Zinc potentiation of the glycine receptor chloride channel is mediated by allosteric pathways

Molecular mechanisms of zinc potentiation were investigated in recombinant human alpha 1 glycine receptors (GlyRs) by whole-cell patch-clamp recording and [H-3]strychnine binding assays. In the wild-type (WT) GlyR, 1 mu M zinc enhanced the apparent binding affinity of the agonists glycine and taurine and reduced their concentrations required for half-maximal activation. Thus, in the WT GlyR, zinc potentiation apparently occurs by enhancing agonist binding. However, analysis of GlyRs incorporating mutations in the membrane-spanning domain M1-M2 and M2-M3 loops, which are both components of the agonist gating mechanism, indicates that most mutations uncoupled zinc potentiation from glycine-gated currents but preserved zinc potentiation of taurine-gated currents. One such mutation in the M2-M3 loop, L274A, abolished the ability of zinc to potentiate taurine binding but did not inhibit zinc potentiation of taurine-gated currents. In this same mutant where taurine acts as a partial agonist, zinc potentiated taurine-gated currents but did not potentiate taurine antagonism of glycine-gated currents, suggesting that zinc interacts selectively with the agonist transduction pathway. The intracellular M246A mutation, which is unlikely to bind zinc, also disrupted zinc potentiation of glycine currents. Thus, zinc potentiation of the GlyR is mediated via allosteric mechanisms that are independent of its effects on agonist binding.

Self-efficacy for refusal mediated by outcome expectancies in the prediction of alcohol-dependence amongst young adults

The present study examined the relative importance of outcome expectancies and self-efficacy [1] in the prediction of alcohol dependence [2] and alcohol consumption in a sample of young adult drinkers drawn from a milieu previously reported as supportive of risky drinking. In predicting alcohol dependence, outcome expectancies were found to mediate self-efficacy and the same pattern was found for both males and females. This suggests that male and female drinkers may become more similar as they progress along the drinking continuum from risky drinking to dependent drinking. However, in women, in comparison to men, a greater array of expectancies and self-efficacy scales were found to predict heavy drinking, as measured by quantity and frequency. These results suggest that heavy drinking women are particularly at risk of developing drinking related complications and that preventative education needs to take into account gender differences.

Modeling the fluid-flow-induced stress and collapse in a dendritic network

An analytical approach to the stress development in the coherent dendritic network during solidification is proposed. Under the assumption that stresses are developed in the network as a result of the friction resisting shrinkage-induced interdendritic fluid flow, the model predicts the stresses in the solid. The calculations reflect the expected effects of postponed dendrite coherency, slower solidification conditions, and variations of eutectic volume fraction and shrinkage. Comparing the calculated stresses to the measured shear strength of equiaxed mushy zones shows that it is possible for the stresses to exceed the strength, thereby resulting in reorientation or collapse of the dendritic network.

Targeting local tissues by transdermal application: Understanding drug physicochemical properties that best exploit protein binding and blood flow effects

The targeting of topically applied drug molecules into tissues below a site of application requires an understanding of the complex interrelationships between the drug, its formulation, the barrier properties of the skin, and the physiological processes occurring below the skin that are responsible for drug clearance from the site, tissue, and/or systemic distribution and eventual elimination. There is still a certain amount of controversy over the ability of topically applied drugs to penetrate into deeper tissues by diffusion or whether this occurs by redistribution in the systemic circulation. The major focus of our work in this area has been in determining how changes in drug structure and physicochemical properties, such as protein binding and lipophilicity, affect drug clearance into the local dermal microcirculation and lymphatics, as well as subsequent distribution into deeper tissues below an application site. The present study outlines our recent thinking on the drug molecule optimal physical attributes, in terms of plasma and tissue partitioning behaviour, that offer the greatest potential for deep tissue targeting. Drug Dev. Res. 46:309-315, 1999. (C) 1999 Wiley-Liss, Inc.

