Voltammetric Studies on Mechanisms of Dopamine Efflux in the Presence of Substrates and Cocaine from Cells Expressing Human Norepinephrine Transporter

Abstract: The effects of substrates m -tyramine and β-phenethylamine, as well as cocaine, on the DA efflux from a cell line stably expressing the human norepinephrine transporter (hNET) were investigated by using rotating disk electrode voltammetry. Both the substrates and cocaine induced apparent DA efflux in a concentration-dependent manner. Their EC₅₀ values for inducing DA efflux were similar to their IC₅₀ values for inhibiting DA uptake. The substrate-induced DA efflux was inhibited by various NET blockers, enhanced by raising the internal [Na⁺] with Na⁺,K⁺-ATPase inhibition, but was insensitive to membrane potential-altering agents valinomycin, veratridine, and high [K⁺]. The initial rate of m -tyramine-induced DA efflux was related to preloaded [DA] in a manner defined by a Michaelis-Menten expression. In contrast, DA efflux in the presence of cocaine displayed a much slower efflux rate, lower efficacy, was not stimulated by elevated internal [Na⁺], and was nonsaturable with preloaded [DA]. Single exponential kinetic analysis of the entire time course of the DA efflux showed that the apparent first-order rate constant for m -tyramine-induced DA efflux declined with increased preloaded [DA], whereas that for the DA efflux in the presence of cocaine was unchanged with varying preloaded [DA]. These results suggest that the substrates stimulate the NET-dependent DA efflux by increasing the accessibility of the NET to internal DA, whereas cocaine "uncovers" NET-independent DA efflux by reducing the accessibility of diffused/leaked external DA to the NET.     

Composition of the cuticle of developing sweet cherry fruit

The composition of wax and cutin from developing sweet cherry (Prunus avium) fruit was studied by GC-MS between 22 and 85 days after full bloom (DAFB). In this and our previous study, fruit mass and surface area increased in a sigmoidal pattern with time, but mass of the cuticular membrane (CM) per unit fruit surface area decreased. On a whole fruit basis, mass of CM increased up to 36 DAFB and remained constant thereafter. At maturity, triterpenes, alkanes and alcohols accounted for 75.6%, 19.1% and 1.2% of total wax, respectively. The most abundant constituents were the triterpenes ursolic (60.0%) and oleanolic acid (7.5%), the alkanes nonacosane (13.0%) and heptacosane (3.0%), and the secondary alcohol nonacosan-10-ol (1.1%). In developing fruit triterpenes per unit area decreased, but alkanes and alcohols remained essentially constant. The cutin fraction of mature fruit consisted of mostly C16 (69.5%) and, to a lower extent, C18 monomers (19.4%) comprising alkanoic, omega-hydroxyacids, alpha,omega-dicarboxylic and midchain hydroxylated acids. The most abundant constituents were 9(10),16-dihydroxy-hexadecanoic acid (53.6%) and 9,10,18-trihydroxy-octadecanoic acid (7.8%). Amounts of C16 and C18 monomers per unit area decreased in developing fruit, but remained approximately constant on a whole fruit basis. Within both classes of monomers, opposing changes occurred. Amounts of hexadecandioic, 16-hydroxy-hexadecanoic, 9(10)-hydroxy-hexadecane-1,16-dioic and 9,10-epoxy-octadecane-1,18-dioic acids increased, but 9,10,18-trihydroxy-octadecanoic and 9,10,18-trihydroxy-octadecenoic acids decreased. There were no qualitative and minor quantitative differences in wax and cutin composition between cultivars at maturity. Our data indicate that deposition of some constituents of wax and cutin ceased during early fruit development.     

Predicting Secchi disk depth from average beam attenuation in a deep,ultra-clear lake

We addressed potential sources of error in estimating the water clarity of mountain lakes by investigating the use of beam transmissometer measurements to estimate Secchi disk depth. The optical properties Secchi disk depth (SD) and beam transmissometer attenuation (BA) were measured in Crater Lake (Crater Lake National Park, Oregon, USA) at a designated sampling station near the maximum depth of the lake. A standard 20 cm black and white disk was used to measure SD. The transmissometer light source had a nearly monochromatic wavelength of 660 nm and a path length of 25 cm. We created a SD prediction model by regression of the inverse SD of 13 measurements recorded on days when environmental conditions were acceptable for disk deployment with BA averaged over the same depth range as the measured SD. The relationship between inverse SD and averaged BA was significant and the average 95% confidence interval for predicted SD relative to the measured SD was ±1.6 m (range = −4.6 to 5.5 m) or ±5.0%. Eleven additional sample dates tested the accuracy of the predictive model. The average 95% confidence interval for these sample dates was ±0.7 m (range = −3.5 to 3.8 m) or ±2.2%. The 1996–2000 time-series means for measured and predicted SD varied by 0.1 m, and the medians varied by 0.5 m. The time-series mean annual measured and predicted SD’s also varied little, with intra-annual differences between measured and predicted mean annual SD ranging from −2.1 to 0.1 m. The results demonstrated that this prediction model reliably estimated Secchi disk depths and can be used to significantly expand optical observations in an environment where the conditions for standardized SD deployments are limited.     

