Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels

Small conductance Ca²⁺-sensitive potassium (SK2) channels are voltage-independent, Ca²⁺-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca²⁺ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3′ terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca²⁺ and Ca²⁺-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca²⁺ influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.     

An “orientation sphere” visualization for examining animal head movements

1. Animal behavior is elicited, in part, in response to external conditions, but understanding how animals perceive the environment and make the decisions that bring about these behavioral responses is challenging.
2. Animal heads often move during specific behaviors and, additionally, typically have sensory systems (notably vision, smell, and hearing) sampling in defined arcs (normally to the front of their heads). As such, head‐mounted electronic sensors consisting of accelerometers and magnetometers, which can be used to determine the movement and directionality of animal heads (where head “movement” is defined here as changes in heading [azimuth] and/or pitch [elevation angle]), can potentially provide information both on behaviors in general and also clarify which parts of the environment the animals might be prioritizing (“environmental framing”).
3. We propose a new approach to visualize the data of such head‐mounted tags that combines the instantaneous outputs of head heading and pitch in a single intuitive spherical plot. This sphere has magnetic heading denoted by “longitude” position and head pitch by “latitude” on this “orientation sphere” (O‐sphere).
4. We construct the O‐sphere for the head rotations of a number of vertebrates with contrasting body shape and ecology (oryx, sheep, tortoises, and turtles), illustrating various behaviors, including foraging, walking, and environmental scanning. We also propose correcting head orientations for body orientations to highlight specific heading‐independent head rotation, and propose the derivation of O‐sphere‐metrics, such as angular speed across the sphere. This should help identify the functions of various head behaviors.
5. Visualizations of the O‐sphere provide an intuitive representation of animal behavior manifest via head orientation and rotation. This has ramifications for quantifying and understanding behaviors ranging from navigation through vigilance to feeding and, when used in tandem with body movement, should provide an important link between perception of the environment and response to it in free‐ranging animals.

     

The effects of Bombyx mori silk strain and extraction time on the molecular and biological characteristics of sericin

Sericin was extracted from three strains of Thai Bombyx mori silk cocoons (white shell Chul1/1, greenish shell Chul3/2, and yellow shell Chul4/2) by a high-pressure and high-temperature technique. The characteristics of sericin extracted from different fractions (15, 45, and 60 min extraction process) were compared. No differences in amino acid composition were observed among the three fractions. For all silk strains, sericin extracted from a 15-min process presented the highest molecular weight. The biological potential of the different sericin samples as a bioadditive for 3T3 fibroblast cells was assessed. When comparing sericin extracted from three silk strains, sericin fractions extracted from Chul4/2 improved cell proliferation, while sericin from Chul 1/1 activated Type I collagen production to the highest extent. This study allows the natural variability of sericin obtained from different sources and extraction conditions to be addressed and provides clues for the selection of sericin sources.     

Immunocytochemical characterisation of endophytic bacteriaAzospirillum brasilense, Herbaspirillum seropedicae, Burkholderia ambifaria andGluconacetobacter diazotrophicus

In the present work an immunocytochemical characterisation of four endophytic bacterial species has been made by using polyclonal antiserum produced against each of the four bacterial strains previously heated at 60 °C. The aim of this researchsito identify common elements among bacteria associated with their endophytic behaviour. Analysis of extracts of each strain by immunoblotting and ELISA confirmed the presence of proteins from different bacterial strains made up of common epitopes. However, antisaproduced againstHerbaspirillum seropedicae andBurkholderia ambifaria show a high number of bands recognised on each extracts, while antisera againstAzospirillum brasilense andGluconacetobacter diazotrophicus show a low number of bands recognised on each extract. Immunogold labelling showed that epitopes are located both on the cell wall and in the cytoplasm; most likely they could be preursor cell wall proteins synthesized inside the cytoplasm and subsequently transported onto cell wall. Finally, the common bands amog bacterial strains revealed by immunoblotting could play a role as active hydrolases involved in host tissue penetration.     

Heterogeneous phenotype of human glioblastoma: In vitro study

Glioblastomas (GBMs) are the most lethal primary brain tumours. Increasing evidence shows that brain tumours contain the population of stem cells, so‐called cancer stem cells (CSCs). Stem cell marker CD133 was reported to identify CSC population in GBM. Further studies have indicated that CD133 negative cells exhibiting similar properties and are able to initiate the tumour, self‐renew and undergo multilineage differentiation. GBM is a highly heterogeneous tumour and may contain different stem cell populations with different functional properties. We characterized five GBM cell lines, established from surgical samples, according to the marker expression, proliferation and differentiation potential. CD133 positive cell lines showed increased proliferation rate in neurosphere condition and marked differentiation potential towards neuronal lineages. Whereas two cell lines low‐expressing CD133 marker showed mesenchymal properties in vitro, that is high proliferation rate in serum condition and differentiation in mesenchymal cell types. Further, we compared therapy resistance capacity of GBM cell lines treated with hydroxyurea. Our results suggest that CSC concept is more complex than it was believed before, and CD133 could not define entire stem cell population within GBM. At least two different subtypes of GBM CSCs exist, which may have different biological characteristics and imply different therapeutic strategies. Copyright © 2013 John Wiley & Sons, Ltd.     

