Sample records for factor type beta

  1. Beta-type transforming growth factor specifies organizational behavior in vascular smooth muscle cell cultures

    PubMed Central

    1987-01-01

    In culture, vascular smooth muscle cells (SMC) grow in a "hill-and- valley" (multilayered) pattern of organization. We have studied the growth, behavioral organization, and biosynthetic phenotype of rat aortic SMC exposed to purified platelet-derived growth regulatory molecules. We show that multilayered growth is not a constitutive feature of cultured SMC, and that beta-type transforming growth factor (TGF-beta) is the primary determinant of multilayered growth and the hill-and-valley pattern of organization diagnostic for SMC in culture. TGF-beta inhibited, in a dose-dependent manner, the serum- or platelet- derived growth factor-mediated proliferation of these cells in two- dimensional culture, but only when cells were plated at subconfluent densities. The ability of TGF-beta to inhibit SMC growth was inversely correlated to plating cell density. When SMC were plated at monolayer density (5 X 10(4) cells/cm2) to allow maximal cell-to-cell contact, TGF-beta potentiated cell growth. This differential response of SMC to TGF-beta may contribute to the hill-and-valley pattern of organization. Unlike its effect on other cell types, TGF-beta did not enhance the synthesis of fibronectin or its incorporation into the extracellular matrix. However, the synthesis of a number of other secreted proteins was altered by TGF-beta treatment. SMC treated with TGF-beta for 4 or 8 h secreted markedly enhanced amounts of an Mr 38,000-D protein doublet whose synthesis is known to be increased by heparin (another inhibitor of SMC growth), suggesting metabolic similarities between heparin- and TGF-beta-mediated SMC growth inhibition. The data suggest that TGF-beta may play an important and complex regulatory role in SMC proliferation and organization during development and after vascular injury. PMID:3475277

  2. A Synopsis of Factors Regulating Beta Cell Development and Beta Cell Mass

    PubMed Central

    Prasadan, Krishna; Shiota, Chiyo; Xiangwei, Xiao; Ricks, David; Fusco, Joseph; Gittes, George

    2016-01-01

    The insulin-secreting beta cells in the endocrine pancreas regulate blood glucose levels, and loss of functional beta cells leads to insulin deficiency, hyperglycemia (high blood glucose) and diabetes mellitus. Current treatment strategies for type-1 (autoimmune) diabetes are islet transplantation, which has significant risks and limitations, or normalization of blood glucose with insulin injections, which is clearly not ideal. The type-1 patients can lack insulin counter-regulatory mechanism; therefore, hypoglycemia is a potential risk. Hence, a cell-based therapy offers a better alternative for the treatment of diabetes. Past research was focused on attempting to generate replacement beta cells from stem cells, however, recently there has been an increasing interest in identifying mechanisms that will lead to the conversion of pre-existing differentiated endocrine cells into beta cells. The goal of this review is to provide an overview of several of the key factors that regulate new beta cell formation (neogenesis) and beta cell proliferation. PMID:27105622

  3. NFI-Ski interactions mediate transforming growth factor beta modulation of human papillomavirus type 16 early gene expression.

    PubMed

    Baldwin, Amy; Pirisi, Lucia; Creek, Kim E

    2004-04-01

    Human papillomaviruses (HPVs) are present in virtually all cervical cancers. An important step in the development of malignant disease, including cervical cancer, involves a loss of sensitivity to transforming growth factor beta (TGF-beta). HPV type 16 (HPV16) early gene expression, including that of the E6 and E7 oncoprotein genes, is under the control of the upstream regulatory region (URR), and E6 and E7 expression in HPV16-immortalized human epithelial cells is inhibited at the transcriptional level by TGF-beta. While the URR contains a myriad of transcription factor binding sites, including seven binding sites for nuclear factor I (NFI), the specific sequences within the URR or the transcription factors responsible for TGF-beta modulation of the URR remain unknown. To identify potential transcription factors and binding sites involved in TGF-beta modulation of the URR, we performed DNase I footprint analysis on the HPV16 URR using nuclear extracts from TGF-beta-sensitive HPV16-immortalized human keratinocytes (HKc/HPV16) treated with and without TGF-beta. Differentially protected regions were found to be located around NFI binding sites. Electrophoretic mobility shift assays, using the NFI binding sites as probes, showed decreased binding upon TGF-beta treatment. This decrease in binding was not due to reduced NFI protein or NFI mRNA levels. Mutational analysis of individual and multiple NFI binding sites in the URR defined their role in TGF-beta sensitivity of the promoter. Overexpression of the NFI family members in HKc/HPV16 decreased the ability of TGF-beta to inhibit the URR. Since the oncoprotein Ski has been shown to interact with and increase the transcriptional activity of NFI and since cellular Ski levels are decreased by TGF-beta treatment, we explored the possibility that Ski may provide a link between TGF-beta signaling and NFI activity. Anti-NFI antibodies coimmunoprecipitated endogenous Ski in nuclear extracts from HKc/HPV16, confirming that NFI

  4. c-Ski overexpression promotes tumor growth and angiogenesis through inhibition of transforming growth factor-beta signaling in diffuse-type gastric carcinoma.

    PubMed

    Kiyono, Kunihiko; Suzuki, Hiroshi I; Morishita, Yasuyuki; Komuro, Akiyoshi; Iwata, Caname; Yashiro, Masakazu; Hirakawa, Kosei; Kano, Mitsunobu R; Miyazono, Kohei

    2009-10-01

    c-Ski, originally identified as a proto-oncogene product, is an important negative regulator of transforming growth factor (TGF)-beta family signaling through interaction with Smad2, Smad3, and Smad4. High expression of c-Ski has been found in some cancers, including gastric cancer. We previously showed that disruption of TGF-beta signaling by dominant-negative TGF-beta type II receptor in a diffuse-type gastric carcinoma model accelerated tumor growth through induction of tumor angiogenesis by decreased expression of the anti-angiogenic factor thrombospondin (TSP)-1. Here, we examined the function of c-Ski in human diffuse-type gastric carcinoma OCUM-2MLN cells. Overexpression of c-Ski inhibited TGF-beta signaling in OCUM-2MLN cells. Interestingly, c-Ski overexpression resulted in extensive acceleration of the growth of subcutaneous xenografts in BALB/c nu/nu female mice (6 weeks of age). Similar to tumors expressing dominant-negative TGF-beta type II receptor, histochemical studies revealed less fibrosis and increased angiogenesis in xenografted tumors expressing c-Ski compared to control tumors. Induction of TSP-1 mRNA by TGF-beta was attenuated by c-Ski in vitro, and expression of TSP-1 mRNA was decreased in tumors expressing c-Ski in vivo. These findings suggest that c-Ski overexpression promotes the growth of diffuse-type gastric carcinoma through induction of angiogenesis.

  5. Interferon beta 2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells.

    PubMed Central

    Gauldie, J; Richards, C; Harnish, D; Lansdorp, P; Baumann, H

    1987-01-01

    One of the oldest and most preserved of the homeostatic responses of the body to injury is the acute phase protein response associated with inflammation. The liver responds to hormone-like mediators by the increased synthesis of a series of plasma proteins called acute phase reactants. In these studies, we examined the relationship of hepatocyte-stimulating factor derived from peripheral blood monocytes to interferon beta 2 (IFN-beta 2), which has been cloned. Antibodies raised against fibroblast-derived IFN-beta having neutralizing activity against both IFN-beta 1 and -beta 2 inhibited the major hepatocyte-stimulating activity derived from monocytes. Fibroblast-derived mediator elicited the identical stimulated response in human HepG2 cells and primary rat hepatocytes as the monocyte cytokine. Finally, recombinant-derived human B-cell stimulatory factor type 2 (IFN-beta 2) from Escherichia coli induced the synthesis of all major acute phase proteins studied in human hepatoma HepG2 and primary rat hepatocyte cultures. These data demonstrate that monocyte-derived hepatocyte-stimulating factor and IFN-beta 2 share immunological and functional identity and that IFN-beta 2, also known as B-cell stimulatory factor and hybridoma plasmacytoma growth factor, has the hepatocyte as a major physiologic target and thereby is essential in controlling the hepatic acute phase response. Images PMID:2444978

  6. Sequence swapping does not result in conformation swapping for the beta4/beta5 and beta8/beta9 beta-hairpin turns in human acidic fibroblast growth factor.

    PubMed

    Kim, Jaewon; Lee, Jihun; Brych, Stephen R; Logan, Timothy M; Blaber, Michael

    2005-02-01

    The beta-turn is the most common type of nonrepetitive structure in globular proteins, comprising ~25% of all residues; however, a detailed understanding of effects of specific residues upon beta-turn stability and conformation is lacking. Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil superfold and contains a total of five beta-hairpin structures (antiparallel beta-sheets connected by a reverse turn). beta-Turns related by the characteristic threefold structural symmetry of this superfold exhibit different primary structures, and in some cases, different secondary structures. As such, they represent a useful system with which to study the role that turn sequences play in determining structure, stability, and folding of the protein. Two turns related by the threefold structural symmetry, the beta4/beta5 and beta8/beta9 turns, were subjected to both sequence-swapping and poly-glycine substitution mutations, and the effects upon stability, folding, and structure were investigated. In the wild-type protein these turns are of identical length, but exhibit different conformations. These conformations were observed to be retained during sequence-swapping and glycine substitution mutagenesis. The results indicate that the beta-turn structure at these positions is not determined by the turn sequence. Structural analysis suggests that residues flanking the turn are a primary structural determinant of the conformation within the turn.

  7. Transforming growth factor-beta controls T helper type 1 cell development through regulation of natural killer cell interferon-gamma.

    PubMed

    Laouar, Yasmina; Sutterwala, Fayyaz S; Gorelik, Leonid; Flavell, Richard A

    2005-06-01

    Interferon-gamma and interleukin 12 produced by the innate arm of the immune system are important regulators of T helper type 1 (T(H)1) cell development, but signals that negatively regulate their expression remain controversial. Here we show that transforming growth factor-beta (TGF-beta) controlled T(H)1 differentiation through the regulation of interferon-gamma produced by natural killer (NK) cells. Blockade of TGF-beta signaling in NK cells caused the accumulation of a large pool of NK cells secreting copious interferon-gamma, responsible for T(H)1 differentiation and protection from leishmania infection. In contrast, blockade of TGF-beta signaling in dendritic cells did not affect dendritic cell homeostasis or interleukin 12 production, thus indicating a previously undescribed demarcation of the function of TGF-beta in NK cells versus dendritic cells.

  8. Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type beta transforming growth factor.

    PubMed

    Xu, W; Angelis, K; Danielpour, D; Haddad, M M; Bischof, O; Campisi, J; Stavnezer, E; Medrano, E E

    2000-05-23

    The c-ski protooncogene encodes a transcription factor that binds DNA only in association with other proteins. To identify co-binding proteins, we performed a yeast two-hybrid screen. The results of the screen and subsequent co-immunoprecipitation studies identified Smad2 and Smad3, two transcriptional activators that mediate the type beta transforming growth factor (TGF-beta) response, as Ski-interacting proteins. In Ski-transformed cells, all of the Ski protein was found in Smad3-containing complexes that accumulated in the nucleus in the absence of added TGF-beta. DNA binding assays showed that Ski, Smad2, Smad3, and Smad4 form a complex with the Smad/Ski binding element GTCTAGAC (SBE). Ski repressed TGF-beta-induced expression of 3TP-Lux, the natural plasminogen activator inhibitor 1 promoter and of reporter genes driven by the SBE and the related CAGA element. In addition, Ski repressed a TGF-beta-inducible promoter containing AP-1 (TRE) elements activated by a combination of Smads, Fos, and/or Jun proteins. Ski also repressed synergistic activation of promoters by combinations of Smad proteins but failed to repress in the absence of Smad4. Thus, Ski acts in opposition to TGF-beta-induced transcriptional activation by functioning as a Smad-dependent co-repressor. The biological relevance of this transcriptional repression was established by showing that overexpression of Ski abolished TGF-beta-mediated growth inhibition in a prostate-derived epithelial cell line.

  9. C/EBP beta regulation of the tumor necrosis factor alpha gene.

    PubMed Central

    Pope, R M; Leutz, A; Ness, S A

    1994-01-01

    Activated macrophages contribute to chronic inflammation by the secretion of cytokines and proteinases. Tumor necrosis factor alpha (TNF alpha) is particularly important in this process because of its ability to regulate other inflammatory mediators in an autocrine and paracrine fashion. The mechanism(s) responsible for the cell type-specific regulation of TNF alpha is not known. We present data to show that the expression of TNF alpha is regulated by the transcription factor C/EBP beta (NF-IL6). C/EBP beta activated the TNF alpha gene promoter in cotransfection assays and bound to it at a site which failed to bind the closely related protein C/EBP alpha. Finally, a dominant-negative version of C/EBP beta blocked TNF alpha promoter activation in myeloid cells. Our results implicate C/EBP beta as an important regulator of TNF alpha by myelomonocytic cells. Images PMID:7929820

  10. Cloning the promoter for transforming growth factor-beta type III receptor. Basal and conditional expression in fetal rat osteoblasts

    NASA Technical Reports Server (NTRS)

    Ji, C.; Chen, Y.; McCarthy, T. L.; Centrella, M.

    1999-01-01

    Transforming growth factor-beta binds to three high affinity cell surface molecules that directly or indirectly regulate its biological effects. The type III receptor (TRIII) is a proteoglycan that lacks significant intracellular signaling or enzymatic motifs but may facilitate transforming growth factor-beta binding to other receptors, stabilize multimeric receptor complexes, or segregate growth factor from activating receptors. Because various agents or events that regulate osteoblast function rapidly modulate TRIII expression, we cloned the 5' region of the rat TRIII gene to assess possible control elements. DNA fragments from this region directed high reporter gene expression in osteoblasts. Sequencing showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding sequences within the 3' region of the promoter co-mapped with multiple transcription initiation sites, DNase I footprints, gel mobility shift analysis, or loss of activity by deletion or mutation. An upstream enhancer was evident 5' proximal to nucleotide -979, and a silencer region occurred between nucleotides -2014 and -2194. Glucocorticoid sensitivity mapped between nucleotides -687 and -253, whereas bone morphogenetic protein 2 sensitivity co-mapped within the silencer region. Thus, the TRIII promoter contains cooperative basal elements and dispersed growth factor- and hormone-sensitive regulatory regions that can control TRIII expression by osteoblasts.

  11. Lewis type 1 antigen synthase (beta3Gal-T5) is transcriptionally regulated by homeoproteins.

    PubMed

    Isshiki, Soichiro; Kudo, Takashi; Nishihara, Shoko; Ikehara, Yuzuru; Togayachi, Akira; Furuya, Akiko; Shitara, Kenya; Kubota, Tetsuro; Watanabe, Masahiko; Kitajima, Masaki; Narimatsu, Hisashi

    2003-09-19

    The type 1 carbohydrate chain, Galbeta1-3GlcNAc, is synthesized by UDP-galactose:beta-N-acetylglucosamine beta1,3-galactosyltransferase (beta3Gal-T). Among six beta3Gal-Ts cloned to date, beta3Gal-T5 is an essential enzyme for the synthesis of type 1 chain in epithelium of digestive tracts or pancreatic tissue. It forms the type 1 structure on glycoproteins produced from such tissues. In the present study, we found that the transcriptional regulation of the beta3Gal-T5 gene is controlled by homeoproteins, i.e. members of caudal-related homeobox protein (Cdx) and hepatocyte nuclear factor (HNF) families. We found an important region (-151 to -121 from the transcription initiation site), named the beta3Gal-T5 control element (GCE), for the promoter activity. GCE contained the consensus sequences for members of the Cdx and HNF families. Mutations introduced into this sequence abolished the transcriptional activity. Four factors, Cdx1, Cdx2, HNF1alpha, and HNF1beta, could bind to GCE and transcriptionally activate the beta3Gal-T5 gene. Transcriptional regulation of the beta3Gal-T5 gene was consistent with that of members of the Cdx and HNF1 families in two in vivo systems. 1) During in vitro differentiation of Caco-2 cells, transcriptional up-regulation of beta3Gal-T5 was observed in correlation with the increase in transcripts for Cdx2 and HNF1alpha. 2) Both transcript and protein levels of beta3Gal-T5 were determined to be significantly reduced in colon cancer. This down-regulation was correlated with the decrease of Cdx1 and HNF1beta expression in cancer tissue. This is the first finding that a glycosyltransferase gene is transcriptionally regulated under the control of homeoproteins in a tissue-specific manner. beta3Gal-T5, controlled by the intestinal homeoproteins, may play an important role in the specific function of intestinal cells by modifying the carbohydrate structure of glycoproteins.

  12. Integrin-mediated transforming growth factor-beta activation regulates homeostasis of the pulmonary epithelial-mesenchymal trophic unit.

    PubMed

    Araya, Jun; Cambier, Stephanie; Morris, Alanna; Finkbeiner, Walter; Nishimura, Stephen L

    2006-08-01

    Trophic interactions between pulmonary epithelial and mesenchymal cell types, known as the epithelial-mesenchymal trophic unit (EMTU), are crucial in lung development and lung disease. Transforming growth factor (TGF)-beta is a key factor in mediating these interactions, but it is expressed in a latent form that requires activation to be functional. Using intact fetal tracheal tissue and primary cultures of fetal tracheal epithelial cells and fibroblasts, we demonstrate that a subset of integrins, alpha(v)beta(6) and alpha(v)beta(8), are responsible for almost all of the TGF-beta activation in the EMTU. Both alpha(v)beta(8) and alpha(v)beta(6) contribute to fetal tracheal epithelial activation of TGF-beta, whereas only alpha(v)beta(8) contributes to fetal tracheal fibroblast activation of TGF-beta. Interestingly, fetal tracheal epithelial alpha(v)beta(8)-mediated TGF-beta activation can be enhanced by phorbol esters, likely because of the increased activity of MT1-MMP, an essential co-factor in alpha(v)beta(8)-mediated activation of TGF-beta. Autocrine alpha(v)beta(8)-mediated TGF-beta activation by fetal tracheal fibroblasts results in suppression of both transcription and secretion of hepatocyte growth factor, which is sufficient to affect phosphorylation of the airway epithelial hepatocyte growth factor receptor, c-Met, as well as airway epithelial proliferation in a co-culture model of the EMTU. These findings elucidate the function and complex regulation of integrin-mediated activation of TGF-beta within the EMTU.

  13. [Factors causing damage and destruction of beta-cells of the islets of Langerhans in the pancreas].

    PubMed

    Anděl, Michal; Němcová, Vlasta; Pavlíková, Nela; Urbanová, Jana; Cecháková, Marie; Havlová, Andrea; Straková, Radka; Večeřová, Livia; Mandys, Václav; Kovář, Jan; Heneberg, Petr; Trnka, Jan; Polák, Jan

    2014-09-01

    Insulin secretion in patients with manifested diabetes mellitus tends to disappear months to decades after the diagnosis, which is a clear sign of a gradual loss of pancreatic islet beta-cells. In our sample of 30 type 2 diabetic patients, whose disease manifested between 30 and 45 years of age, about a half have retained or even increased insulin secretion 30 years later, while the other half exhibit a much diminished or lost insulin secretion. Factors that can damage or destroy beta-cells can be divided into the following groups: Metabolic factors: hyperglycemia and glucotoxicity, lipotoxicity, hypoxia, reactive oxygen species; Pharmacological factors: antimicrobial medication pentamidine, SSRI antidepressants; Factors related to impaired insulin secretion: MODY type diabetes; Environmental toxic factors: rat poison Vacor, streptozotocin, polychlorinated and polybrominated hydrocarbons; Disorders of the exocrine pancreas: tumor infiltration, fibrous infiltration, chronic pancreatitis, cystic fibrosis; Infections, inflammation, autoimmunity, viral factors: Coxsackie viruses, H1N1 influenza, enteroviruses. We are currently working on finding other factors leading to beta-cell damage, studying their effect on apoptosis and necrosis and looking for possible protective factors to prevent this damage. We our increasing knowledge about the mechanisms of beta-cell damage and destruction we come ever closer to suggest measures for their prevention. In this review we offer a brief and simplified summary of some of the findings related to this area.Key words: pancreatic islet beta-cells of Langerhans - factors damaging or destroying beta-cells - insulin secretion.

  14. Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-{beta} responsiveness

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mori, Yasuji; Hinchcliff, Monique; Wu, Minghua

    2008-03-10

    Background: The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-{beta} (TGF-{beta}) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-{beta}, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-{beta}. To explore this notion, we characterized TGF-{beta}-induced activation of fibroblasts from CCN2-null (CCN2{sup -/-}) mouse embryos. Methods: The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-{beta} signal transduction and regulation of collagen genemore » expression were examined in CCN2{sup -/-} MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays. Results: Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2{sup -/-} MEFs was markedly reduced compared to wild type MEFs, TGF-{beta}-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2{sup -/-} MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2. Conclusion: Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-{beta}-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.« less

  15. Low dose radiation interactions with the transformation growth factor (TFG)-beta pathway

    NASA Astrophysics Data System (ADS)

    Maslowski, Amy Jesse

    A major limiting factor for long-term, deep-space missions is the radiation dose to astronauts. Because the dose to the astronauts is a mixed field of low- and high-LET radiation, there is a need to understand the effects of both radiation types on whole tissue; however, there are limited published data on the effects of high-LET (linear-energy-transfer) radiation on tissue. Thus, we designed a perfusion chamber system for rat trachea in order to mimic in vivo respiratory tissue. We successfully maintained the perfused tracheal tissue ex vivo in a healthy and viable condition for up to three days. In addition, this project studied the effects of high-LET Fe particles on the overall transformation growth factor (TGF)-beta response after TGF-beta inactivation and compared the results to the TGF-beta response post x-ray irradiation. It was found that a TGF-beta response could be measured in the perfused tracheal tissue, for x-ray and Fe particle irradiations, despite the high autofluorescent background intrinsic to tissue. However, after comparing the TGF-beta response of x-ray irradiation to High-Z-High-energy (HZE) irradiation, there was not a significant difference in radiation types. The TGF-beta response in x-ray and HZE irradiated perfusion chambers was also measured over time post irradiation. It was found that for 6 hour and 8 hour post irradiation, the TGF-beta response was higher for lower doses of radiation than for higher doses. This is in contrast to the 0 hour fixation which found the TGF-beta response to increase with increased dose. The inverse relationship found for 6 hour and 8 hour fixation times may indicate a threshold response for TGF-beta response; i.e., for low doses, a threshold of dose must be reached for an immediate TGF-beta response, otherwise the tissue responds more slowly to the irradiation damage. This result was unexpected and will require further investigation to determine if the threshold can be determined for the 250 kVp x-rays and

  16. Opposite Smad and chicken ovalbumin upstream promoter transcription factor inputs in the regulation of the collagen VII gene promoter by transforming growth factor-beta.

    PubMed

    Calonge, María Julia; Seoane, Joan; Massagué, Joan

    2004-05-28

    A critical component of the epidermal basement membrane, collagen type VII, is produced by keratinocytes and fibroblasts, and its production is stimulated by the cytokine transforming growth factor-beta (TGF-beta). The gene, COL7A1, is activated by TGF-beta via Smad transcription factors in cooperation with AP1. Here we report a previously unsuspected level of complexity in this regulatory process. We provide evidence that TGF-beta may activate the COL7A1 promoter by two distinct inputs operating through a common region of the promoter. One input is provided by TGF-beta-induced Smad complexes via two Smad binding elements that function redundantly depending on the cell type. The second input is provided by relieving the COL7A1 promoter from chicken ovalbumin upstream promoter transcription factor (COUP-TF)-mediated transcriptional repression. We identified COUP-TFI and -TFII as factors that bind to the TGF-beta-responsive region of the COL7A1 promoter in an expression library screening. COUP-TFs bind to a site between the two Smad binding elements independently of Smad or AP1 and repress the basal and TGF-beta-stimulated activities of this promoter. We provide evidence that endogenous COUP-TF activity represses the COL7A1 promoter. Furthermore, we show that TGF-beta addition causes a rapid and profound down-regulation of COUP-TF expression in keratinocytes and fibroblasts. The results suggest that TGF-beta signaling may exert tight control over COL7A1 by offsetting the balance between opposing Smad and COUP-TFs.

  17. Cellular and molecular mechanisms of chronic rhinosinusitis and potential therapeutic strategies: review on cytokines, nuclear factor kappa B and transforming growth factor beta.

    PubMed

    Phan, N T; Cabot, P J; Wallwork, B D; Cervin, A U; Panizza, B J

    2015-07-01

    Chronic rhinosinusitis is characterised by persistent inflammation of the sinonasal mucosa. Multiple pathophysiological mechanisms are likely to exist. Previous research has focused predominantly on T-helper type cytokines to highlight the inflammatory mechanisms. However, proteins such as nuclear factor kappa B and transforming growth factor beta are increasingly recognised to have important roles in sinonasal inflammation and tissue remodelling. This review article explores the roles of T-helper type cytokines, nuclear factor kappa B and transforming growth factor beta in the pathophysiological mechanisms of chronic rhinosinusitis. An understanding of these mechanisms will allow for better identification and classification of chronic rhinosinusitis endotypes, and, ultimately, improved therapeutic strategies.

  18. Redox-mediated activation of latent transforming growth factor-beta 1

    NASA Technical Reports Server (NTRS)

    Barcellos-Hoff, M. H.; Dix, T. A.; Chatterjee, A. (Principal Investigator)

    1996-01-01

    Transforming growth factor beta 1 (TGF beta) is a multifunctional cytokine that orchestrates response to injury via ubiquitous cell surface receptors. The biological activity of TGF beta is restrained by its secretion as a latent complex (LTGF beta) such that activation determines the extent of TGF beta activity during physiological and pathological events. TGF beta action has been implicated in a variety of reactive oxygen-mediated tissue processes, particularly inflammation, and in pathologies such as reperfusion injury, rheumatoid arthritis, and atherosclerosis. It was recently shown to be rapidly activated after in vivo radiation exposure, which also generates reactive oxygen species (ROS). In the present studies, the potential for redox-mediated LTGF beta activation was investigated using a cell-free system in which ROS were generated in solution by ionizing radiation or metal ion-catalyzed ascorbate reaction. Irradiation (100 Gray) of recombinant human LTGF beta in solution induced 26% activation compared with that elicited by standard thermal activation. Metal-catalyzed ascorbate oxidation elicited extremely efficient recombinant LTGF beta activation that matched or exceeded thermal activation. The efficiency of ascorbate activation depended on ascorbate concentrations and the presence of transition metal ions. We postulate that oxidation of specific amino acids in the latency-conferring peptide leads to a conformation change in the latent complex that allows release of TGF beta. Oxidative activation offers a novel route for the involvement of TGF beta in tissue processes in which ROS are implicated and endows LTGF beta with the ability to act as a sensor of oxidative stress and, by releasing TGF beta, to function as a signal for orchestrating the response of multiple cell types. LTGF beta redox sensitivity is presumably directed toward recovery of homeostasis; however, oxidation may also be a mechanism of LTGF beta activation that can be deleterious during

  19. 17 beta-estradiol and tamoxifen upregulate estrogen receptor beta expression and control podocyte signaling pathways in a model of type 2 diabetes.

    PubMed

    Catanuto, Paola; Doublier, Sophie; Lupia, Enrico; Fornoni, Alessia; Berho, Mariana; Karl, Michael; Striker, Gary E; Xia, Xiaomei; Elliot, Sharon

    2009-06-01

    Diabetic nephropathy remains one of the most important causes of end-stage renal disease. This is particularly true for women from racial/ethnic minorities. Although administration of 17beta-estradiol to diabetic animals has been shown to reduce extracellular matrix deposition in glomeruli and mesangial cells, effects on podocytes are lacking. Given that podocyte injury has been implicated as a factor leading to the progression of proteinuria and diabetic nephropathy, we treated db/db mice, a model of type 2 diabetic glomerulosclerosis, with 17beta-estradiol or tamoxifen to determine whether these treatments reduce podocyte injury and decrease glomerulosclerosis. We found that albumin excretion, glomerular volume, and extracellular matrix accumulation were decreased in these mice compared to placebo treatment. Podocytes isolated from all treatment groups were immortalized and these cell lines were found to express the podocyte markers WT-1, nephrin, and the TRPC6 cation channel. Tamoxifen and 17beta-estradiol treatment decreased podocyte transforming growth factor-beta mRNA expression but increased that of the estrogen receptor subtype beta protein. 17beta-estradiol, but not tamoxifen, treatment decreased extracellular-regulated kinase phosphorylation. These data, combined with improved albumin excretion, reduced glomerular size, and decreased matrix accumulation, suggest that both 17beta-estradiol and tamoxifen may protect podocytes against injury and therefore ameliorate diabetic nephropathy.

  20. 17{beta}-Hydroxysteroid dehydrogenase type 13 is a liver-specific lipid droplet-associated protein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Horiguchi, Yuka; Araki, Makoto; Motojima, Kiyoto

    2008-05-30

    17{beta}-Hydroxysteroid dehydrogenase (17{beta}HSD) type 13 is identified as a new lipid droplet-associated protein. 17{beta}HSD type 13 has an N-terminal sequence similar to that of 17{beta}HSD type 11, and both sequences function as an endoplasmic reticulum and lipid droplet-targeting signal. Localization of native 17{beta}HSD type 13 on the lipid droplets was confirmed by subcellular fractionation and Western blotting. In contrast to 17{beta}HSD type 11, however, expression of 17{beta}HSD type 13 is largely restricted to the liver and is not enhanced by peroxisome proliferator-activated receptor {alpha} and its ligand. Instead the expression level of 17{beta}HSD type 13 in the receptor-null mice wasmore » increased several-fold. 17{beta}HSD type 13 may have a distinct physiological role as a lipid droplet-associated protein in the liver.« less

  1. A study on the relationship between genetic and environmental factors of type 2 diabetes mellitus in humans.

    PubMed

    Chen, Yu; Zhou, Ling; Xu, Yaochu; Shen, Hongbing; Niu, Juying

    2002-05-01

    To study the relationship between the inheritable factor and environmental factors of type 2 diabetes mellitus in humans. A case-control study based on 154 type 2 diabetes mellitus and 130 healthy controls was carried out in Jiangsu Province in 1997. The age, family history of diabetes mellitus, hypertension history, high waist/hip ratio (WHR), high systolic blood pressure, huge fetus history, and the genotype of beta(3)-adrenergic receptor (beta(3)-AR) were the risk factors of type 2 diabetes mellitus; while occupational physical activity was protective factor of type 2 diabetes mellitus. The risk for diabetes mellitus distinctly increased while genetic factor and obesity, beta(3)-AR mutation were coexisting. Type 2 diabetes mellitus is caused by the effect of both genetic and environmental factors.

  2. Effect of botulinum toxin type A on transforming growth factor beta1 in fibroblasts derived from hypertrophic scar: a preliminary report.

    PubMed

    Xiao, Zhibo; Zhang, Fengmin; Lin, Weibin; Zhang, Miaobo; Liu, Ying

    2010-08-01

    Hypertrophic scar is a common dermal disease. Numerous treatments are currently available but they do not always yield excellent therapeutic results. Hence, alternatives are needed. Recent basic and clinical research has shown that botulinum toxin type A (BTXA) has antihypertrophic scar properties but the molecular mechanism for this action is unknown. The aim of this study was to explore the effect of BTXA on transforming growth factor beta1 (TGF-beta1) in fibroblasts derived from hypertrophic scar and further elucidate its actual mechanism. Fibroblasts were isolated from tissue specimens of hypertrophic scar. Fibroblasts were treated with BTXA and the difference in proliferation between treated and nontreated cells was analyzed through the MTT method from the first to the fifth day after treatment. Proteins of TGF-beta1 were checked using ELISA in fibroblasts with BTXA and without BTXA from the first to the fifth day. The growth of the fibroblast treated with BTXA was obviously slower than that of the fibroblast without BTXA treatment (p < 0.01), which showed that BTXA effectively inhibited the growth of fibroblasts. Proteins of TGF-beta1 between fibroblasts with BTXA and fibroblasts without BTXA are statistically significant (p < 0.01). These results suggest that BTXA effectively inhibited the growth of fibroblasts derived from hypertrophic scar and in turn caused a decrease in TGF-beta1 protein, indicating that BTXA-based therapies for hypertrophic scar are promising and worth investigating further.

  3. Cell type-specific regulation of beta2-adrenoceptor mRNA by agonists.

    PubMed

    Danner, S; Lohse, M J

    1997-07-16

    Prolonged agonist stimulation of beta2-adrenoceptors results in receptor down-regulation which is often paralleled by a reduction of the corresponding mRNA. In this study, we investigated the agonist-dependent regulation of beta2-adrenoceptor mRNA in DDT1-MF2 smooth muscle cells and C6 glioma cells. In DDT1-MF2 cells the half-life of the mRNA was 12 h in monolayer compared to 2 h in suspension cultures. Under both conditions, the agonist isoproterenol reduced this half-life by a factor of 2. In contrast, in C6 glioma cells isoproterenol had no effect on the mRNA stability, even though it reduced mRNA levels by approximately 50%. Isoproterenol-induced downregulation of beta2-adrenoceptor mRNA was completely blocked in C6 cells by the presence of a protein synthesis inhibitor, while this was not so in DDT1-MF2-cells. These data show that beta2-adrenoceptor downregulation occurs via cell-type specific mechanisms.

  4. Neutrinoless double beta decay in type I+II seesaw models

    NASA Astrophysics Data System (ADS)

    Borah, Debasish; Dasgupta, Arnab

    2015-11-01

    We study neutrinoless double beta decay in left-right symmetric extension of the standard model with type I and type II seesaw origin of neutrino masses. Due to the enhanced gauge symmetry as well as extended scalar sector, there are several new physics sources of neutrinoless double beta decay in this model. Ignoring the left-right gauge boson mixing and heavy-light neutrino mixing, we first compute the contributions to neutrinoless double beta decay for type I and type II dominant seesaw separately and compare with the standard light neutrino contributions. We then repeat the exercise by considering the presence of both type I and type II seesaw, having non-negligible contributions to light neutrino masses and show the difference in results from individual seesaw cases. Assuming the new gauge bosons and scalars to be around a TeV, we constrain different parameters of the model including both heavy and light neutrino masses from the requirement of keeping the new physics contribution to neutrinoless double beta decay amplitude below the upper limit set by the GERDA experiment and also satisfying bounds from lepton flavor violation, cosmology and colliders.

  5. Beta amyloid and hyperphosphorylated tau deposits in the pancreas in type 2 diabetes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Miklossy, J.; Miller, L.; Qing, H.

    2008-08-25

    Strong epidemiologic evidence suggests an association between Alzheimer disease (AD) and type 2 diabetes. To determine if amyloid beta (A{beta}) and hyperphosphorylated tau occurs in type 2 diabetes, pancreas tissues from 21 autopsy cases (10 type 2 diabetes and 11 controls) were analyzed. APP and tau mRNAs were identified in human pancreas and in cultured insulinoma beta cells (INS-1) by RT-PCR. Prominent APP and tau bands were detected by Western blotting in pancreatic extracts. Aggregated A{beta}, hyperphosphorylated tau, ubiquitin, apolipoprotein E, apolipoprotein(a), IB1/JIP-1 and JNK1 were detected in Langerhans islets in type 2 diabetic patients. A{beta} was co-localized with amylinmore » in islet amyloid deposits. In situ beta sheet formation of islet amyloid deposits was shown by infrared microspectroscopy (SIRMS). LPS increased APP in non-neuronal cells as well. We conclude that A{beta} deposits and hyperphosphorylated tau are also associated with type 2 diabetes, highlighting common pathogenetic features in neurodegenerative disorders, including AD and type 2 diabetes and suggesting that A{beta} deposits and hyperphosphorylated tau may also occur in other organs than the brain.« less

  6. Monocyte production of transforming growth factor beta in long-term hemodialysis: modulation by hemodialysis membranes.

    PubMed

    Mege, J L; Capo, C; Purgus, R; Olmer, M

    1996-09-01

    Cytokines are likely involved in hemodialysis-associated complications such as immunodeficiency and beta 2 microglobulin amyloidosis. Because transforming growth factors beta (TGF beta) exert immunosuppressive effects on lymphocytes, down-modulate monocyte functions, and promote fibrosis, we hypothesize that they participate in the deleterious effects of hemodialysis. We investigated the production of TGF beta 1 and TGF beta 2 by monocytes from controls and patients dialyzed with high-flux cellulose triacetate (CT) and polyacrylonitrile (PAN) membranes. The detection of both TGF beta s required an acidification step, suggesting that they are secreted as latent complexes. The spontaneous production of TGF beta 1 and TGF beta 2 was significantly higher in patients dialyzed with CT or PAN than in controls, but the oversecretion of TGF beta 1 was more sustained in CT-treated patients than in PAN-dialyzed patients. The production of interleukin-6 (IL-6) was increased in both patient groups as compared with controls. In contrast to TGF beta 1, the increase was greater in PAN-treated patients than in CT-treated patients, and the release of tumor necrosis factor alpha (TNF alpha) was increased only in PAN-treated patients. Taken together, our results show that hemodialysis is associated with the oversecretion of monocyte cytokines. Moreover, the type of dialysis membrane specifically affects the balance between the secretion of suppressive cytokines such as TGF beta and that of inflammatory cytokines such as IL-6 and TNF alpha.

  7. Expression of transforming growth factor-beta1, -beta2 and -beta3 in normal and diseased canine mitral valves.

    PubMed

    Aupperle, H; März, I; Thielebein, J; Schoon, H-A

    2008-01-01

    The pathogenesis of chronic valvular disease (CVD) in dogs remains unclear, but activation and proliferation of valvular stromal cells (VSC) and their transdifferentiation into myofibroblast-like cells has been described. These alterations may be influenced by transforming growth factor-beta (TGF-beta), a cytokine involved in extracellular matrix (ECM) regulation and mesenchymal cell differentiation. The present study investigates immunohistochemically the expression of TGF-beta1, -beta2, -beta3 and smooth muscle alpha actin (alpha-SMA) in normal canine mitral valves (MVs) (n=10) and in the valves of dogs with mild (n=7), moderate (n=14) and severe (n=9) CVD. In normal mitral valves there was no expression of alpha-SMA but VSC displayed variable expression of TGF-beta1 (10% of VSC labelled), TGF-beta2 (1-5% labelled) and TGF-beta3 (50% labelled). In mild CVD the affected atrialis contain activated and proliferating alpha-SMA-positive VSC, which strongly expressed TGF-beta1 and -beta3, but only 10% of these cells expressed TGF-beta2. In unaffected areas of the leaflet there was selective increase in expression of TGF-beta1 and -beta3. In advanced CVD the activated subendothelial VSC strongly expressed alpha-SMA, TGF-beta1 and -beta3. Inactive VSC within the centre of the nodules had much less labelling for TGF-beta1 and -beta3. TGF-beta1 labelling was strong within the ECM. These data suggest that TGF-beta plays a role in the pathogenesis of CVD by inducing myofibroblast-like differentiation of VSC and ECM secretion. Changed haemodynamic forces and expression of matrix metalloproteinases (MMPs) may in turn regulate TGF-beta expression.

  8. A new compound heterozygous frameshift mutation in the type II 3{beta}-hydroxysteroid dehydrogenase 3{beta}-HSD gene causes salt-wasting 3{beta}-HSD deficiency congenital adrenal hyperplasia

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, L.; Sakkal-Alkaddour, S.; Chang, Ying T.

    1996-01-01

    We report a new compound heterozygous frameshift mutation in the type II 3{Beta}-hydroxysteroid dehydrogenase (3{beta}-HSD) gene in a Pakistanian female child with the salt-wasting form of 3{Beta}-HSD deficiency congenital adrenal hyperplasia. The etiology for her congenital adrenal hyperplasia was not defined. Although the family history suggested possible 3{beta}-HSd deficiency disorder, suppressed adrenal function caused by excess glucocorticoid therapy in this child at 7 yr of age did not allow hormonal diagnosis. To confirm 3{beta}-HSD deficiency, we sequenced the type II 3{beta}-HSD gene in the patient, her family, and the parents of her deceased paternal cousins. The type II 3{beta}-HSD genemore » region of a putative promotor, exons I, II, III, and IV, and exon-intron boundaries were amplified by PCR and sequenced in all subjects. The DNA sequence of the child revealed a single nucleotide deletion at codon 318 [ACA(Thr){r_arrow}AA] in exon IV in one allele, and two nucleotide deletions at codon 273 [AAA(Lys){r_arrow}A] in exon IV in the other allele. The remaining gene sequences were normal. The codon 318 mutation was found in one allele from the father, brother, and parents of the deceased paternal cousins. The codon 273 mutation was found in one allele of the mother and a sister. These findings confirmed inherited 3{beta}-HSD deficiency in the child caused by the compound heterozygous type II 3{beta}-HSD gene mutation. Both codons at codons 279 and 367, respectively, are predicted to result in an altered and truncated type II 3{beta}-HSD protein, thereby causing salt-wasting 3{beta}-HSD deficiency in the patient. 21 refs., 2 figs., 1 tab.« less

  9. N-terminal tyrosine phosphorylation of caveolin-2 negates anti-proliferative effect of transforming growth factor beta in endothelial cells

    PubMed Central

    Abel, Britain; Willoughby, Cara; Jang, Sungchan; Cooper, Laura; Xie, Leike; Vo-Ransdell, Chi; Sowa, Grzegorz

    2012-01-01

    Here we show that tyrosine phosphorylation of caveolin-2 (Cav-2) negatively regulates the anti-proliferative function of transforming growth factor beta (TGF-beta) in endothelial cells. In contrast to wild-type-Cav-2, retroviral re-expression of Y19/27F-Cav-2 in Cav-2 knockout endothelial cells did not affect anti-proliferative effect of TGF-beta compared to empty vector. Conversely, although less effective than wild-type, re-expression of S23/36A-Cav-2 reduced the effect of TGF-beta compared to empty vector. This differential effect of tyrosine and serine phosphorylation mutants of Cav-2 correlated with TGF-beta-induced Smad3 phosphorylation and transcriptional activation of plasminogen activator inhibitor-1. Thus tyrosine-phosphorylated Cav-2 counteracts anti-proliferative effect of TGF-beta in endothelial cells. PMID:22819829

  10. A theoretical case study of type I and type II beta-turns.

    PubMed

    Czinki, Eszter; Császár, Attila G; Perczel, András

    2003-03-03

    NMR chemical shielding anisotropy tensors have been computed by employing a medium size basis set and the GIAO-DFT(B3LYP) formalism of electronic structure theory for all of the atoms of type I and type II beta-turn models. The models contain all possible combinations of the amino acid residues Gly, Ala, Val, and Ser, with all possible side-chain orientations where applicable in a dipeptide. The several hundred structures investigated contain either constrained or optimized phi, psi, and chi dihedral angles. A statistical analysis of the resulting large database was performed and multidimensional (2D and 3D) chemical-shift/chemical-shift plots were generated. The (1)H(alpha-13)C(alpha), (13)C(alpha-1)H(alpha-13)C(beta), and (13)C(alpha-1)H(alpha-13)C' 2D and 3D plots have the notable feature that the conformers clearly cluster in distinct regions. This allows straightforward identification of the backbone and side-chain conformations of the residues forming beta-turns. Chemical shift calculations on larger For-(L-Ala)(n)-NH(2) (n=4, 6, 8) models, containing a single type I or type II beta-turn, prove that the simple models employed are adequate. A limited number of chemical shift calculations performed at the highly correlated CCSD(T) level prove the adequacy of the computational method chosen. For all nuclei, statistically averaged theoretical and experimental shifts taken from the BioMagnetic Resonance Bank (BMRB) exhibit good correlation. These results confirm and extend our previous findings that chemical shift information from selected multiple-pulse NMR experiments could be employed directly to extract folding information for polypeptides and proteins.

