Cell-free Model Research Articles

Lysis of chromaffin granules by phospholipase A 2-treated plasma membranes : A cell-free model for exocytosis in adrenal medulla

The possible involvement of phospholipase A 2 (PLA 2) in the release of catecholamines was examined in a cell-free model system, using isolated chromaffin granules and plasma membranes of adrenal medulla cells. Plasma membranes treated with PLA 2 in the presence of Ca 2+ caused lysis of chromaffin granules which was also dependent on Ca 2+. This finding suggests that Ca 2+ acts in two steps of exocytosis, namely in the transformation of plasma membranes into a lytic form by PLA 2+ and in the interaction of chromaffin granules with plasma membranes. These findings show good agreement with recently reported findings in leaky adrenal medulla cells, and suggest the involvement of PLA 2+ in the release of catecholamines.

Background levels of DNA damage in the population.

In addition to the dangers of man-made mutagenic substances, there exist a variety of naturally-occurring agents and processes than can cause DNA damage in man. These include exogenous agents, such as sunlight and dietary mutagens, as well as endogenous agents, such as reactive oxygen species which are formed within the cell via normal metabolism. These processes can lead to at least six major types of DNA damage: base loss, base deamination, base alkylation, base dimerization, base oxidation, and single-strand breakage. Based upon in vitro measurements in cell-free model systems or cultured cells, estimates of the rates of production of these lesions have been made for the average human cell: about 26,000 base losses per day, primarily purines lost because of spontaneous hydrolysis of the glycosyl bond; about 350 cytosine deaminations per day; tens of thousands of base alkylations per day, primarily methylations of guanine caused by S-adenosylmethionine; up to 50,000 pyrimidine dimerizations per day, dependent upon exposure to sunlight; and about 100,000 single-strand breaks per day. Based upon measurements of DNA oxidation products in human urine, we estimate that the total rate of formation of all oxidative DNA damage products in man is on the order of a few thousand events per cell per day. In addition, base mispairing can take place in the absence of DNA damage because of tautomeric shifts, base ionization, or base rotation.(ABSTRACT TRUNCATED AT 250 WORDS)

Enrichment of fibrillar cytoplasmic actomyosin in protoplasmic strands of Physarum polycephalum for the production of cell-free models.

The treatment of isolated protoplasmic strands of Physarum polycephalum with 2.5% ethanol in a physiological salt solution under isometric conditions induces the formation of a large amount of mostly longitudinally organized actomyosin fibrils in the endoplasmic channel, a region normally free of actomyosin fibrils. The quantity of fibrillogenesis as well as the concomitant force output during the induced contractures are dependent on the Ca++-content and the temperature of the test solution. The method was developed to optimize the structure of the plasmodial strands before their subsequent transformation into cell-free models by permeabilization and extraction of the strands. Cryosections of plasmodial strands containing cytoplasmic actomyosin fibrils stained with fluorescently labeled phallotoxins offer a further assay for the study of their contraction physiology under cell-free conditions.

Down-regulation of human natural killer activity against tumors by the neutrophil myeloperoxidase system and hydrogen peroxide.

There is growing evidence that natural killer (NK) cells play an important role in immune surveillance against tumors and certain infections. The coexistence of activated neutrophils with lymphocytes in tumor masses and inflammatory tissues suggests the possibility of interaction between secreted neutrophil products and nearby lymphocytes. We examined the susceptibility of lymphocyte NK activity to oxidative injury by the neutrophil myeloperoxidase (MPO) system and H2O2 with the use of a cellfree model system. Exposure of human mononuclear leukocytes (MNL) to MPO, an H2O2-generating system (glucose + glucose oxidase), and a halide (C1- or I-) resulted in marked suppression of MNL-NK activity, as measured by 51Cr release from K562 tumor targets (p less than 0.001). This suppression was dependent on the presence and activity of each system component and was blocked by azide and catalase, but not by heated catalase. In spite of the marked functional suppression of NK activity, MNL viability was more than 95% and target binding frequency was not affected. NK suppression was reversible after 24 hr in culture. The mechanism of suppression was dependent on the amount and rate of H2O2 delivered, and on MNL number. MPO was essential when H2O2 flux was low or when MNL numbers were high. As H2O2 flux increased or MNL numbers decreased, NK suppression gradually became MPO-independent and was mediated by H2O2 alone. The ability of the MPO system to compromise lymphocyte NK function may explain the in vitro inhibition of NK activity of mixed cell populations by the tumor promoter phorbol esters, because these agents are potent stimulants for neutrophil secretion of MPO and H2O2. This study may also provide a possible mechanism for the reported in situ NK activity suppression by adherent phagocytic cells during carcinogenesis in both humans and animals.

