Optimal Velocity and Safety of Discontinuous Conduction through the Heterogeneous Purkinje-Ventricular Junction

Slow and discontinuous wave conduction through nonuniform junctions in cardiac tissues is generally considered unsafe and proarrythmogenic. However, the relationships between tissue structure, wave conduction velocity, and safety at such junctions are unknown. We have developed a structurally and electrophysiologically detailed model of the canine Purkinje-ventricular junction (PVJ) and varied its heterogeneity parameters to determine such relationships. We show that neither very fast nor very slow conduction is safe, and there exists an optimal velocity that provides the maximum safety factor for conduction through the junction. The resultant conduction time delay across the PVJ is a natural consequence of the electrophysiological and morphological differences between the Purkinje fiber and ventricular tissue. The delay allows the PVJ to accumulate and pass sufficient charge to excite the adjacent ventricular tissue, but is not long enough for the source-to-load mismatch at the junction to be enhanced over time. The observed relationships between the conduction velocity and safety factor can provide new insights into optimal conditions for wave propagation through nonuniform junctions between various cardiac tissues.     

2-Methoxy-3-isobutylpyrazine in grape berries and its dependence on genotype

2-Methoxy-3-isobutylpyrazine (MIBP) contributes a bell pepper aroma to many grape cultivars and has a reported aroma threshold of ～2 ng L(-1) in water. The purpose of this study was twofold: (1) develop a procedure using headspace solid phase micro-extraction combined with GC-MS in the selected ion monitoring mode (HS-SPME-GC-MS-SIM) for analysis of MIBP in grape berries, and (2) determine the location of MIBP biosynthesis in grapevines by approach grafting clusters of Vitis vinifera L. cvs Cabernet Sauvignon and Muscat blanc onto each other. The soluble solids and pH of the grape juice/homogenate matrix from different grape berry developmental stages influenced the method precision; therefore, quantification via the method of standard addition was used. Using our developed method, the limit of detection (LOD) and limit of quantitation (LOQ) of MIBP were 0.1 ng L(-1) and 2 ng L(-1), respectively, measured in a model juice and non-MIBP containing Chardonnay juice. Spiked recoveries averaged between 91% and 112% in Cabernet Sauvignon and Pinot noir homogenates and the overall relative standard deviation was less than 10%. The method was used to analyze MIBP in 29 grape cultivars and in fruit from clusters grafted to Cabernet Sauvignon or Muscat vines. Quantifiable levels were found only in Cabernet franc, Cabernet Sauvignon, Merlot, Sauvignon blanc and Semillon, providing information on the genetic connection for the occurrence of MIBP in grapes. No MIBP was detected in the berries of Muscat blanc clusters grafted onto Cabernet Sauvignon vines when sampled at fruit maturity. MIBP was detected in all berries of Cabernet Sauvignon regardless the graft configuration. The data indicate that MIBP or its precursors originate in the berry and its formation depends upon grape genotype.     

Rab11b regulates the trafficking and recycling of the epithelial sodium channel (ENaC)

Expression of the epithelial sodium channel (ENaC) at the apical membrane of cortical collecting duct (CCD) principal cells is modulated by regulated trafficking mediated by vesicle insertion and retrieval. Small GTPases are known to facilitate vesicle trafficking, recycling, and membrane fusion events; however, little is known about the specific Rab family members that modify ENaC surface density. Using a mouse CCD cell line that endogenously expresses ENaC (mpkCCD), the channel was localized to both Rab11a- and Rab11b-positive endosomes by immunoisolation and confocal fluorescent microscopy. Expression of a dominant negative (DN) form of Rab11a or Rab11b significantly reduced the basal and cAMP-stimulated ENaC-dependent sodium (Na(+)) transport. The greatest reduction in Na(+) transport was observed with the expression of DN-Rab11b. Furthermore, small interfering RNA-mediated knockdown of each Rab11 isoform demonstrated the requirement for Rab11b in ENaC surface expression. These data indicate that Rab11b, and to a lesser extent Rab11a, is involved in establishing the constitutive and cAMP-stimulated Na(+) transport in mpkCCD cells.     

