Identification of the linear epitope for Fc-binding on the bovine IgG2 Fc receptor (boFcgamma2R) using synthetic peptides

To identify the linear epitope for Fc-binding on the bovine IgG2 Fc receptor (boFcgamma2R), peptides derived from the membrane-distal extracellular domain (EC1) of boFcgamma2R corresponding to the homologous region of human FcalphaRI were synthesized. Binding of bovine IgG2 to the different peptides was tested by Dot-blot assay, and the peptide showing maximal binding was further modified by truncation and mutation. The minimum effective peptide 82FIGV85 located in the putative F-G loop of the EC1 domain was found to bind bovine IgG2 specifically and inhibit the binding of bovine IgG2 to the receptor. The Phe82, Ile83 and Val85 residues within the linear epitope were shown to be critical for IgG2-binding. Such functional epitope peptide should be very useful for understanding the IgG-Fcgamma interaction and development of FcR-targeting drugs.     

Major contribution of MEK1 to the activation of ERK1/ERK2 and to the growth of LS174T colon carcinoma cells

Mammalian cells express two closely related MEK isoforms, MEK1 and MEK2, upstream of the ERK1/ERK2 MAPK module. Although genetic studies have suggested that MEK1 and MEK2 do not have overlapping functions in vivo, little is known about their specific contribution to the activation of ERKs and to tumor cell proliferation. We used Tet-inducible shRNA to investigate the independent role of MEK1 and MEK2 for the oncogenic and the serum-induced activation of ERK1 and ERK2 in LS174T colon carcinoma cells. We show that MEK1 is the main activator of both ERK1 and ERK2. MEK2 removal has no impact by itself but it can cooperate with MEK1 ablation for the inhibition of ERK1/2 activity. In addition, we show that MEK1 is the critical isoform regulating tumor cell proliferation in vitro and in vivo.     

PTTH-stimulated ERK phosphorylation in prothoracic glands of the silkworm, Bombyx mori: Role of Ca/calmodulin and receptor tyrosine kinase

Our previous studies showed that the prothoracicotropic hormone (PTTH) stimulated extracellular signal-regulated kinase (ERK) phosphorylation in prothoracic glands of Bombyx mori both in vitro and in vivo. In the present study, the signaling pathway by which PTTH activates ERK phosphorylation was further investigated using PTTH, second messenger analogs, and various inhibitors. ERK phosphorylation induced by PTTH was partially reduced in Ca²⁺-free medium. The calmodulin antagonist, calmidazolium, partially inhibited both PTTH-stimulated ERK phosphorylation and ecdysteroidogenesis, indicating the involvement of calmodulin. When the prothoracic glands were treated with agents that directly elevate the intracellular Ca²⁺ concentration [either A23187, thapsigargin, or the protein kinase C (PKC) activator, phorbol 12-myristate acetate (PMA)], a great increase in ERK phosphorylation was observed. In addition, it was found that PTTH-stimulated ecdysteroidogenesis was greatly attenuated by treatment with PKC inhibitors (either calphostin C or chelerythrine C). However, PTTH-stimulated ERK phosphorylation was not attenuated by the above PKC inhibitors, indicating that PKC is not involved in PTTH-stimulated ERK phosphorylation. A potent and specific inhibitor of insulin receptor tyrosine kinase, HNMPA-(AM)₃, greatly inhibited the ability of PTTH to activate ERK phosphorylation and stimulate ecdysteroidogenesis. However, genistein, another tyrosine kinase inhibitor, did not inhibit PTTH-stimulated ERK phosphorylation, although it did markedly attenuate the ability of A23187 to activate ERK phosphorylation. From these results, it is suggested that PTTH-stimulated ERK phosphorylation is only partially Ca²⁺- and calmodulin-dependent and that HNMPA-(AM)₃-sensitive receptor tyrosine kinase is involved in activation of ERK phosphorylation by PTTH.     

Cyclin B1/Cdk1 binds and phosphorylates Filamin A and regulates its ability to cross-link actin

Substantial actin remodelling occurs prior to mitosis as cells alter their shape in preparation for cytokinesis. In mammalian cells, mitosis is initiated by a heterodimer of cyclin B1 and the cyclin dependent kinase 1 (Cdk1) serine/threonine kinase. In this report. we show that human cyclin B1 binds the actin cross-linking protein Filamin-A (FLNa). The proteins co-immunoprecipitate and co-localize in mitotic human cells. We find that cyclin B1/Cdk1 can phosphorylate FLNa in vitro and reduce its ability to gelate actin. We have also identified serine 1436 as one FLNa residue phosphorylated by cyclin B1/Cdk1 in vitro. Our results suggest a role for cyclin B1/Cdk1 in FLNa-dependent actin remodelling.     

