Modification of chromaffin cells with pertussis toxin or N-ethylmaleimide lowers cytoskeletal F-actin and enhances Ca(2+)-dependent secretion.

In an attempt to identify proteins involved in the secretory response, bovine chromaffin cells were modified with N-ethylmaleimide (NEM). NEM concentrations less than 30 microM enhanced norepinephrine secretion evoked by nicotine or by K+ depolarization and increased Ca(2+)-dependent secretion from digitonin-permeabilized cells. Higher concentrations of NEM inhibited secretion. The protein modified by NEM which was responsible for the enhancement of secretory activity appeared to rapidly diffuse out of the digitonin-permeabilized cells. When proteins which diffuse from control digitonin-permeabilized cells were incubated with pertussis toxin and [32P]NAD, several proteins were ADP-ribosylated. However, when proteins from cells preincubated with 30 microM NEM were incubated with pertussis toxin and [32P]NAD, these GTP-binding proteins (G-proteins) were not ADP-ribosylated, which suggests that they were modified in the cell by NEM. Stimulation of norepinephrine secretion by NEM was not additive with that caused by pertussis toxin. Modification of chromaffin cells with pertussis toxin or with 30 microM NEM caused a 40-50% decrease in the amount of cytoskeletal F-actin. This decrease in cytoskeletal F-actin may account for the increase in secretory activity.     

晚期糖基化终产物通过其受体促进内皮细胞分泌IL-8的研究

探讨晚期糖基化终产物(AGE)修饰蛋白对内皮细胞生成白介素8(IL-8)的作用,及晚期糖基化终产物受体(RAGE)在此病理过程中的作用.内皮细胞来自培养的人脐静脉内皮细胞(HUVEC).将内皮细胞与不同浓度的AGE修饰人血清白蛋白(AGE-HSA)在体外共同培养,或以可溶性晚期糖基化终产物受体(sRAGE)对AGE-HSA进行预处理后再与HUVEC共同培养.用蛋白质液相芯片法检测HUVEC培养上清中IL-8水平,并提取细胞RNA,进行RT-PCR反应,检测细胞中IL-8 mRNA的表达水平.结果表明,AGE-HSA以时间和剂量依赖的方式刺激HUVEC生成IL-8,未经修饰的HSA无此作用.AGE-HSA用sRAGE预处理后,刺激HUVEC生成IL-8的作用被抑制,并且此抑制作用呈剂量依赖的方式.AGE-HSA刺激HUVEC使IL-8 mRNA表达增高,未经修饰的HSA无此作用.sRAGE能够阻断AGE-HSA诱导HUVEC表达IL-8mRNA的作用.整个变化趋势与蛋白质水平一致.研究首次证实,AGE-HSA与细胞表面受体RAGE相互作用可刺激内皮细胞分泌IL-8,并上调IL-8 mRNA的表达.这为研究加速型血管病变的发病机制提供了新视角,也为治疗由AGE增多和潴留所引起的病理损害提供了新靶点.     

Cytofluorometric analysis of small intensively fluorescent atrial cells at various stages of postnatal ontogenesis

The number and fluorescence intensity of small intensely fluorescent (SIF) cells in the nerve ganglia of the rat atria were determined after the treatment, using modified Falk's method. The animals were 1, 7, 14, 28 days and 30 months of age. Similar changes were found in the amount of SIF cells and fluorophore content in all the atrial regions. A marked increase in the number of SIF cells and maximum values were determined between the second and fourth week after birth. The first two weeks of age corresponded to the decrease and the forth week to the increase in SIF cells fluorescent intensity. The data of the investigation made it possible to suggest the connection between principal stages of SIF cell pool formation and processes of autonomic ganglia innervation development.     