  11. Epidermal growth factor increases cortisol production and type II 3 beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase expression in human adrenocortical carcinoma cells: evidence for a Stat5-dependent mechanism.

    PubMed

    Feltus, F Alex; Kovacs, William J; Nicholson, Wendell; Silva, Corrine M; Nagdas, Subir K; Ducharme, Nicole A; Melner, Michael H

    2003-05-01

    We tested the ability of epidermal growth factor (EGF) to regulate a key enzyme in the adrenal synthesis of glucocorticoids: human type II 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3 beta HSD). EGF treatment (25 ng/ml) of human adrenocortical carcinoma cells (H295R) resulted in a 5-fold increase in cortisol production and a corresponding 2-fold increase in 3 beta HSD mRNA. Experiments were performed to determine whether EGF is acting through a previously identified signal transducer and activator of transcription 5 (Stat5)-responsive element located from -110 to -118 in the human type II 3 beta HSD promoter. A Stat5 expression construct was cotransfected with a 3 beta HSD-chloramphenol acetyltransferase (CAT) reporter construct comprised of nucleotides -301-->+45 of the human type II 3 beta HSD promoter linked to the CAT reporter gene sequence. The addition of EGF at doses as low as 10 ng/ml resulted in an 11- to 15-fold increase in CAT activity. The introduction of 3-bp point mutations into critical nucleotides in the Stat5 response element obviated the EGF response. Either Stat5a or Stat5b isoforms induced CAT reporter expression upon treatment with EGF. These results demonstrate the ability of EGF to regulate the expression of a critical enzyme (3 beta HSD) in the production of cortisol and suggest a molecular mechanism by which this regulation occurs.

  12. Unraveling the contribution of pancreatic beta-cell suicide in autoimmune type 1 diabetes✩

    PubMed Central

    Jaberi-Douraki, Majid; Schnell, Santiago; Pietropaolo, Massimo; Khadra, Anmar

    2014-01-01

    In type 1 diabetes, an autoimmune disease mediated by autoreactive T-cells that attack insulin-secreting pancreatic beta-cells, it has been suggested that disease progression may additionally require protective mechanisms in the target tissue to impede such auto-destructive mechanisms. We hypothesize that the autoimmune attack against beta-cells causes endoplasmic reticulum stress by forcing the remaining beta-cells to synthesize and secrete defective insulin. To rescue beta-cell from the endoplasmic reticulum stress, beta-cells activate the unfolded protein response to restore protein homeostasis and normal insulin synthesis. Here we investigate the compensatory role of unfolded protein response by developing a multi-state model of type 1 diabetes that takes into account beta-cell destruction caused by pathogenic autoreactive T-cells and apoptosis triggered by endoplasmic reticulum stress. We discuss the mechanism of unfolded protein response activation and how it counters beta-cell extinction caused by an autoimmune attack and/or irreversible damage by endoplasmic reticulum stress. Our results reveal important insights about the balance between beta-cell destruction by autoimmune attack (beta-cell homicide) and beta-cell apoptosis by endoplasmic reticulum stress (beta-cell suicide). It also provides an explanation as to why the unfolded protein response may not be a successful therapeutic target to treat type 1 diabetes. PMID:24831415

  13. [Effects of Valeriana officinalis var. latifolia on expression of transforming growth factor beta 1 in hypercholesterolemic rats].

    PubMed

    Si, Xiao-yun; Jia, Ru-han; Huang, Cong-xin; Ding, Guo-hua; Liu, Hong-yan

    2003-09-01

    To evaluate the effect of Valeriana officinalis var latifolia(VOL) on expression of transforming growth factor beta 1 (TGF-beta 1) in hypercholesterolemic rats and study its possible mechanisms. Dietary-induced hypercholesterolemia was induced in male Wistar rats by given 4% cholesterol and 1% cholic acid diet for 16 weeks. Changes of serum lipid, urinary albumin, renal function and Mesangial matrix index were assessed. Moreover, immunohistochemical stain for TGF-beta 1 and type IV collagen were performed. VOL could reduce the serum levels of total cholesterol, low density lipoprotein, urinary albumin and serum creatinine. Light microscopy and immunohistochemical stain revealed that in the same time of lowing serum lipid, Mesangial matrix index was significantly reduced, accompanied by decreased expression of TGF-beta 1 and type IV collagen. VOL has the protective effect on lipid-induced nephropathy, and the inhibition of TGF-beta 1 expression might be the mechanism of VOL on renal protection.

  14. Progressive loss of sensitivity to growth control by retinoic acid and transforming growth factor-beta at late stages of human papillomavirus type 16-initiated transformation of human keratinocytes.

    PubMed

    Creek, K E; Geslani, G; Batova, A; Pirisi, L

    1995-01-01

    Retinoids (vitamin A and its natural and synthetic derivatives) have shown potential as chemopreventive agents, and diets poor in vitamin A and/or its precursor beta-carotene have been linked to an increased risk of cancer at several sites including the cervix. Human papillomavirus (HPV) plays an important role in the etiology of cervical cancer. We have developed an in vitro model of cancer progression using human keratinocytes (HKc) immortalized by HPV16 DNA (HKc/HPV16). Although immortal, early passage HKc/HPV16, like normal HKc, require epidermal growth factor (EGF) and bovine pituitary extract (BPE) for proliferation and undergo terminal differentiation in response to serum and calcium. However, following prolonged culture, growth factor independent HKc/HPV16 lines that no longer require EGF and BPE can be selected (HKc/GFI). Further selection of HKc/GFI produces lines that are resistant to serum- and calcium- induced terminal differentiation (HKc/DR). HKc/DR, but not early passage HKc/HPV16, are susceptible to malignant conversion following transfection with viral Harvey ras or Herpes simplex virus type II DNA. We have investigated the sensitivity of low to high passage HKc/HPV16 and HKc/GFI to growth control by all-trans-retinoic acid (RA, an active metabolite of vitamin A). Early passage HKc/HPV16 are very sensitive to growth inhibition by RA, and in these cells RA decreases the expression of the HPV16 oncogenes E6 and E7. However, as the cells progress in culture they lose their sensitivity to RA. Growth inhibition by RA may be mediated through the cytokine transforming growth factor-beta (TGF-beta), a potent inhibitor of epithelial cell proliferation. RA treatment of HKc/HPV16 and HKc/GFI results in a dose-and time-dependent induction (maximal of 3-fold) in secreted levels of TGF-beta. Also, Northern blot analysis of mRNA isolated from HKc/HPV16 demonstrated that RA treatment induced TGF-beta 1 and TGF-beta 2 expression about 3- and 50-fold, respectively

  15. Prediction of beta-turns and beta-turn types by a novel bidirectional Elman-type recurrent neural network with multiple output layers (MOLEBRNN).

    PubMed

    Kirschner, Andreas; Frishman, Dmitrij

    2008-10-01

    Prediction of beta-turns from amino acid sequences has long been recognized as an important problem in structural bioinformatics due to their frequent occurrence as well as their structural and functional significance. Because various structural features of proteins are intercorrelated, secondary structure information has been often employed as an additional input for machine learning algorithms while predicting beta-turns. Here we present a novel bidirectional Elman-type recurrent neural network with multiple output layers (MOLEBRNN) capable of predicting multiple mutually dependent structural motifs and demonstrate its efficiency in recognizing three aspects of protein structure: beta-turns, beta-turn types, and secondary structure. The advantage of our method compared to other predictors is that it does not require any external input except for sequence profiles because interdependencies between different structural features are taken into account implicitly during the learning process. In a sevenfold cross-validation experiment on a standard test dataset our method exhibits the total prediction accuracy of 77.9% and the Mathew's Correlation Coefficient of 0.45, the highest performance reported so far. It also outperforms other known methods in delineating individual turn types. We demonstrate how simultaneous prediction of multiple targets influences prediction performance on single targets. The MOLEBRNN presented here is a generic method applicable in a variety of research fields where multiple mutually depending target classes need to be predicted. http://webclu.bio.wzw.tum.de/predator-web/.

  16. Age-related changes in expression of transforming growth factor-beta and receptors in cells of intervertebral discs.

    PubMed

    Matsunaga, Shunji; Nagano, Satoshi; Onishi, Toshiyuki; Morimoto, Norio; Suzuki, Shusaku; Komiya, Setsuro

    2003-01-01

    The authors conducted a study to determine age-related changes in expression of transforming growth factor (TGF)-beta1, -beta2, -beta3, and Type I and Type II receptors in various cells in the nucleus pulposus and anulus fibrosus. Immunolocalization of TGFbetas and Type I and II receptors was examined during the aging process of cervical intervertebral discs in senescence-accelerated mice (SAM). The TGFbeta family has important roles for cellular function of various tissues. Its role in disc aging, however, is unknown. Detailed information on the temporal and spatial localization of TGFbetas and their receptors in discs is required before discussing introduction of them clinically into the intervertebral disc. Three groups of five SAM each were used. The groups of SAM were age 8, 24, and 50 weeks, respectively. Hematoxylin and eosin staining and immunohistochemical study involving specific antibodies for TGFbeta1, -beta2, -beta3, and Types I and II TGF receptors were performed. Intervertebral discs exhibited degenerative change with advancing age. The TGFbetas and their receptors were present in the fibrocartilaginous cells within the anulus fibrosus and notochord-like cells within the nucleus pulposus of young mice. Expression of TGFbetas and Type I and Type II receptors changed markedly in the cells within the anulus fibrosus during the aging process. The TGFbetas and their receptors were present in cells within the nucleus pulposus and the anulus fibrosus of young mice, and their expression decreased with age.

  17. Hepatocyte growth factor and transforming growth factor beta regulate 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression in rat hepatocyte primary cultures.

    PubMed Central

    Joaquin, M; Rosa, J L; Salvadó, C; López, S; Nakamura, T; Bartrons, R; Gil, J; Tauler, A

    1996-01-01

    Hepatocyte growth factor (HGF) and transforming growth factor beta (TGF-beta) are believed to be of major importance for hepatic regeneration after liver damage. We have studied the effect of these growth factors on fructose 2,6-bisphosphate (Fru-2,6-P2) levels and the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-BPase) in rat hepatocyte primary cultures. Our results demonstrate that HGF activates the expression of the 6PF2K/Fru-2,6-BPase gene by increasing the levels of its mRNA. As a consequence of this activation, the amount of 6PF2K/Fru-2,6-BPase protein and 6-phosphofructo-2-kinase activity increased, which was reflected by a rise in Fru-2,6-P2 levels. In contrast, TGF-beta decreased the levels of 6PF2K/Fru-2,6-BPase mRNA, which led to a decrease in the amount of 6PF2K/Fru-2,6-BPase protein and Fru-2,6-P2. The different actions of HGF and TGF-beta on 6PF2K/Fru-2,6-BPase gene expression are concomitant with their effect on cell proliferation. Here we show that, in the absence of hormones, primary cultures of hepatocytes express the F-type isoenzyme. In addition, HGF increases the expression of this isoenzyme, and dexamethasone activates the L-type isoform. HGF and TGF-beta were able to inhibit this activation. PMID:8660288

  18. [Association between Fok I vitamin D receptor (VDR) gene polymorphism and plasmatic concentrations of transforming growth factor-beta1 and interferon gamma in type 1 diabetes mellitus].

    PubMed

    López, Tatiana; García, Diego; Angel, Bárbara; Carrasco, Elena; Codner, Ethel; Ugarte, Francisca; Pérez-Bravo, Francisco

    2008-02-02

    In order to assess whether Fok I vitamin D receptor gene (VDR) polymorphism is involved in the genetic susceptibility of type 1 diabetes, a case-control study was conducted and VDR genotypes were related to serum concentrations of 25(OH) vitamin D and cytokines transforming growth factor beta1 (TGF-beta1) and interferon gamma (INF-gamma). 151 incident cases of type 1 diabetes and 182 non related healthy controls from Santiago were studied for VDR polymorphisms in peripheral blood DNA. Exon 2 (Fok I) segments were amplified by polimerase chain reaction and analyzed by means of restriction fragment length polymorphism to determine each corresponding genotype. Differences for allele, genotype and serological markers as 25(OH) vitamin D, TGF-beta1 and INF-gamma levels distribution between patients and controls were analyzed. Fok I polymorphism distribution analysis showed no differences between patients and controls. Among diabetics, higher levels of TGF-beta1 (median, 282.6 pg/ml; range, 131.8-3,031.4) were observed compared with healthy children (median, 232.2 pg/ml; range, 135.7-506.5) (p < 0.0038). Similar results were observed for INF-gamma concentrations (median, 121.1 pg/ml, and range, 5.3-228.8, in cases, and median, 89.6 pg/ml, and range, 10.9-117.2 in controls) (p < 0.0004). The diabetic carriers of the ff genotype showed low levels of 25(OH) vitamin D compared with the carriers of the F allele: mean (standard deviation), 23.1 (5.9) versus 27.9 (10.3) ng/ml (p < 0.03). A similar result was observed for TGF-beta1 concentrations in diabetic carriers of ff genotype and patients carriers of the F allele (298.5 versus 276.6; p < 0.05). Fok I polymorphism of VDR could have a marginal role in the immunologic disturbance in type 1 diabetes.

  19. Mimicry by asx- and ST-turns of the four main types of beta-turn in proteins.

    PubMed

    Duddy, William J; Nissink, J Willem M; Allen, Frank H; Milner-White, E James

    2004-11-01

    Hydrogen-bonded beta-turns in proteins occur in four categories: type I (the most common), type II, type II', and type I'. Asx-turns resemble beta-turns, in that both have an NH. . .OC hydrogen bond forming a ring of 10 atoms. Serine and threonine side chains also commonly form hydrogen-bonded turns, here called ST-turns. Asx-turns and ST-turns can be categorized into four classes, based on side chain rotamers and the conformation of the central turn residue, which are geometrically equivalent to the four types of beta-turns. We propose asx- and ST-turns be named using the type I, II, I', and II' beta-turn nomenclature. Using this, the frequency of occurrence of both asx- and ST-turns is: type II' > type I > type II > type I', whereas for beta-turns it is type I > type II > type I' > type II'. Almost all type II asx-turns occur as a recently described three residue feature named an asx-nest.

  20. Beta reduction factors for protective clothing at the Oak Ridge National Laboratory

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Franklin, G.L.; Gonzalez, P.L.

    1998-12-31

    Beta reduction factors (f{sub {beta}}) for protective clothing (PC) at the Oak Ridge National Laboratory (ORNL) have been determined for a variety of protective clothing combinations. Data was collected to determine the experimental f{sub {beta}} for several combinations of PCs under laboratory conditions. Radiation dose rates were measured with an open window Bicron{reg_sign} RSO-5 ion chamber for two distinct beta energy groups (E{sub max} = 1.218 {times} 10{sup {minus}13} J(0.860 MeV) and 3.653 {times} 10{sup {minus}13} J (2.280 MeV)). Data points determined, as the ratio of unattenuated (no PCs) to attenuated (PCs), were used to derive a set of equationsmore » using the Microsoft{reg_sign} Excel Linet function. Field comparison tests were then conducted to determine the validity of these beta reduction factors. The f{sub {beta}} from the field tests were significantly less than the experimental f{sub {beta}}, indicating that these factors will yield conservative results.« less

  1. Pin1 promotes transforming growth factor-beta-induced migration and invasion.

    PubMed

    Matsuura, Isao; Chiang, Keng-Nan; Lai, Chen-Yu; He, Dongming; Wang, Guannan; Ramkumar, Romila; Uchida, Takafumi; Ryo, Akihide; Lu, Kunping; Liu, Fang

    2010-01-15

    Transforming growth factor-beta (TGF-beta) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-beta responses. TGF-beta binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-beta-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-beta, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-beta-induced growth-inhibitory responses and a number of TGF-beta/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-beta-mediated migration and invasion. Accordingly, TGF-beta induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-beta-induced migration and invasion of cancer cells.

  2. Transforming growth factor-{beta}-inducible phosphorylation of Smad3.

    PubMed

    Wang, Guannan; Matsuura, Isao; He, Dongming; Liu, Fang

    2009-04-10

    Smad proteins transduce the transforming growth factor-beta (TGF-beta) signal at the cell surface into gene regulation in the nucleus. Upon TGF-beta treatment, the highly homologous Smad2 and Smad3 are phosphorylated by the TGF-beta receptor at the SSXS motif in the C-terminal tail. Here we show that in addition to the C-tail, three (S/T)-P sites in the Smad3 linker region, Ser(208), Ser(204), and Thr(179) are phosphorylated in response to TGF-beta. The linker phosphorylation peaks at 1 h after TGF-beta treatment, behind the peak of the C-tail phosphorylation. We provide evidence suggesting that the C-tail phosphorylation by the TGF-beta receptor is necessary for the TGF-beta-induced linker phosphorylation. Although the TGF-beta receptor is necessary for the linker phosphorylation, the receptor itself does not phosphorylate these sites. We further show that ERK is not responsible for TGF-beta-dependent phosphorylation of these three sites. We show that GSK3 accounts for TGF-beta-inducible Ser(204) phosphorylation. Flavopiridol, a pan-CDK inhibitor, abolishes TGF-beta-induced phosphorylation of Thr(179) and Ser(208), suggesting that the CDK family is responsible for phosphorylation of Thr(179) and Ser(208) in response to TGF-beta. Mutation of the linker phosphorylation sites to nonphosphorylatable residues increases the ability of Smad3 to activate a TGF-beta/Smad-target gene as well as the growth-inhibitory function of Smad3. Thus, these observations suggest that TGF-beta-induced phosphorylation of Smad3 linker sites inhibits its antiproliferative activity.

  3. Effects of type I/type II interferons and transforming growth factor-beta on B-cell differentiation and proliferation. Definition of costimulation and cytokine requirements for immunoglobulin synthesis and expression.

    PubMed

    Estes, D M; Tuo, W; Brown, W C; Goin, J

    1998-12-01

    In this report, we sought to determine the role of selected type I interferons [interferon-alpha (IFN-alpha) and interferon-tau (IFN-tau)], IFN-gamma and transforming growth factor-beta (TGF-beta) in the regulation of bovine antibody responses. B cells were stimulated via CD40 in the presence or absence of B-cell receptor (BCR) cross-linking. IFN-alpha enhanced IgM, IgG2 and IgA responses but did not enhance IgG1 responses. BCR signalling alone was more effective at inducing IgG2 responses with IFN-alpha than dual cross-linking with CD40. Recombinant ovine IFN-tau was less effective at inducing IgG2 responses when compared with IFN-alpha, though IgA responses were similar in magnitude following BCR cross-linking. At higher concentrations, IFN-tau enhanced IgA responses greater than twofold over the levels observed with IFN-alpha. Previous studies have shown that addition of IFN-gamma to BCR or pokeweed mitogen-activated bovine B cells stimulates IgG2 production. However, following CD40 stimulation alone, IFN-gamma was relatively ineffective at stimulating high-rate synthesis of any non-IgM isotype. Dual cross-linking via CD40 and the BCR resulted in decreased synthesis of IgM with a concomitant increase in IgA and similar levels of IgG2 production to those obtained via the BCR alone. We also assessed the effects of endogenous and exogenous TGF-beta on immunoglobulin synthesis by bovine B cells. Exogenous TGF-beta stimulates both IgG2 and IgA production following CD40 and BCR cross-linking in the presence of IL-2. Blocking endogenous TGF-beta did not inhibit the up-regulation of IgG2 or IgA by interferons.

  4. Pin1 down-regulates transforming growth factor-beta (TGF-beta) signaling by inducing degradation of Smad proteins.

    PubMed

    Nakano, Ayako; Koinuma, Daizo; Miyazawa, Keiji; Uchida, Takafumi; Saitoh, Masao; Kawabata, Masahiro; Hanai, Jun-ichi; Akiyama, Hirotada; Abe, Masahiro; Miyazono, Kohei; Matsumoto, Toshio; Imamura, Takeshi

    2009-03-06

    Transforming growth factor-beta (TGF-beta) is crucial in numerous cellular processes, such as proliferation, differentiation, migration, and apoptosis. TGF-beta signaling is transduced by intracellular Smad proteins that are regulated by the ubiquitin-proteasome system. Smad ubiquitin regulatory factor 2 (Smurf2) prevents TGF-beta and bone morphogenetic protein signaling by interacting with Smads and inducing their ubiquitin-mediated degradation. Here we identified Pin1, a peptidylprolyl cis-trans isomerase, as a novel protein binding Smads. Pin1 interacted with Smad2 and Smad3 but not Smad4; this interaction was enhanced by the phosphorylation of (S/T)P motifs in the Smad linker region. (S/T)P motif phosphorylation also enhanced the interaction of Smad2/3 with Smurf2. Pin1 reduced Smad2/3 protein levels in a manner dependent on its peptidyl-prolyl cis-trans isomerase activity. Knockdown of Pin1 increased the protein levels of endogenous Smad2/3. In addition, Pin1 both enhanced the interaction of Smurf2 with Smads and enhanced Smad ubiquitination. Pin1 inhibited TGF-beta-induced transcription and gene expression, suggesting that Pin1 negatively regulates TGF-beta signaling by down-regulating Smad2/3 protein levels via induction of Smurf2-mediated ubiquitin-proteasomal degradation.

  5. Programmed disorders of beta-cell development and function as one cause for type 2 diabetes? The GK rat paradigm.

    PubMed

    Portha, Bernard

    2005-01-01

    Now that the reduction in beta-mass has been clearly established in humans with type 2 diabetes mellitus (T2DM) 1-4, the debate focuses on the possible mechanisms responsible for decreased beta-cell number and impaired beta-cell function and their multifactorial etiology. Appropriate inbred rodent models are essential tools for identification of genes and environmental factors that increase the risk of abnormal beta-cell function and of T2DM. The information available in the Goto-Kakizaki (GK) rat, one of the best characterized animal models of spontaneous T2DM, are reviewed in such a perspective. We propose that the defective beta-cell mass and function in the GK model reflect the complex interactions of three pathogenic players: (1) several independent loci containing genes causing impaired insulin secretion; (2) gestational metabolic impairment inducing a programming of endocrine pancreas (decreased beta-cell neogenesis) which is transmitted to the next generation; and (3) secondary (acquired) loss of beta-cell differentiation due to chronic exposure to hyperglycemia (glucotoxicity). An important message is that the 'heritable' determinants of T2DM are not simply dependant on genetic factors, but probably involve transgenerational epigenetic responses. Copyright (c) 2005 John Wiley & Sons, Ltd.

  6. Cell surface retention sequence binding protein-1 interacts with the v-sis gene product and platelet-derived growth factor beta-type receptor in simian sarcoma virus-transformed cells.

    PubMed

    Boensch, C; Huang, S S; Connolly, D T; Huang, J S

    1999-04-09

    The cell surface retention sequence (CRS) binding protein-1 (CRSBP-1) is a newly identified membrane glycoprotein which is hypothesized to be responsible for cell surface retention of the oncogene v-sis and c-sis gene products and other secretory proteins containing CRSs. In simian sarcoma virus-transformed NIH 3T3 cells (SSV-NIH 3T3 cells), a fraction of CRSBP-1 was demonstrated at the cell surface and underwent internalization/recycling as revealed by cell surface 125I labeling and its resistance/sensitivity to trypsin digestion. However, the majority of CRSBP-1 was localized in intracellular compartments as evidenced by the resistance of most of the 35S-metabolically labeled CRSBP-1 to trypsin digestion, and by indirect immunofluorescent staining. CRSBP-1 appeared to form complexes with proteolytically processed forms (generated at and/or after the trans-Golgi network) of the v-sis gene product and with a approximately 140-kDa proteolytically cleaved form of the platelet-derived growth factor (PDGF) beta-type receptor, as demonstrated by metabolic labeling and co-immunoprecipitation. CRSBP-1, like the v-sis gene product and PDGF beta-type receptor, underwent rapid turnover which was blocked in the presence of 100 microM suramin. In normal and other transformed NIH 3T3 cells, CRSBP-1 was relatively stable and did not undergo rapid turnover and internalization/recycling at the cell surface. These results suggest that in SSV-NIH 3T3 cells, CRSBP-1 interacts with and forms ternary and binary complexes with the newly synthesized v-sis gene product and PDGF beta-type receptor at the trans-Golgi network and that the stable binary (CRSBP-1.v-sis gene product) complex is transported to the cell surface where it presents the v-sis gene product to unoccupied PDGF beta-type receptors during internalization/recycling.

  7. Role for transforming growth factor-beta1 in alport renal disease progression.

    PubMed

    Sayers, R; Kalluri, R; Rodgers, K D; Shield, C F; Meehan, D T; Cosgrove, D

    1999-11-01

    Alport syndrome results from mutations in either the alpha3(IV), alpha4(IV), or alpha5(IV) collagen genes. The disease is characterized by a progressive glomerulonephritis usually associated with a high-frequency sensorineural hearing loss. A mouse model for an autosomal form of Alport syndrome [collagen alpha3(IV) knockout] was produced and characterized. In this study, the model was exploited to demonstrate a potential role for transforming growth factor-beta1 (TGF-beta1) in Alport renal disease pathogenesis. Kidneys from normal and Alport mice, taken at different stages during the course of renal disease progression, were analyzed by Northern blot, in situ hybridization, and immunohistology for expression of TGF-beta1 and components of the extracellular matrix. Normal and Alport human kidney was examined for TGF-beta1 expression using RNase protection. The mRNAs encoding TGF-beta1 (in both mouse and human), entactin, fibronectin, and the collagen alpha1(IV) and alpha2(IV) chains were significantly induced in total kidney as a function of Alport renal disease progression. The induction of these specific mRNAs was observed in the glomerular podocytes of animals with advanced disease. Type IV collagen, laminin-1, and fibronectin were markedly elevated in the tubulointerstitium at 10 weeks, but not at 6 weeks, suggesting that elevated expression of specific mRNAs on Northern blots reflects events associated with tubulointerstitial fibrosis. The concomitant accumulation of mRNAs encoding TGF-beta1 and extracellular matrix components in the podocytes of diseased kidneys may reflect key events in Alport renal disease progression. These data suggest a role for TGF-beta1 in both glomerular and tubulointerstitial damage associated with Alport syndrome.

  8. Determination of transmission factors in beta radiation beams.

    PubMed

    Polo, Ivón Oramas; Caldas, Linda V E

    2018-06-01

    In beta emitters, in order to evaluate the absorbed dose rate at different tissue depths, it is necessary to determine the transmission factors. In this work, the transmission factors determined in beta secondary standard radiation beams are presented. For this purpose, an extrapolation chamber was used. The results obtained were considered acceptable, and they are within the uncertainties in comparison with the values provided by the source calibration certificate. The maximum differences between the results obtained in this work and those from the calibration certificate were 3.3%, 3.8% and 5.9% for 90 Sr/ 90 Y, 85 Kr and 147 Pm sources respectively. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. Transcription factor EGR-1 suppresses the growth and transformation of human HT-1080 fibrosarcoma cells by induction of transforming growth factor beta 1.

    PubMed Central

    Liu, C; Adamson, E; Mercola, D

    1996-01-01

    The early growth response 1 (EGR-1) gene product is a transcription factor with role in differentiation and growth. We have previously shown that expression of exogenous EGR-1 in various human tumor cells unexpectedly and markedly reduces growth and tumorigenicity and, conversely, that suppression of endogenous Egr-1 expression by antisense RNA eliminates protein expression, enhances growth, and promotes phenotypic transformation. However, the mechanism of these effects remained unknown. The promoter of human transforming growth factor beta 1 (TGF-beta 1) contains two GC-rich EGR-1 binding sites. We show that expression of EGR-1 in human HT-1080 fibrosarcoma cells uses increased secretion of biologically active TGF-beta 1 in direct proportion (rPearson = 0.96) to the amount of EGR-1 expressed and addition of recombinant human TGF-beta 1 is strongly growth-suppressive for these cells. Addition of monoclonal anti-TGF-beta 1 antibodies to EGR-1-expressing HT-1080 cells completely reverses the growth inhibitory effects of EGR-1. Reporter constructs bearing the EGR-1 binding segment of the TGF-beta 1 promoter was activated 4- to 6-fold relative to a control reporter in either HT-1080 cells that stably expressed or parental cells cotransfected with an EGR-1 expression vector. Expression of delta EGR-1, a mutant that cannot interact with the corepressors, nerve growth factor-activated factor binding proteins NAB1 and NAB2, due to deletion of the repressor domain, exhibited enhanced transactivation of 2- to 3.5-fold over that of wild-type EGR-1 showing that the reporter construct reflected the appropriate in vivo regulatory context. The EGR-1-stimulated transactivation was inhibited by expression of the Wilms tumor suppressor, a known specific DNA-binding competitor. These results indicate that EGR-1 suppresses growth of human HT-1080 fibrosarcoma cells by induction of TGF-beta 1. Images Fig. 1 Fig. 5 PMID:8876223

  10. A Logical OR Redundancy within the Asx-Pro-Asx-Gly Type 1 {Beta}-Turn Motif

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Jihun; Dubey, Vikash Kumar; Longo, Lian M.

    2008-04-19

    Turn secondary structure is essential to the formation of globular protein architecture. Turn structures are, however, much more complex than either {alpha}-helix or {beta}-sheet, and the thermodynamics and folding kinetics are poorly understood. Type I {beta}-turns are the most common type of reverse turn, and they exhibit a statistical consensus sequence of Asx-Pro-Asx-Gly (where Asx is Asp or Asn). A comprehensive series of individual and combined Asx mutations has been constructed within three separate type I 3:5 G1 bulge {beta}-turns in human fibroblast growth factor-1, and their effects on structure, stability, and folding have been determined. The results show amore » fundamental logical OR relationship between the Asx residues in the motif, involving H-bond interactions with main-chain amides within the turn. These interactions can be modulated by additional interactions with residues adjacent to the turn at positions i + 4 and i + 6. The results show that the Asx residues in the turn motif make a substantial contribution to the overall stability of the protein, and the Asx logical OR relationship defines a redundant system that can compensate for deleterious point mutations. The results also show that the stability of the turn is unlikely to be the prime determinant of formation of turn structure in the folding transition state.« less

  11. The oncoprotein Ski acts as an antagonist of transforming growth factor-beta signaling by suppressing Smad2 phosphorylation.

    PubMed

    Prunier, Celine; Pessah, Marcia; Ferrand, Nathalie; Seo, Su Ryeon; Howe, Philip; Atfi, Azeddine

    2003-07-11

    The phosphorylation of Smad2 and Smad3 by the transforming growth factor (TGF)-beta-activated receptor kinases and their subsequent heterodimerization with Smad4 and translocation to the nucleus form the basis for a model how Smad proteins work to transmit TGF-beta signals. The transcriptional activity of Smad2-Smad4 or Smad3-Smad4 complexes can be limited by the corepressor Ski, which is believed to interact with Smad complexes on TGF-beta-responsive promoters and represses their ability to activate TGF-beta target genes by assembling on DNA a repressor complex containing histone deacetylase. Here we show that Ski can block TGF-beta signaling by interfering with the phosphorylation of Smad2 and Smad3 by the activated TGF-beta type I receptor. Furthermore, we demonstrate that overexpression of Ski induces the assembly of Smad2-Smad4 and Smad3-Smad4 complexes independent of TGF-beta signaling. The ability of Ski to engage Smad proteins in nonproductive complexes provides new insights into the molecular mechanism used by Ski for disabling TGF-beta signaling.

  12. Alterations in the mitochondrial regulatory pathways constituted by the nuclear co-factors PGC-1alpha or PGC-1beta and mitofusin 2 in skeletal muscle in type 2 diabetes.

    PubMed

    Zorzano, Antonio; Hernández-Alvarez, María Isabel; Palacín, Manuel; Mingrone, Geltrude

    2010-01-01

    Muscle mitochondrial metabolism is regulated by a number of factors, many of which are responsible for the transcription of nuclear genes encoding mitochondrial proteins such as PPARdelta, PGC-1alpha or PGC-1beta. Recent evidence indicates that proteins participating in mitochondrial dynamics also regulate mitochondrial metabolism. Thus, in cultured cells the mitochondrial fusion protein mitofusin 2 (Mfn2) stimulates respiration, substrate oxidation and the expression of subunits involved in respiratory complexes. Mitochondrial dysfunction has been reported in skeletal muscle of type 2 diabetic patients. Reduced mitochondrial mass and defective activity has been proposed to explain this dysfunction. Alterations in mitochondrial metabolism may be crucial to account for some of the pathophysiological traits that characterize type 2 diabetes. Skeletal muscle of type 2 diabetic patients shows reduced expression of PGC-1alpha, PGC-1beta, and Mfn2. In addition, a differential response to bilio-pancreatic diversion-induced weight loss in non-diabetic and type 2 diabetic patients has been reported. While non-diabetic morbidly obese subjects showed an increased expression of genes encoding Mfn2, PGC-1alpha, PGC-1beta, PPARdelta or SIRT1 in response to bariatric surgery-induced weight loss, no effect was detected in type 2 diabetic patients. These observations suggest the existence of a heritable component responsible for the abnormal control of the expression of genes encoding for modulators of mitochondrial biogenesis/metabolism, and which may participate in the development of the disease. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. Caveolae are negative regulators of transforming growth factor-beta1 signaling in ureteral smooth muscle cells.

    PubMed

    Stehr, Maximilian; Estrada, Carlos R; Khoury, Joseph; Danciu, Theodora E; Sullivan, Maryrose P; Peters, Craig A; Solomon, Keith R; Freeman, Michael R; Adam, Rosalyn M

    2004-12-01

    The mechanisms underlying ureteral cell regulation are largely unknown. Previous studies have identified lipid rafts/caveolae as regulators of growth stimulatory signals in ureteral smooth muscle cells (USMCs). In this study we determined whether growth inhibitory signaling by transforming growth factor-beta1 (TGF-beta1) is also regulated by caveolae in USMC. Expression of components of the TGF-beta1 signaling axis in USMCs was determined by immunoblot and mRNA analyses. Growth regulatory activity of TGF-beta1 was assessed by H-thymidine incorporation. In select experiments caveolae were disrupted reversibly by cholesterol depletion and replenishment prior to TGF-beta1 treatment. TGF-beta1-responsive gene expression was evaluated using the TGF-beta1 responsive promoter-reporter construct 3TP-Lux. USMCs expressed TGF-beta1, types I and II TGF-beta1 receptors, and the effector Smad-2. TGF-beta1 potently inhibited DNA synthesis in USMCs (IC50 60 pM). TGF-beta1 mediated DNA synthesis inhibition was potentiated following the disruption of caveolae by cholesterol depletion. This effect was reversible with membrane cholesterol restoration. TGF-beta1 stimulated gene activity was augmented by caveolae disruption, while caveolae reformation returned promoter activity to baseline levels. TGF-beta1 is a potent growth inhibitor of USMCs and its activity can be enhanced by caveolae ablation. These findings suggest a role for TGF-beta1 in the growth regulation of normal ureteral cells and implicate caveolar membrane domains in the negative regulation of TGF-beta1 signaling. These studies may be relevant to ureteral pathologies that are characterized by smooth muscle dysplasia.

  14. Effects of hydrostatic pressure and transforming growth factor-beta 3 on adult human mesenchymal stem cell chondrogenesis in vitro.

    PubMed

    Miyanishi, Keita; Trindade, Michael C D; Lindsey, Derek P; Beaupré, Gary S; Carter, Dennis R; Goodman, Stuart B; Schurman, David J; Smith, R Lane

    2006-06-01

    This study examined the effects of intermittent hydrostatic pressure (IHP) and transforming growth factor-beta 3 on chondrogenesis of adult human mesenchymal stem cells (hMSCs) in vitro. Chondrogenic gene expression was determined by quantifying mRNA signal levels for SOX9, a transcription factor critical for cartilage development and the cartilage matrix proteins, aggrecan and type II collagen. Extracellular matrix production was determined by weight and histology. IHP was applied to hMSCs in pellet culture at a level of 10 MPa and a frequency of 1 Hz for 4 h per day for periods of 3, 7, and 14 days. hMSCs responded to addition of TGF-beta 3 (10 ng/mL) with a greater than 10-fold increase (p < 0.01) in mRNA levels for each, SOX9, type II collagen, and aggrecan during a 14-day culture period. Applying IHP in the presence of TGF-beta 3 further increased the mRNA levels for these proteins by 1.9-, 3.3-, and 1.6-fold, respectively, by day 14. Chondrogenic mRNA levels were increased with just exposure to IHP. Extracellular matrix deposition of type II collagen and aggrecan increased in the pellets as a function of treatment conditions and time of culture. This study demonstrated adjunctive effects of IHP on TGF-beta 3-induced chondrogenesis and suggests that mechanical loading can facilitate articular cartilage tissue engineering.

  15. Human T-cell leukemia virus type I oncoprotein Tax represses Smad-dependent transforming growth factor beta signaling through interaction with CREB-binding protein/p300.

    PubMed

    Mori, N; Morishita, M; Tsukazaki, T; Giam, C Z; Kumatori, A; Tanaka, Y; Yamamoto, N

    2001-04-01

    Human T-cell leukemia virus type I (HTLV-I) Tax is a potent transcriptional regulator that can activate or repress specific cellular genes and that has been proposed to contribute to leukemogenesis in adult T-cell leukemia. Previously, HTLV-I- infected T-cell clones were found to be resistant to growth inhibition by transforming growth factor (TGF)-beta. Here it is shown that Tax can perturb Smad-dependent TGF-beta signaling even though no direct interaction of Tax and Smad proteins could be detected. Importantly, a mutant Tax of CREB-binding protein (CBP)/p300 binding site, could not repress the Smad transactivation function, suggesting that the CBP/p300 binding domain of Tax is essential for the suppression of Smad function. Because both Tax and Smad are known to interact with CBP/p300 for the potentiation of their transcriptional activities, the effect of CBP/p300 on suppression of Smad-mediated transactivation by Tax was examined. Overexpression of CBP/p300 reversed Tax-mediated inhibition of Smad transactivation. Furthermore, Smad could repress Tax transcriptional activation, indicating reciprocal repression between Tax and Smad. These results suggest that Tax interferes with the recruitment of CBP/p300 into transcription initiation complexes on TGF-beta-responsive elements through its binding to CBP/p300. The novel function of Tax as a repressor of TGF-beta signaling may contribute to HTLV-I leukemogenesis. (Blood. 2001;97:2137-2144)

  16. Potentiating role of interleukin-1beta (IL-1beta) and IL-1beta type 1 receptors in the medial hypothalamus in defensive rage behavior in the cat.

    PubMed

    Hassanain, M; Bhatt, S; Zalcman, S; Siegel, A

    2005-06-28

    Recently, this laboratory provided evidence that interleukin-1beta (IL-1beta), an immune and brain-derived cytokine, microinjected into the medial hypothalamus, potentiates defensive rage behavior in the cat elicited from the midbrain periaqueductal gray (PAG), and that such effects are blocked by a 5-HT2 receptor antagonist. Since this finding represents the first time that a brain cytokine has been shown to affect defensive rage behavior, the present study replicated and extended these findings by documenting the specific potentiating role played by IL-1beta Type 1 receptor (IL-1RI), and the anatomical relationship between IL-1beta and 5-HT2 receptors in the medial hypothalamus. IL-1beta (10 ng) microinjected into the medial hypothalamus induced two separate phases of facilitation, one at 60 min and another at 180 min, post-injection. In turn, these effects were blocked with pretreatment of the selective IL-1 Type I receptor antagonist (IL-1ra) (10 ng), demonstrating the selectivity of the effects of IL-1beta on medial hypothalamic neurons upon PAG-elicited defensive rage behavior. The next stage of the study utilized immunohistochemical methods to demonstrate that IL-1beta and 5-HT2 receptors were present on the same neurons within regions of the medial hypothalamus where IL-1beta and the IL-1beta receptor antagonists were administered. This provided anatomical evidence suggesting a relationship between IL-1RI and 5-HT2 receptors in the medial hypothalamus that is consistent with the previous pharmacological observations in our laboratory. The overall findings show that activation of IL-1RI in the medial hypothalamus potentiates defensive rage behavior in the cat and that these effects may also be linked to the presence of 5-HT2 receptors on the same groups of neurons in this region of hypothalamus.

  17. Neomycin is a platelet-derived growth factor (PDGF) antagonist that allows discrimination of PDGF alpha- and beta-receptor signals in cells expressing both receptor types.