Exposure to novel environment is characterized by an interaction of D1/NMDA receptors underlined by phosphorylation of the NMDA and AMPA receptor subunits and activation of ERK1/2 signaling, leading to epigenetic changes and gene expression in rat hippocampus

Interactions between dopamine and glutamate receptors are essential for prefrontal cortical (PFC) and hippocampal cognitive functions. The hippocampus has been identified as a detector of a novel stimulus, where an association between incoming information and stored memories takes place. Further to our previous results which showed a strong synergistic interaction of dopamine D1 and glutamate NMDA receptors, the present study is going to investigate the functional status of that interaction in rats, following their exposure to a novel environment. Our results showed that the “spatial” novelty induced in rat hippocampus and PFC (a) a significant increase in phosphorylation of NMDA and AMPA receptor subunits, as well as a robust phosphorylation/activation of ERK1/2 signaling, which are both dependent on the concomitant stimulation of D1/NMDA receptors and are both abolished by habituation procedure, (b) chromatin remodeling events (phosphorylation-acetylation of histone H3) and (c) an increase in the immediate early genes (IEGs) c-Fos and zif-268 expression in the CA1 region of hippocampus, which is dependent on the co-activation of D1/NMDA and acetylcholine muscarinic receptors. In conclusion, our results clearly show that a strong synergistic interaction of D1/NMDA receptor is required for the novelty-induced phosphorylation of NMDA and AMPA receptor subunits and for the robust activation of ERK1/2 signaling, leading to chromatin remodeling events and the expression of the IEGs c-Fos and zif-268, which are involved in the regulation of synaptic plasticity and memory consolidation.     

Domain-dependent modulation of insulin-induced AS160 phosphorylation and glucose uptake by Ca2+/calmodulin-dependent protein kinase II in L6 myotubes

In skeletal muscle, the molecular mechanisms by which insulin stimulates glucose transport remains incompletely understood. Our study investigated the cellular dynamics of intracellular Ca²⁺ mobilisation and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activation on insulin-induced skeletal muscle glucose transport. L6 myotubes were treated without or with insulin [100 nM] for 15 min and subsequently monitored for glucose uptake using isotope-labelled 2-deoxyglucose (I-2DOG), intracellular Ca²⁺ (Ca_i²⁺) release using Fluo-4AM and protein phosphorylation using Western blotting. Acute exposure of myotubes to insulin increased both Akt substrate-160 kDa (AS160) phosphorylation and I-2DOG uptake. Insulin concurrently increased Ca_i²⁺ and activated CaMKII. Exposing myotubes to either BAPTA/AM to sequester Ca_i²⁺ or KN-93 to inhibit CaMKII activity, decreased insulin-induced glucose uptake without affecting AS160 phosphorylation. On the other hand, blocking either calmodulin or the autoregulatory domain of CaMKII blocked the effect of insulin on both AS160 phosphorylation and glucose transport. Likewise, genetic knockdown of CaMKII in myotubes using siRNA completely abolished insulin-mediated glucose uptake. These results illustrate impairments in Ca_i²⁺ mobilisation and CaMKII activation are sufficient to negatively influence insulin-dependent glucose transport by L6 myotubes. Additionally, our results show for the first time that Ca_i²⁺ and domain-dependent CaMKII signalling differentially affect insulin-induced AS160 phosphorylation, and establish that Ca²⁺ and CaMKII are components of the insulin signalling pathway in L6 myotubes.     

Intraspecies variability in Vipera ammodytes ammodytes venom related to its toxicity and immunogenic potential

Vipera ammodytes is the most venomous European snake, whose venom has been used as antigen for immunization of antivenom-producing animals. Same as venom of any other snake, it is a complex mixture of proteins, peptides and other compounds which biochemical and pharmacological variability has been demonstrated at interspecies and intraspecies level. In this work we demonstrated intraspecific variability between 8 venom production batches using both the conventional and the new methodology. Moreover, in contrast to the literature on different venoms' variability, for the first time we were able to select those biochemical differences that are related to and give information on the venom's toxicity and immunogenicity. We have shown that methods quantifying ammodytoxin (the most toxic compound identified so far in the Vipera ammodytes ammodytes venom) content of the venom clearly distinguish between high and low immunogenic venoms.     

Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery

The epithelial and endothelial barriers of the human body are major obstacles for drug delivery to the systemic circulation and to organs with unique environment and homeostasis, like the central nervous system. Several transport routes exist in these barriers, which potentially can be exploited for enhancing drug permeability. Beside the transcellular pathways via transporters, adsorptive and receptor-mediated transcytosis, the paracellular flux for cells and molecules is very limited. While lipophilic molecules can diffuse across the cellular plasma membranes, the junctional complexes restrict or completely block the free passage of hydrophilic molecules through the paracellular clefts. Absorption or permeability enhancers developed in the last 40 years for modifying intercellular junctions and paracellular permeability have unspecific mode of action and the effective and toxic doses are very close. Recent advances in barrier research led to the discovery of an increasing number of integral membrane, adaptor, regulator and signalling proteins in tight and adherens junctions. New tight junction modulators are under development, which can directly target tight or adherens junction proteins, the signalling pathways regulating junctional function, or tight junction associated lipid raft microdomains. Modulators acting directly on tight junctions include peptides derived from zonula occludens toxin, or Clostridium perfringens enterotoxin, peptides selected by phage display that bind to integral membrane tight junction proteins, and lipid modulators. They can reversibly increase paracellular transport and drug delivery with less toxicity than previous absorption enhancers, and have a potential to be used as pharmaceutical excipients to improve drug delivery across epithelial barriers and the blood-brain barrier.