Cytoskeletal mechanisms responsible for invasive migration of neoplastic cells

Cytoskeletal reorganizations, especially alterations of contractile tension generated by the actin-myosin cortex, are of central importance in the development of the phenotype of morphologically transformed neoplastic cells with invasive behavior. These reorganizations can be regarded as genetically determined aberrations of the physiological reactions of normal cells which are responsible for their ability to undergo exploratory migrations, including epithelio-mesenchymal transformations, invasion of matrix by epithelial tubules etc. It is suggested that these physiological and neoplastic transformations are based on Rho-dependent alterations in contractility. A decrease or an increase in contractility may result in the development of distinct types of invasive phenotypes. These contractility-dependent phenotype alterations may be modified by alterations in the expression of other genes, especially of those coding for components of adhesive structures.     

Water and ion balance in rat thymocytes under apoptosis induced with dexamethasone or etoposide. Ion-osmotic model of cell volume decrease

Cell ion and water balance was studied with respect to analysis of the osmotic model of apoptotic volume decrease (AVD) in rat thymocytes under dexamethasone (1 microM, 4-6 h) or etoposide (50 microM, 5 h) treatment. Intracellular water content was determined by measurement of cell buoyant density in continuous Percoll gradient, while intracellular potassium and sodium contents were determined by flame emission analysis. Apoptosis was verified by an increase in cell buoyant density, fluorescence of cells stained with Acridine orange and Ethidium bromide (flow cytometry), by changes in the cell cycle and the appearance of sub-diploid peak in the DNA histogram (flow cytometry), and by a decrease in cell size examined with light microscope. A separate fraction of dense cells with reduced size was found to appear after dexamethasone or etoposide treatment. This fraction was considered as apoptotic. An increase in buoyant density of apoptotic cells corresponded to a decrease in cell water content. In apoptotic cells vs. cells with normal buoyant density, the intracellular potassium content was lower, but sodium content was higher. The sum of potassium and sodium contents was lower in apoptotic cells. Taken into account the loss of anions, associated with the loss of cations, the bulk decrease in ions content has been sufficient to be accounted for cell volume decrease on the basis of the ion-osmotic model.     

Autoradiographic studies of the synthesis of RNA and protein as a function of cell volume in Streptococcus faecium

Mid-exponential-phase cultures were either labeled continuously with tritiated leucine and uracil or pulse-labeled with tritiated leucine. The amount of leucine and uracil incorporated into protein or RNA per cell was determined by grain counts of autoradiographs of cells seen in electron micrographs; the volume of each cell was determined by three-dimensional reconstruction. The average number of autoradiographic grains around cells continuously labeled with uracil and leucine increased linearly with cell volume. In contrast, while the average grain count around cells pulse-labeled with leucine increased in a near-linear fashion over most of the volume classes, less than the expected number of grains were seen around cells in large- and small-size classes. The distribution of grains around cells from both the continuously and pulse-labeled populations could be fit at the 5% confidence level with a Poisson distribution modified to take into consideration the volume distribution of each population of cells analyzed. These findings suggested that large changes in the density of RNA and protein do not occur in most cells as they increase in size; however, there may be decreases in the rate of protein synthesis in some large and small cells. The decrease in the rate of protein synthesis appears consistent with the hypothesis that new sites of envelope growth must be introduced into cells that are close to the division event to restore rapid growth.     

Decreased prostaglandin production by cholesterol-rich macrophages

The regulation of prostaglandin production by macrophages enriched in cholesterol was examined. Mouse peritoneal macrophages were incubated for 18 h with 25 micrograms/ml of human acetyl-LDL (low density lipoprotein) and trace amounts of labeled arachidonic acid. After cholesterol enrichment, the cells were incubated with phorbol 12-myristate 13-acetate (PMA), calcium ionophore, or zymosan to stimulate endogenous arachidonic acid metabolism. A high performance liquid chromatography profile of the eicosanoids released revealed no qualitative differences between unmodified and modified macrophages. Cholesterol-rich cells, however, released less prostacyclin (PGI2) and prostaglandin E2 (PGE2) compared to unmodified cells, and products from the lipoxygenase pathway became the predominant metabolites. A decrease in the synthesis of PGI2 and PGE2 by cholesterol-rich macrophages was confirmed by radioimmunoassay and radiolabeled experiments. The activity of prostaglandin synthetase was modestly increased in the cholesterol-modified macrophages compared to controls. As an estimation of phospholipase activity, the release of labeled arachidonic acid from membrane phospholipids, however, was significantly decreased in cholesterol-rich macrophages. The phosphatidylinositol fraction was particularly resistant to arachidonate release in response to calcium ionophore and PMA in the modified cells. The measurement of membrane phospholipid fatty acid composition before and after calcium ionophore supported the observation that less arachidonate was released by cholesterol-enriched cells in response to the ionophore. Based on these observations, we propose that prostaglandin synthesis from endogenous arachidonate stores is decreased in the cholesterol-rich macrophage. A decrease in agonist-induced activation of the phospholipase activity is proposed as a mechanism for this effect.     

