Involvement of Porin N,N-dicyclohexylcarbodiimide-Reactive Domain in Hexokinase Binding to the Outer Mitochondrial Membrane

The proportion of hexokinase that is bound to the outer mitochondrial membrane is tissue specific and metabolically regulated. This study examined the role of the N,N-dicyclohexylcarbodiimide-binding domain of mitochondrial porin in binding to hexokinase I. Selective proteolytic cleavage of porin protein was performed and peptides were assayed for their, effect on hexokinase I binding to isolated mitochondria. Specificity of DCCD-reactive domain binding to hexokinase I was demonstrated by competition of the peptides for porin binding sites on hexokinase as well as by blockage hexokinase binding by N,N-dicyclohexylcarbodiimide. One of the peptides, designated as 5 kDa (the smallest of the porin peptides, which contains a DCCD-reactive site), totally blocked binding of the enzyme to the mitochondrial membrane, and significantly enhanced the release of the mitochondrially bound enzyme. These experiments demonstrate that there exists a direct and specific interaction between the DCCD-reactive domain of VDAC and hexokinase I. The peptides were further characterized with respect to their effects on certain functional properties of hexokinase I. None had any detectable effect on catalytic properties, including inhibition by glucose 6-phosphate. To evaluate further the outer mitochondrial membranes role in the hexokinase binding, insertion of VDAC was examined using isolated rat mitochondria. Pre-incubation of mitochondria with purified porin strongly increases hexokinase I binding to rat liver mitochondria. Collectively, the results imply that the high hexokinase-binding capability of porin-enriched mitochondria was due to a quantitative difference in binding sites.     

Sodium leak pathway and substrate binding order in the Na+-glucose cotransporter.

The Na+-glucose cotransporter (SGLT1) expressed in Xenopus laevis oocytes was shown to generate a phlorizin-sensitive sodium leak in the absence of sugars. Using the current model for SGLT1, where the sodium leak was presumed to occur after two sodium ions are bound to the free carrier before glucose binding, a characteristic concentration constant (Kc) was introduced to describe the relative importance of the sodium leak versus Na+-glucose cotransport currents. Kc represents the glucose concentration at which the Na+-glucose cotransport current is equal to the sodium leak. As both the sodium leak and the Na+-glucose cotransport current are predicted to occur after the binding of two sodium ions, the model predicted that Kc should be sodium-independent. However, by using a two-microelectrode voltage-clamp technique, the observed Kc was shown to depend strongly on the external sodium concentration ([Na+]o): it was four times higher at 5 mM [Na+]o than at 20 mM [Na+]o. In addition, the magnitude of the sodium leak varied as a function of [Na+]o in a Michaelian fashion, and the sodium affinity constant for the sodium leak was 2-4 times lower than that for cotransport in the presence of low external glucose concentrations (50 or 100 microM), whereas the current model predicted a sigmoidal sodium dependence of the sodium leak and identical sodium affinities for the sodium leak and the Na+-glucose cotransport. These observations indicate that the sodium leak occurs after one sodium ion is associated with the carrier and agree with predictions from a model with the binding order sodium-glucose-sodium. This conclusion was also supported by experiments performed where protons replaced Na+ as a "driving cation."     

Epigallocatechin-3-Gallate Alleviates Salinity-Retarded Seed Germination and Oxidative Stress in Tomato

Journal of Plant Growth Regulation - Salinity is a global environmental problem, restricting crop production in a vast area of agricultural land. Although epigallocatechin-3-gallate (EGCG), the...     

Adsorption-Desorption Characteristics of Polychlorinated Biphenyls on Various Polymers Commonly Found in Laboratories

Adsorption and desorption of Aroclor 1254 (a mixture of polychlorinated biphenyls [PCBs]) from 0.25% (wt/vol) aqueous solutions of Triton X-100 on polymeric materials common in laboratories (red vacuum rubber, latex, Tygon, Norprene, manosil, polyethylene, polypropylene, phenoxy resin, nylon, and Teflon) is described. Teflon, nylon, and phenoxy resins showed the lowest adsorptions, with efficiencies of 3.4, 22.9, and 33.0%, respectively. The other polymers adsorbed more than 90% of Aroclor 1254 under similar conditions. Adsorption of PCBs was found to increase with the lipophilicity of the polymer and to be irreversible on soft polymers. The variation in the adsorption efficiencies of these polymers toward PCBs is apparently related to variations in the chemical composition, electronic properties, and degree of softness of these polymers. The present study showed that Teflon, with less than 4% adsorption and more than 99% desorption of Aroclor 1254, is the best candidate for use in biological laboratory work.     

Familial transmission of 16p trisomy in an infant

Based on four reported cases including the present case, 16p trisomic infants have remarkably similar features. These are severe developmental delay, psychomotor retardation, typical facies, and anomalies of extremities.     

Differential chromosomal fluorescence with 33258 Hoechst

In order to initiate studies on chemical carcinogenesis in an in vitro system analogous to mouse epidermis, primary epidermal cell cultures from perinatal mouse skin were established. A standardized method for the large-scale isolation of epidermal cells from late embryonic or newborn mouse dorsal skin has been developed. The epidermal cells were separated from fibroblasts by two series of discontinuous Ficoll density gradients. Using 80 to 100 animals/experiment, an average yield of 3×10⁶ viable epidermal cells/animal was obtained. The viability of the purified cell suspension exceeded 85%, and the plating efficiency, representing the growing cell fraction 24 h after plating, was about 43%.The cultures started as epithelial monolayers without fibroblast contamination. Their epidermal nature and origin was proved immunologically by an in vivo absorbed rabbit anti-mouse epidermis antiserum. The purity of the epidermal cells was quantitatively determined in trypsinized suspensions by the indirect immunofluorescence test yielding more than 95% epidermal antigen-positive cells. About half of the remaining antigen-negative cells could be identified as melanocytes. These highly purified epidermal cells grew in vitro for 2–3 weeks without dermal constituents or diffusible mesenchymal factors. The monolayers differentiated in culture giving rise to keratinizing cell sheets on top of the proliferating basal layer. By morphological, histochemical and physical methods, it could be evidenced that the differentiation processes in vitro are quite similar to keratinization in vivo.