Febs Letters

doi: 10.1016/S0014-5793(03)00750-6pmid: N/A

doi: 10.1016/S0014-5793(03)00735-Xpmid: N/A

Geng, Xin; Xiao, Lei; Lin, Gu Fa; Hu, Ruiying; Wang, Jin Hu; Rupp, Ralph A.W; Ding, Xiaoyan

doi: 10.1016/S0014-5793(03)00639-2pmid: 12860376

Though the Wnt/β‐catenin signaling pathway is known to play key roles during Xenopus axis specification, whether it signals exclusively through Lef/Tcf transcription factors in this process remains unclear. To investigate this issue, we generated transgenic frog embryos expressing green fluorescent protein (GFP) driven by a Lef/Tcf‐dependent and Wnt/β‐catenin‐responsive promoter. This promoter is highly sensitive and even detects maternal β‐catenin activity prior to the large‐scale transcription of zygotic genes. Unexpectedly, GFP expression was observed only in some, but not all, known Wnt/β‐catenin‐positive territories in Xenopus early development. Furthermore, ubiquitous expression of dominant Lef‐1 protein variants from transgenes revealed that zygotic Lef/Tcf activity is required for the ventroposterior development of Xenopus embryos. In summary, our results suggest that endogenous Wnt/β‐catenin activity does not result in obligatory Lef/Tcf‐dependent gene activation, and that the ventroposteriorizing activity of zygotic Wnt‐8 signaling is mediated by Lef/Tcf proteins.

Eleuteri, Anna Maria; Angeletti, Mauro

doi: 10.1016/S0014-5793(03)00660-4pmid: 12860377

The effect of sodium ion on the inhibition exerted by Cbz‐Leu‐Leu‐Leu‐CHO on the chymotrypsin‐like activity of the 20S proteasome isolated from bovine lung was investigated. The experimental data were analyzed using a standard linkage formalism. The calculated equilibrium affinity constants for the sodium ion binding to the free‐enzyme and the inhibitor‐bound enzyme are compatible to other well‐characterized ion‐involving heterotropic systems. The functional interdependence between the binding events played by the inhibitor and the sodium ion conforms to a heterotropic modulatory mechanism.

Rakus, D.; Pasek, M.; Krotkiewski, H.; Dzugaj, A.

doi: 10.1016/S0014-5793(03)00661-6pmid: 12860378

Real‐time interaction analysis, using the BIAcore biosensor, of rabbit muscle FBPase–aldolase complex revealed apparent binding constant [K Aapp] values of about 4.4×108 M−1. The stability of the complex was down‐regulated by the glycolytic intermediates dihydroxyacetone phosphate and fructose 6‐phosphate, and by the regulator of glycolysis and glyconeogenesis – fructose 2,6‐bisphosphate. FBPase in a complex with aldolase was entirely insensitive to inhibition by physiological concentrations of AMP (I 0.5 was 1.35 mM) and the cooperativity of the inhibition was not observed. The existence of an FBPase–aldolase complex that is insensitive to AMP inhibition explains the possibility of glycogen synthesis from carbohydrate precursors in vertebrates’ myocytes.

De Angelis, Roberta; Iezzi, Simona; Bruno, Tiziana; Corbi, Nicoletta; Di Padova, Monica; Floridi, Aristide; Fanciulli, Maurizio; Passananti, Claudio

doi: 10.1016/S0014-5793(03)00659-8pmid: 12860379

RPB3 is a core subunit of RNA polymerase II (pol II) that, together with the RPB11 subunit, forms the heterodimer considered as a functional counterpart of the bacterial α subunit homodimer involved in promoter recognition. We previously employed the yeast two‐hybrid system and identified an interaction between RPB3 and the myogenic transcription factor myogenin, demonstrating an involvement of this subunit in muscle differentiation. In this paper we report the interaction between RPB3 and another known transcription factor, ATF4. We found that the intensity of the interaction between RPB3 and ATF4 is similar to the one between RPB3 and myogenin. This interaction involves an RPB3 specific region not homologous to the prokaryotic α subunit. We demonstrated that RBP3 is able to enhance ATF4 transactivation, whereas the region of RPB3 (Sud) that contacts ATF4, when used as a dominant negative, markedly inhibits ATF4 transactivation activity. Interestingly, ATF4 protein level, as reported for its partner RPB3, increases during C2C7 cell line muscle differentiation.