Germination and physiological properties of Frankia spores

Spores from four Frankia strains were isolated and purified to homogeneity. The purified spores were biochemically and physiologically characterized and compared to vegetative cells. Frankia spores exhibited low levels of endogenous respiration that were at least ten-fold lower than the endogenous respiration rate of vegetative cells. The macromolecular content of purified spores and vegetative cells differed. One striking difference among the Frankia spores was their total DNA content. From DAPI staining experiments, only 9% of strain ACN1^AG spore population contained DNA. With strains DC12 and EuI1c, 92% and 67% of their spore population contained DNA. The efficiency of spore germination was correlated to the percentage of the spore population containing DNA. These results suggest that the majority of strain ACN1^AG spores were immature or nonviable. The presence of a solidifying agent inhibited the initial stages of spore germination, but had no effect once the process had been initiated. The optimal incubation temperature for spore germination was 25°C and 30°C for strains DC12 and EuI1c, respectively. A mild heat shock increased the efficiency of spore germination, while root extracts also stimulated spore germination. These results suggest that strains DC12 and EuI1c may be suitable strains for further germination and genetic studies.     

From genetics to epigenetics: new insights into keloid scarring

Keloid scarring is a dermal fibroproliferative response characterized by excessive and progressive deposition of collagen; aetiology and molecular pathology underlying keloid formation and progression remain unclear. Genetic predisposition is important in the pathogenic processes of keloid formation, however, environmental factors and epigenetic mechanisms may also play pivotal roles. Epigenetic modification is a recent area of investigation in understanding the molecular pathogenesis of keloid scarring and there is increasing evidence that epigenetic changes may play a role in induction and persistent activation of fibroblasts in keloid scars. Here we have reviewed three epigenetic mechanisms: DNA methylation, histone modification and the role of non‐coding RNAs. We also review the evidence that these mechanisms may play a role in keloid formation ‐ in future, it may be possible that epigenetic markers may be used instead of prognostic or diagnostic markers here. However, there is a significant amount of work required to increase our current understanding of the role of epigenetic modification in keloid disease.     

Layilin,A Novel Talin-binding Transmembrane Protein Homologous with C-type Lectins,is Localized in Membrane Ruffles

Changes in cell morphology and motility are mediated by the actin cytoskeleton. Recent advances in our understanding of the regulators of microfilament structure and dynamics have shed light on how these changes are controlled, and efforts continue to define all the structural and signaling components involved in these processes. The actin cytoskeleton-associated protein talin binds to integrins, vinculin, and actin. We report a new binding partner for talin that we have named layilin, which contains homology with C-type lectins, is present in numerous cell lines and tissue extracts, and is expressed on the cell surface. Layilin colocalizes with talin in membrane ruffles, and is recruited to membrane ruffles in cells induced to migrate in in vitro wounding experiments and in peripheral ruffles in spreading cells. A ten–amino acid motif in the layilin cytoplasmic domain is sufficient for talin binding. We have identified a short region within talin''s amino-terminal 435 amino acids capable of binding to layilin in vitro. This region overlaps a binding site for focal adhesion kinase.     

Thermal effects in stretching of Go-like models of titin and secondary structures

The effect of temperature on mechanical unfolding of proteins is studied using a Go-like model with a realistic contact map and Lennard-Jones contact interactions. The behavior of the I27 domain of titin and its serial repeats is contrasted to that of simple secondary structures. In all cases, thermal fluctuations accelerate the unraveling process, decreasing the unfolding force nearly linearly at low temperatures. However, differences in bonding geometry lead to different sensitivity to temperature and different changes in the unfolding pattern. Due to its special native-state geometry, titin is much more thermally and elastically stable than the secondary structures. At low temperatures, serial repeats of titin show a parallel unfolding of all domains to an intermediate state, followed by serial unfolding of the domains. At high temperatures, all domains unfold simultaneously, and the unfolding distance decreases monotonically with the contact order, that is, the sequence distance between the amino acids that form the native contact.     

