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.     

MyD88-dependent signals are essential for the host immune response in experimental brain abscess

Brain abscesses form in response to a parenchymal infection by pyogenic bacteria, with Staphylococcus aureus representing a common etiologic agent of human disease. Numerous receptors that participate in immune responses to bacteria, including the majority of TLRs, the IL-1R, and the IL-18R, use a common adaptor molecule, MyD88, for transducing activation signals leading to proinflammatory mediator expression and immune effector functions. To delineate the importance of MyD88-dependent signals in brain abscesses, we compared disease pathogenesis using MyD88 knockout (KO) and wild-type (WT) mice. Mortality rates were significantly higher in MyD88 KO mice, which correlated with a significant reduction in the expression of several proinflammatory mediators, including but not limited to IL-1beta, TNF-alpha, and MIP-2/CXCL2. These changes were associated with a significant reduction in neutrophil and macrophage recruitment into brain abscesses of MyD88 KO animals. In addition, microglia, macrophages, and neutrophils isolated from the brain abscesses of MyD88 KO mice produced significantly less TNF-alpha, IL-6, MIP-1alpha/CCL3, and IFN-gamma-induced protein 10/CXCL10 compared with WT cells. The lack of MyD88-dependent signals had a dramatic effect on the extent of tissue injury, with significantly larger brain abscesses typified by exaggerated edema and necrosis in MyD88 KO animals. Interestingly, despite these striking changes in MyD88 KO mice, bacterial burdens did not significantly differ between the two strains at the early time points examined. Collectively, these findings indicate that MyD88 plays an essential role in establishing a protective CNS host response during the early stages of brain abscess development, whereas MyD88-independent pathway(s) are responsible for pathogen containment.     

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.     

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     

TLR2 expression in astrocytes is induced by TNF-alpha- and NF-kappa B-dependent pathways

Astrocytes participate in CNS innate immune responses as evident by their ability to produce a wide array of inflammatory mediators upon exposure to diverse stimuli. Although we have established that astrocytes use TLR2 to signal inflammatory mediator production in response to Staphylococcus aureus, a common etiological agent of CNS infections, the signal transduction pathways triggered by this pathogen and how TLR2 expression is regulated remain undefined. Three disparate inhibitors that block distinct steps in the NF-kappaB pathway, namely SC-514, BAY 11-7082, and caffeic acid phenethyl ester, attenuated NO, TNF-alpha, and CXCL2 release from S. aureus-activated astrocytes. Among these proinflammatory mediators, autocrine/paracrine TNF-alpha was pivotal for augmenting TLR2 expression, since receptor levels were not elevated in astrocytes isolated from TNF-alpha knockout mice upon bacterial exposure. Since TLR2 is critical for signaling astrocytic cytokine production in response to S. aureus, we evaluated the effect of TNF-alpha loss on proinflammatory mediator release. Interestingly, among the molecules assayed, only NO production was significantly attenuated in TNF-alpha knockout astrocytes compared with wild-type cells. Similar results were obtained following LPS treatment, suggesting that TNF-alpha is an important regulator of astrocytic TLR2 expression and NO release in response to diverse microbial stimuli. In addition, NF-kappaB inhibitors attenuated TNF-alpha-induced TLR2 expression in astrocytes. Overall, this study suggests that two important anti-bacterial effector molecules, TLR2 and NO, are regulated, in part, by NF-kappaB-dependent autocrine/paracrine effects of TNF-alpha in astrocytes.     

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.     

Human pyruvate kinase M2: A multifunctional protein

Glycolysis, a central metabolic pathway, harbors evolutionary conserved enzymes that modulate and potentially shift the cellular metabolism on requirement. Pyruvate kinase, which catalyzes the last but rate‐limiting step of glycolysis, is expressed in four isozymic forms, depending on the tissue requirement. M2 isoform (PKM2) is exclusively expressed in embryonic and adult dividing/tumor cells. This tetrameric allosterically regulated isoform is intrinsically designed to downregulate its activity by subunit dissociation (into dimer), which results in partial inhibition of glycolysis at the last step. This accumulates all upstream glycolytic intermediates as an anabolic feed for synthesis of lipids and nucleic acids, whereas reassociation of PKM2 into active tetramer replenishes the normal catabolism as a feedback after cell division. In addition, involvement of this enzyme in a variety of pathways, protein–protein interactions, and nuclear transport suggests its potential to perform multiple nonglycolytic functions with diverse implications, although multidimensional role of this protein is as yet not fully explored. This review aims to provide an overview of the involvement of PKM2 in various physiological pathways with possible functional implications.     

Poly (ADP-ribose) polymerases (PARPs) 1-3 regulate astrocyte activation

Besides their traditional role in maintaining CNS homeostasis, astrocytes also participate in innate immune responses. Indeed, we have previously demonstrated that astrocytes are capable of recognizing bacterial pathogens such as Staphylococcus aureus , a common etiologic agent of CNS infections, and respond with the robust production of numerous proinflammatory mediators. Suppression of Poly (ADP-ribose) polymerase-1 (PARP-1), a DNA repair enzyme, has been shown to attenuate inflammatory responses in several cell types including mixed glial cultures. However, a role for PARP-1 in regulating innate immune responses in purified astrocytes and the potential for multiple PARP family members to cooperatively regulate astrocyte activation has not yet been examined. The synthetic PARP-1 inhibitor PJ-34 attenuated the production of several proinflammatory mediators by astrocytes in response to S. aureus stimulation including nitric oxide, interleukin-1 beta, tumor necrosis factor-alpha, and CCL2. The release of all four mediators was partially reduced in PARP-1 knockout (KO) astrocytes compared to wild-type cells. The residual inflammatory mediator expression detected in PARP-1 KO astrocytes was further blocked with PJ-34, suggesting either non-specific effects of the drug or actions on alternative PARP isoforms. Reduction in PARP-2 or PARP-3 expression by siRNA knock down revealed that these isoforms also contributed to inflammatory mediator regulation in response to S. aureus . Interestingly, the combined targeting of either PARP-1/PARP-2 or PARP-2/PARP-3 attenuated astrocyte inflammatory responses more effectively compared to knock down of either PARP alone, suggesting cooperativity between PARP isoforms. Collectively, these findings suggest that PARPs influence the extent of S. aureus -induced astrocyte activation.