Characterization of a novel melamine-degrading bacterium isolated from a melamine-manufacturing factory in China

Melamine (2,4,6-triamino-1,3,5-triazine, C₃H₆N₆), belonging to the s-triazine family, is an anthropogenic and versatile raw material for a large number of consumer products and its extensive use has resulted in the contamination of melamine in the environment. A novel melamine-degrading bacterium strain CY1 was isolated from a melamine-manufacturing factory in China. The strain is phylogenetically different from the known melamine-degrading bacteria. Approximately, 94 % melamine (initial melamine concentration 4.0 mM, initial cell OD 0.05) was degraded in 10 days without the addition of additional carbon source. High-performance liquid chromatography showed the production of degradation intermediates including ammeline, ammelide, cyanuric acid, biuret, and urea. Kinetic simulation analysis indicated that transformation of urea into ammonia was the rate-limiting step for the degradation process. The melamine–cyanurate complex was formed due to self-assembly of melamine and cyanuric acid during the degradation. The tracking experiment using CY1 cells and ¹³C₃-melamine showed that the CY1 could mineralize s-triazine ring carbon to CO₂. The strain CY1 could also catalyze partial transformation of cyromazine, a cyclopropyl derivative of melamine, to 6-(cyclopropylamino)-[1,3,5]triazine-2,4-diol.     

Hydration effect on proton transfer in melamine−cyanuric acid complex

Self-assembly of melamine-cyanuric acid (MC) leads to urinary tract calculi and renal failure. The hydration effects on molecular geometry, the IR spectra, the frontier molecular orbital, the energy barrier of proton transfer (PT), as well as the stability of MC were explored by density functional theory (DFT) calculations. The intramolecular PT breaks the big π-conjugated ring of melamine or converts the p-π conjugation (:N-C'=O) to π-π conjugation (O=C-N=C') of cyanuric acid. The intermolecular PT varies the coupling between melamine and cyanuric acid from pure hydrogen bonds (N_a…HN_d and NH…O) to the cooperation of cation…anion electrostatic interaction (N_aH⁺…N_d ^-) and two NH…O hydrogen bonds. Distinct IR spectra shifts occur for N_a…HN_d stretching mode upon PT, i.e., blue-shift upon intramolecular PT and red-shift upon intermolecular PT. It is expected that the PT would inhibit the generation of rosette-like structure or one-dimensional tape conformer for the MC complexes. Hydration obviously effects the local geometric structure around the water binding site, as well as the IR spectra of NH…O and N…HN hydrogen bonds. Hydration decreases the intramolecular PT barrier from ~45 kcal mol^-1 in anhydrous complex to ~11.5 kcal mol^-1 in trihydrated clusters. While, the hydration effects on intermolecular PT barrier is slight. The relative stability of MC varies slightly by hydration due to the strong hydrogen bond interaction between melamine and cyanuric acid fragments.

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Graphical Abstract Hydration effect on proton transfer in melamine?cyanuric acid complex

     

Determination of melamine and cyanuric acid in human urine by a liquid chromatography tandem mass spectrometry

Melamine was found to be the etiological factor for the urinary stones epidemic in infants and young children in China in 2008. Urine level of melamine and its analog cyanuric acid may be useful markers for the evaluation of toxic effects. Liquid chromatography tandem mass spectrometry methods for the individual determination of melamine and cyanuric acid in human urine are described. Using isotope labeled internal standards during liquid–liquid extraction, the method was fully validated by verifying specificity, linearity, LLOQ, intra- and inter-assay precision and accuracy, matrix effect, recovery and stability. Calibration curves with good linearity (r = 0.9999) over the concentration range from 10 to 5000 ng/ml, intra-assay precision <10% and inter-assay precision <15%, accuracy between 93.0 and 111.6% were obtained with multiple reaction monitoring mode for melamine and cyanuric acid in human urine. The methods were successfully applied to the analysis of urine samples collected from 86 infants and 110 adults.     

