Purification of real car wash wastewater with complex coagulation/flocculation methods using polyaluminum chloride, polyelectrolyte, clay mineral and cationic surfactant.

In the present study, real car wash wastewater was purified by different coagulation/flocculation methods. As coagulant, polyaluminum chloride ('BOPAC'), conventional iron(III) chloride, iron(III) sulfate, and aluminum(III) chloride were used, while as flocculant non-ionic and anionic polyelectrolytes were investigated. The effects of added clay mineral (Na-bentonite) and cationic surfactant (hexadecyltrimethyl ammonium bromide - 'HTABr') were also investigated. The use of BOPAC was significantly more effective than conventional coagulants. Extra addition of clay mineral was also beneficial in relation to both the sediment volume and sedimentation speed, while polyelectrolyte addition enhanced further the sedimentation. Moreover, the simultaneous addition of HTABr significantly enhanced the color removal efficiency due to the successful in-situ generation of organophilic bentonite. In summary, the application of 100 mg L-1 Na-bentonite with 20 mg L-1 Al3+ (from BOPAC) and 0.5 mg L-1 anionic polyelectrolyte resulted in the efficient reduction of the turbidity (4-6 NTU), the COD (158 mg L-1) and the extractable oil content (4 mg L-1) with efficiencies of 98%, 59%, and 85%, respectively. By applying organophilic bentonite in high concentration (500 mg L-1) with identical concentrations of BOPAC and anionic polyelectrolyte, significant color removal (5 times lower absorbance at λ = 400 nm) and 27% lower sediment volume were achieved.

Investigation of surface and filtration properties of TiO2 coated ultrafiltration polyacrylonitrile membranes.

In the present work, the surface and filtration properties of TiO2 coated polyacrylonitrile ultrafiltration membranes were investigated. The membranes were coated using the physical deposition method. The appropriate TiO2 coverage proved to be 0.3 mg/cm2, which formed a hydrophilic cake layer on the membrane surface. The cleanability without chemicals and the retention of the coated membranes was compared to the neat membrane after model oily wastewater filtration. The cleaning sustained of rinsing with distilled water and ultraviolet (UV) irradiation of the fouled membranes. The coated membranes have better antifouling properties; higher flux values during oily water filtration and by the mentioned cleaning process a significantly better flux recovery can be achieved. The amount of the catalyst and the irradiation time are limiting factors to the effectiveness of the cleaning process. The UV irradiation increases the wettability of the fouled membrane surface by degrading the oil layer. The coating, the continuous use, and the cleaning process do not significantly affect the membrane retention expressed in chemical oxygen demand.

Clearance of dying autophagic cells of different origin by professional and non-professional phagocytes.

MCF-7 cells undergo autophagic death upon tamoxifen treatment. Plated on non-adhesive substratum these cells died by anoikis while inducing autophagy as revealed by monodansylcadaverine staining, elevated light-chain-3 expression and electron microscopy. Both de novo and anoikis-derived autophagic dying cells were engulfed by human macrophages and MCF-7 cells. Inhibition of autophagy by 3-methyladenine abolished engulfment of cells dying through de novo autophagy, but not those dying through anoikis. Blocking exposure of phosphatidylserine (PS) on both dying cell types inhibited phagocytosis by MCF-7 but not by macrophages. Gene expression profiling showed that though both types of phagocytes expressed full repertoire of the PS recognition and signaling pathway, macrophages could evolve during engulfment of de novo autophagic cells the potential of calreticulin-mediated processes as well. Our data suggest that cells dying through autophagy and those committing anoikis with autophagy may engage in overlapping but distinct sets of clearance mechanisms in professional and non-professional phagocytes.

Photodynamic therapy combined with a cysteine proteinase inhibitor synergistically decrease VEGF production and promote tumour necrosis in a rat mammary carcinoma.

