Organophosphorus pesticide degradation product in vitro metabolic stability and time-course uptake and elimination in rats following oral and intravenous dosing.

Levels of urinary dialkylphosphates (DAPs) are currently used as a biomarker of human exposure to organophosphorus insecticides (OPs). It is known that OPs degrade on food commodities to DAPs at levels that approach or exceed those of the parent OP. However, little has been reported on the extent of DAP absorption, distribution, metabolism and excretion. The metabolic stability of O,O-dimethylphosphate (DMP) was assessed using pooled human and rat hepatic microsomes. Time-course samples were collected over 2 h and analyzed by LC-MS/MS. It was found that DMP was not metabolized by rat or pooled human hepatic microsomes. Male Sprague-Dawley rats were administered DMP at 20 mg kg(-1) via oral gavage and i.v. injection. Time-course plasma and urine samples were collected and analyzed by LC-MS/MS. DMP oral bioavailability was found to be 107 ± 39% and the amount of orally administered dose recovered in the urine was 30 ± 9.9% by 48 h. The in vitro metabolic stability, high bioavailability and extent of DMP urinary excretion following oral exposure in a rat model suggests that measurement of DMP as a biomarker of OP exposure may lead to overestimation of human exposure.

A gas chromatograph/resonant electron capture-TOF mass spectrometer for four dimensions of negative ion analytical information.

A prototype gas chromatograph (GC) electron monochromator (EM) reflectron time-of-flight (TOF) mass spectrometer has been constructed and demonstrated to simultaneously record four-dimensional resonant electron capture (REC) mass spectra (m/z, ion-intensity, electron-energy, and retention time) of electron-capturing compounds in real time. Specifically, complete REC mass spectra of all of the components in a mixture of perfluorocarboxylic acids and in a sample of pentafluorobenzyl alcohol were recorded in the GC mode. For each compound, the data enable one to distinguish different electronic states of the molecular ion and different possible decomposition pathways for each state. This new instrument can be used to obtain analytical information unrecognizable by any other mass spectrometric technique from the isomeric species of a variety of electron-capturing structures.

Chloroplast biogenesis. Cell-free transfer of envelope monogalactosylglycerides to thylakoids.

An ATP- and temperature-dependent transfer of monogalactosylglycerides from the chloroplast envelope to the chloroplast thylakoids was reconstituted in a cell-free system prepared from isolated chloroplasts of garden pea (Pisum sativum) or spinach (Spinacia oleracea). Isolated envelope membranes, in which the label was present exclusively in monogalactosylglycerides, were prepared radiolabeled in vitro with [14C]galactose from UDP-[14C]galactose to label galactolipids as the donor. ATP-dependent transfer of radioactivity from donor to unlabeled acceptor thylakoids, immobilized on nitrocellulose strips, was observed. In some experiments linear transfer for longer than 30 min of incubation was facilitated by the addition of stroma proteins but in other experiments stroma was without effect or inhibitory suggesting no absolute requirements for a soluble protein carrier. Transfer was donor specific. No membrane fraction tested (plasma membrane, tonoplast, endoplasmic reticulum, nuclei, Golgi apparatus, mitochondria or thylakoids) (isolated from tissue radiolabeled in vivo with [14C]acetate) other than chloroplast envelopes demonstrated any significant ability to transfer labeled membrane lipids to immobilized thylakoids. Acceptor specificity, while not absolute, showed a 3-10-fold greater ATP-dependent transfer of labeled galactolipids from chloroplast envelopes to immobilized thylakoids than to other leaf membranes. The results provide independent confirmation of the potential for transfer of galactolipids between chloroplast envelopes and thylakoids suggested previously from ultrastructural studies and of the known location of thylakoid galactolipid biosynthetic activities in the chloroplast envelope.

Phosphatidylinositol turnover in isolated soybean membranes stimulated by the synthetic growth hormone 2,4-dichlorophenoxyacetic acid.