Effect of cytochalasins on cytosolic-free calcium concentration and phosphoinositide metabolism in leukocytes

Cytochalasins are routinely used to stimulate a variety of functions in eukaryotic cells even though their precise mode of action remains to be elucidated. In the present work we used the fluorescent Ca2+ indicator quin2 to study the effect of various cytochalasins, cytochalasins A, B, C, D, E (CA, CB, CC, CD, CE) and dihydrocytochalasin B (dhCB) on the intracellular Ca2+ concentration ([Ca2+]i) in various types of leukocytes, viz, neutrophils and lymphocytes. In human neutrophils, cytochalasins increase [Ca2+]i mainly by releasing Ca2+ from membrane-bound, intracellular stores. Thus, in order to readily appreciate the effect of cytochalasins on [Ca2+ )i, these cells must be loaded with low intracellular quin2 concentrations. On the other hand, in peripheral blood lymphocytes, splenocytes and thymocytes, the increase in [Ca2+]i is predominantly due to an increased Ca2+ influx from the extracellular medium. In addition, we found that in neutrophils these drugs prolong the increase in [Ca2+]i induced by chemotactic peptides, probably by increasing the cell permeability to Ca2+. Finally, in thymocytes, cytochalasins potentiate the production of inositol phosphates induced by the polyclonal mitogen concanavalin A (conA).     

Voltage-dependent activation and inactivation of calcium channels in PC12 cells. Correlation with neurotransmitter release

The existence and mechanisms of inactivation of voltage-gated Ca2+ channels are important, but still debatable, physiological problems. By using the Ca2+ indicators quin2 and fura-2, we demonstrate that in PC12 cells voltage-gated Ca2+ channels undergo inactivation dependent on both voltage and [Ca2+]i. Inactivation, however, is never complete and a small number of channels remains open during prolonged depolarization, explaining the steady state elevation of [Ca2+]i observed in cells depolarized with high KCl. A close parallel exists between Ca2+ channel inactivation and the transient nature of neurotransmitter release: secretion is rapidly stimulated during the first 30 s of depolarization, when a transient overshoot in [Ca2+]i can be demonstrated, while it is negligible during the following period, despite the persistence of an elevated [Ca2+]i; predepolarization in Ca2+-free medium and subsequent addition of Ca2+ (a condition which allows the development of the voltage inactivation) abolishes the fast phase of secretion, while not modifying the steady state [Ca2+]i eventually attained; and increases in the intracellular Ca2+ buffering decreases the amplitude of the fast secretion phase induced by KCl without altering the steady state [Ca2+]i. We suggest that localized [Ca2+]i gradients form close to the plasma membrane shortly after depolarization and that the [Ca2+]i reached in these regions is the relevant parameter in the regulation of secretion.     

Monoclonal antibodies against pertussis toxin subunits

Abstract Twenty monoclonal antibodies (mAbs) reacting with cholera toxin (CT) of Vibrio cholerae strain 569B were characterized in cross-section and G_M1 ganglioside inhibition assays. MAbs were characterized by reaction with CT and Escherichia coli heat-labile porcine strain (LT_p) and human strain (LTh) enterotoxins, and by G_M1 ganglioside inhibition of mAb binding. Eight of 10 CT-A specific and 3 of 10 CT-B-specific mAbs cross-reacted with LTh and LTp. G_M1 ganglioside inhibited reactions of the CT-B cross-reacting antibodies. Results showed that these epitodes common to the B subunit of CT and LT are located in or near the G_M1 ganglioside binding region, and that the G_M1 ganglioside-binding region of LT differs from that of CT.     

Fura-2 secretion and sequestration in macrophages. A blocker of organic anion transport reveals that these processes occur via a membrane transport system for organic anions

Fura-2, loaded into J774.2 macrophages as the acetoxymethyl ester, is sequestered into intracellular vacuoles within 90 min after the beginning of the loading at 37 degrees C. The dye is also efficiently secreted from the cells. Sequestration and secretion of fura-2 reduce the accuracy of measurements of cytosolic free Ca2+ concentration in this cell line. Fura-2 is also sequestered and secreted by J774.2 when the dye is loaded into the cytoplasm as the pentapotassium salt by reversible permeabilization of the plasma membrane. Regardless of the mechanism by which fura-2 is loaded into the cytoplasm, both sequestration and secretion are prevented by 2.5 mM probenecid, a blocker of organic anion transport. Probenecid has no effect on resting or stimulated cytosolic free Ca2+ levels or on FcR-mediated phagocytosis. These findings suggest that macrophages express a transport mechanism for the anionic form of fura-2. This transport system is responsible for the clearance of fura-2 from the cytoplasm of this cell type. Furthermore we suggest that use of probenecid to block secretion and intracellular sequestration of fura-2 may overcome problems arising in the application of this Ca2+ indicator to macrophages and perhaps to other cell types.     

The presence of immunoreactive vertebrate bioactive peptide substances in hemocytes of the freshwater snailViviparus ater (gastropoda,prosobranchia)

1. Using an immunocytochemical procedure a wide range of immunoreactive vertebrate bioactive peptides (BAPs) has been found in hemocytes of Viviparus ater: bombesin, calcitonin, CCK-8, CCK-39, GH, glucagon, insulin, oxytocin, neurotensin, secretin, serotonin, somatostatin, substance P, vasopressin, and VIP. 2. No immunostaining was observed for antigastrin and antithyroglobulin antibodies. 3. The presence of BAP-like molecules in hemocytes suggests a correlation between hemocyte and APUD cells and is evidence of a relationship between the neuroendocrine and the immune systems.