Streptococcus pyogenes CAMP factor promotes calcium ion uptake in RAW264.7 cells.

Streptococcus pyogenes is a bacterium that causes systemic diseases such as pharyngitis and toxic shock syndrome. S. pyogenes produces molecules that inhibit the function of the human immune system, thus allowing growth and spread of the pathogen in tissues. It is known that S. pyogenes CAMP factor induces vacuolation in macrophages; however, the mechanism remains unclear. In the current study, the mechanism by which CAMP factor induces vacuolation in macrophages was investigated. CAMP factor was found to induce calcium ion uptake in murine macrophage RAW264.7 cells. In addition, EDTA inhibited calcium ion uptake and vacuolation in the cells. The L-type voltage-dependent calcium ion channel blockers nifedipine and verapamil reduced vacuolation. Furthermore, the phosphoinositide 3-kinase inhibitors LY294002 and wortmannin also inhibited the vacuolation induced by CAMP factor. Fluorescent microscopy revealed that clathrin localized to the vacuoles. These results suggest that the vacuolation is related to calcium ion uptake by RAW264.7 cells via L-type voltage-dependent calcium ion channels. Therefore, it was concluded that the vacuoles induced by S. pyogenes CAMP factor in macrophages are clathrin-dependent endosomes induced by activation of the phosphoinositide 3-kinase signaling pathway through calcium ion uptake.

Purification and partial characterization of oxalate oxidase from leaves of forage Sorghum (Sorghum vulgare var. KH-105) seedlings.

An oxalate oxidase was purified to apparent homogeneity from the leaves of 10-days old seedlings of forage Sorghum (Sorghum vulgare var. KH-105). The enzyme had a Mr of 124 kDa with two identical subunits, an optimum pH of 4.5, optimum temperature of 37 degrees C and activation energy (Ea) of 2.0338 Kcal/mol. The rate of reaction was linear up to 7 min. K(m) value for oxalate was 0.22 mM. The enzyme was stimulated by Cu2+ and inhibited by EDTA, NaCN, diethyldithiocarbamate, Na2SO4, but unaffected by NaCl at 0.1 mM concentration. Although the enzyme was stimulated by flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), UV and visible spectra of the enzyme did not match with that of a flavoprotein. The positive reaction of the enzyme with orcinol-H2SO4 reagent indicated its glycoprotein nature. The superiority of the purified enzyme over earlier reported oxalate oxidases for determination of urinary oxalate has been demonstrated.

Enhanced Production of Fatty Acids via Redirection of Carbon Flux in Marine Microalga Tetraselmis sp.

Lipids in microalgae are energy-rich compounds and considered as an attractive feedstock for biodiesel production. To redirect carbon flux from competing pathways to the fatty acid synthesis pathway of Tetraselmis sp., we used three types of chemical inhibitors that can block the starch synthesis pathway or photorespiration, under nitrogen-sufficient and nitrogen-deficient conditions. The starch synthesis pathway in chloroplasts and the cytosol can be inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 1,2-cyclohexane diamine tetraacetic acid (CDTA), respectively. Degradation of glycine into ammonia during photorespiration was blocked by aminooxyacetate (AOA) to maintain biomass concentration. Inhibition of starch synthesis pathways in the cytosol by CDTA increased fatty acid productivity by 27% under nitrogen deficiency, whereas the blocking of photorespiration in mitochondria by AOA was increased by 35% under nitrogen-sufficient conditions. The results of this study indicate that blocking starch or photorespiration pathways may redirect the carbon flux to fatty acid synthesis.

Electrostatic control of chloroplast coupling factor binding to thylakoid membranes as indicated by cation effects of electron transport and reconstitution of photophosphorylation.

1. Increase in electron transport rate and the decay rate of the 518 nm absorption change, induced by EDTA treatment, is prevented by cations. The order of effectiveness is C3+ > C2+ > C+. 2. In this respect methyl viologen is an effective divalent cation in addition to its action as an electron acceptor. 3. Complete cation irreversible EDTA-induced uncoupling occurs in the dark in 2 min. Light greatly stimulates the rate of uncoupling by EDTA. It is concluded that the uncoupling is due to release of coupling factor I from the thylakoid membrane. 4. Binding of purified coupling factor I to coupling factor I-depleted thylakoids can be achieved with any cation. The order of effectiveness is C3+ > C2+ > C+, reconstituted thylakoids are active in photophosphorylation regardless of the cation used for coupling factor I binding. 5. The marked difference in the concentration requirements for cation effects on 9-aminoacridine fluorescence yield and for prevention of uncoupling by EDTA indicate that coupling factor I and its binding site have a lower surface charge density than the net surface charge density of the thylakoid membrane. 6. It is concluded that coupling factor I binding only occurs when negative charges on coupling factor I and its binding site are electrostatically screened by cations. 7. Previously reported examples of uncoupling by low ionic conditions are discussed in relation to the basic concepts of diffuse electrical layer theory.

