Tyrosine kinase modulation of protein kinase C activity regulates G protein-linked Ca2+ signaling in leukemic hematopoietic cells

We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control of an inducible promoter) and several human leukemia cell lines to study the effect of high tyrosine kinase activity on G protein-coupled receptor (GPCR) agonist-stimulated cellular Ca(2+) release and store-operated Ca(2+) entry (SOCE). After induction of Bcr/Abl expression, GPCR-linked SOCE increased. The effect was reverted in the presence of the specific Abl inhibitor imatinib (1microM) and the Src inhibitor PP2 (10microM). In leukemic cell lines constitutively expressing high tyrosine kinase activity, Ca(2+) transients were reduced by imatinib and/or PP2. Ca(2+) transients were enhanced by specific inhibitors of PKC subtypes and this effect was amplified by tyrosine kinase inhibition in Bcr/Abl expressing TonB210.1 and K562 cells. Under all conditions Ca(2+) transients were essentially blocked by the PKC activator PMA. In Bcr/Abl expressing (but not in native) TonB210.1 cells, tyrosine kinase inhibitors enhanced PKCalpha catalytic activity and PKCalpha co-immunoprecipitated with Bcr/Abl. Unlike native TonB210.1 cells, Bcr/Abl expressing cells showed a high rate of cell death if Ca(2+) influx was reduced by complexing extracellular Ca(2+) with BAPTA. Our data suggest that tonic inhibition of PKC represents a mechanism by which high tyrosine kinase activity can enhance cellular Ca(2+) transients and thus exert profound effects on the proliferation, apoptosis and chemotaxis of leukemic cells.

Ophioluxin, a convulxin-like C-type lectin from Ophiophagus hannah (King cobra) is a powerful platelet activator via glycoprotein VI

Ophioluxin, a potent platelet agonist, was purified from the venom of Ophiophagus hannah (King cobra). Under nonreducing conditions it has a mass of 85 kDa, similar to convulxin, and on reduction gives two subunits with masses of 16 and 17 kDa, slightly larger than those of convulxin. The N-terminal sequences of both subunits are very similar to those of convulxin and other C-type lectins. Ophioluxin induces a pattern of tyrosine-phosphorylated proteins in platelets like that caused by convulxin, when using appropriate concentrations based on aggregation response, because it is about 2-4 times more powerful as agonist than the latter. Ophioluxin and convulxin induce [Ca(2+)](i) elevation both in platelets and in Dami megakaryocytic cells, and each of these C-type lectins desensitizes responses to the other. Convulxin agglutinates fixed platelets at 2 microg/ml, whereas ophioluxin does not, even at 80 microg/ml. Ophioluxin resembles convulxin more than echicetin or alboaggregin B because polyclonal anti-ophioluxin antibodies recognize both ophioluxin and convulxin, but not echicetin, and platelets adhere to and spread on ophioluxin- or convulxin-precoated surfaces in the same way that is clearly different from their behavior on an alboaggregin B surface. Immobilized ophioluxin was used to isolate the glycoprotein VI-Fcgamma complex from resting platelets, which also contained Fyn, Lyn, Syk, LAT, and SLP76. Ophioluxin is the first multiheterodimeric, convulxin-like snake C-type lectin, as well as the first platelet agonist, to be described from the Elapidae snake family.

Mechanism of Ca(v)1.2 channel modulation by the amino terminus of cardiac beta2-subunits

L-type calcium channels are composed of a pore, alpha1c (Ca(V)1.2), and accessory beta- and alpha2delta-subunits. The beta-subunit core structure was recently resolved at high resolution, providing important information on many functional aspects of channel modulation. In this study we reveal differential novel effects of five beta2-subunits isoforms expressed in human heart (beta(2a-e)) on the single L-type calcium channel current. These splice variants differ only by amino-terminal length and amino acid composition. Single-channel modulation by beta2-subunit isoforms was investigated in HEK293 cells expressing the recombinant L-type ion conducting pore. All beta2-subunits increased open probability, availability, and peak current with a highly consistent rank order (beta2a approximately = beta2b > beta2e approximately = beta2c > beta2d). We show graded modulation of some transition rates within and between deep-closed and inactivated states. The extent of modulation correlates strongly with the length of amino-terminal domains. Two mutant beta2-subunits that imitate the natural span related to length confirm this conclusion. The data show that the length of amino termini is a relevant physiological mechanism for channel closure and inactivation, and that natural alternative splicing exploits this principle for modulation of the gating properties of calcium channels.