    PubMed

    Vassbotn, F S; Ostman, A; Siegbahn, A; Holmsen, H; Heldin, C H

    1992-08-05

    The aminoglycoside neomycin has recently been found to affect certain platelet-derived growth factor (PDGF) responses in C3H/10T1/2 C18 fibroblasts. Using porcine aortic endothelial cells transfected with PDGF alpha- or beta-receptors, we explored the possibility that neomycin interferes with the interaction between the different PDGF isoforms and their receptors. We found that neomycin (5 mM) inhibited the binding of 125I-PDGF-BB to the alpha-receptor with only partial effect on the binding of 125I-PDGF-AA; in contrast, the binding of 125I-PDGF-BB to the beta-receptor was not affected by the aminoglycoside. Scatchard analyses showed that neomycin (5 mM) decreased the number of binding sites for PDGF-BB on alpha-receptor-expressing cells by 87%. Together with cross-competition studies with 125I-labeled PDGF homodimers, the effect of neomycin indicates that PDGF-AA and PDGF-BB bind to both common and unique structures on the PDGF alpha-receptor. Neomycin specifically inhibited the autophosphorylation of the alpha-receptor by PDGF-BB, with less effect on the phosphorylation induced by PDGF-AA and no effect on the phosphorylation of the beta-receptor by PDGF-BB. Thus, neomycin is a PDGF isoform- and receptor-specific antagonist that provides a possibility to compare the signal transduction pathways of alpha- and beta-receptors in cells expressing both receptor types. This approach was used to show that activation of PDGF beta-receptors by PDGF-BB mediated a chemotactic response in human fibroblasts, whereas activation of alpha-receptors by the same ligand inhibited chemotaxis.

  18. Predicting beta-turns and their types using predicted backbone dihedral angles and secondary structures.

    PubMed

    Kountouris, Petros; Hirst, Jonathan D

    2010-07-31

    Beta-turns are secondary structure elements usually classified as coil. Their prediction is important, because of their role in protein folding and their frequent occurrence in protein chains. We have developed a novel method that predicts beta-turns and their types using information from multiple sequence alignments, predicted secondary structures and, for the first time, predicted dihedral angles. Our method uses support vector machines, a supervised classification technique, and is trained and tested on three established datasets of 426, 547 and 823 protein chains. We achieve a Matthews correlation coefficient of up to 0.49, when predicting the location of beta-turns, the highest reported value to date. Moreover, the additional dihedral information improves the prediction of beta-turn types I, II, IV, VIII and "non-specific", achieving correlation coefficients up to 0.39, 0.33, 0.27, 0.14 and 0.38, respectively. Our results are more accurate than other methods. We have created an accurate predictor of beta-turns and their types. Our method, called DEBT, is available online at http://comp.chem.nottingham.ac.uk/debt/.

  19. Transforming growth factor-beta regulates mammary carcinoma cell survival and interaction with the adjacent microenvironment.

    PubMed

    Bierie, Brian; Stover, Daniel G; Abel, Ty W; Chytil, Anna; Gorska, Agnieszka E; Aakre, Mary; Forrester, Elizabeth; Yang, Li; Wagner, Kay-Uwe; Moses, Harold L

    2008-03-15

    Transforming growth factor (TGF)-beta signaling has been associated with early tumor suppression and late tumor progression; however, many of the mechanisms that mediate these processes are not known. Using Cre/LoxP technology, with the whey acidic protein promoter driving transgenic expression of Cre recombinase (WAP-Cre), we have now ablated the type II TGF-beta receptor (T beta RII) expression specifically within mouse mammary alveolar progenitors. Transgenic expression of the polyoma virus middle T antigen, under control of the mouse mammary tumor virus enhancer/promoter, was used to produce mammary tumors in the absence or presence of Cre (T beta RII((fl/fl);PY) and T beta RII((fl/fl);PY;WC), respectively). The loss of TGF-beta signaling significantly decreased tumor latency and increased the rate of pulmonary metastasis. The loss of TGF-beta signaling was significantly correlated with increased tumor size and enhanced carcinoma cell survival. In addition, we observed significant differences in stromal fibrovascular abundance and composition accompanied by increased recruitment of F4/80(+) cell populations in T beta RII((fl/fl);PY;WC) mice when compared with T beta RII((fl/fl);PY) controls. The recruitment of F4/80(+) cells correlated with increased expression of known inflammatory genes including Cxcl1, Cxcl5, and Ptgs2 (cyclooxygenase-2). Notably, we also identified an enriched K5(+) dNp63(+) cell population in primary T beta RII((fl/fl);PY;WC) tumors and corresponding pulmonary metastases, suggesting that loss of TGF-beta signaling in this subset of carcinoma cells can contribute to metastasis. Together, our current results indicate that loss of TGF-beta signaling in mammary alveolar progenitors may affect tumor initiation, progression, and metastasis through regulation of both intrinsic cell signaling and adjacent stromal-epithelial interactions in vivo.

  20. Transforming growth factor-beta stimulates wound healing and modulates extracellular matrix gene expression in pig skin. I. Excisional wound model.

    PubMed

    Quaglino, D; Nanney, L B; Kennedy, R; Davidson, J M

    1990-09-01

    The effect of transforming growth factor-beta 1 (TGF-beta 1) on matrix gene expression has been investigated during the process of wound repair, where the formation of new connective tissue represents a critical step in restoring tissue integrity. Split-thickness excisional wounds in the pig were studied by in situ hybridization in order to obtain subjective findings on the activity and location of cells involved in matrix gene expression after the administration of recombinant TGF-beta 1. Data focus on the stimulatory role of this growth factor in granulation tissue formation, on the enhanced mRNA content of collagen types I and III, fibronectin, TGF-beta 1 itself, and on the reduction in stromelysin mRNA, suggesting that increased matrix formation measured after treatment with TGF-beta 1 is due to fibroplasia regulated by the abundance of mRNAs for several different structural, matrix proteins as well as inhibition of proteolytic phenomena elicited by metalloproteinases. These studies reveal elastin mRNA early in the repair process, and elastin mRNA expression is enhanced by administration of TGF-beta 1. Moreover, we show that TGF-beta 1 was auto-stimulating in wounds, accounting, at least in part, for the persistent effects of single doses of this multipotential cytokine.

  1. Targeting dysfunctional beta-cell signaling for the potential treatment of type 1 diabetes mellitus.

    PubMed

    Fenske, Rachel J; Kimple, Michelle E

    2018-03-01

    Since its discovery and purification by Frederick Banting in 1921, exogenous insulin has remained almost the sole therapy for type 1 diabetes mellitus. While insulin alleviates the primary dysfunction of the disease, many other aspects of the pathophysiology of type 1 diabetes mellitus are unaffected. Research aimed towards the discovery of novel type 1 diabetes mellitus therapeutics targeting different cell signaling pathways is gaining momentum. The focus of these efforts has been almost entirely on the impact of immunomodulatory drugs, particularly those that have already received FDA-approval for other autoimmune diseases. However, these drugs can often have severe side effects, while also putting already immunocompromised individuals at an increased risk for other infections. Potential therapeutic targets in the insulin-producing beta-cell have been largely ignored by the type 1 diabetes mellitus field, save the glucagon-like peptide 1 receptor. While there is preliminary evidence to support the clinical exploration of glucagon-like peptide 1 receptor-based drugs as type 1 diabetes mellitus adjuvant therapeutics, there is a vast space for other putative therapeutic targets to be explored. The alpha subunit of the heterotrimeric G z protein (Gα z ) has been shown to promote beta-cell inflammation, dysfunction, death, and failure to replicate in the context of diabetes in a number of mouse models. Genetic loss of Gα z or inhibition of the Gα z signaling pathway through dietary interventions is protective against the development of insulitis and hyperglycemia. The multifaceted effects of Gα z in regards to beta-cell health in the context of diabetes make it an ideal therapeutic target for further study. It is our belief that a low-risk, effective therapy for type 1 diabetes mellitus will involve a multidimensional approach targeting a number of regulatory systems, not the least of which is the insulin-producing beta-cell. Impact statement The expanding

  2. Inhibition of Transforming Growth Factor-Beta1 SignalingAttenuates Ataxia Telangiectasia Mutated Activity in Response toGenotoxic Stress

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kirshner, Julia; Jobling, Michael F.; Pajares, Maria Jose

    2006-01-01

    Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor {beta} (TGF{beta})-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGF{beta} inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgf{beta}I null murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGF{beta} type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced H2AX radiation-induced foci; and increased radiosensitivity compared with TGF{beta} competent cells.more » We determined that loss of TGF{beta} signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGF{beta} restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgf{beta}I, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGF{beta} may be used to advantage in cancer therapy.« less

  3. Regulation of cell growth by redox-mediated extracellular proteolysis of platelet-derived growth factor receptor beta.

    PubMed

    Okuyama, H; Shimahara, Y; Kawada, N; Seki, S; Kristensen, D B; Yoshizato, K; Uyama, N; Yamaoka, Y

    2001-07-27

    Redox-regulated processes are important elements in various cellular functions. Reducing agents, such as N-acetyl-l-cysteine (NAC), are known to regulate signal transduction and cell growth through their radical scavenging action. However, recent studies have shown that reactive oxygen species are not always involved in ligand-stimulated intracellular signaling. Here, we report a novel mechanism by which NAC blocks platelet-derived growth factor (PDGF)-induced signaling pathways in hepatic stellate cells, a fibrogenic player in the liver. Unlike in vascular smooth muscle cells, we found that reducing agents, including NAC, triggered extracellular proteolysis of PDGF receptor-beta, leading to desensitization of hepatic stellate cells toward PDGF-BB. This effect was mediated by secreted mature cathepsin B. In addition, type II transforming growth factor-beta receptor was also down-regulated. Furthermore, these events seemed to cause a dramatic improvement of rat liver fibrosis. These results indicated that redox processes impact the cell's response to growth factors by regulating the turnover of growth factor receptors and that "redox therapy" is promising for fibrosis-related disease.

  4. Induction of myofibroblastic differentiation in vitro by covalently immobilized transforming growth factor-beta(1).

    PubMed

    Metzger, Wolfgang; Grenner, Nadine; Motsch, Sandra E; Strehlow, Rothin; Pohlemann, Tim; Oberringer, Martin

    2007-11-01

    Growth factors are an important tool in tissue engineering. Bone morphogenetic protein-2 and transforming growth factor-beta(1) (TGF-beta(1)) are used to provide bioactivity to surgical implants and tissue substitute materials. Mostly growth factors are used in soluble or adsorbed form. However, simple adsorption of proteins to surfaces is always accompanied by reduced stability and undefined pharmacokinetics. This study aims to prove that TGF-beta(1) can be covalently immobilized to functionalized surfaces, maintaining its ability to induce myofibroblastic differentiation of normal human dermal fibroblasts. In vivo, fibroblasts differentiate to myofibroblasts (MFs) during soft tissue healing by the action of TGF-beta(1). As surfaces for our experiments, we used slides bearing aldehyde, epoxy, or amino groups. For our in vitro cell culture experiments, we used the expression of alpha-smooth muscle actin as a marker for MFs after immunochemical staining. Using the aldehyde and the epoxy slides, we were able to demonstrate the activity of immobilized TGF-beta(1) through a significant increase in MF differentiation rate. A simple immunological test was established to detect TGF-beta(1) on the surfaces. This technology enables the creation of molecular "landscapes" consisting of several factors arranged in a distinct spatial pattern and immobilized on appropriate surfaces.

  5. Loss of c-myc repression coincides with ovarian cancer resistance to transforming growth factor beta growth arrest independent of transforming growth factor beta/Smad signaling.

    PubMed

    Baldwin, Rae Lynn; Tran, Hang; Karlan, Beth Y

    2003-03-15

    Many epithelial carcinomas, including ovarian, are refractory to the antiproliferative effects of transforming growth factor (TGF) beta. In some cancers, TGF-beta resistance has been linked to TGF-beta receptor II (TbetaR-II) and Smad4 mutations; however, in ovarian cancer, the mechanism of resistance remains unclear. Primary ovarian epithelial cell cultures were used as a model system to determine the mechanisms of TGF-beta resistance. To simulate in vivo responses to TGF-beta, primary cultures derived from normal human ovarian surface epithelium (HOSE) and from ovarian carcinomas (CSOC) were grown on collagen I gel, the predominant matrix molecule in the ovarian tumor milieu. When treated with 5 ng/ml TGF-beta for 72 h, HOSE (n = 11) proliferation was inhibited by 20 +/- 21% on average. In contrast, CSOC (n = 10) proliferation was stimulated 5 +/- 10% in response to TGF-beta (a statistically significant difference in response when compared with HOSE; P = 0.001). To dissect the TGF-beta/Smad signaling pathway we used a quantitative RNase protection assay (RPA) for measuring mRNA levels of TGF-beta pathway components in 20 HOSE and 20 CSOC cultures. Basal mRNA levels of TGF-beta receptors I and II, downstream signaling components Smad2, 3, 4, 6, 7, and the transcriptional corepressors Ski and SnoN did not show a statistically significant difference between HOSE and CSOC, and cannot explain their differential susceptibility to TGF-beta-induced cell cycle arrest. To assess functional differences of the TGF-beta pathway in TGF-beta-sensitive HOSE and TGF-beta-resistant CSOC, we measured Smad2/4 and 3/4 complex induction after TGF-beta treatment. HOSE and CSOC showed equivalent Smad2/4 and 3/4 complex induction after TGF-beta exposure for 0, 0.5, 2, and 4 h. It has been proposed that SnoN and Ski are corepressors of the TGF-beta/Smad pathway and undergo TGF-beta-induced degradation followed by reinduction of SnoN mRNA. However, our data show equivalent SnoN degradation

  6. Regulation of pancreatic islet beta-cell mass by growth factor and hormone signaling.

    PubMed

    Huang, Yao; Chang, Yongchang

    2014-01-01

    Dysfunction and destruction of pancreatic islet beta cells is a hallmark of diabetes. Better understanding of cellular signals in beta cells will allow development of therapeutic strategies for diabetes, such as preservation and expansion of beta-cell mass and improvement of beta-cell function. During the past several decades, the number of studies analyzing the molecular mechanisms, including growth factor/hormone signaling pathways that impact islet beta-cell mass and function, has increased exponentially. Notably, somatolactogenic hormones including growth hormone (GH), prolactin (PRL), and insulin-like growth factor-1 (IGF-1) and their receptors (GHR, PRLR, and IGF-1R) are critically involved in beta-cell growth, survival, differentiation, and insulin secretion. In this chapter, we focus more narrowly on GH, PRL, and IGF-1 signaling, and GH-IGF-1 cross talk. We also discuss how these signaling aspects contribute to the regulation of beta-cell proliferation and apoptosis. In particular, our novel findings of GH-induced formation of GHR-JAK2-IGF-1R protein complex and synergistic effects of GH and IGF-1 on beta-cell signaling, proliferation, and antiapoptosis lead to a new concept that IGF-1R may serve as a proximal component of GH/GHR signaling. © 2014 Elsevier Inc. All rights reserved.

  7. Is beta-thalassemia trait a protective factor against ischemic cerebrovascular accidents?

    PubMed

    Karimi, Mehran; Borhani Haghighi, Afshin; Yazdani, Maryam; Raisi, Hamideh; Giti, Rahil; Namazee, Mohammad Reza

    2008-01-01

    In this research, we sought to determine the association between beta-thalassemia trait and ischemic cerebrovascular accident (CVA). In acase-control study, 148 patients with thromboembolic cerebrovascular events were evaluated for the presence of hypertension, diabetes mellitus, hyperlipidemia, and beta-thalassemia trait. A total of 156 age- and sex-matched patients with no cardiac or cerebrovascular diseases, serving as the control group, were also investigated for the above-mentioned risk factors. We found that 6.1% of patients with ischemic CVA and 12.2% of the control group had beta-thalassemia trait (P = .066). In male patients, the negative association between ischemic CVA and presence of beta-thalassemia trait was significant (P = .008). In patients, the prevalence of hypertension was also significantly different between those with and without beta-thalassemia trait (P = .01); those with beta-thalassemia trait had a lower mean blood pressure than those without the trait. beta-Thalassemia trait may have a protective effect against ischemic CVA that might be caused by the lower arterial blood pressure observed in those with this trait.

  8. Determination of correction factors in beta radiation beams using Monte Carlo method.

    PubMed

    Polo, Ivón Oramas; Santos, William de Souza; Caldas, Linda V E

    2018-06-15

    The absorbed dose rate is the main characterization quantity for beta radiation. The extrapolation chamber is considered the primary standard instrument. To determine absorbed dose rates in beta radiation beams, it is necessary to establish several correction factors. In this work, the correction factors for the backscatter due to the collecting electrode and to the guard ring, and the correction factor for Bremsstrahlung in beta secondary standard radiation beams are presented. For this purpose, the Monte Carlo method was applied. The results obtained are considered acceptable, and they agree within the uncertainties. The differences between the backscatter factors determined by the Monte Carlo method and those of the ISO standard were 0.6%, 0.9% and 2.04% for 90 Sr/ 90 Y, 85 Kr and 147 Pm sources respectively. The differences between the Bremsstrahlung factors determined by the Monte Carlo method and those of the ISO were 0.25%, 0.6% and 1% for 90 Sr/ 90 Y, 85 Kr and 147 Pm sources respectively. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. Mediation of wound-related Rous sarcoma virus tumorigenesis by TFG (transforming growth factor)-. beta

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sieweke, M.H.; Bissell, M.J.; Thompson, N.L.

    1990-06-29

    In Rous sarcoma virus (RSV)-infected chickens, wounding leads to tumor formation with nearly 100% frequency in tissues that would otherwise remain tumor-free. Identifying molecular mediators of this phenomenon should yield important clues to the mechanisms involved in RSV tumorigenesis. Immunohistochemical staining showed that TGF-{beta} is present locally shortly after wounding, but not in unwounded controls. In addition, subcutaneous administration of recombinant transforming growth factor {beta}1 (TGF-{beta}1) could substitute completely for wounding in tumor induction. A treatment protocol of four doses of 800 nanograms of TGF-{beta} resulted in v-src-expressing tumors with 100% frequency; four doses of only 10 nanograms still ledmore » to tumor formation in 80% of the animals. This effect was specific, as other growth factors with suggested roles in would healing did not elicit the same response. Epidermal growth factor (EGF) or TGF-{alpha} had no effect, and platelet-derived growth factor (PDGF) or insulin-like growth factor-1 (IGF-1) yielded only occasional tumors after longer latency. TGF-{beta} release during the would-healing response may thus be a critical event that creates a conducive environment for RSV tumorigenesis and may act as a cofactor for transformation in this system. 31 refs., 3 figs., 2 tabs.« less

  10. Selective destruction of mouse islet beta cells by human T lymphocytes in a newly-established humanized type 1 diabetic model

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhao, Yong, E-mail: yongzhao@uic.edu; Guo, Chengshan; Hwang, David

    2010-09-03

    Research highlights: {yields} Establish a human immune-mediated type 1 diabetic model in NOD-scid IL2r{gamma}{sup null} mice. {yields} Using the irradiated diabetic NOD mouse spleen mononuclear cells as trigger. {yields} The islet {beta} cells were selectively destroyed by infiltrated human T cells. {yields} The model can facilitate translational research to find a cure for type 1 diabetes. -- Abstract: Type 1 diabetes (T1D) is caused by a T cell-mediated autoimmune response that leads to the loss of insulin-producing {beta} cells. The optimal preclinical testing of promising therapies would be aided by a humanized immune-mediated T1D model. We develop this model inmore » NOD-scid IL2r{gamma}{sup null} mice. The selective destruction of pancreatic islet {beta} cells was mediated by human T lymphocytes after an initial trigger was supplied by the injection of irradiated spleen mononuclear cells (SMC) from diabetic nonobese diabetic (NOD) mice. This resulted in severe insulitis, a marked loss of total {beta}-cell mass, and other related phenotypes of T1D. The migration of human T cells to pancreatic islets was controlled by the {beta} cell-produced highly conserved chemokine stromal cell-derived factor 1 (SDF-1) and its receptor C-X-C chemokine receptor (CXCR) 4, as demonstrated by in vivo blocking experiments using antibody to CXCR4. The specificity of humanized T cell-mediated immune responses against islet {beta} cells was generated by the local inflammatory microenvironment in pancreatic islets including human CD4{sup +} T cell infiltration and clonal expansion, and the mouse islet {beta}-cell-derived CD1d-mediated human iNKT activation. The selective destruction of mouse islet {beta} cells by a human T cell-mediated immune response in this humanized T1D model can mimic those observed in T1D patients. This model can provide a valuable tool for translational research into T1D.« less

  11. Transforming growth factor-beta and nitrates in epithelial ovarian cancer.

    PubMed

    Khalifa, A; Kassim, S K; Ahmed, M I; Fayed, S T

    1999-12-01

    The role of transforming growth factor-beta (TGF-beta) and nitric oxide (NO) in ovarian neoplasia is still not clear. We studied the expression of TGF-beta by enzyme immunoassay, and nitrates (as a stable end product of NO) in 127 ovarian tissues (36 normal, 37 benign, and 54 malignant). Ploidy status and synthetic phase fraction (SPF) were also assessed by flow cytometry. Mean ranks of TGF-beta, nitrate, and SPF were significant among different groups (X2 = 12.01, P = 0.0025, X2 = 67.42, P = 0.000, X2 = 9.06, P = 0.011 respectively). Nitrate mean ranks were significant among different FIGO stages of the disease (X2 = 17.6, P = 0.000). A significant correlation was shown between TGF-beta, and nitrate levels in all tissues (r = 0.24, P = 0.01), as well as in malignant tissues (r = 0.3, P = 0.026). Cutoff values were determined for both TGF-beta (290 pg/mg protein), and nitrates (310 nmole/mg non protein nitrogenous substances). At these cut-offs, nitrates showed a sensitivity of 93% and 84% specificity for malignant versus normal cases, while TGF-beta had 76% sensitivity, and 82.4% specificity for poor versus good outcome. Patients with epithelial ovarian cancer were followed up for a total of 40 months. Survival analysis showed that patients with TGF-beta above the cut-off had worse prognosis (X2 = 12.69, P = 0.004). The present results suggest that malignant transformation of ovarian tissues is associated with increased TGF-beta and NO production. NO level is related to the development and progression of epithelial ovarian cancer, while high levels of TGF-beta could be of prognostic significance.

  12. Immunocytochemical localization of latent transforming growth factor-beta1 activation by stimulated macrophages

    NASA Technical Reports Server (NTRS)

    Chong, H.; Vodovotz, Y.; Cox, G. W.; Barcellos-Hoff, M. H.; Chatterjee, A. (Principal Investigator)

    1999-01-01

    Transforming growth factor-beta1 (TGF-beta) is secreted in a latent form consisting of mature TGF-beta noncovalently associated with its amino-terminal propeptide, which is called latency associated peptide (LAP). Biological activity depends upon the release of TGF-beta from the latent complex following extracellular activation, which appears to be the key regulatory mechanism controlling TGF-beta action. We have identified two events associated with latent TGF-beta (LTGF-beta) activation in vivo: increased immunoreactivity of certain antibodies that specifically detect TGF-beta concomitant with decreased immunoreactivity of antibodies to LAP. Macrophages stimulated in vitro with interferon-gamma and lipopolysaccharide reportedly activate LTGF-beta via cell membrane-bound protease activity. We show through dual immunostaining of paraformaldehyde-fixed macrophages that such physiological TGF-beta activation is accompanied by a loss of LAP immunoreactivity with concomitant revelation of TGF-beta epitopes. The induction of TGF-beta immunoreactivity colocalized with immunoreactive betaglycan/RIII in activated macrophages, suggesting that LTGF-beta activation occurs on the cell surface. Confocal microscopy of metabolically active macrophages incubated with antibodies to TGF-beta and betaglycan/RIII prior to fixation supported the localization of activation to the cell surface. The ability to specifically detect and localize LTGF-beta activation provides an important tool for studies of its regulation.

  13. Asporin and transforming growth factor-beta gene expression in osteoblasts from subchondral bone and osteophytes in osteoarthritis.

    PubMed

    Sakao, Kei; Takahashi, Kenji A; Arai, Yuji; Saito, Masazumi; Honjyo, Kuniaki; Hiraoka, Nobuyuki; Kishida, Tsunao; Mazda, Osam; Imanishi, Jiro; Kubo, Toshikazu

    2009-11-01

    To clarify the significance of subchondral bone and osteophytes in the pathology of osteoarthritis (OA), we investigated the expression of asporin (ASPN), transforming growth factor-beta1 (TGF-beta1), TGF-beta2, TGF-beta3, and runt-related transcription factor-2 (Runx2) genes involved in bone metabolism. Osteoblasts were isolated from 19 patients diagnosed with knee OA and from 4 patients diagnosed with femoral neck fracture. Osteoblast expression of mRNA encoding ASPN, TGF-beta1, TGF-beta2, TGF-beta3, and Runx2 was analyzed using real-time RT-PCR. Expression of ASPN, TGF-beta1, and TGF-beta3 mRNA in the subchondral bone and osteophytes of OA patients increased compared with that of non-OA patients. The ratio of ASPN to TGF-beta1 mRNA in patients with severe cartilage damage was higher than that in patients with mild cartilage damage. The increased ratio of ASPN mRNA to TGF-beta1 mRNA in patients with severe relative to mild cartilage damage indicates that increased ASPN mRNA expression was significantly associated with the severity of cartilage degeneration. This finding suggests that ASPN may regulate TGF-beta1-mediated factors in the development of OA, which may provide clues as to the underlying pathology of OA.

  14. alpha1B-Adrenergic receptor phosphorylation and desensitization induced by transforming growth factor-beta.

    PubMed Central

    Romero-Avila, M Teresa; Flores-Jasso, C Fabián; García-Sáinz, J Adolfo

    2002-01-01

    Transforming growth factor-beta (TGF-beta) induced alpha(1B)-adrenergic receptor phosphorylation in Rat-1 fibroblasts stably expressing these adrenoceptors. This effect of TGF-beta was rapid, reaching a maximum within 30 min and decreasing thereafter, and concentration-dependent (EC(50) 0.3 pM). The phosphoinositide 3-kinase inhibitors wortmannin and LY294002, and the protein kinase C inhibitors staurosporine, Ro 318220 and bisindolylmaleimide, blocked the effect of this growth factor. alpha(1B)-Adrenergic receptor phosphorylation was associated with desensitization, as indicated by a reduction in the adrenergic-mediated production of [(3)H]inositol phosphates. Phosphorylation of alpha(1B)-adrenergic receptors by TGF-beta was also observed in Cos-1 cells transfected with the receptor. Co-transfection of the dominant-negative mutant of the regulatory subunit of phosphoinositide 3-kinase (Deltap85) inhibited the phosphorylation of alpha(1B)-adrenergic receptors induced by TGF-beta. Our results indicate that activation of TGF-beta receptors induces alpha(1B)-adrenergic receptor phosphorylation and desensitization. The data suggest that phosphoinositide 3-kinase and protein kinase C play key roles in this effect of TGF-beta. PMID:12234252

  15. alpha1B-Adrenergic receptor phosphorylation and desensitization induced by transforming growth factor-beta.

    PubMed

    Romero-Avila, M Teresa; Flores-Jasso, C Fabián; García-Sáinz, J Adolfo

    2002-12-01

    Transforming growth factor-beta (TGF-beta) induced alpha(1B)-adrenergic receptor phosphorylation in Rat-1 fibroblasts stably expressing these adrenoceptors. This effect of TGF-beta was rapid, reaching a maximum within 30 min and decreasing thereafter, and concentration-dependent (EC(50) 0.3 pM). The phosphoinositide 3-kinase inhibitors wortmannin and LY294002, and the protein kinase C inhibitors staurosporine, Ro 318220 and bisindolylmaleimide, blocked the effect of this growth factor. alpha(1B)-Adrenergic receptor phosphorylation was associated with desensitization, as indicated by a reduction in the adrenergic-mediated production of [(3)H]inositol phosphates. Phosphorylation of alpha(1B)-adrenergic receptors by TGF-beta was also observed in Cos-1 cells transfected with the receptor. Co-transfection of the dominant-negative mutant of the regulatory subunit of phosphoinositide 3-kinase (Deltap85) inhibited the phosphorylation of alpha(1B)-adrenergic receptors induced by TGF-beta. Our results indicate that activation of TGF-beta receptors induces alpha(1B)-adrenergic receptor phosphorylation and desensitization. The data suggest that phosphoinositide 3-kinase and protein kinase C play key roles in this effect of TGF-beta.

  16. Maternal breast milk transforming growth factor beta and feeding intolerance in preterm infants

    PubMed Central

    Frost, Brandy L.; Jilling, Tamas; Lapin, Brittany; Maheshwari, Akhil; Caplan, Michael S.

    2015-01-01

    Background Feeding intolerance occurs commonly in the NICU. Breast milk contains a large pool of transforming growth factor-beta (TGF-beta). Few studies describe TGF-beta levels in preterm milk, and the relationship to feeding intolerance (FI) remains unexplored. We measured TGF-beta levels in preterm breast milk to investigate a correlation with FI in preterm infants. Methods Prospective observational trial of 100 mother-infant pairs, enrolling infants born below 32 weeks gestation and less than 1500 grams, and mothers who planned to provide breast milk. TGF-beta levels were measured using ELISA. Infant charts were reviewed for outcomes. Results TGF-beta declined postnatally, most elevated in colostrum (p<0.01). TGF-beta 2 levels were higher than TGF-beta 1 at all time points (p<0.01). Colostrum TGF-beta levels correlated inversely with birth weight (p<0.01) and gestational age (p<0.05). One week TGF-beta 2 levels were reduced in growth-restricted infants with FI (p<0.01). Of infants with NEC, TGF-beta 2 levels appeared low, but small sample size precluded meaningful statistical comparisons. Conclusions TGF-beta levels decline temporally in preterm milk. TGF-beta 1 colostrum levels correlate inversely with birth weight and gestational age. TGF-beta 2 may play a role in FI in growth-restricted infants. The relationship of TGF-beta 2 and NEC merits future investigation. PMID:24995914

  17. Reduced skeletal muscle inhibitor of kappaB beta content is associated with insulin resistance in subjects with type 2 diabetes: reversal by exercise training.

    PubMed

    Sriwijitkamol, Apiradee; Christ-Roberts, Christine; Berria, Rachele; Eagan, Phyllis; Pratipanawatr, Thongchai; DeFronzo, Ralph A; Mandarino, Lawrence J; Musi, Nicolas

    2006-03-01

    Skeletal muscle insulin resistance plays a key role in the pathogenesis of type 2 diabetes. It recently has been hypothesized that excessive activity of the inhibitor of kappaB (IkappaB)/nuclear factor kappaB (NFkappaB) inflammatory pathway is a mechanism underlying skeletal muscle insulin resistance. However, it is not known whether IkappaB/NFkappaB signaling in muscle from subjects with type 2 diabetes is abnormal. We studied IkappaB/NFkappaB signaling in vastus lateralis muscle from six subjects with type 2 diabetes and eight matched control subjects. Muscle from type 2 diabetic subjects was characterized by a 60% decrease in IkappaB beta protein abundance, an indicator of increased activation of the IkappaB/NFkappaB pathway. IkappaB beta abundance directly correlated with insulin-mediated glucose disposal (Rd) during a hyperinsulinemic (40 mU x m(-2) x min(-1))-euglycemic clamp (r = 0.63, P = 0.01), indicating that increased IkappaB/NFkappaB pathway activity is associated with muscle insulin resistance. We also investigated whether reversal of this abnormality could be a mechanism by which training improves insulin sensitivity. In control subjects, 8 weeks of aerobic exercise training caused a 50% increase in both IkappaB alpha and IkappaB beta protein. In subjects with type 2 diabetes, training increased IkappaB alpha and IkappaB beta protein to levels comparable with that of control subjects, and these increments were accompanied by a 40% decrease in tumor necrosis factor alpha muscle content and a 37% increase in insulin-stimulated glucose disposal. In summary, subjects with type 2 diabetes have reduced IkappaB protein abundance in muscle, suggesting excessive activity of the IkappaB/NFkappaB pathway. Moreover, this abnormality is reversed by exercise training.

  18. Pulsational mode-typing in line profile variables. I - Four Beta Cephei stars

    NASA Technical Reports Server (NTRS)

    Campos, A. J.; Smith, M. A.

    1980-01-01

    The detailed variations of line profiles in the Beta Cephei-type variable stars Gamma Pegasi, Beta Cephei, Delta Ceti and Sigma Scorpii are modeled throughout their pulsation cycles in order to classify the dominant pulsation mode as radial or nonradial. High-dispersion Reticon observations of the variables were obtained for the Si III line at 4567 A, and line profiles broadened by radial or nonradial pulsations, rotation and radial-tangential macroturbulence were calculated based on a model atmosphere. It is found that only a radial pulsation mode can reproduce the radial velocity amplitude, changes in line asymmetry and uniform line width observed in all four stars. Results are in agreement with the color-to-light arguments of Stamford and Watson (1978), and suggest that radial pulsation plays the dominant role in the observed variations in most Beta Cephei stars. Evidence for shocks or moving shells is also found in visual line data for Sigma Scorpii and an ultraviolet line of Beta Cephei, together with evidence of smooth, secular period changes in Beta Cephei and Delta Ceti.

  19. A negative feedback control of transforming growth factor-beta signaling by glycogen synthase kinase 3-mediated Smad3 linker phosphorylation at Ser-204.

    PubMed

    Millet, Caroline; Yamashita, Motozo; Heller, Mary; Yu, Li-Rong; Veenstra, Timothy D; Zhang, Ying E

    2009-07-24

    Through the action of its membrane-bound type I receptor, transforming growth factor-beta (TGF-beta) elicits a wide range of cellular responses that regulate cell proliferation, differentiation, and apo ptosis. Many of these signaling responses are mediated by Smad proteins. As such, controlling Smad activity is crucial for proper signaling by TGF-beta and its related factors. Here, we show that TGF-beta induces phosphorylation at three sites in the Smad3 linker region in addition to the two C-terminal residues, and glycogen synthase kinase 3 is responsible for phosphorylation at one of these sites, namely Ser-204. Alanine substitution at Ser-204 and/or the neighboring Ser-208, the priming site for glycogen synthase kinase 3 in vivo activity, strengthened the affinity of Smad3 to CREB-binding protein, suggesting that linker phosphorylation may be part of a negative feedback loop that modulates Smad3 transcriptional activity. Thus, our findings reveal a novel aspect of the Smad3 signaling mechanism that controls the final amplitude of cellular responses to TGF-beta.

  20. Analysis of interleukin (IL)-1 beta and transforming growth factor (TGF)-beta-induced signal transduction pathways in IL-2 and TGF-beta secretion and proliferation in the thymoma cell line EL4.NOB-1.

    PubMed

    Siese, A; Jaros, P P; Willig, A

    1999-02-01

    In the present study we investigated the interleukin (IL)-1beta and transforming growth factor-beta1 (TGF-beta1)-mediated proliferation, and production of IL-2 and TGF-beta, in the murine T-cell line, EL4.NOB-1. This cell line is resistant to TGF-beta concerning growth arrest but not autoinduction or suppression of IL-1-induced IL-2 production. When cocultured with IL-1beta, TGF-beta showed growth-promoting activity that could be antagonized by adding the phosphatidyl choline-dependent phospholipase C (PC-PLC) inhibitor, D609. Using specific enzyme inhibitors of protein kinases (PK) C and A, mitogen-activated protein kinase (MAPK), phospholipase A2 (PLA2), phosphatidylinositol-dependent (PI)-PLC and PC-PLC, we showed that IL-1beta-induced IL-2 synthesis was dependent on all investigated kinases and phospholipases, except PC-PLC. TGF-beta1 was able to inhibit IL-2 synthesis by the activation of PKA and MAPK. The same kinases are involved in TGF-beta autoinduction that is accompanied by a secretion of the active but not the latent growth factor and is antagonized by IL-1beta. Addition of the PI-PLC inhibitor, ET 18OCH3, or the PLA2 inhibitor (quinacrine) alone, resulted in secretion of latent TGF-beta and, in the case of ET 18OCH3, active TGF-beta. These data implicate a role for PI-PLC and PLA2 in the control of latency and secretion. Analysis of specific tyrosine activity and c-Fos expression showed synergistic but no antagonistic effects. These events are therefore not involved in IL- and TGF-beta-regulated IL-2 and TGF-beta production, but might participate in IL-1/TGF-beta-induced growth promotion.

  1. Dynamics of beta-cell turnover: evidence for beta-cell turnover and regeneration from sources of beta-cells other than beta-cell replication in the HIP rat.

    PubMed

    Manesso, Erica; Toffolo, Gianna M; Saisho, Yoshifumi; Butler, Alexandra E; Matveyenko, Aleksey V; Cobelli, Claudio; Butler, Peter C

    2009-08-01

    Type 2 diabetes is characterized by hyperglycemia, a deficit in beta-cells, increased beta-cell apoptosis, and islet amyloid derived from islet amyloid polypeptide (IAPP). These characteristics are recapitulated in the human IAPP transgenic (HIP) rat. We developed a mathematical model to quantify beta-cell turnover and applied it to nondiabetic wild type (WT) vs. HIP rats from age 2 days to 10 mo to establish 1) whether beta-cell formation is derived exclusively from beta-cell replication, or whether other sources of beta-cells (OSB) are present, and 2) to what extent, if any, there is attempted beta-cell regeneration in the HIP rat and if this is through beta-cell replication or OSB. We conclude that formation and maintenance of adult beta-cells depends largely ( approximately 80%) on formation of beta-cells independent from beta-cell duplication. Moreover, this source adaptively increases in the HIP rat, implying attempted beta-cell regeneration that substantially slows loss of beta-cell mass.

  2. MOON for neutrino-less {beta}{beta} decays and {beta}{beta} nuclear matrix elements

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ejiri, H.

    2009-11-09

    The MOON project aims at spectroscopic 0v{beta}{beta} studies with the v-mass sensitivity of 100-30 meV by measuring two beta rays from {sup 100}Mo and/or {sup 82}Se. The detector is a compact super-module of multi-layer PL scintillator plates. R and D works made by the pro to-type MOON-1 and the small PL plate show the possible energy resolution of around {sigma}{approx}2.2%, as required for the mass sensitivity. Nuclear matrix elements M{sup 2v} for 2v{beta}{beta} are shown to be given by the sum {sigma}{sub L}M{sub k} of the 2v{beta}{beta} matrix elements M{sub k} through intermediate quasi-particle states in the Fermi-surface, where Mimore » is obtained experimentally by using the GT(J{sup {pi}} = 1{sup +}) matrix elements of M{sub i}(k) and M{sub f}(k) for the successive single-{beta} transitions through the k-th intermediate state.« less

  3. Protective effects of melittin on transforming growth factor-{beta}1 injury to hepatocytes via anti-apoptotic mechanism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Woo-Ram; Park, Ji-Hyun; Kim, Kyung-Hyun

    Melittin is a cationic, hemolytic peptide that is the main toxic component in the venom of the honey bee (Apis mellifera). Melittin has multiple effects, including anti-bacterial, anti-viral and anti-inflammatory, in various cell types. However, the anti-apoptotic mechanisms of melittin have not been fully elucidated in hepatocytes. Apoptosis contributes to liver inflammation and fibrosis. Knowledge of the apoptotic mechanisms is important to develop new and effective therapies for treatment of cirrhosis, portal hypertension, liver cancer, and other liver diseases. In the present study, we investigated the anti-apoptotic effect of melittin on transforming growth factor (TGF)-{beta}1-induced apoptosis in hepatocytes. TGF-{beta}1-treated hepatocytesmore » were exposed to low doses (0.5 and 1 {mu}g/mL) and high dose (2 {mu}g/mL) of melittin. The low doses significantly protected these cells from DNA damage in TGF-{beta}1-induced apoptosis compared to the high dose. Also, melittin suppressed TGF-{beta}1-induced apoptotic activation of the Bcl-2 family and caspase family of proteins, which resulted in the inhibition of poly-ADP-ribose polymerase (PARP) cleavage. These results demonstrate that TGF-{beta}1 induces hepatocyte apoptosis and that an optimal dose of melittin exerts anti-apoptotic effects against TGF-{beta}1-induced injury to hepatocytes via the mitochondrial pathway. These results suggest that an optimal dose of melittin can serve to protect cells against TGF-{beta}1-mediated injury. - Highlights: > We investigated the anti-apoptotic effect of melittin on TGF-{beta}1-induced hepatocyte. > TGF-{beta}1 induces hepatocyte apoptosis. > TGF-{beta}1-treated hepatocytes were exposed to low doses and high dose of melittin. > Optimal dose of melittin exerts anti-apoptotic effects to hepatocytes.« less

  4. Are genetic variants in the platelet-derived growth factor [beta] gene associated with chronic pancreatitis?

    PubMed

    Muddana, Venkata; Park, James; Lamb, Janette; Yadav, Dhiraj; Papachristou, Georgios I; Hawes, Robert H; Brand, Randall; Slivka, Adam; Whitcomb, David C

    2010-11-01

    Platelet-derived growth factor [beta] (PDGF-[beta]) is a major signal in proliferation and matrix synthesis through activated pancreatic stellate cells, leading to fibrosis of the pancreas. Recurrent acute pancreatitis (RAP) seems to predispose to chronic pancreatitis (CP) in some patients but not others. We tested the hypothesis that 2 known PDGF-[beta] polymorphisms are associated with progression from RAP to CP. We also tested the hypothesis that PDGF-[beta] polymorphisms in combination with environmental risk factors such as alcohol and smoking are associated with CP. Three hundred eighty-two patients with CP (n = 176) and RAP (n = 206) and 251 controls were evaluated. Platelet-derived growth factor [beta] polymorphisms +286 A/G (rs#1800818) seen in 5'-UTR and +1135 A/C (rs#1800817) in first intron were genotyped using single-nucleotide polymorphism polymerase chain reaction approach and confirmed by DNA sequencing. The genotypic frequencies for PDGF-[beta] polymorphisms in positions +286 and +1135 were found to be similar in controls and patients with RAP and CP. There was no difference in genotypic frequencies among RAP, CP, and controls in subjects in the alcohol and smoking subgroups. Known variations in the PDGF-[beta] gene do not have a significant effect on promoting or preventing fibrogenesis in pancreatitis. Further evaluation of this important pathway is warranted.