Complement-mediated regulation of Trichomonas vaginalis infection in mice

Trichomonas vaginalis is a flagellated protozoan which causes trichomoniasis, a sexually transmitted disease of the human genitourinary tract, The importance of the alternative complement pathway in host defence against T. vaginalis was investigated in vitro. Kinetic studies utilising immunofixation following electrophoresis showed that both a strongly and weakly virulent strain of T, vaginalis activated murine serum C3. In vivo studies with congenic-resistant, C5-deficient, B10.D2/OSn- and C5-sufficient, B10.D2/nSn mice showed that the presence of C5 is a significant factor in the innate host resistance to primary infection with a strongly virulent, but not a weakly virulent trichomonad strain.

p75 neurotrophin receptor-mediated neuronal death is promoted by Bcl-2 and prevented by Bcl-x(L)

The p75 neurotrophin receptor (p75NTR) has been shown to mediate neuronal death through an unknown pathway. We microinjected p75NTR expression plasmids into sensory neurons in the presence of growth factors and assessed the effect of the expressed proteins on cell survival. We show that, unlike other members of the TNFR family, p75NTR signals death through a unique caspase-dependent death pathway that does not involve the death domain and is differentially regulated by Bcl-2 family members: the anti-apoptotic molecule Bcl-2 both promoted, and was required for, p75NTR killing, whereas killing was inhibited by its homologue BcI-x(L). These results demonstrate that Bcl-2, through distinct molecular mechanisms, either promotes or inhibits neuronal death depending on the nature of the death stimulus.

Relation of TNF-related apoptosis-inducing ligand (TRAIL) receptor and FLICE-inhibitory protein expression to TRAIL-induced apoptosis of melanoma

Past studies have shown that apoptosis mediated by TNF-related apoptosis-inducing ligand (TRAIL) is regulated by the expression of two death receptors [TRAIL receptor 1 (TRAIL-RI) and TRAIL-R2] and two decoy receptors (TRAIL-R3 and TRAIL-R4) that inhibit apoptosis, In previous studies, me have shown that TRAIL but not other members of the tumor necrosis factor family induce apoptosis in approximately two-thirds of melanoma cell lines. Here, we examined whether the expression of TRAIL-R at the mRNA and protein level in a panel of 28 melanoma cell lines and melanocytes correlated with their sensitivity to TRAIL-induced apoptosis, We report that at least three factors appear to underlie the variability in TRAIL-induced apoptosis. (a) Pour of nine cell lines that were insensitive to TRAIL-induced apoptosis failed to express death receptors, and in two instances, lines were devoid of all TRAIL-Rs. Southern analysis suggested this was due to loss of the genes for the death receptors, (b) Despite the presence of mRNA for the TRAIL-R, some of the lines failed to express TRAIL-R protein on their surface. This was evident for TRAIL-RI and more so for the TRAIL decoy receptors TRAIL-R3 and -R4, Studies on permeabilized cells revealed that the receptors were located within the cytoplasm and redistribution from the cytoplasm may represent a posttranslational control mechanism. (c) Surface expression of TRAIL-RI and -R2 (but not TRAIL-R3 and -R4) showed an overall correlation with TRAIL-induced apoptosis. However, certain melanoma cell lines and clones were relatively resistant to TRAIL-induced apoptosis despite the absence of decoy receptors and moderate levels of TRAIL-RI and -R2 expression. This may indicate the presence of inhibitors within the cells, but resistance to apoptosis could not be correlated with expression of the caspase inhibitor FLICE-inhibitory protein. mRNA for another TRAIL receptor, osteoprotegerin, was expressed in 22 of the melanoma lines but not on melanocytes. Its role in induction of apoptosis remains to be studied. These results appear to have important implications for future clinical studies on TRAIL.

