Metarhodopsin I/metarhodopsin II transition triggers light-induced change in calcium binding at rod disk membranes

The hypothesis of Yoshikami and Hagins that calcium ions act as diffusible transmitter molecules between the photochemistry of rhodopsin and the subsequent electrical events at the outer plasma membrane of rods initiated many investigations on light-stimulated calcium release in vertebrate photoreceptor cells (see refs 2, 3). Although it not seems firmly established that light has some effect on the redistribution of calcium in various disk preparations, reconstituted systems and intact rod outer segments, the physiological significance remained unclear. We previously reported a rapid, light-triggered calcium release from binding sites at the disk membrane in the presence of calcium ionophore A23187 (refs 3, 8). However, there is no evidence for rapid calcium release into the cytosol in the absence of ionophore. On fragmentation of intact rod outer segments, calcium release due to a light-requlated change of calcium binding appeared almost completely abolished. We describe here experiments with sonicated rod outer segments in which the previously observed loss of the calcium release capacity has been prevented. Calcium release in sonicated disks in the presence of A23187 kinetically follows the metarhodopsin I/metarhodopsin II transition (tau 1/2 = 10 ms, activation energy EA = 34 kcal mol-1), suggesting that calcium release is triggered by this photochemical transition.     

Vasorelaxant properties of the endothelium-derived relaxing factor more closely resemble S-nitrosocysteine than nitric oxide

Studies of cultured bovine aortic endothelial cells using quantitative chemiluminescence techniques have shown that the amount of nitric oxide released under basal conditions, or in response to either bradykinin or the calcium ionophore A23187 is insufficient to account for the vasorelaxant activities of the endothelium-derived relaxing factor (EDRF) derived from the same source. This observation contradicts previous suggestions that nitric oxide and EDRF are the same compound, but may be explained if EDRF is a compound that contains nitric oxide within its structure but is a much more potent vasodilator than nitric oxide. Such a molecule could be one of several nitrosothiols which may yield nitric oxide after a one-electron reduction. The present experiments were carried out to test the possibility that the biological activities of the endothelium-derived relaxing factor might more closely resemble those of one of these compounds, S-nitrosocysteine, than nitric oxide. Nitric oxide release from cultured bovine aortic endothelial cells was detected by chemiluminescence and bioassay experiments compared the vasodilator potencies of nitric oxide, S-nitrosocysteine, and EDRF. The results suggest that EDRF is much more likely to be a nitrosylated compound such as a nitrosothiol than authentic nitric oxide.     

Vitamin E modulates the lipoxygenation of arachidonic acid in leukocytes

The arachidonic acid released from cellular phospholipids of specifically stimulated platelets and leukocytes is oxygenated enzymatically by two major pathways. A complex cycloxygenase converts some of the free arachidonic acid to labile endoperoxides that are transformed to prostaglandins, thromboxanes and prostacyclin (PGI2). Lipoxygenases convert part of the arachidonic acid to unstable hydroperoxy-eicosatetraenoic acids (OOHETEs) that are transformed to monohydroxyeicosatetraenoic acids (HETEs), oligohydroxy-eicosatetraenoic or -eicostatrienoic acids such as di-HETEs and tri-HETEs, and, in some instances, more complex humoral mediators, including slow-reacting substances. Both the nature of the HETEs and the ratio of the HETEs to the cyclo-oxygenase products are specific characteristics of each type of cell. In human neutrophils, the sum of the lipoxygenase products 5-HETE, 11-HETE and 5,12-di-HETE substantially exceeds the total amount of PGE2 and other cyclo-oxygenase metabolites that are generated concurrently, and the endogenous lipoxygenase products regulate neutrophil function. The present data indicate that vitamin E (alpha-tocopherol) bidirectionally modulates the activity of the lipoxygenase pathway of human neutrophils in vitro. Normal plasma concentrations of alpha-tocopherol enhance the lipoxygenation of arachidonic acid, whereas higher concentrations of alpha-tocopherol exert a suppressive effect that is consistent with its role as a hydroperoxide scavenger.     