Phospholipids of plant membranes isolated from homogenates of dark-grown hypocotyls of soybean (Glycine max L.) undergo rapid and specific degradative changes. The degradation of phosphatidylinositol (PI) in such membranes is enhanced in the presence of the synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), measured as the hydrolysis of PI or by an enhancement of [3H]inositol incorporation into membrane-associated PI stimulated by Mn2+, but not dependent upon added CTP, Mg2+, or diglyceride. The response is rapid and enhanced by auxin throughout the physiological range of growth-promoting concentrations (optimum at about 7 X 10(-7) M). The growth-inactive 2,4-D analogue, 2,3-dichlorophenoxyacetic acid (2,3-D), is without effect. These findings suggest a cell-free response of isolated membranes to the hormone mediated by a definable enzymatic reaction.

Phosphorylation of membrane-located proteins of soybean in vitro and response to auxin.

Isolated membranes of soybean incorporate (32)P from gamma-[(32)P]ATP in vitro. The incorporation was rapid and did not require added calcium. When displayed on 10% sodium dodecyl sulfate-polyacrylamide gels, several protein bands were revealed. An apparent auxin (2,4-dichlorophenoxyacetic acid) stimulation of (32)P incorporation into material from membrane vesicles insoluble in trichloroacetic acid-perchloric acid may be reflected partly in enhanced incorporation into protein bands with apparent molecular weights of 45,000 and 50,000. Additionally, a low molecular weight component was sometimes observed where incorporation was stimulated 2- to 3-fold by auxin. However, protein-bound radioactivity represented only a small fraction of the total radioactivity of the acid-insoluble material. Other labeled constituents, not retained on the gels, may contribute to the apparent, rapid (10 s or less) auxin response of the isolated membranes. Stimulation of incorporation into the low molecular weight component was given by diglyceride plus calcium, constituents known to augment protein kinase activities in other systems.

Gangliosides and other lipids of hyperplastic liver nodules induced by N-2-fluorenylacetamide.

Hyperplastic liver nodules induced in rats fed the carcinogen N-2-fluorenylacetamide had levels of total, protein-bound, and ganglioside sialic acid above those of controls. Yet, the total lipid content of hyperplastic nodules and surrounding tissue was nearly identical to the lipid content of control liver. Hyperplastic nodules and surrounding liver did not differ from control tissue in phospholipid distribution patterns or in fatty acid composition of total lipids, neutral lipids, and/or polar lipids. Elevated levels of both free and esterified sterols, and a slightly lower level of phospholipid, were found in both hyperplastic nodules and in surrounding tissue as compared to control liver. The increased sterol ester content was largely due to increased amounts of cholesterol oleate. Thin-layer chromatography of ganglioside extracts revealed all gangliosides of control liver. Amounts of tri- and tetrasialoganglioside were reduced and levels of disialoganglioside were increased in modules relative to both control liver and surrounding tissue. The ganglioside alterations may represent at least one significant minimal deviation of hyperplastic nodules toward the malignant phenotype in keeping with the hypothesis that hyperplastic nodules represent a pre malignant form of hyperplasia.

Simulation of action potential propagation in an inhomogeneous sheet of coupled excitable cells.

Cable theory and active equivalent circuits have been used to simulate the propagation of action potentials along a single nerve or muscle fiber by representing the cell as a unidimensional cable composed of isopotential segments. We extended this method to a two-dimensional sheet of cells which in many ways represents the atrium. Our method consisted of solving for the potential profile of a sheet composed of a large number of isopotential membrane patches, each of which was represented by an active equivalent circuit in which the ionic conductances were functions of voltage and time. The patches were arranged in a rectangular array with resistive interconnections that could be varied over the sheet. We used this model to study the effect of various inhomogeneities on conduction velocity and the resulting wave fronts in a sheet of excitable tissue. Some of these inhomogeneities included different effective internal resistances in the x and y directions, preferential pathways, and discrete regions of changing resistive connections. The results showed that very localized changes in membrane properties or cellular interconnections produce changes in the wave front over broad areas. This model provides a method for computing the wave fronts of action potential propagation in any two-dimensional inhomogeneous sheet of coupled excitable cells.