OBJECTIVES: Photodynamic therapy (PDT) and inhibition of cathepsin B proteases by cystatin (cysteine proteinase inhibitor, CPI) are potential new tumour treatment modalities. We have investigated the efficacy of PDT and CPI alone and in combination on a solid mammary carcinoma transplanted into Wistar rats. MATERIALS AND METHODS: Intraperitoneally injected single doses of chlorine e6 or HpD as photosensitizers were excited at 630 nm (90 J/cm(2)). CPI (500 micro g per animal) was injected around the tumour daily during the 8-day treatment. Inoculation of tumour was either on day 1 of the protocol, or 8 days before. On day 8, tumour size was measured, tumour necrosis and vascularization were determined based on haematoxylin and eosin (H&E)-stained sections and serum vascular endothelial growth factor (VEGF) levels measured using an enzyme-linked immunosorbent assay kit. RESULTS: No differences (two-way anova) were found for treatments started with various time lags. At doses where CPI or PDT alone had no or negligible effect, their combination caused a marked (P < 0.001) decrease in serum VEGF, paralleled by a significant decrease in tumour size and number of capillary vessels, and a significant increase in necrosis (up to 80% of the tumour tissue). CONCLUSIONS: The combination of PDT and CPI could be a useful approach in tumour therapy as the two agents appear to be synergistic and probably decrease VEGF production by the tumour tissue.

Thiophosphate-activated phosphorylase kinase as a probe in the regulation of phosphorylase phosphatase.

Rabbit muscle nonactivated phosphorylase kinase (EC 2.7.1.38) is converted to thiophosphate-activated phosphorylase kinase by cyclic AMP dependent protein kinase, Mg2+ and ATP-gamma-S/adenosine-5'-O-(s-thiotriphosphate)/. The formation of thiophosphate-activated phosphorylase kinase wal also observed in the protein-glycogen complex from skeletal muscle. This new form of kinase is resistant to the action of phosphatase and behaves as a competitive inhibitor in the dephosphorylation of phosphorylase alpha by phosphorylase phosphatase (Ki = 0.04 mg per ml). The fact that the inhibitory effect of thiophosphate-activated phosphorylase kinase is 3 times higher than in the case of nonactivated kinase, may explain the transient inhibition of phosphorylase phosphatase in the protein-glycogen complex. The use of activated (phosphorylated) phosphorylase kinase supports this assumption since it causes a delay in the dephosphorylation of phosphorylase alpha, i.e. the conversion of phosphorylase alpha into beta could start only after the dephosphorylation of activated phosphorylase kinase.

Kinetic characterization of rabbit skeletal muscle phosphorylase ab hybrid.

Phosphorylase ab was prepared in vitro by partial phosphorylation of rabbit skeletal muscle phosphorylase b and was isolated by DEAE-Sephacel chromatography. Its phosphorylated and non-phosphorylated subunits could not be distinguished by different affinity to substrates, activators or inhibitors, indicating their coordinated function. In the absence of nucleotide activators, the Km values for Pi and glucose-1-P were 28 mM and 18 mM, respectively. Activity in the presence of 16 mM glucose-1-P was doubled by 10(-4) M AMP or 10(-3) M IMP, mainly by lowering the Km for glucose-1-P. Half-maximum activation was exerted by 2 microM AMP or 0.1 mM IMP. Activation by these nucleotides showed no cooperativity. Glucose exerted competitive inhibition with respect to glucose-1-P, while for the inhibition by glucose-6-P an allosteric mechanism is suggested; the appropriate Ki values were 4.5 mM and 1.5 mM, respectively. The Hill coefficient for glucose-1-P binding was about 1.0, even in the presence of glucose (up to 10 mM), but 10 mM glucose-6-P lowered it to 0.47, indicating a negative heterotropic cooperativity. Effective regulation of the activity of phosphorylase ab by physiological concentrations of Pi, AMP, IMP and glucose-6-P suggests its metabolic control under in vivo condition.