C-Port Flex-A-assisted automated anastomosis for high-flow extracranial-intracranial bypass surgery in patients with symptomatic carotid artery occlusion: a feasibility study. Clinical article

OBJECT: Preliminary experience with the C-Port Flex-A Anastomosis System (Cardica, Inc.) to enable rapid automated anastomosis has been reported in coronary artery bypass surgery. The goal of the current study was to define the feasibility and safety of this method for high-flow extracranial-intracranial (EC-IC) bypass surgery in a clinical series. METHODS: In a prospective study design, patients with symptomatic carotid artery (CA) occlusion were selected for C-Port-assisted high-flow EC-IC bypass surgery if they met the following criteria: 1) transient or moderate permanent symptoms of focal ischemia; 2) CA occlusion; 3) hemodynamic instability; and 4) had provided informed consent. Bypasses were done using a radial artery graft that was proximally anastomosed to the superficial temporal artery trunk, the cervical external, or common CA. All distal cerebral anastomoses were performed on M2 branches using the C-Port Flex-A system. RESULTS: Within 6 months, 10 patients were enrolled in the study. The distal automated anastomosis could be accomplished in all patients; the median temporary occlusion time was 16.6+/-3.4 minutes. Intraoperative digital subtraction angiography (DSA) confirmed good bypass function in 9 patients, and in 1 the anastomosis was classified as fair. There was 1 major perioperative complication that consisted of the creation of a pseudoaneurysm due to a hardware problem. In all but 1 case the bypass was shown to be patent on DSA after 7 days; furthermore, in 1 patient a late occlusion developed due to vasospasm after a sylvian hemorrhage. One-week follow-up DSA revealed transient asymptomatic extracranial spasm of the donor artery and the radial artery graft in 1 case. Two patients developed a limited zone of infarction on CT scanning during the follow-up course. CONCLUSIONS: In patients with symptomatic CA occlusion, C-Port Flex-A-assisted high-flow EC-IC bypass surgery is a technically feasible procedure. The system needs further modification to achieve a faster and safer anastomosis to enable a conclusive comparison with standard and laser-assisted methods for high-flow bypass surgery.

Die gestempelten Bronze-Gewichte aus Himera

Bei Ausgrabungen in der antiken griechischen Stadt Himera an der Nordküste Siziliens wurden zwei Bronzegewichte gefunden. Beide zeigen eine Beprägung , die dasselbe Prägebild wie die Rückseite zeitgleicher Litraprägungen aus Himera wiedergeben. Im Jahr 2010 erschien im Münzhandel zudem ein zuvor unveröffentlichtes Bronzegewicht, beprägt mit einem Hemilitron-Rückseitenstempel aus Himera. Anhand dieser gestempelten Bronzegewichte können – unter Einbezug weiterer Quellen – die im letzten Viertel des 5. Jhs. v. Chr. in Himera üblichen Gewichtssysteme für Bronze nachgezeichnet werden: Neben einer überregionalen, kanonischen Bronze-Litra, die bereits früher im 5. Jh. v. Chr. von ca. 108 g auf ca. 216 g im Gewicht verdoppelt worden war, scheint ein regionaler Litrafuß zu ca. 50 g etabliert gewesen zu sein. Dieser den frühesten Bronzemünzen von Himera, Selinunt und Akragas zugrundeliegende Litrafuß existierte auch dann noch, als die Münzen bereits in stark reduziertem Gewicht geprägt wurden.

"Eventless" InsP 3 -dependent SR-Ca 2+ Release Affecting Atrial Ca 2+ Sparks

Augmented inositol 1,4,5-trisphosphate receptor (InsP3R) function has been linked to a variety of cardiac pathologies, including cardiac arrhythmia. The contribution of inositol 1,4,5-trisphosphate-induced Ca2+ release (IP3ICR) in excitation-contraction coupling (ECC) under physiological conditions, as well as under cellular remodelling, remains controversial. Here we test the hypothesis that local IP3ICR directly affects ryanodine receptor (RyR) function and subsequent Ca2+-induced Ca2+ release in atrial myocytes. IP3ICR was evoked by UV-flash photolysis of caged InsP3 under whole-cell configuration of the voltage-clamp technique in atrial myocytes isolated from C57/BL6 mice. Photolytic release of InsP3 was accompanied by a significant increase in the Ca2+ release event frequency (4.14±0.72 vs. 6.20±0.76 events (100 μm)−1 s−1). These individual photolytically triggered Ca2+ release events were identified as Ca2+ sparks, which originated from RyR openings. This was verified by Ca2+ spark analysis and pharmacological separation between RyR and InsP3R-dependent sarcoplasmic reticulum (SR)-Ca2+ release (2-aminoethoxydiphenyl borate, xestospongin C, tetracaine). Significant SR-Ca2+ flux but eventless SR-Ca2+ release through InsP3R were characterized using SR-Ca2+ leak/SR-Ca2+ load measurements. These results strongly support the idea that IP3ICR can effectively modulate RyR openings and Ca2+ spark probability. We conclude that eventless and highly efficient InsP3-dependent SR-Ca2+ flux is the main mechanism of functional cross-talk between InsP3Rs and RyRs, which may be an important factor in the modulation of ECC sensitivity.

