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PubMed1993

Evidence for predominantly paracellular transport of thyrotropin-releasing hormone across CACO-2 cell monolayers.

Uptake, transport, and metabolism of tripeptide thyrotropin-releasing hormone were examined in the human intestinal epithelial cell line, Caco-2. A linear relationship between rate and concentration was observed for both the uptake and the transport of thyrotropin-releasing hormone across Caco-2 cell monolayers. Transport of thyrotropin-releasing hormone was not affected by the presence of dipeptide glycylsarcosine, amino acid glycine, tripeptide thyrotropin-releasing hormone free acid as well as active transport inhibitors 2,4-dinitrophenol, sodium azide, ouabain, and amiloride. There was no formation of metabolites during the course of thyrotropin-releasing hormone transport across Caco-2 cells. Incubation of Caco-2 cell homogenate with thyrotropin-releasing hormone, however, showed a time-dependent hydrolysis of thyrotropin-releasing hormone and the formation of thyrotropin-releasing hormone free acid. Increased rate of transport in the presence of EDTA indicates a paracellular passive diffusion as the major route for the transport of TRH. The hydrolytic enzyme present in Caco-2 cells appeared to have little or no access to TRH during the transcellular transport across Caco-2 cell monolayers.

2,4-Dinitrophenol↗

PubMed1996

ATP depletion affects NPM translocation and exportation of rRNA from nuclei.

Nucleophosmin/B23 (NPM) is a nucleolar phosphoprotein which shifts from nucleoli to the nucleoplasm in cells treated with certain cytotoxic agents (NPM-translocation). NPM requires GTP for localization into nucleoli (J. Biol. Chem. 268, 5823-5827, 1993). To understand more about NPM's dynamic localization, the effects of lowering ATP on NPM-translocation and rRNA synthesis were studied. When the ATP level in HeLa cells was reduced by sodium azide, NPM-translocation was blocked. Similar results were obtained when ATP was depleted by other agents, suggesting that ATP depletion was responsible for the blocking of NPM-translocation. It was found that newly synthesized rRNA accumulated in the nuclei during ATP-depletion. Significantly larger than normal nucleoli were also observed. These results indicate that NPM may be involved in the transportation of newly synthesized ribosomes.

2,4-Dinitrophenol↗

PubMed1989

Effect of cellular function inhibitors on the infection of Frp/3 cells by hepatitis A virus.

The effect of some cellular function inhibitors on hepatitis A virus (HAV) adsorption and on the successive events of infection in a monkey cell line (Frp/3 cells) was investigated. Treatments of Frp/3 cells with colcemide, vinblastine and cytochalasin D, which affect cytoskeleton organization, indicated that neither microtubules nor microfilaments play an important role in the early events of HAV infection. Monensin, which acts as an ionophore on intracellular vesicle compartments inhibited HAV infection probably at the uncoating step. Inhibition of viral replication to a different degree was observed with both inhibitors of oxidative phosphorylation, such as dinitrophenol and sodium azide, and with an inhibitor of glycolysis, 2-deoxy-D-glucose. However, none of these compounds significantly affected the early steps of infection, thus demonstrating that HAV replication is largely dependent upon cell energy.

Azides↗

PubMed1991

Microphotometric determination of enzymes in brain sections. IV. Isocitrate dehydrogenases.