梅花鹿鹿茸不同产品中氨基酸含量的比较

通过对二杠鹿茸、三杈鹿茸、鹿茸片、鹿茸血、鹿角、鹿角盘等鹿茸产品中氨基酸含量的测定研究,结果表明:鹿茸血中氨基酸含量最高,鹿茸片中的氨基酸含量次之,三杈鹿茸中的氨基酸含量高于二杠鹿茸中氨基酸含量,鹿花盘中的含量高于鹿角中的氨基酸含量,从而为鹿茸这一动物性中药材资源的功能评价、药理作用提供科学理论依据。     

Saline-induced changes of epicuticular waxy layer on the Puccinellia tenuiflora and Oryza sativa leave surfaces

Background

The epicuticular waxy layer of plant leaves enhances the extreme environmental stress tolerance. However, the relationship between waxy layer and saline tolerance was not established well. The epicuticular waxy layer of rice (Oryza sativa L.) was studied under the NaHCO₃ stresses. In addition, strong saline tolerance Puccinellia tenuiflora was chosen for comparative studies.

Results

Scanning electron microscope (SEM) images showed that there were significant changes in waxy morphologies of the rice epicuticular surfaces, while no remarkable changes in those of P. tenuiflora epicuticular surfaces. The NaHCO₃-induced morphological changes of the rice epicuticular surfaces appeared as enlarged silica cells, swollen corns-shapes and leaked salt columns under high stress. Energy dispersive X-ray (EDX) spectroscopic profiles supported that the changes were caused by significant increment and localization of [Na⁺] and [Cl⁻] in the shoot. Atomic absorption spectra showed that [Na⁺]_shoot/[Na⁺]_root for P. tenuiflora maintained stable as the saline stress increased, but that for rice increased significantly.

Conclusion

In rice, NaHCO₃ stress induced localization and accumulation of [Na⁺] and [Cl⁻] appeared as the enlarged silica cells (MSC), the swollen corns (S-C), and the leaked columns (C), while no significant changes in P. tenuiflora.     

The area composita of adhering junctions connecting heart muscle cells of vertebrates. II. Colocalizations of desmosomal and fascia adhaerens molecules in the intercalated disk

Using immunofluorescence histochemistry and immunoelectron microscopy on sections through myocardiac tissues of diverse mammalian (human, cow, rat, mouse) and fish species we show that both desmosomal and fascia adhaerens proteins identified by gel electrophoresis and immunoblot occur in the area composita, the by far major type of plaque-bearing junctions of the intercalated disks (IDs) connecting cardiomyocytes. Specifically, we demonstrate that desmoplakin and the other desmosomal proteins occur in these junctions, together with N-cadherin, cadherin-11, alpha- and beta-catenin as well as vinculin, afadin and proteins p120(ctn), ARVCF, p0071, and ZO-1, suggestive of colocalization. We conclude that the predominant type of adhering junction present in IDs is a junction sui generis, termed area composita, that is characterized by an unusually high molecular complexity and an intimate association of molecules of both ensembles, the desmosomal one and the fascia adhaerens category. We discuss possible myocardium-specific, complex-forming interactions between members of the two ensembles and the relevance of our findings for the formation and functioning of the heart and for the understanding of hereditary and other cardiomyopathies. We further propose to use this highly characteristic area composita ensemble of molecules as cardiomyocyte markers for the monitoring of cardiomyogenesis, cardiomyocyte regeneration and possible cardiomyocyte differentiation from mesenchymal stem cells.     

Optimizing the rotor design for controlled-shear affinity filtration using computational fluid dynamics

Controlled shear affinity filtration (CSAF) is a novel integrated processing technology that positions a rotor directly above an affinity membrane chromatography column to permit protein capture and purification directly from cell culture. The conical rotor is intended to provide a uniform and tunable shear stress at the membrane surface that inhibits membrane fouling and cell cake formation by providing a hydrodynamic force away from and a drag force parallel to the membrane surface. Computational fluid dynamics (CFD) simulations are used to show that the rotor in the original CSAF device (Vogel et al., 2002) does not provide uniform shear stress at the membrane surface. This results in the need to operate the system at unnecessarily high rotor speeds to reach a required shear stress of at least 0.17 Pa at every radial position of the membrane surface, compromising the scale-up of the technology. Results from CFD simulations are compared with particle image velocimetry (PIV) experiments and a numerical solution for low Reynolds number conditions to confirm that our CFD model accurately describes the hydrodynamics in the rotor chamber of the CSAF device over a range of rotor velocities, filtrate fluxes, and (both laminar and turbulent) retentate flows. CFD simulations were then carried out in combination with a root-finding method to optimize the shape of the CSAF rotor. The optimized rotor geometry produces a nearly constant shear stress of 0.17 Pa at a rotational velocity of 250 rpm, 60% lower than the original CSAF design. This permits the optimized CSAF device to be scaled up to a maximum rotor diameter 2.5 times larger than is permissible in the original device, thereby providing more than a sixfold increase in volumetric throughput.     

High-yield synthesis of wax esters catalysed by modified <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase

Candida rugosa lipase (CRL) was applied in a non-solvent esterification reaction to yield twelve wax esters. All products were obtained in nearly 100% yield for 10 h at 50°C when immobilized PEG₂₀₀₀-activated C. rugosa lipase was added to the reaction mixture. The surfactant had also a beneficial effect on the stability of the biocatalytic preparation with 83% of its activity conserved after the seventh run of repeated batch reactions.