Shared requirement for dynein function and intact microtubule cytoskeleton for normal surface expression of cardiac potassium channels

Potassium channels at the cardiomyocyte surface must eventually be internalized and degraded, and changes in cardiac potassium channel expression are known to occur during myocardial disease. It is not known which trafficking pathways are involved in the control of cardiac potassium channel surface expression, and it is not clear whether all cardiac potassium channels follow a common pathway or many pathways. In the present study we have surveyed the role of retrograde microtubule-dependent transport in modulating the surface expression of several cardiac potassium channels in ventricular myocytes and heterologous cells. The disruption of microtubule transport in rat ventricular myocytes with nocodazole resulted in significant changes in potassium currents. A-type currents were enhanced 1.6-fold at +90 mV, rising from control densities of 20.9 +/- 2.8 to 34.0 +/- 5.4 pA/pF in the nocodazole-treated cells, whereas inward rectifier currents were reduced by one-third, perhaps due to a higher nocodazole sensitivity of Kir channel forward trafficking. These changes in potassium currents were associated with a significant decrease in action potential duration. When expressed in heterologous human embryonic kidney (HEK-293) cells, surface expression of Kv4.2, known to substantially underlie A-type currents in rat myocytes, was increased by nocodazole, by the dynein inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, and by p50 overexpression, which specifically interferes with dynein motor function. Peak current density was 360 +/- 61.0 pA/pF in control cells and 658 +/- 94.5 pA/pF in cells overexpressing p50. The expression levels of Kv2.1, Kv3.1, human ether-a-go-go-related gene, and Kir2.1 were similarly increased by p50 overexpression in this system. Thus the regulation of potassium channel expression involves a common dynein-dependent process operating similarly on the various channels.     

Sterilization of ginseng using a high pressure CO2 at moderate temperatures

The aim of this study was to determine the feasibility of using high pressure CO2 for sterilization of Ginseng powder, as an alternative method to conventional techniques such as gamma-irradiation and ethylene oxide. The Ginseng sample used in this study was originally contaminated with fungi and 5 x 10(7) bacteria/g that was not suitable for oral use. This is the first time that high pressure CO2 has been used for the sterilization of herbal medicine to decrease the total aerobic microbial count (TAMC) and fungi. The effect of the process duration, operating pressure, temperature, and amount of additives on the sterilization efficiency of high pressure CO2 were investigated. The process duration was varied over 15 h; the pressure between 100 and 200 bar and the temperature between 25 and 75 degrees C. A 2.67-log reduction of bacteria in the Ginseng sample was achieved after long treatment time of 15 h at 60 degrees C and 100 bar, when using neat carbon dioxide. However, the addition of a small quantity of water/ethanol/H2O2 mixture, as low as 0.02 mL of each additive/g Ginseng powder, was sufficient for complete inactivation of fungi within 6 h at 60 degrees C and 100 bar. At these conditions the bacterial count was decreased from 5 x 10(7) to 2.0 x 10(3) TAMC/g complying with the TGA standard for orally ingested products. A 4.3 log reduction in bacteria was achieved at 150 bar and 30 degrees C, decreasing the TAMC in Ginseng sample to 2,000, below the allowable limit. However, fungi still remained in the sample. The complete inactivation of both bacteria and fungi was achieved within 2 h at 30 degrees C and 170 bar using 0.1 mL of each additive/g Ginseng. Microbial inactivation at this low temperature opens an avenue for the sterilization of many thermally labile pharmaceutical and food products that may involve sensitive compounds to gamma-radiation and chemically reactive antiseptic agents.     

Synthesis, characterization and the thermodynamic study of intermediate sitting-atop (i-SAT) complexes of free base meso-tetraarylporphyrins with InCl3

The reaction of free base para-substituted meso-tetraarylporphyrins (H₂T(p-X)PP, X = H, OMe, Me, and Cl) with indium(III) chloride in CHCl₃ and mild conditions produced intermediate sitting-atop (i-SAT) complexes, [InCl₂(H₂T(p-X)PP)]InCl₄, as sole products. In the proposed structures of these complexes, four pyrrole rings are tilted alternatively up and down the porphyrin plane. This distortion makes suitable orientation of lone pairs of two pyrrolenine nitrogens for electron donation to an indium center of cation. The 1:2 (porphyrin:indium) formation constant of resulting i-SAT complexes were calculated by the computer fitting of the complexes absorbance versus mole ratio data based on appropriate equations. Thermodynamic parameters, ΔG⁰, ΔH⁰, and ΔS⁰, have been determined and the influence of electron donation of the para-substituted aryl groups in the free base porphyrins on the stability of the complexes is discussed.     