Expanding the Cyanuric Acid Hydrolase Protein Family to the Fungal Kingdom

The known enzymes that open the s-triazine ring, the cyanuric acid hydrolases, have been confined almost exclusively to the kingdom Bacteria and are all homologous members of the rare cyanuric acid hydrolase/barbiturase protein family. In the present study, a filamentous fungus, Sarocladium sp. strain CA, was isolated from soil by enrichment culturing using cyanuric acid as the sole source of nitrogen. A reverse-genetic approach identified a fungal cyanuric acid hydrolase gene composed of two exons and one intron. The translated spliced sequence was 39 to 53% identical to previously characterized bacterial cyanuric acid hydrolases. The sequence was used to generate a gene optimized for expression in Escherichia coli and encoding an N-terminally histidine-tagged protein. The protein was purified by nickel affinity and anion-exchange chromatography. The purified protein was shown by ¹³C nuclear magnetic resonance (¹³C-NMR) to produce carboxybiuret as the product, which spontaneously decarboxylated to yield biuret and carbon dioxide. The protein was very narrow in substrate specificity, showing activity only with cyanuric acid and N-methyl cyanuric acid. Barbituric acid was an inhibitor of enzyme activity. Sequence analysis identified genes with introns in other fungi from the Ascomycota that, if spliced, are predicted to encode proteins with cyanuric acid hydrolase activity. The Ascomycota cyanuric acid hydrolase homologs are most closely related to cyanuric acid hydrolases from Actinobacteria.     

Ovarian Toxicity in Female Rats after Oral Administration of Melamine or Melamine and Cyanuric Acid

Although the toxicity of melamine to the kidneys and testes is well known, few studies have investigated the effects of melamine on female reproductive organs. Therefore, this study explores the effects of oral administration melamine or melamine and cyanuric acid for 28 days on the ovaries of female rats. Rats that were exposed to the mixture exhibited reduced ovarian and uterine weights, a shorter estrous cycle, and reduced serum estrogen and progesterone levels compared to rats that were exposed to melamine and control rats. Furthermore, morphological analysis revealed pathological changes in the ovaries of rats exposed to melamine or the mixture, such as more atretic follicles and necrosis of oocytes and granulosa cells. TUNEL staining revealed that the exposed groups had a higher proportion of TUNEL-positive granulosa cells than the control group, and the mRNA expressions of SOD1, GPX1, GPX2, P450scc, 17β-HSD I, and 17β-HSD II were reduced in the exposure groups compared with the control group. These results indicated that exposure to melamine alone or to the melamine-cyanuric acid mixture could damage the ovaries in rats.     

