Apoptotic effects of over-expressed estrogen receptor-beta on LoVo colon cancer cell is mediated by p53 signalings in a ligand-dependent manner

Epidemiologic studies reported that the prevalence of hereditary non-polyposis colon cancer (HNPCC) in male is about 1.5-fold higher than that in female. Decreases in circulatory estradiol (E2) have been reported to downregulate the expression of E2 receptor (ER) and significantly increase the risk of colorectal cancer. Patients that received E2 replacement therapy were found to have a reduction in the incidence of colon adenoma and carcinoma. Furthermore, significant decreases in the expression of ER have been found in colorectal cancer specimens. These data strongly suggest the protective roles of E2 and ER against colorectal cancer. However, the mechanisms remain unexplored. LoVo cells were transient transfected to overexpress ER-beta, DNA fragmentation and caspase activity assay were performed to evaluate apoptotic effects. Western blotting was used to evaluate protein levels, and luciferase activity assay to measure the TNF-alpha promoter activity. Our data clearly demonstrated that E2 and ER-beta alone could upregulate p21 and p27 proteins, which further activate caspase-8 and caspase-9 to induce apoptosis in LoVo cell, and the ER-beta. effects were enhanced by E2. However, overexpressed ER-beta did not influence the expression and promoter activity of TNF-alpha. In addition, E2 and overexpressed ER-beta downregulated the beta-catenin proteins which cause the downregulation of its target genes, cyclin D1 and Rb, to inhibit the cell cycle and cell proliferation. The results indicate that overexpressed ER-beta may induce LoVo cell apoptosis and anti-proliferation by increasing p53 signaling in a ligand-dependent manner, and without hTNF-alpha involvement. Efforts aiming at enhancing ER-beta expression and/or activity may prove to be an attractive alternative therapy against colorectal cancer.     

Bioactivity assay of extracts from Calocedrus macrolepis var. formosana bark

Alcoholic extracts from bark of Calocedrus macrolepis var. formosana Florin (Cupressaceae) were extracted successively using n-hexane, dichloromethane, ethyl acetate, 1-butanol and water, which gave 34.8%, 34.1%, 24.1%, 3.3% and 3.7% soluble fractions, respectively. Antioxidation activity of these fractions by DPPH assay and dissimilar IC50 values of the DPPH showed that ethyl acetate fraction had the best antioxidant activity; its IC50 was 2.6 microg/ml. Analyses of the composition and anti-inflammatory activity of the subfractions from n-C6H14 fraction showed that the T3 and H5ppt had the best anti-inflammatory activity in LPS-stimulated murine macrophage J774A. 1 cells, respectively; moreover, their major constituent was sugiol (T3 37.1%, H5ppt 81.1%), which at dosages of 10 microg/ml inhibited proIL-1beta protein production completely. Furthermore, the T1 also exhibited anti-inflammatory activity, and its major constituent was ferruginol (above 85.6%).     

N-3 polyunsaturated fatty acids decrease levels of doxorubicin-induced reactive oxygen species in cardiomyocytes -- involvement of uncoupling protein UCP2

Background

Use of the chemotherapeutic drug doxorubicin (DOX) is associated with serious cardiotoxicity, as it increases levels of reactive oxygen species (ROS). N-3 polyunsaturated fatty acid dietary supplements can be of benefit to patients undergoing cancer therapy. The aims of this study were to determine whether DOX-induced cardiotoxicity is related to mitochondrial uncoupling proteins and whether eicosapentaenoic acid (EPA, C20:5 n-3) or docosahexaenoic acid (DHA, C22:6 n-3) affects DOX-induced cardiomyocyte toxicity.

Results

Treatment of H9C2 cells with DOX resulted in decreased cell viability and UCP2 expression. Treatment with 100 μM EPA or 50 μM DHA for 24 h resulted in a maximal mitochondria concentration of these fatty acids and increased UCP2 expression. Pretreatment with 100 μM EPA or 50 μM DHA prevented the DOX-induced decrease in UCP2 mRNA and protein levels, but these effects were not seen with EPA or DHA and DOX cotreatment. In addition, the DOX-induced increase in ROS production and subsequent mitochondrial membrane potential change (∆ψ) were significantly attenuated by pretreatment with EPA or DHA.

Conclusion

EPA or DHA pre-treatment inhibits the DOX-induced decrease in UCP2 expression, increase in ROS production, and subsequent mitochondrial membrane potential change that contribute to the cardiotoxicity of DOX.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0101-3) contains supplementary material, which is available to authorized users.     