A polyvinyl alcohol-(PVA) containing incubation medium was adapted for the microphotometric determination (kinetic and end-point measurements) of the activities of NAD- and NADP-linked isocitrate dehydrogenases (ICDHs) in cryostat sections of the rat hippocampus. The following incubation medium is recommended for the quantification of NAD- and NADP- (differences in brackets) ICDHs: 100 mM DL-isocitrate, 10 mM sodium azide, 5 mM (4 mM) nitroblue tetrazolium (NBT), 7 mM NAD (4 mM NADP), 10 mM magnesium chloride, 0.25 mM phenazine methosulfate (PMS), with or without 5 mM ADP (without ADP), 23% PVA in 0.05 M Hepes buffer; the final pH was 7.5. With these incubation media a linear response of the reactions lasted at least 20 min. In kinetic and end-point measurements the same level of activities was demonstrable. The use of NaN3 (as a blocker of the respiratory chain) and PMS (as artificial electron carrier) was indispensible for the transfer of all reduction equivalents in the dehydrogenase reactions to the tetrazolium salt NBT. Furthermore, the activation by magnesium ions and the need of PVA to avoid diffusion artefacts of the loosely bound ICDHs were clearly shown. It is concluded that the quantification of ICDHs in situ could be a valuable tool for neurochemical investigations because ICDHs play a role not only in the substrate flux through the tricarboxylic acid cycle but also in providing alpha-ketoglutarate for the formation of glutamate which is an important amino acid in the brain.

Animals↗

PubMed1987

Studies of phagocytosis in chronic granulomatous disease.

Abnormal phagocyte function in chronic granulomatous disease (CGD) is associated with decreased bactericidal activity. Ingestion of serum-opsonized organisms is reported to be normal in these patients. We previously showed that in CGD the expression of C3b receptors (CR1) on polymorphonuclear leukocytes (PMNs) is significantly depressed. In this study, we compared the phagocytic activity of the PMNs from normal healthy controls with that of CGD patients and one individual with myeloperoxidase (MPO) deficiency. The ingestion of sheep erythrocytes (E) by PMNs adherent to a glass surface was examined; the E were coated either with excess IgG (E-IgG) or with C3b plus limited IgG (EAC3b-IgG). The PMNs, both in CGD and in MPO deficiency, ingested E-IgG and EAC3b-IgG at levels markedly above normal. C3b-coated erythrocytes were not phagocytosed. Preincubating the PMNs with sodium azide, which blocks MPO, or catalase, a scavenger of H2O2, caused a marked increase in phagocytosis by normal PMNs. Azide had a variable effect on PMN activity in CGD and no effect on the activity in the subject with MPO deficiency. Even in the presence of azide, the ingestion of EAC3b-IgG by the PMNs from the CGD patients was significantly greater than that seen in paired normals [mean phagocytic index (PI), 2.13 for CGD vs. 1.48 for normals; P less than 0.05 by the paired sample t test]. Similar results were obtained with ingestion of E-IgG. Notably, ingestion of serum-opsonized Candida organisms (relatively nondegradable particles) was markedly above normal with CGD PMNs and, in normal PMNs, azide treatment also evoked an increase. In addition, rosette formation of the adhered PMNs with E-IgG was enhanced with CGD and the azide-treated normal PMNs. We demonstrated that this increased activity was not the result of increased Fc receptor (FcR) number, as determined from the binding of a monoclonal anti-FcR antibody. Both the E-IgG rosette formation and the ingestion by CGD PMNs were abrogated in the presence of an H2O2-generating system. In contrast, the phagocytic activity of MPO-deficient PMNs was not altered by exogenous H2O2. These findings suggest that cellular products generated by the H2O2-MPO-halide system down-regulate the rosette-forming and phagocytic activity of PMNs from normal healthy individuals, but not that from CGD and MPO-deficient patients.

Azides↗

PubMed1991

Mechanism of cell protrusion formation in electrical field: the role of actin.