Bacterial Ammeline Metabolism via Guanine Deaminase

Melamine toxicity in mammals has been attributed to the blockage of kidney tubules by insoluble complexes of melamine with cyanuric acid or uric acid. Bacteria metabolize melamine via three consecutive deamination reactions to generate cyanuric acid. The second deamination reaction, in which ammeline is the substrate, is common to many bacteria, but the genes and enzymes responsible have not been previously identified. Here, we combined bioinformatics and experimental data to identify guanine deaminase as the enzyme responsible for this biotransformation. The ammeline degradation phenotype was demonstrated in wild-type Escherichia coli and Pseudomonas strains, including E. coli K12 and Pseudomonas putida KT2440. Bioinformatics analysis of these and other genomes led to the hypothesis that the ammeline deaminating enzyme was guanine deaminase. An E. coli guanine deaminase deletion mutant was deficient in ammeline deaminase activity, supporting the role of guanine deaminase in this reaction. Two guanine deaminases from disparate sources (Bradyrhizobium japonicum USDA 110 and Homo sapiens) that had available X-ray structures were purified to homogeneity and shown to catalyze ammeline deamination at rates sufficient to support bacterial growth on ammeline as a sole nitrogen source. In silico models of guanine deaminase active sites showed that ammeline could bind to guanine deaminase in a similar orientation to guanine, with a favorable docking score. Other members of the amidohydrolase superfamily that are not guanine deaminases were assayed in vitro, and none had substantial ammeline deaminase activity. The present study indicated that widespread guanine deaminases have a promiscuous activity allowing them to catalyze a key reaction in the bacterial transformation of melamine to cyanuric acid and potentially contribute to the toxicity of melamine.Ammeline is an intermediate in the bacterial metabolism of melamine (Fig. ​(Fig.1).1). Melamine has become internationally recognized as a chemical adulterant in pet foods and infant formula that caused morbidity and mortality in pets and children (12). In pets, where more than 1,000 deaths have been attributed to melamine poisoning, the composition of the causal kidney precipitate was found to be a 1:1 complex of melamine-cyanuric acid (2, 25). In human babies, melamine-uric acid cocrystals have been identified (11). Feeding animals a mixture of melamine and cyanuric acid or a mixture of melamine, ammeline, ammelide, and cyanuric acid was found to produce acute kidney disease (5). Melamine and cyanuric acid are known to form a highly insoluble, hydrogen-bonded network (33) that can precipitate in the kidneys, causing kidney failure. Since bacterial metabolism of melamine generates cyanuric acid (6, 7, 14), it is possible that bacterial melamine metabolism could contribute to melamine toxicity in some cases.Open in a separate windowFIG. 1.The known metabolic pathway in bacteria for transforming melamine to cyanuric acid.Bacteria metabolize melamine by sequential deamination (4, 6, 7, 14, 30) to ammeline, ammelide, and cyanuric acid (Fig. ​(Fig.1).1). The genes and enzymes involved in the deamination of melamine and ammelide are known. Melamine deaminases (TriA and TrzA) have been purified and characterized (20, 28). The enzymes AtzC (34) and TrzC (7) were shown to be capable of ammelide deamination. Although ammeline deamination has been observed in a large number of microbial strains (37), the genes and enzymes involved in bacterial ammeline deamination have remained obscure. Many of the bacteria and fungi that were shown to deaminate ammeline did not deaminate melamine or ammelide (37), indicating that these ammeline deaminating enzymes have not evolved as a component of the melamine degradation pathway.Enzymes functioning in the metabolism of the s-triazine herbicide atrazine are related to some of the enzymes in the melamine pathway. TriA (melamine deaminase) is related to AtzA (28, 31) and TrzN (35), enzymes that catalyze the dechlorination of atrazine. AtzB, which catalyzes the second step in the atrazine metabolic pathway, was reported to also deaminate ammeline as a side reaction, but the rate of the reaction was not measured in that study (27).The enzymes involved in melamine and ammelide deamination, along with other enzymes acting on s-triazine herbicides, are all members of the amidohydrolase superfamily (32, 36). These enzymes typically contain one or two metal ions that are involved in activating water for nucleophilic displacement reactions. A significant number of amidohydrolase superfamily members catalyze deamination reactions with nitrogen heterocyclic ring substrates. In this context, we specifically analyzed the amidohydrolase enzymes in ammeline-metabolizing bacteria to identify the enzyme responsible for the activity. Molecular genetic, biochemical, and in silico data support the hypothesis that guanine deaminase functions as the principal ammeline deaminase activity of bacteria. This has implications for enzyme catalytic promiscuity and understanding the bacterial metabolism of melamine. The latter may be relevant to melamine toxicity in humans and animals.     