Milnes, James T; Dempsey, Christopher E; Ridley, John M; Crociani, Olivia; Arcangeli, Annarosa; Hancox, Jules C; Witchel, Harry J

doi: 10.1016/S0014-5793(03)00662-8pmid: 12860380

The scorpion toxin peptide BeKm‐1 was synthesised by fluorenylmethoxycarbonyl solid phase chemistry and folded by air oxidation. The peptide's effects on heterologous human ether‐a‐go‐go‐related gene potassium current (I HERG) in HEK293 cells were assessed using ‘whole‐cell’ patch clamp. Blockade of I HERG by BeKm‐1 was concentration‐dependent, temperature‐dependent, and rapid in onset and reversibility. Blockade also exhibited inverse voltage dependence, inverse dependence on duration of depolarisation, and reverse use‐ and frequency‐dependence. Blockade by BeKm‐1 and recombinant ergtoxin, another scorpion toxin known to block HERG, differed in their recovery from HERG current inactivation elicited by strong depolarisation and in their ability to block HERG when the channels were already activated. We conclude that synthetic BeKm‐1 toxin blocks HERG preferentially through a closed (resting) state channel blockade mechanism, although some open channel blockade also occurs.

Shumilina, Ekaterina V; Khaitlina, Sofia Yu; Morachevskaya, Elena A; Negulyaev, Yuri A

doi: 10.1016/S0014-5793(03)00663-Xpmid: 12860381

The role of G proteins in regulation of non‐voltage‐gated Na+ channels in human myeloid leukemia K562 cells was studied by inside‐out patch‐clamp method. Na+ channels were activated by non‐hydrolyzable analog of guanosine triphosphate (GTP), GTPγS, known to activate both heterotrimeric and small G proteins. Channel activity was not affected by aluminum fluoride that indiscriminately activates heterotrimeric G proteins. The effect of GTPγS was prevented by phalloidin and by G‐actin, both interfering with actin disassembly, which indicates that GTPγS‐induced channel activation was likely due to microfilament disruption. GTPγS‐activated channels were inactivated by polymerizing actin. These data show, for the first time, that small G proteins can regulate Na+ channels, and an intracellular mechanism mediating their effect involves actin cytoskeleton rearrangements.

Zima, Aleksey V; Copello, Julio A; Blatter, Lothar A

doi: 10.1016/S0014-5793(03)00664-1pmid: 12860382

The effects of nicotinamide‐adenine dinucleotides (NADH, NAD+, NADPH) on ryanodine receptor channels (RyRs) from cardiac and skeletal muscle were compared in planar bilayers. NADH decreased cardiac RyR activity, which was counteracted by NAD+. In contrast, NADH and NAD+ both activated skeletal RyRs. NADPH/NADP+ were without effect on cardiac and skeletal RyRs. In the presence of ATP, NADH inhibition of cardiac RyRs remained. Differently, in the presence of ATP both NADH and NAD+ were ineffective as skeletal RyR agonists, suggesting interactions with the ATP binding site(s) of the channels. The results suggest that the direct effect of cytosolic NADH is physiologically important for the modulation of cardiac RyRs.

Park, Jin-Yong; Jeong, Seong-Woo; Perez-Reyes, Edward; Lee, Jung-Ha

doi: 10.1016/S0014-5793(03)00665-3pmid: 12860383

Although T‐type Ca2+ channels have been implicated in numerous physiological functions, their regulations by protein kinases have been obscured by conflicting reports. We investigated the effects of protein kinase C (PKC) on Cav3.2 T‐type channels reconstituted in Xenopus oocytes. Phorbol‐12‐myristate‐13‐acetate (PMA) strongly enhanced the amplitude of Cav3.2 channel currents (∼3‐fold). The augmentation effects were not mimicked by 4α‐PMA, an inactive stereoisomer of PMA, and abolished by preincubation with PKC inhibitors. Our findings suggest that PMA upregulates Cav3.2 channel activity via activation of oocyte PKC.