A toxicokinetic model to assess the risk of azinphosmethyl exposure in humans through measures of urinary elimination of alkylphosphates. (1/724)

Azinphosmethyl (APM) is one of the most common insecticides used in fruit farming. The object of this paper is to develop a quick and practical test for assessing the risk for humans coming into contact with APM. It has been shown that the principal component of occupational and/or accidental exposure is through the skin (C. A. Franklin et al., 1981, J. Toxicol. Environ. Health 7, 715-731), but our approach is applicable to exposures via any route or a combination of routes. The method proposed in the present paper can accommodate a single-event exposure or repeated exposures over long periods. Urinary alkylphosphate (AP) metabolites are reliable bioindicators of the presence of APM in the body; they are easily accessible and can be used to estimate APM body burden. We developed a simple toxicokinetic model to link the time varying APM body burden to absorbed doses and to rates of elimination in the form of AP urinary metabolites. Using this model and data available in the literature, we are able to propose a "no observed adverse effect level" (NOAEL) for APM body levels and for corresponding absorbed doses. We have established that after a single exposure, the safe limit corresponding to the NOAEL is reached at a cumulative 0.215 mumoles AP/kg bw eliminated in urine in the first 24 hours following the beginning of exposure. For repeated daily exposures at steady state, the corresponding urinary AP metabolite level is equal to a cumulative 0.266 mumoles AP/kg bw eliminated per 24 hours.  (+info)

Mechanistic alternatives in phosphate monoester hydrolysis: what conclusions can be drawn from available experimental data? (2/724)

Phosphate monoester hydrolysis reactions in enzymes and solution are often discussed in terms of whether the reaction pathway is associative or dissociative. Although experimental results for solution reactions have usually been considered as evidence for the second alternative, a closer thermodynamic analysis of observed linear free energy relationships shows that experimental information is consistent with the associative, concerted and dissociative alternatives.  (+info)

EnvZ-independent phosphotransfer signaling pathway of the OmpR-mediated osmoregulatory expression of OmpC and OmpF in Escherichia coli. (3/724)

The Escherichia coli EnvZ-OmpR regulatory system is a paradigm of intracellular signal transduction mediated by the well-documented phosphotransfer mechanism, by which the expression of the major outer membrane proteins, OmpC and OmpF, is regulated in response to the medium osmolarity. Although it is clear that the EnvZ histidine(His)-kinase is the major player in the phosphorylation of OmpR, it has been assumed for some time that there may be an alternative phospho-donor(s) that can phosphorylate OmpR under certain in vitro and in vivo conditions. In this study, to address this long-standing issue, extensive genetic studies were done with certain mutant alleles, including delta envZ, delta(ackA-pta), and delta sixA, as well as delta ompR. Here, for the first time, genetic evidence is provided that, in addition to EnvZ, acetyl phosphate and an as yet unidentified sensor His-kinase can serve as alternative in vivo phospho-donors for OmpR, even in the envZ+ background. A model for the alternative phosphotransfer signaling pathway involved in the phosphorylation of OmpR is proposed.  (+info)

Vitamin B6 biosynthesis: formation of pyridoxine 5'-phosphate from 4-(phosphohydroxy)-L-threonine and 1-deoxy-D-xylulose-5-phosphate by PdxA and PdxJ protein. (4/724)

In Escherichia coli the coenzyme pyridoxal 5'-phosphate (PLP) is synthesised de novo by a pathway that is thought to involve the condensation of 4-(phosphohydroxy)-L-threonine and 1-deoxy-D-xylulose, catalysed by the enzymes PdxA and PdxJ, to form either pyridoxine (vitamin B6) or pyridoxine 5'-phosphate (PNP). Here we show that incubation of PdxJ with PdxA, 4-(phosphohydroxy)-L-threonine, NAD and 1-deoxy-D-xylulose-5-phosphate, but not 1-deoxy-D-xylulose, results in the formation of PNP. The PNP formed was characterised by (i) cochromatography with an authentic standard, (ii) conversion to pyridoxine by alkaline phosphatase treatment, and (iii) UV and fluorescence spectroscopy. Furthermore, when [2-(14)C]1-deoxy-D-xylulose-5-phosphate was used as a substrate, the radioactivity was incorporated into PNP. These results clarify the previously unknown role of PdxJ in the de novo PLP biosynthetic pathway. The sugar used as substrate by PdxJ is 1-deoxy-D-xylulose-5-phosphate rather than the previously assumed 1-deoxy-D-xylulose. The first vitamin B6 vitamer synthesised is PNP, and not pyridoxine.  (+info)

Conservation of nitrogen in cattle feedlot waste with urease inhibitors. (5/724)

