Efficacy of natamycin for control of growth and ochratoxin A production by Aspergillus carbonarius strains under different environmental conditions

AIMS: To examine the efficacy of natamycin produced by Streptomyces natalensis against strains of Aspergillus carbonarius growth and ochratoxin A (OTA) production under different environmental factors on a grape juice-based medium. METHODS AND RESULTS: Detailed studies in the range 0-20 ng ml(-1) for control of growth and ochratoxin production by strains of A. carbonarius at 0.98, 0.96 and 0.94 water availabilities (a(w)) and 15-25 degrees C on a fresh red grape extract medium were examined. Inhibition of growth was depending on temperature and a(w) level. At 15 degrees C, 5-10 ng ml(-1) natamycin was effective in reducing growth almost completely. However, at 20-25 degrees C and all the three a(w) levels, growth was only slightly inhibited by 5-10 ng ml(-1) natamycin. There were strain differences with regard to inhibition of OTA production. At 15 degrees C and 0.98 a(w), 10 ng ml(-1) was required to inhibit production by >90%. However, at 0.96 and 0.94 a(w), almost complete inhibition occurred. At 20 degrees C, OTA production was only significantly inhibited by 10 ng ml(-1) natamycin at 0.94 a(w). At 0.96 and 0.98 a(w), some inhibition occurred with 5-10 ng ml(-1), but greater concentrations would be required for effective inhibition. At 25 degrees C, 5 ng ml(-1) was effective at all a(w) levels. However, at 15 degrees C and 25 degrees C and a wide range of a(w) levels, natamycin effectively controlled OTA production. CONCLUSIONS: Natamycin appears to be a very effective for controlling growth and OTA production by strains of A. carbonarius over a range of a(w) and temperature conditions on grape-based media. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first detailed study to demonstrate the impact of natamycin against A. carbonarius. This study suggests that use of natamycin at 50-100 ng ml(-1) can give complete inhibition of growth of A. carbonarius and OTA production over a range of environmental conditions. Natamycin could be an important component of a system to prevent OTA contamination of wine as well during the drying and production of vine fruits.     

Selective inhibition of uracil tRNA methylases of E. coli by ethionine.

L-ethionine has been found to inhibit uracil tRNA methylating enzymes in vitro under conditions where methylation of other tRNA bases is unaffected. No selective inhibitor for uracil tRNA methylases has been identified previously. 15 mM L-ethionine or 30 mM D,L-ethionine caused about 40% inhibition of tRNA methylation catalyzed by enzyme extracts from E. coli B or E. coli M3S (mixtures of methylases for uracil, guanine, cytosine, and adenine) but did not inhibit the activity of preparations from an E. coli mutant that lacks uracil tRNA methylase. Analysis of the 14CH3 bases in methyl-deficient E. coli tRNA after its in vitro methylation with E. coli B3 enzymes in the presence or absence of ethionine showed that ethionine inhibited 14CH3 transfer to uracil in tRNA, but did not diminish significantly the 14CH3 transfer to other tRNA bases. Under similar conditions 0.6 mM S-adenosylethionine and 0.2 mM ethylthioadenosine inhibited the overall tRNA base methylating activity of E. coli B preparations about 50% but neither of these ethionine metabolites preferentially inhibited uracil methylation. Ethionine was not competitive with S-adenosyl methionine. Uracil methylation was not inhibited by alanine, valine, or ethionine sulfoxide. It is suggested that the thymine deficiency that we found earlier in tRNA from ethionine-treated E. coli B cells, resulted from base specific inhibition by the amino acid, ethionine, of uracil tRNA methylation in vivo.     

Characterization of glucocorticoid inhibition of antigen-induced inositolphosphate formation by rat basophilic leukemia cells: possible involvement of phosphatases.

