Effect of 3,3',4',5-tetrachlorosalicylanilide on reduction of excess sludge and nitrogen removal in biological wastewater treatment process

A metabolic uncoupler, 3,3',4',5-tetrachlorosalicylanilide (TCS), was used to reduce excess sludge production in biological wastewater treatment processes. Batch experiments confirmed that 0.4 mg/l of TCS reduced the aerobic growth yield of activated sludge by over 60%. However, the growth yield remained virtually constant even at the increased concentrations of TCS when cultivations were carried out under the anoxic condition. Reduction of sludge production yield was confirmed in a laboratory-scale anoxic-oxic process operated for 6 months. However, it was found that ammonia oxidation efficiency was reduced by as much as 77% in the presence of 0.8 mg/l of TCS in the batch culture. Similar results were also obtained through batch inhibition tests with activated sludges and by bioluminescence assays using a recombinant Nitrosomonas europaea (pMJ217). Because of this inhibitory effect of TCS on nitrification, the TCS-fed continuous system failed to remove ammonia in the influent. When TCS feeding was stopped, the nitrification yield of the process was resumed. Therefore, it seems to be necessary to assess the nitrogen content of wastewater if TCS is used for reducing sludge generation.     

The interaction of ions with phosphatidylcholine bilayers

The interaction of lanthanides and other cations with phosphatidylcholine bilayers present as single bilayer vesicles in ²H₂O has been investigated in terms of stoichiometry, apparent binding constants and environmental conditions.Lanthanides are shown to form 2 : 1 (molar ratio) phosphatidylcholine to metal ion complexes.The apparent binding constant $\text{K}{}_{\mathrm{<mtext>b</mtext>}}$ varies as a function of the quantity of metal ion bound and as a function of the Cl^? concentration. The apparent binding constant at “zero loading” is $\text{K}{}_0\text{= 1.25 · 10}{}^4\text{L}{}^2\text{·}\text{M}{}^{\mathrm{?}}\text{at}\text{0.15}\text{M KCl}$. It decreases exponentially with increased “loading” expressed as the molar ratio of metal ion bound to effective phosphatidylcholine concentration and increases exponential with Cl^? concentration.The interaction of lanthanides and divalent cations such as Ca²⁺ and Mg²⁺ is independent of pH in the pH range 3–7⁺ and 3–10 respectively, but is sensitive to the nature of the anion. The presence of anions enhances the interaction with polyvalent cations, the chaotropic anions showing the largest effect. The order of enhancement is $\text{Cl}{}^{\mathrm{?}}\text{< Br}{}^{\mathrm{?}}\text{< NO}{}_3{}^{\mathrm{?}}\text{< SCN}{}^{\mathrm{?}}\text{< I}{}^{\mathrm{?}}\text{< ClO}{}_4{}^{\mathrm{?}}$. The nature of the monovalent counterion (cation) has little effect on the enhanced binding of lanthanides in the presence of the above anions.The affinity of other polyvalent cations for phosphatidylcholine bilayers has been determined by competition with lanthanides. The physiologically important divalent cations Ca²⁺ and Mg²⁺ both bind less strongly (by about an order of magnitude) to the lipid surface. The order of binding of cations reflects direct binding to the phosphodiester group, with UO₂²⁺ showing the highest affinity.     

Transverse motion of spin-labeled 3,3',5-triiodo-L-thyronine in phospholipid bilayers

Using electron spin resonance stop-flow technique, the transverse motion (flip-flop) of 3-([alpha-carboxy-4-(4-hydroxy-3-iodophenoxy)-3,5- diiodophenethyl]carbamoyl)-2,2,5,5-tetramethyl-3-pyrrolin (T3-SL) in dipalmitoyl L-alpha-phosphosphatidylcholine (DPPC) membranes was evaluated. At 22 degrees C, the electron spin resonance spectra of T3-SL in DPPC vesicles were compared before and after the addition of sodium ascorbate, a membrane impermeable reducing agent. The addition of ascorbate reduces the signal amplitude by 67% in 3 min but yields no further reduction for at least 60 min. These results indicate that T3-SL does not flip-flop at any appreciable rate in the membranes. This finding suggests that once partitioned into the membrane, T3 remains in the outer half of the lipid bilayer, thus reducing the possibility that T3 enters the cell by passive diffusion.     

