Differential effects of stretch and compression on membrane currents and [Na]c in ventricular myocytes

Mechano-electrical feedback was studied in the single ventricular myocytes. A small fraction (approximately 10%) of the cell surface could be stretched or compressed by a glass stylus. Stretch depolarised, shortened the action potential and induced extra systoles. Stretch activated non-selective cation currents (I_ns) showed a linear voltage dependence, a reversal potential of 0 mV, a pure cation selectivity, and were blocked by 8 μM Gd³⁺ or 30 μM streptomycin. Stretch reduced Ca²⁺ and K⁺ (I_K) currents. Local compression of broadwise attached cells activated I_K but not I_ns. Cytochalasin D or colchicin, thought to disrupt the cytoskeleton, suppressed the mechanosensitivity of I_ns and I_K. During stretch, the cytosolic sodium concentration increased with spatial heterogeneities, local hotspots with [Na⁺]_c>24 mM appeared close to surface membrane and t-tubules (pseudoratiometric imaging using Sodium Green fluorescence). Electronprobe microanalysis confirmed this result and indicated that stretch increased total sodium [Na] in cell compartments such as mitochondria, nuclear envelope and nucleus. Our results obtained by local stretch differ from those obtained by end-to-end stretch (literature). We speculate that channels may be activated not only by axial but also by shear stress, and, that stretch can activate channels outside the deformed sarcomeres via second messenger.     

Cladogenesis and reticulation in the Hawaiian endemic mints (Lamiaceae)

The Hawaiian endemic mints, which comprise 58 species of dry‐fruited Haplostachys and fleshy‐fruited Phyllostegia and Stenogyne, represent a major island radiation that likely originated from polyploid hybrid ancestors in the temperate North American Stachys lineage. In contrast with considerable morphological and ecological diversity among taxa, sequence variation in the nrDNA 5S non‐transcribed spacer was found to be remarkably low, which when analyzed using standard parsimony resulted in a lack of phylogenetic resolution among accessions of insect‐pollinated Phyllostegia and bird‐pollinated Stenogyne. However, many within‐individual nucleotide polymorphisms were observed, and under the assumption that they could contain phylogenetic information, these ambiguities were recoded as new character states. Substantially more phylogenetic structure was obtained with these data, including the resolution of most Stenogyne species into a monophyletic group with an apparent recent origin on O’ahu (3.0 My) or the Maui Nui island complex (2.2 My). Subsequent diversification appears to have involved multiple inter‐island dispersal events. Intergeneric placements for a few morphotypes, seemingly misplaced within either Phyllostegia or Stenogyne, may indicate reticulation as one polymorphism‐generating force. For a finer scale exploration of hybridization, preliminary AFLP fragment data were examined among putative hybrids of Stenogyne microphylla and S. rugosa from Mauna Kea, Hawai’i, that had been identified based on morphology. Cladistic analysis (corroborated by multivariate correspondence analysis) showed the morphologically intermediate individuals to group in a strongly supported monophyletic clade with S. microphylla. Therefore, reticulation could be both historic and active in Stenogyne, and perhaps a force of general importance in the evolution of the Hawaiian mints. The relatively greater extent of lineage‐sorted polymorphisms in Stenogyne may indicate selective differentiation from other fleshy‐fruited taxa, perhaps through the agency of highly specialized bird pollinators that restricted gene flow with other Hawaiian mint morphotypes.     

Human exposure to mycotoxins in Egypt

This investigation examined the exposure of Egyptian infants to Aflatoxin M₁ (AfM₁) and of lactating mothers to Aflatoxin B₁, using AfM₁ in human milk as a biomarker for exposure to AfB₁. The presence of ochratoxin A (OA) in human milk was also investigated to determine the levels of infants exposure to OA from human milk. The results indicated that AfM₁ was found in 66 (55 %) of 120 human milk samples with a mean of 0.3 ± 0.53 ng/mL (range 0.02 to 2.09 ng/mL). OA was found in 43 (35.8 %) of 120 human milk samples with a mean of 21.1 ± 13.7 ng/mL (range 5.07 to 45.01 ng/mL), which will cause a daily intake of OA from human milk exceeding the suggested tolerable dose of 5 ng/kg_-1 of OA body weight. On the other side AfM₁ was found in 25 % of blood samples (5 out of 20 samples), at a mean of 1.18 ng/mL, but it was detected only in one urine sample (1 out of 20 samples). OA was detected only in 2 out of 13 blood samples (15.4 %) with an average 3.67 ng/mL. Whereas OA was not detected in all analyzed urine samples.     

