Distribution of RNA binding protein MOEP19 in the oocyte cortex and early embryo indicates pre-patterning related to blastomere polarity and trophectoderm specification

We report the cloning and characterization of MOEP19, a novel 19 kDa RNA binding protein that marks a defined cortical cytoplasmic domain in oocytes and provides evidence of mammalian oocyte polarity and a form of pre-patterning that persists in zygotes and early embryos through the morula stage. MOEP19 contains a eukaryotic type KH-domain, typical of the KH-domain type I superfamily of RNA binding proteins, and both recombinant and native MOEP19 bind polynucleotides. By immunofluorescence, MOEP19 protein was first detected in primary follicles throughout the ooplasm. As oocytes expanded in size during oogenesis, MOEP19 increased in concentration. MOEP19 localized in the ovulated egg and early zygote as a symmetrical spherical cortical domain underlying the oolemma, deep to the zone of cortical granules. MOEP19 remained restricted to a cortical cytoplasmic crescent in blastomeres of 2-, 4- and 8-cell embryos. The MOEP19 domain was absent in regions underlying cell contacts. In morulae, the MOEP19 domain was found at the apex of outer, polarized blastomeres but was undetectable in blastomeres of the inner cell mass. In early blastocysts, MOEP19 localized in both mural and polar trophectoderm and a subset of embryos showed inner cell mass localization. MOEP19 concentration dramatically declined in late blastocysts. When blastomeres of 4- to 8-cell stages were dissociated, the polarized MOEP19 domain assumed a symmetrically spherical localization, while overnight culture of dissociated blastomeres resulted in formation of re-aggregated embryos in which polarity of the MOEP19 domain was re-established at the blastomere apices. MOEP19 showed no evidence of translation in ovulated eggs, indicating that MOEP19 is a maternal effect gene. The persistence during early development of the MOEP19 cortical oocyte domain as a cortical crescent in blastomers suggests an intrinsic pre-patterning in the egg that is related to the apical-basolateral polarity of the embryo. Although the RNAs bound to MOEP19 are presently unknown, we predict that the MOEP19 domain directs RNAs essential for normal embryonic development to specific locations in the oocyte and early embryo.     

Alcohol and nicotine consumption exacerbates choroidal neovascularization by modulating the regulation of complement system

The objective of the present study was to investigate the effect of alcohol and nicotine consumption on the pathogenesis of choroidal neovascularization (CNV) in rats after laser-photocoagulation. Confocal microscopic analysis demonstrated an increase in CNV complex size in rats fed with alcohol (2.3-fold), nicotine (1.9-fold), and the combination of alcohol and nicotine (2.7-fold) compared with the control groups. Immunohistochemical analysis revealed that alcohol and nicotine consumption increased MAC deposition and VEGF expression in laser spots. Expression of CD59 by RT-PCR and Western blot was drastically reduced in the animals that were fed with alcohol, nicotine and alcohol and nicotine compared to those fed with water alone and this was associated with exacerbation of CNV.     

Photochemistry of dyes and fluorochromes used in biology and medicine: some physicochemical background and current applications

An overview of the basic principles of photochemistry is presented to facilitate discussion of fluorescence, quenching and quantum yields. These topics in turn provide the foundation for an account of fluorescence spectroscopy and its application to microscopy. A brief overview of light microscopy and the application of fluorescence microscopy is given. The influences of molecular features, such as aromatic character and substitution patterns, on color and fluorescence are described. The concept of color fading is considered with particular reference to its effect on microscopic preparations. A survey of representative fluorescent probes is provided, and their sensitivity, application, and limitations are described. The phototoxicity of fluorescent molecules is discussed using biomembranes and DNA as examples of targets of toxicity. Photodynamic therapy, a relatively new clinical application of phototoxicity, is described. Both anticancer and antimicrobial applications are noted, and an assessment is given of the current ideas on the ideal physicochemical properties of the sensitizing agents for such applications.     

Comparative study of catalase-peroxidases (KatGs)

Catalase-peroxidases or KatGs from seven different organisms, including Archaeoglobus fulgidus,Bacillus stearothermophilus, Burkholderia pseudomallei, Escherichia coli, Mycobacterium tuberculosis, Rhodobacter capsulatus and Synechocystis PCC 6803, have been characterized to provide a comparative picture of their respective properties. Collectively, the enzymes exhibit similar turnover rates with the catalase and peroxidase reactions varying between 4900 and 15,900 s⁻¹ and 8-25 s⁻¹, respectively. The seven enzymes also exhibited similar pH optima for the peroxidase (4.25-5.0) and catalase reactions (5.75), and high sensitivity to azide and cyanide with IC₅₀ values of 0.2-20 μM and 50-170 μM, respectively. The K_Ms of the enzymes for H₂O₂ in the catalase reaction were relatively invariant between 3 and 5 mM at pH 7.0, but increased to values ranging from 20 to 225 mM at pH 5, consistent with protonation of the distal histidine (pKa approximately 6.2) interfering with H₂O₂ binding to Cpd I. The catalatic k_cat was 2- to 3-fold higher at pH 5 compared to pH 7, consistent with the uptake of a proton being involved in the reduction of Cpd I. The turnover rates for the INH lyase and isonicotinoyl-NAD synthase reactions, responsible for the activation of isoniazid as an anti-tubercular drug, were also similar across the seven enzymes, but considerably slower, at 0.5 and 0.002 s⁻¹, respectively. Only the NADH oxidase reaction varied more widely between 10⁻⁴ and 10⁻² s⁻¹ with the fastest rate being exhibited by the enzyme from B. pseudomallei.     

