Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Properties of unsaturated phospholipid bilayers: Effect of cholesterol

Properties of hydrated unsaturated phosphatidylcholine (PC) lipid bilayers containing 40 mol % cholesterol and of pure PC bilayers have been studied. Various methods were applied, including molecular dynamics simulations, self-consistent field calculations, and the pulsed field gradient nuclear magnetic resonance technique. Lipid bilayers were composed of 18:0/18:1(n-9)cis PC, 18:0/18:2(n-6)cis PC, 18:0/18:3(n-3)cis PC, 18:0/20:4(n-6)cis PC, and 18:0/22:6(n-3)cis PC molecules. Lateral self-diffusion coefficients of the lipids in all these bilayers, mass density distributions of atoms and atom groups with respect to the bilayer normal, the C-H and C-C bond order parameter profiles of each phospholipid hydrocarbon chain with respect to the bilayer normal were calculated. It was shown that the lateral self-diffusion coefficient of PC molecules of the lipid bilayer containing 40 mol % cholesterol is smaller than that for a corresponding pure PC bilayer; the diffusion coefficients increase with increasing the degree of unsaturation of one of the PC chains in bilayers of both types (i.e., in pure bilayers or in bilayers with cholesterol). The presence of cholesterol in a bilayer promoted the extension of saturated and polyunsaturated lipid chains. The condensing effect of cholesterol on the order parameters was more pronounced for the double C=C bonds of polyunsaturated chains than for single C-C bonds of saturated chains.     

Comparative mechanical unfolding studies of spectrin domains R15, R16 and R17

Spectrins belong to repetitive three-helix bundle proteins that have vital functions in multicellular organisms and are of potential value in nanotechnology. To reveal the unique physical features of repeat proteins we have studied the structural and mechanical properties of three repeats of chicken brain α-spectrin (R15, R16 and R17) at the atomic level under stretching at constant velocities (0.01, 0.05 and 0.1?Å·ps^?1) and constant forces (700 and 900?pN) using molecular dynamics (MD) simulations at T?=?300?K. 114 independent MD simulations were performed and their analysis has been done. Despite structural similarity of these domains we have found that R15 is less mechanically stable than R16, which is less stable than R17. This result is in agreement with the thermal unfolding rates. Moreover, we have observed the relationship between mechanical stability, flexibility of the domains and the number of aromatic residues involved in aromatic clusters.     

Size-spectra as indicators of the effects of fishing on coral reef fish assemblages

The data requirements and resources needed to develop multispecies indicators of fishing impacts are often lacking and this is particularly true for coral reef fisheries. Size-spectra, relationships between abundance and body-size class, regardless of taxonomy, can be calculated from simple sizeabundance data. Both the slope and the mid-point height of the relationship can be compared at different fishing intensities. Here, we develop size-spectra for reef fish assemblages using body size- abundance data collected by underwater visual census in each of ten fishing grounds across a known gradient of fishing intensity in the Kadavu Island group, Fiji. Slopes of the size-spectra became steeper (F_9,69=3.20, p<0.01) and the height declined (F_9,69=15.78, p<0.001) with increasing fishing intensity. Regressions of numbers of individuals per size class across grounds were negative for all size classes, although the slope was almost zero for the smallest size class. Response to exploitation of each size class category was greatest for larger fish. Steepening of the slope with increasing fishing intensity largely resulted from reductions in the relative abundance of large fish and not from the ecological release of small fish following depletion of their predators. The slope and height of the size-spectrum appear to be good indicators of fishing effects on reef fish assemblages.     

Chordin knockdown enhances the osteogenic differentiation of human mesenchymal stem cells

Introduction

Bone morphogenetic proteins (BMPs) are critical growth factors in the osteogenic differentiation of progenitor cells during development in embryos and fracture repair in adults. Although recombinant BMPs are in use clinically, their clinical efficiency needs to be improved. The biological activities of BMPs are naturally regulated by extracellular binding proteins. The specific hypotheses tested in this study were as follows: the BMP inhibitor chordin is produced endogenously during the osteogenic differentiation of human mesenchymal stem cells (MSCs); and blockade of the activity of the BMP inhibitor increases the rate of osteogenic differentiation of human MSCs in vitro.

