Rgnef (p190RhoGEF) Knockout Inhibits RhoA Activity, Focal Adhesion Establishment, and Cell Motility Downstream of Integrins

Cell migration is a highly regulated process that involves the formation and turnover of cell-matrix contact sites termed focal adhesions. Rho-family GTPases are molecular switches that regulate actin and focal adhesion dynamics in cells. Guanine nucleotide exchange factors (GEFs) activate Rho-family GTPases. Rgnef (p190RhoGEF) is a ubiquitous 190 kDa GEF implicated in the control of colon carcinoma and fibroblast cell motility.Rgnef exon 24 floxed mice (Rgnef(flox)) were created and crossed with cytomegalovirus (CMV)-driven Cre recombinase transgenic mice to inactivate Rgnef expression in all tissues during early development. Heterozygous Rgnef(WT/flox) (Cre+) crosses yielded normal Mendelian ratios at embryonic day 13.5, but Rgnef(flox/flox) (Cre+) mice numbers at 3 weeks of age were significantly less than expected. Rgnef(flox/flox) (Cre+) (Rgnef-/-) embryos and primary mouse embryo fibroblasts (MEFs) were isolated and verified to lack Rgnef protein expression. When compared to wildtype (WT) littermate MEFs, loss of Rgnef significantly inhibited haptotaxis migration, wound closure motility, focal adhesion number, and RhoA GTPase activation after fibronectin-integrin stimulation. In WT MEFs, Rgnef activation occurs within 60 minutes upon fibronectin plating of cells associated with RhoA activation. Rgnef-/- MEF phenotypes were rescued by epitope-tagged Rgnef re-expression.Rgnef-/- MEF phenotypes were due to Rgnef loss and support an essential role for Rgnef in RhoA regulation downstream of integrins in control of cell migration

Phospholipase D signaling: orchestration by PIP2 and small GTPases

Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of the versatile lipid second messenger, phosphatidic acid (PA), which is involved in fundamental cellular processes, including membrane trafficking, actin cytoskeleton remodeling, cell proliferation and cell survival. PLD activity can be dramatically stimulated by a large number of cell surface receptors and is elaborately regulated by intracellular factors, including protein kinase C isoforms, small GTPases of the ARF, Rho and Ras families and, particularly, by the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 is well known as substrate for the generation of second messengers by phospholipase C, but is now also understood to recruit and/or activate a variety of actin regulatory proteins, ion channels and other signaling proteins, including PLD, by direct interaction. The synthesis of PIP2 by phosphoinositide 5-kinase (PIP5K) isoforms is tightly regulated by small GTPases and, interestingly, by PA as well, and the concerted formation of PIP2 and PA has been shown to mediate receptor-regulated cellular events. This review highlights the regulation of PLD by membrane receptors, and describes how the close encounter of PLD and PIP5K isoforms with small GTPases permits the execution of specific cellular functions

Phase-shift-controlled three-level converter with reduced voltage stress featuring ZVS over the full operation range

The simplicity of phase-shift control at fixed switching frequency and 50% duty-cycle operation is fully exploited by the proposed converter topology. The transistor voltages are clamped to only 50% of the dc input, the dc bus capacitive dividers being naturally stabilized. Furthermore, zero-voltage switching for all switches is guaranteed from no-load to full-load conditions, that is to say, from zero to nominal output voltage and from zero to nominal load current. As such, the proposed topology is an excellent candidate for demanding applications as compact battery chargers for electric vehicles. Experimental results obtained from a 400-80-V/0-360-V/2-kW/100-kHz prototype support the theoretical analysis

Phase-shift-controlled three-level converter with reduced voltage stress featuring ZVS over the full operation range

The simplicity of phase-shift control at fixed switching frequency and 50% duty-cycle operation is fully exploited by the proposed converter topology. The transistor voltages are clamped to only 50% of the dc input, the dc bus capacitive dividers being naturally stabilized. Furthermore, zero-voltage switching for all switches is guaranteed from no-load to full-load conditions, that is to say, from zero to nominal output voltage and from zero to nominal load current. As such, the proposed topology is an excellent candidate for demanding applications as compact battery chargers for electric vehicles. Experimental results obtained from a 400-80-V/0-360-V/2-kW/100-kHz prototype support the theoretical analysis