Feedlot cattle normally retain less than 20% of their dietary nitrogen intake. Sixty to 80% of the nitrogen excreted is normally lost through volatilization of ammonia, which is primarily generated from urea. This loss of ammonia nitrogen pollutes the environment and creates an unfavorable ratio of nitrogen to phosphorous (N:P) in the waste for crop growth. Two urease inhibitors, cyclohexylphosphoric triamide (CHPT) and N-(n-butyl) thiophosphoric triamide (NBPT) were evaluated for their ability to reduce the rate of urea hydrolysis in beef cattle feedlot pens. Initially, a total of six pens were used, two pens per treatment, with approximately 70 cattle per pen, and a single topical application of CHPT or NBPT at 20 mg/kg of manure. Essentially no urea was found in untreated pens. However, with CHPT treatment, 2 g of urea/kg of dry manure accumulated by d 4, and all gradually disappeared by d 11; NBPT conserved 3 and 3.5 g of urea/kg by d 4 and 9, respectively, and it had disappeared by d 14 (treatment [trt] x day, P = .003). A second study involved application of NBPT weekly for 6 wk. This caused urea to accumulate to a peak concentration of 17 g/kg of manure by d 30 (trt x day2, P = .001). Once the treatment was stopped the urea concentration began to decrease. When the NBPT was applied weekly, the concentration of ammonia in the waste was less for the treated pens (trt x day, P = .01), the total nitrogen was greater (trt x day, P = .04), pH tended to be lower (trt x day, P = .10), and the total volatile acids were not different (trt x day, P = .51) from untreated pens. We concluded that urease inhibitors could be used to control ammonia emissions from animal wastes, prevent environmental damage, and produce a more balanced (N:P) fertilizer from manure.  (+info)

Calcium dependence of Pi phosphorylation of sarcoplasmic reticulum Ca2+-ATPase at low water content: water dependence of the E2-->E1 conversion. (6/724)

Enzymes entrapped in reverse micelles can be studied in low-water environments that have the potential of restricting conformational mobility in specific steps of the reaction cycle. Sarcoplasmic reticulum Ca2+-ATPase was incorporated into a reverse-micelle system (TPT) composed of toluene, phospholipids, Triton X-100 and varying amounts of water (0.5-7%, v/v). Phosphorylation of the Ca2+-ATPase by ATP required the presence of both water and Ca2+ in the micelles. No phosphoenzyme (EP) was detected in the presence of EGTA. Phosphorylation by Pi (inorganic phosphate) in the absence of Ca2+ was observed at water content below that necessary for phosphorylation by ATP. In contrast to what is observed in a totally aqueous medium, EP formed by Pi was partially resistant to dephosphorylation by Ca2+. However, the addition of non-radioactive Pi to the EP already formed caused a rapid decrease in radiolabelled enzymes, as expected for the isotopic dilution, indicating the existence of an equilibrium (E+Pi<-->EP). Phosphorylation by Pi also occurred in TPT containing millimolar Ca2+ concentrations in a range of water concentrations (2-5% v/v). The substrates p-nitrophenyl phosphate, acetyl phosphate, ATP and GTP increased the EP level under these conditions. These results suggest that: (1) the rate of conversion of the ATPase conformer E2 into E1 is greatly reduced at low water content, so that E2-->E1 becomes the rate-limiting step of the catalytic cycle; and (2) in media of low water content, Pi can phosphorylate both E1Ca and E2. Thus, the effect of enzyme hydration is complex and involves changes in the phosphorylation reaction at the catalytic site, in the equilibrium between E2 and E1 conformers, and in their specificity for substrates.  (+info)

Formation of adenosine 5'-tetraphosphate from the acyl phosphate intermediate: a difference between the MurC and MurD synthetases of Escherichia coli. (7/724)

The mechanism of the Mur synthetases of peptidoglycan biosynthesis is thought to involve in each case the successive formation of an acyl phosphate and a tetrahedral intermediate. The existence of the acyl phosphates for the MurC and MurD enzymes from Escherichia coli was firmly established by their in situ reduction by sodium borohydride followed by acid hydrolysis, yielding the corresponding amino alcohols. Furthermore, it was found that MurD, but not MurC, catalyses the synthesis of adenosine 5'-tetraphosphate from the acyl phosphate, thereby substantiating its existence and pointing out a difference between the two enzymes.  (+info)

Stimulated activity of human topoisomerases IIalpha and IIbeta on RNA-containing substrates. (8/724)

Eukaryotic topoisomerase II is a dimeric nuclear enzyme essential for DNA metabolism and chromosome dynamics. Central to the activities of the enzyme is its ability to introduce transient double-stranded breaks in the DNA helix, where the two subunits of the enzyme become covalently attached to the generated 5'-ends through phosphotyrosine linkages. Here, we demonstrate that human topoisomerases IIalpha and IIbeta are able to cleave ribonucleotide-containing substrates. With suicide substrates, which are partially double-stranded molecules containing a 5'-recessed strand, cleavage of both strands was stimulated approximately 8-fold when a ribonucleotide rather than a deoxyribonucleotide was present at the scissile phosphodiester of the recessed strand. The existence of a ribonucleotide at the same position in a normal duplex substrate also enhanced topoisomerase II-mediated cleavage, although to a lesser extent. The enzyme covalently linked to the 5'-ribonucleotide in the cleavage complex efficiently performed ligation, and ligation occurred equally well to acceptor molecules terminated by either a 3'-ribo- or deoxyribonucleotide. Besides the enhanced topoisomerase II-mediated cleavage of ribonucleotide-containing substrates, cleavage of such substrates could be further stimulated by ATP or antitumor drugs. In conclusion, the observed in vitro activities of the human topoisomerase II isoforms indicate that the enzymes can operate on RNA or RNA-containing substrates and thus might possess an intrinsic RNA topoisomerase activity, as has previously been demonstrated for Escherichia coli topoisomerase III.  (+info)