Structural and electronic properties of the liver fluke hemoglobin heme cavity by nuclear magnetic resonance: hemin isotope labeling

Reconstitution of liver fluke (Dicrocoelium dendriticum) apo-hemoglobin with hemins selectively deuterated at specific positions has permitted the assignment of several heme resonances in the proton nuclear magnetic resonance spectrum of the Met-aquo and Met-cyano forms of the holoprotein. It was established that in the Met-aquo form the meso protons resonate at positions characteristic of a six-co-ordinated in-plane iron. From this, we deduced that the Met-aquo species retains a bound water molecule at pH values as low as 4.5. The orientation of the proximal histidine imidazole ring with respect to the heme group in the cavity was determined through the identification of the heme methyl signals and the analysis of the hyperfine shift pattern in the Met-cyano hemoglobin proton nuclear magnetic resonance spectrum. Compared to sperm whale myoglobin, the heme appears to be rotated by 180 degrees about the alpha, gamma meso-axis. Protein isomers with the heme group in a reversed orientation were not detected, even shortly after reconstitution. In the Met-cyano form, the resonances most affected by the Bohr transition were shown to arise from the heme propionates.     

Cell death induced by Pteris semipinnata L. is associated with p53 and oxidant stress in gastric cancer cells

In this study, we demonstrated that Ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) had stronger cytotoxicity against MKN-45, a gastric cancer cell line bearing wild-type p53 than MKN-28, another gastric cancer cell line containing missense mutation in p53. The rapid increase of ROS level was involved in the mechanism of cytotoxicity. Classical features of apoptosis induced by 5F were observed in MKN-45 cells only or more significant in MKN-45 cells than MKN-28 cells. Translocation of Bax from cytosol to mitochondria, reduction of delta psi m and DNA fragmentation were induced by 5F in the p53-dependent manner. We conclude that the expression of Bax and its downstream molecules requires the presentation of a wild-type p53 in the cells treated by 5F.     

Automated analysis of three-dimensional stress echocardiography

Real-time three-dimensional (3D) ultrasound imaging has been proposed as an alternative for two-dimensional stress echocardiography for assessing myocardial dysfunction and underlying coronary artery disease. Analysis of 3D stress echocardiography is no simple task and requires considerable expertise. In this paper, we propose methods for automated analysis, which may provide a more objective and accurate diagnosis. Expert knowledge is incorporated via statistical modelling of patient data. Methods for identifying anatomical views, detecting endocardial borders, and classification of wall motion are described and shown to provide favourable results. We also present software developed especially for analysis of 3D stress echocardiography in clinical practice. Interobserver agreement in wall motion scoring is better using the dedicated software (96%) than commercially available software not dedicated for this purpose (79%). The developed tools may provide useful quantitative and objective parameters to assist the clinical expert in the diagnosis of left ventricular function.     

In Vivo Quantification Reveals Extensive Natural Variation in Mitochondrial Form and Function in Caenorhabditis briggsae

We have analyzed natural variation in mitochondrial form and function among a set of Caenorhabditis briggsae isolates known to harbor mitochondrial DNA structural variation in the form of a heteroplasmic nad5 gene deletion (nad5Δ) that correlates negatively with organismal fitness. We performed in vivo quantification of 24 mitochondrial phenotypes including reactive oxygen species level, membrane potential, and aspects of organelle morphology, and observed significant among-isolate variation in 18 traits. Although several mitochondrial phenotypes were non-linearly associated with nad5Δ levels, most of the among-isolate phenotypic variation could be accounted for by phylogeographic clade membership. In particular, isolate-specific mitochondrial membrane potential was an excellent predictor of clade membership. We interpret this result in light of recent evidence for local adaptation to temperature in C. briggsae. Analysis of mitochondrial-nuclear hybrid strains provided support for both mtDNA and nuclear genetic variation as drivers of natural mitochondrial phenotype variation. This study demonstrates that multicellular eukaryotic species are capable of extensive natural variation in organellar phenotypes and highlights the potential of integrating evolutionary and cell biology perspectives.     

Modified Long Wavelength Approximation for the Optical Response of a Graded-Index Plasmonic Nanoparticle

The optical response of graded-index spherical metallic nanoparticles is studied in the modified long wavelength approximation with electrodynamic effects accounted for to the lowest order of the inverse of the wavelength. An effective-medium approach is adopted which leads to the conclusion that the first-order dynamical effects will enter mainly via the polarizability and not the effective dielectric function of the system. Numerical studies using various graded Drude functions show that these effects are not only significant for particles of large sizes but can also be appreciable for smaller particles with varying index profile.     