Methods

Human MSCs were derived from bone marrow from an iliac crest aspirate and from patients undergoing hip hemiarthroplasty. The MSCs were induced down the osteogenic pathway using standard osteogenic differentiation media, and expressions of BMP-2 and chordin were determined by gene expression analysis. During osteogenic differentiation, chordin knockdown was induced using RNA interference. Osteogenic differentiation was assessed by measuring the expression of alkaline phosphatase and calcium deposition. The differences in expression of osteogenic makers between groups were compared by analysis of variance, followed by Gabriel post hoc test.

Results

We demonstrate the expression of BMP-2 and chordin in human MSCs during osteogenic differentiation. Knockdown of chordin by RNA interference in vitro resulted in a significant increase in the expression of the osteogenic marker alkaline phosphatase and the deposition of extracellular mineral, in response to osteogenic stimulation.

Conclusion

We conclude that endogenously produced chordin constrains the osteogenic differentiation of human MSCs. The targeting of BMP inhibitors, such as chordin, may provide a novel strategy for enhancing bone regeneration.     

Promoter Hypermethylation in Tumor Suppressing Genes p16 and FHIT and Their Relationship with Estrogen Receptor and Progesterone Receptor Status in Breast Cancer Patients from Northern India

BACKGROUND: Aberrant DNA methylation has been recognized in human breast carcinogenesis as a common molecular alteration associated with the loss of expression of a number of key regulatory genes. The present study was undertaken to determine whether methylation and expression of p16 and FHIT genes would correlate with the estrogen receptor (ER) and progesterone receptor (PR) status. METHODS: Methylation-specific polymerase chain reaction, messenger RNA (mRNA) expression analysis, immunohistochemistry, and Western blot analysis were performed to study the methylation of p16 and FHIT genes in 351 pairs of malignant/normal breast tissues. We examined the expression of ER and PR in those specimens by immunohistochemistry. Mutations of p16 and FHIT genes in tumors were detected by direct sequencing. RESULTS: The frequency of hypermethylation was 31.9% and 36.8% in p16 and FHIT genes, respectively, and showed significant harmony in concordant hypermethylation (P < .0001). In postmenopausal patients, methylation frequency in both genes is significantly higher in poorly and moderately differentiated tumors. Loss of protein expression of p16 and FHIT in 77 and 74 tumors, respectively, is associated with their methylation status in premenopausal women. CONCLUSION: We did not find any significant differences in tumor-related gene methylation patterns relevant to both ER and PR status of breast tumors.     

The dynamic heterogeneity of the phospholipid bilayer and diffusion of molecules at the interface

The method of steered molecular dynamics was developed to evaluate the kinetic parameters of the penetration of molecules through the interface. Heterogeneous microviscosity of a membrane was calculated by the example of a hydrated 1-palmitoyl-2-oleyl-sn-glycero-3-phosphatidylcholine bilayer. The influence of the chemical nature of the penetrant molecule on its penetration through the membrane was studied.     

Molecular simulation of Tyr105 phosphorylated pyruvate kinase M2 to understand its structure and dynamics