The interaction of phosphatidylcholine bilayers with Triton X-100

The interaction of multilamellar phosphatidylcholine vesicles with the non-ionic detergent Triton X-100 has been studied under equilibrium conditions, specially in the sub-lytic range of surfactant concentrations. Equilibrium was achieved in less than 24 h. Estimations of detergent binding to bilayers, using [3H]Triton X-100, indicate that the amphiphile is incorporated even at very low concentrations (below its critical micellar concentration); a dramatic increase in the amount of bound Triton X-100 occurs at detergent concentrations just below those producing membrane solubilization. Solubilization occurs at phospholipid/detergent molar ratios near 0.65 irrespective of lipid concentration. The perturbation produced by the surfactant in the phospholipid bilayer has been studied by differential scanning calorimetry, NMR and Fourier-transform infrared spectroscopy. At low detergent concentration (lipid/detergent molar ratios above 3), a reduction in 2H-NMR quadrupolar splitting occurs, suggesting a decrease in the static order of the acyl chains; the same effect is detected by Fourier-transform infrared spectroscopy in the form of blue shifts of the methylene stretching vibration bands. Simultaneously, the enthalpy variation of the main phospholipid phase transition is decreased by about a third with respect to its value in the pure lipid/water system. For phospholipid/detergent molar ratios between 3 and 1, the decrease in lipid static order does not proceed any further; rather an increase in fluidity is observed, characterized by a marked decrease in the midpoint transition temperature of the gel-to-fluid phospholipid transition. At the same time an isotropic component is apparent in both 31P-NMR and 2H-NMR spectra, and a new low-temperature endotherm is detected in differential scanning calorimetric traces. When phospholipid and Triton X-100 are present at equimolar ratios some bilayer structure persists, as judged from calorimetric observations, but NMR reveals only one-component isotropic signals. At lipid/detergent molar ratios below unity, the NMR lines become narrower, the main (lamellar) calorimetric endotherm tends to vanish and solubilization occurs.     

Lipid-protein interaction. The incorporation of myelin proteolipid apoprotein into phosphatidylcholine bilayers

Bovine myelin proteolipid apoprotein (PLA), obtained in high yield and purity by a novel ultrafiltration procedure, has been used to study the perturbations produced by this protein on phosphatidylcholine bilayers, using infrared spectroscopy, nuclear magnetic resonance and fluorescence polarisation. PLA interacts with phospholipids in a similar manner to other intrinsic proteins. For bilayers in the fluid state, the fatty-acyl chain static order, as measured by deuterium NMR, is slightly increased in the presence of the protein, except at very high PLA concentrations. Phosphorus NMR reveals some perturbation of the phospholipid polar group by PLA, but to a smaller degree than occurs with other intrinsic proteins. An increase in static order above tc (the onset temperature for gel-to-fluid transition) is also detected by infrared spectroscopy. Studies using steady-state polarisation of diphenylhexatriene fluorescence indicate that the microviscosity of the bilayer increases as a function of the protein mole fraction. From these data an estimation of the average number of lipids perturbed per protein monomer has been made, and a figure of 37 phospholipid molecules determined. The data are compatible with a picture of a hydrophobic polypeptide, perturbing the phospholipids close to it, but allowing rapid (greater than 10(4) s-1) exchange with all the lipid molecules in the system.     