  5. The association of GSK3 beta with E2F1 facilitates nerve growth factor-induced neural cell differentiation.

    PubMed

    Zhou, Fangfang; Zhang, Long; Wang, Aijun; Song, Bo; Gong, Kai; Zhang, Lihai; Hu, Min; Zhang, Xiufang; Zhao, Nanming; Gong, Yandao

    2008-05-23

    It is widely acknowledged that E2F1 and GSK3beta are both involved in the process of cell differentiation. However, the relationship between E2F1 and GSK3beta in cell differentiation has yet to be discovered. Here, we provide evidence that in the differentiation of PC12 cells induced by nerve growth factor (NGF), GSK3beta was increased at both the mRNA and protein levels, whereas E2F1 at these two levels was decreased. Both wild-type GSK3beta and its kinase-defective mutant GSK3beta KM can inhibit E2F1 by promoting its ubiquitination through physical interaction. In addition, the colocalization of GSK3beta and E2F1 and their subcellular distribution, regulated by NGF, were observed in the process of PC12 differentiation. At the tissue level, GSK3beta colocalized and interacted with E2F1 in mouse hippocampus. Furthermore, GSK3beta facilitated neurite outgrowth by rescuing the promoter activities of Cdk inhibitors p21 and p15 from the inhibition caused by E2F1. To summarize, our findings suggest that GSK3beta can promote the ubiquitination of E2F1 via physical interaction and thus inhibit its transcription activity in a kinase activity independent manner, which plays an important role in the NGF-induced PC12 differentiation.

  6. Peroxisome proliferator-activated receptor gamma and transforming growth factor-beta pathways inhibit intestinal epithelial cell growth by regulating levels of TSC-22.

    PubMed

    Gupta, Rajnish A; Sarraf, Pasha; Brockman, Jeffrey A; Shappell, Scott B; Raftery, Laurel A; Willson, Timothy M; DuBois, Raymond N

    2003-02-28

    Peroxisome proliferator-activated receptor gamma (PPARgamma) and transforming growth factor-beta (TGF-beta) are key regulators of epithelial cell biology. However, the molecular mechanisms by which either pathway induces growth inhibition and differentiation are incompletely understood. We have identified transforming growth factor-simulated clone-22 (TSC-22) as a target gene of both pathways in intestinal epithelial cells. TSC-22 is member of a family of leucine zipper containing transcription factors with repressor activity. Although little is known regarding its function in mammals, the Drosophila homolog of TSC-22, bunched, plays an essential role in fly development. The ability of PPARgamma to induce TSC-22 was not dependent on an intact TGF-beta1 signaling pathway and was specific for the gamma isoform. Localization studies revealed that TSC-22 mRNA is enriched in the postmitotic epithelial compartment of the normal human colon. Cells transfected with wild-type TSC-22 exhibited reduced growth rates and increased levels of p21 compared with vector-transfected cells. Furthermore, transfection with a dominant negative TSC-22 in which both repressor domains were deleted was able to reverse the p21 induction and growth inhibition caused by activation of either the PPARgamma or TGF-beta pathways. These results place TSC-22 as an important downstream component of PPARgamma and TGF-beta signaling during intestinal epithelial cell differentiation.

  7. [Association of beta 3-adrenergic receptor gene with obesity in patients with type 2 diabetes mellitus].

    PubMed

    Chen, Y; Xu, Y; Zhou, L

    2001-09-01

    To investigate the association between the mutation of beta 3-adrenergoc receptor gene and obesity in patients with type 2 diabetes. Body mass, waist-hip ratio, blood pressure and blood lipids were measured in 154 type 2 diabetic patients. Polymerase chain reaction and the restriction fragment length polymorphism analysis were used to determine the wild, heterozygous and homozygous forms of beta 3-adrenergoc receptor gene. The frequency of the Trp64Arg mutation was 42.5% and the frequency of Arg64 allele was 22.6%. The mutation frequency of the genetic types was significantly different between the obese and non-obese type 2 diabetes mellitus patients. The body mass, systolic blood pressure, diastolic blood pressure, HDL-cholesterol were significantly different, when those with Trp64Arg heterozygous were compared with those with Trp64 homozygous. The genetic mutation of beta 3-adrenegoc receptor in patients with type 2 diabetes is probably related to obesity.

  8. The pleiotropic roles of transforming growth factor beta inhomeostasis and carcinogenesis of endocrine organs.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fleisch, Markus C.; Maxwell, Christopher A.; Barcellos-Hoff,Mary-Helen

    2006-01-13

    Transforming growth factor beta (TGF-beta) is a ubiquitous cytokine that plays a critical role in numerous pathways regulating cellular and tissue homeostasis. TGF-beta is regulated by hormones and is a primary mediator of hormone response in uterus, prostate and mammary gland. This review will address the role of TGF-beta in regulating hormone dependent proliferation and morphogenesis. The subversion of TGF-beta regulation during the processes of carcinogenesis, with particular emphasis on its effects on genetic stability and epithelial to mesenchymal transition (EMT), will also be examined. An understanding of the multiple and complex mechanisms of TGF-beta regulation of epithelial function, andmore » the ultimate loss of TGF-beta function during carcinogenesis, will be critical in the design of novel therapeutic interventions for endocrine-related cancers.« less

  9. Nuclear factor erythroid 2-related factor 2 deletion impairs glucose tolerance and exacerbates hyperglycemia in type 1 diabetic mice.

    PubMed

    Aleksunes, Lauren M; Reisman, Scott A; Yeager, Ronnie L; Goedken, Michael J; Klaassen, Curtis D

    2010-04-01

    The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) induces a battery of cytoprotective genes after oxidative stress. Nrf2 aids in liver regeneration by altering insulin signaling; however, whether Nrf2 participates in hepatic glucose homeostasis is unknown. Compared with wild-type mice, mice lacking Nrf2 (Nrf2-null) have lower basal serum insulin and prolonged hyperglycemia in response to an intraperitoneal glucose challenge. In the present study, blood glucose, serum insulin, urine flow rate, and hepatic expression of glucose-related genes were quantified in male diabetic wild-type and Nrf2-null mice. Type 1 diabetes was induced with a single intraperitoneal dose (200 mg/kg) of streptozotocin (STZ). Histopathology and serum insulin levels confirmed depleted pancreatic beta-cells in STZ-treated mice of both genotypes. Five days after STZ, Nrf2-null mice had higher blood glucose levels than wild-type mice. Nine days after STZ, polyuria occurred in both genotypes with more urine output from Nrf2-null mice (11-fold) than wild-type mice (7-fold). Moreover, STZ-treated Nrf2-null mice had higher levels of serum beta-hydroxybutyrate, triglycerides, and fatty acids 10 days after STZ compared with wild-type mice. STZ reduced hepatic glycogen in both genotypes, with less observed in Nrf2-null mice. Increased urine output and blood glucose in STZ-treated Nrf2-null mice corresponded with enhanced gluconeogenesis (glucose-6-phosphatase and phosphoenolpyruvate carboxykinase)- and reduced glycolysis (pyruvate kinase)-related mRNA expression in their livers. Furthermore, the Nrf2 activator oltipraz lowered blood glucose in wild-type but not Nrf2-null mice administered STZ. Collectively, these data indicate that the absence of Nrf2 worsens hyperglycemia in type I diabetic mice and Nrf2 may represent a therapeutic target for reducing circulating glucose levels.

  10. Transforming growth factor-beta1 mediates cellular response to DNA damage in situ

    NASA Technical Reports Server (NTRS)

    Ewan, Kenneth B.; Henshall-Powell, Rhonda L.; Ravani, Shraddha A.; Pajares, Maria Jose; Arteaga, Carlos; Warters, Ray; Akhurst, Rosemary J.; Barcellos-Hoff, Mary Helen

    2002-01-01

    Transforming growth factor (TGF)-beta1 is rapidly activated after ionizing radiation, but its specific role in cellular responses to DNA damage is not known. Here we use Tgfbeta1 knockout mice to show that radiation-induced apoptotic response is TGF-beta1 dependent in the mammary epithelium, and that both apoptosis and inhibition of proliferation in response to DNA damage decrease as a function of TGF-beta1 gene dose in embryonic epithelial tissues. Because apoptosis in these tissues has been shown previously to be p53 dependent, we then examined p53 protein activation. TGF-beta1 depletion, by either gene knockout or by using TGF-beta neutralizing antibodies, resulted in decreased p53 Ser-18 phosphorylation in irradiated mammary gland. These data indicate that TGF-beta1 is essential for rapid p53-mediated cellular responses that mediate cell fate decisions in situ.

  11. The effect of pasteurization on transforming growth factor alpha and transforming growth factor beta 2 concentrations in human milk.

    PubMed

    McPherson, R J; Wagner, C L

    2001-01-01

    Transforming growth factor alpha (TGF-alpha) and beta 2 (TGF-beta2) are present in human milk and are involved in growth differentiation and repair of neonatal intestinal epithelia. Heat treatment at 56 degrees C has been shown effective for providing safe banked donor milk, with good retention of other biologically active factors. The purpose of our study was to determine the effect of heat sterilization on TGF-alpha and TGF-beta2 concentrations in human milk. Twenty milk samples were collected from 20 lactating mothers in polypropylene containers and frozen at -20 degrees C for transport or storage. Before heat treatment by holder pasteurization, the frozen milk was thawed and divided into 1-mL aliquots. All samples were heated in an accurately regulated water bath until a holding temperature was achieved, then held for 30 minutes using constant agitation. Holding temperature ranged from 56.5 degrees C to 56.9 degrees C. The milk was then stored at 4 degrees C overnight for analysis the following day. The concentration of TGF-alpha was measured by radioimmunoassay. Mean concentration +/- SD of TGF-alpha in raw milk samples was 119+/-50 pg/mL, range 57 to 234. The mean concentration +/- SD of TGF-alpha in heat treated samples was 113+/-50 pg/mL, range 51 to 227. TGF-alpha concentration was minimally affected by pasteurization, with an overall loss of 6.1%. Of 19 samples, 4 had increased and 15 had decreased concentrations after pasteurization (mean percent SEM: 94%+/-7% of raw milk, range 72%+/-107%). The concentration of acid-activated TGF-beta2 was measured by enzyme-linked immunosorbent assay. Mean concentration +/- SD of TGF-beta2 in raw milk samples was 5624+/-5038 pg/mL, range 195 to 15480. The mean concentration +/- SD of TGF-beta2 in heat-treated samples was 5073+/-4646 pg/mL, range 181 to 15140. TGF-beta2 survived with relatively little loss (0.6%): of 18 samples, 11 had increased and 7 had decreased concentrations after pasteurization (mean percent

  12. Phospholipase C-mediated hydrolysis of phosphatidylcholine is a target of transforming growth factor beta 1 inhibitory signals.

    PubMed Central

    Diaz-Meco, M T; Dominguez, I; Sanz, L; Municio, M M; Berra, E; Cornet, M E; Garcia de Herreros, A; Johansen, T; Moscat, J

    1992-01-01

    Cell growth and tumor transformation can be restrained in certain cell systems by the action of transforming growth factor beta (TGF-beta). It has been established that the mechanism whereby TGF-beta 1 inhibits cell growth does not interfere with the triggering of early mitogenic signal transduction mechanisms. Phospholipase C-catalyzed hydrolysis of phosphatidylcholine (PC) is a relatively late step in the cascade activated by growth factors. Therefore, conceivably activation of phospholipase C-catalyzed hydrolysis of PC could be the target of TGF-beta 1 action. In the study reported here, we demonstrate that TGF-beta 1 inhibits the coupling of ras p21 to the activation of PC hydrolysis, which appears to be critical for the antiproliferative effects of TGF-beta 1. Images PMID:1309592

  13. Metabolic Stress and Compromised Identity of Pancreatic Beta Cells.

    PubMed

    Swisa, Avital; Glaser, Benjamin; Dor, Yuval

    2017-01-01

    Beta cell failure is a central feature of type 2 diabetes (T2D), but the molecular underpinnings of the process remain only partly understood. It has been suggested that beta cell failure in T2D involves massive cell death. Other studies ascribe beta cell failure to cell exhaustion, due to chronic oxidative or endoplasmic reticulum stress leading to cellular dysfunction. More recently it was proposed that beta cells in T2D may lose their differentiated identity, possibly even gaining features of other islet cell types. The loss of beta cell identity appears to be driven by glucotoxicity inhibiting the activity of key beta cell transcription factors including Pdx1, Nkx6.1, MafA and Pax6, thereby silencing beta cell genes and derepressing alternative islet cell genes. The loss of beta cell identity is at least partly reversible upon normalization of glycemia, with implications for the reversibility of T2D, although it is not known if beta cell failure reaches eventually a point of no return. In this review we discuss current evidence for metabolism-driven compromised beta cell identity, key knowledge gaps and opportunities for utility in the treatment of T2D.

  14. Pleiotrophin regulates lung epithelial cell proliferation and differentiation during fetal lung development via beta-catenin and Dlk1.

    PubMed

    Weng, Tingting; Gao, Li; Bhaskaran, Manoj; Guo, Yujie; Gou, Deming; Narayanaperumal, Jeyaparthasarathy; Chintagari, Narendranath Reddy; Zhang, Kexiong; Liu, Lin

    2009-10-09

    The role of pleiotrophin in fetal lung development was investigated. We found that pleiotrophin and its receptor, protein-tyrosine phosphatase receptor beta/zeta, were highly expressed in mesenchymal and epithelial cells of the fetal lungs, respectively. Using isolated fetal alveolar epithelial type II cells, we demonstrated that pleiotrophin promoted fetal type II cell proliferation and arrested type II cell trans-differentiation into alveolar epithelial type I cells. Pleiotrophin also increased wound healing of injured type II cell monolayer. Knockdown of pleiotrophin influenced lung branching morphogenesis in a fetal lung organ culture model. Pleiotrophin increased the tyrosine phosphorylation of beta-catenin, promoted beta-catenin translocation into the nucleus, and activated T cell factor/lymphoid enhancer factor transcription factors. Dlk1, a membrane ligand that initiates the Notch signaling pathway, was identified as a downstream target of the pleiotrophin/beta-catenin pathway by endogenous dlk1 expression, promoter assay, and chromatin immunoprecipitation. These results provide evidence that pleiotrophin regulates fetal type II cell proliferation and differentiation via integration of multiple signaling pathways including pleiotrophin, beta-catenin, and Notch pathways.

  15. Role of transcription factor KLF11 and its diabetes-associated gene variants in pancreatic beta cell function

    PubMed Central

    Neve, Bernadette; Fernandez-Zapico, Martin E.; Ashkenazi-Katalan, Vered; Dina, Christian; Hamid, Yasmin H.; Joly, Erik; Vaillant, Emmanuel; Benmezroua, Yamina; Durand, Emmanuelle; Bakaher, Nicolas; Delannoy, Valerie; Vaxillaire, Martine; Cook, Tiffany; Dallinga-Thie, Geesje M.; Jansen, Hans; Charles, Marie-Aline; Clément, Karine; Galan, Pilar; Hercberg, Serge; Helbecque, Nicole; Charpentier, Guillaume; Prentki, Marc; Hansen, Torben; Pedersen, Oluf; Urrutia, Raul; Melloul, Danielle; Froguel, Philippe

    2005-01-01

    KLF11 (TIEG2) is a pancreas-enriched transcription factor that has elicited significant attention because of its role as negative regulator of exocrine cell growth in vitro and in vivo. However, its functional role in the endocrine pancreas remains to be established. Here, we report, for the first time, to our knowledge, the characterization of KLF11 as a glucose-inducible regulator of the insulin gene. A combination of random oligonucleotide binding, EMSA, luciferase reporter, and chromatin immunoprecipitation assays shows that KLF11 binds to the insulin promoter and regulates its activity in beta cells. Genetic analysis of the KLF11 gene revealed two rare variants (Ala347Ser and Thr220Met) that segregate with diabetes in families with early-onset type 2 diabetes, and significantly impair its transcriptional activity. In addition, analysis of 1,696 type 2 diabetes mellitus and 1,776 normoglycemic subjects show a frequent polymorphic Gln62Arg variant that significantly associates with type 2 diabetes mellitus in North European populations (OR = 1.29, P = 0.00033). Moreover, this variant alters the corepressor mSin3A-binding activity of KLF11, impairs the activation of the insulin promoter and shows lower levels of insulin expression in pancreatic beta cells. In addition, subjects carrying the Gln62Arg allele show decreased plasma insulin after an oral glucose challenge. Interestingly, all three nonsynonymous KLF11 variants show increased repression of the catalase 1 promoter, suggesting a role in free radical clearance that may render beta cells more sensitive to oxidative stress. Thus, both functional and genetic analyses reveal that KLF11 plays a role in the regulation of pancreatic beta cell physiology, and its variants may contribute to the development of diabetes. PMID:15774581

  16. BetaTPred: prediction of beta-TURNS in a protein using statistical algorithms.

    PubMed

    Kaur, Harpreet; Raghava, G P S

    2002-03-01

    beta-turns play an important role from a structural and functional point of view. beta-turns are the most common type of non-repetitive structures in proteins and comprise on average, 25% of the residues. In the past numerous methods have been developed to predict beta-turns in a protein. Most of these prediction methods are based on statistical approaches. In order to utilize the full potential of these methods, there is a need to develop a web server. This paper describes a web server called BetaTPred, developed for predicting beta-TURNS in a protein from its amino acid sequence. BetaTPred allows the user to predict turns in a protein using existing statistical algorithms. It also allows to predict different types of beta-TURNS e.g. type I, I', II, II', VI, VIII and non-specific. This server assists the users in predicting the consensus beta-TURNS in a protein. The server is accessible from http://imtech.res.in/raghava/betatpred/

  17. Immunohistochemical detection of active transforming growth factor-beta in situ using engineered tissue

    NASA Technical Reports Server (NTRS)

    Barcellos-Hoff, M. H.; Ehrhart, E. J.; Kalia, M.; Jirtle, R.; Flanders, K.; Tsang, M. L.; Chatterjee, A. (Principal Investigator)

    1995-01-01

    The biological activity of transforming growth factor-beta 1 (TGF-beta) is governed by dissociation from its latent complex. Immunohistochemical discrimination of active and latent TGF-beta could provide insight into TGF-beta activation in physiological and pathological processes. However, evaluation of immunoreactivity specificity in situ has been hindered by the lack of tissue in which TGF-beta status is known. To provide in situ analysis of antibodies to differentiate between these functional forms, we used xenografts of human tumor cells modified by transfection to overexpress latent TGF-beta or constitutively active TGF-beta. This comparison revealed that, whereas most antibodies did not differentiate between TGF-beta activation status, the immunoreactivity of some antibodies was activation dependent. Two widely used peptide antibodies to the amino-terminus of TGF-beta, LC(1-30) and CC(1-30) showed marked preferential immunoreactivity with active TGF-beta versus latent TGF-beta in cryosections. However, in formalin-fixed, paraffin-embedded tissue, discrimination of active TGF-beta by CC(1-30) was lost and immunoreactivity was distinctly extracellular, as previously reported for this antibody. Similar processing-dependent extracellular localization was found with a neutralizing antibody raised to recombinant TGF-beta. Antigen retrieval recovered cell-associated immunoreactivity of both antibodies. Two antibodies to peptides 78-109 showed mild to moderate preferential immunoreactivity with active TGF-beta only in paraffin sections. LC(1-30) was the only antibody tested that discriminated active from latent TGF-beta in both frozen and paraffin-embedded tissue. Thus, in situ discrimination of active versus latent TGF-beta depends on both the antibody and tissue preparation. We propose that tissues engineered to express a specific form of a given protein provide a physiological setting in which to evaluate antibody reactivity with specific functional forms of a

  18. Functional cloning of the proto-oncogene brain factor-1 (BF-1) as a Smad-binding antagonist of transforming growth factor-beta signaling.

    PubMed

    Rodriguez, C; Huang, L J; Son, J K; McKee, A; Xiao, Z; Lodish, H F

    2001-08-10

    Using the plasminogen activator inhibitor (PAI) promoter to drive the expression of a reporter gene (mouse CD2), we devised a system to clone negative regulators of the transforming growth factor-beta (TGF-beta) signaling pathway. We infected a TGF-beta-responsive cell line (MvLu1) with a retroviral cDNA library, selecting by fluorescence-activated cell sorter single cells displaying low PAI promoter activity in response to TGF-beta. Using this strategy we cloned the proto-oncogene brain factor-1 (BF-1). BF-1 represses the PAI promoter in part by associating with both unphosphorylated Smad3 (in the cytoplasm) and phosphorylated Smad3 (in the nucleus), thus preventing its binding to DNA. BF-1 also associates with Smad1, -2, and -4; the Smad MH2 domain binds to BF-1, and the C-terminal segment of BF-1 is uniquely and solely required for binding to Smads. Further, BF-1 represses another TGF-beta-induced promoter (p15), it up-regulates a TGF-beta-repressed promoter (Cyclin A), and it reverses the growth arrest caused by TGF-beta. Our results suggest that BF-1 is a general inhibitor of TGF-beta signaling and as such may play a key role during brain development.

  19. Inhibitory effects of hepatocyte growth factor and interleukin-6 on transforming growth factor-beta1 mediated vocal fold fibroblast-myofibroblast differentiation.

    PubMed

    Vyas, Bimal; Ishikawa, Keiko; Duflo, Suzy; Chen, Xia; Thibeault, Susan L

    2010-05-01

    The role of myofibroblasts in vocal fold scarring has not been extensively studied, partly because of the lack of a robust in vitro model. The objective of this investigation was to develop and characterize a myofibroblast in vitro model that could be utilized to investigate the molecular mechanism of myofibroblast differentiation and function in injured vocal fold tissue. Differentiation of human primary vocal fold fibroblasts (hVFFs) to myofibroblasts was stimulated with 5, 10, or 20 ng/mL of recombinant transforming growth factor-beta1 (TGF-beta1). Cultures were analyzed by immunofluorescence and Western blotting, with an alpha-smooth muscle actin (alpha-SMA) antibody used as a myofibroblast marker. Normal rabbit vocal folds were treated with 10 ng/mL of TGF-beta1 for 7 days for in vivo corroboration. The effects of interleukin-6 (IL-6) and hepatocyte growth factor (HGF) on myofibroblast differentiation were studied with Western blots. The hVFFs demonstrated positive alpha-SMA labeling in cells stimulated by 10 and 20 ng/mL TGF-beta1, indicating that hVFFs were capable of differentiation to myofibroblasts. Transforming growth factor-beta1 induced the largest increase in alpha-SMA at 10 ng/mL on day 5 of treatment. Both HGF and IL-6 suppressed the expression of TGF-beta1-induced alpha-SMA. Our work characterizes a useful in vitro model of TGF-beta1-mediated vocal fold fibroblast-myofibroblast differentiation. The extent of differentiation appears to be attenuated by HGF, suggesting a potential mechanism to support prior work indicating that HGF plays a protective role in reducing scar formation in vocal fold injuries. Paradoxically, IL-6, which has been shown to play a profibrotic role in dermal studies, also attenuated the TGF-beta1 response.

  20. Immune Interventions to Preserve Beta Cell Function in Type 1 Diabetes

    PubMed Central

    Ehlers, Mario R.

    2015-01-01

    Type 1 diabetes (T1D) is a chronic autoimmune disease that leads to destruction of pancreatic beta cells, lifelong dependence on insulin, and increased morbidity and mortality from diabetes-related complications. Preservation of residual beta cells at diagnosis is a major goal because higher levels of endogenous insulin secretion are associated with better short- and long-term outcomes. Over the past 3 decades, a variety of immune interventions have been evaluated in the setting of new-onset T1D, including nonspecific immunosuppression, pathway-specific immune modulation, antigen-specific therapies, and cellular therapies. To date, no single intervention has produced durable remission off-therapy in the majority of treated patients, but the field has gained valuable insights into disease mechanisms and potential immunologic correlates of success. In particular, T cell-directed therapies, including therapies that lead to partial depletion or modulation of effector T (Teff) cells and preservation or augmentation of regulatory T (Treg) cells, have shown the most success and will likely form the backbone of future approaches. The next phase will see evaluation of rational combinations, comprising one or more of the following: a Teff-depleting or modulating drug, a cytokine-based tolerogenic (Treg-promoting) agent, and an antigen-specific component. The long-term goal is to reestablish immunologic tolerance to beta cells, thereby preserving residual beta cells early after diagnosis or enabling restoration of beta cell mass from autologous stem cells or induced neogenesis in patients with established T1D. PMID:26225763

  1. Embryonic expression of the transforming growth factor beta ligand and receptor genes in chicken.

    PubMed

    Cooley, James R; Yatskievych, Tatiana A; Antin, Parker B

    2014-03-01

    Transforming growth factor-beta (TGFβ) signaling regulates a myriad of biological processes during embryogenesis, in the adult, and during the manifestation of disease. TGFβ signaling is propagated through one of three TGFβ ligands interacting with Type I and Type II receptors, and Type III co-receptors. Although TGFβ signaling is regulated partly by the combinatorial expression patterns of TGFβ receptors and ligands, a comprehensive gene expression analysis has not been published. Here we report the embryonic mRNA expression patterns in chicken embryos of the canonical TGFβ ligands (TGFB1, TGFB2, and TGFB3) and receptors (TGFBR1, TGFBR2, TGFBR3), plus the Activin A receptor, type 1 (ACVR1) and co receptor Endoglin (ENG) that also transduce TGFβ signaling. TGFB ligands and receptors show dynamic and frequently overlapping expression patterns in numerous embryonic cell layers and structures. Integrating expression information identifies combinations of ligands and receptors that are involved in specific developmental processes including somitogenesis, cardiogenesis and vasculogenesis. Copyright © 2013 Wiley Periodicals, Inc.

  2. Transforming growth factor beta mediates the progesterone suppression of an epithelial metalloproteinase by adjacent stroma in the human endometrium.

    PubMed Central

    Bruner, K L; Rodgers, W H; Gold, L I; Korc, M; Hargrove, J T; Matrisian, L M; Osteen, K G

    1995-01-01

    Unlike most normal adult tissues, cyclic growth and tissue remodeling occur within the uterine endometrium throughout the reproductive years. The matrix metalloproteinases (MMPs), a family of structurally related enzymes that degrade specific components of the extracellular matrix are thought to be the physiologically relevant mediators of extracellular matrix composition and turnover. Our laboratory has identified MMPs of the stromelysin family in the cycling human endometrium, implicating these enzymes in mediating the extensive remodeling that occurs in this tissue. While the stromelysins are expressed in vivo during proliferation-associated remodeling and menstruation-associated endometrial breakdown, none of the stromelysins are expressed during the progesterone-dominated secretory phase of the cycle. Our in vitro studies of isolated cell types have confirmed progesterone suppression of stromal MMPs, but a stromal-derived paracrine factor was found necessary for suppression of the epithelial-specific MMP matrilysin. In this report, we demonstrate that transforming growth factor beta (TGF-beta) is produced by endometrial stroma in response to progesterone and can suppress expression of epithelial matrilysin independent of progesterone. Additionally, we find that an antibody directed against the mammalian isoforms of TGF-beta abolishes progesterone suppression of matrilysin in stromal-epithelial cocultures, implicating TGF-beta as the principal mediator of matrilysin suppression in the human endometrium. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7638197

  3. Transforming growth factor-beta1 accelerates resorption of a calcium carbonate biomaterial in periodontal defects.

    PubMed

    Koo, Ki-Tae; Susin, Cristiano; Wikesjö, Ulf M E; Choi, Seong-Ho; Kim, Chong-Kwan

    2007-04-01

    In a previous study, recombinant human transforming growth factor-beta1 (rhTGF-beta(1)) in a calcium carbonate carrier was implanted into critical-size, supraalveolar periodontal defects under conditions for guided tissue regeneration (GTR) to study whether rhTGF-beta(1) would enhance or accelerate periodontal regeneration. The results showed minimal benefits of rhTGF-beta(1), and a clear account for this could not be offered. One potential cause may be that the rhTGF-beta(1) formulation was biologically inactive. Several growth or differentiation factors have been suggested to accelerate degradation of biomaterials used as carriers. The objective of this study was to evaluate possible activity of rhTGF-beta(1) on biodegradation of the calcium carbonate carrier. rhTGF-beta(1) in a putty-formulated particulate calcium carbonate carrier was implanted into critical-size, supraalveolar periodontal defects under conditions for GTR in five beagle dogs. Contralateral defects received the calcium carbonate carrier combined with GTR without rhTGF-beta(1) (control). The animals were euthanized at week 4 post-surgery and block biopsies of the defect sites were collected for histologic and histometric analysis. Radiographs were obtained at defect creation and weeks 2 and 4 after defect creation. No statistically significant differences were observed in new bone formation (bone height and area) among the treatments. However, total residual carrier was significantly reduced in sites receiving rhTGF-beta(1) compared to control (P = 0.04). Similarly, carrier density was considerably reduced in sites receiving rhTGF-beta(1) compared to control; the difference was borderline statistically significant (P = 0.06). Within the limitations of the study, it may be concluded that rhTGF-beta(1) accelerates biodegradation of a particulate calcium carbonate biomaterial, indicating a biologic activity of the rhTGF-beta(1) formulation apparently not encompassing enhanced or accelerated

  4. Environmental Risk Factors and Type 1 Diabetes: Past, Present, and Future.

    PubMed

    Butalia, Sonia; Kaplan, Gilaad G; Khokhar, Bushra; Rabi, Doreen M

    2016-12-01

    Type 1 diabetes is an autoimmune condition that results from the destruction of the insulin-producing beta cells of the pancreas. The excess morbidity and mortality resulting from its complications, coupled with its increasing incidence, emphasize the importance of better understanding the causes of this condition. Over the past several decades, a substantive amount of work has been done and, although many advances have occurred in identifying disease-susceptibility genes, there has been a lag in understanding the environmental triggers. Several putative environmental risk factors have been proposed, including infections, dietary factors, air pollution, vaccines, location of residence, family environment and stress. However, most of these factors have been inconclusive, thus supporting the need for further study into the causes of type 1 diabetes. Copyright © 2016 Canadian Diabetes Association. Published by Elsevier Inc. All rights reserved.

  5. MicroRNA-26a modulates transforming growth factor beta-1-induced proliferation in human fetal lung fibroblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Xiaoou; Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan; Liu, Lian

    2014-11-28

    Highlights: • Endogenous miR-26a inhibits TGF-beta 1 induced proliferation of lung fibroblasts. • miR-26a induces G1 arrest through directly targeting 3′-UTR of CCND2. • TGF indispensable receptor, TGF-beta R I, is regulated by miR-26a. • miR-26a acts through inhibiting TGF-beta 2 feedback loop to reduce TGF-beta 1. • Collagen type I and connective tissue growth factor are suppressed by miR-26a. - Abstract: MicroRNA-26a is a newly discovered microRNA that has a strong anti-tumorigenic capacity and is capable of suppressing cell proliferation and activating tumor-specific apoptosis. However, whether miR-26a can inhibit the over-growth of lung fibroblasts remains unclear. The relationship betweenmore » miR-26a and lung fibrosis was explored in the current study. We first investigated the effect of miR-26a on the proliferative activity of human lung fibroblasts with or without TGF-beta1 treatment. We found that the inhibition of endogenous miR-26a promoted proliferation and restoration of mature miR-26a inhibited the proliferation of human lung fibroblasts. We also examined that miR-26a can block the G1/S phase transition via directly targeting 3′-UTR of CCND2, degrading mRNA and decreasing protein expression of Cyclin D2. Furthermore, we showed that miR-26a mediated a TGF-beta 2-TGF-beta 1 feedback loop and inhibited TGF-beta R I activation. In addition, the overexpression of miR-26a also significantly suppressed the TGF-beta 1-interacting-CTGF–collagen fibrotic pathway. In summary, our studies indicated an essential role of miR-26a in the anti-fibrotic mechanism in TGF-beta1-induced proliferation in human lung fibroblasts, by directly targeting Cyclin D2, regulating TGF-beta R I as well as TGF-beta 2, and suggested the therapeutic potential of miR-26a in ameliorating lung fibrosis.« less

  6. An animal source for the ROB-1 beta-lactamase of Haemophilus influenzae type b.

    PubMed

    Medeiros, A A; Levesque, R; Jacoby, G A

    1986-02-01

    The most common cause of ampicillin resistance in Haemophilus influenzae type b is production of TEM-1 beta-lactamase; however, a novel enzyme with a similar substrate profile but a quite different isoelectric point has also been described. This beta-lactamase, designated ROB-1, has not been found previously in any other organism. In a survey of 46 ampicillin-resistant H. influenzae type b isolates, we found a second human isolate that produces ROB-1 and discovered that ampicillin-resistant isolates of the porcine pathogen Haemophilus pleuropneumoniae also produced ROB-1. In both Haemophilus species ROB-1 production was determined by plasmids that had considerable DNA sequence homology. However, the ROB-1 and TEM-1 beta-lactamase genes were not related. Our findings suggest that this form of ampicillin resistance has an animal reservoir and that conditions fostering its prevalence in animal strains may play a role in the spread of resistance to human pathogens.

  7. Osteoblast gene expression is differentially regulated by TGF-beta isoforms.

    PubMed

    Fagenholz, P J; Warren, S M; Greenwald, J A; Bouletreau, P J; Spector, J A; Crisera, F E; Longaker, M T

    2001-03-01

    The transforming growth factor beta (TGF-beta) superfamily encompasses a number of important growth factors including several TGF-beta isoforms, the bone morphogenetic proteins, activins, inhibins, and growth and differentiation factors. TGF-beta 1, -beta 2, and -beta 3 are three closely related isoforms that are widely expressed during skeletal morphogenesis and bone repair. Numerous studies suggest that each isoform has unique in vivo functions; however, the effects of these TGF-beta isoforms on osteoblast gene expression and maturation have never been directly compared. In the current study, we treated undifferentiated neonatal rat calvaria osteoblast-enriched cell cultures with 2.5 ng/ml of each TGF-beta isoform and analyzed gene expression at 0, 3, 6, and 24 hours. We demonstrated unique isoform-specific regulation of endogenous TGF-beta 1 and type I collagen mRNA transcription. To assess the effects of extended TGF-beta treatment on osteoblast maturation, we differentiated osteoblast cultures in the presence of 2.5 ng/ml of each TGF-beta isoform. Analysis of collagen I, alkaline phosphatase, and osteocalcin demonstrated that each TGF-beta isoform uniquely suppressed the transcription of these osteoblast differentiation markers. Interestingly, TGF-beta isoform treatment increased osteopontin expression in primary osteoblasts after 4 and 10 days of differentiation. To our knowledge, these data provide the first direct comparison of the effects of the TGF-beta isoforms on osteoblast gene expression in vitro. Furthermore, these data suggest that TGF-beta isoforms may exert their unique in vivo effects by differentially regulating osteoblast cytokine secretion, extracellular matrix production, and the rate of cellular maturation.

  8. Metabolic Stress and Compromised Identity of Pancreatic Beta Cells

    PubMed Central

    Swisa, Avital; Glaser, Benjamin; Dor, Yuval

    2017-01-01

    Beta cell failure is a central feature of type 2 diabetes (T2D), but the molecular underpinnings of the process remain only partly understood. It has been suggested that beta cell failure in T2D involves massive cell death. Other studies ascribe beta cell failure to cell exhaustion, due to chronic oxidative or endoplasmic reticulum stress leading to cellular dysfunction. More recently it was proposed that beta cells in T2D may lose their differentiated identity, possibly even gaining features of other islet cell types. The loss of beta cell identity appears to be driven by glucotoxicity inhibiting the activity of key beta cell transcription factors including Pdx1, Nkx6.1, MafA and Pax6, thereby silencing beta cell genes and derepressing alternative islet cell genes. The loss of beta cell identity is at least partly reversible upon normalization of glycemia, with implications for the reversibility of T2D, although it is not known if beta cell failure reaches eventually a point of no return. In this review we discuss current evidence for metabolism-driven compromised beta cell identity, key knowledge gaps and opportunities for utility in the treatment of T2D. PMID:28270834

  9. A polymorphic region in the human transcription factor AP-2beta gene is associated with specific personality traits.

    PubMed

    Damberg, M; Garpenstrand, H; Alfredsson, J; Ekblom, J; Forslund, K; Rylander, G; Oreland, L

    2000-03-01

    Transcription factor AP-2beta is implicated in playing an important role during embryonic development of different parts of the brain, eg, midbrain, hindbrain, spinal cord, dorsal and cranial root ganglia.1,2 The gene encoding AP-2beta contains a polymorphic region which includes a tetranucleotide repeat of [CAAA] four or five times, located in intron 2 between nucleotides 12593 and 12612.3 Since the midbrain contains structures important for variables such as mood and personality, we have investigated if the AP-2beta genotype is associated with personality traits estimated by the Karolinska Scales of Personality (KSP). Identification of transcription factor genes as candidate genes in psychiatric disorders is a novel approach to further elucidate the genetic factors that, together with environmental factors, are involved in the expression of specific psychiatric phenotypes. The AP-2beta genotype and KSP scores were determined for 137 Caucasian volunteers (73 females and 64 males). The personality traits muscular tension, guilt, somatic anxiety, psychastenia and indirect aggression were significantly associated with the specific AP-2beta genotype, albeit with significant difference between genders. Based on this result the human AP-2beta gene seems to be an important candidate gene for personality disorders. Moreover, the present results suggest that the structure of the intron 2 region of the AP-2beta gene is one factor that contributes to development of the constitutional component of specific personality traits.

  10. Expression and structural features of endoglin (CD105), a transforming growth factor beta1 and beta3 binding protein, in human melanoma.

    PubMed Central

    Altomonte, M.; Montagner, R.; Fonsatti, E.; Colizzi, F.; Cattarossi, I.; Brasoveanu, L. I.; Nicotra, M. R.; Cattelan, A.; Natali, P. G.; Maio, M.

    1996-01-01

    Human endoglin (CD105) is a member of the transforming growth factor beta (TGF-beta) receptor family that binds TGF-beta1 and -beta3, but not TGF-beta2, on human endothelial cells. Immunohistochemical analyses demonstrated that CD105 is expressed on normal and neoplastic cells of the melanocytic lineage. The anti-CD105 MAb, MAEND3, stained 50, 25 and 34% of intradermal naevi, primary and metastatic melanomas investigated, respectively, and nine out of 12 melanoma cell lines. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that CD105 expressed by melanoma cells consists of a homodimeric protein with an apparent molecular weight of 180 and 95 kDa under non-reducing and reducing conditions. Cross-linking of 125I-labelled TGF-beta1 to melanoma cells, Mel 97, by disuccinimidyl suberate (DSS) demonstrated that CD105 expressed on pigmented cells binds TGF-beta1; the pattern of binding of TGF-beta1 to melanoma cells was found to be similar to that of human umbilical vein endothelial cells. The addition of exogenous, bioactive TGF-beta1 significantly (P<0.05) inhibited the growth of CD105-positive melanoma cells, Mel 97, but did not affect that of CD105-negative melanoma cells, F0-1. These data, altogether, demonstrate that CD105 is expressed on pigmented cells and might play a functionally relevant role in the biology of human melanoma cells by regulating their sensitivity to TGF-betas. Images Figure 1 Figure 3 Figure 4 PMID:8932339

  11. Association of T-cell reactivity with beta-cell function in recent onset type 1 diabetes patients.

    PubMed

    Pfleger, Christian; Meierhoff, Guido; Kolb, Hubert; Schloot, Nanette C

    2010-03-01

    The aim of the current study was to investigate whether autoantigen directed T-cell reactivity relates to beta-cell function during the first 78 weeks after diagnosis of type 1 diabetes. 50 adults and 49 children (mean age 27.3 and 10.9 years respectively) with recent onset type 1 diabetes who participated in a placebo-controlled trial of immune intervention with DiaPep277 were analyzed. Secretion of interferon (IFN)-gamma, interleukin (IL)-5, IL-13 and IL-10 by single peripheral mononuclear cells (PBMC) upon stimulation with islet antigens GAD65, heat shock protein 60 (Hsp60) protein-tyrosine-phosphatase-like-antigen (pIA2) or tetanus toxoid (TT) was determined applying ELISPOT; beta-cell function was evaluated by glucagon stimulated C-peptide. Multivariate regression analysis was applied. In general, number of islet antigen-reactive cells decreased over 78 weeks in both adults and children, whereas reactivity to TT was not reduced. In addition, there was an association between the quality of immune cell responses and beta-cell function. Overall, increased responses by IFN-gamma secreting cells were associated with lower beta-cell function whereas IL-5, IL-13 and IL-10 cytokine responses were positively associated with beta-cell function in adults and children. Essentially, the same results were obtained with three different models of regression analysis. The number of detectable islet-reactive immune cells decreases within 1-2 years after diagnosis of type 1 diabetes. Cytokine production by antigen-specific PBMC reactivity is related to beta-cell function as measured by stimulated C-peptide. Cellular immunity appears to regress soon after disease diagnosis and begin of insulin therapy. Copyright 2009 Elsevier Ltd. All rights reserved.