A numerical study of pore-fluid, thermal and mass flow iu fluid-saturated porous rock basins

We present a numerical methodology for the study of convective pore-fluid, thermal and mass flow in fluid-saturated porous rock basins. lit particular, we investigate the occurrence and distribution pattern of temperature gradient driven convective pore-fluid flow and hydrocarbon transport in the Australian North West Shelf basin. The related numerical results have demonstrated that: (1) The finite element method combined with the progressive asymptotic approach procedure is a useful tool for dealing with temperature gradient driven pore-fluid flow and mass transport in fluid-saturated hydrothermal basins; (2) Convective pore-fluid flow generally becomes focused in more permeable layers, especially when the layers are thick enough to accommodate the appropriate convective cells; (3) Large dislocation of strata has a significant influence off the distribution patterns of convective pore;fluid flow, thermal flow and hydrocarbon transport in the North West Shelf basin; (4) As a direct consequence of the formation of convective pore-fluid cells, the hydrocarbon concentration is highly localized in the range bounded by two major faults in the basin.

T cell-mediated and non-specific inflammatory mechanisms contribute to the skin pathology of HPV 16 E6E7 transgenic mice

One of three lines of mice transgenic for the E6 and E7 genes of human papillomavirus type 16 (HPV16) expressed from an alpha A-crystallin promoter also expresses the transgene ectopically in the skin. This line, designated alpha ACE6E7#19, develops skin disease from 3 months of age, characterised by epidermal hyperplasia and eventual skin loss. Administration of complete Freund's adjuvant (CFA) to alpha ACE6E7#19 mice, but not to nontransgenic littermate controls, induced local epidermal hyperplasia which was histologically similar to the spontaneously arising skin pathology. Local application of 2,4-dinitrochlorobenzene (DNCB) to DNCB-sensitised aACE6E7#19 mice, but not DNCB-sensitised controls, also induced hyperplasia. Treatment with cyclosporin A (CsA) or systemic depletion of CD4+ cells significantly reduced the incidence of skin disease. These data suggest that local inflammation, and cytokines produced by T helper cells, contribute to the induction of hyperplastic skin disease in alpha ACE6E7#19 mice. Spontaneous skin disease with similar histological appearance, frequency, age of onset and severity in alpha ACE6E7#19 mice was observed in scid-/- aACE6E7#19 mice, despite immune paresis. Antigen-specific immune responses and T-cell cytokines a re therefore not necessary for the induction of skin disease. We propose that epidermal hyperplasia associated with HPV16 E6 and E7 expression in skin is accelerated by local secretion of pro-inflammatory cytokines, whose production can be enhanced by activated CD4+ T cells.

Finite element analysis of flow patterns near geological lenses in hydrodynamic and hydrothermal systems

We use theoretical and numerical methods to investigate the general pore-fluid flow patterns near geological lenses in hydrodynamic and hydrothermal systems respectively. Analytical solutions have been rigorously derived for the pore-fluid velocity, stream function and excess pore-fluid pressure near a circular lens in a hydrodynamic system. These analytical solutions provide not only a better understanding of the physics behind the problem, but also a valuable benchmark solution for validating any numerical method. Since a geological lens is surrounded by a medium of large extent in nature and the finite element method is efficient at modelling only media of finite size, the determination of the size of the computational domain of a finite element model, which is often overlooked by numerical analysts, is very important in order to ensure both the efficiency of the method and the accuracy of the numerical solution obtained. To highlight this issue, we use the derived analytical solutions to deduce a rigorous mathematical formula for designing the computational domain size of a finite element model. The proposed mathematical formula has indicated that, no matter how fine the mesh or how high the order of elements, the desired accuracy of a finite element solution for pore-fluid flow near a geological lens cannot be achieved unless the size of the finite element model is determined appropriately. Once the finite element computational model has been appropriately designed and validated in a hydrodynamic system, it is used to examine general pore-fluid flow patterns near geological lenses in hydrothermal systems. Some interesting conclusions on the behaviour of geological lenses in hydrodynamic and hydrothermal systems have been reached through the analytical and numerical analyses carried out in this paper.