Reactivation of NBD-phallacidin-labelled actomyosin fibrils in cryosections of Physarum polycephalum: A new cell-free model

Application of ATP to cryosections of plasmodial strands from Physarum polycephalum leads to an isotonic contraction of the cytoplasmic actomyosin fibrils: when the fibrils are labelled with NBD-phallacidin, their contraction can be observed in the fluorescence microscope. While performing contraction, the fibrils separate into many small units and the formerly continuous fibrils exhibit the appearance of beaded chains. The possibility of visualizing directly the contraction of cytoplasmic actomyosin fibrils in the fluorescence microscope represents a favourable condition for the study of their physiological contraction mechanism, because this new and convenient cell-free model offers in situ contractile structures that are non-denatured and non-extracted.

Effects of molybdate and endogenous inhibitors on steroid-receptor inactivation, transformation, and translocation.

To exert their specific effects on the regulation of gene expression, steroid honnones must first bind to cytoplasmic receptors that are subsequently trans­ located to specific acceptor sites in the cell nucleus. Prior to this translocation, the steroid-receptor complex is transfonned by a temperature-dependent pro­ cess to a state that can bind to nuclear components. This general model of binding, followed by receptor transfonnation and translocation to the nucleus, was originally proposed in 1968 on the basis of studies in intact cells (25, 37). The initial events in this pathway, honnone binding and receptor transfonna­ tion, can be readily studied under cell-free conditions. Although clearly valid cell-free models of nuclear translocation have not been developed, binding of transfonned receptors to nuclei or to DNA-cellulose can be assayed in a quantitative manner and is employed as an operational method of assaying translocation of receptors that have been transfonned under cell-free condi­ tions. Both group VI-A transition-metal oxyanions (42, 66, 92) and a small, heat-stable endogenous factor (7, 9, 24, 43, 77, 79, 80) have been found to

Neutrophils kill pulmonary endothelial cells by a hydrogen-peroxide-dependent pathway. An in vitro model of neutrophil-mediated lung injury.

Neutrophil-mediated injury to lung parenchymal cells has been proposed as an important step in the pathogenesis of many acute and chronic lung disorders. As an in vitro model of neutrophil-mediated injury, this study used activated human neutrophils as effector cells in an 18-h cytotoxicity assay with 51Cr-labeled bovine pulmonary artery endothelial cells serving as target cells. Neutrophils effectively injured pulmonary endothelial cells, expressed as cytotoxic index (CI), of 63.8 +/- 5.4, and this injury could be significantly reduced by several agents, including 1% dimethyl sulfoxide (CI, 51.3 +/- 3.7), 50 micrograms/ml ascorbic acid (CI, 40.8 +/- 4.7), and especially 1,100 U/ml catalase (CI, 14.3 +/- 4.1). As cell-free models of neutrophil-mediated endothelial cell injury, H2O2 (30 microM), O2- (generated by 0.5 mU xanthine oxidase), and the myeloperoxidase-dependent (0.32 U) hypohalite ion were each capable of injuring the target cells with CI of 6.21 +/- 2.8, 53.6 +/- 5.3, and 21.2 +/- 1.5, respectively. Catalase was effective in reducing the injurious effect of each of these oxidant-generating systems (p less than 0.01, all comparisons), confirming the important role for H2O2 in the mediation of this injury. The data indicate that neutrophils are capable of killing pulmonary endothelial cells by a pathway largely dependent on the generation of H2O2, and suggest the possibility that removal of H2O2 from the alveolar structures in subjects with these disorder might be an effective future therapeutic approach.