Effect of cyclosporin A on the membrane potential and Ca2+ level of human lymphoid cell lines and mouse thymocytes.

The effect of the immunosuppressive cyclosporin A (CsA) on the cytosolic free Ca2+ concentration ([Ca2+]i) and membrane potential of human B and T lymphoblastoid cells and mouse thymocytes was studied in order to reveal some features of the early stage of drug-cell interaction. Cytosolic free Ca2+ concentration of the cells was measured by spectrofluorimetry using indo-1 and quin2 fluorescent calcium indicators. Membrane potential was monitored in a flow cytometer with oxonol dye. CsA applied at 2-20 micrograms/ml final concentrations caused a dose-dependent, rapid, transient rise of [Ca2+]i in all cell types. This effect could be blocked by chelating the extracellular Ca2+ with EGTA but was not sensitive to Ca2+ channel blockers verapamil and nifedipine or K+ channel blocker 4-aminopyridine. A possible explanation for the calcium mobilizing effect of CsA is an ionophore-like mode of action at the cell membrane level. Besides directly interfering with mitogenic signals, the elevation of [Ca2+]i could be responsible for an initial hyperpolarization observed in CsA-treated T lymphocytes. This hyperpolarization, however, was not detectable in B lymphoblastoid cells. A further difference between B and T cells was the diverse pattern of depolarization following CsA treatment. This variance in the behaviour of T and B lymphocytes and the diversity of membrane transport systems in its background could account for the different final outcome of the drug-cell interaction.

Identification of a 200 kDa polypeptide as type 3 phosphatidylinositol 4-kinase from bovine brain by partial protein and cDNA sequencing.

Two phosphatidylinositol 4-kinase isozymes, type 3 and type 2, have been separated on hydroxylapatite after solubilizing bovine brain microsomes with Triton X-114. Employing a newly developed renaturation procedure following SDS-PAGE, we demonstrate that a 200 kDa polypeptide carries the enzyme activity of this type 3 isoform. Chromatography on hydroxylapatite, Heparin-Sepharose, Superdex 200 and finally SDS-PAGE results in an approximately 30,000-fold purification. Tryptic peptides generated from the 200 kDa polypeptide after SDS-PAGE have been sequenced and the obtained data have been used for constructing and synthesizing degenerated oligonucleotides. Polymerase chain reaction as well as screening of cDNA libraries allowed several clones to be isolated from which a 4.7 kb contiguous sequence can be built up. The open reading frame covers 4.4 kb with a 0.3 kb untranslated 3' end which yields a deduced amino acid sequence of 1,467 amino acids. The C-terminal part of ca. 300 amino acids represents the catalytic domain. Sequence alignment of this domain with the mammalian counterpart, the human type 2 phosphatidylinositol 4-kinase, the yeast kinases STT4 and PIK1, as well as with the catalytic domains of bovine, human, mouse and yeast phosphatidylinositol 3-kinases reveals a high degree of identity: 26 of these approximately 300 amino acids are invariable in all of these eight catalytic domains. Five motifs indicate nuclear localization and DNA binding properties of the enzyme. Two leucine zipper motifs (amino acids 358-386, 862-882) are detectable. Furthermore, a helix loop helix motif (amino acids 716-729) as well as two nuclear localization signals (amino acids 838-854, 345-349) indicate the presence of the type 3 isoform in the nucleus.

Functional expression and characterisation of a new human phosphatidylinositol 4-kinase PI4K230.