An unlabeled antibody macromolecule technique using hemocyanin for the identification of type B and type C retrovirus envelope and cell surface antigens by correlative fluorescence, transmission electron, and scanning electron microscopy.

The present resolution (75-100 A) of the conventional scanning electron microscope (SEM) and its ability to image the surfaces of large numbers of whole cells in situ permit the approach of problems such as viral and cell surface antigen localization by immunological labeling with visual markers. Identification of virus and cell surface antigens in situ has been accomplished in indirect reactions by unconjugated markers. Hemocyanin (Hcy) from whelk, Busycon canniculatum, has been developed as an immunospecific marker for virion and cell surface labeling in the electron microscope. Its size (30 x 50 nm) and distinct cylindrical shape permit easy visualization in the SEM and the transmission electron microscope (TEM). The Hcy method involves the preparation of antisera to Hcy in appropriate hosts for use in an unlabeled antibody macromolecule procedure based exclusively on antigen-antibody affinity to couple the macromolecule to the antigen site. Further correlative data from fluorescence microscopy can be obtained from similarly labeled samples by binding fluorescein to the bridging antibodies used in the Hcy technique. The usefulness of the Hcy marker system was demonstrated by employing highly specific antisera to the major envelope and cell surface glycoprotein (gp70) of Rauscher murine leukemia virus (R-MuLV), a type C retrovirus. The antiserum was shown to bind to the virion and cell surfaces of virus-infected cells in the homologous virus-infected cell system. It also demonstrated the expression of R-MuLV gp70-related antigens on a murine cell line Mm5mt/c1 which produces mouse mammary tumor virus (MuMTV), a type B retrovirus. Furthermore, when used in the Hcy marker system the anti-gp70 serum was able to distinguish type B from type C budding virus on the same cell. Methods for the preparation of immunoreagents and labeling of cells are discussed.     

Mechanobiological dysregulation of the epidermis and dermis in skin disorders and in degeneration

During growth and development, the skin expands to cover the growing skeleton and soft tissues by constantly responding to the intrinsic forces of underlying skeletal growth as well as to the extrinsic mechanical forces from body movements and external supports. Mechanical forces can be perceived by two types of skin receptors: (1) cellular mechanoreceptors/mechanosensors, such as the cytoskeleton, cell adhesion molecules and mechanosensitive (MS) ion channels, and (2) sensory nerve fibres that produce the somatic sensation of mechanical force. Skin disorders in which there is an abnormality of collagen [e.g. Ehlers–Danlos syndrome (EDS)] or elastic (e.g. cutis laxa) fibres or a malfunction of cutaneous nerve fibres (e.g. neurofibroma, leprosy and diabetes mellitus) are also characterized to some extent by deficiencies in mechanobiological processes. Recent studies have shown that mechanotransduction is crucial for skin development, especially hemidesmosome maturation, which implies that the pathogenesis of skin disorders such as bullous pemphigoid is related to skin mechanobiology. Similarly, autoimmune diseases, including scleroderma and mixed connective tissue disease, and pathological scarring in the form of keloids and hypertrophic scars would seem to be clearly associated with the mechanobiological dysfunction of the skin. Finally, skin ageing can also be considered as a degenerative process associated with mechanobiological dysfunction. Clinically, a therapeutic strategy involving mechanoreceptors or MS nociceptor inhibition or acceleration together with a reduction or augmentation in the relevant mechanical forces is likely to be successful. The development of novel approaches such as these will allow the treatment of a broad range of cutaneous diseases.     

You stay,but I Hop: Host shifting near and far co‐dominated the evolution of Enchenopa treehoppers

The importance and prevalence of phylogenetic tracking between hosts and dependent organisms caused by co‐evolution and shifting between closely related host species have been debated for decades. Most studies of phylogenetic tracking among phytophagous insects and their host plants have been limited to insects feeding on a narrow range of host species. However, narrow host ranges can confound phylogenetic tracking (phylogenetic tracking hypothesis) with host shifting between hosts of intermediate relationship (intermediate hypothesis). Here, we investigated the evolutionary history of the Enchenopa binotata complex of treehoppers. Each species in this complex has high host fidelity, but the entire complex uses hosts across eight plant orders. The phylogenies of E. binotata were reconstructed to evaluate whether (1) tracking host phylogeny; or (2) shifting between intermediately related host plants better explains the evolutionary history of E. binotata. Our results suggest that E. binotata primarily shifted between both distant and intermediate host plants regardless of host phylogeny and less frequently tracked the phylogeny of their hosts. These findings indicate that phytophagous insects with high host fidelity, such as E. binotata, are capable of adaptation not only to closely related host plants but also to novel hosts, likely with diverse phenology and defense mechanisms.     