An intense alternating current electrical field that imposes membrane-applied force on the cell surface can induce formation of cell protrusions (Popov, S.V. and Margolis, L.B. (1988) J. Cell Sci. 90, 379-389). This technique has been used to investigate the role of actin in the cell protrusion formation. Platinum replicas of the cytoskeleton were prepared to characterize the organization of the cytoskeleton in external force-induced protrusions. Bundles of microfilaments were found in the processes. A specific inhibitor of actin polymerization, cytochalasin B, as well as inhibitors of ATP synthesis (sodium azide and carbonyl m-chlorophenylhydrazone) did not change the morphology of electrical field-generated protrusions, revealed by scanning electron microscopy. However, organization of the cytoskeleton inside the processes changed drastically using these inhibitors. The results of these experiments demonstrate that (i) Membrane-applied force is sufficient to produce native-like cell protrusions, even in conditions where activity of the cytoskeleton is inhibited; (ii) Actin microfilaments can be organized into bundles directly under the action of membrane-applied force. The significance of these observations to cell protrusion formation under normal physiological conditions is discussed.

Actin Cytoskeleton↗

PubMed1984

Guanosine triphosphate catabolism in purine nucleoside phosphorylase deficient human B lymphoblastoid cells.

GTP catabolism induced by sodium azide or deoxyglucose was studied in purine nucleoside phosphorylase (PNP) deficient human B lymphoblastoid cells. In PNP deficient cells, as in control cells, guanylate was both dephosphorylated and deaminated but dephosphorylation was the major pathway. Only nucleosides were excreted during GTP catabolism by PNP deficient cells and the main product was guanosine. The level of nucleoside excretion was largely affected by intracellular orthophosphate (Pi) level. In contrast, normal cells excreted nucleosides only at low Pi level while at high Pi levels, purine bases (guanine and hypoxanthine) were exclusively excreted. PNP deficiency had no effect on the extent of GMP deamination.

Azides↗

PubMed1988

Efficient inhibition of endogenous peroxidase without antigen denaturation in immunohistochemistry.

The immunoperoxidase technique is commonly used for the antigenic characterization of cells and tissues. However, the occurrence of endogenous peroxidase-positive cells frequently complicates such evaluations. In some instances it is impossible to distinguish between these cells and antibody-stained cells. Methods so far described for inhibiting the endogenous peroxidase, are not always satisfactory - either the endogenous peroxidase is not satisfactorily blocked, or the cellular antigens lose their reactivity. A new two-step method is described which involves transient inhibition of endogenous peroxidase by sodium azide and hydrogen peroxide and a final blocking of this enzyme by lowering the pH to 2.3, while antigens remain undamaged. This method may be of value in immunohistochemical studies of various tissues, especially inflammatory tissues which contain many endogenous peroxidase-positive cells.

Animals↗

PubMed1985

Assessment of attachment, ingestion, and killing of Escherichia coli by bovine polymorphonuclear cells with combined micromethods.

A set of microassays separately measuring attachment, ingestion, and overall killing of Escherichia coli by bovine granulocytes was devised and its analytical potential used to test the effect of drugs which block intracellular killing: sodium azide, phenylbutazone, chloroquine phosphate were all inactive, suggesting that O2-dependent systems were not the sole pathway involved in the killing of E.coli by granulocytes. The microtechniques were also used to investigate the opsonic requirements for phagocytosis of two E.coli strains. Absorption of normal bovine serum with the homologous and the heterologous strains showed that specific antibodies were necessary to induce attachment of bacteria to phagocytes. Once bound to granulocytes, the unencapsulated strain P4 was engulfed, whereas for the encapsulated strain B117, complement was required for the internalization step of phagocytosis. With immune serum the need for complement was not absolute.

Animals↗

PubMed1995

A practical method for preparing peroxynitrite solutions of low ionic strength and free of hydrogen peroxide.