  12. Latent transforming growth factor beta1 activation in situ: quantitative and functional evidence after low-dose gamma-irradiation

    NASA Technical Reports Server (NTRS)

    Ehrhart, E. J.; Segarini, P.; Tsang, M. L.; Carroll, A. G.; Barcellos-Hoff, M. H.; Chatterjee, A. (Principal Investigator)

    1997-01-01

    The biological activity of transforming growth factor beta1 (TGF-beta) is controlled by its secretion as a latent complex in which it is noncovalently associated with latency-associated peptide (LAP). Activation is the extracellular process in which TGF-beta is released from LAP, and is considered to be a primary regulatory control. We recently reported rapid and persistent changes in TGF-beta immunoreactivity in conjunction with extracellular matrix remodeling in gamma-irradiated mouse mammary gland. Our hypothesis is that these specific changes in immunoreactivity are indicative of latent TGF-beta activation. In the present study, we determined the radiation dose response and tested whether a functional relationship exists between radiation-induced TGF-beta and collagen type III remodeling. After radiation exposures as low as 0.1 Gy, we detected increased TGF-beta immunoreactivity in the mammary epithelium concomitant with decreased LAP immunostaining, which are events consistent with activation. Quantitative image analysis demonstrated a significant (P=0.0005) response at 0.1 Gy without an apparent threshold and a linear dose response to 5 Gy. However, in the adipose stroma, loss of LAP demonstrated a qualitative threshold at 0.5 Gy. Loss of LAP paralleled induction of collagen III immunoreactivity in this tissue compartment. We tested whether TGF-beta mediates collagen III expression by treating animals with TGF-beta panspecific monoclonal antibody, 1D11.16, administered i.p. shortly before irradiation. Radiation-induced collagen III staining in the adipose stroma was blocked in an antibody dose-dependent manner, which persisted through 7 days postirradiation. RNase protection assay revealed that radiation-induced elevation of total gland collagen III mRNA was also blocked by neutralizing antibody treatment. These data provide functional confirmation of the hypothesis that radiation exposure leads to latent TGF-beta activation, support our interpretation of the

  13. Effect of transforming growth factor-beta and growth differentiation factor-5 on proliferation and matrix production by human bone marrow stromal cells cultured on braided poly lactic-co-glycolic acid scaffolds for ligament tissue engineering.

    PubMed

    Jenner, J M G Th; van Eijk, F; Saris, D B F; Willems, W J; Dhert, W J A; Creemers, Laura B

    2007-07-01

    Tissue engineering of ligaments based on biomechanically suitable biomaterials combined with autologous cells may provide a solution for the drawbacks associated with conventional graft material. The aim of the present study was to investigate the contribution of recombinant human transforming growth factor beta 1 (rhTGF-beta1) and growth differentiation factor (GDF)-5, known for their role in connective tissue regeneration, to proliferation and matrix production by human bone marrow stromal cells (BMSCs) cultured onto woven, bioabsorbable, 3-dimensional (3D) poly(lactic-co-glycolic acid) scaffolds. Cells were cultured for 12 days in the presence or absence of these growth factors at different concentrations. Human BMSCs attached to the suture material, proliferated, and synthesized extracellular matrix rich in collagen type I and collagen III. No differentiation was demonstrated toward cartilage or bone tissue. The addition of rhTGF-beta1 (1-10 ng/mL) and GDF-5 (10-100 ng/mL) increased cell content (p < 0.05), but only TGF-beta1 also increased total collagen production (p < 0.05) and collagen production per cell, which is a parameter indicating differentiation. In conclusion, stimulation with rhTGF-beta1, and to a lesser extent with GDF-5, can modulate human BMSCs toward collagenous soft tissue when applied to a 3D hybrid construct. The use of growth factors could play an important role in the improvement of ligament tissue engineering.

  14. Plasmid-mediated AmpC-type beta-lactamase isolated from Klebsiella pneumoniae confers resistance to broad-spectrum beta-lactams, including moxalactam.

    PubMed Central

    Horii, T; Arakawa, Y; Ohta, M; Ichiyama, S; Wacharotayankun, R; Kato, N

    1993-01-01

    Klebsiella pneumoniae NU2936 was isolated from a patient and was found to produce a plasmid-encoded beta-lactamase (MOX-1) which conferred resistance to broad spectrum beta-lactams, including moxalactam, flomoxef, ceftizoxime, cefotaxime, and ceftazidime. Resistance could be transferred from K. pneumoniae NU2936 to Escherichia coli CSH2 by conjugation with a transfer frequency of 5 x 10(-7). The structural gene of MOX-1 (blaMOX-1) was cloned and expressed in E. coli HB101. The MIC of moxalactam for E. coli HB101 producing MOX-1 was > 512 micrograms/ml. The apparent molecular mass and pI of this enzyme were calculated to be 38 kDa and 8.9, respectively. Hg2+ and Cu2+ failed to block enzyme activity, and the presence of EDTA in the reaction buffer did not reduce the enzyme activity. However, clavulanate and cloxacillin, serine beta-lactamase inhibitors, inhibited the enzyme activity competitively (Kis = 5.60 and 0.35 microM, respectively). The kinetic study of MOX-1 suggested that it effectively hydrolyzed broad-spectrum beta-lactams. A hybridization study confirmed that blaMOX-1 is encoded on a large resident plasmid (pRMOX1; 180 kb) of strain NU2936. By deletion analysis, the functional region was localized within a 1.2-kb region of the plasmid. By amino acid sequencing, 18 of 33 amino acid residues at the N terminus of MOX-1 were found to be identical to those of Pseudomonas aeruginosa AmpC. These findings suggest that MOX-1 is a plasmid-mediated AmpC-type beta-lactamase that provides enteric bacteria resistance to broad-spectrum beta-lactams, including moxalactam. Images PMID:8517725

  15. Glucocorticoids enhance activation of the human type II 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase gene.

    PubMed

    Feltus, F Alex; Cote, Stephanie; Simard, Jacques; Gingras, Sebastien; Kovacs, William J; Nicholson, Wendell E; Clark, Barbara J; Melner, Michael H

    2002-09-01

    Glucocorticoids indirectly alter adrenocortical steroid output through the inhibition of ACTH secretion by the anterior pituitary. However, previous studies suggest that glucocorticoids can directly affect adrenocortical steroid production. Therefore, we have investigated the ability of glucocorticoids to affect transcription of adrenocortical steroid biosynthetic enzymes. One potential target of glucocorticoid action in the adrenal is an enzyme critical for adrenocortical steroid production: 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD). Treatment of the adrenocortical cell line (H295R) with the glucocorticoid agonist dexamethasone (DEX) increased cortisol production and 3beta-HSD mRNA levels alone or in conjunction with phorbol ester. This increase in 3beta-HSD mRNA was paralleled by increases in Steroidogenic Acute Regulatory Protein (StAR) mRNA levels. The human type II 3beta-HSD promoter lacks a consensus palindromic glucocorticoid response element (GRE) but does contain a Stat5 response element (Stat5RE) suggesting that glucocorticoids could affect type II 3beta-HSD transcription via interaction with Stat5. Transfection experiments show enhancement of human type II 3beta-HSD promoter activity by coexpression of the glucocorticoid receptor (GR) and Stat5A and treatment with 100nM dexamethasone. Furthermore, removal of the Stat5RE either by truncation of the 5' flanking sequence in the promoter or introduction of point mutations to the Stat5RE abolished the ability of DEX to enhance 3beta-HSD promoter activity. These studies demonstrate the ability of glucocorticoids to directly enhance the expression of an adrenal steroidogenic enzyme gene albeit independent of a consensus palindromic glucocorticoid response element.

  16. Arsenic Exposure and Calpain-10 Polymorphisms Impair the Function of Pancreatic Beta-Cells in Humans: A Pilot Study of Risk Factors for T2DM

    PubMed Central

    Díaz-Villaseñor, Andrea; Cruz, Laura; Cebrián, Arturo; Hernández-Ramírez, Raúl U.; Hiriart, Marcia; García-Vargas, Gonzálo; Bassol, Susana; Sordo, Monserrat; Gandolfi, A. Jay; Klimecki, Walter T.; López-Carillo, Lizbeth; Cebrián, Mariano E.; Ostrosky-Wegman, Patricia

    2013-01-01

    The incidence of type 2 diabetes mellitus (T2DM) is increasing worldwide and diverse environmental and genetic risk factors are well recognized. Single nucleotide polymorphisms (SNPs) in the calpain-10 gene (CAPN-10), which encodes a protein involved in the secretion and action of insulin, and chronic exposure to inorganic arsenic (iAs) through drinking water have been independently associated with an increase in the risk for T2DM. In the present work we evaluated if CAPN-10 SNPs and iAs exposure jointly contribute to the outcome of T2DM. Insulin secretion (beta-cell function) and insulin sensitivity were evaluated indirectly through validated indexes (HOMA2) in subjects with and without T2DM who have been exposed to a gradient of iAs in their drinking water in northern Mexico. The results were analyzed taking into account the presence of the risk factor SNPs SNP-43 and -44 in CAPN-10. Subjects with T2DM had significantly lower beta-cell function and insulin sensitivity. An inverse association was found between beta-cell function and iAs exposure, the association being more pronounced in subjects with T2DM. Subjects without T2DM who were carriers of the at-risk genotype SNP-43 or -44, also had significantly lower beta-cell function. The association of SNP-43 with beta-cell function was dependent on iAs exposure, age, gender and BMI, whereas the association with SNP-44 was independent of all of these factors. Chronic exposure to iAs seems to be a risk factor for T2DM in humans through the reduction of beta-cell function, with an enhanced effect seen in the presence of the at-risk genotype of SNP-43 in CAPN-10. Carriers of CAPN-10 SNP-44 have also shown reduced beta-cell function. PMID:23349674

  17. Discovery of Novel Drugs To Improve Bone Health in Neurofibromatosis Type 1: The Wnt/Beta-Catenin Pathway in Fracture Repair and Pseudarthrosis

    DTIC Science & Technology

    2014-06-01

    Bone Health in Neurofibromatosis Type 1: The Wnt/Beta-Catenin Pathway in Fracture Repair and Pseudarthrosis PRINCIPAL INVESTIGATOR...Award Number: W81XWH-13-1-0113 TITLE: Discovery of Novel Drugs To Improve Bone Health in Neurofibromatosis Type 1: The Wnt/Beta-Catenin...31 May 2014 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Discovery of Novel Drugs To Improve Bone Health in Neurofibromatosis Type 1: The Wnt/Beta

  18. Ultrasound measurement of peripheral endothelial dysfunction in type 2 diabetic patients: correlation with risk factors

    PubMed Central

    Bosevski, Marijan; Georgievska-Ismail, Ljubica

    2010-01-01

    The purpose of the study was to assess the endothelial dysfunction (ED) in type 2 diabetic patients ultrasonographicaly and estimate the correlation of ED with glycemia and other cardio-metabolic risk factors. 171 patient (age 60,0 + 8,5 years) with diagnosed type 2 diabetes and coronary artery disease (CAD) were randomly included in a cross sectional study. B-mode ultrasound system with a linear transducer of 7.5 MHz was used for evaluation of flow-mediated vasodilation in brachial artery (FMV). FMV was presented as a change of brachial artery diameter at rest and after limb ischemia, previously provoked by cuff inflation. Peripheral ED was found in 77,2% (132 patients). Multivariate logistic regression model defined: age (OR 1,071, 95% CI 1,003 1,143) and plasma cholesterol (OR 4,083 95%CI 1,080 17,017) as determinants for ED. Linear multivariate analysis presented duration of diabetes (Beta 0,173, Sig 0,024), and glycemia (Beta 0,132, Sig 0,044) to be associated independently with FMV value. Estimated factors influencing FMV might be potential therapeutic targets for presented endothelial dysfunction in type 2 diabetic patients with coronary artery disease. PMID:20507285

  19. Expression of collagen and related growth factors in rat tendon and skeletal muscle in response to specific contraction types.

    PubMed

    Heinemeier, K M; Olesen, J L; Haddad, F; Langberg, H; Kjaer, M; Baldwin, K M; Schjerling, P

    2007-08-01

    Acute exercise induces collagen synthesis in both tendon and muscle, indicating an adaptive response in the connective tissue of the muscle-tendon unit. However, the mechanisms of this adaptation, potentially involving collagen-inducing growth factors (such as transforming growth factor-beta-1 (TGF-beta-1)), as well as enzymes related to collagen processing, are not clear. Furthermore, possible differential effects of specific contraction types on collagen regulation have not been investigated. Female Sprague-Dawley rats were subjected to 4 days of concentric, eccentric or isometric training (n = 7-9 per group) of the medial gastrocnemius, by stimulation of the sciatic nerve. RNA was extracted from medial gastrocnemius and Achilles tendon tissue 24 h after the last training bout, and mRNA levels for collagens I and III, TGF-beta-1, connective tissue growth factor (CTGF), lysyl oxidase (LOX), metalloproteinases (MMP-2 and -9) and their inhibitors (TIMP-1 and 2) were measured by Northern blotting and/or real-time PCR. In tendon, expression of TGF-beta-1 and collagens I and III (but not CTGF) increased in response to all types of training. Similarly, enzymes/factors involved in collagen processing were induced in tendon, especially LOX (up to 37-fold), which could indicate a loading-induced increase in cross-linking of tendon collagen. In skeletal muscle, a similar regulation of gene expression was observed, but in contrast to the tendon response, the effect of eccentric training was significantly greater than the effect of concentric training on the expression of several transcripts. In conclusion, the study supports an involvement of TGF-beta-1 in loading-induced collagen synthesis in the muscle-tendon unit and importantly, it indicates that muscle tissue is more sensitive than tendon to the specific mechanical stimulus.

  20. Activation of the canonical beta-catenin pathway by histamine.

    PubMed

    Diks, Sander H; Hardwick, James C; Diab, Remco M; van Santen, Marije M; Versteeg, Henri H; van Deventer, Sander J H; Richel, Dick J; Peppelenbosch, Maikel P

    2003-12-26

    Histamine signaling is a principal regulator in a variety of pathophysiological processes including inflammation, gastric acid secretion, neurotransmission, and tumor growth. We report that histamine stimulation causes transactivation of a T cell factor/beta-catenin-responsive construct in HeLa cells and in the SW-480 colon cell line, whereas histamine did not effect transactivation of a construct containing the mutated response construct FOP. On the protein level, histamine treatment increases phosphorylation of glycogen synthase kinase 3-beta in HeLa cells, murine macrophages, and DLD-1, HT-29, and SW-480 colon cell lines. Furthermore, histamine also decreases the phosphorylated beta-catenin content in HeLa cells and murine macrophages. Finally, pharmacological inhibitors of the histamine H1 receptor counteracted histamine-induced T cell factor/beta-catenin-responsive construct transactivation and the dephosphorylation of beta-catenin in HeLa cells and in macrophages. We conclude that the canonical beta-catenin pathway acts downstream of the histamine receptor H1 in a variety of cell types. The observation that inflammatory molecules, like histamine, activate the beta-catenin pathway may provide a molecular explanation for a possible link between inflammation and cancer.

  1. Beta-diversity of ectoparasites at two spatial scales: nested hierarchy, geography and habitat type.

    PubMed

    Warburton, Elizabeth M; van der Mescht, Luther; Stanko, Michal; Vinarski, Maxim V; Korallo-Vinarskaya, Natalia P; Khokhlova, Irina S; Krasnov, Boris R

    2017-06-01

    Beta-diversity of biological communities can be decomposed into (a) dissimilarity of communities among units of finer scale within units of broader scale and (b) dissimilarity of communities among units of broader scale. We investigated compositional, phylogenetic/taxonomic and functional beta-diversity of compound communities of fleas and gamasid mites parasitic on small Palearctic mammals in a nested hierarchy at two spatial scales: (a) continental scale (across the Palearctic) and (b) regional scale (across sites within Slovakia). At each scale, we analyzed beta-diversity among smaller units within larger units and among larger units with partitioning based on either geography or ecology. We asked (a) whether compositional, phylogenetic/taxonomic and functional dissimilarities of flea and mite assemblages are scale dependent; (b) how geographical (partitioning of sites according to geographic position) or ecological (partitioning of sites according to habitat type) characteristics affect phylogenetic/taxonomic and functional components of dissimilarity of ectoparasite assemblages and (c) whether assemblages of fleas and gamasid mites differ in their degree of dissimilarity, all else being equal. We found that compositional, phylogenetic/taxonomic, or functional beta-diversity was greater on a continental rather than a regional scale. Compositional and phylogenetic/taxonomic components of beta-diversity were greater among larger units than among smaller units within larger units, whereas functional beta-diversity did not exhibit any consistent trend regarding site partitioning. Geographic partitioning resulted in higher values of beta-diversity of ectoparasites than ecological partitioning. Compositional and phylogenetic components of beta-diversity were higher in fleas than mites but the opposite was true for functional beta-diversity in some, but not all, traits.

  2. Increased T cell recruitment to the CNS after amyloid beta 1-42 immunization in Alzheimer's mice overproducing transforming growth factor-beta 1.

    PubMed

    Buckwalter, Marion S; Coleman, Bronwen S; Buttini, Manuel; Barbour, Robin; Schenk, Dale; Games, Dora; Seubert, Peter; Wyss-Coray, Tony

    2006-11-01

    Immunotherapy targeting the amyloid beta (Abeta) peptide is a novel therapy under investigation for the treatment of Alzheimer's disease (AD). A clinical trial using Abeta(1-42) (AN1792) as the immunogen was halted as a result of development of meningoencephalitis in a small number of patients. The cytokine TGF-beta1 is a key modulator of immune responses that is increased in the brain in AD. We show here that local overexpression of TGF-beta1 in the brain increases both meningeal and parenchymal T lymphocyte number. Furthermore, TGF-beta1 overexpression in a mouse model for AD [amyloid precursor protein (APP) mice] leads to development of additional T cell infiltrates when mice were immunized at a young but not old age with AN1792. Notably, only mice overproducing both Abeta (APP mice) and TGF-beta1 experienced a rise in T lymphocyte number after immunization. One-third of infiltrating T cells were CD4 positive. We did not observe significant differences in B lymphocyte numbers in any of the genotypes or treatment groups. These results demonstrate that TGF-beta1 overproduction in the brain can promote T cell infiltration, in particular after Abeta(1-42) immunization. Likewise, levels of TGF-beta1 or other immune factors in brains of AD patients may influence the response to Abeta(1-42) immunization.

  3. High accuracy prediction of beta-turns and their types using propensities and multiple alignments.

    PubMed

    Fuchs, Patrick F J; Alix, Alain J P

    2005-06-01

    We have developed a method that predicts both the presence and the type of beta-turns, using a straightforward approach based on propensities and multiple alignments. The propensities were calculated classically, but the way to use them for prediction was completely new: starting from a tetrapeptide sequence on which one wants to evaluate the presence of a beta-turn, the propensity for a given residue is modified by taking into account all the residues present in the multiple alignment at this position. The evaluation of a score is then done by weighting these propensities by the use of Position-specific score matrices generated by PSI-BLAST. The introduction of secondary structure information predicted by PSIPRED or SSPRO2 as well as taking into account the flanking residues around the tetrapeptide improved the accuracy greatly. This latter evaluated on a database of 426 reference proteins (previously used on other studies) by a sevenfold crossvalidation gave very good results with a Matthews Correlation Coefficient (MCC) of 0.42 and an overall prediction accuracy of 74.8%; this places our method among the best ones. A jackknife test was also done, which gave results within the same range. This shows that it is possible to reach neural networks accuracy with considerably less computional cost and complexity. Furthermore, propensities remain excellent descriptors of amino acid tendencies to belong to beta-turns, which can be useful for peptide or protein engineering and design. For beta-turn type prediction, we reached the best accuracy ever published in terms of MCC (except for the irregular type IV) in the range of 0.25-0.30 for types I, II, and I' and 0.13-0.15 for types VIII, II', and IV. To our knowledge, our method is the only one available on the Web that predicts types I' and II'. The accuracy evaluated on two larger databases of 547 and 823 proteins was not improved significantly. All of this was implemented into a Web server called COUDES (French acronym

  4. Beta-blocking agents in patients with insulin resistance: effects of vasodilating beta-blockers.

    PubMed

    Jacob, S; Balletshofer, B; Henriksen, E J; Volk, A; Mehnert, B; Löblein, K; Häring, H U; Rett, K

    1999-01-01

    Essential hypertension is--at least in many subjects--associated with a decrease in insulin sensitivity, while glycaemic control is (still) normal. It seems that in hypertensive patients, two major functions of insulin are impaired: there is insulin resistance of peripheral glucose uptake (primarily skeletal muscle) and insulin resistance of insulin-stimulated vasodilation. In view of some retrospective data and meta-analyses, which showed a less than expected reduction in coronary events (coronary paradox), the metabolic side effects of the antihypertensive treatment have received more attention. Many groups have shown that conventional antihypertensive treatment, both with beta-blockers and/or diuretics, decreases insulin sensitivity by various mechanisms. While low-dose diuretics seem to be free of these metabolic effects, there is no evidence for this in the beta-adrenergic blockers. However, recent metabolic studies evaluated the effects of vasodilating beta-blockers, such as dilevalol, carvedilol and celiprolol, on insulin sensitivity and the atherogenic risk factors. None of them decreased insulin sensitivity, as has been described for the beta-blockers with and without beta1 selectivity. This supports the idea that peripheral vascular resistance and peripheral blood flow play a central role in mediating the metabolic side effects of the beta-blocking agents, as the vasodilating action (either via beta2 stimulation or alpha1-blockade) seems to more than offset the detrimental effects of the blockade of beta (or beta1) receptors. Further studies are needed to elucidate the relevance of the radical scavenging properties of these agents and their connection to their metabolic effects. Therefore, the beneficial characteristics of these newer beta-adrenoreceptor blockers suggest that the vasodilating beta-blocking agents could be advantageous for hypertensive patients with insulin resistance or type 2 diabetes.

  5. Beta-and gamma-turns in proteins revisited: a new set of amino acid turn-type dependent positional preferences and potentials.

    PubMed

    Guruprasad, K; Rajkumar, S

    2000-06-01

    The number of beta-turns in a representative set of 426 protein three-dimensional crystal structures selected from the recent Protein Data Bank has nearly doubled and the number of gamma-turns in a representative set of 320 proteins has increased over seven times since the previous analysis. Beta-turns (7153) and gamma-turns (911) extracted from these proteins were used to derive a revised set of type-dependent amino acid positional preferences and potentials. Compared with previous results, the preference for proline, methionine and tryptophan has increased and the preference for glutamine, valine, glutamic acid and alanine has decreased for beta-turns. Certain new amino acid preferences were observed for both turn types and individual amino acids showed turn-type dependent positional preferences. The rationale for new amino acid preferences are discussed in the light of hydrogen bonds and other interactions involving the turns. Where main-chain hydrogen bonds of the type NH(i + 3) --> CO(i) were not observed for some beta-turns, other main-chain hydrogen bonds or solvent interactions were observed that possibly stabilize such beta-turns. A number of unexpected isolated beta-turns with proline at i + 2 position were also observed. The NH(i + 2) --> CO(i) hydrogen bond was observed for almost all gamma-turns. Nearly 20% classic gamma-turns and 43% inverse gamma-turns are isolated turns.

  6. Signal transduction by beta1 integrin receptors in human chondrocytes in vitro: collaboration with the insulin-like growth factor-I receptor.

    PubMed

    Shakibaei, M; John, T; De Souza, P; Rahmanzadeh, R; Merker, H J

    1999-09-15

    We have examined the mechanism by which collagen-binding integrins co-operate with insulin-like growth factor-I (IGF-I) receptors (IGF-IR) to regulate chondrocyte phenotype and differentiation. Adhesion of chondrocytes to anti-beta1 integrin antibodies or collagen type II leads to phosphorylation of cytoskeletal and signalling proteins localized at focal adhesions, including alpha-actinin, vinculin, paxillin and focal adhesion kinase (FAK). These stimulate docking proteins such as Shc (Src-homology collagen). Moreover, exposure of collagen type II-cultured chondrocytes to IGF-I leads to co-immunoprecipitation of Shc protein with the IGF-IR and with beta1, alpha1 and alpha5 integrins, but not with alpha3 integrin. Shc then associates with growth factor receptor-bound protein 2 (Grb2), an adaptor protein and extracellular signal-regulated kinase. The expression of the docking protein Shc occurs only when chondrocytes are bound to collagen type II or integrin antibodies and increases when IGF-I is added, suggesting a collaboration between integrins and growth factors in a common/shared biochemical signalling pathway. Furthermore, these results indicate that focal adhesion assembly may facilitate signalling via Shc, a potential common target for signal integration between integrin and growth-factor signalling regulatory pathways. Thus, the collagen-binding integrins and IGF-IR co-operate to regulate focal adhesion components and these signalling pathways have common targets (Shc-Grb2 complex) in subcellular compartments, thereby linking to the Ras-mitogen-activated protein kinase signalling pathway. These events may play a role during chondrocyte differentiation.

  7. Determination of the efficiency of commercially available dose calibrators for beta-emitters.

    PubMed

    Valley, Jean-François; Bulling, Shelley; Leresche, Michel; Wastiel, Claude

    2003-03-01

    The goals of this investigation are to determine whether commercially available dose calibrators can be used to measure the activity of beta-emitting radionuclides used in pain palliation and to establish whether manufacturer-supplied calibration factors are appropriate for this purpose. Six types of commercially available dose calibrators were studied. Dose calibrator response was controlled for 5 gamma-emitters used for calibration or typically encountered in routine use. For the 4 most commonly used beta-emitters ((32)P, (90)Sr, (90)Y, and (169)Er) dose calibrator efficiency was determined in the syringe geometry used for clinical applications. Efficiency of the calibrators was also measured for (153)Sm and (186)Re, 2 beta-emitters with significant gamma-contributions. Source activities were traceable to national standards. All calibrators measured gamma-emitters with a precision of +/-10%, in compliance with Swiss regulatory requirements. For beta-emitters, dose calibrator intrinsic efficiency depends strongly on the maximal energy of the beta-spectrum and is notably low for (169)Er. Manufacturer-supplied calibration factors give accurate results for beta-emitters with maximal beta-energy in the middle-energy range (1 MeV) but are not appropriate for use with low-energy ((169)Er) or high-energy ((90)Y) beta-emitters. beta-emitters with significant gamma-contributions behave like gamma-emitters. Commercially available dose calibrators have an intrinsic efficiency that is sufficient for the measurement of beta-emitters, including beta-emitters with a low maximum beta-energy. Manufacturer-supplied calibration factors are reliable for gamma-emitters and beta-emitters in the middle-energy range. For low- and high-energy beta-emitters, the use of manufacturer-supplied calibration factors introduces significant measurement inaccuracy.

  8. Inverse expression of estrogen receptor-beta and nuclear factor-kappaB in urinary bladder carcinogenesis.

    PubMed

    Kontos, Stylianos; Kominea, Athina; Melachrinou, Maria; Balampani, Eleni; Sotiropoulou-Bonikou, Georgia

    2010-09-01

    To investigate the expression of nuclear factor-kappaB (NF-kappaB) and estrogen receptor-beta (ER-beta) signalling pathways in bladder urothelial carcinoma according to clinicopathological features, in order to elucidate their role during carcinogenesis. Immunohistochemical methodology was carried out on formalin-fixed, paraffin-embedded sections from urinary bladder carcinomas of 140 patients (94 males and 46 females) who underwent transurethral resection of bladder neoplasms. Correlations between ER-beta and NF-kappaB, and tumor grade and T-stage were evaluated, along with demographic data, sex and age. A significant decrease in ER-beta expression in the nucleus of bladder cells during loss of cell differentiation (r(s) = -0.61, P-value < 0.001, test of trend P-value = 0.003) and in muscle invasive carcinomas (T2-T4; test of trend P-value < 0.001) was found. p65 Subunit of NF-kappaB was expressed in the nucleus and in the cytoplasm of bladder epithelial cells. A strong positive association between tumor grade and nuclear expression of NF-kappaB was shown. No correlation between NF-kappaB, nuclear or cytoplasmic staining, with T-stage was observed. An inverse correlation between ER-beta and nuclear p65 immunoreactivity was observed (r(s) = -0.45, P-value < 0.001). There was no correlation with demographic data. Our immunohistochemical study suggests the possible inverse regulation of NF-kappaB and ER-beta transcription factor during bladder carcinogenesis. Selective ER-beta agonists and agents, inhibitors of NF-kappaB, might represent a possible new treatment strategy for bladder urothelial tumors.

  9. Reversal of acute and chronic synovial inflammation by anti-transforming growth factor beta.

    PubMed

    Wahl, S M; Allen, J B; Costa, G L; Wong, H L; Dasch, J R

    1993-01-01

    Transforming growth factor beta (TGF-beta) induces leukocyte recruitment and activation, events central to an inflammatory response. In this study, we demonstrate that antagonism of TGF-beta with a neutralizing antibody not only blocks inflammatory cell accumulation, but also tissue pathology in an experimental model of chronic erosive polyarthritis. Intraarticular injection of monoclonal antibody 1D11.16, which inhibits both TGF-beta 1 and TGF-beta 2 bioactivity, into animals receiving an arthropathic dose of bacterial cell walls significantly inhibits arthritis. Inhibition was observed with a single injection of 50 micrograms antibody, and a 1-mg injection blocked acute inflammation > 75% compared with the contralateral joints injected with an irrelevant isotype control antibody (MOPC21) as quantitated by an articular index (AI = 0.93 +/- 0.23 for 1D11.16, and AI = 4.0 +/- 0 on day 4; p < 0.001). Moreover, suppression of the acute arthritis achieved with a single injection of antibody was sustained into the chronic, destructive phase of the disease (on day 18, AI = 0.93 +/- 0.07 vs. AI = 2.6 +/- 0.5; p < 0.01). The decreased inflammatory index associated with anti-TGF-beta treatment was consistent with histopathologic and radiologic evidence of a therapeutic response. These data implicate TGF-beta as a profound agonist not only in the early events responsible for synovial inflammation, but also in the chronicity of streptococcal cell wall fragment-induced inflammation culminating in destructive pathology. Interrupting the cycle of leukocyte recruitment and activation with TGF-beta antagonists may provide a mechanism for resolution of chronic destructive lesions.

  10. Reversal of acute and chronic synovial inflammation by anti- transforming growth factor beta

    PubMed Central

    1993-01-01

    Transforming growth factor beta (TGF-beta) induces leukocyte recruitment and activation, events central to an inflammatory response. In this study, we demonstrate that antagonism of TGF-beta with a neutralizing antibody not only blocks inflammatory cell accumulation, but also tissue pathology in an experimental model of chronic erosive polyarthritis. Intraarticular injection of monoclonal antibody 1D11.16, which inhibits both TGF-beta 1 and TGF-beta 2 bioactivity, into animals receiving an arthropathic dose of bacterial cell walls significantly inhibits arthritis. Inhibition was observed with a single injection of 50 micrograms antibody, and a 1-mg injection blocked acute inflammation > 75% compared with the contralateral joints injected with an irrelevant isotype control antibody (MOPC21) as quantitated by an articular index (AI = 0.93 +/- 0.23 for 1D11.16, and AI = 4.0 +/- 0 on day 4; p < 0.001). Moreover, suppression of the acute arthritis achieved with a single injection of antibody was sustained into the chronic, destructive phase of the disease (on day 18, AI = 0.93 +/- 0.07 vs. AI = 2.6 +/- 0.5; p < 0.01). The decreased inflammatory index associated with anti-TGF-beta treatment was consistent with histopathologic and radiologic evidence of a therapeutic response. These data implicate TGF-beta as a profound agonist not only in the early events responsible for synovial inflammation, but also in the chronicity of streptococcal cell wall fragment-induced inflammation culminating in destructive pathology. Interrupting the cycle of leukocyte recruitment and activation with TGF-beta antagonists may provide a mechanism for resolution of chronic destructive lesions. PMID:8418203

  11. Transforming growth factor-beta in the chicken fundal layers: an immunohistochemical study.

    PubMed

    Mathis, Ute; Schaeffel, Frank

    2010-06-01

    In the chicken model of myopia, it has first been shown that imposing defocus to the retina results in active remodelling of the sclera which, in turn, results in axial length changes of the eye. Transforming growth factor-beta (TGF-beta) is one of the scleral growth modulators but its cellular localization in the fundal layers, colocalization and function are not well known. The aim of the current study was to investigate the cellular distribution of the three isoforms TGF-beta1, 2 and 3 by immunohistochemical labelling. Furthermore, the effects of visual experience that induces refractive errors on TGF-beta2 labelling were examined. Transversal cryostat sections of the fundal layers were analyzed by indirect immunofluorescent labelling and cell counts. Visual experience was changed by having the chicks wear either diffusers, or positive or negative lenses of 7D power in front of the right eyes for various periods of time. Left eyes served as uncovered controls. All TGF-beta isoforms were localized in both scleral layers. In choroid, diffuse labelling of all isoforms was found. In retina, TGF-beta1 and 3 were detected in bipolar, amacrine and ganglion cells and TGF-beta2 in amacrine and ganglion cells. To further characterize these cells, double-labelling with known amacrine and bipolar cell markers was performed (calbindin, cellular retinoic acid binding protein (CRABP), Islet1, Lim3 and protein kinase C (PKC)). TGF-beta1, 2 and 3 could be colocalized with calbindin and CRABP in single amacrine cells. TGF-beta1-positive bipolar cells were immunoreactive to Lim3. TGF-beta1 and 3 were never colocalized with PKC in bipolar cells. Also, colocalization with peptides known to be involved in myopia development in chicks, such as glucagon, or vasointestinal polypeptide and the key enzyme for dopamine synthesis, tyrosine hydroxylase, was not observed. Lenses or diffusers, worn by the chicks for various periods of time, had no effect on TGF-beta2 immunoreactivity in

  12. Improvement of macrophage dysfunction by administration of anti-transforming growth factor-beta antibody in EL4-bearing hosts.

    PubMed

    Maeda, H; Tsuru, S; Shiraishi, A

    1994-11-01

    An experimental therapy for improvement of macrophage dysfunction caused by transforming growth factor-beta (TGF-beta) was tried in EL4 tumor-bearing mice. TGF-beta was detected in cell-free ascitic fluid from EL4-bearers, but not in that from normal mice, by western blot analysis. The ascites also showed growth-suppressive activity against Mv1Lu cells, and the suppressive activity was potentiated by transient acidification. To investigate whether the functions of peritoneal macrophages were suppressed in EL4-bearers, the abilities to produce nitric oxide and tumor necrosis factor-alpha (TNF-alpha) upon lipopolysaccharide (LPS) stimulation were measured. Both abilities of macrophages in EL4-bearing mice were suppressed remarkably on day 9, and decreased further by day 14, compared with non-tumor-bearing controls. TGF-beta activity was abrogated by administration of anti-TGF-beta antibody to EL4-bearing mice. While a large amount of TGF-beta was detected in ascitic fluid from control EL4-bearers, little TGF-beta was detectable in ascites from EL4-bearers given anti-TGF-beta antibody. Furthermore, while control macrophages exhibited little or no production of nitric oxide and TNF-alpha on LPS stimulation in vitro, macrophages from EL4-bearers administered with anti-TGF-beta antibody showed the same ability as normal macrophages. These results clearly indicate that TGF-beta contributes to macrophage dysfunction and that the administration of specific antibody for TGF-beta reverses macrophage dysfunction in EL4-bearing hosts.

  13. Transforming growth factor-beta inhibits human antigen-specific CD4+ T cell proliferation without modulating the cytokine response.

    PubMed

    Tiemessen, Machteld M; Kunzmann, Steffen; Schmidt-Weber, Carsten B; Garssen, Johan; Bruijnzeel-Koomen, Carla A F M; Knol, Edward F; van Hoffen, Els

    2003-12-01

    Transforming growth factor (TGF)-beta has been demonstrated to play a key role in the regulation of the immune response, mainly by its suppressive function towards cells of the immune system. In humans, the effect of TGF-beta on antigen-specific established memory T cells has not been investigated yet. In this study antigen-specific CD4(+) T cell clones (TCC) were used to determine the effect of TGF-beta on antigen-specific proliferation, the activation status of the T cells and their cytokine production. This study demonstrates that TGF-beta is an adequate suppressor of antigen-specific T cell proliferation, by reducing the cell-cycle rate rather than induction of apoptosis. Addition of TGF-beta resulted in increased CD69 expression and decreased CD25 expression on T cells, indicating that TGF-beta is able to modulate the activation status of in vivo differentiated T cells. On the contrary, the antigen-specific cytokine production was not affected by TGF-beta. Although TGF-beta was suppressive towards the majority of the T cells, insensitivity of a few TCC towards TGF-beta was also observed. This could not be correlated to differential expression of TGF-beta signaling molecules such as Smad3, Smad7, SARA (Smad anchor for receptor activation) and Hgs (hepatocyte growth factor-regulated tyrosine kinase substrate). In summary, TGF-beta has a pronounced inhibitory effect on antigen-specific T cell proliferation without modulating their cytokine production.

  14. Determination of transmission factors for beta radiation using Al 2O 3:C commercial OSL dosimeters

    NASA Astrophysics Data System (ADS)

    Pinto, T. N. O.; Caldas, L. V. E.

    2010-07-01

    In recent years, the optically stimulated luminescence (OSL) technique has been used in personal dosimetry, and aluminum oxide (Al 2O 3:C) has become a very useful material for this technique. The objective of this work was the determination of the transmission factors for beta radiation using Al 2O 3:C commercial dosimeters and the OSL method. The obtained results were similar to the transmission factors reported in the beta source calibration certificates.

  15. Transforming growth factor-beta 1 (TGF-beta1) promotes IL-2 mRNA expression through the up-regulation of NF-kappaB, AP-1 and NF-AT in EL4 cells.

    PubMed

    Han, S H; Yea, S S; Jeon, Y J; Yang, K H; Kaminski, N E

    1998-12-01

    Transforming growth factor beta1 (TGF-beta1) has been previously shown to modulate interleukin 2 (IL-2) secretion by activated T-cells. In the present studies, we determined that TGF-beta1 induced IL-2 mRNA expression in the murine T-cell line EL4, in the absence of other stimuli. IL-2 mRNA expression was significantly induced by TGF-beta1 (0.1-1 ng/ml) over a relatively narrow concentration range, which led to the induction of IL-2 secretion. Under identical condition, we examined the effect of TGF-beta1 on the activity of nuclear factor AT (NF-AT), nuclear factor kappaB (NF-kappaB), activator protein-1 (AP-1) and octamer, all of which contribute to the regulation of IL-2 gene expression. Electrophoretic mobility shift assays showed that TGF-beta1 markedly increased NF-AT, NF-kappaB and AP-1 binding to their respective cognate DNA binding sites, whereas octamer binding remained constant, as compared with untreated cells. Employing a reporter gene expression system with p(NF-kappaB)3-CAT, p(NF-AT)3-CAT and p(AP-1)3-CAT, TGF-beta1 treatment of transfected EL4 cells induced a dose-related increase in chloramphenicol acetyltransferase activity that correlated well with the DNA binding profile found in the electrophoretic mobility shift assay studies. These results show that TGF-beta1, in the absence of any additional stimuli, up-regulates the activity of key transcription factors involved in IL-2 gene expression, including NF-AT, NF-kappaB and AP-1, to help promote IL-2 mRNA expression by EL4 cells.

  16. Inhibition of glycogen synthase kinase 3beta during heart failure is protective.

    PubMed

    Hirotani, Shinichi; Zhai, Peiyong; Tomita, Hideharu; Galeotti, Jonathan; Marquez, Juan Pablo; Gao, Shumin; Hong, Chull; Yatani, Atsuko; Avila, Jesús; Sadoshima, Junichi

    2007-11-26

    Glycogen synthase kinase (GSK)-3, a negative regulator of cardiac hypertrophy, is inactivated in failing hearts. To examine the histopathological and functional consequence of the persistent inhibition of GSK-3beta in the heart in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative GSK-3beta (Tg-GSK-3beta-DN) and tetracycline-regulatable wild-type GSK-3beta. GSK-3beta-DN significantly reduced the kinase activity of endogenous GSK-3beta, inhibited phosphorylation of eukaryotic translation initiation factor 2B epsilon, and induced accumulation of beta-catenin and myeloid cell leukemia-1, confirming that GSK-3beta-DN acts as a dominant negative in vivo. Tg-GSK-3beta-DN exhibited concentric hypertrophy at baseline, accompanied by upregulation of the alpha-myosin heavy chain gene and increases in cardiac function, as evidenced by a significantly greater Emax after dobutamine infusion and percentage of contraction in isolated cardiac myocytes, indicating that inhibition of GSK-3beta induces well-compensated hypertrophy. Although transverse aortic constriction induced a similar increase in hypertrophy in both Tg-GSK-3beta-DN and nontransgenic mice, Tg-GSK-3beta-DN exhibited better left ventricular function and less fibrosis and apoptosis than nontransgenic mice. Induction of the GSK-3beta transgene in tetracycline-regulatable wild-type GSK-3beta mice induced left ventricular dysfunction and premature death, accompanied by increases in apoptosis and fibrosis. Overexpression of GSK-3beta-DN in cardiac myocytes inhibited tumor necrosis factor-alpha-induced apoptosis, and the antiapoptotic effect of GSK-3beta-DN was abrogated in the absence of myeloid cell leukemia-1. These results suggest that persistent inhibition of GSK-3beta induces compensatory hypertrophy, inhibits apoptosis and fibrosis, and increases cardiac contractility and that the antiapoptotic effect of GSK-3beta inhibition is mediated by myeloid cell leukemia-1. Thus

  17. TGF-{beta} receptors, in a Smad-independent manner, are required for terminal skeletal muscle differentiation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Droguett, Rebeca; Cabello-Verrugio, Claudio; Santander, Cristian

    2010-09-10

    Skeletal muscle differentiation is strongly inhibited by transforming growth factor type {beta} (TGF-{beta}), although muscle formation as well as regeneration normally occurs in an environment rich in this growth factor. In this study, we evaluated the role of intracellular regulatory Smads proteins as well as TGF-{beta}-receptors (TGF-{beta}-Rs) during skeletal muscle differentiation. We found a decrease of TGF-{beta} signaling during differentiation. This phenomenon is explained by a decline in the levels of the regulatory proteins Smad-2, -3, and -4, a decrease in the phosphorylation of Smad-2 and lost of nuclear translocation of Smad-3 and -4 in response to TGF-{beta}. No changemore » in the levels and inhibitory function of Smad-7 was observed. In contrast, we found that TGF-{beta}-R type I (TGF-{beta}-RI) and type II (TGF-{beta}-RII) increased on the cell surface during skeletal muscle differentiation. To analyze the direct role of the serine/threonine kinase activities of TGF-{beta}-Rs, we used the specific inhibitor SB 431542 and the dominant-negative form of TGF-{beta}-RII lacking the cytoplasmic domain. The TGF-{beta}-Rs were important for successful muscle formation, determined by the induction of myogenin, creatine kinase activity, and myosin. Silencing of Smad-2/3 expression by specific siRNA treatments accelerated myogenin, myosin expression, and myotube formation; although when SB 431542 was present inhibition in myosin induction and myotube formation was observed, suggesting that these last steps of skeletal muscle differentiation require active TGF-{beta}-Rs. These results suggest that both down-regulation of Smad regulatory proteins and cell signaling through the TGF-{beta} receptors independent of Smad proteins are essential for skeletal muscle differentiation.« less

  18. Beta 2 toxigenic Clostridium perfringens type A colitis in a three-day-old foal.

    PubMed

    Hazlett, Murray J; Kircanski, Jasmina; Slavic, Durda; Prescott, John F

    2011-03-01

    Beta 2 (β2)-toxigenic Clostridium perfringens type A was recovered in large numbers from the intestine of a neonatal foal with colitis. The foal had been treated with gentamicin. Necropsy revealed marked distension of cecum and colon with watery, rust-colored homogeneous fluid and gastric infarction. Microscopic colonic lesions were superficial necrosis of 50% of the colonic mucosal surface and scattered 1-3-mm ulcers with subjacent neutrophilic infiltration and large Gram-positive bacilli in the necrotic mucosa. Beta-2 toxin was demonstrated in the lesions by immunohistochemical staining.

