Determination of the stability constants of mercury(II) complexes of mixed donor macrobicyclic encapsulating ligands

The reactions of mercury(II) with the mixed donor encapsulating ligands 3,6,16-trithia-6,11,19-triazabicyclo[6.6.6]icosane (AMN(3)S(3)sar) and 1-amino-8-methyl-6,19-dithia-3,10,13,16-tetraazabicyclo[6.6.6]icosane (AMN(4)S(2)sar) have been studied. NMR ligand-ligand competition experiments with the ligands 1,4,8,11-tetraazaeyclotetradecane ([14]aneN(4)), 1-thia-4,7,10-triazacyclododecane ([12]aneN(3)S) and ethylenediaminetetraacetic acid (EDTA) with AMN(3)S(3)sar and Hg(II) indicated that [14]aneN(4) would be an appropriate competing ligand for the, determination of the Hg(II) stability constant. Calculations indicated the ratio of concentrations of AMN3S3sar, [14]aneN(4) and Hg(II) required for the determination of the stability constant ranged from 1:1:1 to 1:5:1. Refinement of the titration curves yielded log(10)K[Hg(AMN(3)S(3)sar)](2+) = 17.7. A similar competition titration resulted in the determination of the stability constant for the AMN(4)S(2)sar system as log(10)K[Hg(AMN(4)S(2)sar)](2+) = 19.5. The observed binding constants for the mixed N/S donor systems and the hexaaza analogues sar (3,6,10,13,16,19-hexaazabicyclo [6.6.6]icosane) and diamsar (1,8-diamino-3,6,10,13,16,19 -hexazabicyclo [6.6.6] icosane (log(10)K-[Hg(diamsar)](2+) = 26.4; log(10)K[Hg(sar)](2+) = 28.1) differ by approximately ten orders of magnitude. The difference is ascribed not to a cryptate effect but to a mismatch in the Hg-N and Hg-S bond lengths in the N/S systems.

Genotoxicity of inorganic mercury salts based on disturbed microtubule function

This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg2+ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg2+ inhibits microtubule assembly at concentrations above 1 muM, and inhibition is complete at about 10 muM. In this range, the tubulin assembly is fully ( up to 6 muM) or partially (similar to 6 - 10 muM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect-concentration for inhibition of microtubule assembly in vitro was 1 muM Hg2+, the IC50 5.8 muM. Mercury(II) salts at the IC50 concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a gliding assay'' mimicking intracellular movement and transport processes in vitro, HgCl2 affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 muM and a complete inhibition is reached at 1 muM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 muM HgCl2. Between 15 and 20 muM HgCl2 there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 muM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg2+, with some preponderance of aneugenicity. A morphological effect of high Hg2+ concentrations ( 100 muM HgCl2) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg2+ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg2+ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.

Direct continuities between cisternae at different levels of the Golgi complex in glucose-stimulated mouse islet beta cells

Direct continuity between the membranes of cisternae in the Golgi complex in mammalian cells rarely has been observed; when seen, its documentation has been equivocal. Here we have used dual-axis electron microscope tomography to examine the architecture of the Golgi in three dimensions at approximate to6-nm resolution in rapidly frozen, freeze-substituted murine cells that make and secrete insulin in response to glucose challenge. Our data show three types of direct connections between Golgi cisternae that are normally distinct from one another. These connections all bypass interceding cisternae. We propose that when pancreatic beta cells are stimulated to synthesize and secrete insulin rapidly in vivo, such connections provide a continuous lumen that facilitates the rapid transit of large amounts of newly made protein for secretion. The heterotypic fusion of cisternae, even transiently, raises important questions about the molecular mechanisms that (i) facilitate the fusion/fission of cisternal membranes and control the directionality and specificity of such events, and (it) retain Golgi processing enzymes at specific places within individual cisternae when two cisternae at different levels in the Golgi have fused, maintaining the sequential processing hierarchy that is a hallmark of Golgi organization.