A self-optimizing discretization scheme for 2D boundary element calculations

A self-optimizing discretization adjustment scheme is described for boundary element calculations. The aim is to obtain high accuracy with limited computer resources. Examples will be given of two-dimensional static field calculations, where small capacitances are accurately determined in presence of others with three up to five orders larger values. To demonstrate the power of the scheme, the simplest basis function is used: a constant charge density per boundary segment. Several results are compared to exact calculations, and fast convergence and good agreement are demonstrated

A high-switching-frequency flyback converter in resonant mode

The demand of miniaturization of power systems has accelerated the research on high-switching-frequency power converters. A flyback converter in resonant mode that features low switching losses, less transformer losses, and low switching noise at high switching frequency is investigated in this paper as an alternative to a conventional quasi-resonant (QR) flyback topology to increase power density. In order to find a compromise between magnet size, electromagnetic interference (EMI), and efficiency, the concept utilizes the resonant behavior between transformer leakage inductance and snubber capacitor to achieve near-zero-voltage switching at both turn-on and turn-off of the primary switch, low core loss due to a continuous transformer magnetizing current, and reduced EMI due to low di/dt and dv/dt values. Meanwhile, the concept uses the regenerative snubber to recycle the transformer leakage energy with two snubber diodes and one snubber capacitor. The proposed concept has been validated on a 340-kHz 65-W prototype. Compared to the conventional QR flyback converter operating at the same switching frequency, the proposed concept has 2% efficiency improvement and better EMI performance

Prediction of Common-Mode Currents on Cables Connected to a Multilayer Printed Circuit Board

Common-mode currents through cables connected to printed circuit boards (PCBs) are often the dominant cause interference. Therefore, Bersier proposed in 1983 precompliance tests where a common-mode current is measured or injected though each cable attached to the equipment under test. This relatively simple method has been proven to be accurate and reliable according to many engineers. We model such a test for a PCB and describe the coupling between the signal circuits on a PCB and the circuits formed by the attached cables by means of a transmission-line approach. Demonstration PCBs with linear devices and digital logic showed that for frequencies up to about 700 MHz calculations and measurements on a 20 cm long PCB agreed within 8 dB. Keywords--- Physical Design, Printed Circuit Board (PCB) Design, EMC, Common-mode Currents. I. Introduction All electrical and electronic equipment radiates electromagnetic (EM) waves and is susceptible to external EM waves. With the increasing speed ..

Essential role of type I alpha phosphatidylinositol 4-phosphate 5-kinase in neurite remodeling

AbstractRapid neurite remodeling is fundamental to nervous system development and plasticity [1] and is regulated by Rho family GTPases that signal f-actin reorganization in response to various receptor ligands. Neuronal N1E-115 cells show dramatic neurite retraction and cell rounding in response to serum factors such as lysophosphatidic acid (LPA), sphingosine-1 phosphate (S1P), and thrombin, due to activation of the RhoA-Rho kinase pathway [2]. Type I phosphatidylinositol 4-phosphate 5-kinases (PIPkinase), which regulate cellular levels of PtdIns(4,5)P2[3], have been suggested as targets of the RhoA-Rho kinase pathway [4, 5] able to modulate cytoskeletal dynamics [6, 7]. Here, we show that the introduction of Type Iα PIPkinase into N1E-115 cells leads to cell rounding and complete inhibition of neurite outgrowth, perhaps through the dissociation of vinculin and the destabilization of focal adhesions. This occurs independently of RhoA, Rho kinase, and the activation of actomyosin contraction. Strikingly, expression of kinase-dead PIPkinase promotes the outgrowth of neurites, which fail to retract in response to LPA, S1P, thrombin, or active RhoA. Moreover, neurite retraction in response to an endogenous neuronal guidance cue, Semaphorin3A, was also dependent on Type Iα PIPkinase. Our results suggest an essential role for a Type I PIPkinase during neurite retraction in response to a number of diverse stimuli