  19. Characterization and membrane organization of beta 1----3- and beta 1----4-galactosyltransferases from human colonic adenocarcinoma cell lines Colo 205 and SW403: basis for preferential synthesis of type 1 chain lacto-series carbohydrate structures.

    PubMed

    Holmes, E H

    1989-05-01

    Evidence indicates that activation of a beta 1----3N-acetylglucosaminyltransferase is responsible for accumulation of large quantities of lacto-series tumor-associated antigens in human colonic adenocarcinomas. Expression of type 1 and 2 core chain derivatives characterize human colonic adenocarcinomas, whereas normal adult colonic epithelial cells express detectable quantities of only type 1 chain derivatives. The basis for preferential synthesis of type 1 chain lacto-series carbohydrate structures characteristic of normal colonic mucosa and human colonic adenocarcinoma Colo 205 cells has been studied. The beta 1----3- and beta 1----4galactosyltransferase enzymes associated with synthesis of type 1 and 2 core chain structures, respectively, have been separated from a Triton X-100 solubilized membrane fraction of Colo 205 cells by chromatography on an alpha-lactalbumin-Sepharose column and their properties studied. Optimal transfer of beta 1----3-linked galactose to acceptor Lc3 occurred in the presence of 0.1% Triton CF-54 with Triton X-100 providing 75% of maximal activity. The enzyme was active over a broad pH range from 6.5 to 7.5 and had a near absolute requirement for Mn2+. The Km values for donor UDPgalactose and acceptor Lc3 were determined to be 48 and 13 microM, respectively. In contrast, the beta 1----4galactosyltransferase required taurodeoxycholate for maximal activity and the Km for Lc3 was found to be 20-fold higher than that for the beta 1----3-specific enzyme under the same assay conditions. Studies with membrane-bound beta 1----3- and beta 1----4galactosyltransferases as found in Golgi-rich membrane fractions of SW403 and Colo 205 adenocarcinoma cells showed that preferential synthesis of type 1 chain structures occurs under conditions similar to those in vivo for biosynthesis of lacto-series core chains. The results suggest that both the higher affinity of the beta 1----3galactosyltransferase for acceptor Lc3 and the membrane organizational

  20. PKB/Akt modulates TGF-beta signalling through a direct interaction with Smad3.

    PubMed

    Remy, Ingrid; Montmarquette, Annie; Michnick, Stephen W

    2004-04-01

    Transforming growth factor beta (TGF-beta) has a major role in cell proliferation, differentiation and apoptosis in many cell types. Integration of the TGF-beta pathway with other signalling cascades that control the same cellular processes may modulate TGF-beta responses. Here we report the discovery of a new functional link between TGF-beta and growth factor signalling pathways, mediated by a physical interaction between the serine-threonine kinase PKB (protein kinase B)/Akt and the transcriptional activator Smad3. Formation of the complex is induced by insulin, but inhibited by TGF-beta stimulation, placing PKB-Smad3 at a point of convergence between these two pathways. PKB inhibits Smad3 by preventing its phosphorylation, binding to Smad4 and nuclear translocation. In contrast, Smad3 does not inhibit PKB. Inhibition of Smad3 by PKB occurs through a kinase-activity-independent mechanism, resulting in a decrease in Smad3-mediated transcription and protection of cells against TGF-beta-induced apoptosis. Consistently, knockdown of the endogenous PKB gene with small-interfering RNA (siRNA) has the opposite effect. Our results suggest a very simple mechanism for the integration of signals arising from growth-factor- and TGF-beta-mediated pathways.

  1. 17-Beta-estradiol inhibits transforming growth factor-beta signaling and function in breast cancer cells via activation of extracellular signal-regulated kinase through the G protein-coupled receptor 30.

    PubMed

    Kleuser, Burkhard; Malek, Daniela; Gust, Ronald; Pertz, Heinz H; Potteck, Henrik

    2008-12-01

    Breast cancer development and breast cancer progression involves the deregulation of growth factors leading to uncontrolled cellular proliferation, invasion and metastasis. Transforming growth factor (TGF)-beta plays a crucial role in breast cancer because it has the potential to act as either a tumor suppressor or a pro-oncogenic chemokine. A cross-communication between the TGF-beta signaling network and estrogens has been postulated, which is important for breast tumorigenesis. Here, we provide evidence that inhibition of TGF-beta signaling is associated with a rapid estrogen-dependent nongenomic action. Moreover, we were able to demonstrate that estrogens disrupt the TGF-beta signaling network as well as TGF-beta functions in breast cancer cells via the G protein-coupled receptor 30 (GPR30). Silencing of GPR30 in MCF-7 cells completely reduced the ability of 17-beta-estradiol (E2) to inhibit the TGF-beta pathway. Likewise, in GPR30-deficient MDA-MB-231 breast cancer cells, E2 achieved the ability to suppress TGF-beta signaling only after transfection with GPR30-encoding plasmids. It is most interesting that the antiestrogen fulvestrant (ICI 182,780), which possesses agonistic activity at the GPR30, also diminished TGF-beta signaling. Further experiments attempted to characterize the molecular mechanism by which activated GPR30 inhibits the TGF-beta pathway. Our results indicate that GPR30 induces the stimulation of the mitogen-activated protein kinases (MAPKs), which interferes with the activation of Smad proteins. Inhibition of MAPK activity prevented the ability of E2 from suppressing TGF-beta signaling. These findings are of great clinical relevance, because down-regulation of TGF-beta signaling is associated with the development of breast cancer resistance in response to antiestrogens.

  2. 11beta-Hydroxysteroid dehydrogenase Type 1: genetic polymorphisms are associated with Type 2 diabetes in Pima Indians independently of obesity and expression in adipocyte and muscle.

    PubMed

    Nair, S; Lee, Y H; Lindsay, R S; Walker, B R; Tataranni, P A; Bogardus, C; Baier, L J; Permana, P A

    2004-06-01

    The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) modulates tissue-specific glucocorticoid concentrations by generating active cortisol. We have shown that adipose tissue 11beta-HSD1 mRNA levels were associated with adiposity and insulinaemia. Here we conducted further expression and genetic association studies in Pima Indians. The 11beta-HSD1 mRNA concentrations were measured in abdominal subcutaneous adipocytes (n=61) and skeletal muscle tissues (n=64). Single nucleotide polymorphisms in the HSD11B1 gene were genotyped in a larger group of full-blooded Pima Indians. Two representative SNPs (SNP1, n=706; SNP5, n=839) were associated with Type 2 diabetes mellitus (p=0.01), although neither SNP was associated with obesity. Among subjects with normal glucose tolerance, SNP1 (n=127) and SNP5 (n=159) were associated with insulin-mediated glucose uptake rates (p=0.03 and p=0.04), and SNP1 was further associated with fasting, 30-min, and 2-h plasma insulin concentrations (p=0.002, p=0.002 and p=0.03). Adipocyte 11beta-HSD1 mRNA concentrations were correlated positively with adiposity and insulinaemia, and were additionally negatively correlated with insulin-mediated glucose uptake rates; nevertheless, the adipocyte 11beta-HSD1 expression did not correlate with genotypes of the donors. The muscle 11beta-HSD1 mRNA concentrations did not correlate with any anthropometric or metabolic variables. We confirmed that adipocyte 11beta-HSD1 mRNA concentrations were associated with adiposity, and showed that genetic variations in the HSD11B1 gene were associated with Type 2 diabetes mellitus, plasma insulin concentrations and insulin action, independent of obesity. The variable adipose expression might not be a primary consequence of these HSD11B1 SNPs. Therefore, it is possible that the HSD11B1 gene is under tissue-specific regulation, and has tissue-specific consequences.

  3. Structural Characterization of the Boca/Mesd Maturation Factors for LDL-Receptor-Type beta Propeller Domains

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    M Collins; W Hendrickson

    2011-12-31

    Folding and trafficking of low-density lipoprotein receptor (LDLR) family members, which play essential roles in development and homeostasis, are mediated by specific chaperones. The Boca/Mesd chaperone family specifically promotes folding and trafficking of the YWTD {beta} propeller-EGF domain pair found in the ectodomain of all LDLR members. Limited proteolysis, NMR spectroscopy, analytical ultracentrifugation, and X-ray crystallography were used to define a conserved core composed of a structured domain that is preceded by a disordered N-terminal region. High-resolution structures of the ordered domain were determined for homologous proteins from three metazoans. Seven independent protomers reveal a novel ferrodoxin-like superfamily fold withmore » two distinct {beta} sheet topologies. A conserved hydrophobic surface forms a dimer interface in each crystal, but these differ substantially at the atomic level, indicative of nonspecific hydrophobic interactions that may play a role in the chaperone activity of the Boca/Mesd family.« less

  4. 3-Phosphoinositide-dependent PDK1 negatively regulates transforming growth factor-beta-induced signaling in a kinase-dependent manner through physical interaction with Smad proteins.

    PubMed

    Seong, Hyun-A; Jung, Haiyoung; Kim, Kyong-Tai; Ha, Hyunjung

    2007-04-20

    We have reported previously that PDK1 physically interacts with STRAP, a transforming growth factor-beta (TGF-beta) receptor-interacting protein, and enhances STRAP-induced inhibition of TGF-beta signaling. In this study we show that PDK1 coimmunoprecipitates with Smad proteins, including Smad2, Smad3, Smad4, and Smad7, and that this association is mediated by the pleckstrin homology domain of PDK1. The association between PDK1 and Smad proteins is increased by insulin treatment but decreased by TGF-beta treatment. Analysis of the interacting proteins shows that Smad proteins enhance PDK1 kinase activity by removing 14-3-3, a negative regulator of PDK1, from the PDK1-14-3-3 complex. Knockdown of endogenous Smad proteins, including Smad3 and Smad7, by transfection with small interfering RNA produced the opposite trend and decreased PDK1 activity, protein kinase B/Akt phosphorylation, and Bad phosphorylation. Moreover, coexpression of Smad proteins and wild-type PDK1 inhibits TGF-beta-induced transcription, as well as TGF-beta-mediated biological functions, such as apoptosis and cell growth arrest. Inhibition was dose-dependent on PDK1, but no inhibition was observed in the presence of an inactive kinase-dead PDK1 mutant. In addition, confocal microscopy showed that wild-type PDK1 prevents translocation of Smad3 and Smad4 from the cytoplasm to the nucleus, as well as the redistribution of Smad7 from the nucleus to the cytoplasm in response to TGF-beta. Taken together, our results suggest that PDK1 negatively regulates TGF-beta-mediated signaling in a PDK1 kinase-dependent manner via a direct physical interaction with Smad proteins and that Smad proteins can act as potential positive regulators of PDK1.

  5. Density-dependent induction of apoptosis by transforming growth factor-beta 1 in a human ovarian carcinoma cell line.

    PubMed

    Mathieu, C; Jozan, S; Mazars, P; Côme, M G; Moisand, A; Valette, A

    1995-01-01

    Transforming growth factor-beta 1 inhibited proliferation of a human ovarian carcinoma cell line (NIH-OVCAR-3). The inhibition of NIH-OVCAR-3 cell proliferation was accompanied by a decrease in clonogenic potential, evidenced by the reduced ability of TGF-beta 1-treated NIH-OVCAR-3 cells to form colonies on a plastic substratum. This rapid decrease of clonogenic potential, which was detected 6 h after addition of TGF-beta 1 was dose-dependent (IC50 = 4 pM). Fluorescence microscopy of DAPI-stained cells supported by electron-microscopic examination showed that TGF-beta 1 induced chromatin condensation and nuclear fragmentation. In addition, oligonucleosomal-sized fragments were detected in the TGF-beta 1-treated cells. These features indicated that TGF-beta 1 induced NIH-OVCAR-3 cell death by an apoptosis-like mechanism. This TGF-beta 1 apoptotic effect was subject to modulation by cell density. It was observed that an increase in cell density (up to 20 x 10(3) cells/cm2) protected NIH-OVCAR-3 cells against apoptosis induced by TGF-beta 1. Conditioned medium from high-density cultures of NIH-OVCAR-3 cells did not inhibit apoptosis induced by TGF-beta 1 on NIH-OVCAR-3 cells cultured at low density, suggesting that the protective effect of cell density was not related to the cell secretion of a soluble survival factor.

  6. The beta Burr type X distribution properties with application.

    PubMed

    Merovci, Faton; Khaleel, Mundher Abdullah; Ibrahim, Noor Akma; Shitan, Mahendran

    2016-01-01

    We develop a new continuous distribution called the beta-Burr type X distribution that extends the Burr type X distribution. The properties provide a comprehensive mathematical treatment of this distribution. Further more, various structural properties of the new distribution are derived, that includes moment generating function and the rth moment thus generalizing some results in the literature. We also obtain expressions for the density, moment generating function and rth moment of the order statistics. We consider the maximum likelihood estimation to estimate the parameters. Additionally, the asymptotic confidence intervals for the parameters are derived from the Fisher information matrix. Finally, simulation study is carried at under varying sample size to assess the performance of this model. Illustration the real dataset indicates that this new distribution can serve as a good alternative model to model positive real data in many areas.

  7. Preadipocyte 11beta-hydroxysteroid dehydrogenase type 1 is a keto-reductase and contributes to diet-induced visceral obesity in vivo.

    PubMed

    De Sousa Peixoto, R A; Turban, S; Battle, J H; Chapman, K E; Seckl, J R; Morton, N M

    2008-04-01

    Glucocorticoid excess promotes visceral obesity and cardiovascular disease. Similar features are found in the highly prevalent metabolic syndrome in the absence of high levels of systemic cortisol. Although elevated activity of the glucocorticoid-amplifying enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) within adipocytes might explain this paradox, the potential role of 11beta-HSD1 in preadipocytes is less clear; human omental adipose stromal vascular (ASV) cells exhibit 11beta-dehydrogenase activity (inactivation of glucocorticoids) probably due to the absence of cofactor provision by hexose-6-phosphate dehydrogenase. To clarify the depot-specific impact of 11beta-HSD1, we assessed whether preadipocytes in ASV from mesenteric (as a representative of visceral adipose tissue) and sc tissue displayed 11beta-HSD1 activity in mice. 11beta-HSD1 was highly expressed in freshly isolated ASV cells, predominantly in preadipocytes. 11beta-HSD1 mRNA and protein levels were comparable between ASV and adipocyte fractions in both depots. 11beta-HSD1 was an 11beta-reductase, thus reactivating glucocorticoids in ASV cells, consistent with hexose-6-phosphate dehydrogenase mRNA expression. Unexpectedly, glucocorticoid reactivation was higher in intact mesenteric ASV cells despite a lower expression of 11beta-HSD1 mRNA and protein (homogenate activity) levels than sc ASV cells. This suggests a novel depot-specific control over 11beta-HSD1 enzyme activity. In vivo, high-fat diet-induced obesity was accompanied by increased visceral fat preadipocyte differentiation in wild-type but not 11beta-HSD1(-/-) mice. The results suggest that 11beta-HSD1 reductase activity is augmented in mouse mesenteric preadipocytes where it promotes preadipocyte differentiation and contributes to visceral fat accumulation in obesity.

  8. DACH1 inhibits transforming growth factor-beta signaling through binding Smad4.

    PubMed

    Wu, Kongming; Yang, Ying; Wang, Chenguang; Davoli, Maria A; D'Amico, Mark; Li, Anping; Cveklova, Kveta; Kozmik, Zbynek; Lisanti, Michael P; Russell, Robert G; Cvekl, Ales; Pestell, Richard G

    2003-12-19

    The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, we hypothesized that DACH1 might play a similar cellular function. Herein, DACH1 was found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-beta (TGF-beta)-induced apoptosis. DACH1 repressed TGF-beta induction of AP-1 and Smad signaling in gene reporter assays and repressed endogenous TGF-beta-responsive genes by microarray analyses. DACH1 bound to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localized with NCoR in nuclear dotlike structures. NCoR enhanced DACH1 repression, and the repression of TGF-beta-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH was sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibited the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-beta signaling by interacting with NCoR and Smad4.

  9. Distribution of virulence factors and association with emm polymorphism or isolation site among beta-hemolytic group G Streptococcus dysgalactiae subspecies equisimilis.

    PubMed

    Lo, Hsueh-Hsia; Cheng, Wei-Shan

    2015-01-01

    Distribution of virulence factors and association with emm polymorphism or isolation site among beta-hemolytic group G Streptococcus dysgalactiae subspecies equisimilis. Streptococcus dysgalactiae subspecies equisimilis (SDSE), the dominant human pathogenic species among group G streptococci, is the causative agent of several invasive and non-invasive diseases worldwide. However, limited information is available about the distribution of virulence factors among SDSE isolates, or their association with emm types and the isolation sites. In this study, 246 beta-hemolytic group G SDSE isolates collected in central Taiwan between February 2007 and August 2011 were under investigation. Of these, 66 isolates were obtained from normally sterile sites and 180 from non-sterile sites. emm typing revealed 32 types, with the most prevalent one being stG10.0 (39.8%), followed by stG245.0 (15.4%), stG840.0 (12.2%), stG6.1 (7.7%), and stG652.0 (4.1%). The virulence genes lmb (encoding laminin-binding protein), gapC (glyceraldehyde 3-phosphate dehydrogenase), sagA (streptolysin S), and hylB (hyaluronidase) existed in all isolates. Also, 99.2% of the isolates possessed slo (streptolysin O) and scpA (C5a peptidase) genes. In addition, 72.8%, 14.6%, 9.4%, and 2.4% of the isolates possessed the genes ska (streptokinase), cbp (putative collagen-binding protein, SDEG_1781), fbp (putative fibronectin-binding protein, SDEG_0161), and sicG (streptococcal inhibitor of complement), respectively. The only superantigen gene detected was spegg (streptococcus pyrogenic exotoxin G(dys) ), which was possessed by 74.4% of the isolates; these isolates correlated with non-sterile sites. Positive correlations were observed between the following emm types and virulence genes: stG10.0 and stG840.0 with spegg, stG6.1 and stG652.0 with ska, and stG840.0 with cbp. On the other hand, negative correlations were observed between the following: stG245.0, stG6.1, and stG652.0 types with spegg, stG10.0 with ska

  10. Inhibition of Interferon-beta Responses in Multiple Sclerosis Immune Cells Associated With High-Dose Statins

    PubMed Central

    Feng, Xuan; Han, Diana; Kilaru, Bharat K.; Franek, Beverly S.; Niewold, Timothy B.; Reder, Anthony T.

    2014-01-01

    Objective To determine whether statins affect type 1 interferon responses in relapsing-remitting multiple sclerosis (RRMS). Design Study effects of atorvastatin on type 1 interferon responses in Jurkat cells, mononuclear cells (MNCs) from therapy-naive patients with RRMS in vitro, and MNCs from interferon-treated RRMS patients in vivo in 4 conditions: no drug, statin only, interferon-beta only, and statin added on to interferon-beta therapy. Patients The study examined clinically stable patients with RRMS: 21 therapy-naive patients and 14 patients receiving interferon-beta with a statin. Interventions Statin effects on in vitro and in vivo interferon-beta–induced STAT1 transcription factor activation, expression of interferon-stimulated proteins in MNCs, and serum type 1 interferon activity. Results In vitro, atorvastatin dose dependently inhibited expression of interferon-stimulated P-Y-STAT1 by 44% (P< .001), interferon regulatory factor 1 protein by 30% (P= .006), and myxovirus resistance 1 protein by 32% (P=.004) compared with no-statin control in MNCs from therapy-naive RRMS patients. In vivo, 9 of 10 patients who received high-dose statins (80 mg) had a significant reduction in interferon-beta therapy–induced serum interferon-α/β activity, whereas only 2 of 4 patients who received medium-dose statins (40 mg) had reductions. High-dose add-on statin therapy significantly blocked interferon-beta function, with less P-Y-STAT1 transcription factor activation, and reduced myxovirus resistance 1 protein and viperin protein production. Medium doses of statins did not change STAT1 activation. Conclusions High-dose add-on statin therapy significantly reduces interferon-beta function and type 1 interferon responses in RRMS patients. These data provide a putative mechanism for how statins could counteract the beneficial effects of interferon-beta and worsen disease. PMID:22801747

  11. Quercetin induces insulin secretion by direct activation of L-type calcium channels in pancreatic beta cells

    PubMed Central

    Bardy, G; Virsolvy, A; Quignard, J F; Ravier, M A; Bertrand, G; Dalle, S; Cros, G; Magous, R; Richard, S; Oiry, C

    2013-01-01

    Background and Purpose Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca2+]i) in beta cells, in the absence of any co-stimulating factor. Experimental Approach Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca2+]i were measured using the ratiometric fluorescent Ca2+ indicator Fura-2. Ca2+ channel currents were recorded with the whole-cell patch-clamp technique. Key Results Quercetin concentration-dependently increased insulin secretion and elevated [Ca2+]i. These effects were not modified by the SERCA inhibitor thapsigargin (1 μmol·L−1), but were nearly abolished by the L-type Ca2+ channel antagonist nifedipine (1 μmol·L−1). Similar to the L-type Ca2+ channel agonist Bay K 8644, quercetin enhanced the L-type Ca2+ current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca2+]i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 μmol·L−1), with the two drugs having cumulative effects on [Ca2+]i. Conclusions and Implications Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca2+ influx through an interaction with L-type Ca2+ channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin's mechanism of action on insulin secretion. PMID:23530660

  12. Transcriptional activation of mouse mast cell Protease-7 by activin and transforming growth factor-beta is inhibited by microphthalmia-associated transcription factor.

    PubMed

    Funaba, Masayuki; Ikeda, Teruo; Murakami, Masaru; Ogawa, Kenji; Tsuchida, Kunihiro; Sugino, Hiromu; Abe, Matanobu

    2003-12-26

    Previous studies have revealed that activin A and transforming growth factor-beta1 (TGF-beta1) induced migration and morphological changes toward differentiation in bone marrow-derived cultured mast cell progenitors (BMCMCs). Here we show up-regulation of mouse mast cell protease-7 (mMCP-7), which is expressed in differentiated mast cells, by activin A and TGF-beta1 in BMCMCs, and the molecular mechanism of the gene induction of mmcp-7. Smad3, a signal mediator of the activin/TGF-beta pathway, transcriptionally activated mmcp-7. Microphthalmia-associated transcription factor (MITF), a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, and heart and skeletal muscle, inhibited Smad3-mediated mmcp-7 transcription. MITF associated with Smad3, and the C terminus of MITF and the MH1 and linker region of Smad3 were required for this association. Complex formation between Smad3 and MITF was neither necessary nor sufficient for the inhibition of Smad3 signaling by MITF. MITF inhibited the transcriptional activation induced by the MH2 domain of Smad3. In addition, MITF-truncated N-terminal amino acids could associate with Smad3 but did not inhibit Smad3-mediated transcription. The level of Smad3 was decreased by co-expression of MITF but not of dominant-negative MITF, which resulted from proteasomal protein degradation. The changes in the level of Smad3 protein were paralleled by those in Smad3-mediated signaling activity. These findings suggest that MITF negatively regulates Smad-dependent activin/TGF-beta signaling in a tissue-specific manner.

  13. Beta vulgaris crop types: Genomic signatures of selection (GSS) using next generation sequencing of pooled samples

    USDA-ARS?s Scientific Manuscript database

    Beta vulgaris crop types represent highly diverged populations with distinct phenotypes resulting from long-term selection. Differential end use in the crop types includes: leaf quality (chard/leaf beet), root enlargement and biomass, (table beet, fodder beet, sugar beet), and secondary metabolite a...

  14. Factors that influence the beta-diversity of spider communities in northwestern Argentinean Grasslands

    PubMed Central

    Rodriguez-Artigas, Sandra M.; Ballester, Rodrigo

    2016-01-01

    Beta-diversity, defined as spatial replacement in species composition, is crucial to the understanding of how local communities assemble. These changes can be driven by environmental or geographic factors (such as geographic distance), or a combination of the two. Spiders have been shown to be good indicators of environmental quality. Accordingly, spiders are used in this work as model taxa to establish whether there is a decrease in community similarity that corresponds to geographic distance in the grasslands of the Campos & Malezales ecoregion (Corrientes). Furthermore, the influence of climactic factors and local vegetation heterogeneity (environmental factors) on assemblage composition was evaluated. Finally, this study evaluated whether the differential dispersal capacity of spider families is a factor that influences their community structure at a regional scale. Spiders were collected with a G-Vac from vegetation in six grassland sites in the Campos & Malezales ecoregion that were separated by a minimum of 13 km. With this data, the impact of alpha-diversity and different environmental variables on the beta-diversity of spider communities was analysed. Likewise, the importance of species replacement and nesting on beta-diversity and their contribution to the regional diversity of spider families with different dispersion capacities was evaluated. The regional and site-specific inventories obtained were complete. The similarity between spider communities declined as the geographic distance between sites increased. Environmental variables also influenced community composition; stochastic events and abiotic forces were the principal intervening factors in assembly structure. The differential dispersal capacity of spider groups also influenced community structure at a regional scale. The regional beta-diversity, as well as species replacement, was greater in high and intermediate vagility spiders; while nesting was greater in spiders with low dispersion

  15. Molecular biology of the 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene family.

    PubMed

    Simard, Jacques; Ricketts, Marie-Louise; Gingras, Sébastien; Soucy, Penny; Feltus, F Alex; Melner, Michael H

    2005-06-01

    The 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase (3beta-HSD) isoenzymes are responsible for the oxidation and isomerization of Delta(5)-3beta-hydroxysteroid precursors into Delta(4)-ketosteroids, thus catalyzing an essential step in the formation of all classes of active steroid hormones. In humans, expression of the type I isoenzyme accounts for the 3beta-HSD activity found in placenta and peripheral tissues, whereas the type II 3beta-HSD isoenzyme is predominantly expressed in the adrenal gland, ovary, and testis, and its deficiency is responsible for a rare form of congenital adrenal hyperplasia. Phylogeny analyses of the 3beta-HSD gene family strongly suggest that the need for different 3beta-HSD genes occurred very late in mammals, with subsequent evolution in a similar manner in other lineages. Therefore, to a large extent, the 3beta-HSD gene family should have evolved to facilitate differential patterns of tissue- and cell-specific expression and regulation involving multiple signal transduction pathways, which are activated by several growth factors, steroids, and cytokines. Recent studies indicate that HSD3B2 gene regulation involves the orphan nuclear receptors steroidogenic factor-1 and dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1). Other findings suggest a potential regulatory role for STAT5 and STAT6 in transcriptional activation of HSD3B2 promoter. It was shown that epidermal growth factor (EGF) requires intact STAT5; on the other hand IL-4 induces HSD3B1 gene expression, along with IL-13, through STAT 6 activation. However, evidence suggests that multiple signal transduction pathways are involved in IL-4 mediated HSD3B1 gene expression. Indeed, a better understanding of the transcriptional factors responsible for the fine control of 3beta-HSD gene expression may provide insight into mechanisms involved in the functional cooperation between STATs and nuclear receptors as

  16. Transforming growth factor-beta production in anti-glomerular basement membrane disease in the rabbit.

    PubMed Central

    Coimbra, T.; Wiggins, R.; Noh, J. W.; Merritt, S.; Phan, S. H.

    1991-01-01

    The purpose of this study was to assay for the presence of collagen synthesis stimulatory activity in the kidney during immune-induced renal injury that results in severe fibrosis in both glomerular and interstitial compartments. A model of antiglomerular basement (anti-GBM) disease in the rabbit was induced on day 0 by the injection of anti-GBM antibody and renal cortex tissues were then sampled at various time points. Only conditioned media prepared from diseased renal cortical samples showed collagen synthesis stimulatory activity when tested on rabbit mesangial cells. The activity had an estimated molecular weight range of 16 to 25 kd and was neutralized by antibody to transforming growth factor-beta (TGF-beta). A standard assay for TGF-beta using a mink lung epithelial cell line confirmed the increase in TGF-beta activity in conditioned media of diseased cortex from day 7 and day 14 animals, which was not significantly activated by previous acidification. This suggests that most of the TGF-beta present in renal conditioned media was in the active form. The increase in renal cortical secretion of active TGF-beta was accompanied by increases in renal cortical TGF-beta mRNA content on days 4 and 7 after induction, with subsequent return to control levels. A similar increase in TGF-beta activity was present in nonacidified conditioned media of purified glomeruli from diseased days 7 and 14 animals, which was also accompanied by significant increases in TGF-beta mRNA. However with acidification no significant differences were noted between control and diseased samples, suggesting the presence of substantial latent TGF-beta activity in control glomerular conditioned media. These same control-conditioned media contained inhibitor activity for added exogenous TGF-beta. These results support the conclusion that the association between increased TGF-beta secretion and increased renal cortical collagen synthesis in this model is consistent with a role for this cytokine

  17. Early-Life Origins of Type 2 Diabetes: Fetal Programming of the Beta-Cell Mass

    PubMed Central

    Portha, Bernard; Chavey, Audrey; Movassat, Jamileh

    2011-01-01

    A substantial body of evidence suggests that an abnormal intrauterine milieu elicited by maternal metabolic disturbances as diverse as undernutrition, placental insufficiency, diabetes or obesity, may program susceptibility in the fetus to later develop chronic degenerative diseases, such as obesity, hypertension, cardiovascular diseases and diabetes. This paper examines the developmental programming of glucose intolerance/diabetes by disturbed intrauterine metabolic condition experimentally obtained in various rodent models of maternal protein restriction, caloric restriction, overnutrition or diabetes, with a focus on the alteration of the developing beta-cell mass. In most of the cases, whatever the type of initial maternal metabolic stress, the beta-cell adaptive growth which normally occurs during gestation, does not take place in the pregnant offspring and this results in the development of gestational diabetes. Therefore gestational diabetes turns to be the ultimate insult targeting the offspring beta-cell mass and propagates diabetes risk to the next generation again. The aetiology and the transmission of spontaneous diabetes as encountered in the GK/Par rat model of type 2 diabetes, are discussed in such a perspective. This review also discusses the non-genomic mechanisms involved in the installation of the programmed effect as well as in its intergenerational transmission. PMID:22110471

  18. Cellular localization and expression of template-activating factor I in different cell types.

    PubMed

    Nagata, K; Saito, S; Okuwaki, M; Kawase, H; Furuya, A; Kusano, A; Hanai, N; Okuda, A; Kikuchi, A

    1998-05-01

    Template-activating factors I (TAF-I) alpha and beta have been identified as chromatin remodeling factors from human HeLa cells. TAF-I beta corresponds to the protein encoded by the set gene, which was found in an acute undifferentiated leukemia as a fusion version with the can gene via chromosomal translocation. To determine the localization of TAF-I, we raised both polyclonal and monoclonal antibodies against TAF-I. The proteins that react to the antibodies are present not only in human cells but also in mouse, frog, insect, and yeast cells. The mouse TAF-I homologue is ubiquitous in a variety of tissue cells, including liver, kidney, spleen, lung, heart, and brain. It is of interest that the amounts of TAF-I alpha and beta vary among hemopoietic cells and some specific cell types do not contain TAF-I alpha. The level of the TAF-I proteins does not change significantly during the cell cycle progression in either HeLa cells synchronized with an excess concentration of thymidine or NIH 3T3 cells released from the serum-depleted state. TAF-I is predominantly located in nuclei, while TAF-I that is devoid of its acidic region, the region which is essential for the TAF-I activity, shows both nuclear and cytoplasmic localization. The localization of TAF-I in conjunction with the regulation of its activity is discussed.

  19. The corepressor CtBP interacts with Evi-1 to repress transforming growth factor beta signaling.

    PubMed

    Izutsu, K; Kurokawa, M; Imai, Y; Maki, K; Mitani, K; Hirai, H

    2001-05-01

    Evi-1 is a zinc finger nuclear protein whose inappropriate expression leads to leukemic transformation of hematopoietic cells in mice and humans. This was previously shown to block the antiproliferative effect of transforming growth factor beta (TGF-beta). Evi-1 represses TGF-beta signaling by direct interaction with Smad3 through its first zinc finger motif. Here, it is demonstrated that Evi-1 represses Smad-induced transcription by recruiting C-terminal binding protein (CtBP) as a corepressor. Evi-1 associates with CtBP1 through one of the consensus binding motifs, and this association is required for efficient inhibition of TGF-beta signaling. A specific inhibitor for histone deacetylase (HDAc) alleviates Evi-1-mediated repression of TGF-beta signaling, suggesting that HDAc is involved in the transcriptional repression by Evi-1. This identifies a novel function of Evi-1 as a member of corepressor complexes and suggests that aberrant recruitment of corepressors is one of the mechanisms for Evi-1-induced leukemogenesis.

  20. Pilot-Reported Beta-Blockers Identified by Forensic Toxicology Analysis of Postmortem Specimens.

    PubMed

    Canfield, Dennis V; Dubowski, Kurt M; Whinnery, James M; Forster, Estrella M

    2018-01-01

    This study compared beta-blockers reported by pilots with the medications found by postmortem toxicology analysis of specimens received from fatal aviation accidents between 1999 and 2015. Several studies have compared drugs using the standard approach: Compare the drug found by toxicology analysis with the drug reported by the pilot. This study uniquely examined first the pilot-reported medication and then compared it to that detected by toxicology analysis. This study will serve two purposes: (i) to determine the capability of a toxicology laboratory to detect reported medications, and (ii) to identify pilots with medications below detectable limits. All information required for this study was extracted from the Toxicology Data Base system and was searched using ToxFlo or SQL Server Management Studio. The following information was collected and analyzed: pilot-reported trade and/or generic drug, date specimens received, time of accident, type of aviation operations (CFR), state, pilot level, age, class of medical, specimen type, specimen concentration, dose reported, frequency reported associated with the accident, quantity reported, National Transportation Safety Board (NTSB) accident event number, and all NTSB reports. There were 319 pilots that either reported taking a beta-blocker or were found to be taking a beta-blocker by postmortem toxicology analysis. Time of death, therapeutic concentration and specimen type were found to be factors in the ability of the laboratory to detect beta-blockers. Beta-blockers taken by pilots will, in most cases, be found by a competent postmortem forensic toxicology laboratory at therapeutic concentrations. The dose taken by the pilot was not found to be a factor in the ability of the laboratory to identify beta-blockers. Time of dose, route of administration, specimen tested and therapeutic concentration of the drug were found to be factors in the ability of the laboratory to identify beta-blockers in postmortem specimens

  1. Changes in beta cell function during the proximate post-diagnosis period in persons with type 1 diabetes.

    PubMed

    DiMeglio, Linda A; Cheng, Peiyao; Beck, Roy W; Kollman, Craig; Ruedy, Katrina J; Slover, Robert; Aye, Tandy; Weinzimer, Stuart A; Bremer, Andrew A; Buckingham, Bruce

    2016-06-01

    Prior studies examining beta-cell preservation in type 1 diabetes have predominantly assessed stimulated C-peptide concentrations approximately 10 wk after diagnosis. We examined whether earlier assessments might aid in prediction of beta cell function over time. Using data from a multi-center randomized trial assessing the effect of intensive diabetes management initiated within 1 wk of diagnosis, we assessed which clinical factors predicted 90-min mixed-meal tolerance test (MMTT) stimulated C-peptide values obtained 2 and 6 wk after diagnosis. We also studied associations of these factors with C-peptide values at 1- and 2-year post-diagnosis. Data from intervention and control groups were pooled. Among 67 study participants (mean age 13.3 ± 5.7 yr, range 7.8-45.7 yr) in multivariable analyses, C-peptide increased from baseline to 2 wks and then 6 wk. C-peptide levels at these times were significantly correlated with 1- and 2-yr C-peptide concentrations (all p < 0.001), with the strongest observed associations between 6-wk C-peptide and the 1- and 2-yr values (r = 0.66 and r = 0.61, respectively). In multivariable analyses, greater baseline and 6-wk C-peptide, and older age independently predicted greater 1- and 2-yr C-peptide concentrations. C-peptide assessments close to diagnosis were predictive of subsequent C-peptide production. Our data demonstrate a clear increase in C-peptide over the initial 6 wk after diabetes diagnosis followed by a plateau. Our data do not suggest that MMTT assessments performed closer to diagnosis than 6 wk would improve prediction of subsequent residual beta cell function. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Bioactive molecules in milk and their role in health and disease: the role of transforming growth factor-beta.

    PubMed

    Donnet-Hughes, A; Duc, N; Serrant, P; Vidal, K; Schiffrin, E J

    2000-02-01

    Human breast milk is rich in nutrients, hormones, growth factors and immunoactive molecules, which influence the growth, development and immune status of the newborn infant. Although several of these factors are also present in bovine milk, the greater susceptibility of the formula-fed infant to infection and disease and the development of allergy is often attributed to the reduced level of protective factors in milk formulas. Nevertheless, modifying manufacturing processes may preserve the biological activity of some bioactive molecules in end products. Transforming growth factor (TGF)-beta is one such molecule. TGF-beta is a polypeptide, which has been described in both human and bovine milk. It is implicated in many processes, including epithelial cell growth and differentiation, development, carcinogenesis and immune regulation. The present article discusses the biological activity of TGF-beta2 that has been preserved and activated in a cow's milk-based product. More specifically, it addresses possible mechanisms of action in the intestinal lumen and speculates on how milk products containing naturally occurring TGF-beta2 could be exploited in functional foods for the infant or as therapies for specific intestinal diseases.

  3. beta-catenin mediates insulin-like growth factor-I actions to promote cyclin D1 mRNA expression, cell proliferation and survival in oligodendroglial cultures.

    PubMed

    Ye, Ping; Hu, Qichen; Liu, Hedi; Yan, Yun; D'ercole, A Joseph

    2010-07-01

    By promoting cell proliferation, survival and maturation insulin-like growth factor (IGF)-I is essential to the normal growth and development of the central nervous system. It is clear that IGF-I actions are primarily mediated by the type I IGF receptor (IGF1R), and that phosphoinositide 3 (PI3)-Akt kinases and MAP kinases signal many of IGF-I-IGF1R actions in neural cells, including oligodendrocyte lineage cells. The precise downstream targets of these signaling pathways, however, remain to be defined. We studied oligodendroglial cells to determine whether beta-catenin, a molecule that is a downstream target of glycogen synthase kinase-3beta (GSK3beta) and plays a key role in the Wnt canonical signaling pathway, mediates IGF-I actions. We found that IGF-I increases beta-catenin protein abundance within an hour after IGF-I-induced phosphorylation of Akt and GSK3beta. Inhibiting the PI3-Akt pathway suppressed IGF-I-induced increases in beta-catenin and cyclin D1 mRNA, while suppression of GSK3beta activity simulated IGF-I actions. Knocking-down beta-catenin mRNA by RNA interference suppressed IGF-I-stimulated increases in the abundance of cyclin D1 mRNA, cell proliferation, and cell survival. Our data suggest that beta-catenin is an important downstream molecule in the PI3-Akt-GSK3beta pathway, and as such it mediates IGF-I upregulation of cyclin D1 mRNA and promotion of cell proliferation and survival in oligodendroglial cells. Copyright 2010 Wiley-Liss, Inc.

  4. Etiopathogenesis of type 1 diabetes mellitus: prognostic factors for the evolution of residual β cell function

    PubMed Central

    2009-01-01

    Type 1A diabetes mellitus (T1ADM) is a progressive autoimmune disease mediated by T lymphocytes with destruction of beta cells. Up to now, we do not have precise methods to assess the beta cell mass, "in vivo" or "ex-vivo". The studies about its genetic susceptibility show strong association with class II antigens of the HLA system (particularly DQ). Others genetics associations are weaker and depend on the population studied. A combination of precipitating events may occur at the beginning of the disease. There is a silent loss of immune-mediated beta cells mass which velocity has an inverse relation with the age, but it is influenced by genetic and metabolic factors. We can predict the development of the disease primarily through the determination of four biochemically islet auto antibodies against antigens like insulin, GAD65, IA2 and Znt8. Beta cell destruction is chronically progressive but at clinical diagnosis of the disease a reserve of these cells still functioning. The goal of secondary disease prevention is halt the autoimmune attack on beta cells by redirecting or dampening the immune system. It is remains one of the foremost therapeutic goals in the T1ADM. Glycemic intensive control and immunotherapeutic agents may preserve beta-cell function in newly diagnosed patients with T1ADM. It may be assessed through C-peptide values, which are important for glycemic stability and for the prevention of chronic complications of this disease. This article will summarize the etiopathogenesis mechanisms of this disease and the factors can influence on residual C-peptide and the strategies to it preservation. PMID:19961609

  5. Cyclic stretching force selectively up-regulates transforming growth factor-beta isoforms in cultured rat mesangial cells.

    PubMed Central

    Riser, B. L.; Cortes, P.; Heilig, C.; Grondin, J.; Ladson-Wofford, S.; Patterson, D.; Narins, R. G.