Metabolite mean transit times in the liver as predicted by various models of hepatic elimination

Predicted area under curve (AUC), mean transit time (MTT) and normalized variance (CV2) data have been compared for parent compound and generated metabolite following an impulse input into the liver, Models studied were the well-stirred (tank) model, tube model, a distributed tube model, dispersion model (Danckwerts and mixed boundary conditions) and tanks-in-series model. It is well known that discrimination between models for a parent solute is greatest when the parent solute is highly extracted by the liver. With the metabolite, greatest model differences for MTT and CV2 occur when parent solute is poorly extracted. In all cases the predictions of the distributed tube, dispersion, and tasks-in-series models are between the predictions of the rank and tube models. The dispersion model with mixed boundary conditions yields identical predictions to those for the distributed tube model (assuming an inverse gaussian distribution of tube transit times). The dispersion model with Danckwerts boundary conditions and the tanks-in series models give similar predictions to the dispersion (mixed boundary conditions) and the distributed tube. The normalized variance for parent compound is dependent upon hepatocyte permeability only within a distinct range of permeability values. This range is similar for each model but the order of magnitude predicted for normalized variance is model dependent. Only for a one-compartment system is the MIT for generated metabolite equal to the sum of MTTs for the parent compound and preformed metabolite administered as parent.

Relative dispersions of intra-albumin transit times across rat and elasmobranch perfused livers, and implications for intra- and inter-species scaling of hepatic clearance using microsomal data

It is recognized that vascular dispersion in the liver is a determinant of high first-pass extraction of solutes by that organ. Such dispersion is also required for translation of in-vitro microsomal activity into in-vivo predictions of hepatic extraction for any solute. We therefore investigated the relative dispersion of albumin transit times (CV2) in the livers of adult and weanling rats and in elasmobranch livers. The mean and normalized variance of the hepatic transit time distribution of albumin was estimated using parametric non-linear regression (with a correction for catheter influence) after an impulse (bolus) input of labelled albumin into a single-pass liver perfusion. The mean +/- s.e. of CV2 for albumin determined in each of the liver groups were 0.85 +/- 0.20 (n = 12), 1.48 +/- 0.33 (n = 7) and 0.90 +/- 0.18 (n = 4) for the livers of adult and weanling rats and elasmobranch livers, respectively. These CV2 are comparable with that reported previously for the dog and suggest that the CV2 Of the liver is of a similar order of magnitude irrespective of the age and morphological development of the species. It might, therefore, be justified, in the absence of other information, to predict the hepatic clearances and availabilities of highly extracted solutes by scaling within and between species livers using hepatic elimination models such as the dispersion model with a CV2 of approximately unity.

Commentary: Using the convection-dispersion model and transit time density functions in the analysis of organ distribution kinetics

The convection-dispersion model and its extended form have been used to describe solute disposition in organs and to predict hepatic availabilities. A range of empirical transit-time density functions has also been used for a similar purpose. The use of the dispersion model with mixed boundary conditions and transit-time density functions has been queried recently by Hisaka and Sugiyanaa in this journal. We suggest that, consistent with soil science and chemical engineering literature, the mixed boundary conditions are appropriate providing concentrations are defined in terms of flux to ensure continuity at the boundaries and mass balance. It is suggested that the use of the inverse Gaussian or other functions as empirical transit-time densities is independent of any boundary condition consideration. The mixed boundary condition solutions of the convection-dispersion model are the easiest to use when linear kinetics applies. In contrast, the closed conditions are easier to apply in a numerical analysis of nonlinear disposition of solutes in organs. We therefore argue that the use of hepatic elimination models should be based on pragmatic considerations, giving emphasis to using the simplest or easiest solution that will give a sufficiently accurate prediction of hepatic pharmacokinetics for a particular application. (C) 2000 Wiley-Liss Inc. and the American Pharmaceutical Association J Pharm Sci 89:1579-1586, 2000.

