M. De Bock

Connexin 43 hemichannels contribute to the propagation of apoptotic cell death in a rat C6 glioma cell model

Gap junctions (GJs) have been demonstrated to communicate cell death signals from apoptotic to healthy cells, thereby spatially extending apoptosis. Before being incorporated into GJs, hemichannels (hemi-GJs) are normally closed but recent evidence suggests that they can be opened by various messengers and conditions, thereby forming a pore through which molecules can enter or leave the cell potentially leading to cell death. The aim of this study was to determine the contribution of GJs and hemichannels in the communication of apoptosis toward surrounding cells. We induced apoptosis in C6 glioma cells stably transfected with connexin (Cx)43, with cytochrome C (cytC) using in situ electroporation and found that healthy surrounding cells underwent apoptotic transformation. Work with various cell death markers, wild-type (WT) and Cx43-expressing cells, inhibitors of GJs and/or hemichannels, and Cx43 gene silencing showed that GJs contribute to the spread of apoptosis in a zone next to where apoptosis was triggered whereas hemichannels also promoted cell death beyond this area. Buffering cytoplasmic Ca2+ changes inhibited the spread of apoptosis in both cases. We conclude that Cx43 hemichannels, in concert with their GJ counterparts, play a role in communicating cytC-induced apoptotic cell death messages.

Transfer of IP 3 through gap junctions is critical, but not sufficient, for the spread of apoptosis

Decades of research have indicated that gap junction channels contribute to the propagation of apoptosis between neighboring cells. Inositol 1,4,5-trisphosphate (IP3) has been proposed as the responsible molecule conveying the apoptotic message, although conclusive results are still missing. We investigated the role of IP3 in a model of gap junction-mediated spreading of cytochrome C-induced apoptosis. We used targeted loading of high-molecular-weight agents interfering with the IP3 signaling cascade in the apoptosis trigger zone and cell death communication zone of C6-glioma cells heterologously expressing connexin (Cx)43 or Cx26. Blocking IP3 receptors or stimulating IP3 degradation both diminished the propagation of apoptosis. Apoptosis spread was also reduced in cells expressing mutant Cx26, which forms gap junctions with an impaired IP3 permeability. However, IP3 by itself was not able to induce cell death, but only potentiated cell death propagation when the apoptosis trigger was applied. We conclude that IP3 is a key necessary messenger for communicating apoptotic cell death via gap junctions, but needs to team up with other factors to become a fully pro-apoptotic messenger.

Prevalence of ‘Candidatus Helicobacter suis’ in pigs of different ages

Samples from the antrum and fundus of the stomachs of 457 pigs from 22 different herds were screened for the presence of ‘Candidatus Helicobacter suis’ by pcr, and samples from the antrum and/or fundus of 222 of the stomachs were tested for urease activity. The prevalence of the infection was very low before weaning, increased rapidly after weaning and reached 90 per cent in the adult boars and sows. The agreement between the results obtained with the pcr test and the urease test was very good for some age groups and sampling sites, but poor for other age groups and sampling sites.

Experimental Infection of Pigs with ‘ Candidatus Helicobacter suis’

Abstract‘Candidatus Helicobacter suis’ is a spiral-shaped bacterium that colonizes the stomach of more than 60% of slaughter pigs. The role of ‘Candidatus Helicobacter suis’ in gastric disease of pigs is still unclear. Experimental studies in pigs are lacking because this bacterium is unculturable until now. An inoculation protocol using ‘Candidatus Helicobacter suis’ infected mouse stomach homogenate was used to reproduce the infection in pigs. Control animals were inoculated using negative mouse stomach homogenate. Pigs were inoculated three times with one-week intervals and euthanized 6 weeks post inoculation. Tissue samples were taken from different mucosal stomach regions to detect ‘Candidatus Helicobacter suis’ by PCR and urease test. Mucosal inflammation was evaluated on formalin-fixed tissue samples. Lesions in the pars oesophagea were scored macroscopically. Infection was succesful in all challenged animals, with the antrum and the fundus being predominantly positive. Infection was associated with infiltration of lymphocytes and plasma cells in the antral mucosa, evolving to follicular gastritis. No apparent inflammation of the fundic stomach region was detected in the infected animals. A clear link between ‘Candidatus Helicobacter suis’ and pars oesophageal lesions could not be found.

Validation of Symbolic Expressions in Circuit Analysis E-Learning

Symbolic circuit analysis is a cornerstone of electrical engineering education. Solving a suitable set of selected problems is essential to developing professional skills in the field. A new method is presented for automatic validation of circuit equations representing a students intermediate steps in the solving process. Providing this immediate feedback may strongly enhance the training effects. The new method was embedded in a Web-based e-learning system and has proved to be useful in circuit analysis training, both at an introductory level and for more advanced problems in analog electronics.