    1996-01-01

    Glomerular distention from increased intraglomerular pressure stretches mesangial cells (MCs). Stretching MCs in culture stimulates extracellular matrix accumulation, suggesting that this may be a mechanism for glomerular hypertension-associated glomerulosclerosis. We examined whether mechanical stretching serves as a stimulus for the synthesis and activation of the prosclerotic molecule transforming growth factor (TGF)-beta, thus providing a potential system for auto-induction of extracellular matrix. Rat MCs cultured on flexible-bottom plates were subjected to cyclic stretching for up to 3 days and then assayed for TGF-beta mRNA, secretion of TGF-beta, and localization of active TGF-beta by immunostaining. MCs contained mRNA for all three mammalian isoforms of TGF-beta. Cyclic stretching for 36 hours increased TGF-beta1 and TGF-beta3 mRNA levels approximately twofold, without altering the levels of TGF-beta2 mRNA. This was followed at 48 to 72 hours by the increased secretion of both latent and active TGF-beta1. Latent, but not active, TGF-beta3 secretion also increased whereas the levels of TGF-beta2 were unaffected by mechanical force. The stretching force in this system is unequally distributed over the culture membrane. Localization of active TGF-beta by immunostaining demonstrated that the quantity of cell-associated cytokine across the culture was directly proportional to the zonal amplitude of the stretching force. These results demonstrate that stretching force stimulates MCs to selectively release and activate TGF-beta1. This mechanical induction of TGF-beta1 may help explain the increased extracellular matrix associated with intraglomerular hypertension. Images Figure 1 Figure 3 PMID:8669477

  6. Deletion of the human beta-globin LCR 5'HS4 or 5'HS1 differentially affects beta-like globin gene expression in beta-YAC transgenic mice.

    PubMed

    Fedosyuk, Halyna; Peterson, Kenneth R

    2007-01-01

    A 213 kb human beta-globin locus yeast artificial chromosome (beta-YAC) was modified by homologous recombination to delete 2.9 kb of cross-species conserved sequence similarity encompassing the LCR 5' hypersensitive site (HS) 4 (Delta5'HS4 beta-YAC). In three transgenic mouse lines, completion of the gamma- to beta-globin switch during definitive erythropoiesis was delayed relative to wild-type beta-YAC mice. In addition, quantitative per-copy human beta-like globin mRNA levels were similar to wild-type beta-YAC transgenic lines, although beta-globin gene expression was slightly decreased in the day 12 fetal liver of Delta5'HS4 beta-YAC mice. A 0.8 kb 5'HS1 fragment was similarly deleted in the YAC. Three Delta5'HS1 beta-YAC transgenic lines were established. epsilon-globin gene expression was markedly reduced, approximately 16 fold, during primitive erythropoiesis compared to wild-type beta-YAC mice, but gamma-globin expression levels were unaffected. However, during the fetal stage of definitive erythropoiesis, gamma-globin gene expression was decreased approximately 4 fold at day 12 and approximately 5 fold at day 14. Temporal developmental expression profiles of the beta-like globin genes were unaffected by deletion of 5'HS1. Decreased expression of the epsilon- and gamma-globin genes is the first phenotype ascribed to a 5'HS1 mutation in the human beta-globin locus, suggesting that this HS does indeed have a role in LCR function beyond simply a combined synergism with the other LCR HSs.

  7. Regeneration of hyaline articular cartilage with irradiated transforming growth factor beta1-producing fibroblasts.

    PubMed

    Song, Sun U; Hong, Young-Jin; Oh, In-Suk; Yi, Youngsuk; Choi, Kyoung Baek; Lee, Jung Woo; Park, Kwang-Won; Han, Jeoung-Uk; Suh, Jun-Kyu; Lee, Kwan Hee

    2004-01-01

    The regeneration of hyaline articular cartilage by cell-mediated gene therapy using transforming growth factor beta(1) (TGF-beta(1))-producing fibroblasts (NIH 3T3-TGF-beta(1)) has been reported previously. In this study, we investigated whether TGF-beta(1)-producing fibroblasts irradiated with a lethal dose of radiation are still capable of inducing the regeneration of hyaline articular cartilage. NIH 3T3TGF-beta(1) fibroblasts were exposed to doses of 20, 40, or 80 Gy, using a irradiator, and then injected into artificially made partial defects on the femoral condyle of rabbit knee joints. The rabbits were killed 3 or 6 weeks postinjection and hyaline articular cartilage regeneration was evaluated by histological and immunohistochemical staining (n = 5 per each group). Irradiated NIH 3T3-TGFbeta(1) fibroblasts started to die rapidly 3 days after irradiation; moreover, the kinetics of their viability were similar regardless of the radiation intensity. TGF-beta1 expression, measured by ELISA, showed that the TGF-beta(1) protein produced from the irradiated cells peaked 5 days after irradiation and thereafter declined rapidly. Complete filling of the defect with reparative tissue occurred in all the groups, although variations were observed in terms of the nature of the repair tissue. Histological and immunohistochemical staining of the repair tissue showed that the tissue newly formed by irradiated NIH 3T3-TGF-beta(1) fibroblasts after exposure to 20 Gy had hyaline cartilage-like characteristics, as was observed in the nonirradiated controls. On the other hand, the repair tissue formed by NIH 3T3-TGF-beta(1) fibroblasts irradiated with 40 or 80 Gy showed more fibrous cartilage-like tissue. These results suggest that TGF-beta(1)-producing fibroblasts irradiated up to a certain level of lethal dose (i.e., 20 Gy) are able to induce normal-appearing articular cartilage in vivo. Therefore, irradiated heterologous cell-mediated TGF-beta(1) gene therapy may be clinically

  8. Different small, acid-soluble proteins of the alpha/beta type have interchangeable roles in the heat and UV radiation resistance of Bacillus subtilis spores.

    PubMed Central

    Mason, J M; Setlow, P

    1987-01-01

    Spores of Bacillus subtilis strains which carry deletion mutations in one gene (sspA) or two genes (sspA and sspB) which code for major alpha/beta-type small, acid-soluble spore proteins (SASP) are known to be much more sensitive to heat and UV radiation than wild-type spores. This heat- and UV-sensitive phenotype was cured completely or in part by introduction into these mutant strains of one or more copies of the sspA or sspB genes themselves; multiple copies of the B. subtilis sspD gene, which codes for a minor alpha/beta-type SASP; or multiple copies of the SASP-C gene, which codes for a major alpha/beta-type SASP of Bacillus megaterium. These findings suggest that alpha/beta-type SASP play interchangeable roles in the heat and UV radiation resistance of bacterial spores. Images PMID:3112127

  9. Deficiency of a beta-arrestin-2 signal complex contributes to insulin resistance.

    PubMed

    Luan, Bing; Zhao, Jian; Wu, Haiya; Duan, Baoyu; Shu, Guangwen; Wang, Xiaoying; Li, Dangsheng; Jia, Weiping; Kang, Jiuhong; Pei, Gang

    2009-02-26

    Insulin resistance, a hallmark of type 2 diabetes, is a defect of insulin in stimulating insulin receptor signalling, which has become one of the most serious public health threats. Upon stimulation by insulin, insulin receptor recruits and phosphorylates insulin receptor substrate proteins, leading to activation of the phosphatidylinositol-3-OH kinase (PI(3)K)-Akt pathway. Activated Akt phosphorylates downstream kinases and transcription factors, thus mediating most of the metabolic actions of insulin. Beta-arrestins mediate biological functions of G-protein-coupled receptors by linking activated receptors with distinct sets of accessory and effecter proteins, thereby determining the specificity, efficiency and capacity of signals. Here we show that in diabetic mouse models, beta-arrestin-2 is severely downregulated. Knockdown of beta-arrestin-2 exacerbates insulin resistance, whereas administration of beta-arrestin-2 restores insulin sensitivity in mice. Further investigation reveals that insulin stimulates the formation of a new beta-arrestin-2 signal complex, in which beta-arrestin-2 scaffolds Akt and Src to insulin receptor. Loss or dysfunction of beta-arrestin-2 results in deficiency of this signal complex and disturbance of insulin signalling in vivo, thereby contributing to the development of insulin resistance and progression of type 2 diabetes. Our findings provide new insight into the molecular pathogenesis of insulin resistance, and implicate new preventive and therapeutic strategies against insulin resistance and type 2 diabetes.

  10. Sintering of beta-type alumina bodies using alpha-alumina encapsulation

    DOEpatents

    McEntire, Bryan J.; Virkar, Anil V.

    1981-01-01

    A method of sintering a shaped green, beta-type alumina body comprising: (A) inserting said body into an open chamber prepared by exposing the interior surface of a container consisting essentially of at least about 50 weight percent of alpha-alumina and a remainder of other refractory material to a sodium oxide or sodium oxide producing environment; (B) sealing the chamber; and heating the chamber with the shaped body encapsulated therein to a temperature and for a time necessary to sinter said body to the desired density. The encapsulation chamber prepared as described above is also claimed.

  11. CCAAT-binding factor regulates expression of the beta1 subunit of soluble guanylyl cyclase gene in the BE2 human neuroblastoma cell line

    NASA Technical Reports Server (NTRS)

    Sharina, Iraida G.; Martin, Emil; Thomas, Anthony; Uray, Karen L.; Murad, Ferid

    2003-01-01

    Soluble guanylyl cyclase (sGC) is a cytosolic enzyme producing the intracellular messenger cyclic guanosine monophosphate (cGMP) on activation with nitric oxide (NO). sGC is an obligatory heterodimer composed of alpha and beta subunits. We investigated human beta1 sGC transcriptional regulation in BE2 human neuroblastoma cells. The 5' upstream region of the beta1 sGC gene was isolated and analyzed for promoter activity by using luciferase reporter constructs. The transcriptional start site of the beta1 sGC gene in BE2 cells was identified. The functional significance of consensus transcriptional factor binding sites proximal to the transcriptional start site was investigated by site deletions in the 800-bp promoter fragment. The elimination of CCAAT-binding factor (CBF) and growth factor independence 1 (GFI1) binding cores significantly diminished whereas deletion of the NF1 core elevated the transcription. Electrophoretic mobility-shift assay (EMSA) and Western analysis of proteins bound to biotinated EMSA probes confirmed the interaction of GFI1, CBF, and NF1 factors with the beta1 sGC promoter. Treatment of BE2 cells with genistein, known to inhibit the CBF binding to DNA, significantly reduced protein levels of beta1 sGC by inhibiting transcription. In summary, our study represents an analysis of the human beta1 sGC promoter regulation in human neuroblastoma BE2 cells and identifies CBF as a critically important factor in beta1 sGC expression.

  12. A critical role for transcription factor Smad4 in T cell function independent of transforming growth factor beta receptor signaling

    PubMed Central

    Gu, Ai-Di; Zhang, Song; Wang, Yunqi; Xiong, Hui; Curtis, Thomas A.; Wan, Yisong Y.

    2014-01-01

    Summary Transforming growth factor-beta (TGF-β) suppresses T cell function to maintain self-tolerance and to promote tumor immune evasion. Yet how Smad4, a transcription factor component of TGF-β signaling, regulates T cell function remains unclear. Here we have demonstrated an essential role for Smad4 in promoting T cell function during autoimmunity and anti-tumor immunity. Smad4 deletion rescued the lethal autoimmunity resulting from transforming growth factor-beta receptor (TGF-βR) deletion and compromised T-cell-mediated tumor rejection. While Smad4 was dispensable for T cell generation, homeostasis and effector function, it was essential for T cell proliferation following activation in vitro and in vivo. The transcription factor Myc was identified to mediate Smad4-controlled T cell proliferation. This study thus reveals a requirement of Smad4 for T-cell-mediated autoimmunity and tumor rejection, which is beyond the current paradigm. It highlights a TGF-βR-independent role for Smad4 in promoting T cell function, autoimmunity and anti-tumor immunity. PMID:25577439

  13. Troglitazone stimulates {beta}-arrestin-dependent cardiomyocyte contractility via the angiotensin II type 1{sub A} receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tilley, Douglas G., E-mail: douglas.tilley@jefferson.edu; Center for Translational Medicine, Thomas Jefferson University; Nguyen, Anny D.

    2010-06-11

    Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists are commonly used to treat cardiovascular diseases, and are reported to have several effects on cardiovascular function that may be due to PPAR{gamma}-independent signaling events. Select angiotensin receptor blockers (ARBs) interact with and modulate PPAR{gamma} activity, thus we hypothesized that a PPAR{gamma} agonist may exert physiologic effects via the angiotensin II type 1{sub A} receptor (AT1{sub A}R). In AT1{sub A}R-overexpressing HEK 293 cells, both angiotensin II (Ang II) and the PPAR{gamma} agonist troglitazone (Trog) enhanced AT1{sub A}R internalization and recruitment of endogenous {beta}-arrestin1/2 ({beta}arr1/2) to the AT1{sub A}R. A fluorescence assay to measure diacylglycerolmore » (DAG) accumulation showed that although Ang II induced AT1{sub A}R-G{sub q} protein-mediated DAG accumulation, Trog had no impact on DAG generation. Trog-mediated recruitment of {beta}arr1/2 was selective to AT1{sub A}R as the response was prevented by an ARB- and Trog-mediated {beta}arr1/2 recruitment to {beta}1-adrenergic receptor ({beta}1AR) was not observed. In isolated mouse cardiomyocytes, Trog increased both % and rate of cell shortening to a similar extent as Ang II, effects which were blocked with an ARB. Additionally, these effects were found to be {beta}arr2-dependent, as cardiomyocytes isolated from {beta}arr2-KO mice showed blunted contractile responses to Trog. These findings show for the first time that the PPAR{gamma} agonist Trog acts at the AT1{sub A}R to simultaneously block G{sub q} protein activation and induce the recruitment of {beta}arr1/2, which leads to an increase in cardiomyocyte contractility.« less

  14. SNP analyses of growth factor genes EGF, TGF{beta}-1, and HGF reveal haplotypic association of EGF with autism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Toyoda, Takao; Thanseem, Ismail; Kawai, Masayoshi

    Autism is a pervasive neurodevelopmental disorder diagnosed in early childhood. Growth factors have been found to play a key role in the cellular differentiation and proliferation of the central and peripheral nervous systems. Epidermal growth factor (EGF) is detected in several regions of the developing and adult brain, where, it enhances the differentiation, maturation, and survival of a variety of neurons. Transforming growth factor-{beta} (TGF{beta}) isoforms play an important role in neuronal survival, and the hepatocyte growth factor (HGF) has been shown to exhibit neurotrophic activity. We examined the association of EGF, TGF{beta}1, and HGF genes with autism, in amore » trio association study, using DNA samples from families recruited to the Autism Genetic Resource Exchange; 252 trios with a male offspring scored for autism were selected for the study. Transmission disequilibrium test revealed significant haplotypic association of EGF with autism. No significant SNP or haplotypic associations were observed for TGF{beta}1 or HGF. Given the role of EGF in brain and neuronal development, we suggest a possible role of EGF in the pathogenesis of autism.« less

  15. The Jak-STAT pathway stimulated by interferon alpha or interferon beta.

    PubMed

    Horvath, Curt M

    2004-11-23

    Type I interferons, such as interferon alpha and interferon beta (IFN-alpha and beta), signal through a Janus kinase (Jak) to signal transduction and activator of transcription (STAT) pathway to stimulate gene expression. In response to ligand binding, the receptors dimerize, Jaks phosphorylate STAT1 and STAT2, which then dimerize and interact with a third transcriptional regulator IFN regulatory factor 9 (IRF9) to stimulate gene expression. IFN-alpha is the main innate antiviral cytokine and is essential for effective immune response to viral infection. The animation shows activation of STAT-responsive gene expression in response to type I IFNs.

  16. Transforming growth factor beta-independent shuttling of Smad4 between the cytoplasm and nucleus.

    PubMed

    Pierreux, C E; Nicolás, F J; Hill, C S

    2000-12-01

    Smad4 plays a pivotal role in all transforming growth factor beta (TGF-beta) signaling pathways. Here we describe six widely expressed alternatively spliced variants of human Smad4 with deletions of different exons in the linker, the region of Smad4 that separates the two well-conserved MH1 and MH2 domains. All these Smad4 variants form complexes with activated Smad2 and Smad3 and are incorporated into DNA-binding complexes with the transcription factor Fast-1, regardless of the amount of linker they contain. However, sequences encoded by exons 5 to 7 in the linker are essential for transcriptional activation. Most importantly, our observation that different Smad4 isoforms have different subcellular localizations has led us to the identification of a functional CRM1-dependent nuclear export signal in the Smad4 linker and a constitutively active nuclear localization signal in the N-terminal MH1 domain. In the absence of TGF-beta signaling, we conclude that Smad4 is rapidly and continuously shuttling between the nucleus and the cytoplasm, the distribution of Smad4 between the nucleus and the cytoplasm being dictated by the relative strengths of the nuclear import and export signals. We demonstrate that inhibition of CRM1-mediated nuclear export by treatment of cells with leptomycin B results in endogenous Smad4 accumulating very rapidly in the nucleus. Endogenous Smad2 and Smad3 are completely unaffected by leptomycin B treatment, indicating that the nucleocytoplasmic shuttling is specific for Smad4. We propose that, upon TGF-beta signaling, complex formation between Smad4 and activated Smad2 or -3 leads to nuclear accumulation of Smad4 through inhibition of its nuclear export. We demonstrate that after prolonged TGF-beta signaling Smad2 becomes dephosphorylated and Smad2 and Smad4 accumulate back in the cytoplasm.

  17. Disruption of transforming growth factor-beta signaling by curcumin induces gene expression of peroxisome proliferator-activated receptor-gamma in rat hepatic stellate cells.

    PubMed

    Zheng, Shizhong; Chen, Anping

    2007-01-01

    Activation of hepatic stellate cells (HSC), the major effectors of hepatic fibrogenesis, is coupled with sequential alterations in gene expression, including an increase in receptors for transforming growth factor-beta (TGF-beta) and a dramatic reduction in the peroxisome proliferator-activated receptor-gamma (PPAR-gamma). The relationship between them remains obscure. We previously demonstrated that curcumin induced gene expression of PPAR-gamma in activated HSC, leading to reducing cell proliferation, inducing apoptosis and suppressing expression of extracellular matrix genes. The underlying molecular mechanisms are largely unknown. We recently observed that stimulation of PPAR-gamma activation suppressed gene expression of TGF-beta receptors in activated HSC, leading to the interruption of TGF-beta signaling. This observation supported our assumption of an antagonistic relationship between PPAR-gamma activation and TGF-beta signaling in HSC. In this study, we further hypothesize that TGF-beta signaling might negatively regulate gene expression of PPAR-gamma in activated HSC. The present report demonstrates that exogenous TGF-beta1 inhibits gene expression of PPAR-gamma in activated HSC, which is eliminated by the pretreatment with curcumin likely by interrupting TGF-beta signaling. Transfection assays further indicate that blocking TGF-beta signaling by dominant negative type II TGF-beta receptor increases the promoter activity of PPAR-gamma gene. Promoter deletion assays, site-directed mutageneses, and gel shift assays localize two Smad binding elements (SBEs) in the PPAR-gamma gene promoter, acting as curcumin response elements and negatively regulating the promoter activity in passaged HSC. The Smad3/4 protein complex specifically binds to the SBEs. Overexpression of Smad4 dose dependently eliminates the inhibitory effects of curcumin on the PPAR-gamma gene promoter and TGF-beta signaling. Taken together, these results demonstrate that the interruption of TGF-beta

  18. Inhibition of GSK-3beta ameliorates hepatic ischemia-reperfusion injury through GSK-3beta/beta-catenin signaling pathway in mice.

    PubMed

    Xia, Yong-Xiang; Lu, Ling; Wu, Zheng-Shan; Pu, Li-Yong; Sun, Bei-Cheng; Wang, Xue-Hao

    2012-06-01

    Glycogen synthase kinase (GSK)-3beta/beta-catenin signaling regulates ischemia-reperfusion (I/R)-induced apoptosis and proliferation, and inhibition of GSK-3beta has beneficial effects on I/R injury in the heart and the central nervous system. However, the role of this signaling in hepatic I/R injury remains unclear. The present study aimed to investigate the effects and mechanism of GSK-3beta/beta-catenin signaling in hepatic I/R injury. Male C57BL/6 mice (weighing 22-25 g) were pretreated with either SB216763, an inhibitor of GSK-3beta, or vehicle. These mice were subjected to partial hepatic I/R. Blood was collected for test of alanine aminotransferase (ALT), and liver specimen for assays of phosphorylation at the Ser9 residue of GSK-3beta, GSK-3beta activity, axin 2 and the anti-apoptotic factors Bcl-2 and survivin, as well as the proliferative factors cyclin D1 and proliferating cell nuclear antigen, and apoptotic index (TUNEL). Real-time PCR, Western blotting and immunohistochemical staining were used. SB216763 increased phospho-GSK-3beta levels and suppressed GSK-3beta activity (1880+/-229 vs 3280+/-272 cpm, P<0.01). ALT peaked at 6 hours after reperfusion. Compared with control, SB216763 decreased ALT after 6 hours of reperfusion (4451+/-424 vs 7868+/-845 IU/L, P<0.01), and alleviated hepatocyte necrosis and vacuolization. GSK-3beta inhibition led to the accumulation of beta-catenin in the cytosol (0.40+/-0.05 vs 1.31+/-0.11, P<0.05) and nucleus (0.62+/-0.14 vs 1.73+/-0.12, P<0.05), beta-catenin further upregulated the expression of axin 2. Upregulation of GSK-3beta/beta-catenin signaling increased Bcl-2, survivin and cyclin D1. Serological and histological analyses showed that SB216763 alleviated hepatic I/R-induced injury by reducing apoptosis (1.4+/-0.2% vs 3.6+/-0.4%, P<0.05) and enhanced liver proliferation (56+/-8% vs 19+/-4%, P<0.05). Inhibition of GSK-3beta ameliorates hepatic I/R injury through the GSK-3beta/beta-catenin signaling pathway.

  19. Mouse Balb/c3T3 cell mutant with low epidermal growth factor receptor activity: induction of stable anchorage-independent growth by transforming growth factor. beta

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kuratomi, Y.; Ono, M.; Yasutake, C.

    1987-01-01

    A mutant clone (MO-5) was originally isolated as a clone resistant to Na/sup +//K/sup +/ ionophoric antibiotic monensin from mouse Balb/c3T3 cells. MO-5 was found to show low receptor-endocytosis activity for epidermal growth factor (EGF):binding activity for EGF in MO-5 was less than one tenth of that in Balb/c3T3. Anchorage-independent growth of MO-5 was compared to that of Balb/c3T3 when assayed by colony formation capacity in soft agar. Coadministration of EGF and TGF-..beta.. efficiently enhanced anchorage-independent growth of normal rat kidney (NRK) cells, but neither factor alone was competent to promote the anchorage-independent growth. The frequency of colonies appearing inmore » soft agar of MO-5 or Balb/c3T3 was significantly enhanced by TGF-..beta.. while EGF did not further enhance that of MO-5 or Balb/c3T3. Colonies of Balb/c3T3 formed in soft agar in the presence of TGF-..beta.. showed low colony formation capacity in soft agar in the absence of TGF-..beta... Colonies of MO-5 formed by TGF-..beta.. in soft agar, however, showed high colony formation capacity in soft agar in the absence of TGF-..beta... Pretreatment of MO-5 with TGF-..beta.. induced secretion of TGF-..beta..-like activity from the cells, while the treatment of Balb/c3T3 did not induce the secretion of a significant amount of TGF-..beta..-like activity. The loss of EGF-receptor activity in the stable expression and maintenance of the transformed phenotype in MO-5 is discussed.« less

  20. 14-3-3 sigma and 14-3-3 zeta plays an opposite role in cell growth inhibition mediated by transforming growth factor-beta 1.

    PubMed

    Hong, Hye-Young; Jeon, Woo-Kwang; Bae, Eun-Jin; Kim, Shin-Tae; Lee, Ho-Jae; Kim, Seong-Jin; Kim, Byung-Chul

    2010-03-01

    The expression of 14-3-3 proteins is dysregulated in various types of cancer. This study was undertaken to investigate the effects of 14-3-3 zeta and 14-3-3 sigma on cell growth inhibition mediated by transforming growth factor-beta 1 (TGF-beta1). Mouse mammary epithelial cells (Eph4) that are transformed with oncogenic c-H-Ras (EpRas) and no longer sensitive to TGF-beta1-mediated growth inhibition displayed increased expression of 14-3-3 zeta and decreased expression of 14-3-3 sigma compared with parental Eph4 cells. Using small interfering RNA-mediated knockdown and overexpression of 14-3-3 sigma or 14-3-3 zeta, we showed that 14-3-3 sigma is required for TGF-beta1-mediated growth inhibition whereas 14-3-3 zeta negatively modulates this growth inhibitory response. Notably, overexpression of 14-3-3 zeta increased the level of Smad3 protein that is phosphorylated at linker regions and cannot mediate the TGF-beta1 growth inhibitory response. Consistent with this finding, mutation of the 14-3-3 zeta phosphorylation sites in Smad3 markedly reduced the 14-3-3 zeta-mediated inhibition of TGF-beta1-induced p15 promoter-reporter activity and cell cycle arrest, suggesting that these residues are critical targets of 14-3-3 zeta in the suppression of TGF-beta1-mediated growth. Taken together, our findings indicate that dysregulation of 14-3-3 sigma or 14-3-3 zeta contributes to TGF-beta1 resistance in cancer cells.

  1. Effects of concomitant use of fibroblast growth factor (FGF)-2 with beta-tricalcium phosphate ({beta}-TCP) on the beagle dog 1-wall periodontal defect model

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Anzai, Jun, E-mail: anzai_jun@kaken.co.jp; Department of Periodontology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871; Kitamura, Masahiro, E-mail: kitamura@dent.osaka-u.ac.jp

    Research highlights: {yields} Concomitant use of FGF-2 and {beta}-TCP (an osteo-conductive scaffold) significantly promotes periodontal regeneration in the severe periodontitis model (1-wall defect model) of beagle dog. {yields} FGF-2 enhanced new bone formation via {beta}-TCP at the defects. {yields} In particular, FGF-2 dramatically regenerated new periodontal ligament and cementum formations at the defects, that is one of the most important healing outcomes during the process of periodontal regeneration. {yields} Epithelial downgrowth (undesirable wound healing) was decreased by administration of FGF-2. {yields} This manuscript indicates for the first time that concomitant use of FGF-2 and {beta}-TCP is efficacious in regenerating periodontalmore » tissue following severe destruction of the tissue by progression of periodontitis. -- Abstract: The effects of concomitant use of fibroblast growth factor-2 (FGF-2) and beta-tricalcium phosphate ({beta}-TCP) on periodontal regeneration were investigated in the beagle dog 1-wall periodontal defect model. One-wall periodontal defects were created in the mesial portion of both sides of the mandibular first molars, and 0.3% FGF-2 plus {beta}-TCP or {beta}-TCP alone was administered. Radiographic evaluation was performed at 0, 3, and 6 weeks. At 6 weeks, the periodontium with the defect site was removed and histologically analyzed. Radiographic findings showed that co-administration of FGF-2 significantly increased bone mineral contents of the defect sites compared with {beta}-TCP alone. Histologic analysis revealed that the length of the regenerated periodontal ligament, the cementum, distance to the junctional epithelium, new bone height, and area of newly formed bone were significantly increased in the FGF-2 group. No abnormal inflammatory response or ankylosis was observed in either group. These findings indicate the efficacy of concomitant use of FGF-2 and {beta}-TCP as an osteoconductive material for periodontal

  2. Cross-talk between Smad and p38 MAPK signalling in transforming growth factor {beta} signal transduction in human glioblastoma cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dziembowska, Magdalena; Danilkiewicz, Malgorzata; Wesolowska, Aleksandra

    2007-03-23

    Transforming growth factor-beta (TGF-{beta}) is a multifunctional cytokine involved in the regulation of cell proliferation, differentiation, and survival. Malignant tumour cells often do not respond to TGF-{beta} by growth inhibition, but retain responsiveness to cytokine in regulating extracellular matrix deposition, cell adhesion, and migration. We demonstrated that TGF-{beta}1 does not affect viability or proliferation of human glioblastoma T98G, but increases transcriptional responses exemplified by induction of MMP-9 expression. TGF-{beta} receptors were functional in T98G glioblastoma cells leading to SMAD3/SMAD4 nuclear translocation and activation of SMAD-dependent promoter. In parallel, a selective activation of p38 MAPK, and phosphorylation of its substrates: ATF2more » and c-Jun proteins were followed by a transient activation of AP-1 transcription factor. Surprisingly, an inhibition of p38 MAPK with a specific inhibitor, SB202190, abolished TGF-inducible activation of Smad-dependent promoter and decreased Smad2 phosphorylation. It suggests an unexpected interaction between Smad and p38 MAPK pathways in TGF-{beta}1-induced signalling.« less

  3. Type I Glanzmann thrombasthenia caused by an apparently silent beta3 mutation that results in aberrant splicing and reduced beta3 mRNA.

    PubMed

    Xie, Jingli; Pabón, Dina; Jayo, Asier; Butta, Nora; González-Manchón, Consuelo

    2005-05-01

    We report a novel genetic defect in a patient with type I Glanzmann thrombasthenia. Flow cytometry analysis revealed undetectable levels of platelet glycoproteins alphaIIb and beta3, although residual amounts of both proteins were detectable in immunoblotting analysis. Sequence analysis of reversely transcribed platelet beta3 mRNA showed a 100-base pair deletion in the 3'-boundary of exon 11, that results in a frame shift and appearance of a premature STOP codon. Analysis of the corresponding genomic DNA fragment revealed the presence of a homozygous C1815T transition in exon 11. The mutation does not change the amino acid residue but it creates an ectopic consensus splice donor site that is used preferentially, causing splicing out of part of exon 11. The parents of the proband, heterozygous for this mutation, were asymptomatic and had reduced platelet content of alphaIIbbeta3. PCR-based relative quantification of beta3 mRNA failed to detect the mutant transcript in the parents and showed a marked reduction in the patient. The results suggest that the thrombasthenic phenotype is, mainly, the result of the reduced availability of beta3-mRNA, most probably due to activation of the nonsense-mediated mRNA decay mechanism. They also show the convenience of analyzing both genomic DNA and mRNA, in order to ascertain the functional consequences of single nucleotide substitutions.

  4. Gonadal Identity in the Absence of Pro-Testis Factor SOX9 and Pro-Ovary Factor Beta-Catenin in Mice1

    PubMed Central

    Nicol, Barbara; Yao, Humphrey H.-C.

    2015-01-01

    Sex-reversal cases in humans and genetic models in mice have revealed that the fate of the bipotential gonad hinges upon the balance between pro-testis SOX9 and pro-ovary beta-catenin pathways. Our central query was: if SOX9 and beta-catenin define the gonad's identity, then what do the gonads become when both factors are absent? To answer this question, we developed mouse models that lack either Sox9, beta-catenin, or both in the somatic cells of the fetal gonads and examined the morphological outcomes and transcriptome profiles. In the absence of Sox9 and beta-catenin, both XX and XY gonads progressively lean toward the testis fate, indicating that expression of certain pro-testis genes requires the repression of the beta-catenin pathway, rather than a direct activation by SOX9. We also observed that XY double knockout gonads were more masculinized than their XX counterpart. To identify the genes responsible for the initial events of masculinization and to determine how the genetic context (XX vs. XY) affects this process, we compared the transcriptomes of Sox9/beta-catenin mutant gonads and found that early molecular changes underlying the XY-specific masculinization involve the expression of Sry and 21 SRY direct target genes, such as Sox8 and Cyp26b1. These results imply that when both Sox9 and beta-catenin are absent, Sry is capable of activating other pro-testis genes and drive testis differentiation. Our findings not only provide insight into the mechanism of sex determination, but also identify candidate genes that are potentially involved in disorders of sex development. PMID:26108792

  5. Expression and regulation of aromatase and 17 beta-hydroxysteroid dehydrogenase type 4 in human THP 1 leukemia cells.

    PubMed

    Jakob, F; Homann, D; Adamski, J

    1995-12-01

    Estradiol is active in proliferation and differentiation of sex-related tissues like ovary and breast. Glandular steroid metabolism was for a long time believed to dominate the estrogenic milieu around any cell of the organism. Recent reports verified the expression of estrogen receptors in "non-target" tissues as well as the extraglandular expression of steroid metabolizing enzymes. Extraglandular steroid metabolism proved to be important in the brain, skin and in stromal cells of hormone responsive tumors. Aromatase converts testosterone into estradiol and androstenedione into estrone, thereby activating estrogen precursors. The group of 17 beta-hydroxysteroid dehydrogenases catalyzes the oxidation and/or reduction of the forementioned compounds, e.g. estradiol/estrone, thereby either activating or inactivating estradiol. Aromatase is expressed and regulated in the human THP 1 myeloid leukemia cell line after vitamin D/GMCSF-propagated differentiation. Aromatase expression is stimulated by dexamethasone, phorbolesters and granulocyte/macrophage stimulating factor (GMCSF). Exons I.2 and I.4 are expressed in PMA-stimulated cells only, exon I.3 in both PMA- and dexamethasone-stimulated cells. Vitamin D-differentiated THP 1 cells produce a net excess of estradiol in culture supernatants, if testosterone is given as aromatase substrate. In contrast, the 17 beta-hydroxysteroid dehydrogenase type 4 (17 beta-HSD 4) is abundantly expressed in unstimulated THP 1 cells and is further stimulated by glucocorticoids (2-fold). The expression is unchanged after vitamin D/GMCSF-propagated differentiation. 17 beta-HSD 4 expression is not altered by phorbolester treatment in undifferentiated cells but is abolished after vitamin D-propagated differentiation along with downregulation of beta-actin. Protein kinase C activation therefore appears to dissociate the expression of aromatase and 17 beta-HSD 4 in this differentiation stage along the monocyte/phagocyte pathway of THP 1

  6. Promotion of mouse fibroblast collagen gene expression by mast cells stimulated via the Fc epsilon RI. Role for mast cell-derived transforming growth factor beta and tumor necrosis factor alpha

    PubMed Central

    1994-01-01

    Chronic allergic diseases and other disorders associated with mast cell activation can also be associated with tissue fibrosis, but a direct link between mast cell mediator release and fibroblast collagen gene expression has not been established. Using in situ hybridization, we show that the elicitation of an IgE-dependent passive cutaneous anaphylaxis (PCA) reaction in mice results in a transient, but marked augmentation of steady state levels of type alpha-1 (I) collagen mRNA in the dermis. While peak levels of collagen mRNA expression in the skin are observed 16-24 h after mast cell activation, substantial numbers of dermal cells are strongly positive for collagen mRNA at 1 and 2 h after antigen challenge, before circulating inflammatory cells are recruited into the tissues. Furthermore, experiments in mast cell- reconstituted or genetically mast cell-deficient WBB6F1-W/Wv mice demonstrate that the increased expression of collagen mRNA at sites of PCA reactions is entirely mast cell dependent. In vitro studies show that the supernatants of mouse serosal mast cells activated via the Fc epsilon RI markedly increase type alpha-1 (I) collagen mRNA levels in mouse embryonic skin fibroblasts, and also upregulate collagen secretion by these cells. The ability of mast cell supernatants to induce increased steady state levels of collagen mRNA in mouse skin fibroblasts is markedly diminished by absorption with antibodies specific for either of two mast cell-derived cytokines, transforming growth factor beta (TGF-beta 1) or tumor necrosis factor alpha (TNF- alpha), and is eliminated entirely by absorption with antibodies against both cytokines. Taken together, these findings demonstrate that IgE-dependent mouse mast cell activation can induce a transient and marked increase in steady state levels of type alpha-1 (I) collagen mRNA in dermal fibroblasts and that mast cell-derived TGF-beta 1 and TNF-alpha importantly contribute to this effect. PMID:7964480

  7. Biochemical basis of type IB (E1beta ) mutations in maple syrup urine disease. A prevalent allele in patients from the Druze kindred in Israel.

    PubMed

    Wynn, R M; Chuang, J L; Sansaricq, C; Mandel, H; Chuang, D T

    2001-09-28

    Maple syrup urine disease (MSUD) is a metabolic disorder associated with often-fatal ketoacidosis, neurological derangement, and mental retardation. In this study, we identify and characterize two novel type IB MSUD mutations in Israeli patients, which affect the E1beta subunit in the decarboxylase (E1) component of the branched-chain alpha-ketoacid dehydrogenase complex. The recombinant mutant E1 carrying the prevalent S289L-beta (TCG --> TTG) mutation in the Druze kindred exists as a stable inactive alphabeta heterodimer. Based on the human E1 structure, the S289L-beta mutation disrupts the interactions between Ser-289-beta and Glu-290-beta', and between Arg-309-beta and Glu-290-beta', which are essential for native alpha(2)beta(2) heterotetrameric assembly. The R133P-beta (CGG --> CCG) mutation, on the other hand, is inefficiently expressed in Escherichia coli as heterotetramers in a temperature-dependent manner. The R133P-beta mutant E1 exhibits significant residual activity but is markedly less stable than the wild-type, as measured by thermal inactivation and free energy change of denaturation. The R133P-beta substitution abrogates the coordination of Arg-133-beta to Ala-95-beta, Glu-96-beta, and Ile-97-beta, which is important for strand-strand interactions and K(+) ion binding in the beta subunit. These findings provide new insights into folding and assembly of human E1 and will facilitate DNA-based diagnosis for MSUD in the Israeli population.

  8. Chronic inflammation associated with hepatitis C virus infection perturbs hepatic transforming growth factor beta signaling, promoting cirrhosis and hepatocellular carcinoma.

    PubMed

    Matsuzaki, Koichi; Murata, Miki; Yoshida, Katsunori; Sekimoto, Go; Uemura, Yoshiko; Sakaida, Noriko; Kaibori, Masaki; Kamiyama, Yasuo; Nishizawa, Mikio; Fujisawa, Junichi; Okazaki, Kazuichi; Seki, Toshihito

    2007-07-01

    Many patients with chronic hepatitis caused by hepatitis C virus (HCV) infection develop liver fibrosis with high risk for hepatocellular carcinoma (HCC), but the mechanism underling this process is unclear. Conversely, transforming growth factor beta (TGF-beta) activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), which convert the mediator Smad3 into two distinctive phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). Whereas the TbetaRI/pSmad3C pathway suppresses epithelial cell growth by upregulating p21(WAF1) transcription, JNK/pSmad3L-mediated signaling promotes extracellular matrix deposition, partly, by upregulating plasminogen activator inhibitor 1 (PAI-1). We studied the domain-specific Smad3 phosphorylation in biopsy specimens representing chronic hepatitis, cirrhosis, or HCC from 100 patients chronically infected with HCV, and correlated Smad3 phosphorylation with clinical course. As HCV-infected livers progressed from chronic hepatitis through cirrhosis to HCC, hepatocytic pSmad3L/PAI-1 increased with fibrotic stage and necroinflammatory grade, and pSmad3C/p21(WAF1) decreased. Of 14 patients with chronic hepatitis C with strong hepatocytic pSmad3L positivity, 8 developed HCC within 12 years; only 1 of 12 showing little pSmad3L positivity developed HCC. We further sought molecular mechanisms in vitro. JNK activation by the pro-inflammatory cytokine interleukin-1beta stimulated the pSmad3L/PAI-1 pathway in facilitating hepatocytic invasion, in the meantime reducing TGF-beta-dependent tumor-suppressive activity by the pSmad3C/p21(WAF1) pathway. These results indicate that chronic inflammation associated with HCV infection shifts hepatocytic TGF-beta signaling from tumor-suppression to fibrogenesis, accelerating liver fibrosis and increasing risk for HCC.

  9. Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Dang; Fang, Liurong; Luo, Rui

    2010-08-13

    Research highlights: {yields} FMDV L{sup pro} inhibits poly(I:C)-induced IFN-{alpha}1/{beta} mRNA expression. {yields} L{sup pro} inhibits MDA5-mediated activation of the IFN-{alpha}1/{beta} promoter. {yields} L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes. {yields} L{sup pro} inhibits IFN-{alpha}1/{beta} promoter activation by decreasing IRF-3/7 in protein levels. {yields} The ability to process eIF-4G of L{sup pro} is not necessary to inhibit IFN-{alpha}1/{beta} activation. -- Abstract: The leader proteinase (L{sup pro}) of foot-and-mouth disease virus (FMDV) has been identified as an interferon-{beta} (IFN-{beta}) antagonist that disrupts the integrity of transcription factor nuclear factor {kappa}B (NF-{kappa}B). In this study, we showed that the reductionmore » of double stranded RNA (dsRNA)-induced IFN-{alpha}1/{beta} expression caused by L{sup pro} was also associated with a decrease of interferon regulatory factor 3/7 (IRF-3/7) in protein levels, two critical transcription factors for activation of IFN-{alpha}/{beta}. Furthermore, overexpression of L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, and RANTES. Screening L{sup pro} mutants indicated that the ability to process eIF-4G of L{sup pro} is not required for suppressing dsRNA-induced activation of the IFN-{alpha}1/{beta} promoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-{kappa}B, L{sup pro} also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.« less

  10. Relationship between subclinical rejection and genotype, renal messenger RNA, and plasma protein transforming growth factor-beta1 levels.

    PubMed

    Hueso, Miguel; Navarro, Estanis; Moreso, Francesc; Beltrán-Sastre, Violeta; Ventura, Francesc; Grinyó, Josep M; Serón, Daniel

    2006-05-27

    Transforming growth factor (TGF)-beta(1) is increased in allograft rejection and its production is associated with single nucleotide polymorphisms (SNPs). The contribution of SNPs at codons 10 and 25 of the TGF-beta(1) gene to renal allograft damage was assessed in 6-month protocol biopsies and their association with TGF-beta(1) production. TGF-beta(1) genotypes were evaluated by polymerase chain reaction (PCR)/restriction fragment length polymorphism. Intragraft TGF-beta(1) messenger RNA (mRNA) was measured by real-time PCR and TGF-beta(1) plasma levels were assessed by enzyme-linked immunosorbent assay. Eighty consecutive patients were included. Allele T at codon 10 (risk ratio, 6.7; P = 0.02) and an episode of acute rejection before protocol biopsy (risk ratio, 6.2; P = 0.01) were independent predictors of subclinical rejection (SCR). TGF-beta(1) plasma levels, but not those of TGF-beta(1) mRNA, were increased in patients with SCR (2.59 ng/mL +/- 0.91 [n = 22] vs. 2.05 ng/mL +/- 0.76 [n = 43]; P = 0.01). There was no association between allele T and TGF-beta(1) plasma or intragraft levels. Allele T at codon 10 of the TGF-beta(1) gene is associated with a higher incidence of SCR.