The suppressive effect of glucocorticoids (GC) upon antigen-induced phosphatidylinositol phospholipase C (PI-PLC) activity and inositol phosphate formation by rat basophilic leukemia cells (RBL-2H3) has been characterized. Addition of antigen for a period of 1-30 min enhanced production of [3H]inositol monophosphate (IP1), inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3) by about 5-10-fold. Pretreatment with hydrocortisone (HC) reduced formation of the various inositol phosphates (IPs) and degradation of phosphatidylinositol 4,5-bisphosphate (PIP2) by an average of 50%. Maximal inhibition of hydrolysis of PIP2 and reduction in stimulation of IP3 formation was reached after 4 h of preincubation with 2.10(-6) M of HC. Cycloheximide and RU486, a GC receptor antagonist, completely prevented the inhibitory effect of HC on IP formation. Other GC, dexamethasone (DEX) and triamcinolone (each at 2.10(-7) M) markedly suppressed antigen induced IP3 production, while aldosterone and sex steroids such as estradiol and progesterone (each at 2.10(-6) M) were virtually inactive. Antigen-stimulated phosphorylation of a 18 kDa and other proteins was inhibited by about 60% following pretreatment with the GC. This inhibition was in turn prevented by cycloheximide. DEX also doubled the activity of cellular acid phosphatase activity. The results suggest that the inhibitory effect of GC is specific, receptor-mediated, dependent on protein synthesis and possibly mediated by protein phosphatase activity.     

Specific inhibition by prostaglandin D2 and its metabolites of lysozyme synthesis in mouse macrophage-like cell line, Mm-1

The cultured mouse macrophage-like cell line Mm-1 synthesizes and secretes lysozyme continuously like normal macrophages. Culture of the cells in the presence of prostaglandin D2 for 3 days strongly inhibited their production of lysozyme activity. Prostaglandin D2 caused dose-dependent inhibition of the activity: 1 X 10(-6) M prostaglandin D2 caused about 50% inhibition. Inhibition by prostaglandin D2 was not related to cytotoxicity and was reversible. The rate of synthesis of lysozyme protein was measured by culturing Mm-1 cells with radioactive amino acids and then immunoprecipitating the protein. At the concentrations used, prostaglandin D2 inhibited the synthesis of lysozyme dose-dependently, but did not suppress the synthesis of total protein. Of the various types of prostaglandin, only prostaglandin D2 inhibited the production of lysozyme in Mm-1 cells. Moreover, prostaglandin D2 did not inhibit the production of other lysosomal enzymes, such as acid proteinase, acid phosphatase and beta-glucuronidase, and did not affect Fc receptors on the cell surface, adherence of cells to the culture dish or the cell morphology. These results indicate that prostaglandin D2 specifically inhibits the synthesis of lysozyme in Mm-1 cells. When Mm-1 cells were cultured for 3 days in the presence of the ethyl acetate extract from the culture medium in which Mm-1 cells had been cultured with prostaglandin D2 for 3 days, the production of lysozyme activity of Mm-1 cells was also markedly inhibited by the extract. After the incubation of prostaglandin D2 for 3 days with Mm-1 cells, less than 10% of the initial prostaglandin D2 remained and two major metabolites appeared. These results suggest that the metabolites of prostaglandin D2 were involved in the inhibitory action of prostaglandin D2 in Mm-1 cells.     

Pitfalls in the use of lead nitrate for the histochemical demonstration of adenylate cyclase activity