Human p38 mitogen-activated protein kinase inhibitor drugs inhibit Plasmodium falciparum replication

We recently demonstrated that human p38 mitogen-activated protein kinase (MAPK) inhibitors reduced in vitro and in vivo replication of the protozoan parasites Toxoplasma gondii and Encephalitozoon cuniculi. In this study, we assessed the efficacy of five p38 MAPK inhibitors to block the replication of Plasmodium falciparum in human erythrocytes cultured ex vivo and demonstrate that the pyridinylimidazole RWJ67657 and the pyrrolobenzimidazole RWJ68198 reduced P. falciparum replication, yielded trophozoites that were greatly diminished in size at 24 h, and that these two agents interfered with stage differentiation. Interestingly, the chloroquine-resistant strain W2 was significantly more sensitive to these drugs than was the chloroquine-sensitive strain HB3. These results suggest that pyridinylimidazoles and pyrrolobenzimidazoles designed to inhibit human p38 MAPK activation can be developed to treat malaria.     

Subunit–subunit interactions are weakened in mutant forms of acetohydroxy acid synthase insensitive to valine inhibition

In acetohydroxy acid synthase from Streptomyces cinnamonensis mutants affected in valine regulation, the impact of mutations on interactions between the catalytic and the regulatory subunits was examined using yeast two-hybrid system. Mutations in the catalytic and the regulatory subunits were projected into homology models of the respective proteins. Two changes in the catalytic subunit, E139A (α domain) and ΔQ217 (β domain), both located on the surface of the catalytic subunit dimer, lowered the interaction with the regulatory subunit. Three consecutive changes in the N-terminal part of the regulatory subunit were examined. Changes G16D and V17D in a loop and adjacent α-helix of ACT domain affected the interaction considerably, indicating that this region might be in contact with the catalytic subunit during allosteric regulation. In contrast, the adjacent mutation L18F did not influence the interaction at all. Thus, L18 might participate in valine binding or conformational change transfer within the regulatory subunits. Shortening of the regulatory subunit to 107 residues reduced the interaction essentially, suggesting that the C-terminal part of the regulatory subunit is also important for the catalytic subunit binding.     

Two-stage dependence for 1-methyladenine induced reinitiation of meiotic maturation in starfish oocytes

The maturation hormone 1-methyladenine (1-MA) causes meiotic resumption of prophase arrested immature starfish oocytes. Continuous exposure to ≥ 0.5 µM 1-MA causes germinal vesicle breakdown (GVBD) in ∼ 20 min, but oocytes pretreated for > 30 min with a subthreshold dose of 1-MA undergo GVBD much faster (∼ 10 min) when they are exposed to 1 µM 1-MA. Furthermore, a very low subthreshold 1-MA suffices to start the maturation process: oocytes exposed to 0.005 µM 1-MA for up to 10 min followed by 1 µM 1-MA is equivalent to continuous exposure to 1 µM 1-MA. These dose and timing relationships indicate that there is a two-stage dependence on 1-MA. A possible explanation for this dependence is that there are two processes involved: an initial process that is triggered by a low dose of 1-MA, and a second process that cannot start until the first process is completed and is stimulated by a higher dose of 1-MA. These subthreshold 1-MA effects on GVBD timing are not directly coupled to changes in calcium physiology that also occur during maturation. Subthreshold 1-MA was found to cause a transient accumulation of Cdc2/cyclin B into the nucleus. The two-stage dependence indicates that there are unsuspected features in this well-studied pathway leading to GVBD. In the animal, this hormone dependence may help to synchronize maturation throughout all parts of the ovary.