Mechanism for 3,3',4',5-tetrachlorosalicylanilide-induced activation of sarcoplasmic reticulum ATPase

Effects of 3,3',4',5-tetrachlorosalicylanilide (TCS), a lipophilic weak acid, on Ca2+ uptake and ATP hydrolysis by the sarcoplasmic reticulum calcium pump were characterized to obtain insight into the possible role of hydrophobic portions of the Ca2+-ATPase in the catalytic mechanism of the enzyme. TCS exhibited both the stimulatory and inhibitory effects on the calcium pump activities depending on its concentration. At optimal concentrations, it increased these activities by up to 5-fold at pH 7.0 and 6 degrees C. Analysis of partial reactions of ATP hydrolysis by the purified ATPase revealed that TCS accelerated Ca2+ release from the ADP-sensitive phosphoenzyme up to 6-fold, whereas it affected other reaction steps to a much less extent, indicating that the site of the stimulatory action of TCS is rather specific in terms of the reaction sequence. These effects of TCS became less prominent at higher temperatures, although the enzyme-TCS interactions as detected in the direct binding experiment or by measurement of quenching of protein fluorescence were not affected by a similar change in temperature. The TCS effects were also dependent on pH of the 8.0 suggested that the protonated form of TCS is responsible for both the stimulatory and inhibitory effects of the drug. These results, taken together with those obtained previously with a spin-labeled probe (Barratt, M. D., and Weaver, A. C. (1979) Biochim. Biophys. Acta 555, 337-348), may suggest that TCS stimulates the calcium pump activity through its effect on the lipid bilayer, although its direct action on hydrophobic portion(s) of the ATPase protein cannot be ruled out.     

Non-covalent interaction of 2', 4', 5', 7'-tetrabromo-4, 5, 6, 7-tetrachlorofluorescein with proteins and its application

The interactions of 2', 4', 5', 7'-tetrabromo-4, 5, 6, 7-tetrachlorofluorescein (TBTCF) with BSA, ovalbumin (OVA) and poly-L-lysine (PLYS) at pH 3.70 have been investigated by combination of the spectral correction technique and the Langmuir isothermal adsorption. The active connection actions such as ion pairs, van der Waals' force, hydrogen bond, hydrophobic bond were proposed to explain the non-covalent interaction between TBTCF and BSA, OVA and PLYS. Effects of the electrolyte and high temperature indicated that union of the active connections between TBTCF and BSA and OVA was too firm to be destroyed. The relationship between the binding number of TBTCF and variety fraction of the amino acid residues was analyzed. The binding number of TBTCF depended on the number of positively charged amino acid residues. The other amino acid residues surrounded and seized TBTCF by hydrogen bonds and hydrophobic bonds when the electrostatic attraction pulled TBTCF to link protein. In addition, a novel method named the absorbance ratio difference was established for determination of protein in trace level and was applied with much higher sensitivity than the ordinary method.     

The influence of 3,3',5-triiodo-L-thyronine on human haematopoiesis

OBJECTIVES: Thyroid hormones mediate many physiological and developmental functions in humans. The role of the 3,3',5-triiodo-L-thyronine (T3) in normal human haematopoiesis at the cellular and molecular levels has not been determined. In this study, it was revealed that the human haematopoietic system might be directly depended on T3 influence. MATERIALS AND METHODS: We detected the TRalpha1 and TRbeta1 gene expression at the mRNA level in human cord blood, peripheral blood and bone marrow CD34(+)-enriched progenitor cells, using the RT-PCR method. Furthermore, we performed Western blotting to prove TRalpha1 and TRbeta1 expression occurs at the protein level in human cord blood, peripheral blood and bone marrow CD34(+) cells. In addition, the examined populations of cells were exposed in serum-free conditions to increasing doses of T3 and were subsequently investigated for clonogenic growth of granulocyte-macrophage colony-forming unit and erythrocyte burst-forming unit in methylcellulose cultures, and for the level of apoptosis, by employing annexin V staining and the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling method. We investigated expression levels of apoptosis-related Bax and antiapoptotic Bcl-2 and Bcl-x(L) genes in the examined cells. RESULTS: We found that exposure to higher and lower than normal concentration of thyroid hormone significantly influenced clonogenecity and induced apoptosis in human haematopoietic progenitor cells. CONCLUSIONS: This study expands the understanding of the role of thyroid disorders in normal human haematopoiesis and indicates a direct influence of T3 on this process.     