The biochemistry of the lead histochemical technique for demonstrating adenylate cyclase was studied. The enzyme activity of fat cell plasma membranes, using 5'-adenylyl-imidodiphosphate (AMP-PNP) as substrate, was completely inhibited at 1 times 10- minus 4 M Pb(NO3)2 and yet at 4 times 10- minus 3 M Pb(NO3)2 precipitate could be demonstrated by electron microscopy on both sides of plasma membrane vesicles. No lead-diphosphoimide or lead-phosphate precipitate could be visualized by electron microscopy when the lead was reduced to a level (2 times 10- minus 5 M) which caused only 50% inhibition of the enzyme. A solubility product coefficient of 1 times 10- minus 10 M was found necessary to allow precipitation of lead-phosphate complex in the adenylate cyclase medium. Varying the ratio of substrate or dextran relative to the lead failed to protect the inhibition of the enzyme. Increasing concentrations of beta-mercaptoethanol restored the basal and stimulated activity of adenylate cyclase but also prevented the precipitation reaction. Lead at 2 times 10- minus 3 M caused the nonenzymatic hydrolysis of AMP-PNP, resulting in the production of small but significant quantities of cyclic AMP and substantial amounts of AMP. This hydrolysis was inhibited by alloxan but unaffected by dextran of NaF. The adenylate cyclase activity of pancreatic islet homogenates and of fat pad capillaries was completely inhibited by lead concentrations equal to or less than those used in histochemical studies (Howell, S. L., and M. Whitfield. 1972. J. Histochem. Cytochem. 20:873-879. and Wagner, R. C., P. Kreiner, R. J. Barrnett, and M. W. Bitensky. 1972. Proc. Natl. Acad. Sci. U.S.A. 69:3175-3179.). The present study shows that the lead histochemical method cannot be used for localization of adenylate cyclase because of the inhibition of the enzyme and artifacts produced by high lead concentrations and the inability to produce a visible precipitate at low lead concentrations which only partially inhibit the enzyme.     

Embryonic palatal responses to teratogens in serum-free organ culture.

This study examines development of rat, mouse, and human embryonic palates in submerged, serum-free organ culture. The concentration-response profiles for retinoic acid (RA), triamcinolone (TRI), hydrocortisone (HC), dexamethasone (DEX), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were examined and the mechanisms of clefting in vitro were compared to observed in vivo responses. Craniofacial regions were dissected on gestational day (GD) 12 for mice and GD 14 for rat, and cultured for 3-4 days in Bigger's BGJb medium in flasks flushed with 50% O2, 45% N2, 5% CO2. Growth and fusion of secondary palates were scored under a dissecting microscope. In serum-free control medium, mouse and rat palatal fusion occurred within the 4-day culture period. Supplementing with fetal bovine serum (FBS) in excess of 1% interfered with growth and fusion in control medium. RA significantly inhibited fusion of mouse and rat palates at 5 x 10(-9) and 1 x 10(-10) M, respectively, with RA-induced clefting related to abnormal proliferation and differentiation of medial epithelia. In contrast, glucocorticoid-induced clefting was due to concentration-dependent inhibition of shelf growth. TRI significantly inhibited fusion at 4 x 10(-5) M, and 1 x 10(-4) M DEX or HC, inhibited fusion of 19 and 42% of shelves, respectively. The response rate for DEX in the presence of 1% FBS was increased (42% unfused). TCDD clefting was due to altered medial epithelial differentiation and 1 x 10(-8) M TCDD affected 36% of CD-1 mouse, 23% of C57BL/6N mouse, and 47% of F344 rat palates. When the medium was supplemented with 1% FBS, selenium, transferrin, and additional glutamine, the response of C57BL/6N embryos increased to 75%. This rate is similar to that reported for Trowell's-type cultures with IMEM:F12 medium and 1% FBS. The increased responsiveness to DEX or TCDD in the presence of serum suggests that an unknown factor in serum may be required for full activity. Three human embryonic palatal explants (GD 52 or 53) were cultured for 3-6 days and fused during culture. The present study demonstrates that serum-free organ culture supports development of mouse, rat, and human palatal explants. The present study demonstrates the capacity of this organ culture system to model palatogenesis for several species, and to distinguish between various mechanisms of clefting as presented through selected model compounds. This model should be useful for exploring mechanisms of activity at a cellular and molecular level.     

The interactions of triethyltin with rat glutathione-S-transferases A, B and C. Enzyme-inhibition and equilibrium-dialysis studies.

