KCNE4 juxtamembrane region is required for interaction with calmodulin and for functional suppression of KCNQ1

Voltage-gated potassium (K(V)) channels, such as KCNQ1 (K(V)7.1), are modulated by accessory subunits and regulated by intracellular second messengers. Accessory subunits belonging to the KCNE family exert diverse functional effects on KCNQ1, have been implicated in the pathogenesis of various genetic disorders of heart rhythm, and contribute to transducing intracellular signaling events into changes in K(V) channel activity. We investigated the interactions between calmodulin (CaM), the ubiquitous Ca(2+)-transducing protein that binds and confers Ca(2+) sensitivity to the biophysical properties of KCNQ1, and KCNE4. These studies were motivated by the observed similarities between the suppression of KCNQ1 function by pharmacological disruption of KCNQ1-CaM interactions and the effects of KCNE4 co-expression on the channel. We determined that KCNE4, but not KCNE1, can biochemically interact with CaM and that this interaction is Ca(2+)-dependent and requires a tetraleucine motif in the juxtamembrane region of the KCNE4 C terminus. Furthermore, disruption of the KCNE4-CaM interaction either by mutagenesis of the tetraleucine motif or by acute Ca(2+) chelation impairs the ability of KCNE4 to inhibit KCNQ1. Our findings have potential relevance to KCNQ1 regulation both by KCNE accessory subunits and by an important intracellular signaling molecule.     

Cloning of B cells from autoimmune MRL-lpr/lpr and MRL.xid mice

The relationship between colony formation (cloning) of B cells and their activation in murine autoimmunity was investigated in MRL-lpr/lpr and MRL.xid mice. Cells from MRL-lpr/lpr mice showed similar requirements for in vitro growth as normal CBA/J and BALB/c cells, with maximal colony formation in the presence of the supporting factors lipopolysaccharide and sheep red blood cells. The frequency of colony-forming cells from MRL-lpr/lpr spleens or hapten-specific B-cell preparations was slightly greater than the two normal control strains, with this difference significant only for a comparison of BALB/c and MRL-lpr/lpr spleens. In contrast, MRL-lpr/lpr mice bearing the xid gene for B-cell immunodeficiency (MRL.xid) had markedly reduced B-cell colony formation. These mice nevertheless expressed anti-DNA antibodies, although at levels reduced from that of MRL-lpr/lpr controls. These results indicate that enhanced in vitro colony formation need not accompany B-cell hyperactivity in murine autoimmune disease and that autoantibody production can occur in mice with impairment in this growth property.     

Pelagostrobilidium neptuni (Montagnes and Taylor, 1994) and Strombidium biarmatum nov. spec. (Ciliophora, Oligotrichea): phylogenetic position inferred from morphology, ontogenesis, and gene sequence data

Morphological data from life, protargol impregnation, and scanning electron microscopy were combined with genetic data not only to describe the marine plankton ciliates Pelagostrobilidium neptuni (Montagnes and Taylor, 1994) Petz, Song, and Wilbert, 1995 and Strombidium biarmatum nov. spec., but also to elucidate their phylogenetic relationships. Additionally, the ontogenesis of P. neptuni was studied and the diagnosis of the genus Pelagostrobilidium was improved due to further data from the newly affiliated species P. epacrum (Lynn and Montagnes, 1988) nov. comb. (basionym: Strobilidium epacrum Lynn and Montagnes, 1988). The phylogenetic analysis of the small subunit ribosomal RNA genes matched the morphologic and ontogenetic assigning of P. neptuni to the choreotrichid family Strobilidiidae. The considerable genetic distance of d = 0.074 between P. neptuni and Strobilidium caudatum corroborated the morphological differences and thus the maintenance of the genus Pelagostrobilidium. Strombidium biarmatum nov. spec. is a typical member of the genus, except for the two types of extrusomes ("trichites"): ~12 × 0.5 μm, needle-shaped ones attached anterior to the girdle kinety and ~6 × 0.5 μm, rod-shaped ones at the distal end of the intermembranellar ridges. Its flask-shaped resting cysts have several strong spines. In accordance with the morphologic data, S. biarmatum is placed within the order Oligotrichida by gene sequence analysis. The great genetic distances within the oligotrichids support the diversity found in morphologic and ontogenetic studies.     