The reaction of ozone (approximately 5% in oxygen) with sodium azide (0.02-0.2 M in water) at pH 12 and 0-4 degrees C is shown to yield concentrated, stable peroxynitrite solutions of up to 80 mM. The product of this reaction is identified based on a broad absorption spectrum with a maximum around 302 nm and by its first-order rate of decomposition (k = 0.40 +/- 0.01 s-1 at pH 7.05 and 25 degrees C). These peroxynitrite solutions can be obtained essentially free of hydrogen peroxide (detection limit 1 microM) and only traces of azide (detection limit 0.1 mM). They are low in ionic strength and have a pH of about 12 but without buffering capacity; therefore, they can be adjusted to any pH by addition of buffer. These preparations of peroxynitrite frozen at -20 degrees C show negligible decomposition for about 3 weeks of storage and follow a first-order decomposition with a halflife of about 7 days at refrigerator temperatures (approximately 5 degrees C). These preparations give reactions that are characteristic of peroxynitrite. For example, at pH 7.0, they react with L-tyrosine to give a 7.3 mol % yield of nitrotyrosine(s), and with dimethyl sulfoxide to give a 8.2 mol % yield of formaldehyde, based on starting peroxynitrite concentration.

Azides↗

PubMed1997

Binding of acetaldehyde to rat gastric mucosa during ethanol oxidation.

Acetaldehyde, the first product of ethanol metabolism, has previously been shown to form potentially harmful adducts with various proteins. The aim of this study was to investigate whether acetaldehyde--either exogenous or metabolically derived--binds to gastric mucosal proteins. Homogenized rat gastric mucosa was incubated with various concentrations of radiolabeled acetaldehyde or ethanol for different time periods. Acetaldehyde-protein adducts were determined by a liquid scintillation counter. In addition, mucosa was incubated with nonlabeled ethanol, and the acetaldehyde formed was measured by using headspace gas chromatography. Incubation of gastric mucosa with (14C)-acetaldehyde led to a concentration- and time-dependent radiolabeling of mucosal proteins. Formation of acetaldehyde adducts occurred relatively rapidly within 30 minutes and even at low acetaldehyde levels (5 micromol/L). Stable adducts represented 77% +/- 5% (mean +/- SEM) of the total adducts formed. In the presence of ethanol, acetaldehyde production and adduct formation took place in a concentration- and time-dependent manner. 4-Methylpyrazole and sodium azide inhibited acetaldehyde production to 7% +/- 1% of control and decreased the amount of acetaldehyde adducts to 55% +/- 8%. Enhanced acetaldehyde formation (to 420% +/- 50%) was clearly reflected in increased adduct formation (550% +/- 110%). In conclusion, both exogenous and endogenous acetaldehyde binds to gastric mucosal proteins in vitro. Gastric mucosal acetaldehyde production and the consequent adduct formation could be a pathogenetic factor behind ethanol-associated gastric injury.

Acetaldehyde↗

PubMed1996

Relationship between mitochondrial dysfunction and toxicity of propyl gallate in isolated rat hepatocytes.

The relationship between cytotoxicity and mitochondrial dysfunction caused by propyl gallate (PG) has been studied in hepatocytes freshly prepared from fasted rats. Hepatocytes isolated from fasted (18 h) rats were significantly more susceptible to the toxicity of PG than hepatocytes from fed rats. The addition of fructose (15 mM), an alternative carbohydrate source, to hepatocyte suspensions resulted in the prevention of PG (1 mM)-induced cell killing accompanied by decrease in intracellular ATP loss during a 3 h-incubation period. Despite this, fructose did not completely prevent an abrupt loss of intracellular glutathione caused by PG, but effectively inhibited the loss of protein thiol levels. Fructose elicited a concentration (0.5-20mM)-dependent protection against the cytotoxicity of 1.5 mM PG. The incubation of hepatocytes with sodium azide (4 mM), an inhibitor of oxidative phosphorylation, enhanced the toxicity induced by PG (1 mM), but coincubation with fructose delayed the onset of toxicity. Neither azide alone nor fructose plus azide did affect the cell viability during the incubation period. Furthermore, the addition of 2 mM salicylamide, nontoxic to hepatocytes during the incubation period, enhanced PG (1 mM)-induced cytotoxicity and decreased the loss of free PG. These results indicate that the onset of cytotoxicity caused by PG may depend on the intracellular energy status and that mitochondria are critical target for the compound. In addition, the toxicity caused by the inhibition of mitochondrial ATP synthesis is related to the concentration of PG remaining in cell suspensions.