Purified glutathione(GSH)-S-transferases A, B and C from rat liver are inhibited by triethyltin (SnEt3). With 1-chloro-2,4-dinitro benzene (CDNB) as the limiting substrate the inhibition is competitive in each case. At a GSH concentration of 5 . 10(-3) M the inhibition constants for transferases A and C at 25 degrees C are similar and very low, 3.2 . 10(-8) M and 5.6 . 10(-8) M respectively, whereas for transferase B the inhibition constant is 3.5 . 10(-5) M. Equilibrium-dialysis experiments carried out at 4 degrees C in the absence of GSH give apparent dissociation constants of 7.1 . 10(-4) M and 3.4 . 10(-4) M for transferases A and B respectively, but if 5 . 10(-3) M glutathione is included in the dialysis solutions these values fall to 2.0 . 10(-7) M and 2.6 . 10(-5) M, which are within an order of magnitude of the kinetic Ki-values. Chromatographic experiments with Sephadex G-10 show that GSH and SnEt3 interact in aqueous solution under the conditions of the enzyme-kinetic and equilibrium-dialysis experiments. It is suggested that the inhibited enzymes are in the form of ternary complexes, enzyme-GSH-SnEt3, in which GSH and SnEt3 may or may not interact directly; or are possibly quaternary complexes, enzyme-(GSH)2-SnEt3. SnEt3 could be valuable as a selective inhibitor of transferases A and C in mixtures of the three transferases.     

细胞DNA低甲基化是否为化学致癌启动过程的必经途径

There seems to be a current view that the inhibition of DNA methylation may be a mechanism of initiation of carcinogenesis or one of the steps required in the carcinogenic process. However, because of the deficiencies in research works on cellular level, there is a long way to make such a general relationship between carcinogen and the inhibition of cellular DNA methylation. In this paper the effects of some chemical carcinogens on methylation of newly replicated DNA in human FL cells was analyzed by comparing the weight average length (Lw) of the DNA digests after complete digestion by restriction endonuclease Hpa II. It was observed that two non-genotoxic carcinogens (5-azacytidine and L-ethionine) and two genotoxic carcinogens (MNNG and aflatoxin B1) used in this study all caused obvious cytotoxicity on FL cells at the test concentration. Five days after the termination of 5-azacytidine treatment (2 x 10(-6) M, 24 hours), Lw (kb) of the Hpa II digests of cellular DNA was smaller than that of control (8.0 +/- 0.1 vs 10.9 +/- 1.0, P less than 0.01), the Lw change rate was -27%. When DNA was analyzed from FL cells after 9 days continuous treatment of L-ethionine (2 x 10 M), the digestibility of Hpa II was also increased as compared with that of the control, the Lw values (kb) showed a decrease of about 8% (9.8 +/- 0.3 vs 10.6 +/- 0.3, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat liver cysteine dioxygenase (cysteine oxidase). Further purification, characterization, and analysis of the activation and inactivation

Rat liver cysteine dioxygenase has been purified to homogeneity. It is a single subunit protein having a molecular weight of 22,500 +/- 1,000, with a pI of 5.5. The enzyme purified was catalytically inactive and activated by anaerobic incubation with either L-cysteine or its analogues such as carboxymethyl-L-cysteine, carboxyethyl-L-cysteine, S-methyl-L-cysteine, D-cysteine, cysteamine, N-acetyl-L-cysteine, and DL-homocysteine. The enzyme thus activated with L-cysteine was rapidly inactivated under aerobic condition. This rapid inactivation was observed at 0 degrees C where no formation of either the reaction product cysteine sulfinate or the autoxidation product of cysteine, cystine, was detected. Further analysis shows that the inactivation of the activated enzyme was due to oxygen but unrelated to either the presence of substrate, enzyme turnover or accumulation of inhibitor produced during assay. A distinct rat liver cytoplasmic protein, called protein-A, could completely prevented the enzyme from the aerobic inactivation. The loss of activity during assay in the absence of protein-A was shown to be a first order decay process. From the plots of log(deltaproduct/min) versus time, the initial velocity (VO) and the velocity at 7 min (V7) were obtained. The apparent Km value for L-cysteine in the absence of protein-A was calculated from the initial velocity as 4.5 X 10(-4)M. Protein-A did not alter the apparent Km value for L-cysteine. The chelating agents such as o-phenanthroline, alpha,alpha'-dipyridyl, bathophenanthroline, 8-hydroxyquinoline, EGTA, and EDTA strongly inhibited the enzyme activity when these chelating agents were added before preactivation. The purified cystein dioxygenase contains 1 atom of iron per mol of enzyme protein. By the activation procedure, the enzyme became less susceptible to the heat denaturation, the inhibitory effects of chelating agents and the tryptic digestion.     