Fixed Dystonia in Complex Regional Pain Syndrome: a Descriptive and Computational Modeling Approach

Background  

Complex regional pain syndrome (CRPS) may occur after trauma, usually to one limb, and is characterized by pain and disturbed blood flow, temperature regulation and motor control. Approximately 25% of cases develop fixed dystonia. Involvement of dysfunctional GABAergic interneurons has been suggested, however the mechanisms that underpin fixed dystonia are still unknown. We hypothesized that dystonia could be the result of aberrant proprioceptive reflex strengths of position, velocity or force feedback.     

Der Nachweis des Cholins in der Pflanze

Zusammenfassung Es wurden in der vorliegenden Arbeit Methoden ausgearbeitet, die einen raschen mikrochemischen Nachweis des Cholins ermöglichen. Mit diesen Methoden wurden über 100 Spezies aus den verschiedensten Pflanzenfamilien auf ihren Cholingehalt sowie auf den Cholingehalt ihrer Organe geprüft und es wurde das Cholin überall, in Stengeln, Blättern, Blüten, Holz, Rinde, Wurzeln usw. gefunden. Kein Cholin wurde nur in den drei untersuchten Flechten (Evernia prunastri, Parmelia sulcata undParmelia perforata) gefunden.Orientierende Versuche über die Veränderungen des Cholingehaltes von keimenden Samen und von Blättern im Laufe einer Nacht geben Anhaltspunkte für starke physiologische Verschiebungen im Cholingehalt.Nach Beendigung der Ausarbeitung einer quantitativen Methode zur Bestimmung von Cholin in kleinen Mengen Ausgangsmaterial sollen die angedeuteten Fragen auf exakter quantitativer Grundlage näher untersucht werden.     

Rat adipocyte lipoprotein lipase activity is inhibited by theophylline and stimulated by inosine through adenosine-independent mechanisms

Incubation of rat adipose tissue or isolated rat adipocytes with high (50 mM) but not with low concentrations (0.5 mM) of theophylline results in a decrease of lipoprotein lipase (LPL) activity. This effect is not altered by the addition of adenosine deaminase, indicating that the decrease of adipose LPL activity by theophylline is not due to the competition of theophylline with adenosine. On the contrary, incubation of isolated fat cells with adenosine (0.1 – 100 μM) results in an increase of the intracellular form of LPL activity. As this effect is also observed in cells incubated with adenosine deaminase (40 mU/ml) or with inosine (0.1 – 100 μM) but not in cells incubated with the adenosine analog N⁶-phenylisopropyladenosine, it is concluded that the increase in the intracellular form of LPL found after incubation with adenosine is not due to adenosine $\text{per se}$ but to inosine generated from the breakdown of endogenous adenosine by adenosine deaminase.     

Sequencing of high G+C microbial genomes using the ultrafast pyrosequencing technology

Next generation pyrosequencing of high G + C content genomes still poses problems to automated sequencing and assembly processes which necessitates cost and time intensive manual work in order to finish such genomes completely. The sequencing of the high G + C actinomycete Actinoplanes sp. SE50/110 was performed with standard pyrosequencing technology (454 Life Sciences) and revealed a high number of gaps. The reasons for the introduction of gaps were analyzed on a previously known 41 kb long DNA reference sequence from Actinoplanes sp. SE50/110, hosting the acarbose biosynthesis gene cluster. Mapping of the sequencing results on the reference gene cluster sequence revealed a fragmentation into 30 contiguous sequences of different lengths. The gaps between these sequences were characterized by extremely low read coverage which strongly correlated with the G + C content in the gap regions in a negative manner. Furthermore, the gap-sequences contained strong stem-loop structures which hindered the amplification of these sequences during the emulsion PCR. Being significantly underrepresented or absent in the subsequent sequencing process, these sequences lead to weakly or uncovered genomic regions which forces the assembly algorithm to output multiple contiguous sequences instead of one finished genome. However, by applying a different pyrosequencing protocol, it was possible to sequence the complete acarbose biosynthesis gene cluster. The changes to the protocol include longer read length and addition of chemicals to the amplification chemistry, which reduces the self-annealing of DNA fragments during the amplification process and enables the complete reconstruction of high G + C content genomes without manual intervention.