Possible association of alcohol tolerance with increased synaptic Ca2+ sensitivity

The inhibitory effect of ethanol on neurotransmitter release has been suggested to be due to either reduced Ca2+ entry or increased removal of free intracellular Ca2+ from the synapse. The use of the Ca2+ ionophore, A23187, to allow direct access of external Ca2+ to the presynaptic interior should help to determine which of these two factors is the more important, as ethanol should inhibit A23187-induced release of transmitter only if increased Ca2+ removal from the synapse is important. Here we show in rat striatal slices that, although 3H-dopamine release evoked by depolarization with 40 mM K+ is inhibited by 50 mM ethanol, the release evoked by A23187 is enhanced by the presence of ethanol in vitro. The results suggest that ethanol reduces depolarization-induced transmitter release by reducing Ca2+ entry to the presynaptic terminal. However, for brain slices taken from rats made tolerant to ethanol, 3H-dopamine release in the absence of ethanol showed altered characteristics; both K+ depolarization and A23187 released a significantly greater fraction of 3H-dopamine from these slices than from controls. Thus tolerance to the inhibitory effect of ethanol on release may develop by a mechanism involving increased sensitivity of the terminal to Ca2+ entry.     

Activated human eosinophils generate SRS-A leukotrienes following IgG-dependent stimulation

Eosinophils, a class of granular leukocytes, are prominent in many inflammatory processes, particularly in asthma, certain allergic diseases and during infections with helminthic parasites. Following incubation with the Ca ionophore A23187 (refs 1-4) (a non-physiological agent which circumvents membrane calcium-gating mechanisms), eosinophils generate large amounts of sulphidopeptide leukotrienes, potent inducers of smooth muscle constriction and mucus production. These are now known to represent the activity previously termed 'slow-reacting substance of anaphylaxis' (SRS-A) but attempts to identify a physiological stimulus for SRS-A production by eosinophils have so far been unsuccessful. The cells contain recognized receptors for IgG (Fc) and it is known that they adhere to, and can be activated by, contact with the surface of large organisms such as helminthic larvae. We show here that eosinophils, particularly when activated, produce sulphidopeptide leukotrienes after contact with large particles coated with IgG.     

Homogeneous interferon-inducing 22K factor is related to endogenous pyrogen and interleukin-1

In vitro stimulation of mononuclear cells from human peripheral blood with mitogens causes the release of factors (monokines and lymphokines) which possess distinct biological activities. One such factor, termed 22K, can induce production of human beta-interferon (HuIFN-beta) in cultured human fibroblasts, thereby rendering these cells resistant to virus infection. Here we report the complete purification and partial sequencing (39 N-terminal amino acids) of this factor, whose relative molecular mass was estimated by SDS-polyacrylamide gel electrophoresis to be 17,000 (17K). In addition to an antiviral effect, the pure protein exhibits several other biological activities. Most significantly, intravenous (i.v.) injection of the factor in rabbits caused fever and granulopenia at doses of 0.1-1 microgram per kg, effects which we attribute to a monokine called endogenous pyrogen (EP). In vitro, the protein was scored as positive in a LAF (lymphocyte-activating factor) assay at 0.1-1 ng ml-1. LAF and EP are considered to be members of one family of monokines, called interleukin-1 (IL-1). For this reason, and also because the amino-acid sequence of the 22K factor is at least partially homologous to a complementary DNA-derived IL-1 sequence, we postulate that the 22K factor also belongs to the IL-1 family.     

Identification and isolation of a membrane protein necessary for leukotriene production

Several inflammatory diseases, including asthma, arthritis and psoriasis are associated with the production of leukotrienes by neutrophils, mast cells and macrophages. The initial enzymatic step in the formation of leukotrienes is the oxidation of arachidonic acid by 5-lipoxygenase (5-LO) to leukotriene A4. Osteosarcoma cells transfected with 5-LO express active enzyme in broken cell preparations, but no leukotriene metabolites are produced by these cells when stimulated with the calcium ionophore A23187, indicating that an additional component is necessary for cellular 5-LO activity. A new class of indole leukotriene inhibitor has been described that inhibits the formation of cellular leukotrienes but has no direct inhibitory effect on soluble 5-LO activity. We have now used these potent agents to identify and isolate a novel membrane protein of relative molecular mass 18,000 which is necessary for cellular leukotriene synthesis.     

