit either phospholipase C (PLC) or phospholipase A2 (PLA2) could explain the antisecretory activity of this drug.     

Vibrio cholerae: cholera toxin

The bacterial protein toxin of Vibrio cholerae, cholera toxin, is a major agent involved in severe diarrhoeal disease. Cholera toxin is a member of the AB toxin family and is composed of a catalytically active heterodimeric A-subunit linked with a homopentameric B-subunit. Upon binding to its receptor, GM0(1), cholera toxin is internalized and transported in a retrograde manner through the Golgi to the ER, where it is retrotranslocated to the cytosol. Here, cholera toxin reaches its intracellular target, the basolaterally located adenylate cyclase which becomes constitutively activated after toxin-induced mono-ADP-ribosylation of the regulating G(S)-protein. Elevated intracellular cAMP levels provoke loss of water and electrolytes which is manifested as the typical diarrhoea. The cholera toxin B-subunit displays the capacity to fortify immune responses to certain antigens, to act as a carrier and to be competent in inducing immunological tolerance. These unique features make cholera toxin a promising tool for immunologists.     

昆虫滞育光周期理论的再思考

任何地方一年中的每一天都有一个特定的光照长度,日期和光周期变化存在固定的对应关系。昆虫在一定的日子滞育,自然也就是在特定的光照长度下滞育,但是并不意味着光周期必然就是滞育的主要诱因。分析光周期的几个重要特点,笔者认为光周期不一定是所有昆虫滞育开始及结束的最主要的或实质性的诱因。     

p97 Is in a complex with cholera toxin and influences the transport of cholera toxin and related toxins to the cytoplasm

Certain protein toxins, including cholera toxin, ricin, and Pseudomonas aeruginosa exotoxin A, are transported to the lumen of the endoplasmic reticulum where they retro-translocate across the endoplasmic reticulum membrane to enter the cytoplasm. The mechanism of retrotranslocation is poorly understood but may involve the endoplasmic reticulum-associated degradation pathway. The AAA ATPase p97 (also called valosin-containing protein) participates in the retro-translocation of cellular endoplasmic reticulum-associated degradation substrates and is therefore a candidate to participate in the retrotranslocation of protein toxins. To investigate whether p97 functions in toxin delivery to the cytoplasm, we measured the sensitivity to toxins of cells expressing either wild-type p97 or a dominant ATPase-defective p97 mutant under control of a tetracycline-inducible promoter. The rate at which cholera toxin and related toxins entered the cytoplasm was reduced in cells expressing the ATPase-defective p97, suggesting that the toxins might interact with p97. To detect interaction, the cholera toxin A chain was immunoprecipitated from cholera toxin-treated Vero cells, and co-immunoprecipitation of p97 was assessed by immunoblotting. The immunoprecipitates contained both cholera toxin A chain and p97, evidence that the two proteins are in a complex. Altogether, these results provide functional and structural evidence that p97 participates in the transport of cholera toxin to the cytoplasm.