Microstructure of dibenzo-18-crown-6 (DB18C6) and DB18C6/Li⁺ complex in different solvents (water, methanol, chloroform, and nitrobenzene) have been analyzed using radial distribution function (RDF), coordination number (CN), and orientation profiles, in order to identify the role of solvents on complexation of DB18C6 with Li⁺, using molecular dynamics (MD) simulations. In contrast to aqueous solution of LiCl, no clear solvation pattern is found around Li⁺ in the presence of DB18C6. The effect of DB18C6 has been visualized in terms of reduction in peak height and shift in peak positions of g_Li-Ow. The appearance of damped oscillations in velocity autocorrelation function (VACF) of complexed Li⁺ described the high frequency motion to a “rattling” of the ion in the cage of DB18C6. The solvent-complex interaction is found to be higher for water and methanol due to hydrogen bond (HB) interactions with DB18C6. However, the stability of DB18C6/Li⁺ complex is found to be almost similar for each solvent due to weak complex-solvent interactions. Further, Li⁺ complex of DB18C6 at the liquid/liquid interface of two immiscible solvents confirm the high interfacial activity of DB18C6 and DB18C6/Li⁺ complex. The complexed Li⁺ shows higher affinity for water than organic solvents; still they remain at the interface rather than migrating toward water due to higher surface tension of water as compared to organic solvents. These simulation results shed light on the role of counter-ions and spatial orientation of species in pure and hybrid solvents in the complexation of DB18C6 with Li⁺. Graphical Abstract

DB18C6/Li⁺ complex in pure solvents (water, methanol, chloroform, and nitrobenzene) and water/nitrobenzene interface     

Effect of cholesterol on the structure and dynamic properties of unsaturated phospholipid bilayers

Molecular dynamics (MD) computer simulations of five different hydrated unsaturated phosphatidylcholine lipid bilayers built up by 18:0/18:1(n-9)cis PC, 18:0/18:2(n-6)cis PC, 18:0/18:3(n-3)cis PC, 18:0/20:4(n-6)cis PC, and 18:0/22:6(n-3)cis PC molecules with 40 mol% cholesterol, and the same five pure phosphatidylcholine bilayers have been performed at 303 K. The simulation box of a lipid bilayer contained 96 phosphatidylcholines, 64 cholesterols, and 3840 water molecules (48 phosphatidylcholine molecules and 32 cholesterols per layer and 24 water molecules per phospholipid or cholesterol in each case). The lateral self-diffusion coefficients of the lipids in these systems and mass density profiles with respect to the bilayer normal have been analyzed. It has been found that the lateral diffusion coefficients of phosphatidylcholine molecules increase with increasing number of double bonds in one of the lipid chains, both in pure bilayers and in bilayers with cholesterol. It has been found as well that the lateral diffusion coefficient of phosphatidylcholine molecules of a lipid bilayer with 40 mol% cholesterol is smaller than that for the corresponding pure phosphatidylcholine bilayer.     

Mitotic chromosome condensation in the sperm nucleus during post fertilization maturation division in urechis eggs

Changes in the morphology of the sperm nucleus in the egg cytoplasm are mong the immediate events in nucleocytoplasmic interactions during early embryogenesis. Soon after its entrance into the egg cytoplasm, the sperm nucleus of various organisms increases in size with the transformation of condensed chromatin to a diffuse state, resembling the chromatin of an interphase nucleus (2, 13, 15, 16). This is followed by a close association or fusion of male and female pronuclei (2, 13, 15, 16). Cytoplasmic influences on nuclear morphology have also been demonstrated clearly in nuclear transplantation and cell fusion studies (10, 11). Reactivation of the nucleus, such as the transplanted brain nucleus in Xenopus egg cytoplasm or the hen erythrocyte nucleus in interphase cytoplasm of HeLa cells, is accompanied by nuclear enlargement and chromatin dispersion (10, 11). However, premature mitotic-like chromosome condensation takes place in the nuclei of sperm or interphase cells fused with mitotic cells (9, 12). Thus, chromosome dispersion and condensation seem to depend on the state of the cytoplasm in which the nucleus is present. These observations imply that the initial morphological changes in the sperm nucleus after fertilization may very well be dependent on the state of maturation of eggs at the time of sperm entry. Unfertilized eggs of Urechis caupo, a marine echiuroid worm, are stored at the diakinesis stage. These eggs complete maturation division after insemination and this is followed by fusion of male and female pronuclei (5, 8). Therefore, Urechis caupo is a suitable organism in which to study the response of the sperm nucleus to the changing state of the egg cytoplasm during and after postfertilization maturation division.