Glutamate stimulates inositol phosphate formation in striatal neurones

The major excitatory amino acids, glutamate (Glu) and aspartate (Asp), are thought to act at three receptor subtypes in the mammalian central nervous system (CNS). These are termed quisqualate (QA), N-methyl-D-aspartate (NMDA) and kainate (KA) receptors according to the specific agonist properties of these compounds revealed by electrophysiological studies. Although Glu has been shown to stimulate cyclic GMP formation in brain slices, direct regulation of second messenger systems (cyclic AMP, Ca2+ or inositol phosphates) subsequent to activation of excitatory amino-acid receptors, has not been extensively studied. Here we demonstrate that in striatal neurones, excitatory amino acids, but not inhibitory or non-neuroactive amino acids, induce a three- to fourfold increase in inositol mono-, di- and triphosphate (IP, IP, IP) formation with the relative potency QA greater than Glu greater than NMDA, KA. The Glu-evoked formation of inositol phosphates appears to result principally from actions at QA as well as NMDA receptors on striatal neurones. Our results suggest that excitatory amino acids stimulate inositol phosphate formation directly, rather than indirectly by the evoked release and subsequent actions of adenosine or acetylcholine.     

Inhibition of histamine secretion from mast cells

Histamine secretion from mast cells may be inhibited by elevated intracellular levels of cyclic AMP and by several anti-allergic drugs. These compounds are claimed to act directly on the calcium-gating mechanism activated by the anaphylactic reaction, preventing influx of Ca2+ from the external environment and so blocking exocytosis. To examine this hypothesis further, we have compared here the histamine secretion induced by immunoglobulin E-directed ligands in the presence and absence of added calcium and by the ionophore A23187. Exocytosis evoked by these former agents was originally considered to be almost totally dependent on extracellular calcium but recent studies have shown otherwise. In the absence of added cation, the agents act by mobilizing membrane-bound or intracellular stores of calcium. We show that here that a variety of anti-allergic drugs are potent inhibitors in the conditions used, suggesting that alternative explanations for their action must be sought.     

Role of Ca(2+)-dependent regulator protein in intestinal secretion

AFTER exposure to secretagogues the small intestine changes from a tissue that absorbs fluid and electrolyte from lumen to blood into a tissue that secretes electrolyte and fluid into the lumen(1-4). It has been shown that this secretion results from an increase in the passive Cl(-) permeability of the mucosal border, which permits Nad to leak passively from the lateral intercellular spaces, where it is present at hypertonic concentrations(5), into the mucosal bathing solution. Na(+) and water, electroosmotically coupled to Na(+) movement, leak through the tight junctions(1,2), and Cl(-) leaks through relatively anhydrous anion-selective channels, induced withira the mucosal border by secretagogues. The increased reflux of NaCl from the lateral intercellular space accounts for both the apparent decrease in electroneutral NaCl uptake across the mucosal border induced by secretagogues and the apparent increase in active CP secretion and short-circuit current(3,6,7). We have investigated the mechanism by which intestinal secretagogues increase passive Cl(-) permeability and thereby cause secretion. Cl(-) permeability is increased by several secretagogues, some of which, such as theophylline and choleragen, increase intracellular cyclic AMP concentration, and others, such as A23187, the Ca(2+) ionophore, or carbachol, do not(8). Thus there has been no known common mode of secretory induction. To investigate this problem we used two drugs that prevent intestinal secretion in vitro, RMI 12330A (Richardson Merrell), and the antipsychotic pheno-thiazine trifluoperazine (Stelazine, Smith, Kline and French). RMI 12330A prevents secretion by inhibiting choleragen-induced adenylyl cyclase activity(9). Stelazine inhibits phosphodiesterase in tissues(11,12) by preventing the activation of the enzyme by Ca(2+)-dependent regulator protein, CDR. We report here that it also inhibits Cl(-) secretion and binds to CDR.     

Reconstruction of human embryos derived from somatic cells

Reconstruction of human nuclear transfer embryos is a necessary step of therapeutic cloning. In this study we injected somatic cell nuclei into M Ⅱ oocytes and activated reconstructed oocytes with calcium ionophore A23187 (CaA) and 6-dimethylaminopurine (6-DMAP). After oocyteactivation and 2PN formation, we removed the female PN.By using this method, we avoided the application of DNA fluorescent stain and ultraviolet light for oocyte enucleation,and over elimination of ooplasm was also mitigated. Some reconstructed embryos developed into the blastocyst stage in vitro.     