Uncovering the regeneration strategies of zebrafish organs: a comprehensive systems biology study on heart,cerebellum, fin,and retina regeneration

Background

Regeneration is an important biological process for the restoration of organ mass, structure, and function after damage, and involves complex bio-physiological mechanisms including cell differentiation and immune responses. We constructed four regenerative protein-protein interaction (PPI) networks using dynamic models and AIC (Akaike’s Information Criterion), based on time-course microarray data from the regeneration of four zebrafish organs: heart, cerebellum, fin, and retina. We extracted core and organ-specific proteins, and proposed a recalled-blastema-like formation model to uncover regeneration strategies in zebrafish.

Results

It was observed that the core proteins were involved in TGF-β signaling for each step in the recalled-blastema-like formation model and TGF-β signaling may be vital for regeneration. Integrins, FGF, and PDGF accelerate hemostasis during heart injury, while Bdnf shields retinal neurons from secondary damage and augments survival during the injury response. Wnt signaling mediates the growth and differentiation of cerebellum and fin neural stem cells, potentially providing a signal to trigger differentiation.

Conclusion

Through our analysis of all four zebrafish regenerative PPI networks, we provide insights that uncover the underlying strategies of zebrafish organ regeneration.

     

Isolation of single-site Escherichia coli mutants deficient in thiamine and 4-thiouridine syntheses: identification of a nuvC mutant

A method is described to rapidly select and classify many independent near-UV irradiation-resistant Escherichia coli mutants, which include tRNA modification and RNA synthesis control mutants. One class of these mutants was found to be simultaneously deficient in thiamine biosynthesis and in the ability to modify uridine in tRNA to 4-thiouridine, known to be the target for near-UV irradiation. These mutants were found to be unable to make thiazole, a thiamine precursor. The addition of thiazole restores the thiamine deficiency but does not render the cells near-UV irradiation sensitive. In vitro studies on one of these mutants indicated a deficiency in protein factor C (nuvC), required for the 4-thiouridine modification of tRNA. In P1 transduction, the thiazole marker cotransduced with the histidine marker, which places the thiazole marker between 42 and 46 min on the E. coli chromosome map. Both thiamine production and 4-thiouridine production were resumed by 87% of the spontaneous reversions, suggesting a single-point mutation. Our results indicate that we have isolated nuvC mutants and that the nuvC polypeptide is involved in two functions, tRNA modification and thiazole biosynthesis.     

Synthesis and characterization of a new bioactivated paramagnetic gadolinium(III) complex [Gd(DOTA-FPG)(H2O)] for tracing gene expression

A smart contrast agent for magnetic resonance imaging (MRI) can be used to exploit an enzymatic activity specific to the tissue or disease state signified by converting an MRI-inactivated agent to an activated MRI agent. In this study, a beta-galactopyranose-containing gadolinium(III) complex [Gd(DOTA-FPG)(H 2O)] was designed, synthesized, and characterized as being potentially suitable for a bioactivated MRI contrast agent. The (17)O NMR experiments were conducted to estimate the water exchange rate k e x 298 and rotational correlation time tau R 298 . The k ex 298 value of [Gd(DOTA-FPG)(H 2O)] is similar to that of [Gd(DO3A-bz-NO 2)(H 2O)]. The rotational correlation time value of [Gd(DOTA-FPG)(H 2O)] is dramatically longer than that of [Gd(DOTA)(H 2O)] (-) Relaxometric studies show that the percentage change in the T 1 value of [Gd(DOTA-FPG)(H 2O)] decreases dramatically in the presence of beta-galactosidase and human serum albumin. The T(1) change percentage of [Gd(DOTA-FPG)(H 2O)] (60%) is significantly higher than those of Egad and gadolinium(III)-1-(4-(2-(1-(4,7,10-triscarboxymethyl-(1,4,7,10-tetraazacyclododecyl)))-ethylcarbamoyloxymethyl)-2-nitrophenyl)-beta- d-glucopyronuronate. The signal intensity of the MR image for [Gd(DOTA-FPG)(H 2O)] in the presence of human serum albumin and beta-galactosidase (2670 +/- 210) is significantly higher than that of [Gd(DOTA-FPG)(H 2O)] in the sodium phosphate buffer solution (1490 +/- 160). In addition, the MR images show a higher-intensity enhancement in CT26/beta-gal tumor with beta-galactosidase gene expression but not for the CT26 tumor without beta-galactosidase gene expression. We conclude that [Gd(DOTA-FPG)(H 2O)] is a suitable candidate for a bioactivated MRI contrast agent in tracing gene expression.