Properties of gonadotropin-stimulable adenylyl cyclase of the human corpus luteum: regulation of hormonal responsiveness by guanyl nucleotide and magnesium ion.

We studied the properties of a gonadotropin-responsive adenylyl cyclase in membrane preparations obtained from human corpus luteum and explored the nature of guanyl nucleotide and Mg ion involvement in activation of the enzyme. Maximal adenylyl cyclase activity required ATP concentrations of 1-2.5 mM, total MgCl2 concentrations of 8-10 mM, and 1 mM EDTA. Optimal hCG responsiveness, however, required lower (approximately 5 mM) MgCl2 concentrations. Both GTP and its hydrolysis-resistant analog guanyl 5'-yl imidodiphosphate [GMP-P(NH)P] increased enzyme activity, but the response to each nucleotide had distinct characteristics. The rate and the extent of activation were greater in the presence of GMP-P(NH)P than in that of GTP. Moreover, enzyme activation by GMP-P(NH)P was hysteretic in nature, requiring about 8 min to reach steady state velocity in the absence of hormonal stimuli. The slow rate of activation by GMP-P(NH)P was accelerated by either hCG or increases (3-10 mM) in the concentration of MgCl2. Thus, both gonadotropin and Mg ion are inherently antihysteretic in the human luteal adenylyl cyclase system. Basal and hCG stimulation were under the control of guanine nucleotides. Dose-response curves showed that the apparent activation constants for GTP and GMP-P(NH)P were 0.30 and 0.51 microM, respectively; these values did not shift after the addition of hCG. At a higher concentration of guanyl nucleotides (1000 microM), basal and hCG-stimulated activities were markedly reduced, suggesting bimodal regulation of the enzyme by the nucleotides. We also found that enzyme responsiveness to prostaglandin E2 was small and that, in contrast to a number of other nonprimate species, adenylyl cyclase from the human corpus luteum was not stimulated by isoproterenol. Taken together, these data support the usefulness of the cell-free model for studying the role of adenylyl cyclase in the regulation of luteal function in the human.

Ultrastructural localization of 5'-nucleotidase in guinea pig neutrophils based upon the use of cerium as capturing agent.

A cytochemical method for 5'-nucleotidase localization in which cerium serves as the capture agent in order to enzymatically detect liberated inorganic phosphate has been developed. The method has been established in cell-free model systems and in guinea pig neutrophils where 5'-nucleotidase is restricted to the plasmalemma as an ectoenzyme. This cerium-based method gives better results for ultrastructural localization of 5'-nucleotidase than conventional lead-based methods.

Ultrastructural localization of several phosphatases with cerium.

Cerium ions have been used as the capture agent for inorganic phosphate released during the enzymatic hydrolysis of phosphate-containing substrates by a variety of phosphatases. Cerium phosphate reaction product accumulation is proportional to the amount of enzyme present in a cell-free model system. Ultrastructurally, cerium phosphate reaction product appears as a very fine electron-dense precipitate. Cerium appears to be a better capture agent for inorganic phosphate than lead in that reaction product is usually more uniform and more consistently reproducible when cerium is used. Furthermore, nonspecific deposits of reaction product that are commonly encountered in lead-based phosphatase reactions are virtually nonexistent when cerium is the capture agent.