Pyridoxal isonicotinoyl hydrazone inhibits iron-induced ascorbate oxidation and ascorbyl radical formation.

Previous work from our laboratory demonstrated that pyridoxal isonicotinoyl hydrazone (PIH) has in vitro antioxidant activity against iron plus ascorbate-induced 2-deoxyribose degradation due to its ability to chelate iron; the resulting Fe(III)-PIH(2) complex is supposedly unable to catalyze oxyradical formation. A putative step in the antioxidant action of PIH is the inhibition of Fe(III)-mediated ascorbate oxidation, which yields the Fenton reagent Fe(II) [Biochim. Biophys. Acta 1523 (2000) 154]. In this work, we demonstrate that PIH inhibits Fe(III)-EDTA-mediated ascorbate oxidation (measured at 265 nm) and the formation of ascorbyl radical (in electron paramagnetic resonance (EPR) studies). The efficiency of PIH against ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation was dose dependent and directly proportional to the period of preincubation of PIH with Fe(III)-EDTA. The efficiency of PIH in inhibiting ascorbate oxidation and ascorbyl radical formation was also inversely proportional to the Fe(III)-EDTA concentration in the media. When EDTA was replaced by the weaker iron ligand nitrilotriacetic acid (NTA), PIH was much more effective in preventing ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation. Moreover, the replacement of EDTA with citrate, a physiological chelator with a low affinity for iron, also resulted in PIH having a higher efficiency in inhibiting iron-mediated ascorbate oxidation and 2-deoxyribose degradation. These results demonstrate that PIH removes iron from EDTA (or from either NTA or citrate), forming an iron-PIH complex that cannot induce ascorbate oxidation effectively, thus inhibiting iron-mediated oxyradical formation. These results are of pharmacological relevance because PIH has been considered for experimental chelating therapy in iron-overload diseases.

Effect of EDTA and Fe-EDTA complex concentration on TCF Kraft mill effluent degradability. Batch and continuous treatments.

The effect of ethylenediaminetetracetic acid (EDTA) and Fe-EDTA complex on synthetic totally chlorine-free (TCF) effluent degradability in batch and continuously operating reactors was evaluated. Under batch treatment, the addition of EDTA and Fe-EDTA complex was studied in the range of 80 to 320 mg l(-1). Under continuously operated reactors, the Fe-EDTA complex concentration varied from 20 to 80 mg l(-1), and the hydraulic retention time (HRT) varied from 48 to 24 h. Sludge oxygen uptake rate (OUR) and chemical oxygen demand (COD) removal decreased when EDTA concentration increased in the influent under batch treatment; however, this inhibitory effect was reduced by the addition of Fe-EDTA complex. Without the addition of EDTA, COD removal decreased from 71% to 8%. The most efficient EDTA removal treatment (almost 10%) was the treatment of 80 mg l(-1) Fe-EDTA. Under continuously operated reactors, COD removal was greater than 57% in the synthetic TCF effluent with a Fe-EDTA concentration that varied from 20 to 80 mg l(-1); however, EDTA removal was lower than 25% in all cases. Synthetic TCF effluent with a Fe -EDTA concentration higher than 80 mg l(-1) could not be treated by the activated sludge treatment due to EDTA's inhibitory effect on the sludge.

Experimental study on the inhibition of biological reduction of Fe(III)EDTA in NO(x) absorption solution.

Scrubbing of NO(x) from the gas phase with Fe(II)EDTA has been shown to be highly effective. A new biological method can be used to convert NO to N(2) and regenerate the chelating agent Fe(II)EDTA for continuous NO absorption. The core of this biological regeneration is how to effectively simultaneous reduce Fe(III)EDTA and Fe(II)EDTA-NO, two mainly products in the ferrous chelate absorption solution. The biological reduction rate of Fe(III)EDTA plays a main role for the NO(x) removal efficiency. In this paper, a bacterial strain identified as Klebsiella Trevisan sp. was used to demonstrate an inhibition of Fe(III)EDTA reduction in the presence of Fe(II)EDTA-NO. The competitive inhibition experiments indicted that Fe(II)EDTA-NO inhibited not only the growth rate of the iron-reduction bacterial strain but also the Fe(III)EDTA reduction rate. Cell growth rate and Fe(III)EDTA reduction rate decreased with increasing Fe(II)EDTA-NO concentration in the solution.