Intracardiac atrial signal amplitude in congenital and acquired complete heart block

Background

Good and reliable atrial sensing is a fundamental part of atrioventricular (AV) synchrony in dual chamber pacemakers. Due to the floating nature of atrial sensing electrode in single pass dual chamber pacemakers (VDD) compared with two-lead dual chamber pacemakers (DDD), they are more prone to atrial under-sensing and the resulting loss of AV synchrony. We hypothesized that there is a relation between the chronicity of AV block and the amplitude of intracardiac atrial signal amplitudes (IASA).

Methods

Detailed demographic, electrocardiographic and echocardiographic data were recorded in 34 consecutive patients with congenital and acquired complete heart block (CHB). The intracardiac atrial signal amplitudes (IASA) were recorded at implantation time, 48 hours and 2 months post-implantation and compared between the two groups of patients.

Results

The mean age of the study group was 38.73±12.53 years (congenital: 30.08±11.07, acquired: 47.38±6.5). There were no important differences in left atrial or ventricular sizes and in P-wave amplitude in lead II, but the IASA was significantly higher in the congenital group at implantation time (5.21±1.86 vs. 3.38±0.84 mV, P<0.001) and during the follow-up.

Conclusion

The intracardiac atrial signal amplitudes were higher in congenital CHB compared with the acquired CHB. Chronicity (and may be the congenital type) of CHB may be an affecting factor in case selection for VDD pacemaker implantation.     

Structural studies of SNARE complex and its interaction with complexin by molecular dynamics simulation

Since neurotransmitter releasing into the synaptic space delivers electrical signals from presynaptic neural cell to the postsynaptic cell, neurotransmitter secretion must be much orchestrated. Crowded intracellular vesicles involving neurotransmitters present a question of the how secretory vesicles fuse onto the plasma membrane in a fast synchronized fashion. Complexin is one of the most experimentally studied proteins that regulate assembly of fusogenic four‐helix SNARE complex to synchronized neurotransmitter secretion. We used MD simulation to investigate the interaction of complexin with the neural SNARE complex in detail. Our results show that the SNARE complex interacts with the complexin central helix by forming salt bridges and hydrogen bonds. Complexin also can interact with the Q‐SNARE complex instead of synaptobrevin to decrease the Q‐SNARE flexibility. The complexin alpha‐accessory helix and the C‐terminal region of synaptobrevin can interact with the same region of syntaxin. Although the alpha‐accessory helix aids the tight binding of the central helix to the SNARE complex, its proximity with synaptobrevin causes the destabilization of syntaxin and Sn1 helices. This study suggests that the alpha‐accessory helix of complexin can be an inhibiting factor for membrane fusion by competing with synaptobrevin for binding to the Q‐SNARE complex. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 560–570, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Divergent Cortical Generators of MEG and EEG during Human Sleep Spindles Suggested by Distributed Source Modeling

Background

Sleep spindles are ∼1-second bursts of 10–15 Hz activity, occurring during normal stage 2 sleep. In animals, sleep spindles can be synchronous across multiple cortical and thalamic locations, suggesting a distributed stable phase-locked generating system. The high synchrony of spindles across scalp EEG sites suggests that this may also be true in humans. However, prior MEG studies suggest multiple and varying generators.

Methodology/Principal Findings

We recorded 306 channels of MEG simultaneously with 60 channels of EEG during naturally occurring spindles of stage 2 sleep in 7 healthy subjects. High-resolution structural MRI was obtained in each subject, to define the shells for a boundary element forward solution and to reconstruct the cortex providing the solution space for a noise-normalized minimum norm source estimation procedure. Integrated across the entire duration of all spindles, sources estimated from EEG and MEG are similar, diffuse and widespread, including all lobes from both hemispheres. However, the locations, phase and amplitude of sources simultaneously estimated from MEG versus EEG are highly distinct during the same spindles. Specifically, the sources estimated from EEG are highly synchronous across the cortex, whereas those from MEG rapidly shift in phase, hemisphere, and the location within the hemisphere.

Conclusions/Significance

The heterogeneity of MEG sources implies that multiple generators are active during human sleep spindles. If the source modeling is correct, then EEG spindles are generated by a different, diffusely synchronous system. Animal studies have identified two thalamo-cortical systems, core and matrix, that produce focal or diffuse activation and thus could underlie MEG and EEG spindles, respectively. Alternatively, EEG spindles could reflect overlap at the sensors of the same sources as are seen from the MEG. Although our results generally match human intracranial recordings, additional improvements are possible and simultaneous intra- and extra-cranial measures are needed to test their accuracy.     