Mouse fetal kidneys in serum-free organ culture: effects of epidermal growth factor and hydrocortisone.

1. The present study was undertaken to determine whether epidermal growth factor (EGF, 100 ng/ml) or hydrocortisone (HC, 10(-8)-10(-5) M) directly influence proliferation and differentiation of mouse fetal kidney maturing in serum-free organ culture. 2. Addition of EGF to the medium significantly stimulated DNA synthesis after 2 and 5 days of culture. Labelled nuclei were mainly localized in the mesenchymal tissue. Protein synthesis remained unchanged. Activities of three hydrolases, markers of brush border differentiation, were reduced. 3. Hydrocortisone (HC), at all concentrations used, significantly inhibited DNA synthesis. Labelled nuclei were distributed in various cell populations of both control and treated explants. Protein synthesis was stimulated by 10(-7) M after 5 days of culture. Hydrolase activities were slightly modified by HC treatment. 4. The present results indicate that EGF stimulates whereas HC decreases proliferation. Both factors have regulatory effects on brush border maturation. 5. Thus, this culture model is a valuable tool for the study of nephrogenesis.     

Indomethacin inhibition of glutathione S-transferases

Indomethacin inhibited rat liver glutathione S-transferases (EC 2.5.1.18). Its inhibition was non-competitive with respect to 3,4-dichloronitrobenzene with an apparent Ki of 5.3 X 10(-5) M and uncompetitive with respect to glutathione with an apparent Ki of 4.0 X 10(-5) M. 4-Chlorobenzoic acid and 5-methoxy-2-methylindole-3-acetic acid, two metabolites of indomethacin, were weak inhibitors of the enzymes. On the other hand, meclofenamic acid was a competitive inhibitor of the enzymes with an apparent Ki of 3.0 X 10(-4) M. Possible significance of these findings in arachidonic acid metabolism is discussed.     

Kinetics of ochratoxin A production in different Aspergillus species

Kinetics of ochratoxin A production was examined in a number of ochratoxin producing isolates representing different sections of the Aspergillus genus. Both weak and high ochratoxin producers were tested using immunochemical or high-performance liquid chromatograhic methods. All isolates were found to produce the highest amounts of ochratoxin A after 7-10 days of incubation. Ochratoxin production varied between 30 - 5 x l0(5) ng ml(-1) among the Aspergillus isolates tested. The A. albertensis and A. melleus isolates examined were found to produce ochratoxin A constitutively. A. albertensis produced the highest amounts of ochratoxin A at 30 degrees C after 7 days' incubation in YES liquid medium. Ergosterol content and ochratoxin production of A. albertensis cultures were in good correlation.     

Effects of several unusual sulfur-containing amino acids on rat liver cystathionine-gamma-lyase.