The influence of cholesterol on the interaction between N-dodecyl-N,N-dimethyl-N-benzylammonium halides and phosphatidylcholine bilayers

Effects of N-dodecyl-N,N-dimethyl-N-benzylammonium halides (DBeAX) on thermotropic phase behavior of phosphatidylcholine/cholesterol bilayers as well as on 1H NMR spectra were studied. The surfactants were added either to the water phase or directly to the lipid phase (a mixed film was formed). The benzyl group, opposite to liposomes without cholesterol, is not incorporated into the bilayer in the gel state but only in the liquid state. All the halides DBeAX (particularly the chloride DBeAC) showed greater ability to destabilize the membrane structure in the presence than in the absence of cholesterol. The interaction of DBeAX with DPPC/cholesterol bilayers and subsequent changes in the phospholipid bilayer organization depended on the kind of counterion. The strongest effects were observed for chloride (most electronegative ion) and for iodide (largest ion). The effects of chloride and bromide on phase transition and 1H NMR spectra in the presence and absence of cholesterol were opposite. This is discussed in terms of the influence of counterions on the pair cholesterol-DPPC interactions.     

Metabolic uncoupling of Shewanella oneidensis MR-1, under the influence of excess substrate and 3, 3', 4', 5-tetrachlorosalicylanilide (TCS)

The dissociation between catabolism and anabolism is generally termed as metabolic uncoupling. Experimentally, metabolic uncoupling is characterized by a reduction in the observed biomass yield. This condition can be brought about by: (a) excess-substrate (as measured by S(0)/X(0)), and (b) addition of chemical uncouplers such as 3, 3', 4', 5-Tetrachlorosalicylanilide (TCS). An empirical model is proposed to quantify the uncoupling effects of both excess-substrate and uncoupler addition on the microbial cultures. Metabolic uncoupling of Shewanella oneidensis MR-1, under the influence of excess pyruvate and TCS, has been modeled using the proposed expression. The degree of uncoupling was measured as a fractional reduction in theoretical maximum observed yield. Excess-substrate was observed to successively reduce biomass yield as substrate concentration was increased. In the presence of TCS, conflicting trends were obtained for number yield and protein yield. This could, in part, be attributed to the observed increase in cellular protein content upon addition of TCS. Excess-substrate conditions dominated uncoupling, as compared to uncoupler addition. However, these two approaches were found to have additive effects and could, in conjunction, be employed to control biomass growth during microbial processes such as subsurface bioremediation and activated sludge treatment.     

The effect of 3,3',5-triiodo-L-thyronine on the renal handling of citrate.

The effects of a 500 mug injection of T3 on the renal handling of citrate by the albino rat was studied by measuring citrate synthase activity, NADP-isocitrate dehydrogenase activity, and plasma, kidney, and urine citrate concentrations 12, 18, 24, 36, and 48 hr after injection. Kidney citrate synthase activity of the T3-injected rats was significantly lower than the controls in the 24- and 36-hr treatment groups, while NADP-IDH activity was significantly lowered only in the 36-hr treatment group. The injection of T3 resulted in hypercitricemia in the 12-, 18-, and 48-hr experimental animals while there was no significant change in citrate between the control values and treated values in the 24- and 36-hr experiments. There was no significant change in renal citrate levels in any of the treatment groups and hypercitrauria was not observed. The results of the present study suggest that T3 can control citrate utilization by increasing the levels of circulating citrate and then increasing the utilization of citrate by the kidney. This is facilitated by a decrease in NADP-IDH activity resulting in a decrease in biosynthesis and a decrease in citrate synthase activity resulting in a decrease in FFA metabolism. It is proposed that this system functions in providing fuel (citrate) for the increased Krebs cycle flux occurring in hyperthyroidism.     