By constructing DNA probes we have identified and cloned a human PtdIns 4-kinase, PI4K230, corresponding to a mRNA of 7.0 kb. The cDNA encodes a protein of 2044 amino acids. The C-terminal part of ca. 260 amino acids represents the catalytic domain which is highly conserved in all recently cloned PtdIns 4-kinases. N-terminal motifs indicate multiple heterologous protein interactions. Human PtdIns 4-kinase PI4K230 expressed in vitro exhibits a specific activity of 58 micromol mg-1min-1. The enzyme expressed in Sf9 cells is essentially not inhibited by adenosine, it shows a high Km for ATP of about 300 microM and it is half-maximally inactivated by approximately 200 nM wortmannin. These data classify this enzyme as type 3 PtdIns 4-kinase. Antibodies raised against the N-terminal part moderately activate and those raised against the C-terminal catalytic domain inhibit the enzymatic activity. The coexistence of two different type 3 PtdIns 4-kinases, PI4K92 and PI4K230, in several human tissues, including brain, suggests that these enzymes are involved in distinct basic cellular functions.

Calcium channels in PDGF-stimulated A172 cells open after intracellular calcium release and are not voltage-dependent.

Using laser image cytometry and Indo-1 fluorescence, we investigated the intracellular free Ca2+ concentration ([Ca2+]i) of confluent A172 human glioblastoma cells stimulated by the BB homodimer of platelet-derived growth factor (PDGF-BB). The shape of the calcium transients and the delay time between stimulation and the beginning of the transient varied considerably. The percentage of responsive cells, the peak [Ca2+]i and the duration of the response were directly related to PDGF-BB dose, while the delay time was inversely related; the maximal response occurred at a PDGF-BB concentration of 20 ng/ml. Studies with EGTA and inorganic calcium-channel blockers (Ni2+, La3+) showed that the increase of [Ca2+]i resulted from initial release of intracellular stores and subsequent calcium influx across the plasma membrane. Opening of calcium channels in the plasma membrane, monitored directly by studying Mn2+ quenching of Indo-1 fluorescence, was stimulated by PDGF-BB and blocked by La3+; the opening occurred 55 +/- 10 s after the initial increase in [Ca2+]i. Therefore, in these tumor cells, intracellular release always occurs before channel opening in the plasma membrane. Depolarization of cells with high extracellular [K+] did not generally induce calcium transients but did decrease calcium influx. L-type calcium-channel blockers (verapamil, nifedipine, and diltiazem) had little or no effect on the calcium influx induced by PDGF-BB. These results indicate that PDGF-BB induces calcium influx by a mechanism independent of voltage-sensitive calcium channels in A172 human glioblastoma cells.

The ATP-binding site of brain phosphatidylinositol 4-kinase PI4K230 as revealed by 5'-p-fluorosulfonylbenzoyladenosine.

The ATP-binding site of purified bovine brain phosphatidylinositol 4-kinase 230 (PI4K230) was studied by its reaction with 5'-p-fluorosulfonylbenzoyladenosine (FSBA), an ATP-like alkylating reagent. Four hundred to eight hundred micromolar FSBA inactivated PI4K230 specifically with apparently first-order kinetics and resulted in 50% loss of enzyme activity in 36--130 min. The specificity of the reaction with FSBA was demonstrated by the lack of inactivation with 5'-p-fluorosulfonylbenzoyl chloride and by protection with ATP and ATP analogues against inactivation. Most ATP analogues competed with FSBA inactivation in order of their increasing hydrophobicity, parallel to their inhibitory potency in activity measurements. The specific binding of FSBA to PI4K230 was demonstrated also by Western-blot experiments. These results suggest that FSBA-reactive group(s) involved in the enzyme activity are located near to the ATP-binding site in a hydrophobic region of native PI4K230. Experiments with site-directed mutagenesis indicate that the conserved Lys-1792 plays essential role in the enzyme activity and serves as one target of affinity labelling by FSBA. Prevention of both Lys-1792-directed and Lys-1792-independent binding of FSBA by Cibacron Blue 3GA suggest that these sites are located spatially close to each other.

Regulatory subunit of type II cAMP-dependent protein kinase as substrate and inhibitor of protein phosphatase-1 and -2A.