1. The mode of inhibition of rat liver cystathionine-gamma-lyase [L-cystathionine cysteine-lyase (deaminating), EC 4.4.1.1] was studied by using several unusual sulfur-containing amino acids newly found in this laboratory. Some cysteine conjugates (CMC, Beta-CEC, HCETC and HCPC) inhibited noncompetitively both homoserine dehydratase and diaminopropionate ammonia-lyase activities, and competitively gamma-cystathionase activity. CMTC exhibited a mixed type inhibition on both homoserine dehydratase and gamma-cystathionase activities, and a noncompetitive inhibition on the diaminopropionate ammonia-lyase activity. Some homocysteine conjugates (CMHC, beta-CEHC and HCEHC) inhibited competitively both the activity of homoserine dehydratase and of gamma-cystathionase, and exhibited a mixed type inhibition on the diaminopropionate ammonia-lyase activity. beta-CEC, CMHC and beta-CEHC were also effective inhibitors to cysteine desulfhydrase activity. 2. Among the other amino acids tested, DL-homocysteine and D-cysteine, irrespective of their concentration, exhibited a mixed type inhibition on the homoserine dehydratase activity. However, they promoted gamma-cystathionase activity at their lower concentrations and inhibited at their higher concentrations, more so than cystathionine. DL-alpha-Aminobutyric acid was a weak competitive inhibitor of the homoserine dehydratase, gamma-cystathionase and diaminopropionate ammonia-lyase activities. DL-alpha-Aminopimeric acid has the same chain length as beta-CEC, CMHC and CMTC, but it showed a very weak inhibitory effect compared with the latter sulfur-containing compounds. L-Methionine, DL-methionine sulfoxide, L-ethionine, L-cysteic acid, L-aspartic acid, L-asparagine, L-glutamic acid, L-glutamine, D-alanine, beta-alanine, L-ornithine and L-lysine had little or no effect on any activities of the enzyme preparation. These results were discussed in relation to the catalytic center of cystathionine-gamma-lyase.     

Prostaglandin inhibition of testosterone production induced by luteinizing hormone, dibutyryl cyclic AMP or 3-isobutyl-1-methyl xanthine in dispersed rat testicular interstitial cells.

Testicular interstitial cells were utilized to study the effects of prostaglandins (PG) on in vitro testosterone production and to examine the role of cyclic adenosine-3',5'-monophosphate (cAMP) in this process. Testosterone production was assessed after 3 hour incubations while cAMP accumulation was examined after a 0.5 hour incubation period. Testosterone and cAMP were measured by radioimmunoassay. None of the PGs tested (PGA, PGA2, PGB1, PGE1, PGE2, PGF1alpha PGF2alpha) altered basal testosterone production when present in incubates at concentrations of 1.3 X 10(-8) M to 1.3 X 10(-4). However, at concentrations of 1.3 X 10(-4) M all of these PGs were capable of decreasing Luteinizing Hormone (LH; 100ng)-induced testosterone production. The inhibition of LH-induced testosterone production by the B, E and F series PGs was less pronounced than that for the A series. PGA1 and PGA2 exhibited 80% and 95% inhibition, respectively, at 1.3 X 10(4) M. The inhibitory action of 4 X 10(5) M PGA1 or PGA2 was evident within 30 minutes. Preincubation of interstitial cells with indomethacin (10(-5) or 10(-6) M) for 30 minutes did not alter subsequent basal or LH (100ng)-induced testosterone production. Accumulation of cAMP was stimulated by LH (10 microgram) or by PGs (1.3 X 10(-4) M PGA1, PGA2, PGB1, PGE1 or PGF2alpha). The PG-induced cAMP accumulation thus occurred at concentrations where LH-stimulated testosterone production was inhibited. Furthermore, PGA1 and PGA2 (1.3 X 10(-4) M) inhibited testosterone production induced by either 3-isobutyl-1-methyl xanthine (MIX; 10(-4) M or 10(-3) M) or dibutyryl cAMP (dbcAMP; 10(-4) M or 10(-3) M). These results indicate that PGs can block testosterone production by a direct effect on testicular interstitial cells and suggest that PGs exert their inhibitory action distal to stimulation of cAMP formation. PGs do not appear to play a role in the mechanism of LH action.     