On the validity of the dispersion model of hepatic drug elimination when intravascular transit time densities are long-tailed

The dispersion model with mixed boundary conditions uses a single parameter, the dispersion number, to describe the hepatic elimination of xenobiotics and endogenous substances. An implicit a priori assumption of the model is that the transit time density of intravascular indicators is approximated by an inverse Gaussian distribution. This approximation is limited in that the model poorly describes the tail part of the hepatic outflow curves of vascular indicators. A sum of two inverse Gaussian functions is proposed as ail alternative, more flexible empirical model for transit time densities of vascular references. This model suggests that a more accurate description of the tail portion of vascular reference curves yields an elimination rate constant (or intrinsic clearance) which is 40% less than predicted by the dispersion model with mixed boundary conditions. The results emphasize the need to accurately describe outflow curves in using them as a basis for determining pharmacokinetic parameters using hepatic elimination models. (C) 1997 Society for Mathematical Biology.

Relative dispersion of intravascular transit times during isolated human limb perfusions for recurrent melanoma

Aims We have characterized the relative dispersion of vascular and extravascular markers in the limbs of three patients undergoing isolated limb perfusions with the cytotoxic melphalan for recurrent malignant melanoma both before and after melphalan dosing. Methods A bolus of injectate containing [Cr-51] labelled red blood cells, [C-14]-sucrose and [H-3]-water was injected into an iliac or femoral artery and outflow samples collected at 1 s intervals by a fraction collector. The radioactivity due to each isotype was analysed by either gamma [Cr-51] or beta [C-14 and H-3] counting. The moments of the outflow fraction-time profiles were estimated by a nonparametric (numerical integration) method and a parametric model (sum of two inverse Gaussian functions). Results The availability, mean transit time and normalised variance (CV2) obtained for labelled red blood cells, sucrose and water were similar before and after melphalan dosing and with the two methods of calculation but varied between the patients. Conclusions The vascular space is not well-stirred but characterized by a CV2 similar that reported previously for in situ rat hind limb and rat liver perfusions. A flow-limited blood-tissue exchange was observed for the permeating indicators. Administration of melphalan did not influence the distribution characteristics of the indicators.

Separation of mercury (II), lead (II), and silver (I) from aqueous solution using an aminopolycarboxylate silica

A quartz crystal microbalance modified by the attachment of silica particles derivatized with the aminopolycarboxylate ligand N-[(3-trimethoxysilyl)propyl]ethylenediamine-N,N',N'-triacetic acid has been employed to assess conditions under which mercury (II), lead (II), and silver (I) nitrates may be separated in aqueous solution. The separation protocol, which involved removal of Hg(II), as [HgI4](2-), and Pb(II) with H+ was successfully applied to a batchwise separation of the 3 metal ions.

Simultaneous observations of ionospheric quasiperiodic scintillations from short and long meridional baselines using VHF transmissions from transit satellites

For the first time it was possible to observe regular quasiperiodic scintillations (QPS) in VHF radio-satellite transmissions from orbiting satellites simultaneously at short (2.1 km) and long (121 km) meridional baselines in the vicinity of a typical mid-latitude station (Brisbane; 27.5degreesS and 152.9degreesE geog. and 35.6degrees invar.lat.), using three sites (St. Lucia-S, Taringa-T in Brisbane and Boreen Pt.-B, north of Brisbane). A few pronounced quasiperiodic (QP) events were recorded showing unambiguous regular structures at the sites which made it possible to deduce a time displacement of the regular fading minimum at S, T and B. The QP structure is highly dependent on the geometry of the ray-path from a satellite to the observer which is manifested as a change of a QP event from symmetrical to non-symmetrical for stations separated by 2.1 km, and to a radical change in the structure of the event over a distance of 121 km. It is suggested the short-duration intense QP events are due to a Fresnel diffraction (or a reflection mechanism) of radio-satellite signals by a single ionospheric irregularity in a form of an ellipsoid with a large ionization gradient along the major axis. The structure of a QP event depends on the angle of viewing of the irregular blob from a radio-satellite. In view of this it is suggested that the reported variety of the ionization formation, responsible for different types of QPS, is only apparent but not real. (C) 2003 Elsevier Science Ltd. All rights reserved.

