Lumican is a major proteoglycan component of the bone matrix.

MC3T3-E1 mouse calvaria cells are a clonal population of committed osteoprogenitors that in the presence of appropriate supplements form a mineralized bone matrix. The development of the MC3T3-E1 cells can be divided into three major stages, namely, proliferation, differentiation, and mineralization. Recently, using the cDNA microarray technology we found lumican to be abundantly expressed during the mineralization and differentiation stages of the MC3T3-E1 development and not during the proliferation stage. Lumican has been shown to play essential roles in regulating collagen fibril formation in different extracellular matrices but its expression in the developing bone matrix remains elusive. By examining the expression profile of this gene during the different stages of MC3T3-E1 development, utilizing the 'real-time' PCR technology, we observed that the expression of lumican increases as the osteoblast culture differentiates and matures, suggesting that lumican may be involved in regulating collagen fibrillogenesis in bone matrices. Using immunostaining, we observed that during the early embryonic development of mouse (E11 to E13), lumican is mainly expressed in the cartilaginous matrices. However, in the older embryos (E14 to E16), the expression of lumican is more prominent in the developing bone matrices. Our data suggest that lumican is a significant proteoglycan component of bone matrix, which is secreted by differentiating and mature osteoblasts only and therefore it can be used as a marker to distinguish proliferating pre-osteoblasts from the differentiating osteoblasts.     

Kinetics in signal transduction pathways involving promiscuous oligomerizing receptors can be determined by receptor specificity: apoptosis induction by TRAIL

Here we show by computer modeling that kinetics and outcome of signal transduction in case of hetero-oligomerizing receptors of a promiscuous ligand largely depend on the relative amounts of its receptors. Promiscuous ligands can trigger the formation of nonproductive receptor complexes, which slows down the formation of active receptor complexes and thus can block signal transduction. Our model predicts that increasing the receptor specificity of the ligand without changing its binding parameters should result in faster receptor activation and enhanced signaling. We experimentally validated this hypothesis using the cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its four membrane-bound receptors as an example. Bypassing ligand-induced receptor hetero-oligomerization by receptor-selective TRAIL variants enhanced the kinetics of receptor activation and augmented apoptosis. Our results suggest that control of signaling pathways by promiscuous ligands could result in apparent slow biological kinetics and blocking signal transmission. By modulating the relative amount of the different receptors for the ligand, signaling processes like apoptosis can be accelerated or decelerated and even inhibited. It also implies that more effective treatments using protein therapeutics could be achieved simply by altering specificity.     

Depression,Sex and Gender Roles in Older Adult Populations: The International Mobility in Aging Study (IMIAS)

Objectives

To assess the associations between gender roles and depression in older men and women and whether gender roles are independent risk factors for depression.

Methods

International cross-sectional study of adults between 65 and 74 years old (n = 1,967). Depression was defined by a score of 16 or over in the Center for Epidemiologic Studies Depression Scale (CES-D). A validated 12-item Bem Sex Role Inventory (BSRI) was used to classify participants in gender roles (Masculine, Feminine, Androgynous, and Undifferentiated) using research site medians of femininity and masculinity as cut-off points. Poisson regressions were fitted to estimate the prevalence ratios (PR) of depression for each gender role compared to the masculine role, adjusting for sex, sufficiency of income, education, marital status, self-rated health, and chronic conditions.

Results

Among men, 31.2% were androgynous, 26% were masculine, 14.4% were feminine, and 28.4% were undifferentiated; among women, the corresponding percentages were 32.7%, 14.9%, 27%, and 25.4%. Both in men and in women, depressive symptoms (CES-D≥16) were more prevalent in those endorsing the undifferentiated type, compared to masculine, feminine or androgynous groups. However, after adjusting for potential confounders, compared to the masculine group only those endorsing the androgynous role were 28% less likely to suffer from depression: PR of 0.72 (95% CI: 0.55–0.93). In fully adjusted models, prevalence rates of depression were not different from masculine participants in the two other gender groups of feminine and undifferentiated.

Conclusions

Androgynous roles were associated with lower rates of depression in older adults, independently of being a man or a woman.     

Genotyping by apyrase-mediated allele-specific extension

This report describes a single-step extension approach suitable for high-throughput single-nucleotide polymorphism typing applications. The method relies on extension of paired allele-specific primers and we demonstrate that the reaction kinetics were slower for mismatched configurations compared with matched configurations. In our approach we employ apyrase, a nucleotide degrading enzyme, to allow accurate discrimination between matched and mismatched primer-template configurations. This apyrase-mediated allele-specific extension (AMASE) protocol allows incorporation of nucleotides when the reaction kinetics are fast (matched 3′-end primer) but degrades the nucleotides before extension when the reaction kinetics are slow (mismatched 3′-end primer). Thus, AMASE circumvents the major limitation of previous allele-specific extension assays in which slow reaction kinetics will still give rise to extension products from mismatched 3′-end primers, hindering proper discrimination. It thus represents a significant improvement of the allele-extension method. AMASE was evaluated by a bioluminometric assay in which successful incorporation of unmodified nucleotides is monitored in real-time using an enzymatic cascade.     