Adenosine Triphosphate↗

PubMed1997

Effect of energy shortage and oxidative stress on amyloid precursor protein metabolism in COS cells.

The present study investigates the influence of energy related metabolic stress on amyloid precursor protein (APP) non-amyloidogenic secretory processing in COS cells. The effect of glucose deprivation on soluble APP (sAPP) secretion has been evaluated: incubation of COS cells with 50 mM 2-deoxy-D-glucose (2-DG) in glucose free medium was able to reduce sAPP secretion (-26%). Sodium azide (NaN3), an inhibitor of cytochrome c oxidase (complex IV of the mitochondrial electron transfer chain) decreased sAPP release in a concentration dependent way (maximum -75%). Treatment of COS cells with the antioxidant glutathione (GSH) fully antagonized the inhibitory effect of azide (1 mM) and elicited sAPP release over basal level. These results suggest that the inhibition of energy metabolism can influence APP processing leading to a decreased secretion of non-amyloidogenic fragments of APP.

Amyloid beta-Protein Precursor↗

PubMed1997

Mucochloric acid action on phi X174 DNA: a comparison to other chlorine-substituted 2(5H)-furanones.

The action of the drinking water mutagen 3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA) on the plasmid phi X174 converted this initially closed circular, supercoiled (SC) DNA to its relaxed (R) and linear (L) forms. Kinetic analysis of this process gave results coinciding with a sequential, two-step cleavage model whereby the SC was cleaved to the R form, which in its turn was cleaved to the L form. The actions of two additional chlorine-substituted 2(5H)-furanones, reduced MCA (RMCA) and the C-5 isopropyl ether of MCA (MCA-IPE), were compared to that of MCA. Incubation of SC-phi X174 with RMCA gave R form only while MCA-IPE had virtually no effect. Likewise, neither methyl methanesulfonate nor sodium azide cleaved SC-phi X174 to its L form, under conditions whereby MCA caused the formation of L form. Also, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) caused formation of R form, but no apparent L form. Increasing concentration of glutathione diminished cleavage of SC phi X174 by MCA, but increased cleavage for MX. The DNA cleavage action of MCA was unique, among the several stronger and weaker mutagens investigated, in its action phi X174.

Azides↗

PubMed1986

Ca2+ compartments in saponin-skinned cultured vascular smooth muscle cells.

A method for saponin skinning of primary cultured rat aortic smooth muscle cells was established. The saponin-treated cells could be stained with trypan blue and incorporated more 45Ca2+ than the nontreated cells under the same conditions. At low free Ca2+ concentration, greater than 85% of 45Ca2+ uptake into the skinned cells was dependent on the extracellularly supplied MgATP. In the intact cells, both caffeine and norepinephrine increased 45Ca2+ efflux. In the skinned cells, caffeine increased 45Ca2+ efflux, whereas norepinephrine did not. The caffeine-releasable 45Ca2+ uptake fraction in the skinned cells appeared at 3 X 10(-7) M Ca2+, increased gradually with the increase in free Ca2+ concentration, and reached a plateau at 1 X 10(-5) M Ca2+. The 45Ca2+ uptake fraction, which was significantly suppressed by sodium azide, appeared at 1 X 10(-5) M Ca2+ and increased monotonically with increasing free Ca2+ concentration. The results suggest that the caffeine-sensitive Ca2+ store, presumably the sarcoplasmic reticulum, plays a physiological role by releasing Ca2+ in response to norepinephrine or caffeine and by buffering excessive Ca2+. The 45Ca2+ uptake by mitochondria appears too insensitive to be important under physiological conditions.

Animals↗

PubMed1982

Cultivation of Bacteroides fragilis in tissue culture.