Prediction of the changes in thermodynamic stability of proteins caused by single amino acid substitutions

Prediction of the effect of amino acid substitutions on the thermodynamic stability of proteins is of great importance for studies into the molecular mechanisms underlying the abnormal function of mutant proteins, interpretation of genotyping results, and purposeful design of modified proteins with improved biomedical and biotechnological properties. A set of methods was developed for predicting the changes in free energy (ΔΔG) of mutant proteins containing single substitutions using the information only about protein primary structure or also about the spatial structure. A modified KRAB algorithm was used; its higher accuracy in predicting the changes in the thermodynamic stability of mutant proteins compared with the other known methods designed for solving this problem is demonstrated. Distribution of the positions in the sequence of Malayan pit viper venom protein (kistrin) where the substitutions decrease or increase kistrin stability is analyzed. The substitutions at most positions conserved in the disintegrin family decrease the stability of this protein, except for several positions whose conservation can be determined by functional significance.     

Changes in membrane functions during short-term starvation of Vibrio fluvialis strain NCTC 11328

The marine bacterium Vibrio fluvialis strain NCTC 11328 responded to starvation conditions by forming ultramicrocells of dwarf bacteria. The viability of starved cells began to decrease after 2–3 days. During this time the respiratory potential of the bacteria decreased by four- or fivefold, most probably as a result of a decrease in the specific activity of NADH and succinate dehydrogenases. Although respiratory potential in starving cells was lower than in growing cells, bacteria starved for 1 or 2 days maintained a proton motive force that was slightly larger than that of growing bacteria. Starved bacteria contained substantial concentrations of ATP although the UTP and GTP concentrations were much lower in starved than in growing cells. Two or three proteins that were not present in membranes of growing cells, were evident in the membranes of starved bacteria.Abbreviations MMS modified Morita's salts - MMSGC modified Morita's salts plus 20 mM glucose and 0.1% (w/v) casamino acids - MMST modified Morita's salts buffered with 50 mM tricine, (pH 8.5) - NM broth nutrient modified Morita's salts - CFU colony-forming unit - TPP tetraphenylphosphonium - STM 0.1 M tricine, (pH 8.0) plus 0.25 M sucrose and 0.02 M magnesium acetate - DCPIP dichlorophenolindophenol - CCCP carbonyl cyanidem-chlorophenylhydrazone - PMF proton motive force     

CONCANAVALIN A-INDUCED AGGLUTINATION AND BINDING OF CON A TO THE DIFFERENTIATING CELLS OF DICTYOSTELIUM DISCOIDEUM

Changes in agglutinability of Dictyostelium discoideum cells with Concanavalin A (Con A) during the course of development were investigated. The agglutinability of the cells was assayed under conditions where no spontaneous cell agglutination occurred. It was found that there was a progressive decrease in Con A-induced agglutinability during development: a decrease to half from exponentially growing cells to preaggregation cells, and to sixth in disaggregated slug cells. Pronase-BAL treatment of preaggregation cells did not enhance their agglutinability with Con A. The amounts of sites available for binding Con A were determined with preaggregation and slug cells. Cells were incubated at 4°C and in the presence of NaN₃ to avoid possible endocytosis of Con A. No significant differences in numbers of Con A-binding sites per unit area of cell surface was detected among preaggregation cells, those treated with pronase and BAL and cells disaggregated from slugs by similar treatment. It was thus concluded that the decrease in Con A-induced agglutinability during development is not attributable to changes in the numbers of Con A-binding sites.     