Disulfide bond formation through Cys186 facilitates functionally relevant dimerization of trimeric hyaluronan-binding protein 1 (HABP1)/p32/gC1qR.

Hyaluronan-binding protein 1 (HABP1), a ubiquitous multifunctional protein, interacts with hyaluronan, globular head of complement component 1q (gC1q), and clustered mannose and has been shown to be involved in cell signalling. In vitro, this recombinant protein isolated from human fibroblast exists in different oligomeric forms, as is evident from the results of various independent techniques in near-physiological conditions. As shown by size-exclusion chromatography under various conditions and glutaraldehyde cross-linking, HABP1 exists as a noncovalently associated trimer in equilibrium with a small fraction of a covalently linked dimer of trimers, i.e. a hexamer. The formation of a covalently-linked hexamer of HABP1 through Cys186 as a dimer of trimers is achieved by thiol group oxidation, which can be blocked by modification of Cys186. The gradual structural transition caused by cysteine-mediated disulfide linkage is evident as the fluorescence intensity increases with increasing Hg(2+) concentration until all the HABP1 trimer is converted into hexamer. In order to understand the functional implication of these transitions, we examined the affinity of the hexamer for different ligands. The hexamer shows enhanced affinity for hyaluronan, gC1q, and mannosylated BSA compared with the trimeric form. Our data, analyzed with reference to the HABP1/p32 crystal structure, suggest that the oligomerization state and the compactness of its structure are factors that regulate its function.     

Use of the random amplif|ed polymorphic DNA (RAPD) assay for the detection of DNA damage and mutations: possible implications of confounding factors

The aim of this study was to evaluate the potential of the random amplified polymorphic DNA (RAPD) assay to qualitatively detect the kinetics of benzo[a]pyrene (B[Ma]P)-induced DNA effects in the water flea Daphnia magna exposed to 25 and 50 µg l^-1 B[a]P for 7 and 6 days, respectively. Mortality was recorded on a daily basis in both experiments, and RAPD analysis was performed on samples collected every day following isolation of genomic DNA. The main changes occurring in RAPD profiles produced by the population of Daphnia magna exposed to 25 and 50µg l^-1 B[a]P was a decrease and increase in band intensity, respectively. Most of the changes occurring in the RAPD patterns were likely to be the result of B[a]P-induced DNA damage (B[a]P DNA adducts, oxidized bases, DNA breakages) and/or mutations (point mutations and large rearrangements). In addition, reproducible changes also occurred in the profiles generated by control Daphnia magna. The results lead us to suggest that, in addition to B[a]P-induced DNA damage and mutations, factors such as variation in gene expression, steady levels of genetic alterations and changes in metabolic processes could induce some changes in RAPD patterns. Nevertheless, our data suggest that DNA damage and mutations appear to be the main factors influencing RAPD patterns. This study also emphasizes that unexpected variation in control profiles is not always associated with artefacts.     

Comparative Study of Seed Protein Profiles in the Genus Pisum

Seed protein profiles of 24 wild and cultivated taxa of Pisum have been compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis. No consistent differences were detected either among wild taxa or between wild and cultivated taxa. This shows that Pisum forms a single-species complex on the basis of seed protein profiles.     

Are Global and Regional Improvements in Life Expectancy and in Child,Adult and Senior Survival Slowing?

Improvements in life expectancy have been considerable over the past hundred years. Forecasters have taken to applying historical trends under an assumption of continuing improvements in life expectancy in the future. A linear mixed effects model was used to estimate the trends in global and regional rates of improvements in life expectancy, child, adult, and senior survival, in 166 countries between 1950 and 2010. Global improvements in life expectancy, including both child and adult survival rates, decelerated significantly over the study period. Overall life expectancy gains were estimated to have declined from 5.9 to 4.0 months per year for a mean deceleration of -0.07 months/year²; annual child survival gains declined from 4.4 to 1.6 deaths averted per 1000 for a mean deceleration of -0.06 deaths/1000/year²; adult survival gains were estimated to decline from 4.8 to 3.7 deaths averted per 1000 per year for a mean deceleration of -0.08 deaths/1000/year². Senior survival gains however increased from 2.4 to 4.2 deaths averted per 1000 per year for an acceleration of 0.03 deaths/1000/year². Regional variation in the four measures was substantial. The rates of global improvements in life expectancy, child survival, and adult survival have declined since 1950 despite an increase in the rate of improvements among seniors. We postulate that low-cost innovation, related to the last half-century progress in health–primarily devoted to children and middle age, is reaping diminishing returns on its investments. Trends are uneven across regions and measures, which may be due in part to the state of epidemiological transition between countries and regions and disparities in the diffusion of innovation, accessible only in high-income countries where life expectancy is already highest.