Tryptophan hydroxylase from mouse mastocytoma P-815. Reversible activation by ethylenediaminetetraacetate.

Tryptophan hydroxylase from mouse mastocytoma P-815 is activated by ethylenediaminetetraacetate (EDTA). The activation proceeds in a pH-, temperature-, and time-dependent manner and leads to a 30-fold higher activity at maximum. The optimal EDTA concentration is 10 microns. The activation requires solely EDTA with desalted crude enzyme solution in the absence of any cellular metabolites. There are no indications that the activation is due to proteolysis, modification of protein-bound sulfhydryl groups or other covalent modifications such as phosphorylation and methylation. In the presence of appropriate stabilizing agents, the activated state is maintained after the removal of EDTA by gel-filtration. The activation is reversible under conditions in which bound metal(s) is dissociated from the complex with EDTA. These results imply that the role of EDTA is metal chelation. A model is proposed in which the enzyme has at least two interconvertible states, the activated state and ground state, corresponding to the free and metal-bound forms, respectively. The metal is probably derived from the cell. The assay method for tryptophan hydroxylase utilized a rapid and sensitive (5 pmol/injection) determination of 5-hydroxytryptophan by high performance liquid chromatography.

Reactivation of the EDTA-treated arginase from rat and calf liver.

1. The anionic calf liver arginase, like the cationic rat liver enzyme, is inactivated by EDTA-treatment. The activity is fully restored by Mn2+. A smaller effect is observed with Cd2+, Ni2+ and Co2+. 2. The EDTA-inactivated calf liver arginase, unlike the rat liver enzyme, does not dissociate into subunits, and its mol.wt. (120 000) is unchanged. 3. The reactivation of rat liver arginase subunits (mol.wt. 30 000) by Ni2+ is accompanied, similarly as in the case of Mn2+, by reassociation to the form of mol.wt. 120 000, i.e. the same as for the native enzyme. 4. It is suggested that Mn2+ in arginase is bound at the active site and at the site responsible for maintenance of the oligomeric structure. In calf liver enzyme this binding site is inaccessible to the chelating agent.

Characterization of hemorrhagic principles from Trimeresurus gramineus snake venom.

In addition to alpha-fibrinogenase (hemorrhagin I, HR1), a potent hemorrhagic principle (hemorrhagin II, HR2) was purified from Trimeresurus gramineus venom. It was homogeneous as judged by SDS-polyacrylamide gel electrophoresis. HR2 was a single peptide chain containing 10% carbohydrate with a molecular weight of 81,500. It possessed 669 amino acid residues per molecule, while HR1 contained only 203 amino acid residues per molecule with a molecular weight of 23,500. Both hemorrhagins possessed proteolytic activities toward fibrinogen, casein and azocoll. However, the proteolytic activities of HR1 were much more potent than those of HR2. They were devoid of TAME-esterase and phospholipase A2 activities which were found in crude venom. beta-Mercaptoethanol and antivenin completely inhibited the hemorrhagic activities of HR1 and HR2, while epsilon-aminocaproic acid, trasylol, p-bromophenacyl bromide, phenylmethanesulfonyl fluoride and soybean trypsin inhibitor did not. EDTA completely inhibited the hemorrhagic, fibrinogenolytic and caseinolytic activities of HR1. EDTA also completely inhibited the caseinolytic and fibrinogenolytic activities of HR2, but only partially inhibited its hemorrhagic activity. Subsequent addition of Zn2+ (5 mM) reversed the EDTA-induced inhibitory effect on the hemorrhagic activity of HR1. However, ZN2+ did not reverse the EDTA-induced inhibitory effect on the HR2-induced hemorrhagic activity. These hemorrhagins were found to be ZN2+-containing metalloproteinases. Therefore, the hemorrhagic activity of HR1 seems to be related to its proteolytic activity while that of HR2 seems to be unrelated to its proteolytic activity.

The effect of jararhagin, a metalloproteinase from Bothrops jararaca venom, on pro-inflammatory cytokines released by murine peritoneal adherent cells.