Membrane fatty acid composition and endothelial cell functional properties

In order to study the influence of endothelial cell fatty acid composition on various membrane related parameters, several in vitro methods were developed for manipulating the fatty acid content of human endothelial cell membranes. Changes in membrane fatty acid profile were induced by using fatty acid modified lipoproteins or free fatty acids. The largest changes in endothelial fatty acid composition were obtained by culturing the cells in media supplemented with specific free fatty acids. An increase in arachidonic acid content of endothelial phospholipids was induced by supplementation with saturated fatty acids or with arachidonic acid itself. A decrease in arachidonic acid content was obtained by supplementation with other unsaturated fatty acids. Under the experimental conditions used endothelial cells showed a low desaturase activity and a high elongase activity. Considerable alterations in membrane fatty acid composition did not greatly influence certain membrane related parameters such as polymorphonuclear leukocyte adherence and endothelial cell procoagulant activity. In general, for fatty acid modified endothelial cells an association between endogenous arachidonic acid content and total production of eicosanoids was found. This study demonstrates that considerable changes in membrane fatty acid profile affect endothelial cell arachidonic acid metabolism, but it also illustrates homeostasis at the level of endothelial cell functional activity.     

Intracellular predominance of the pyridoxal 5'-phosphate form of aspartate aminotransferase in Escherichia coli B and reversible transformation of this form by extracellular substances

The intracellular proportion of the pyridoxal 5'-phosphate form of aspartate aminotransferase to the total enzyme in E. coli B cells was determined by a newly devised method, dependent on selective inactivation of the intracellular pyridoxal 5'-phosphate form of the enzyme by extracellularly added sodium borohydride. A large portion (80-99%) of the intracellular aspartate aminotransferase was in pyridoxal 5'-phosphate form in both natural and synthetic medium-grown bacterial cells. The intracellular predominancy of pyridoxal 5'-phosphate did not vary during the growth of bacteria and during incubation of bacterial cells in various kinds of buffers with different pH values. In contrast, the saturation levels generally used to describe in vivo the proportions of the apo and holo vitamin B6-dependent enzymes did not reflect the intracellular amount of the pyridoxal 5'-phosphate (holo) form of aspartate aminotransferase probably because the intracellular pyridoxal 5'-phosphate form was changed to an apo form by the disruption of bacterial cells for preparing crude extract. Various extracellularly-added vitamin B6 antagonists decreased the intracellular amount of pyridoxal 5'-phosphate without decrease in the total intracellular activity of the enzyme. The modified forms were stable in E. coli B cells and reversed into pyridoxal 5'-phosphate form by incubation of the antagonist-treated cells in the buffer containing pyridoxal. The present results showed that the sodium borohydride reduction method can be used for further analysis of the in vivo interaction of pyridoxal 5'-phosphate and apoaspartate aminotransferase. The fact that about 50% of the intracellular pyridoxal 5'-phosphate form was changed to a modified form without impairment of cell growth in the presence of 4-deoxypyridoxine, and that about 50% of intracellular modified aspartate aminotransferase was reversed to pyridoxal 5'-phosphate by the removal of antagonist followed by incubation suggested that there exists characteristically 2 different fractions of pyridoxal 5'-phosphate forms of aspartate aminotransferase in E. coli cells.     

Substitution of the insulin receptor transmembrane domain with that of glycophorin A inhibits insulin action.

To study the role of transmembrane (TM) domains interactions in the activation of the insulin receptor, we have replaced the insulin receptor TM domain with that of glycophorin A (GpA), an erythrocyte protein that spontaneously forms detergent-resistant dimers through TM-TM interactions. Insulin receptor cDNA sequences with the TM domain replaced by that of GpA were constructed and stably transfected in CHO cells. Insulin binding to cells and solubilized receptors was not modified. Electrophoresis after partial reduction of disulfide bonds revealed an altered structure for the soluble chimeric receptors, seen as an altered mobility apparently due to increased interactions between the beta subunits of the receptor. Insulin signaling was markedly decreased for cells transfected with chimeric receptors compared with cells transfected with normal receptors. A decrease in insulin-induced receptor kinase activity was observed for solubilized chimeric receptors. In conclusion, substitution by the native GpA TM domain of the insulin receptor results in structurally modified chimeric receptors that are unable to transmit the insulin signal properly. It is hypothesized that this substitution may impose structural constraints that prevent the proper changes in conformation necessary for activation of the receptor kinase. Other mutants modifying the structure or the membrane orientation of the glycophorin A TM domain are required to better understand these constraints.     