Les villages préhistoriques des bords des lacs circum-alpins entre le Ve et IVe millénaire av. J.-C.

Connus sous le nom populaire de palafittes, les habitats préhistoriques construits sur les rives des lacs subalpins du Néolithique à l’aube de l’âge du Fer (entre 5300 et 700 av. J.-C.) offrent des informations exceptionnelles sur l’évolution culturelle d’une importante région européenne, grâce à la préservation remarquable des matériaux organiques, en particulier du bois. À partir de la deuxième moitié du XXe siècle, le perfectionnement des techniques de fouille subaquatiques et de la dendrochronologie permettront la construction d’un schéma chronologique précis pour l’Europe nord-alpine. Les recherches contribueront à des observations d’ordre écologique à l’échelle locale et régionale et à l’identification des rythmes de développement des villages. Sous l’égide de l'UNESCO, les années 2010 verront la constitution d’un inventaire vaste et uniforme des sites préhistoriques des lacs circumalpins, classés Patrimoine culturel mondial en juin 2011.

Nonclassical mechanisms of progesterone action in the brain: I. Protein kinase C activation in the hypothalamus of female rats.

The modulation of gene regulation by progesterone (P) and its classical intracellular regulation by progestin receptors in the brain, resulting in alterations in physiology and behavior has been well studied. The mechanisms mediating the short latency effects of P are less well understood. Recent studies have revealed rapid nonclassical signaling action of P involving the activation of intracellular signaling pathways. We explored the involvement of protein kinase C (PKC) in P-induced rapid signaling in the ventromedial nucleus of the hypothalamus (VMN) and preoptic area (POA) of the rat brain. Both the Ca2+-independent (basal) PKC activity representing the activation of PKC by the in vivo treatments and the Ca+2-dependent (total) PKC activity assayed in the presence of exogenous cofactors in vitro were determined. A comparison of the two activities demonstrated the strength and temporal status of PKC regulation by steroid hormones in vivo. P treatment resulted in a rapid increase in basal PKC activity in the VMN but not the POA. Estradiol benzoate priming augmented P-initiated increase in PKC basal activity in both the VMN and POA. These increases were inhibited by intracerebroventricular administration of a PKC inhibitor administered 30 min prior to P. The total PKC activity remained unchanged demonstrating maximal PKC activation within 30 min in the VMN. In contrast, P regulation in the POA significantly attenuated total PKC activity +/- estradiol benzoate priming. These rapid changes in P-initiated PKC activity were not due to changes in PKC protein levels or phosphorylation status.

Molecular signals in B lymphocyte activation: The role of intracellular calcium ion concentration, protein kinase C activation, and autocrine interleukin-2

Calcium ionophore, ionomycin, and phorbol myristate acetate (PMA) were used to activate rabbit peripheral blood B cells to study the role of increased intracellular calcium ion concentration ( (Ca$\sp2+\rbrack\sb{\rm i}$), protein kinase C (PKC) activation, and autocrine interleukin (IL-2) in inducing cell cycle entry and maintaining activation to DNA synthesis. When stimulated with a combination of ionomycin and PMA the B cells produced a soluble factor that supported the IL-2 dependent cell line, CTLL-2. The identity of the factor was established as IL-2 and its source was proved to be B cells in further experiments. Absorption studies and limiting dilution analysis indicated that IL-2 produced by B cells can act as an autocrine growth factor. Next, the effect of complete and incomplete signalling on B lymphocyte activation leading to cell cycle entry, IL-2 production, functional IL-2 receptor (IL-2R) expression, and DNA synthesis was examined. It was observed that cell cycle entry could be induced by signals provided by each reagent alone, but IL-2 production, IL-2R expression, and progression to DNA synthesis required activation with both reagents. Incomplete activation with ionomycin or PMA alone altered the responsiveness of B cells to further stimulation only in the case of ionomycin, and the unresponsiveness of these cells was apparently due to a lack of functional IL-2R expression on these cells, even though IL-2 production was maintained. The requirement of IL-2 for maintenance of activation to DNA synthesis was then investigated. The hypothesis that IL-2, acts in late G$\sb1$ and is required for DNA synthesis in B cells was supported by comparing IL-2 production and DNA synthesis in peripheral blood cells and purified B cells, kinetic analysis of these events in B cells, effects of anti-IL-2 antibody and PKC inhibitors, and by the response of G$\sb1$ B cells. Additional signals transduced by the interaction of autocrine IL-2 and functional IL-2 receptor on rabbit B cells were found to be necessary to drive these cells to S phase, after initial activation caused by simultaneous increase in (Ca$\sp2+\rbrack\sb{\rm i}$ and PKC activation had induced cell cycle entry, IL-2 production, and functional IL-2 receptor expression. ^