Human and murine cytomegalovirus evasion of cytotoxic T lymphocyte and natural killer cell-mediated immune responses

Herpesviruses, such as human and murine cytomegalovirus, possess an impressive array of genes believed to assist in virus survival against the host immune response. In this review, we cover the rapidly growing area of cytomegalovirus evasion of cellular immunity, specifically cytotoxic T lymphocytes and natural killer cells. The proposed mechanisms of action of viral proteins involved in blocking peptide presentation to CD8(+) T cells, namely, interference with peptide generation, inhibition of peptide assembly with class I MHC and retention/destabilization of class I MHC complexes, are described. In addition, recent evidence implicating the viral class I MHC-like proteins as inhibitors of natural killer cell-mediated clearance is reviewed, (C) 1998 Academic Press.

m144, a murine cytomegalovirus (MCMV)-encoded major histocompatibility complex class I homologue, confers tumor resistance to natural killer cell-mediated rejection

Until now, it has been unclear whether murine cytomegalovirus (MCMV)-encoded protein m144 directly regulates natural killer (NK) cell effector function and whether the effects of m144 are only strictly evident in the context of MCMV infection. We have generated clones of the transporter associated with antigen processing (TAP)-2-deficient RMA-S T lymphoma cell line and its parent cell line, RMA, that stably express significant and equivalent levels of m144. In vivo NK cell-mediated rejection of RMA-S-m144 lymphomas was reduced compared with rejection of parental or mock-transfected RMA-S clones, indicating the ability of m144 to regulate NK cell-mediated responses in vivo. Significantly, the accumulation of NK cells in the peritoneum was reduced in mice challenged with RMA-S-m144, as was the lytic activity of NK cells recovered from the peritoneum. Expression of m144 on RMA-S cells also conferred resistance to cytotoxicity mediated in vitro by interleukin 2-activated adherent spleen NK cells. In summary, the data demonstrate that m144 confers some protection from NK cell effector function mediated in the absence of target cell class I expression, but that in vivo the major effect of m144 is to regulate NK cell accumulation and activation at the site of immune challenge.

Increasing the efficiency of solute leaching: impacts of flow interruption with drainage of the "preferential flow paths"

Most soils contain preferential flow paths that can impact on solute mobility. Solutes can move rapidly down the preferential flow paths with high pore-water velocities, but can be held in the less permeable region of the soil matrix with low pore-water velocities, thereby reducing the efficiency of leaching. In this study, we conducted leaching experiments with interruption of the flow and drainage of the main flow paths to assess the efficiency of this type of leaching. We compared our experimental results to a simple analytical model, which predicts the influence of the variations in concentration gradients within a single spherical aggregate (SSA) surrounded by preferential flow paths on leaching. We used large (length: 300 mm, diameter: 216 mm) undisturbed field soil cores from two contrasting soil types. To carry out intermittent leaching experiments, the field soil cores were first saturated with tracer solution (CaBr2), and background solution (CaCl2) was applied to mimic a leaching event. The cores were then drained at 25- to 30-cm suction to empty the main flow paths to mimic a dry period during which solutes could redistribute within the undrained region. We also conducted continuous leaching experiments to assess the impact of the dry periods on the efficiency of leaching. The flow interruptions with drainage enhanced leaching by 10-20% for our soils, which was consistent with the model's prediction, given an optimised equivalent aggregate radius for each soil. This parameter quantifies the time scales that characterise diffusion within the undrained region of the soil, and allows us to calculate the duration of the leaching events and interruption periods that would lead to more efficient leaching. Application of these methodologies will aid development of strategies for improving management of chemicals in soils, needed in managing salts in soils, in improving fertiliser efficiency, and in reclaiming contaminated soils. (C) 2000 Elsevier Science B.V. All rights reserved.

Strain-dependent fluid flow defined through rock mass classification schemes

Strain-dependent hydraulic conductivities are uniquely defined by an environmental factor, representing applied normal and shear strains, combined with intrinsic material parameters representing mass and component deformation moduli, initial conductivities, and mass structure. The components representing mass moduli and structure are defined in terms of RQD (rock quality designation) and RMR (rock mass rating) to represent the response of a whole spectrum of rock masses, varying from highly fractured (crushed) rock to intact rock. These two empirical parameters determine the hydraulic response of a fractured medium to the induced-deformations The constitutive relations are verified against available published data and applied to study one-dimensional, strain-dependent fluid flow. Analytical results indicate that both normal and shear strains exert a significant influence on the processes of fluid flow and that the magnitude of this influence is regulated by the values of RQD and RMR.