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.     

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.     

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.     

Effect of Zeitgeber Intensity Reduction on a Simulated Dual-Oscillator Human Circadian System: Classical and Dynamic Analysis

The two-oscillator model of human circadian rhythmicity was analyzed when a zeitgeber relative intensity of 1, 0.5, or 0.1 was introduced into the equations. Fourier analysis was compared with dynamic analysis such as attractor reconstruction or Liapunov exponent calculation. After a 50 or 90% reduction in zeitgeber intensity, the dynamics of the system became equivalent and differed significantly from those of a system with maximal zeitgeber intensity. When 10% aleatory noise was added to the data, the analysis was still applicable, and the results obtained were essentially the same as in the absence of noise. Dynamic analysis could thus provide a distinct classification for periodic data, based on the type of analysis.     

Effect of a melatonin implant on the circadian variation of plasma prolactin and rectal temperature in newborn sheep.

Plasma prolactin and rectal temperature show a circadian rhythm in newborn sheep raised under continuous light. Melatonin lowers the concentration of plasma prolactin but it is not known if it affects its circadian rhythm. To detect whether melatonin acts on the circadian system we studied the effect of a subcutaneous melatonin implant in the circadian rhythms of prolactin and rectal temperature in newborn lambs raised under continuous light. We placed catheters in the pedal artery and vein in 9 newborn lambs (2-5 days of age). A subcutaneous melatonin implant was placed in 4 of the lambs at 9-12 days of age. Blood samples and rectal temperature measurements were obtained hourly for a period of 24 h, 11-15 days after the implant, at 20-27 days of age. To avoid interferences of heparin in our melatonin assay, serum melatonin concentration was measured before and during the implant in three additional newborns. Prolactin and melatonin were measured by RIA. Melatonin concentrations were 52.8 +/- 45.9 pg/ml (day) and 315.5 +/- 77.0 pg/ml (night) before treatment (SEM, P less than 0.001), and increased to 594.1 +/- 54.5 pg/ml after placing the implant (there was no difference in melatonin concentration between day and night during the time that the implant was in place). Melatonin had no effect on rectal temperature or its rhythm, but decreased basal plasma prolactin concentration (control: 97.5 +/- 11.3 ng/ml; treated: 25.1 +/- 2.4 ng/ml, P less than 0.001) and abolished the prolactin circadian rhythm, (Cosinor analysis): control: log prolactin (ng/ml) = 1.8 + 0.26 cos 15 (t - 11.16), p = 0.05; treated: log prolactin (ng/ml) = 1.2 + 0.14 cos 15 (t - 9.43), P = 0.36.     

Presence of a drifting algal bed in the Mar Piccolo basin,Taranto (Ionian Sea,Southern Italy)

A survey is reported of the drifting algal community in Mar Piccolo, a polluted basin subject to sewage outlets. The key role was played by a few key species, mainly floridean red algae.     

Isolation and partial characterization of a protein kinase NII from wheat germ chromatin

A protein kinase, type NII, has been purified from wheat germ chromatin. The enzyme, which uses both ATP and GTP as phosphoryl donors, catalyzes the phosphorylation of casein, phosvitin and E. coli RNA polymerase, but not of histone proteins. Polypeptide bands at 46 kDa, 37 kDa and 25 kDa were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autophosphorylation of the 25 kDa subunit was observed following incubation of the purified kinase with (-³²P)ATP and (-³²P)GTP.