同型半胱氨酸对钙离子激活状态下内皮细胞eNOS活性及NO含量的影响

研究同型半胱氨酸(HCY)在钙离子导体(A23187)刺激下,对内皮细胞内皮型一氧化氮合酶(eNOS)活性及一氧化氮(NO)含量的影响及机制。收集人脐静脉内皮细胞(HUVEC)进行细胞培养,取第二代细胞分4组:对照组,HCY组,A23187组和HCY A23187组。继续培养24h后,测定细胞培养液中NO、丙二醛(MDA)含量和eNOS、超氧化物歧化酶(SOD)活性。结果显示:1)与对照组相比,HCY组培养液中NO含量降低、eNOS活性减弱、MDA含量升高、SOD活力增强(P<0.05);A23187组NO含量升高、eNOS活性增强、MDA含量降低、SOD活力增强(P<0.05)。2)与A23187组相比,HCY A23187组NO含量降低、eNOS活性减弱、MDA含量升高(P<0.05),HCY不影响A23187激活状态下SOD活力的升高(P>0.05)。结论:HCY在静息及钙离子导体(A23187)激活状态下,使HUVEC中NO含量降低e、NOS活性减弱、MDA含量升高,SOD活力代偿性升高。其机制可能与HCY导致的氧化应激抑制eNOS活性及降低NO的生物利用度有关。     

A common sequence of calcium and pH signals in the mitogenic stimulation of eukaryotic cells

When normal quiescent (G0) cells are stimulated by mitogens to enter the cell cycle, the metabolic derepression which occurs is similar in a variety of cells. The mechanisms initiating these responses and their relationship to subsequent progression through G1 to DNA synthesis in S phase, however, are generally undefined. The clearest evidence has been obtained in sea urchin eggs, where fertilization by sperm causes a rapid, transient increase in the concentration of free cytoplasmic Ca2+ [(Ca]i), followed by a sustained increase in cytoplasmic pH (pHi). It has been demonstrated clearly that these ionic responses are obligatory for progression to DNA synthesis by the normal pathway after fertilization, although the Ca2+ signal can be bypassed by parthenogenetic agents which elevate directly pHi (for example, NH+4 ions). These observations raise the questions of whether other eukaryotic cells show the same sequence of ionic responses when stimulated by mitogens and whether such signals are an obligatory component of their mitogenic pathways. We show here that a common sequence of [Ca]i and pHi responses occurs in both quiescent mouse thymocytes and Swiss 3T3 fibroblasts stimulated by appropriate mitogens. Furthermore, 'opportunistic' mitogens (those that do not act on the cells in vivo, such as concanavalin A (Con A), the Ca2+ ionophore A23187 and 12-o-tetradecanoyl phorbol 13-acetate CTPA] that are mitogenic for both mouse thymocytes and 3T3 fibroblast, each produce characteristic ionic responses that are the same in both types of cell.     

Protein kinase C regulation of the receptor-coupled calcium signal in histamine-secreting rat basophilic leukaemia cells

It has been proposed that protein kinase C mediates cellular responses evoked by external stimuli, leading to alterations in internal free calcium concentrations. We have shown previously that histamine-secreting rat basophilic leukaemia cells (RBL-2H3), which degranulate on aggregation of the receptors for immunoglobulin IgE, contain a Ca2+- and phospholipid-dependent protein kinase (kinase C). The partially purified enzyme is activated directly by the tumour-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In intact RBL cells, TPA potentiates histamine release induced by the Ca2+-ionophore A23187 (similar to the synergy reported for platelets, neutrophils and rat peritoneal mast cells). Although TPA at concentrations below 15 nM synergizes with the antigen, higher TPA concentrations inhibit secretion. This selective inhibition suggested that kinase C is involved in both the activation and termination of the secretory process. To examine this possibility, we have determined the effect of TPA on changes in free cytosolic Ca2+ concentration during antigen-induced release. We report here that TPA completely blocks the increase in Ca2+ concentration induced by antigen. Our results strongly suggest that protein kinase C is involved in the regulation of receptor-dependent Ca2+ signalling.     

对成都地区10株黏细菌菌株酶活性的初步研究

为了从黏细菌中分离各种酶类,通过各种选择培养基对成都地区10株黏细菌菌株的酶活性进行了检测.研究发现,菌株CX-1为高产纤维素酶菌株、菌株CL-7为高产淀粉酶菌株、菌株CC-1为高产蛋白酶菌株、菌株CL-1为高产壳聚糖酶及脂肪酸氧化分解酶类菌株、菌株CL-5为高产脂肪酶菌株.本研究为下一步从这些黏细菌中提取特定的酶类提供了实验依据.     