The dissociated regulatory subunit (RII) of autophosphorylated cAMP-dependent protein kinase II was dephosphorylated by the catalytic subunits of protein phosphatase-1 and -2A (phosphatase-1c and -2Ac) and by a high-Mr polycation-dependent form of phosphatase-2A (2Ao) with Km values of 5, 0.3 and 1 microM, respectively. Dissociation of protein kinase by cAMP preferentially increased the dephosphorylation of RII by phosphatase-1c, whereas polycations (histone Hl or polybrene) markedly stimulated phosphatase-2Ac and -2Ao even in the absence of cAMP. Thiophosphorylated RII inhibited the dephosphorylation of phosphorylase a by these phosphatases with half-maximum inhibitory concentrations of 0.1-0.36 microM.

Synthesis and enzymatic activity of some new purine ring system analogues of adenosine 3',5'-cyclic monophosphate.

A series of novel adenosine 3',5'-cyclic monophosphate (cAMP) analogues, as well as their 6-deamino and 6-nitro derivatives, were synthesized where the purine ring was replaced by indazole, benzotriazole, and benzimidazole. The 3',5'-cyclic monophosphates of indazole and benzotriazole ribofuranosides, where the sugar-phosphate moiety is attached to the N-2 nitrogen atoms of the heterocycles, were also prepared. The biological efficiency of the analogues was tested by their ability to activate purified cAMP-dependent protein kinase I (PK-I) from rabbit skeletal muscle and cAMP-dependent protein kinase II (PK-II) from bovine heart. Each cyclic nucleotide is capable of activating both PK isozymes in half-maximum concentrations (Ka) ranging from 2.0 x 10(-8) to 4.8 x 10(-6) M. The cyclic phosphate of N-1-beta-D-ribofuranosylindazole (13) proved to be a very poor activator for both PK-I and PK-II, but when indazole binds by N-2 to ribose or when the hydrogen atom at C-4 is substituted by a nitro or amino group, activities of the analogues increase considerably. The activating potencies of benzotriazole derivatives are similar to that of cAMP, irrespective of the C-4 substituents. The Ka' values of cyclic nucleotides containing benzimidazole were found to be higher for PK-II than for PK-I; e.g. the activity of 4-nitro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphate (32) is nearly 20 times as high for PK-II than for PK-I.

Synthesis of new cyclitol compounds that influence the activity of phosphatidylinositol 4-kinase isoform, PI4K230.

The synthesis, chemical derivatization, and investigation of the inhibitory properties of novel cyclitol derivatives on the phosphatidylinositol 4-kinase enzymes PI4K55 and PI4K230 involved in the phosphatidylinositol cycle are reported. Some of the prepared cyclitol derivatives (i.e. 9, 11, 12, and 14) proved to be very powerful and specific irreversible inhibitors of PI4K230 at or below a concentration of 1 mM.

Partially phosphorylated phosphorylase in the rat heart after beta-receptor stimulation in vivo.

The formation of the phosphorylase ab hybrid and its further transformation into phosphorylase a has been demonstrated in the rat heart after different periods of i.v. isoproterenol administration. Phosphorylase ab hybrid was determined in the presence of AMP and/or caffeine. Only the partially phosphorylated phosphorylase was found in the control rat hearts and its activity was 30% of the total phosphorylase. The phosphorylase ab hybrid was disclosed particularly after small isoproterenol doses (0.031-0.062 microgram.kg-1) and at short time interval (15 s) after its administration. Higher isoproterenol doses (0.25-0.5 microgram.kg-1) changed the partially phosphorylated phosphorylase to phosphorylase a (58%) after a longer time interval (40 s). The phosphorylase ab hybrid was revealed even at the maximal rate of stimulation. The formation of the phosphorylase ab hybrid in the rat heart in vivo appears to be of physiological significance. Our results confirmed the earlier suggestion that the -AMP/+AMP activity ratio reflects the percentage proportion of the phosphorylated subunits of phosphorylase but not of the activated phosphorylase molecules.

The role of autophosphorylation of cAMP-dependent protein kinase II in the inhibition of protein phosphatase-1.