Thermostable Cyanuric Acid Hydrolase from Moorella thermoacetica ATCC 39073

Cyanuric acid, a metabolic intermediate in the degradation of many s-triazine compounds, is further metabolized by cyanuric acid hydrolase. Cyanuric acid also accumulates in swimming pools due to the breakdown of the sanitizing agents di- and trichloroisocyanuric acid. Structurally stable cyanuric acid hydrolases are being considered for usage in pool water remediation. In this study, cyanuric acid hydrolase from the thermophile Moorella thermoacetica ATCC 39073 was cloned, expressed in Escherichia coli, and purified to homogeneity. The recombinant enzyme was found to have a broader temperature range and greater stability, at both elevated and low temperatures, than previously described cyanuric acid hydrolases. The enzyme had a narrow substrate specificity, acting only on cyanuric acid and N-methylisocyanuric acid. The M. thermoacetica enzyme did not require metals or other discernible cofactors for activity. Cyanuric acid hydrolase from M. thermoacetica is the most promising enzyme to use for cyanuric acid remediation applications.s-Triazine compounds have diverse applications as herbicides, resins, and disinfectants. The s-triazine herbicides, such as atrazine, help to promote high-yield, sustainable agriculture. Melamine, or triamino-s-triazine, is a high-volume industrial chemical. Melamine-based polymers have outstanding thermosetting properties, ideal for their use in kitchen utensils and plates, as high-pressure laminates such as Formica, and as whiteboards. Di- and tri-chloroisocyanuric acids find widespread application as disinfectants, algicides, and bactericides. The chlorinated isocyanuric acids are used in wastewater treatment, in the textile industry as bleaching compounds, and in preventing and curing diseases in husbandry and fisheries. A major use of these compounds is for swimming pool chlorination. They have outstanding performance for maintaining a high, stable chlorine content by dissolving slowly in water, allowing a continuous metered dosing of chlorine.Degradation of these and other s-triazine compounds results in the production of cyanuric acid (Fig. ​(Fig.1).1). Cyanuric acid has come under increased scrutiny because of its potential involvement in comediating toxicity resulting from the ingestion of melamine (10). Recently, melamine has been found in adulterated pet food and baby formula. Melamine and its metabolite cyanuric acid cocrystallize at low concentrations and are implicated in acute renal failure in cats that have consumed adulterated food products (10). Cyanuric acid degradation is also of interest from the perspective of environmental remediation. The use of di- or trichloroisocyanuric acid in pool water results in spontaneous chemical dechlorination that disinfects the water but also produces, as a by-product, large amounts of cyanuric acid. High levels of cyanuric acid perturb the equilibrium, thus preventing dechlorination by additional chlorinated isocyanuric acid, such that disinfection is not achieved. As a result, swimming pools must be emptied and refilled, using water and causing discharge issues. It would be desirable to remediate pool water in situ, conserving water, saving money, and extending pool water use. In this context, there is a need to better understand cyanuric acid degradation and to identify highly stable biological catalysts to use for this purpose.Open in a separate windowFIG. 1.Atrazine, ametryn, trichloroisocyanuric acid, and melamine are all metabolized via cyanuric acid that is transformed to biuret by the action of cyanuric acid hydrolases.Microbial enzymatic degradation of cyanuric acid has been studied previously (3, 4, 8, 18). Two distinct but homologous enzymes, AtzD from Pseudomonas sp. strain ADP (8) and TrzD from Pseudomonas sp. strain NRRLB-12227 (now called Acidovorax avenae subsp. citrulli) (11), have been studied in detail. These enzymes, known as cyanuric acid hydrolases, catalyze the conversion of cyanuric acid to biuret (Fig. ​(Fig.1).1). Biuret is not considered toxic to humans and degrades more readily than cyanuric acid.Barbiturase is the only protein known to be homologous to cyanuric acid hydrolase that has a defined and different physiological function. Barbiturase catalyzes the conversion of barbituric acid to ureidomalonic acid in organisms that catabolize pyrimidines by the oxidative pathway. Barbiturase is unstable at 4°C in the absence of ethylene glycol and dithiothreitol (DTT). Furthermore, activity is completely lost when the protein is maintained at 55°C for 30 min (20). AtzD and TrzD are relatively stable at 4°C, but they lose activity when frozen (our unpublished data). Moreover, the thermostability properties of AtzD and TrzD are not well studied, but these enzymes are derived from mesophilic bacteria. In this context, we initiated a search to identify a stable cyanuric acid hydrolase. Enzymes that are more stable in response to temperature changes are more stable in response to many environmental factors. Thus, a thermostable enzyme would be most applicable to pool water and other remediation efforts.We employed bioinformatic techniques that identified a cyanuric acid hydrolase homolog in Moorella thermoacetica ATCC 39073, an anaerobic, acetogenic bacterium that is able to grow at 65°C. The gene was cloned into E. coli, the protein was expressed at high levels, the recombinant E. coli strain degraded cyanuric acid, and the enzyme was obtained in homogeneous form by a convenient one-step purification. The enzyme''s function as a cyanuric acid hydrolase was confirmed, and it was shown to be significantly more stable than other known members of the cyanuric acid protein family.     

Protective Effects of Ferulic Acid on High Glucose-Induced Protein Glycation,Lipid Peroxidation,and Membrane Ion Pump Activity in Human Erythrocytes

Ferulic acid (FA) is the ubiquitous phytochemical phenolic derivative of cinnamic acid. Experimental studies in diabetic models demonstrate that FA possesses multiple mechanisms of action associated with anti-hyperglycemic activity. The mechanism by which FA prevents diabetes-associated vascular damages remains unknown. The aim of study was to investigate the protective effects of FA on protein glycation, lipid peroxidation, membrane ion pump activity, and phosphatidylserine exposure in high glucose-exposed human erythrocytes. Our results demonstrated that FA (10-100 μM) significantly reduced the levels of glycated hemoglobin (HbA1c) whereas 0.1-100 μM concentrations inhibited lipid peroxidation in erythrocytes exposed to 45 mM glucose. This was associated with increased glucose consumption. High glucose treatment also caused a significant reduction in Na⁺/K⁺-ATPase activity in the erythrocyte plasma membrane which could be reversed by FA. Furthermore, we found that FA (0.1-100 μM) prevented high glucose-induced phosphatidylserine exposure. These findings provide insights into a novel mechanism of FA for the prevention of vascular dysfunction associated with diabetes.     