Author index

The stearoyl-coenzyme A desaturase system of L-M cells, grown as monolayers, was examined in microsomal membranes that contained 8.2% phosphatidylisopropylethanolamine, an unnatural phospholipid analog. Desaturation of both [1-¹⁴C]stearic acid by whole cells and [1-¹⁴C]stearoyl-coenzyme A by cell-free homogenates, or microsomes, was decreased to about 40% of control levels in cells that had been grown for 24 h in the presence of 10 mmN-isopropylethanolamine. No decrease in microsomal NADH- or NADPH-dependent cytochrome c reductase activities or the level of cytochrome b₅ was found in the L-M cells that had been treated for 24 h with N-isopropylethanolamine. Although amino acid transport into L-M cells was not affected by treatment with N-isopropylethanolamine, protein synthesis was decreased by about 30%. These results indicate that the decrease in stearoyl-coenzyme A desaturation in the modified membranes is specifically associated with the terminal oxidase activity (cyanide-sensitive factor) of the desaturase enzyme complex.     

Mechanism of action of aryl hydrocarbon receptor antagonists: inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1A1 gene expression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 gene expression as determined by increased CYP1A1 mRNA levels and ethoxyresorufin O-deethylase (EROD) activity in mouse Hepa 1c1c7, rat hepatoma H-4II E and human Hep G2 cancer cell lines. In contrast, treatment of these cell lines with either alpha-naphthoflavone (alpha NF) or 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) at concentrations as high as 10(-6) M resulted in only minimal induction of CYP1A1 mRNA levels or EROD activity. Cotreatment of the cells with 10(-9) M TCDD plus different concentrations (10(-8)-10(-6) M) of MCDF or alpha NF resulted in a concentration-dependent decrease in TCDD-induced CYP1A1 mRNA levels and EROD activity in the three cell lines. Moreover, using 10(-9) M [3H]TCDD, it was shown that the alpha NF- and MCDF-mediated antagonism of TCDD-induced CYP1A1 gene expression was paralleled by a decrease in levels of the nuclear [3H]TCDD-Ah receptor complex as determined by velocity sedimentation analysis of the nuclear extracts. The binding of nuclear extracts from the treated cells to a synthetic consensus dioxin responsive element (DRE) (a 26-mer) was determined by gel retardation studies using 32P-DRE. In cells treated with 10(-9) M TCDD or TCDD plus 10(-8)-10(-6) M alpha NF, the concentration-dependent decrease in TCDD-induced CYP1A1 gene expression by alpha NF was also paralleled by decreased levels of a retarded band associated with the nuclear Ah receptor-DRE complex. In contrast, the results of the gel shift assay of nuclear extracts treated with 10(-9) M TCDD or TCDD plus 10(-8)-10(-6) M MCDF indicated that there were relatively high levels of nuclear MCDF-Ah receptor complex in the cells co-treated with TCDD plus the antagonist but this was not accompanied by induced CYP1A1 gene expression. The results suggest that alpha NF and possibly MCDF compete with TCDD for cytosolic Ah receptor binding sites; however, MCDF may also inhibit the induction response by competing for and/or partially inactivating genomic binding sites.     