The capacity of animal cells to support the growth of obligate anaerobes was shown by inoculating Bacteroides fragilis and Bacteroides thetaiotaomicron into cultures of Vero, HeLa, Chinese hamster ovary, and Chinese hamster lung cells. From an extremely small inoculum (nearly 1 cfu), B. fragilis multiplied up to 10(8) cfu/ml during aerobic cultivation in L-15 medium containing kanamycin sulfate (50 micrograms/ml) and 5% fetal bovine serum (total depth, 3-5 mm). Infecting bacteria formed a cluster on the cell monolayer and permitted the concomitant growth of infected Vero cells. Treatment of Vero cells with an inhibitor of respiration (sodium azide) arrested the bacterial growth reversibly. In contrast, an uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, enhanced the growth of B.fragilis. By use of the thin oxygen electrode, low oxygen pressure was detected in the vicinity of the cultured Vero cells. Thus, respirating animal cells seem to provide a microenvironment with low enough oxygen pressure for the growth of some anaerobes.

2,4-Dinitrophenol↗

PubMed1990

Regulation of activity of an ATP-dependent protease, Clp, by the amount of a subunit, ClpA, in the growth of Escherichia coli cells.

The activity of an ATP-dependent protease, Clp, was examined in Escherichia coli SG1110 (lon-) in various growth phases. The ATP-dependent proteolytic activity (Clp activity) in a crude extract of the cells changed with the growth phase. Cells in the early exponential growth phase showed the lowest activity, but then the activity increased dramatically with cell growth. The highest Clp activity was found in the cells in the late exponential and early stationary phases, however, the activity returned to the original level on prolonged culturing. These changes in Clp activity were closely correlated to the amount of one of the components of Clp, Clp A, which was quantitated immunochemically with antibodies against the Clp A protein. However, the amount of the other component of Clp, Clp P, did not change with the growth phase. These results suggest that the activity of Clp in the cells is regulated by the amount of Clp A in various growth phases. We next examined the effect of the cellular ATP level on Clp activity, because ATP is a cofactor for Clp protease in vitro. The addition of dinitrophenol (DNP) and sodium azide reduced the intracellular concentration of ATP, but had no effect on the Clp activity or the level of the Clp A protein when these drugs were added to the culture at the stationary phase. On the other hand, these drugs elevated both the Clp activity and the Clp A amount in exponentially growing cells, whose cellular ATP level was also reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP-Dependent Proteases↗

PubMed1989

Adherence of multiple serovars of Chlamydia trachomatis to a common receptor on HeLa and McCoy cells is mediated by thermolabile protein(s).

Several aspects of the adherence of purified elementary bodies (EB) of Chlamydia trachomatis to HeLa and to McCoy cells were examined using different techniques, including an ELISA. Serovar-specific, biotinylated monoclonal antibodies were used to detect cell-bound chlamydiae. In addition, purified chlamydiae were biotinylated and their adherence properties were studied. The assays were done at 4 degrees C to exclude the energy-dependent internalization of the cell-bound EB and host-cell membrane recycling that occur at 37 degrees C. Saturation kinetics were routinely observed at 4 degrees C, and the rate of adherence remained linear for approximately 60 min. Lineweaver-Burk analysis of the kinetics data showed that adherence of any one serovar was competitively inhibited by other serovars of C. trachomatis. This competition for the same receptor on the two alternative hosts, HeLa and McCoy, was also seen when the adherence assays were done at 37 degrees C in the presence of sodium azide, an energy poison that inhibits endocytosis of cell-bound chlamydiae. Chlamydiae exposed to 56 degrees C for 5 min, or treated with low doses of trypsin, failed to exhibit competitive inhibition, having suffered considerable loss of the ability to adhere to host-cells. These data suggest that heat- and trypsin-labile chlamydial moieties participate in the adherence reaction, and that oculo-genital serovars of C. trachomatis, including that of lymphogranuloma venereum, attach to the same receptor on the host-cell membrane.

Azides↗