A novel mixed-mode solid phase extraction for simultaneous determination of melamine and cyanuric acid in food by hydrophilic interaction chromatography coupled to tandem mass chromatography

Utilizing a solid phase extraction column (MCT) containing mixed hydrophilic functional gel and cation exchange sorbent, a sensitive and rapid HPLC–MS/MS method for simultaneously determining the residues of melamine (MEL) and cyanuric acid (CYA) in human foodstuffs was developed. MEL and CYA in egg, pork, liver, kidney and pork, shrimp, sausage casing, honey, soybean milk, soybean powder and dairy product were extracted using acetonitrile/water, defatted with hexane and isolated using MCT solid phase extraction column. The residues were separated upon a hydrophilic interaction liquid chromatography (HILIC) column and analyzed by electrospray ionization under negative–positive switched mode on a triplequadrupole mass spectrometry. The selected reaction monitoring was performed on [M+H]⁺ of m/z 127.9 to provide the transition of 127 > 85 and 127 > 68 (MEL) while the [M−H]⁻ of m/z 127.1 was selected as the precursor ion for CYA resulting in product ions m/z 85 and 42. Isotope labeled internal standard (¹⁵N₃-MEL and ¹³C₃-CYA) and matrix-matched calibration were both used to observe the recovery to be 70.0–129.6% and 70.0–128.9% with RSD of 1.4–23.3% and 1.5–21.7% for MEL and CYA, respectively (n = 6). All the LODs and LOQs of MEL and CYA were less than 39.4 and 99.1 μg kg⁻¹, respectively, in 18 matrices, which were sensitive enough for quantitative analysis. This method has been proven effective in simultaneous determination of melamine and cyanuric acid when inspecting unknown and positive samples.     

Methionine transport in the malaria parasite Plasmodium falciparum

The intraerythrocytic malaria parasite, Plasmodium falciparum, derives amino acids from the digestion of host cell haemoglobin. However, it also takes up amino acids from the extracellular medium. Isoleucine is absent from adult human haemoglobin and an exogenous source of isoleucine is essential for parasite growth. An extracellular source of methionine is also important for the normal growth of at least some parasite strains. In this study we have characterised the uptake of methionine by P. falciparum-infected human erythrocytes, and by parasites functionally isolated from their host cells by saponin-permeabilization of the erythrocyte membrane. Infected erythrocytes take up methionine much faster than uninfected erythrocytes, with the increase attributable to the flux of this amino acid via the New Permeability Pathways induced by the parasite in the erythrocyte membrane. Having entered the infected cell, methionine is taken up by the intracellular parasite via a saturable, temperature-dependent process that is independent of ATP, Na⁺ and H⁺. Substrate competition studies, and comparison of the transport of methionine with that of isoleucine and leucine, yielded results consistent with the hypothesis that the parasite has at its surface one or more transporters which mediate the flux into and out of the parasite of a broad range of neutral amino acids. These transporters function most efficiently when exchanging one neutral amino acid for another, thus providing a mechanism whereby the parasite is able to import important exogenous amino acids in exchange for surplus neutral amino acids liberated from the digestion of host cell haemoglobin.     