Proteasome activation and nNOS down-regulation in neuroblastoma cells expressing a Cu,Zn superoxide dismutase mutant involved in familial ALS

Reactive oxygen and nitrogen species have emerged as predominant effectors of neurodegeneration. We demonstrated that expression of the fully active G93A Cu,Zn superoxide dismutase mutant in neuroblastoma cells is associated with an increased level of oxidatively modified proteins, in terms of carbonylated residues. A parallel increase in proteasome activity was detected and this was mandatory in order to assure cell viability. In fact, proteasome inhibition by lactacystin or MG132 resulted in programmed cell death. Nitrosative stress was not involved in the oxidative unbalance, as a decrease in neuronal nitric oxide production and down-regulation of neuronal nitric oxide synthase (nNOS) level were detected. The nNOS down-regulation was correlated to increased proteolytic degradation by proteasome, because comparable levels of nNOS were detected in G93A and parental cells upon treatment with lactacystin. The altered rate of proteolysis observed in G93A cells was specific for nNOS as Cu,Zn superoxide dismutase (Cu,Zn SOD) degradation by proteasome was influenced neither by its mutation nor by increased proteasome activity. Treatment with the antioxidant 5,5'-dimethyl-1-pyrroline N-oxide resulted in inhibition of protein oxidation and decrease in proteasome activity to the basal levels. Overall these results confirm the pro-oxidant activity of G93A Cu,Zn SOD mutant and, at the same time, suggest a cross-talk between reactive oxygen and nitrogen species via the proteasome pathway.     

3'-Deoxy-3'-fluoro analogs of core trimers of 2-5A: effect on lytic activity of human NK-lymphocytes]

The effect of core trimers, (2'-5')-analogues of oligoadenylic acid containing 9-(3-deoxy-3-fluoro-beta-D-xylofuranosyl)adenine (AF) and 3'-deoxy-3'-fluoroadenosine (AF) in various positions of the oligomer chain, on the lytic activity of human natural killer cells (NK cells) was studied in three different ways. The cellular cytotoxicity was determined using a highly sensitive nonradioactive approach employing a chelate europium-diethylenetriamino-pentaacetic acid complex (Eu-DTPA). It was shown that all fluorodeoxyanalogues enhance the lytic activity of intact NK lymphocytes, which follows from the lysis rate constant k2. At the same time, the substitution of either the central adenosine fragment or (to a greater extent) the 5'-terminal residue of (2'-5')A3 with AF causes a decrease in the number of active NK cells, which, unlike the case of the natural core trimer, leads to a loss of the capacity to increase the activity of NK. By contrast, isomeric ribo-analogues. (2'-5')(AF)A2 and (2'-5')A(AF)A, and trimers with the 2'(3')-terminal nucleotide substituted by AF or AF increased the activity of NK cells with an effectiveness close to or higher than the natural trimer (2'-5')A3. Inasmuch as isomeric xylo- and ribo-3'-deoxy-3'-fluoroanalogues of (2'-5')A3 are stereochemically modified oligomers, the data unambiguously suggest that the spatial structure of these trimers affects the increase in the lytic activity of NK cells.     

Comparison of methods for enumeration of selected coliforms exposed to ozone

mT7 medium performed no better than m-Endo medium in enumerating cells of Escherichia coli and Citrobacter freundii exposed to ozone. Also, there was no difference in the plate count of heterotrophic bacteria in ozonated raw water determined on modified Henrici agar or R2A agar. Statistically significant differences were seen between bacteria and the type of water in which they were suspended during ozonation.     

Comparison of methods for enumeration of selected coliforms exposed to ozone.

mT7 medium performed no better than m-Endo medium in enumerating cells of Escherichia coli and Citrobacter freundii exposed to ozone. Also, there was no difference in the plate count of heterotrophic bacteria in ozonated raw water determined on modified Henrici agar or R2A agar. Statistically significant differences were seen between bacteria and the type of water in which they were suspended during ozonation.     

The absolute configuration of prunioside A from Spiraea prunifolia and biological activities of related compounds

The stereochemistry of prunioside A isolated from Spiraea prunifolia was determined by chemical transformations and NMR spectral data analysis. The configurations at C-5 and C-6 were determined to be 5S and 6R by application of the modified Mosher's method, CD analysis, and 13C NMR spectroscopic data analysis of an acetonide derivative. Other compounds related to prunioside A have inhibitory effects on the synthesis of nitric oxide in LPS-stimulated macrophage-like RAW 264.7 cells.