Protein phosphorylation and the exocytosis-like interaction between isolated adrenal medullary plasma membranes and chromaffin granules

The interaction between isolated adrenal medullary plasma membranes and chromaffin granules has been proposed as a cell-free model for exocytosis. Phosphorylation experiments showed that isolated chromaffin granules as well as isolated plasma membranes contain protein kinases and phosphate accepting membranous proteins. Upon joint incubation however, the chromaffin granule-located proteins are preferentially phosphorylated. β-ν-methylene-ATP, a non-hydrolysable analogue, was able to reduce both the plasma membrane-induced release of the soluble chromaffin granular content and the phosphate incorporation into the protein fraction. The results of these experiments on a cell-free model system fit in the hypothesis originating from work on several types of intact cells that the exocytotic event is linked with protein phosphorylation.

Development of a cell-free model for compound exocytosis using components of the chromaffin cell

A study of exocytosis and the possible role of chromaffin granules in adrenal medulla secretion is presented. This review describes a system that may recreate Ca 2+-dependent membrane contact and fusion events that occur in exocytosis. The nature and role of synexin and other compounds in this process are discussed.

Contractile and structural reactions to impediments of Ca2+-homeostasis in Physarum polycephalum.

A combined application of 5 mM KCN and 19 microM Ca++-ionophore A-23187 leads to pronounced contractures of plasmodial strands of Physarum polycephalum. The appearance of the contractures is independent of the amount of Ca++ in the external medium. Tensiometric registrations of longitudinal contraction activity (isometric regime) reveal an average tension increase of 50 mp compared with the preceding tension level before the addition of KCN and ionophore. This high force output during the contracture coincides with a pronounced increase in the number of cytoplasmic actomyosin fibrils. Their ultrastructure is seen as a high lateral density of strictly parallel arranged F-actin filaments; the state of cytoplasmic actomyosin during this isometric contracture corresponds to the ultrastructure of isometrically contracted fibrils during the normal contraction-relaxation cycle of this organism. A simultaneous impediment of respiration and Ca++ homeostasis strongly favours a shift of the actin equilibrium to the high polymeric side in the form of fibrils and may thus be used as a preparatory step improving the specimens in the context of other investigations, e.g., for immunocytochemical investigations or for the preparation of cell-free models to be reactivated after extraction procedures.

ChemInform Abstract: STRUCTURE‐ACTIVITY CORRELATIONS OF CYTOCHALASINS. NOVEL HALOGENATED AND RELATED CYTOCHALASIN C AND D DERIVATIVES

AbstractDie Bromierung von Cytochalasin D (CD) (I) mit Phenyltrimethylammoniumtribromid liefert das 6,12‐Dibromo‐CD (II).

Structure-activity correlations of cytochalasins. Novel halogenated and related cytochalasin C and D derivatives

A series of halogenated and related analogues of cytochalasin C (CC) and D (CD) has been synthesized, and the biological activities of the analogues as inhibitors in a cell-free contractility model system obtained from Ehrlich ascites tumor cells were evaluated. The reaction sequence involved treatment of CD with phenyltrimethylammonium perbromide to give 6,12-dibromo-CD (2), dehydrohalogenation of 2 to 12-bromo-CC (3), and the subsequent conversions of 3 to 12-azido- (4), 12-iodo- (5), and 12-cyano-CC (6). The ID50 values for 5, 3, 4, 2, and 6 are 6.0, 7.4, 8.8, 45, and 77 X 10(-7) M, respectively, in comparison to ca. 2.8 X 10(-7) M for the parental compounds. The potential cell and molecular biological applications of these compounds are delineated.

A role for sialic acid containing substrates in the exocytosis-like in vitro interaction between adrenal medullary plasma membranes and chromaffin granules

The “in vitro” interaction between bovine adrenal medullary plasma membranes and chromaffin granules has recently been proposed as a putative cell-free model for exocytosis because calcium ions specifically control the plasma membrane-induced release of 10 −7 and 10 −5 M. Addition of ruthenium red or pretreatment with neuraminidase gradually blocks this interaction indicating that sialic acid containing substrates may be of major importance. These observations and similar results obtained by other authors working on different systems suggest a role for sialic acid containing moieties in exocytosis.