Leukotriene C(4) (LTC(4)) does not share a cellular efflux mechanism with cGMP: characterisation of cGMP transport by uptake to inside-out vesicles from human erythrocytes

The transport of cGMP out of cells is energy requiring and has characteristics compatible with an ATP-energised anion pump. In the present study a model with inside-out vesicles from human erythrocytes was employed for further characterisation of the cGMP transporter. The uptake of leukotriene C(4) (LTC(4)), a substrate for multidrug resistance protein (MRP), was concentration-dependently inhibited by the leukotriene antagonist MK571 (IC(50)=110+/-20 nM), but cGMP was unable to inhibit LTC(4) uptake. Oxidised glutathione (GSSG) and glutathione S-conjugates caused a concentration-dependent inhibition of [(3)H]cGMP uptake with IC(50) of 2200+/-700 microM for GSSG, 410+/-210 microM for S-(p-nitrobenzyl)glutathione and 37+/-16 microM for S-decylglutathione, respectively. Antioxidants such as reduced glutathione and dithiothreitol did not influence transport for concentrations up to 100 microM, but both inhibited cGMP uptake with approx. 25% at 1 mM. The cGMP pump was sensitive to temperature without activity below 20 degrees C. The transport of cGMP was dependent on pH with maximal activity between pH 8.0 and 8.5. Calcium caused a concentration-dependent inhibition with IC(50) of 43+/-12 microM. Magnesium gave a marked activation in the range between 1 and 20 mM with maximum effect at 10 mM. The other divalent cations, Mn(2+) and Co(2+), were unable to substitute Mg(2+), but caused some activation at 1 mM. EDTA and EGTA stimulated cGMP transport concentration-dependently with 50% and 100% above control at 100 microM, respectively. The present study shows that the cGMP pump has properties compatible with an organic anion transport ATPase, without affinity for the MRP substrate LTC(4). However, the blockade of the cGMP transporter by glutathione S-conjugates suggests it is one of several GS-X pumps.     

Hydrocortisone inhibits antigen-induced rise in intracellular free calcium concentration and abolishes leukotriene C4 production in leukemic basophils

Antigenic stimulation of rat basophilic leukemia cells (RBL-3H3) elevates intracellular free Ca2+ concentration ([Ca2+]i) and induces production of leukotriene C4 (LTC4). This model was used to examine the role of Ca2+ in LTC4 formation, and inhibition by hydrocortisone (HC). HC, at a physiological concentration (2 x 10(-7) M), selectively prevented the stimulatory effect of the antigen on LTC4 production whereas the response to calcium ionophore (A23187) remained unimpaired. The inhibition by HC was time-dependent: half maximal response was reached at 2 hour and maximal response at 3 hours. Addition of arachidonic acid (3 micrograms/ml) did not overcome the inhibitory action of HC. An elevated [Ca2+]i is known to be essential for the activation of both 5-lipoxygenase and phospholipase A2. The stimulatory effect of the antigen on LTC4 production was abolished when the cells were incubated in Ca2+-deficient medium. Likewise, calcium ionophore stimulation shows dependence on extracellular Ca2+. Half maximal stimulation by the antigen and calcium ionophore was observed at external Ca2+ concentration of 150 microM and 40 microM respectively. Treatment with HC largely prevented the antigen-induced rise in [Ca2+]i, measured by Quin 2. In addition, HC reduced by 70% the accumulation of 45Ca2+ induced by the antigen. Collectively, these results demonstrate for the first time that HC reduces antigen-induced elevation of [Ca2+]i, and this may be associated with the inhibitory action of HC on LTC4 formation. This property could be partly responsible for the antiallergic and antiinflammatory activities of HC.     

Regulation of cyclic AMP level and lactic acid production in Ehrlich ascites tumor cells.

1. Quercetin (3.3',4',5,7-pentahydroxy flavone) at the concentration of 10(-4) M, as well as 2-10(-2) M theophylline and 1.5 - 10(-4) M prostaglandin E2 caused maximal rise of cyclic AMP in Ehrlich ascites tumor cells. 2. No additional increase of cyclic AMP level in these cells was found when both quercetin (10(-4) M) and theophylline (2-10(-2) M) were present in the incubation medium, while combination of quercetin (10(-4) M) and prostaglandin E2 (1.5 - 10(-4) M) has a synergistic effect on the level of cyclic AMP. 3. Degradation of cyclic AMP by homogenate of Ehrlich ascites tumor cells was inhibited by both quercetin and theophylline. 4. Quercetin, and to a smaller but significant extent theophylline, inhibited the lactic acid production in Ehrlich ascites tumor cells while prostaglandin E2 did not change the glycolytic rate in these cells. No synergistic inhibitory effect on lactic acid production was found when combinations of quercetin and prostaglandin E2, quercetin and theophylline or prostaglandin E2 and theophylline were tested. 5. Treatment of Ehrlich ascites tumor cells with dextran sulfate abolished the inhibitory effect of quercetin on lactic acid production, while the effect of the bioflavonoid on cyclic AMP levels was not altered.     