Bioinformatic analyses of mammalian 5'-UTR sequence properties of mRNAs predicts alternative translation initiation sites

Background  

Utilization of alternative initiation sites for protein translation directed by non-AUG codons in mammalian mRNAs is observed with increasing frequency. Alternative initiation sites are utilized for the synthesis of important regulatory proteins that control distinct biological functions. It is, therefore, of high significance to define the parameters that allow accurate bioinformatic prediction of alternative translation initiation sites (aTIS). This study has investigated 5'-UTR regions of mRNAs to define consensus sequence properties and structural features that allow identification of alternative initiation sites for protein translation.     

Novel dense CO2 technique for beta-galactosidase immobilization in polystyrene microchannels

In this study we design new fabrication techniques and demonstrate the potential of using dense CO2 for facilitating crucial steps in the fabrication of polymeric lab-on-a-chip microdevices by embedding biomolecules at temperatures well below the polymer's glass transition temperature (T(g)). These new techniques are environmentally friendly and done without the use of a clean room. Carbon dioxide at 40 degrees C and between 4.48 and 6.89 MPa was used to immobilize the biologically active molecule, beta-galactosidase (beta-gal), on the surface of polystyrene microchannels. To our knowledge, this is the first time dense CO2 has been used to directly immobilize an enzyme in a microchannel. beta-gal activity was maintained and shown via a fluorescent reaction product, after enzyme immobilization and microchannel capping by the designed fabrication steps at 40 degrees C and pressures up to 6.89 MPa.     

The pituitary adenylate cyclase-activating polypeptide modulates glutamatergic calcium signalling: investigations on rat suprachiasmatic nucleus neurons

Circadian rhythms generated by the hypothalamic suprachiasmatic nucleus (SCN) are synchronized with the external light/dark cycle by photic information transmitted directly from the retina via the retinohypothalamic tract (RHT). The RHT contains the neurotransmitters glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP), which code chemically for 'light' or 'darkness' information, respectively. We investigated interactions of PACAP and glutamate by analysing effects on the second messenger calcium in individual SCN neurons using the Fura-2 technique. PACAP did not affect NMDA-mediated calcium increases, but influenced signalling cascades of non-NMDA glutamate receptors, which in turn can regulate NMDA receptors. On the one hand, PACAP amplified/induced glutamate-dependent calcium increases by interacting with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate signalling. This was not related to direct PACAPergic effects on the second messengers cAMP and calcium. On the other hand, PACAP reduced/inhibited calcium increases elicited by glutamate acting on metabotropic receptors. cAMP analogues mimicked this inhibition. Most neurons displaying PACAPergic neuromodulation were immunoreactive for vasoactive intestinal polypeptide, which is a marker for retinorecipient SCN neurons. The observed PACAPergic effects provide a broad range of interactions that allow a fine-tuning of the endogenous clock by the integration of 'light' and 'darkness' information on the level of single SCN neurons.     

Intrinsic susceptibility of rhesus macaque peripheral CD4(+) T cells to simian immunodeficiency virus in vitro is predictive of in vivo viral replication

Previous studies with simian immunodeficiency virus (SIV) infection of rhesus macaques suggested that the intrinsic susceptibility of peripheral blood mononuclear cells (PBMC) to infection with SIV in vitro was predictive of relative viremia after SIV challenge. The present study was conducted to evaluate this parameter in a well-characterized cohort of six rhesus macaques selected for marked differences in susceptibility to SIV infection in vitro. Rank order relative susceptibility of PBMC to SIVsmE543-3-infection in vitro was maintained over a 1-year period of evaluation. Differential susceptibility of different donors was maintained in CD8(+) T-cell-depleted PBMC, macrophages, and CD4(+) T-cell lines derived by transformation of PBMC with herpesvirus saimiri, suggesting that this phenomenon is an intrinsic property of CD4(+) target cells. Following intravenous infection of these macaques with SIVsmE543-3, we observed a wide range in plasma viremia which followed the same rank order as the relative susceptibility established by in vitro studies. A significant correlation was observed between plasma viremia at 2 and 8 weeks postinoculation and in vitro susceptibility (P < 0.05). The observation that the two most susceptible macaques were seropositive for simian T-lymphotropic virus type 1 may suggests a role for this viral infection in enhancing susceptibility to SIV infection in vitro and in vivo. In summary, intrinsic susceptibility of CD4(+) target cells appears to be an important factor influencing early virus replication patterns in vivo that should be considered in the design and interpretation of vaccine studies using the SIV/macaque model.