Synergistic anticancer activity of valproate combined with nicotinamide enhances anti-proliferation response and apoptosis in MIAPaca2 cells

Histone deacetylase is strongly associated with epigenetic regulation and carcinogenesis, and its inhibitors can induce cell cycle arrest and apoptosis of the cancer cells. In this study we aimed to examine the antiproliferative effects a combination of the valproate with nicotinamide in MIAPaca2 cell line. We revealed that valproate acted in a synergistic/additive with nicotinamide to inhibit the proliferation and induction of apoptosis in MIAPaca2 cancer cell line. MIAPaca2 was treated with various concentrations of valproate. The MTT assay and colony formation in soft agar indicated that valproate at 0.5 mM, when used alone weakly, suppressed proliferation of cells (37 ± 3.02 %) whereas the combination treatment of valproate + nicotinamide significantly suppressed cell proliferation (58 ± 3.5 %). The effect of nicotinamide at 25 mM on cell proliferation and cell colonization induced 50 % apoptosis of MIAPaca2 cells. To identify the anti-proliferation and apoptotic effects of valproate and nicotinamide we performed flow cytometric and microscopic analyses. The results indicated significant apoptosis induction and nuclear morphological alterations greater than when valproate was used alone. Furthermore, western blot analyses was performed to study the role of acetyl-histone H3 levels, and quantitative RNA expression analyses were performed on expression of thrombospondin (TSP) and maspin genes in MIAPaca2. We found that the combination treatment of valproate + nicotinamide enhanced the expression of maspin and TSP genes and the biological response of the cell line was correlated with the increase of histone H3 acetylation after nicotinamide and valproate application. Together our findings indicate that valproate which act as inhibitor of cell proliferation and inducer of apoptosis in human cancer MIAPaca2 cells when used in combination with nicotinamide makes it a potentially good candidate for new anticancer drug development.     

Characterization of desnutrin functional domains: critical residues for triacylglycerol hydrolysis in cultured cells

Murine desnutrin/human ATGL is a triacylglycerol (TAG) hydrolase with a predicted catalytic dyad within an α-β hydrolase fold in the N-terminal region. In humans, mutations resulting in C-terminal truncation cause neutral lipid storage disease with myopathy. To identify critical functional domains, we measured TAG breakdown in cultured cells by mutated or truncated desnutrin. In vitro, C-terminally truncated desnutrin displayed an even higher apparent V_max than the full-length form without changes in K_m, which may be explained by our finding of an interaction between the C- and N-terminal domains. In live cells, however, C-terminally truncated adenoviral desnutrin had lower TAG hydrolase activity. We investigated a role for the phosphorylation of C-terminal S406 and S430 residues but found that these were not necessary for TAG breakdown or lipid droplet localization in cells. The predicted N-terminal active sites, S47 and D166, were both critical for TAG hydrolysis in live cells and in vitro. We also identified two overlapping N-terminal motifs that predict lipid substrate binding domains, a glycine-rich motif (underlined) and an amphipathic α-helix (bold) within amino acid residues 10–24 (ISFAGCGFLGVYHIG). G14, F17, L18, and V20, but not G16 and G19, were important for TAG hydrolysis, suggesting a potential role for the amphipathic α-helix in TAG binding. This study identifies for the first time critical sites in the N-terminal region of desnutrin and reveals the requirement of the C-terminal region for TAG hydrolysis in cultured cells.     

Mediators of endoplasmic reticulum stress-induced apoptosis

The efficient functioning of the endoplasmic reticulum (ER) is essential for most cellular activities and survival. Conditions that interfere with ER function lead to the accumulation and aggregation of unfolded proteins. ER transmembrane receptors detect the onset of ER stress and initiate the unfolded protein response (UPR) to restore normal ER function. If the stress is prolonged, or the adaptive response fails, apoptotic cell death ensues. Many studies have focused on how this failure initiates apoptosis, as ER stress-induced apoptosis is implicated in the pathophysiology of several neurodegenerative and cardiovascular diseases. In this review, we examine the role of the molecules that are activated during the UPR in order to identify the molecular switch from the adaptive phase to apoptosis. We discuss how the activation of these molecules leads to the commitment of death and the mechanisms that are responsible for the final demise of the cell.     

Design of Localized Surface Plasmon Resonance (LSPR) Biosensor for Immunodiagnostic of E. coli O157:H7 Using Gold Nanoparticles Conjugated to the Chicken Antibody

E. coli O157:H7 is one of the most important pathogens in food-borne diseases and is the main cause of the pseudo pandemic development of hemorrhagic colitis and hemolytic uremic syndrome. Also E. coli O157:H7 is the most common serotype of Shiga-toxin-producing E. coli. Traditional methods for detecting E. coli O157:H7 are expensive, time-consuming, and less sensitive. A method with high sensitivity and high-resolution optical detection is utilizes the LSPR property of spherical gold nanoparticles (GNP). In this work, we constructed a novel nano-bio probe to detect E. coli O157:H7 by synthesizing citrate gold nanoparticle conjugated (non-covalent bond) with specific chicken anti-E. coli O157:H7 antibody (IgY) by changing the pH of the nanoparticles’ environment. UV-visible and DLS methods were used to confirm the bonding between the antibody and nanoparticles and the LSPR sensitivity of the nano-bio probe was evaluated by ELISA method. We could optically detect this bacterium in less than 2 h by measuring the LSPR band λ max shifts of GNPs. The sensitivity of this novel biosensor was determined by about 10 CFU/ml, using the LSPR property of spherical gold nanoparticles. So that, the LSPR λ max red shifted from 530 to 543 nm in presence of 10 CFU bacterium. In conclusion, this nano biosensor can be used to detect this important pathogen among the clinical specimens.