Dissection of the karyopherin alpha nuclear localization signal (NLS)-binding groove: functional requirements for NLS binding

Classical protein import, mediated by the binding of a classical nuclear localization signal (NLS) to the NLS receptor, karyopherin/importin alpha, is the most well studied nuclear transport process. Classical NLSs are either monopartite sequences that contain a single cluster of basic amino acids (Lys/Arg) or bipartite sequences that contain two clusters of basic residues separated by an unconserved linker region. We have created mutations in conserved residues in each of the three NLS-binding sites/regions in Saccharomyces cerevisiae karyopherin alpha (SRP1). For each mutant we have analyzed binding to both a monopartite and a bipartite NLS cargo in vitro. We have also expressed each karyopherin alpha mutant in vivo as the only cellular copy of the NLS receptor and examined the impact on cell growth and import of both monopartite and bipartite NLS-containing cargoes. Our results reveal the functional significance of specific residues within karyopherin alpha for NLS cargo binding. A karyopherin alpha variant with a mutation in the major NLS-binding site exhibits decreased binding to both monopartite and bipartite NLS cargoes, and this protein is not functional in vivo. However, we also find that a karyopherin alpha variant with a mutation in the minor NLS-binding site, which shows decreased binding only to bipartite NLS-containing cargoes, is also not functional in vivo. This suggests that the cell is dependent on the function of at least one bipartite NLS cargo that is imported into the nucleus by karyopherin alpha. Our experiments also reveal functional importance for the linker-binding region. This study provides insight into how changes in binding to cellular NLS sequences could impact cellular function. In addition, this work has led to the creation of conditional alleles of karyopherin alpha with well characterized defects in NLS binding that will be useful for identifying and characterizing novel NLS cargoes.     

PAX6 haplotypes are associated with high myopia in Han chinese

Background

The paired box 6 (PAX6) gene is considered as a master gene for eye development. Linkage of myopia to the PAX6 region on chromosome 11p13 was shown in several studies, but the results for association between myopia and PAX6 were inconsistent so far.

Methodology/Principal Findings

We genotyped 16 single nucleotide polymorphisms (SNPs) in the PAX6 gene and its regulatory regions in an initial study for 300 high myopia cases and 300 controls (Group 1), and successfully replicated the positive results with another independent group of 299 high myopia cases and 299 controls (Group 2). Five SNPs were genotyped in the replication study. The spherical equivalent of subjects with high myopia was ≤−8.0 dioptres. The PLINK package was used for genetic data analysis. No association was found between each of the SNPs and high myopia. However, exhaustive sliding-window haplotype analysis highlighted an important role for rs12421026 because haplotypes containing this SNP were found to be associated with high myopia. The most significant results were given by the 4-SNP haplotype window consisting of rs2071754, rs3026393, rs1506 and rs12421026 (P = 3.54×10⁻¹⁰, 4.06×10⁻¹¹ and 1.56×10⁻¹⁸ for Group 1, Group 2 and Combined Group, respectively) and the 3-SNP haplotype window composed of rs3026393, rs1506 and rs12421026 (P = 5.48×10⁻¹⁰, 7.93×10⁻¹² and 6.28×10⁻²³ for the three respective groups). The results remained significant after correction for multiple comparisons by permutations. The associated haplotyes found in a previous study were also successfully replicated in this study.

Conclusions/Significance

PAX6 haplotypes are associated with susceptibility to the development of high myopia in Chinese. The PAX6 locus plays a role in high myopia.     

The role of carbohydrate moieties of cholecystokinin receptors in cholecystokinin octapeptide binding: alteration of binding data by specific lectins

Cholecystokinin (CCK) receptors on rat pancreatic acini have been demonstrated to be glycoproteins. In order to study whether their carbohydrate moieties play a role in ligand binding, membrane preparations (adjusted to 0.2 mg protein me) were incubated with 20 pM 125-I-CCK octapeptide (125I-CCK8) for 4 h at 30 degrees C in the presence of lectins with different sugar specificities. Concanavalin A, soy-bean agglutinin, and peanut agglutinin in concentrations up to 1 mM did not alter specific 125I-CCK8 binding. Ulex europeus lectin I showed a dose-dependent enhancement of CCK binding up to 150% of controls at a concentration of 1 mM. Wheat-germ agglutinin (WGA) was the only lectin found to have an inhibitory effect. Inhibition was dose-dependent, with maximal reduction attained at 42 nM, but CCK binding was only partially inhibited to 66.2 +/- 4.4%. Inhibition by WGA was prevented by the presence of N-acetyl-D-glucosamine or N,N',N"-triacetylchitotriose, sugars that are specific for WGA. The inhibitory effect of WGA was not due to an increase in non-specific binding, increased CCK degradation, or CCK binding to WGA. Binding data indicated that the presence of WGA resulted in a decrease in receptor affinity (Kd = 567 +/- 191 v. 299 +/- 50 pM). No significant change in the number of available binding sites was observed. This suggests that WGA is not binding to the active binding site. It is conceivable that binding of WGA to N-acetyl-D-glucosamine or its polymers can lead to a conformational change in the receptor protein, and that this carbohydrate moiety is essential for optimal receptor-ligand interaction.