The KCNQ1 (Kv7.1) COOH terminus, a multitiered scaffold for subunit assembly and protein interaction

The Kv7 subfamily of voltage-dependent potassium channels, distinct from other subfamilies by dint of its large intracellular COOH terminus, acts to regulate excitability in cardiac and neuronal tissues. KCNQ1 (Kv7.1), the founding subfamily member, encodes a channel subunit directly implicated in genetic disorders, such as the long QT syndrome, a cardiac pathology responsible for arrhythmias. We have used a recombinant protein preparation of the COOH terminus to probe the structure and function of this domain and its individual modules. The COOH-terminal proximal half associates with one calmodulin constitutively bound to each subunit where calmodulin is critical for proper folding of the whole intracellular domain. The distal half directs tetramerization, employing tandem coiled-coils. The first coiled-coil complex is dimeric and undergoes concentration-dependent self-association to form a dimer of dimers. The outer coiled-coil is parallel tetrameric, the details of which have been elucidated based on 2.0 A crystallographic data. Both coiled-coils act in a coordinate fashion to mediate the formation and stabilization of the tetrameric distal half. Functional studies, including characterization of structure-based and long QT mutants, prove the requirement for both modules and point to complex roles for these modules, including folding, assembly, trafficking, and regulation.     

The detection of age-, gender-, and region-specific changes in mouse brain tocopherol levels via the application of different validated HPLC methods

The aging process clearly increases the demand for antioxidant protection, especially in the brain, involving that provided by α-tocopherol (αT). However, little is known about the age-related changes in brain αT levels and the influencing effect of gender on it, in human or murine samples as well. Accordingly, the aim of the current study was to detect age-, gender- and region-specific changes in αT concentrations in mouse brain tissue and to assess the influencing effect of plasma αT levels on it. Female and male C57BL/6 mice at the ages of 6, 16 and 66 weeks (n?=?9 in each group) were applied. αT levels were determined with high performance liquid chromatography (HPLC) from the striatum, cortex, hippocampus, cerebellum, brainstem and from plasma samples. A detailed validation process was carried out for the applied HPLC method as well. The results demonstrated that brain αT levels significantly increased in the striatum, cortex, and hippocampus with aging in both genders, but in a more pronounced way in females with an increasing magnitude of this difference. In case of the cerebellum, a moderate elevation could be detected only in females, whereas in case of the brainstem there was no significant change in αT level. With regard to plasma samples, no clear trend could be identified. The current study is the first to present age-dependent gender-specific changes in αT level in certain brain regions of the C57Bl/6 mouse strain, and may provide meaningful information for future therapeutic studies targeting aging-related processes.     

Potential application of glycerol in the production of plant beneficial microorganisms

Journal of Industrial Microbiology & Biotechnology - This review highlights the importance of research for development of biofertilizer and biocontrol products based on the use of glycerol for...     

Circadian rhythm in muscarinic receptor subtypes in rat forebrain

The binding of different ligands to muscarinic receptors in the central nervous system is regulated by several factors. Among these are the administration of drugs, disease, ontogeny or aging. Studies carried out in rat brains have demonstrated changes in the density of the muscarinic receptors at different times of the day. These changes might be related to variations in the circadian rhythms. In this work we have studied the binding of the [3H]-N-methyl-escopolamine, the agonist carbachol and the antagonist pirenzepine to muscarinic receptors in rat forebrains at 10.00, 14.00, 18.00, 22.00, 02.00 and 06.00 hr. We have observed changes in the density of muscarinic receptors but not changes in affinity to the radioligand. The Bmax values obtained by saturation studies were maximum at 14.00 hr and minimum at 02.00 hr (P less than 0.05 Mann-Whitney's test). Inhibition studies in the presence of the non-selective agonist carbachol and the selective antagonist pirenzepine, at the same time-points, did not show statistically significant changes in the Bmax values. These data indicate that changes in the Bmax values are only observed in the total population of muscarinic receptors and are not due to modifications in the subtypes of muscarinic receptors nor to the different affinity states of agonist binding.     

GD1b-Derived Gangliosides Modulate Fc��RI Endocytosis in Mast Cells

The role of the mast cell–specific gangliosides in the modulation of the endocytic pathway of FcεRI was investigated in RBL-2H3 cells and in the ganglioside-deficient cell lines, E5 and D1. MAb BC4, which binds to the α subunit of FcεRI, was used in the analysis of receptor internalization. After incubation with BC4-FITC for 30 min, endocytic vesicles in RBL-2H3 and E5 cells were dispersed in the cytoplasm. After 1 hr, the endocytic vesicles of the RBL-2H3 cells had fused and formed clusters, whereas in the E5 cells, the fusion was slower. In contrast, in D1 cells, the endocytic vesicles were smaller and remained close to the plasma membrane even after 3 hr of incubation. When incubated with BC4-FITC and subsequently imunolabeled for markers of various endocytic compartments, a defect in the endocytic pathway in the E5 and D1 cells became evident. In the D1 cells, this defect was observed at the initial steps of endocytosis. Therefore, the ganglioside derivatives from GD1b are important in the endocytosis of FcεRI in mast cells. Because gangliosides may play a role in mast cell–related disease processes, they provide an attractive target for drug therapy and diagnosis.