Studies towards the large scale chemical synthesis of the precursors of ribonucleosides-3',4',5',5'-2H4 and -2',3',4',5',5'-2H5

A summary delineating the large scale synthetic studies to prepare labeled precursors of ribonucleosides-3',4',5',5'-2H4 and -2',3',4',5',5'-2H5 from D-glucose is presented. The recycling of deuterium-labeled by-products has been devised to give a high overall yield of the intermediates and an expedient protocol has been elaborated for the conversion of 3-O-benzyl-alpha,beta-D-allofuranose-3,4-d2 6 to 1-O-methyl-3-O-benzyl-2-O-t-butyldimethylsilyl-alpha,beta-D-ribofuranose-3,4,5,5'-d4 16 (precursor of ribonucleosides-3',4',5',5'-2H4) or to 1-O-methyl-3,5-di-O-benzyl-alpha,beta-D-ribofuranose-3,4,5,5'-d4 18 (precursor of ribonucleosides-3',4',5',5'-2H4).     

Physicochemical studies of the interaction of the lipoheptapeptide surfactin with lipid bilayers of L-alpha-dimyristoyl phosphatidylcholine

To understand the biological action of surfactin from Bacillus subtilis we investigated its effects on the phase transition of L-alpha-dimyristoyl phosphatidylcholine (DMPC)-vesicles from the crystalline to the fluid state using differential scanning calorimetry; light scattering; small angle neutron scattering and cryo-electron microscopy. DSC-thermograms revealed two phase transition peaks. Light scattering profiles showed two branches with characteristic hysteresis phenomena. With both techniques the same values of the phase transition temperatures T(m1) and T(m2) of 23.5 and 23 degrees C were obtained indicating two forms of DMPC-surfactin aggregates which could be visualized by cryo-electron microscopy. Until 4 mol% surfactin the vesicular form predominated, but was accompanied by bilayered membrane fragments by increasing the biosurfactant concentrations. At surfactin concentrations higher than 15 mol% smaller DMPC-surfactin micelles of ellipsoidal conformation were formed, as demonstrated by small angle neutron scattering. In addition, by "Poor Man's" temperature-jump-relaxation spectroscopy slow transients in the phase transition of vesicular DMPC-surfactin aggregates with relaxation times of 20-30 s were detected which presumably indicate the slow dissipation of intermediate lipid-and surfactin domains formed after the main phase transition on the way to the fluid state. This process is accelerated by surfactin.     

Fluorescence probe studies of the interaction of ubiquinone-10 and ubiquinol-10 with phosphatidylcholine bilayers

Ubiquinone-10 and ubiquinol-10 were incorporated into dipalmitoylphosphatidylcholine vesicles and their interaction with the phospholipids was monitored by fluorescence measurements of diphenylhexatriene used as a probe. It was found that ubiquinone-10 did not perturb the phospholipid thermotropic pretransition but ubiquinol-10 was able to do so. Although, in ethanolic solution, ubiquinone-10 was a better quencher of diphenylhexatriene than ubiquinol-10, when incorporated into phospholipid multibilayers and at temperatures above Tc, ubiquinone-10 produced a smaller decrease in the intensity of the fluorescence probe than ubiquinol-10. Furthermore, the fluorescence anisotropy of the probe was significantly increased by ubiquinol-10 but not by ubiquinone-10. It was concluded that both forms of coenzyme Q have different localizations in the phospholipid bilayer.     

The interaction of N-oleylethanolamine with phospholipid bilayers

Long chain acylamides of ethanolamine were previously found to increase in the infarcted canine myocardium. Subsequent in vitro experiments established a number of interesting biological and physiological properties of these compounds including alteration of rabbit skeletal sarcoplasmic reticulum function and inhibition of permeability dependent calcium release from heart mitochondria. These results suggested an interaction between the N-acylethanolamines and biological membranes. In the present work we show that the most potent species in previous studies, N-oleylethanolamine, forms stable complexes with phospholipid vesicles, lowers diphenylhexatriene polarization ratios in dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine uni- and multilamellar bilayer vesicles, and also produces a concentration dependent decrease in the phase transitions of these lipid structures. In addition studies with parinaric acids also suggested that N-oleylethanolamine partitions preferentially into more fluid areas of the bilayer. The results are discussed in terms of possible effects on biological membranes.