5—（1—苯基—5—甲基—1，2，3—三唑—4—基）—4—芳基—1，2，4—三唑—3—硫基乙酸的合成和抗菌活性

合成一系列新的5-（1-苯基-5-甲基-1,2,3-唑-4-基）-4-芳基-1,2,4-三唑-3-硫基乙酸,所有化合物均经元素分析和波谱数据予以鉴定,对代表性化合物作了抗菌活性测试,结果表明它们都表现出不同程度的抗菌活性。     

Interferon inhibits transformation by murine sarcoma viruses before integration of provirus

Neoplastic transformation by C-type retroviruses requires synthesis of a DNA copy (the provirus) of the RNA genome and its integration into the host cell DNA. We have previously shown that interferon (IFN) can stably prevent transformation of murine fibroblasts by the Kirsten strain of murine sarcoma virus (KiMSV), a murine leukaemia virus (MLV). A series of cell clones (IFN clones), isolated in the presence of IFN (10(4) U ml-1) from cultures of NIH-3T3 cells which had been treated with IFN, and then infected with KiMSV (KiMLV) in conditions where every cell was infected, were shown to be phenotypically untransformed. These untransformed cells did not produce virus or contain rescuable KiMSV. However, cells isolated using an identical procedure, but in the absence of IFN, were uniformly transformed and all produced KiMSV (KiMLV) or contained rescuable KiMSV. It was concluded that IFN either prevents synthesis or integration of the provirus, or else that in the presence of IFN the provirus is integrated such that it is not expressed. We now show that five representative clones contain no detectable KiMSV proviral DNA, and also that the initial stages of infection by KiMSV (KiMLV) are inhibited by IFN treatment. IFN seems to act before integration, preventing either the synthesis or the integration of proviral DNA.     

Metabolic stabilization of endplate acetylcholine receptors regulated by Ca2+ influx associated with muscle activity.

During formation of the neuromuscular junction, acetylcholine receptors in the endplate membrane become metabolically stabilized under neural control, their half-life increasing from about 1 day to about 10 days. The metabolic stability of the receptors is regulated by the electrical activity induced in the muscle by innervation. We report here that metabolic stabilization of endplate receptors but not of extrajunctional receptors can be induced in the absence of muscle activity if muscles are treated with the calcium ionophore A23187. Acetylcholine receptor stabilization was also induced by culturing non-stimulated muscle in elevated K+ with the Ca2+ channel activator (+)-SDZ202-791. Conversely, activity-dependent receptor stabilization is prevented in muscle stimulated in the presence of the Ca2+ channel blockers (+)-PN200-110 or D-600. Treatment of muscles with ryanodine, which induces Ca2+ release from the sarcoplasmic reticulum in the absence of activity, does not cause stabilization of junctional receptors. Evidently, muscle activity induces metabolic acetylcholine receptor stabilization by way of an influx of Ca2+ ions through dihydropyridine-sensitive Ca2+ channels in the endplate membrane, whereas Ca2+ released from the sarcoplasmic reticulum is ineffective in this developmental process.     

四种注射用氨基酸原料细菌内毒素的检查

通过鲎试剂的干扰试验,研究注射用氨基酸原料中细菌内毒素检查法的可行性。依据中国药典2000年版中细菌内毒素检查法进行操作。在2~16倍稀释级下,L-缬氨酸(c=2.5%)和L-异亮氨酸(c=2.5%)对鲎试剂无干扰,检测细菌内毒素的鲎试剂灵敏度小于等于0.5 EU.mL-1,而在16倍稀释级范围内,L-脯氨酸(c=4.5%)和L-亮氨酸(c=2.0%)对鲎试剂检查法有抑制作用。实验表明注射用L-异亮氨酸和L-缬氨酸用灵敏度为0.5 EU.mL-1的鲎试剂检查其细菌内毒素方法可行,可以代替家兔法检查热原。供注射用L-缬氨酸细菌内毒素应小于0.04 EU.mg-1,显著高于中国药典2005年版的规定。     

Is an early calcium flux necessary to stimulate lymphocytes?

Concentrations of concanavalin A or the calcium ionophore A23187 that are optimal for the transformation of pig or mouse lymphocytes do not normally cause a measurable increase in calcium influx compared with unstimulated cells. If the cells are treated with the mitogens in conditions where a measurable increase in calcium influx occurs, no stimulation of the cells can occur while the flux is maintained. If an early influx of extracellular calcium is necessary for stimulation, then a much smaller increase in the total concentration of cellular calcium than reported previously is sufficient to allow the entry of lymphocytes into the cell cycle.