Anti-inflammatory drugs, prostanoid and proteoglycan production by cultured bovine articular chondrocytes

The effect of various anti-inflammatory drugs on the production of prostaglandins E2 and F2 alpha, 6 keto PGF1 alpha and thromboxane B2 by bovine articular chondrocytes was measured by radioimmunoassay. While indomethacin and meclofenamic acid caused a dose-dependent inhibition of all prostanoids measured, the effects of hydrocortisone and colchicine varied with respect to different prostanoids. Hydrocortisone (10(-7)M - 10(-13)M) both in the presence and absence of added arachidonic acid, resulted in an inhibition of prostaglandins E2 and F2 alpha, and to a lesser extent, 6 keto PGF 1 alpha, but TxB2 production was only slightly inhibited by the drug in the absence of arachidonic acid and markedly increased in its presence. Colchicine (10(-7)M-10(-3)M) had the opposite effect, causing an inhibition of TxB2 and stimulating PGE2 and 6 keto PGF1 alpha production. These findings suggest that certain anti-inflammatory drugs may, in addition to their action on phospholipase A2 and cyclo-oxygenases, exert potent effects at the level of the different synthetases. In order to see whether these alterations in relative prostanoid levels affected proteoglycan metabolism, the effect of anti-inflammatory drugs on proteoglycan synthesis by cultured chondrocytes was tested using 35SO4 labeling methodology. The results showed that at the concentrations tested (10(-5)M to 10(-7)M), indomethacin, dexamethasone, hydrocortisone and colchicine inhibited 35SO4 incorporation into newly synthesized proteoglycan molecules both in the presence (10(-6)M) and absence of exogenous arachidonic acid. In the same concentration range chloroquine had no effect. These results do not support the hypothesis of direct prostanoid involvement in the modulation of proteoglycan synthesis in articular cartilage.     

Interaction of some Pd(II) complexes with Na+ / K+-ATPase: inhibition, kinetics, prevention and recovery

The aim of this work was to investigate the influence of [PdCl4]2-, [PdCl(dien)]+ and [PdCl(Me4dien)]+ complexes on Na+ / K+-ATPase activity. The dose-dependent inhibition curves were obtained in all cases. IC50 values determined by Hill analysis were 2.25 x 10(-5) M, 1.21 x 10(-4) M and 2.36 x 10(-4) M, respectively. Na+ / K+-ATPase exhibited typical Michelis-Menten kinetics in the presence of Pd(II) complexes. Kinetic parameters (Vmax, Km) derived using Eadie-Hofstee transformation indicated a noncompetitive type of Na+ / K+-ATPase inhibition. The inhibitor constants (Ki) were determined from Dixon plots. The order of complex affinity for binding with Na+ / K+-ATPase, deducted from Ki values, was [PdCl4]2- > [PdCl(dien)]+ > [PdCl(Me4dien)]+. The results indicated that the potency of Pd(II) complexes to inhibit Na+/ K +-ATPase activity depended strongly on ligands of the related compound. Furthermore, the ability of SH-donor ligands, L-cysteine and glutathione, to prevent and recover the Pd(II) complexes-induced inhibition of Na+ / K+-ATPase was examined. The addition of 1 mM L-cysteine or glutathione to the reaction mixture before exposure to Pd(II) complexes prevented the inhibition by increasing the IC50 values by one order of magnitude. Moreover, the inhibited enzymatic activity was recovered by addition of SH-donor ligands in a concentration-dependent manner.