1. The inhibition of the catalytic subunit of protein phosphatase-1 (PP-1c) by the regulatory subunit of cAMP-dependent protein kinase II (RII) was studied. 2. Phosphorylation or thiophosphorylation of RII increased its inhibitory potency up to 4- and 6-fold and rendered it competitive with respect to the substrate of PP-1c, phosphorylase a. The Ki values for thiophospho-RII and phospho-RII were 200 and 500 nM, respectively. 3. Though PP-1c was able to release phosphate from phospho-RII, its activity once incubated with phospho-RII, remained inhibited even 80% of the phosphate was released from phospho-RII. 4. The catalytic subunit of cAMP-dependent protein kinase was effective in suspending the inhibition employed either before or after the addition of phospho-RII to PP-1c. 5. No exclusive bindings of thiophospho-RII and heat-stable protein inhibitors to the PP-1c could be proved by double inhibition studies, however some synergism was observed in their effect.

Role of SH groups in the activity of pig phosphorylase b isoenzymes.

The reaction of phosphorylases from rabbit skeletal muscle, pig skeletal muscle and pig heart with DTNB and the effects of AMP and glucose-6-phosphate on the above reaction were examined and compared. The SH groups of pig heart-specific phosphorylase b were found to be the same as those of rabbit skeletal muscle. Pig skeletal muscle phosphorylase b differed from the foregoing enzymes in that its slowly reacting SH groups played a minor role in the catalytic activity and AMP and glucose-6-phosphate affected neither the DTNB reaction nor the small decrease of activity during the reaction.

Properties of skeletal muscle phosphorylase-protein complexes.

Frontal gel filtration studies on muscle extract and mixture of purified enzymes have verified the existence of a protein-complex between phosphorylase (alpha-1,4-glucan: orthophosphate glycosyltransferase EC 2.4.1.1.) and phosphorylase kinase. The complex has an apparent molecular weight of 750 000 daltons. The complex formation depends on the protein concentration and the presence of Ca2+. Removal of Ca2+ with EGTA results in the dissociation of the complex. A regulatory role may be attributed to Ca2+ since the concentration of free Ca2+ changes in skeletal muscle through the effect of hormonal or electrical stimulation. Strong association was also detected between phosphorylase kinase and phosphorylase phosphatase. The transient inhibition of phosphorylase phosphatase can be explained by this interaction.

Ligand effects on the dephosphorylation of heart and skeletal muscle specific phosphorylases.

Pseudo first order rate constants were determined for the dephosphorylation of heart and skeletal muscle specific phosphorylase a isoenzymes isolated from rabbit and pig using rabbit muscle phosphorylase phosphatase (mol. wt 34,000). The rate constants determined in the absence of ligands, were 4-5 fold lower for heart specific phosphorylases than for skeletal muscle specific ones. Glucose 6-phosphate (0.5-1 mM) enhances the rate of dephosphorylation of heart specific isophosphorylases 3-fold and suspends inhibition by 10(-5) M AMP, however, it has no significant effect on the dephosphorylation of skeletal muscle specific enzymes under the same conditions. Our data support characteristic functional differences between heart and skeletal muscle specific phosphorylases both in rabbit and pig.

Ligand effects on the activity of the a form of various mammalian heart specific isophosphorylases.

1. The allosteric sensitivity of heart specific and skeletal muscle specific phosphorylase a-s from rabbit, pig, bovine and dog was compared. 2. 0.2-0.3 mM glucose 6-phosphate exerts 50% inhibition on the heart specific phosphorylase a-s without any inhibitory effect on the skeletal muscle specific ones. 3. AMP decreases the Km for substrates (glucose 1-phosphate, or Pi) of all heart specific phosphorylase a-s. It also enhances the Vmax of pig and bovine phosphorylase a-s 2-fold, but has no effect on the Vmax of rabbit and dog enzymes.