A Cell-free Model for Protein-Lipid Interactions in Exocytosis: Aggregation and Fusion of Chromaffin Granules in the Presence of Calcium, Synexin, and CIS-Unsaturated Fatty Acids

A Cell-free Model for Protein-Lipid Interactions in Exocytosis: Aggregation and Fusion of Chromaffin Granules in the Presence of Calcium, Synexin, and CIS-Unsaturated Fatty Acids

In vitro interaction between bovine adrenal medullary cell membranes and chromaffin granules: specific control by Ca2+.

The control of the in vitro interaction between bovine adrenal medullary plasma membranes and chromaffin granules by calcium has been studied. This interaction, which has previously been shown to result in the release of the soluble granular content, is a possible cell-free model for exocytosis. The plasma membrane-induced catecholamine release was stimulated when the [Ca2+] exceeded 2x10(-7) M. A maximal release was reached at 10(-5) M with a half maximal response around 10(-6)M. Mg2+ was not able to stimulate the system in the absence of Ca2+. These data suggest a high specificity of the calcium controlled exocytotic mechanism and the absence of an antagonism by high concentrations (10(-5)-10(-1) M) of magnesium at the exocytotic site itself.

Susceptibility of Leishmania to oxygen intermediates and killing by normal macrophages.

This study demonstrates that the promastigote form of virulent Leishmania donovani and Leishmania tropica are both deficient in endogenous enzymatic scavengers of H(2)0(2) (catalase, glutathione peroxidase) and susceptible to low fluxes of H(2)O(2) in a cell-free model. In addition, the killing of promastigotes by H(2)0(2) is markedly enhanced in the presence of a peroxidase and halide. Promastigotes also readily trigger the macrophage oxidative burst including the generation of H(2)0(2), and most intracellular promastigotes are killed within 18 h by unstimulated normal resident cells. Catalase, but not scavengers or quenchers of O(2)(-), OHx, or (1)O(2), protected promastigotes in a cell-free xanthine oxidase microbicidal system, and catalase also partially inhibited the leishmanicidal activity of resident macrophages. Thus, amongst various oxygen intermediates, H(2)0(2) alone appeared to be both necessary and sufficient for promastigote killing. Depriving macrophages of exogenous glucose, which inhibits the generation of oxygen intermediates, achieved effects similar to catalase treatment. These observations directly contrast with the intracellular parasite, T. gondii which is richly endowed with catalase and glutathione peroxidase, highly resistant to H(2)0(2), and requires products of O(2)(-)-H(2)0(2) interaction for effective oxidative killing. Toxoplasmas also fail to trigger the respiratory burst of normal macrophages, and readily multiply within these cells (1-5). Macrophages first activated by in vivo or in vitro immunologic stimuli, however, display an enhanced capacity to generate oxygen intermediates beyond O(2)(-) and H(2)0(2), and are able to kill toxoplasmas or inhibit their intracellular replication (1, 2). These studies illustrate the wide spectrum of susceptibility to oxidative products which appears to exist for virulent intracellular protozoans, and indicate that such differences may be reflected in contrasting fates of parasites within cell-free oxidative environments and the cytoplasm of normal resident macrophages. In addition, these observations also demonstrate that nonactivated phagocytes may display effective microbicidal activity against certain intracellular pathogens utilizing an oxygen-dependent mechanism.

A cell-free model of the encephalomyocarditis virus-induced inhibition of host cell protein synthesis

When preincubated extracts from Krebs-II cells were supplemented with total poly(A)-containing RNA isolated from encephalomyocarditis virus-infected Krebs-II cells, two phenomena characteristic of EMC virus infection of these cells in vivo were observed: (i) preferential translation of viral rather than cellular, mRNA, and (ii) a general inhibition of protein synthesis, relative to the synthesis in samples where the poly(A)-containing RNA from uninfected cells served as the template. The first effect could not be explained by an irreversible functional inactivation of cellular templates and seemed to result from a direct interference of viral RNA with the translation of cellular mRNA. The second effect was due primarily to the presence of double-stranded RNA.