Adhesion of Annexin 7 Deficient Erythrocytes to Endothelial Cells

Annexin 7 deficiency has previously been shown to foster suicidal death of erythrocytes or eryptosis, which is triggered by increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and characterized by cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface. Eryptosis following increase of [Ca²⁺]_i by Ca²⁺ ionophore ionomycin, osmotic shock or energy depletion was more pronounced in erythrocytes from annexinA7-deficient mice (anxA7^−/−) than in erythrocytes from wild type mice (anxA7^+/+). As phosphatidylserine exposure is considered to mediate adhesion of erythrocytes to the vascular wall, the present study explored adhesion of erythrocytes from anx7^−/− and anx7^+/+-mice following increase of [Ca²⁺]_i by Ca²⁺ ionophore ionomycin (1 µM for 30 min), hyperosmotic shock (addition of 550 mM sucrose for 2 hours) or energy depletion (removal of glucose for 12 hours). Phosphatidylserine exposing erythrocytes were identified by annexin V binding, cell volume estimated from forward scatter in FACS analysis and adhesion to human umbilical vein endothelial cells (HUVEC) utilizing a flow chamber. As a result, ionomycin, sucrose addition and glucose removal all triggered phosphatidylserine-exposure, decreased forward scatter and enhanced adhesion of erythrocytes to human umbilical vein endothelial cells (HUVEC), effects significantly more pronounced in anx7^−/− than in anx7^+/+-erythrocytes. Following ischemia, morphological renal injury was significantly higher in anx7^−/− than in anx7^+/+-mice. The present observations demonstrate that enhanced eryptosis of annexin7 deficient cells is paralleled by increased adhesion of erythrocytes to the vascular wall, an effect, which may impact on microcirculation during ischemia.     

Inactivation of fatty acid amide hydrolase protects against ischemic reperfusion injury-induced renal fibrogenesis

Although cannabinoid receptors (CB) are recognized as targets for renal fibrosis, the roles of endogenous cannabinoid anandamide (AEA) and its primary hydrolytic enzyme, fatty acid amide hydrolase (FAAH), in renal fibrogenesis remain unclear. The present study used a mouse model of post-ischemia-reperfusion renal injury (PIR) to test the hypothesis that FAAH participates in the renal fibrogenesis. Our results demonstrated that PIR showed upregulated expression of FAAH in renal proximal tubules, accompanied with decreased AEA levels in kidneys. Faah knockout mice recovered the reduced AEA levels and ameliorated PIR-triggered increases in blood urea nitrogen, plasma creatinine as well as renal profibrogenic markers and injuries. Correspondingly, a selective FAAH inhibitor, PF-04457845, inhibited the transforming growth factor-beta 1 (TGF-β1)–induced profibrogenic markers in human proximal tubular cell line (HK-2 cells) and mouse primary cultured tubular cells. Knockdown of FAAH by siRNA in HK-2 cells had similar effects as PF-04457845. Tubular cells isolated from Faah^?/? mice further validated the protection against TGF-β1–induced damages. The CB 1 or CB2 receptor antagonist and exogenous FAAH metabolite arachidonic acid failed to reverse the protective effects of FAAH inactivation in HK-2 cells. However, a substrate-selective inhibitor of AEA-cyclooxygenase-2 (COX-2) pathway significantly suppressed the anti-profibrogenic actions of FAAH inhibition. Further, the AEA-COX-2 metabolite, prostamide E2 exerted anti-fibrogenesis effect. These findings suggest that FAAH activation and the consequent reduction of AEA contribute to the renal fibrogenesis, and that FAAH inhibition protects against fibrogenesis in renal cells independently of CB receptors via the AEA-COX-2 pathway by the recovery of reduced AEA.     

PERMEABILITY OF ERYTHROCYTES TO RADIOACTIVE POTASSIUM

The diffusion coefficients for the exchange of potassium across the membrane of erythrocytes of humans, rats, and rabbits have been determined by the use of artificially radioactive potassium, both into and out of the erythrocytes both in vitro and in vivo. The diffusion coefficients found in minutes^–1 were 0.2 to 0.25 x 10^–3 for human, 0.32 to 0.665 x 10^–3 for rabbits, and 1.0 x 10^–3 for rat erythrocytes. Rabbit erythrocytes appear to be more permeable in vivo. Reasons are advanced to explain the failure of earlier workers to demonstrate appreciable exchange of potassium in erythrocytes.     

Hypochlorous acid inhibits glutathione S-conjugate export from human erythrocytes

It was found that the hypochlorous acid (HOCl) inhibits the active efflux of glutathione S-conjugates, 2,4-dinitrophenyl-S-glutathione (DNP-SG, c_50%=258±24 μM HOCl) and bimane-S-glutathione (B-SG, c_50%=125±16 μM HOCl) from human erythrocytes, oxidises intracellular reduced glutathione (the ratio [HOCl]/[GSH]_oxidized=4) and inhibits basal as well as 2,4-dinitrophenol- (DNP) and 2,4-dinitrophenyl-S-glutathione (DNP-SG)-stimulated Mg²⁺-ATPase activities of erythrocyte membranes. Multidrug resistance-associated protein (MRP1) mediates the active export of glutathione S-conjugates in mammalian cells, including human erythrocytes. A direct impairment of erythrocyte membrane MRP by hypochlorous acid was shown by electrophoresis and immunoblotting (c_50%=478±36 μM HOCl). The stoichiometry of the MRP/HOCl reaction was 1:1. These results demonstrate that MRP can be one of the cellular targets for the inflammatory mediator hypochlorous acid.     