Dynamic Localization of the Swe1 Regulator Hsl7 During the Saccharomyces cerevisiae Cell Cycle

In Saccharomyces cerevisiae, entry into mitosis requires activation of the cyclin-dependent kinase Cdc28 in its cyclin B (Clb)-associated form. Clb-bound Cdc28 is susceptible to inhibitory tyrosine phosphorylation by Swe1 protein kinase. Swe1 is itself negatively regulated by Hsl1, a Nim1-related protein kinase, and by Hsl7, a presumptive protein-arginine methyltransferase. In vivo all three proteins localize to the bud neck in a septin-dependent manner, consistent with our previous proposal that formation of Hsl1-Hsl7-Swe1 complexes constitutes a checkpoint that monitors septin assembly. We show here that Hsl7 is phosphorylated by Hsl1 in immune-complex kinase assays and can physically associate in vitro with either Hsl1 or Swe1 in the absence of any other yeast proteins. With the use of both the two-hybrid method and in vitro binding assays, we found that Hsl7 contains distinct binding sites for Hsl1 and Swe1. A differential interaction trap approach was used to isolate four single-site substitution mutations in Hsl7, which cluster within a discrete region of its N-terminal domain, that are specifically defective in binding Hsl1. When expressed in hsl7Delta cells, each of these Hsl7 point mutants is unable to localize at the bud neck and cannot mediate down-regulation of Swe1, but retains other functions of Hsl7, including oligomerization and association with Swe1. GFP-fusions of these Hsl1-binding defective Hsl7 proteins localize as a bright perinuclear dot, but never localize to the bud neck; likewise, in hsl1Delta cells, a GFP-fusion to wild-type Hsl7 or native Hsl7 localizes to this dot. Cell synchronization studies showed that, normally, Hsl7 localizes to the dot, but only in cells in the G1 phase of the cell cycle. Immunofluorescence analysis and immunoelectron microscopy established that the dot corresponds to the outer plaque of the spindle pole body (SPB). These data demonstrate that association between Hsl1 and Hsl7 at the bud neck is required to alleviate Swe1-imposed G2-M delay. Hsl7 localization at the SPB during G1 may play some additional role in fine-tuning the coordination between nuclear and cortical events before mitosis.     

Stereodesign of chiral para-substituted calix[4]arenes

According to the hypothesis that the chirality of molecule hosts is a cause of their enantioselectivity, the chirality of para-substituted calix[4]arenes was analyzed quantitatively. The relationship between types of para-substituents and the dissymmetry of the 2D (two-dimensional) entrance into the cavity and the whole 3D (three-dimensional) cavity of calix[4]arenes was studied by means of the enantiomer dissimilarity factor (EDF) method for quantitative evaluation of molecular chirality. The design of the most chiral, and probably enantioselective, para-substituted calix[4]arenes was planned such that all four substituents should be different and the two largest should be near each other (adjacent). It was, on the other hand, shown that the 2D chiral entrance determines chirality of the whole 3D structures of these molecules. This phenomenon is interpreted as an example of the chirality transition from 2D into 3D space.Electronic Supplementary Material available.     

Crystal Structure of Two Anti-Porphyrin Antibodies with Peroxidase Activity

We report the crystal structures at 2.05 and 2.45 Å resolution of two antibodies, 13G10 and 14H7, directed against an iron(III)-αααβ-carboxyphenylporphyrin, which display some peroxidase activity. Although these two antibodies differ by only one amino acid in their variable λ-light chain and display 86% sequence identity in their variable heavy chain, their complementary determining regions (CDR) CDRH1 and CDRH3 adopt very different conformations. The presence of Met or Leu residues at positions preceding residue H101 in CDRH3 in 13G10 and 14H7, respectively, yields to shallow combining sites pockets with different shapes that are mainly hydrophobic. The hapten and other carboxyphenyl-derivatized iron(III)-porphyrins have been modeled in the active sites of both antibodies using protein ligand docking with the program GOLD. The hapten is maintained in the antibody pockets of 13G10 and 14H7 by a strong network of hydrogen bonds with two or three carboxylates of the carboxyphenyl substituents of the porphyrin, respectively, as well as numerous stacking and van der Waals interactions with the very hydrophobic CDRH3. However, no amino acid residue was found to chelate the iron. Modeling also allows us to rationalize the recognition of alternative porphyrinic cofactors by the 13G10 and 14H7 antibodies and the effect of imidazole binding on the peroxidase activity of the 13G10/porphyrin complexes.     

Separation of meso and racemic 2,3-butanediol by aqueous liquid chromatography

Fermentation of xylose by Klebsiella pneumoniae (ATCC 8724) producers meso and nonmeso 2,3-butaneodiol. The enzyme Kinetic of 2,3-butanediol stereoisomer formation from acetone is currently under study in our laboratory. Modeling of these kinetics requires resolution of meso and racemic 2,3-butanediol and positive identification of these resolved components. We report their resolution by aqueous liquid chromatography on both an analytical and a preparative scale. The resolved stereoisomer were identified by a combination of gas chromatography, gas chromatography/mass spectroscopy, ¹³C-NMR spectroscopy, optical activity, and, melting points of the m-dinitrobenzoyl eaters of meso and racemic 2,3-butanediol. An aqueous liquid chromatographic technique for resolving and qualifying major components of a butanediol fermentation mixture in 40 min is presented.