Fluoxetine suppresses calcium signaling in human T lymphocytes through depletion of intracellular calcium stores.

Selective serotonin reuptake inhibitors, such as fluoxetine, have recently been shown to exert anti-inflammatory and immunosuppressive effects. Although the effects on cytokine secretion, proliferation and viability of T lymphocytes have been extensively characterized, little is known about the mechanism behind these effects. It is well known that Ca(2+) signaling is an important step in the signaling transduction pathway following T cell receptor activation. Therefore, we investigated if fluoxetine interferes with Ca(2+) signaling in Jurkat T lymphocytes. Fluoxetine was found to suppress Ca(2+) signaling in response to T cell receptor activation. Moreover, fluoxetine was found to deplete intracellular Ca(2+) stores, thereby leaving less Ca(2+) available for release upon IP3- and ryanodine-receptor activation. The Ca(2+)-modifying effects of fluoxetine are not related to its capability to block the serotonin transporter, as even a large excess of 5HT did not abolish the effects. In conclusion, these data show that fluoxetine decreases IP3- and ryanodine-receptor mediated Ca(2+) release in Jurkat T lymphocytes, an effect likely to be at the basis of the observed immunosuppression.

Inhibiting connexin channels protects against cryopreservation-induced cell death in human blood vessels

OBJECTIVES Cryopreserved blood vessels are being increasingly employed in vascular reconstruction procedures but freezing/thawing is associated with significant cell death that may lead to graft failure. Vascular cells express connexin proteins that form gap junction channels and hemichannels. Gap junction channels directly connect the cytoplasm of adjacent cells and may facilitate the passage of cell death messengers leading to bystander cell death. Two hemichannels form a gap junction channel but these channels are also present as free non-connected hemichannels. Hemichannels are normally closed but may open under stressful conditions and thereby promote cell death. We here investigated whether blocking gap junctions and hemichannels could prevent cell death after cryopreservation. MATERIALS AND METHODS Inclusion of Gap27, a connexin channel inhibitory peptide, during cryopreservation and thawing of human saphenous veins and femoral arteries was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays and histological examination. RESULTS We report that Gap27 significantly reduces cell death in human femoral arteries and saphenous veins when present during cryopreservation/thawing. In particular, smooth muscle cell death was reduced by 73% in arteries and 71% in veins, while endothelial cell death was reduced by 32% in arteries and 51% in veins. CONCLUSIONS We conclude that inhibiting connexin channels during cryopreservation strongly promotes vascular cell viability.

A Describing Function Study of Saturated Quantization and Its Application to the Stability Analysis of Multi-Bit Sigma Delta Modulators

Just as their single-bit counterparts, multi-bit sigma delta modulators exhibit nonlinear behavior due to the presence of the quantizer in the loop. In the multi-bit case this is caused by the fact that any quantizer has a limited output range and hence gives an implicit saturation effect. Due to this, any multi-bit modulator is prone to modulator overloading. Unfortunately, until now, designers had to rely on extensive time-domain simulations to predict the overloading level, because there is no adequate analytical theory to model this effect.

A double-sampling cross noise-coupled split ΣΔ-modulation A/D converter with 80 dB SNR

This paper presents the design of a double-sampling split ΣΔ-modulation analog-to-digital converter with cross noise-coupling. Double-sampling is used to achieve high conversion speed and low power consumption. To tackle the problem of quantization noise folding due to path mismatch, a fully floating bilinear integrator is used. Then the quantization noise is cross-coupled between two identical ΣΔ-modulators. This increases the effective noise shaping order of this structure with one. The final design is an optimized second order double-sampling split ΣΔ-modulator with third order noise shaping through cross noise-coupling. This design is simulated at transistor level in an 0.18-µm CMOS process for 80 dB SNR and 5MHz bandwidth.

Design of an integrated analog controller for a class-D audio amplifier

An integrated analog controller for a self-oscillating class-D audio power amplifier is designed in a 0.35 µm CMOS technology for a 3.3 Volt power supply. It is intended to be used with an external output stage and passive filter, for medium power applications of upto a few 100 Watts. The controller was optimized with regard to its loop gain to suppress the distortion of the output stage. In typical commercially available output stages, the distortion is dominated by dead time effects and the THD can be as low as 20 dB. The controller uses self-oscillation to generate the carrier. To control the self-oscillation a second order phase shift network is embedded in the loop. To increase the loop gain a fifth-order loop filter is added. For a switching frequency of 400kHz the controller achieves a loop gain of 51 dB, nearly flat over the audio band. For reasons of flexibility, the order of the controller is made programmable, as well as the dead time and the delay in the loop. Full spice simulations of the controller combined with an external 120 Watt output stage indicate that a THD of upto 80 dB (better than 0.01%) can be obtained even under the worst case condition of a dead time of 50 ns.