Disturbed microtubule function and induction of micronuclei by chelate complexes of mercury(II)

Interactions of mercury(II) with the microtubule network of cells may lead to genotoxicity. Complexation of mercury(II) with EDTA is currently being discussed for its employment in detoxification processes of polluted sites. This prompted us to re-evaluate the effects of such complexing agents on certain aspects of mercury toxicity, by examining the influences of mercury(H) complexes on tubulin assembly and kinesin-driven motility of microtubules. The genotoxic effects were studied using the micronucleus assay in V79 Chinese hamster fibroblasts. Mercury(II) complexes with EDTA and related chelators interfered dose-dependently with tubulin assembly and microtubule motility in vitro. The no-effect-concentration for assembly inhibition was 1muM of complexed Hg(II), and for inhibition of motility it was 0.05 muM, respectively. These findings are supported on the genotoxicity level by the results of the micronucleus assay, with micronuclei being induced dose-dependently starting at concentrations of about 0.05 muM of complexed Hg(II). Generally, the no-effect-concentrations for complexed mercury(II) found in the cell-free systems and in cellular assays (including the micronucleus test) were identical with or similar to results for mercury tested in the absence of chelators. This indicates that mercury(II) has a much higher affinity to sulfhydryls of cytoskeletal proteins than to this type of complexing agents. Therefore, the suitability of EDTA and related compounds for remediation of environmental mercury contamination or for other detoxification purposes involving mercury has to be questioned. (C) 2004 Elsevier B.V. All rights reserved.

Use of pulse transit time to distinguish respiratory events from tidal breathing in sleeping children

Study objectives: Currently, esophageal pressure monitoring is the "gold standard" measure for inspiratory efforts, hut its invasive nature necessitates a better tolerated and noninvasive method to be used on children. Pulse transit time (PTT) has demonstrated its potential as a noninvasive surrogate marker for inspiratory efforts. The principle velocity determinant of PTT is the change in stiffness of the arterial wall and is inversely correlated to BP. Moreover, PTT has been shown to identify changes in inspiratory effort via the BP fluctuations induced by negative pleural pressure swings. In this study, the capability of PTT to classify respiratory, events during sleep as either central or obstructive in nature was investigated. Setting and participants: PTT measure was used in adjunct to routine overnight polysomnographic studies performed on 33 children (26 boys and 7 girls; mean +/- SD age, 6.7 +/- 3.9 years). The accuracy of PTT measurements was then evaluated against scored corresponding respiratory events in the polysomnography recordings. Results: Three hundred thirty-four valid respiratory events occurred and were analyzed. One hundred twelve obstructive events (OEs) showed a decrease in mean PTT over a 10-sample window that had a probability of being correctly ranked below the baseline PTT during tidal breathing of 0.92 (p < 0.005); 222 central events (CEs) showed a decrease in the variance of PTT over a 10-sample window that had a probability of being ranked below the baseline PTT of 0.94 (p < 0.005). This indicates that, at a sensitivity of 0.90, OEs can be detected with a specificity of 0.82 and CEs can be detected with a specificity of 0.80. Conclusions: PTT is able to categorize CEs and OEs accordingly in the absence of motion artifacts, including hypopneas. Hence, PTT shows promise to differentiate respiratory, events accordingly and can be an important diagnostic tool in pediatric respiratory sleep studies.< 0.005); 222 central events (CEs) showed a decrease in the variance of PTT over a 10-sample window that had a probability of being ranked below the baseline PTT of 0.94 (p < 0.005). This indicates that, at a sensitivity of 0.90, OEs can be detected with a specificity of 0.82 and CEs can be detected with a specificity of 0.80. Conclusions: PTT is able to categorize CEs and OEs accordingly in the absence of motion artifacts, including hypopneas. Hence, PTT shows promise to differentiate respiratory, events accordingly and can be an important diagnostic tool in pediatric respiratory sleep studies.');"