The Differential Effects of 12-O-Tetradecanoylphorbol-13-acetate on the Gap Junctions and Connexins of the Developing Mammalian Lens

AbstractEpithelial cells in primary ovine lens cultures express the gap junction proteins connexin43 (Cx43) and connexin49 (Cx49; a.k.a. MP70), a homologue of mouse connexin50. In contrast, lens cultures of differentiated, fiber-like cells (termed lentoid cells) express Cx49 and connexin46 (Cx46), but not Cx43. To investigate the regulation of lens cell gap junctions by protein kinase C (PKC), differentiating lens cultures were treated with the PKC activator 12-O-tetradecanoylphorbol-13-acetate (β-TPA). Within 10 min, β-TPA significantly inhibited the transfer of Lucifer Yellow dye between epithelial, but not lentoid, cells. This inhibition was correlated with the phosphorylation of Cx43 and was followed by the gradual disappearance of Cx43 from cell interfaces. The protein kinase inhibitor staurosporine prevented Cx43 phosphorylation and the loss of Cx43 from intercellular junctions. Following treatment of cultures with β-TPA for 2–6 hr, Cx49 disappeared from epithelial cell interfaces, and by 24 hr of β-TPA treatment, levels of Cx49 detected on immunoblots of purified epithelial membrane fractions had also diminished significantly. The β-TPA-induced loss of Cx49 both from regions of epithelial cell contact and from isolated membranes was correlated with the disappearance of Cx49 mRNA. In contrast to the epithelial connexins, the lentoid connexins Cx49 and Cx46 were unaffected by even extended β-TPA treatment. In spite of lentoid dye transfer being refractory to β-TPA, significant levels of PKC-α (a β-TPA-sensitive isoform) were detected in the lentoid cell. The response of lens gap junctions to β-TPA depends upon the stage of differentiation and the complement of connexins expressed. The contrasting effects of β-TPA on Cx43 and Cx49 in lens epithelial cells indicate a fundamental difference in the regulation of these connexin proteins in the developing mammalian lens

THE COUPLING BETWEEN PRONATION AND TIBIAL INTERNAL ROTATION IS DIFFERENT IN RUNNER’S WITH ANTERIOR KNEE PAIN

The purpose of this study was to compare the coupling of eversion to tibial internal rotation in runners with (n=19) and without anterior knee pain (n=17). This relationship was captured using both the traditional EV-TIR ratio and continuously using vector coding. Using vector coding, runners experiencing AKP were found to have a significantly different coupling relationship between 40 and 50% of stance. In contrary, as a result of using a singular value to describe this coupling relationship, traditional EV-TIR ratios were not sensitive enough to detect these differences. Future studies evaluating this coupling relationship should consider using continuous techniques or calculating the EV-TIR ratio over smaller periods of stance

A Physically Based Compact Model of Partially Depleted MOSFETs for Analog Circuit Stimulation

In this paper, the Southampton Thermal Analogue (STAG) compact model for partially depleted (PD) silicon-on-insulator (SOI) MOSFETs is presented. The model uses a single expression to model the channel current, thereby ensuring continuous transition between all operating regions. Furthermore, care has been taken to ensure that this expression is also infinitely differentiable, resulting in smooth and continuous conductances and capacitances as well as higher order derivatives. Floating-body effects, which are particular to PD SOI and which are of concern to analog circuit designers in this technology, are well modeled. Small geometry effects such as channel length modulation (CLM), drain-induced barrier lowering (DIBL), charge sharing, and high field mobility effects have also been included. Self-heating (SH) effects are much more apparent in SOI devices than in equivalent bulk devices. These have been modeled in a consistent manner, and the implementation in SPICE3f5 gives the user an additional thermal node which allows internal device temperature rises to be monitored and also accommodates the modeling of coupled heating between separate devices. The model has been successfully used to simulate a variety of circuits which commonly cause problems with convergence. Due to its inherent robustness, the model can normally achieve convergence without recourse to the setting of initial nodal voltage estimates

Ser364 of connexin43 and the upregulation of gap junction assembly by cAMP

The assembly of gap junctions (GJs) is a process coordinated by growth factors, kinases, and other signaling molecules. GJ assembly can be enhanced via the elevation of cAMP and subsequent stimulation of connexon trafficking to the plasma membrane. To study the positive regulation of GJ assembly, fibroblasts derived from connexin (Cx)43 knockout (KO) and wild-type (WT) mice were transfected with WT Cx43 (WTCx43) or mutant Cx43. GJ assembly between untransfected WT fibroblasts or stably transfected WTCx43/KO fibroblasts was increased two- to fivefold by 8Br-cAMP, and this increase could be blocked by inhibition of cAMP-dependent protein kinase (PKA) or truncation of the Cx43 COOH terminus (CT). Although serine 364 (S364) of the Cx43 CT was determined to be a major site of phosphorylation, the molar ratio of Cx43 phosphorylation was not increased by 8Br-cAMP. Importantly, GJ assembly between either S364ECx43/KO or S364ECx43/WT fibroblasts was stimulated by 8Br-cAMP, but that between S364ACx43/KO or S364PCx43/KO fibroblasts was not stimulated, indicating that phosphorylation or a negative charge at S364 is required for enhancement of GJ assembly by cAMP. Furthermore, GJ assembly between S364ACx43/WT fibroblasts could be stimulated by 8Br-cAMP, but could not be between S364PCx43/WT fibroblasts. Thus, S364PCx43 interferes with enhanced GJ assembly when coexpressed with WTCx43

Diversity in protein–protein interactions of connexins: emerging roles

AbstractGap junctions, specialised membrane structures that mediate cell-to-cell communication in almost all tissues, are composed of channel-forming integral membrane proteins termed connexins. The activity of these intercellular channels is closely regulated, particularly by intramolecular modifications as phosphorylations of proteins by protein kinases, which appear to regulate the gap junction at several levels, including assembly of channels in the plasma membrane, connexin turnover as well as directly affecting the opening and closure (“gating”) of channels. The regulation of membrane channels by protein phosphorylation/dephosphorylation processes commonly requires the formation of a multiprotein complex, where pore-forming subunits bind to auxiliary proteins (e.g. scaffolding proteins, catalytic and regulatory subunits), that play essential roles in channel localisation and activity, linking signalling enzymes, substrates and effectors into a structure frequently anchored to the cytoskeleton. The present review summarises the up-to-date progress regarding the proteins capable of interacting or at least of co-localising with connexins and their functional importance