The uptake of cyclic AMP by human erythrocytes and its effect on membrane phosphorylation.

The effects of time and cyclic AMP concentration on cyclic AMP uptake and membrane phosphorylation were studied using intact human erythrocytes. The rate of uptake of cyclic [³H]AMP was nearly linear with respect to cyclic AMP concentration. The amount taken up was small compared to the extracellular cyclic AMP concentration, but was sufficient to significantly increase the intracellular cyclic AMP concentration. Incubation with cyclic AMP resulted in increased incorporation of ³²P_i into several phosphorylated membrane peptides of the intact erythrocytes. Although cyclic AMP altered the distribution of radioactivity among the membrane components, the total amount of incorporation was not increased. The effect of cyclic AMP on phosphorylation of membrane peptides was observed with extracellular cyclic AMP concentrations as low as 1 μm and was most pronounced in incubations of 1 to 4 h. These results indicate that cyclic AMP can enter erythrocytes in sufficient amounts to alter the activity of cyclic AMP-dependent protein kinases, or to alter the rate of turnover of certain phosphorylated membrane peptides.     

Enzymatic Degradation of Chlorodiamino-s-Triazine

2-Chloro-4,6-diamino-s-triazine (CAAT) is a metabolite of atrazine biodegradation in soils. Atrazine chlorohydrolase (AtzA) catalyzes the dechlorination of atrazine but is unreactive with CAAT. In this study, melamine deaminase (TriA), which is 98% identical to AtzA, catalyzed deamination of CAAT to produce 2-chloro-4-amino-6-hydroxy-s-triazine (CAOT). CAOT underwent dechlorination via hydroxyatrazine ethylaminohydrolase (AtzB) to yield ammelide. This represents a newly discovered dechlorination reaction for AtzB. Ammelide was subsequently hydrolyzed by N-isopropylammelide isopropylaminohydrolase to produce cyanuric acid, a compound metabolized by a variety of soil bacteria.     

The Relationship between Cell Membrane Potassium Ion Transport and Glycolysis : The effect of ethacrynic acid

Cell membrane transport of K⁺ stimulates the rate of glycolysis in Ehrlich ascites tumor cells. A study of the characteristics of this relationship indicates that the stimulation occurs under anaerobic as well as under aerobic conditions. The data suggest that glycolysis is stimulated by a K⁺ transport mechanism that is coupled to Na⁺ transport because the effect is blunted or abolished when the principal intracellular ion is lithium or choline. This stimulus to glycolysis is blocked by ouabain and ethacrynic acid, agents that have been shown to inhibit monovalent cation transport in erythrocytes. In contrast to the action of ouabain, glycolysis is inhibited by ethacrynic acid in Ehrlich ascites tumor cells in the absence of cell membrane K⁺ transport. In studies with ghost-free hemolysates of human erythrocytes and with cytosol prepared from Ehrlich ascites tumor cells, ethacrynic acid significantly blocks lactate formation from fructose diphosphate demonstrating the direct inhibitory effect of this agent on one or more enzymes of the Embden-Meyerhof pathway. Since ethacrynic acid has no influence on lactate formation in intact erythrocytes utilizing an endogenous substrate, the presumptive site of inhibition is proximal to the 3-phosphoglycerate level.     