The release of pro-inflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) from murine peritoneal adherent cells (MPAC) was studied after exposure to jararhagin, a metalloproteinase/disintegrin isolated from Bothrops jararaca venom. MPACs were treated with LPS (lipopolysaccharide), jararhagin, or EDTA-inactivated jararhagin for up to 24h. Following incubation, the culture supernatant was assayed by ELISA for the presence of cytokines, while the cells were analysed for viability and cytokine mRNA expression. The cells exposed to native jararhagin released TNF-alpha and IL-1beta after 4 and 24h respectively. When MPACs were exposed to Jararhagin treated with EDTA, TNF-alpha and IL-1beta production was sustained throughout the culture period and IL-6 production was observed. TNF-alpha, IL-6 and IL-1beta mRNA were detected 4h after stimulation with either native or EDTA-treated jararhagin. Addition of jararhagin to LPS stimulated cells resulted in a dramatic decrease in the release of IL-6 and TNF-alpha. RT-PCR showed that this inhibition does not occur at the transcriptional level and further experiments showed that jararhagin degraded soluble cytokines by proteolytic activity. This study suggests that jararhagin induces TNF-alpha, IL-1beta and IL-6 expression, which may be rapidly degraded by its proteolytic activity.

Nonchelational cell growth inhibition by EDTA.

The ability of the dinitrilotetraacetates EDTA, CDTA, and EGTA to inhibit the growth of rat C6 glioma cells was not proportional to their chelational stability constants, suggesting a nonchelational mechanism of action. Ion antagonization studies supported this hypothesis. Growth inhibition did not appear to be caused by an EDTA-ion coordination complex. The chemical structure of the DNTAs suggests that they might act by: increasing zeta potential electronegativity and altering local physical properties; macromolecular crosslinking; and disrupting hydrogen bonds and hydrophobic interactions. Interestingly, their ability to inhibit growth paralleled their hydrophobic surface areas.

Matrix metalloproteinase secretion is stimulated by TGF-beta in cultured lens epithelial cells.

PURPOSE: To determine if TGF-beta regulates the expression of metalloproteinases in chick lens annular pad cells. METHODS: The activity of secreted matrix metalloproteinases was examined with gelatin zymography in primary cultures exposed to TGF-beta. RESULTS: Metalloproteinases with electrophoretic mobilities corresponding to MMP2 and MMP9 were tentatively identified. Activated, processed forms of the two metalloproteinases were also observed. Plasminogen activators potentially capable of initiating metalloproteinase cascades were concomitantly elicited. Metalloproteinase secretion was shown to be specific for TGF-beta stimulation and independent of substrate composition. CONCLUSIONS: These results indicate that TGF-beta-mediated processes could be responsible for localized lens capsular heterogeneity, establishing a substrate suitable for cell migration or the release of matrix-bound factors which influence the terminal differentiation of lens cells.

[Ca++-dependent immunosuppressive effect of ethylenediaminetetraacetic acid (EDTA)]. / Ca++-zavisimyi immunodepressivnyi (effekt) etilendiamintetrauksusnoi kisloty (EDTA).

Subcutaneous injection of a sum total dose of 6.44.10(-5) M of ethylenediaminetetraacetic acid (EDTA), which binds divalent cations, inhibits the process of accumulation of antibody-forming cells in the spleen of mice (CBA X C57BL) F1 hybrids, immunizated with sheep red blood cells. In a dose of 5.2.10(-5) M CaCl2 eliminated the immunodepressive effect of EDTA almost completely. It was assumed that EDTA action was produced through the Ca++-controlled regulation mechanism of one of the following processes: the interaction between T- and B-lymphocytes; proliferation and differentiation of T- and B-cells in the course of the immune response to sheep red blood cells. A more complex mechanism of EDTA action on the above-mentioned processes is not excluded, however.

DNA interaction studies of ethylenediaminetetraaceticacid food additive and selenium effect in DNA cleavage-inhibition.

The interaction of native calf thymus DNA with ethylenediaminetetraaceticacid (EDTA) in Tris-HCl buffer at pH = 7.8 (at this pH EDTA forms a disodium salt) was investigated. EDTA is widely used in medicine, food technology, and the chemical industry. The DNA binding mode of EDTA was monitored by absorption spectrophotometry, circular dichrosim, viscometry, and gel electrophoresis. Ultraviolet spectra of DNA showed small hyperchromicity with increase in EDTA concentration. The circular dichrosim signals at 245 and 280 nm indicated structural changes in DNA structure, and no significant effect on DNA viscosity was observed in the presence of increasing amounts of EDTA. Results are indicative of an outside, nonintercalative binding mode of EDTA to DNA. Moreover, gel electrophoresis studies showed considerable oxidative cleavage of plasmid DNA by EDTA. Mechanistic aspects of the chemical nuclease reactions were investigated using the OH radical quencher dimethylsulfoxide. In the presence of selenium, DNA cleavage by EDTA was inhibited.