Effects of Melamine and Cyanuric Acid on Embryo‐Fetal Development in Rats

After the outbreak of acute renal failure associated with melamine‐contaminated pet food, melamine and melamine‐related compounds have become of great interest from a toxicologic perspective. We investigated the potential effects of melamine in combination with cyanuric acid (M + CA, 1:1) on pregnant dams and embryo‐fetal development in rats. M + CA was orally administered to pregnant rats from gestational days 6 through 19 at doses of 0, 3, 10, and 30 mg/kg/day of both melamine and cyanuric acid. Maternal toxicity of rats administered 30 mg/kg/day M + CA was manifested as increased incidences of clinical signs and death; gross pathologic findings; higher blood urea nitrogen and creatinine levels; lower body weight gain and food intake; decreased thymus weight; and increased heart, lung, and kidney weights. Histopathological examinations revealed an increase in the incidence of congestion, tubular necrosis/degeneration, crystals, casts, mineralization, inflammatory cells in tubules, tubular dilation, and atrophy of glomeruli in maternal kidneys, whereas fetal kidneys did not show any histopathological changes. Developmental toxicity included a decrease in fetal (28%) and placental weights and a delay in fetal ossification (n = 7). Increased incidence of gross and histopathological changes in the maternal kidney was also found in the middle dose group (n = 12). No treatment‐related maternal or developmental effects were observed in the low dose group (n = 12). Under these experimental conditions, M + CA is embryotoxic at an overt maternotoxic dose in rats and the no‐observed‐adverse‐effect level of M + CA is considered to be 3 mg/kg/day for pregnant dams and 10 mg/kg/day for embryo‐fetal development.     

The interaction of human erythrocyte band 3 with cytoskeletal components

Band 3 of the human erythrocyte is involved in anion transport and binding of the cytoskeleton to the membrane bilayer. Human erythrocytes were treated to incorporate varying concentrations of DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid) a non-penetrating, irreversible inhibitor of anion transport, and both functions of Band 3 were analyzed. The rate of efflux of ³⁵SO₄. was measured and the binding of cytoskeletal components to the membrane was evaluated by extracting the membranes with 0.1 n NaOH and analyzing for the peptides remaining with the membrane. It was found that 0.1 n NaOH extracts all the extrinsic proteins from membranes of untreated cells, while, in the case of the membranes from cells treated with DIDS, a portion of the cytoskeletal components, spectrin (Bands 1 and 2) and Band 2.1 (ankyrin, syndein) remain with the membrane. The amount of these cytoskeletal components remaining with the membrane depends on the concentrations of DIDS incorporated. The effect of DIDS on the extractability of the spectrin-Band 2.1 complex correlates well with DIDS inhibition of anion transport (r = 0.91). At DIDS concentrations which completely inhibit anion transport, about 10% of total spectrin-Band 2.1 complex remains unextracted. Another anion-transport inhibitor, pyridoxal phosphate, has no effect on binding of the cytoskeleton to the membrane. On the other hand, digestion of DIDS-pretreated intact erythrocytes with Pronase, chymotrypsin, or trypsin releases the tight binding of Band 3 to cytoskeleton on the inside of the membrane. Since trypsin does not hydrolyze Band 3 the data suggest that a second membrane protein which is trypsin sensitive may be involved with Band 3 in cytoskeletal binding.     

Bacterial Cyanuric Acid Hydrolase for Water Treatment

Di- and trichloroisocyanuric acids are widely used as water disinfection agents, but cyanuric acid accumulates with repeated additions and must be removed to maintain free hypochlorite for disinfection. This study describes the development of methods for using a cyanuric acid-degrading enzyme contained within nonliving cells that were encapsulated within a porous silica matrix. Initially, three different bacterial cyanuric acid hydrolases were compared: TrzD from Acidovorax citrulli strain 12227, AtzD from Pseudomonas sp. strain ADP, and CAH from Moorella thermoacetica ATCC 39073. Each enzyme was expressed recombinantly in Escherichia coli and tested for cyanuric acid hydrolase activity using freely suspended or encapsulated cell formats. Cyanuric acid hydrolase activities differed by only a 2-fold range when comparing across the different enzymes with a given format. A practical water filtration system is most likely to be used with nonviable cells, and all cells were rendered nonviable by heat treatment at 70°C for 1 h. Only the CAH enzyme from the thermophile M. thermoacetica retained significant activity under those conditions, and so it was tested in a flowthrough system simulating a bioreactive pool filter. Starting with a cyanuric acid concentration of 10,000 μM, more than 70% of the cyanuric acid was degraded in 24 h, it was completely removed in 72 h, and a respike of 10,000 μM cyanuric acid a week later showed identical biodegradation kinetics. An experiment conducted with water obtained from municipal swimming pools showed the efficacy of the process, although cyanuric acid degradation rates decreased by 50% in the presence of 4.5 ppm hypochlorite. In total, these experiments demonstrated significant robustness of cyanuric acid hydrolase and the silica bead materials in remediation.