  11. Fermentable metabolite of Zymomonas mobilis controls collagen reduction in photoaging skin by improving TGF-beta/Smad signaling suppression.

    PubMed

    Tanaka, Hiroshi; Yamaba, Hiroyuki; Kosugi, Nobuhiko; Mizutani, Hiroshi; Nakata, Satoru

    2008-04-01

    Solar ultraviolet (UV) irradiation causes damages on human skin and premature skin aging (photoaging). UV-induced reduction of type I collagen in dermis is widely considered primarily induction of wrinkled appearance of photoaging skin. Type I procollagen synthesis is reduced under UV irradiation by blocking transforming growth factor-beta (TGF-beta)/Smad signaling; more specifically, it is down-regulation of TGF-beta type II receptor (T beta RII). Therefore, preventing UV-induced loss of T beta RII results decreased type I collagen reduction in photoaging skin. Zymomonas mobilis is an alcohol fermentable, gram-negative facultative anaerobic bacterium whose effect on skin tissue is scarcely studied. We investigated the protective effects of fermentable metabolite of Z. mobilis (FM of Z. mobilis) against reduction of type I procollagen synthesis of UV-induced down-regulation of T beta RII in human dermal fibroblasts FM of Z. mobilis was obtained from lyophilization of bacterium culture supernatant. The levels of T beta RII and type I procollagen mRNA in human dermal fibroblasts were measured by quantitative real-time RT-PCR, and T beta RII protein levels were assayed by western blotting. T beta RII, type I procollagen, and type I collagen proteins in human dermal fibroblasts or hairless mouse skin were detected by immunostaining. FM of Z. mobilis inhibited down regulation of T beta RII mRNA, and protein levels in UVB irradiated human dermal fibroblasts consequently recover reduced type I procollagen synthesis. These results indicate UVB irradiation inhibits type I procollagen synthesis by suppression of TGF-beta/Smad signaling pathway, and FM of Z. mobilis has inhibitory effect on UVB-induced reduction of type I procollagen synthesis. While short period UVB irradiation decreased both T beta RII and type I procollagen protein levels in hairless mouse skin, topical application of FM of Z. mobilis prevented this decrease. Wrinkle formation in hairless mouse skin

  12. Association between Plasma Levels of Transforming Growth Factor-beta1, IL-23 and IL-17 and the Severity of Autism in Egyptian Children

    ERIC Educational Resources Information Center

    Hashim, Haitham; Abdelrahman, Hadeel; Mohammed, Doaa; Karam, Rehab

    2013-01-01

    It has been recently shown that dysregulation of transforming growth factor-beta1 (TGF-beta1), IL-23 and IL-17 has been identified as a major factor involved in autoimmune disorders. Based on the increasing evidence of immune dysfunction in autism the aim of this study was to measure serum levels of TGF-beta1, IL-23 and IL-17 in relation to the…

  13. Transforming growth factor-{beta} inhibits CCAAT/enhancer-binding protein expression and PPAR{gamma} activity in unloaded bone marrow stromal cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ahdjoudj, S.; Kaabeche, K.; Holy, X.

    2005-02-01

    The molecular mechanisms regulating the adipogenic differentiation of bone marrow stromal cells in vivo remain largely unknown. In this study, we investigated the regulatory effects of transforming growth factor beta-2 (TGF-{beta}2) on transcription factors involved in adipogenic differentiation induced by hind limb suspension in rat bone marrow stromal cells in vivo. Time course real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis of gene expression showed that skeletal unloading progressively increases the expression of CCAAT/enhancer-binding protein (C/EBP){alpha} and C/EBP{beta} {alpha} at 5 days in bone marrow stromal cells resulting in increased peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}2) transcripts at 7 days. TGF-{beta}2more » administration in unloaded rats corrected the rise in C/EBP{alpha} and C/EBP{beta} transcripts induced by unloading in bone marrow stromal cells. This resulted in inhibition of PPAR{gamma}2 expression that was associated with increased Runx2 expression. Additionally, the inhibition of C/EBP{alpha} and C/EBP{beta} expression by TGF-{beta}2 was associated with increased PPAR{gamma} serine phosphorylation in bone marrow stromal cells, a mechanism that inhibits PPAR{gamma} transactivating activity. The sequential inhibitory effect of TGF-{beta}2 on C/EBP{alpha}, C/EBP{beta}, and PPAR{gamma}2 resulted in reduced LPL expression and abolition of bone marrow stromal cell adipogenic differentiation, which contributed to prevent bone loss induced by skeletal unloading. We conclude that TGF-{beta}2 inhibits the excessive adipogenic differentiation of bone marrow stromal cells induced by skeletal unloading by inhibiting C/EBP{alpha}, C/EBP{beta}, and PPAR{gamma} expression and activity, which provides a sequential mechanism by which TGF-{beta}2 regulates adipogenic differentiation of bone marrow stromal cells in vivo.« less

  14. Discovery of Novel Drugs to Improve Bone Health in Neurofibromatosis Type 1: The Wnt/Beta-Catenin Pathway in Fracture Repair and Pseudarthrosis

    DTIC Science & Technology

    2015-08-01

    AWARD NUMBER: W81XWH-13-1-0113 TITLE: Discovery of Novel Drugs To Improve Bone Health in Neurofibromatosis Type 1: The Wnt/Beta-Catenin...Discovery of Novel Drugs To Improve Bone Health in Neurofibromatosis Type 1: The Wnt/Beta-Catenin Pathway in Fracture Repair and Pseudarthrosis 5a...AVAILABILITY STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Patients with Neurofibromatosis (NF1

  15. Application of factor analysis of infrared spectra for quantitative determination of beta-tricalcium phosphate in calcium hydroxylapatite.

    PubMed

    Arsenyev, P A; Trezvov, V V; Saratovskaya, N V

    1997-01-01

    This work represents a method, which allows to determine phase composition of calcium hydroxylapatite basing on its infrared spectrum. The method uses factor analysis of the spectral data of calibration set of samples to determine minimal number of factors required to reproduce the spectra within experimental error. Multiple linear regression is applied to establish correlation between factor scores of calibration standards and their properties. The regression equations can be used to predict the property value of unknown sample. The regression model was built for determination of beta-tricalcium phosphate content in hydroxylapatite. Statistical estimation of quality of the model was carried out. Application of the factor analysis on spectral data allows to increase accuracy of beta-tricalcium phosphate determination and expand the range of determination towards its less concentration. Reproducibility of results is retained.

  16. Critical Role of Transforming Growth Factor Beta in Different Phases of Wound Healing

    PubMed Central

    Pakyari, Mohammadreza; Farrokhi, Ali; Maharlooei, Mohsen Khosravi; Ghahary, Aziz

    2013-01-01

    Significance This review highlights the critical role of transforming growth factor beta (TGF-β)1–3 within different phases of wound healing, in particular, late-stage wound healing. It is also very important to identify the TGF-β1–controlling factors involved in slowing down the healing process upon wound epithelialization. Recent Advances TGF-β1, as a growth factor, is a known proponent of dermal fibrosis. Several strategies to modulate or regulate TGF's actions have been thoroughly investigated in an effort to create successful therapies. This study reviews current discourse regarding the many roles of TGF-β1 in wound healing by modulating infiltrated immune cells and the extracellular matrix. Critical Issues It is well established that TGF-β1 functions as a wound-healing promoting factor, and thereby if in excess it may lead to overhealing outcomes, such as hypertrophic scarring and keloid. Thus, the regulation of TGF-β1 in the later stages of the healing process remains as critical issue of which to better understand. Future Directions One hypothesis is that cell communication is the key to regulate later stages of wound healing. To elucidate the role of keratinocyte/fibroblast cross talk in controlling the later stages of wound healing we need to: (1) identify those keratinocyte-released factors which would function as wound-healing stop signals, (2) evaluate the functionality of these factors in controlling the outcome of the healing process, and (3) formulate topical vehicles for these antifibrogenic factors to improve or even prevent the development of hypertrophic scarring and keloids as a result of deep trauma, burn injuries, and any type of surgical incision. PMID:24527344

  17. Role of transforming growth factor-beta (TGF) beta in the physiopathology of rheumatoid arthritis.

    PubMed

    Gonzalo-Gil, Elena; Galindo-Izquierdo, María

    2014-01-01

    Transforming growth factor-beta (TGF-β) is a cytokine with pleiotropic functions in hematopoiesis, angiogenesis, cell proliferation, differentiation, migration and apoptosis. Although its role in rheumatoid arthritis is not well defined, TGF-β activation leads to functional immunomodulatory effects according to environmental conditions. The function of TGF-β in the development of arthritis in murine models has been extensively studied with controversial results. Recent findings point to a non-relevant role for TGF-β in a mice model of collagen-induced arthritis. The study of TGF-β on T-cell responses has shown controversial results as an inhibitor or promoter of the inflammatory response. This paper presents a review of the role of TGF-β in animal models of arthritis. Copyright © 2013 Elsevier España, S.L. All rights reserved.

  18. Up-regulated transcriptional repressors SnoN and Ski bind Smad proteins to antagonize transforming growth factor-beta signals during liver regeneration.

    PubMed

    Macias-Silva, Marina; Li, Wei; Leu, Julia I; Crissey, Mary Ann S; Taub, Rebecca

    2002-08-09

    Transforming growth factor-beta (TGF-beta) functions as an antiproliferative factor for hepatocytes. However, for unexplained reasons, hepatocytes become resistant to TGF-beta signals and can proliferate despite the presence of TGF-beta during liver regeneration. TGF-beta is up-regulated during liver regeneration, although it is not known whether it is active or latent. TGF-beta activity may be examined by assessing Smad activation, a downstream signaling pathway. Smad pathway activation during liver regeneration induced by partial hepatectomy or CC4 injury was examined by assessing the levels of phospho-Smad2 and Smad2-Smad4 complexes. We found that Smad proteins were slightly activated in quiescent liver, but that their activation was further enhanced in regenerating liver. Interestingly, TGF-beta/Smad pathway inhibitors (SnoN and Ski) were up-regulated during regeneration, and notably, SnoN was induced mainly in hepatocytes. SnoN and Ski are transcriptional repressors that may render some cells resistant to TGF-beta via binding Smad proteins. Complexes between SnoN, Ski, and the activated Smad proteins were detected from 2 to 120 h during the major proliferative phase in regenerating liver. Inhibitory complexes decreased after liver mass restitution (5-15 days), suggesting that persistently activated Smad proteins might participate in returning the liver to a quiescent state. Our data show that active TGF-beta/Smad signals are present during regeneration and suggest that SnoN/Ski induction might explain hepatocyte resistance to TGF-beta during the proliferative phase.

  19. Dual role for the latent transforming growth factor-beta binding protein in storage of latent TGF-beta in the extracellular matrix and as a structural matrix protein

    PubMed Central

    1995-01-01

    The role of the latent TGF-beta binding protein (LTBP) is unclear. In cultures of fetal rat calvarial cells, which form mineralized bonelike nodules, both LTBP and the TGF-beta 1 precursor localized to large fibrillar structures in the extracellular matrix. The appearance of these fibrillar structures preceded the appearance of type I collagen fibers. Plasmin treatment abolished the fibrillar staining pattern for LTBP and released a complex containing both LTBP and TGF-beta. Antibodies and antisense oligonucleotides against LTBP inhibited the formation of mineralized bonelike nodules in long-term fetal rat calvarial cultures. Immunohistochemistry of fetal and adult rat bone confirmed a fibrillar staining pattern for LTBP in vivo. These findings, together with the known homology of LTBP to the fibrillin family of proteins, suggest a novel function for LTBP, in addition to its role in matrix storage of latent TGF-beta, as a structural matrix protein that may play a role in bone formation. PMID:7593177

  20. Small Molecular Inhibitor of Transforming Growth Factor-{beta} Protects Against Development of Radiation-Induced Lung Injury

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Anscher, Mitchell S.; Thrasher, Bradley; Zgonjanin, Larisa

    2008-07-01

    Purpose: To determine whether an anti-transforming growth factor-{beta} (TGF-{beta}) type 1 receptor inhibitor (SM16) can prevent radiation-induced lung injury. Methods and Materials: One fraction of 28 Gy or sham radiotherapy (RT) was administered to the right hemithorax of Sprague-Dawley rats. SM16 was administered in the rat chow (0.07 g/kg or 0.15 g/kg) beginning 7 days before RT. The rats were divided into eight groups: group 1, control chow; group 2, SM16, 0.07 g/kg; group 3, SM16, 0.15 g/kg; group 4, RT plus control chow; group 5, RT plus SM16, 0.07 g/kg; group 6, RT plus SM16, 0.15 g/kg; group 7,more » RT plus 3 weeks of SM16 0.07 g/kg followed by control chow; and group 8, RT plus 3 weeks of SM16 0.15 g/kg followed by control chow. The breathing frequencies, presence of inflammation/fibrosis, activation of macrophages, and expression/activation of TGF-{beta} were assessed. Results: The breathing frequencies in the RT plus SM16 0.15 g/kg were significantly lower than the RT plus control chow from Weeks 10-22 (p <0.05). The breathing frequencies in the RT plus SM16 0.07 g/kg group were significantly lower only at Weeks 10, 14, and 20. At 26 weeks after RT, the RT plus SM16 0.15 g/kg group experienced a significant decrease in lung fibrosis (p = 0.016), inflammatory response (p = 0.006), and TGF-{beta}1 activity (p = 0.011). No significant reduction was found in these measures of lung injury in the group that received SM16 0.7g/kg nor for the short-course (3 weeks) SM16 at either dose level. Conclusion: SM16 at a dose of 0.15 g/kg reduced functional lung damage, morphologic changes, inflammatory response, and activation of TGF-{beta} at 26 weeks after RT. The data suggest a dose response and also suggest the superiority of long-term vs. short-term dosing.« less

  1. Association of transforming growth factor-beta1 gene polymorphism in the development of Epstein-Barr virus-related hematologic diseases.

    PubMed

    Hatta, Kanako; Morimoto, Akira; Ishii, Eiichi; Kimura, Hiroshi; Ueda, Ikuyo; Hibi, Shigeyoshi; Todo, Shinjiro; Sugimoto, Tohru; Imashuku, Shinsaku

    2007-11-01

    Epstein-Barr virus (EBV) is etiologically associated with various hematologic disorders, including primary acute infectious mononucleosis (IM), hemophagocytic lymphohistiocytosis (EBV-HLH), chronic active EBV infection (CAEBV) and malignant lymphomas. Although cytokines play a central role in EBV-related immune responses, the exact mechanisms causing different clinical responses remain unclear. In this study, the pattern of cytokine gene polymorphisms was comparatively analyzed in EBV-related diseases. Eighty-nine patients with EBV-related disease were analyzed; 30 with IM, 28 with EBV-HLH and 31 with CAEBV. Eighty-one EBV-seropositive healthy adults were also used as controls. Associations with polymorphisms of various cytokines, including interleukin (IL)-1 alpha and IL-1 beta were evaluated. The gene polymorphisms were typed by polymerase chain reaction with sequence-specific primers. A significant difference of polymorphisms was found for transforming growth factor (TGF)-beta1; the frequency of TGF-beta1 codon 10 C allele was significantly higher in patients with EBV-related diseases than in controls (p<0.001). The difference was significant in patients with IM or HLH (p<0.001), but not in those with CAEBV (p=0.127), compared with controls. As regards other cytokines, the frequency of the IL-1 alpha -889 C allele was significantly lower in patients with IM than in controls (p<0.05). Our results suggests that TGF-beta1 codon 10 C allele plays a role in the development of EBV-related diseases and that the IL-1 alpha -889 C allele may be involved in response failure and sequential progression into the development of HLH.

  2. Factors affecting the outcome of trabeculectomy: an analysis based on combined data from two phase III studies of an antibody to transforming growth factor beta2, CAT-152.

    PubMed

    Grehn, Franz; Holló, Gábor; Khaw, Peng; Overton, Barry; Wilson, Rosamund; Vogel, Roger; Smith, Zaid

    2007-10-01

    To determine the factors affecting trabeculectomy success. Retrospective analysis of 2 randomized controlled trials comparing an antibody against transforming growth factor beta2 (TGF-beta2) with vehicle (placebo) for prevention of fibrosis after trabeculectomy, in which there was no significant difference between the treatment groups. Data were from patients (n = 726) with a diagnosis of primary open-angle glaucoma, chronic angle-closure glaucoma, pseudoexfoliative glaucoma, or pigmentary glaucoma (PG) who had an intraocular pressure (IOP) > 21 mmHg and visual field or optic disc changes characteristic of glaucoma and were taking the maximum tolerated dose of medication before trabeculectomy. Patients had trabeculectomy and 4 subconjunctival injections of a human monoclonal antibody to TGF-beta2 (CAT-152) or a placebo. The definition of trabeculectomy success in the protocols was an IOP between 6 and 16 mmHg inclusive at months 6 and 12. Analyses of success used factors identified by ophthalmic experts. Covariates analyzed included patient age, black race, gender, time since diagnosis, primary diagnosis, country, diabetes, mean defect, cup-to-disc (C/D) ratio, suture type, anesthetic, flap type, IOP at listing for surgery, suture release/lysis, needling, reformed anterior chamber, wound leak, severe bleb vascularity, and bleb microcysts. A stepwise logistic regression model found the following predictors of treatment success: PG (odds ratio [OR], 4.11; 95% confidence interval [CI], 1.41-11.99), high C/D ratio (OR, 2.84; 95% CI, 1.15-6.99), and use of a corneal traction suture (OR, 1.67; 95% CI, 1.09-2.56). A negative relationship was found for black race (OR, 0.28; 95% CI, 0.13-0.62); treatment in France (OR, 0.35; 95% CI, 0.17-0.70), Sweden (OR, 0.17; 95% CI, 0.05-0.58), Spain (OR, 0.37; 95% CI, 0.21-0.68), Poland (OR, 0.53; 95% CI, 0.32-0.88), or Hungary (OR, 0.14; 95% CI, 0.06-0.34); and suture release/lysis (OR, 0.34; 95% CI, 0.22-0.53). The effect of needling

  3. Regeneration of hyaline cartilage by cell-mediated gene therapy using transforming growth factor beta 1-producing fibroblasts.

    PubMed

    Lee, K H; Song, S U; Hwang, T S; Yi, Y; Oh, I S; Lee, J Y; Choi, K B; Choi, M S; Kim, S J

    2001-09-20

    Transforming growth factor beta (TGF-beta) has been considered as a candidate for gene therapy of orthopedic diseases. The possible application of cell-mediated TGF-beta gene therapy as a new treatment regimen for degenerative arthritis was investigated. In this study, fibroblasts expressing active TGF-beta 1 were injected into the knee joints of rabbits with artificially made cartilage defects to evaluate the feasibility of this therapy for orthopedic diseases. Two to 3 weeks after the injection there was evidence of cartilage regeneration, and at 4 to 6 weeks the cartilage defect was completely filled with newly grown hyaline cartilage. Histological analyses of the regenerated cartilage suggested that it was well integrated with the adjacent normal cartilage at the sides of the defect and that the newly formed tissue was indeed hyaline cartilage. Our findings suggest that cell-mediated TGF-beta 1 gene therapy may be a novel treatment for orthopedic diseases in which hyaline cartilage damage has occurred.

  4. Transforming growth factor (TGF. beta. ) decreases the proliferation of human bone marrow fibroblasts by inhibiting the platelet-derived growth factor (PDGF) binding

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bryckaert, M.C.; Tobelem, G.; Lindroth, M.

    1988-12-01

    Human bone marrow fibroblasts were cultivated and characterized by immunofluorescent staining and electron microscopy. Their interactions with PDGF and TGF{beta} were studied. While a positive intracellular antifibronectin staining was observed, the cultured cells were not labeled with specific antibodies toward factor VIII von Willebrand factor (F VIII/vWF), desmin, and macrophage antigen. The binding of pure human PDGF to the cultured bone marrow fibroblasts was investigated. Addition of an excess of unlabeled PDGF decreased the binding to 75 and 80%, which means that the nonspecific binding represented 20-25% of total binding, whereas epidermal growth factor (EGF) had no effect. Two classesmore » of sites were detected by Scatchard analysis. The stimulation of DNA synthesis of PDGF was quantified by ({sup 3}H)thymidine incorporation. The results suggested that PDGF and TGF{beta} could modulate the growth of bone marrow fibroblasts.« less

  5. Effect of transforming growth factor-beta1 on decorin expression and muscle morphology during chicken embryonic and posthatch growth and development.

    PubMed

    Li, X; Velleman, S G

    2009-02-01

    During skeletal muscle development, transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of muscle cell proliferation and differentiation, as well as a regulator of extracellular matrix (ECM) production. Decorin, a member of the small leucine-rich ECM proteoglycans, binds to TGF-beta1 and modulates TGF-beta1-dependent cell growth stimulation or inhibition. The expression of decorin can be regulated by TGF-beta1 during muscle proliferation and differentiation. How TGF-beta1 affects decorin and muscle growth, however, has not been well documented in vivo. The present study investigated the effect of TGF-beta1 on decorin expression and intracellular connective tissue development during skeletal muscle growth. Exogenous TGF-beta1 significantly decreased the number of myofibers in a given area at both 1 d and 6 wk posthatch. The TGF-beta1-treated muscle had a significant decrease in decorin mRNA expression at embryonic day (ED) 10, whereas protein amounts decreased at 17 ED and 1 d posthatch compared to the control muscle. Decorin was localized in both the endomysium and perimysium in the control pectoralis major muscle. Transforming growth factor-beta1 reduced decorin in both the endomysium and perimysium from 17 ED to 6 wk posthatch. Compared to the control muscle, the perimysium space in the pectoralis major muscle was dramatically decreased by TGF-beta1 during embryonic development through posthatch growth. Because decorin regulates collagen fibrillogenesis, a major component of the ECM, the reduction of decorin by TGF-beta1 treatment may cause the irregular formation of collagen fibrils, leading to the decrease in endomysium and perimysium space. The results from the current study suggest that the effect of TGF-beta1 on decorin expression and localization was likely associated with altered development of the perimysium and the regulation of muscle fiber development.

  6. Risk factors for persistent gestational trophoblastic neoplasia.

    PubMed

    Kuyumcuoglu, Umur; Guzel, Ali Irfan; Erdemoglu, Mahmut; Celik, Yusuf

    2011-01-01

    This retrospective study evaluated the risk factors for persistent gestational trophoblastic disease (GTN) and determined their odds ratios. This study included 100 cases with GTN admitted to our clinic. Possible risk factors recorded were age, gravidity, parity, size of the neoplasia, and beta-human chorionic gonadotropin levels (beta-hCG) before and after the procedure. Statistical analyses consisted of the independent sample t-test and logistic regression using the statistical package SPSS ver. 15.0 for Windows (SPSS, Chicago, IL, USA). Twenty of the cases had persistent GTN, and the differences between these and the others cases were evaluated. The size of the neoplasia and histopathological type of GTN had no statistical relationship with persistence, whereas age, gravidity, and beta-hCG levels were significant risk factors for persistent GTN (p < 0.05). The odds ratios (95% confidence interval (CI)) for age, gravidity, and pre- and post-evacuation beta-hCG levels determined using logistic regression were 4.678 (0.97-22.44), 7.315 (1.16-46.16), 2.637 (1.41-4.94), and 2.339 (1.52-3.60), respectively. Patient age, gravidity, and beta-hCG levels were risk factors for persistent GTN, whereas the size of the neoplasia and histopathological type of GTN were not significant risk factors.

  7. Preliminary crystallographic analysis of mouse Elf3 C-terminal DNA-binding domain in complex with type II TGF-[beta] receptor promoter DNA

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Agarkar, Vinod B.; Babayeva, Nigar D.; Rizzino, Angie

    2010-10-08

    Ets proteins are transcription factors that activate or repress the expression of genes that are involved in various biological processes, including cellular proliferation, differentiation, development, transformation and apoptosis. Like other Ets-family members, Elf3 functions as a sequence-specific DNA-binding transcriptional factor. A mouse Elf3 C-terminal fragment (amino-acid residues 269-371) containing the DNA-binding domain has been crystallized in complex with mouse type II TGF-{beta} receptor promoter (TR-II) DNA. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 42.66, b = 52, c = 99.78 {angstrom}, and diffracted to a resolution of 2.2 {angstrom}.

  8. Parental history and risk of type 2 diabetes in overweight Latino adolescents: a longitudinal analysis.

    PubMed

    Kelly, Louise A; Lane, Christianne J; Weigensberg, Marc J; Koebnick, Corinna; Roberts, Christian K; Davis, Jaimie N; Toledo-Corral, Claudia M; Shaibi, Gabriel Q; Goran, Michael I

    2007-10-01

    The purpose of this article was to examine metabolic risk factors for type 2 diabetes in children and adolescents as a function of maternal versus paternal family history of type 2 diabetes and to examine whether differences in these risk factors emerge during adolescent growth. A total of 247 overweight Latino children (baseline age = 11.1 +/- 1.7 years) with a parental history of type 2 diabetes were followed annually for 5 years (2.2 +/- 1.2 observations/child) with measures of insulin sensitivity, acute insulin response to glucose, and disposition index. Longitudinal linear mixed-effects modeling was used to evaluate the influence of maternal versus paternal family history of type 2 diabetes on changes in diabetes risk factors over age. Insulin sensitivity and the disposition index decreased over age (beta = -0.052 and beta = -0.033, P < 0 0.01). Acute insulin response to glucose and fasting and 2-h glucose increased (beta = 0.019, beta = 0.002, and beta = 0.003, P < 0.01). Declines in insulin sensitivity were significantly greater in participants whose maternal grandmothers had a history of type 2 diabetes (beta = -0.03, P = 0.03). Declines in the disposition index (beta = -0.02, P = 0.04) and increases in fasting glucose were significantly influenced by a maternal history of type 2 diabetes (beta = 0.60, P < 0.05). Maternal but not paternal family history for diabetes may have a significant impact on insulin dynamics, becoming more pronounced during growth in overweight Latino adolescents. Further research is clearly warranted.

  9. Amyloid beta peptide as a physiological modulator of neuronal 'A'-type K+ current.

    PubMed

    Plant, Leigh D; Webster, Nicola J; Boyle, John P; Ramsden, Martin; Freir, Darragh B; Peers, Chris; Pearson, Hugh A

    2006-11-01

    Control of neuronal spiking patterns resides, in part, in the type and degree of expression of voltage-gated K(+) channel subunits. Previous studies have revealed that soluble forms of the Alzheimer's disease associated amyloid beta protein (Abeta) can increase the 'A'-type current in neurones. In this study, we define the molecular basis for this increase and show that endogenous production of Abeta is important in the modulation of Kv4.2 and Kv4.3 subunit expression in central neurones. A-type K(+) currents, and Kv4.2 and Kv4.3 subunit expression, were transiently increased in cerebellar granule neurones by the 1-40 and 1-42 forms of Abeta (100nM, 2-24h). Currents through recombinant Kv4.2 channels expressed in HEK293 cells were increased in a similar fashion to those through the native channels. Increases in 'A'-type current could be prevented by the use of cycloheximide and brefeldin A, indicating that protein expression and trafficking processes were altered by Abeta, rather than protein degredation. Endogenous Abeta production in cerebellar granule neurones was blocked using inhibitors of either gamma- or beta-secretase and resulted in decreased K(+) current. Crucially this could be prevented by co-application of exogenous Abeta (1nM), however, no change in Kv4.2 or Kv4.3 subunit expression occurred. These data show that Abeta is a modulator of Kv4 subunit expression in neurones at both the functional and the molecular level. Thus Abeta is not only involved in Alzheimer pathology, but is also an important physiological regulator of ion channel expression and hence neuronal excitability.

  10. The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus.

    PubMed Central

    Gustafson, G; Armour, S L

    1986-01-01

    The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus (BSMV) has been determined. The sequence is 3289 nucleotides in length and contains four open reading frames (ORFs) which code for proteins of Mr 22,147 (ORF1), Mr 58,098 (ORF2), Mr 17,378 (ORF3), and Mr 14,119 (ORF4). The predicted N-terminal amino acid sequence of the polypeptide encoded by the ORF nearest the 5'-end of the RNA (ORF1) is identical (after the initiator methionine) to the published N-terminal amino acid sequence of BSMV coat protein for 29 of the first 30 amino acids. ORF2 occupies the central portion of the coding region of RNA beta and ORF3 is located at the 3'-end. The ORF4 sequence overlaps the 3'-region of ORF2 and the 5'-region of ORF3 and differs in codon usage from the other three RNA beta ORFs. The coding region of RNA beta is followed by a poly(A) tract and a 238 nucleotide tRNA-like structure which are common to all three BSMV genomic RNAs. Images PMID:3754962

  11. Essential role of mitochondrial antiviral signaling, IFN regulatory factor (IRF)3, and IRF7 in Chlamydophila pneumoniae-mediated IFN-beta response and control of bacterial replication in human endothelial cells.

    PubMed

    Buss, Claudia; Opitz, Bastian; Hocke, Andreas C; Lippmann, Juliane; van Laak, Vincent; Hippenstiel, Stefan; Krüll, Matthias; Suttorp, Norbert; Eitel, Julia

    2010-03-15

    Chlamydophila pneumoniae infection of the vascular wall as well as activation of the transcription factor IFN regulatory factor (IRF)3 have been linked to development of chronic vascular lesions and atherosclerosis. The innate immune system detects invading pathogens by use of pattern recognition receptors, some of which are able to stimulate IRF3/7 activation and subsequent type I IFN production (e. g., IFN-beta). In this study, we show that infection of human endothelial cells with C. pneumoniae-induced production of IFN-beta, a cytokine that so far has been mainly associated with antiviral immunity. Moreover, C. pneumoniae infection led to IRF3 and IRF7 nuclear translocation in HUVECs and RNA interference experiments showed that IRF3 and IRF7 as well as the mitochondrial antiviral signaling (MAVS) were essential for IFN-beta induction. Finally, C. pneumoniae replication was enhanced in endothelial cells in which IRF3, IRF7, or MAVS expression was inhibited by small interfering RNA and attenuated by IFN-beta treatment. In conclusion, C. pneumoniae infection of endothelial cells activates an MAVS-, IRF3-, and IRF7-dependent signaling, which controls bacterial growth and might modulate development of vascular lesions.

  12. Down-regulation of connective tissue growth factor by inhibition of transforming growth factor beta blocks the tumor-stroma cross-talk and tumor progression in hepatocellular carcinoma.

    PubMed

    Mazzocca, Antonio; Fransvea, Emilia; Dituri, Francesco; Lupo, Luigi; Antonaci, Salvatore; Giannelli, Gianluigi

    2010-02-01

    Tumor-stroma interactions in hepatocellular carcinoma (HCC) are of key importance to tumor progression. In this study, we show that HCC invasive cells produce high levels of connective tissue growth factor (CTGF) and generate tumors with a high stromal component in a xenograft model. A transforming growth factor beta (TGF-beta) receptor inhibitor, LY2109761, inhibited the synthesis and release of CTGF, as well as reducing the stromal component of the tumors. In addition, the TGF-beta-dependent down-regulation of CTGF diminished tumor growth, intravasation, and metastatic dissemination of HCC cells by inhibiting cancer-associated fibroblast proliferation. By contrast, noninvasive HCC cells were found to produce low levels of CTGF. Upon TGF-beta1 stimulation, noninvasive HCC cells form tumors with a high stromal content and CTGF expression, which is inhibited by treatment with LY2109761. In addition, the acquired intravasation and metastatic spread of noninvasive HCC cells after TGF-beta1 stimulation was blocked by LY2109761. LY2109761 interrupts the cross-talk between cancer cells and cancer-associated fibroblasts, leading to a significant reduction of HCC growth and dissemination. Interestingly, patients with high CTGF expression had poor prognosis, suggesting that treatment aimed at reducing TGF-beta-dependent CTGF expression may offer clinical benefits. Taken together, our preclinical results indicate that LY2109761 targets the cross-talk between HCC and the stroma and provide a rationale for future clinical trials.

  13. Structural and sequence features of two residue turns in beta-hairpins.

    PubMed

    Madan, Bharat; Seo, Sung Yong; Lee, Sun-Gu

    2014-09-01

    Beta-turns in beta-hairpins have been implicated as important sites in protein folding. In particular, two residue β-turns, the most abundant connecting elements in beta-hairpins, have been a major target for engineering protein stability and folding. In this study, we attempted to investigate and update the structural and sequence properties of two residue turns in beta-hairpins with a large data set. For this, 3977 beta-turns were extracted from 2394 nonhomologous protein chains and analyzed. First, the distribution, dihedral angles and twists of two residue turn types were determined, and compared with previous data. The trend of turn type occurrence and most structural features of the turn types were similar to previous results, but for the first time Type II turns in beta-hairpins were identified. Second, sequence motifs for the turn types were devised based on amino acid positional potentials of two-residue turns, and their distributions were examined. From this study, we could identify code-like sequence motifs for the two residue beta-turn types. Finally, structural and sequence properties of beta-strands in the beta-hairpins were analyzed, which revealed that the beta-strands showed no specific sequence and structural patterns for turn types. The analytical results in this study are expected to be a reference in the engineering or design of beta-hairpin turn structures and sequences. © 2014 Wiley Periodicals, Inc.

  14. A dynamic alpha-beta inter-subunit agonist signaling complex is a novel feedback mechanism for regulating L-type Ca2+ channel opening.

    PubMed

    Zhang, Rong; Dzhura, Igor; Grueter, Chad E; Thiel, William; Colbran, Roger J; Anderson, Mark E

    2005-09-01

    L-type Ca2+ channels are macromolecular protein complexes in neurons and myocytes that open in response to cell membrane depolarization to supply Ca2+ for regulating gene transcription and vesicle secretion and triggering cell contraction. L-type Ca2+ channels include a pore-forming alpha and an auxiliary beta subunit, and alpha subunit openings are regulated by cellular Ca2+ through a mechanism involving the Ca2+-sensing protein calmodulin (CaM) and CaM binding motifs in the alpha subunit cytoplasmic C terminus. Here we show that these CaM binding motifs are "auto-agonists" that increase alpha subunit openings by binding the beta subunit. The CaM binding domains are necessary and sufficient for the alpha subunit C terminus to bind the beta subunit in vitro, and excess CaM blocks this interaction. Addition of CaM binding domains to native cardiac L-type Ca2+ channels in excised cell membrane patches increases openings, and this agonist effect is prevented by excess CaM. Recombinant LTCC openings are also increased by exogenous CaM binding domains by a mechanism requiring the beta subunit, and excess CaM blocks this effect. Thus, the bifunctional ability of the alpha subunit CaM binding motifs to competitively associate with the beta subunit or CaM provides a novel paradigm for feedback control of cellular Ca2+ entry.

  15. Topological side-chain classification of beta-turns: ideal motifs for peptidomimetic development.

    PubMed

    Tran, Tran Trung; McKie, Jim; Meutermans, Wim D F; Bourne, Gregory T; Andrews, Peter R; Smythe, Mark L

    2005-08-01

    Beta-turns are important topological motifs for biological recognition of proteins and peptides. Organic molecules that sample the side chain positions of beta-turns have shown broad binding capacity to multiple different receptors, for example benzodiazepines. Beta-turns have traditionally been classified into various types based on the backbone dihedral angles (phi2, psi2, phi3 and psi3). Indeed, 57-68% of beta-turns are currently classified into 8 different backbone families (Type I, Type II, Type I', Type II', Type VIII, Type VIa1, Type VIa2 and Type VIb and Type IV which represents unclassified beta-turns). Although this classification of beta-turns has been useful, the resulting beta-turn types are not ideal for the design of beta-turn mimetics as they do not reflect topological features of the recognition elements, the side chains. To overcome this, we have extracted beta-turns from a data set of non-homologous and high-resolution protein crystal structures. The side chain positions, as defined by C(alpha)-C(beta) vectors, of these turns have been clustered using the kth nearest neighbor clustering and filtered nearest centroid sorting algorithms. Nine clusters were obtained that cluster 90% of the data, and the average intra-cluster RMSD of the four C(alpha)-C(beta) vectors is 0.36. The nine clusters therefore represent the topology of the side chain scaffold architecture of the vast majority of beta-turns. The mean structures of the nine clusters are useful for the development of beta-turn mimetics and as biological descriptors for focusing combinatorial chemistry towards biologically relevant topological space.

  16. Factors determining insulin requirements in women with type 1 diabetes mellitus during pregnancy: a review

    PubMed Central

    McIntyre, Harold David; Callaway, Leonie

    2014-01-01

    Most women with type 1 diabetes mellitus (T1DM) have increased insulin requirements during pregnancy. However, a minority of women have a fall in insulin requirements. When this occurs in late gestation, it often provokes concern regarding possible compromise of the feto-placental unit. In some centres, this is considered as an indication for delivery, including premature delivery. There are, however, many other factors that affect insulin requirements in pregnancy in women with type 1 diabetes mellitus and the decline in insulin requirements may represent a variant of normal pregnancy. If there is no underlying pathological process, expedited delivery in these women is not warranted and confers increased risks to the newborn. We will explore the factors affecting insulin requirements in gestation in this review. We will also discuss some novel concepts regarding beta-cell function in pregnancy. PMID:27512424

  17. Mechanisms of stimulation of interleukin-1 beta and tumor necrosis factor-alpha by Mycobacterium tuberculosis components.

    PubMed Central

    Zhang, Y; Doerfler, M; Lee, T C; Guillemin, B; Rom, W N

    1993-01-01

    The granulomatous immune response in tuberculosis is characterized by delayed hypersensitivity and is mediated by various cytokines released by the stimulated mononuclear phagocytes, including tumor necrosis factor-alpha (TNF alpha) and IL-1 beta. We have demonstrated that Mycobacterium tuberculosis cell wall component lipoarabinomannan (LAM), mycobacterial heat shock protein-65 kD, and M. tuberculosis culture filtrate, devoid of LPS as assessed by the Amebocyte Lysate assay, stimulate the production of TNF alpha and IL-1 beta proteins and mRNA from mononuclear phagocytes (THP-1 cells). The effect of LAM on the release of these cytokines was specific, as only LAM stimulation was inhibited by anti-LAM monoclonal antibody. Interestingly, we found that LAM and Gram-negative bacterial cell wall-associated endotoxin LPS may share a similar mechanism in their stimulatory action as demonstrated by inhibition of TNF alpha and IL-1 beta release by monoclonal antibodies to CD14. Anti-CD14 monoclonal antibody MY4 inhibited both TNF alpha and IL-1 beta release with LAM and LPS but no effect was observed with other mycobacterial proteins. An isotype antibody control did not inhibit release of cytokines under the same experimental conditions. M. tuberculosis and its components upregulated IL-1 beta and TNF alpha mRNAs in THP-1 cells. Nuclear run-on assay for IL-1 beta demonstrated that LAM increased the transcription rate. The induction of IL-1 beta was regulated at the transcriptional level, in which these stimuli acted through cis-acting element(s) on the 5' flanking region of the IL-1 beta genomic DNA. M. tuberculosis cell wall component LAM acts similarly to LPS in activating mononuclear phagocyte cytokine TNF alpha and IL-1 beta release through CD14 and synthesis at the transcriptional level; both cytokines are key participants in the host immune response to tuberculosis. Images PMID:7683696

  18. Suppression of transforming growth factor-beta-induced apoptosis through a phosphatidylinositol 3-kinase/Akt-dependent pathway.

    PubMed

    Chen, R H; Su, Y H; Chuang, R L; Chang, T Y

    1998-10-15

    Insulin and insulin receptor substrate 1 (IRS-1) are capable of protecting liver cells from apoptosis induced by transforming growth factor-beta1 (TGF-beta). The Ras/mitogen-activated protein kinase (MAP kinase) and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways are both activated upon insulin stimulation and can protect against apoptosis under certain circumstances. We investigated which of these pathways is responsible for the protective effect of insulin on TGF-beta-induced apoptosis. An activated Ras, although elicited a strong mitogenic effect, could not protect Hep3B cells from TGF-beta-induced apoptosis. Furthermore, PD98059, a selective inhibitor of MEK, did not suppress the antiapoptotic effect of insulin. In contrast, the PI 3-kinase inhibitor, LY294002, efficiently blocked the effect of insulin. Protection against TGF-beta-induced apoptosis conferred by PI 3-kinase was further verified by stable transfection of an activated PI 3-kinase. Downstream targets of PI 3-kinase involved in this protection was further investigated. An activated Akt mimicked the antiapoptotic effect of insulin, whereas a dominant-negative Akt inhibited such effect. However, rapamycin, the p70S6 kinase inhibitor, had no effect on the protectivity of insulin against TGF-beta-induced apoptosis, suggesting that the antiapoptotic target of PI 3-kinase/Akt pathway is independent or lies upstream of the p70S6 kinase. The mechanism by which PI 3-kinase/Akt pathway interferes with the apoptotic signaling of TGF-beta was explored. Activation of PI 3-kinase did not lead to a suppression of Smad hetero-oligomerization or nuclear translocation but blocked TGF-beta-induced caspase-3-like activity. In summary, the PI 3-kinase/Akt pathway, but not the Ras/MAP kinase pathway, protects against TGF-beta-induced apoptosis by inhibiting a step downstream of Smad but upstream of caspase-3.

  19. Molecular cloning and characterization of beluga whale (Delphinapterus leucas) interleukin-1beta and tumor necrosis factor-alpha.

    PubMed Central

    Denis, F; Archambault, D

    2001-01-01

    Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are cytokines produced primarily by monocytes and macrophages with regulatory effects in inflammation and multiple aspects of the immune response. As yet, no molecular data have been reported for IL-1beta and TNF-alpha of the beluga whale. In this study, we cloned and determined the entire cDNA sequence encoding beluga whale IL-1beta and TNF-alpha. The genetic relationship of the cytokine sequences was then analyzed with those from several mammalian species, including the human and the pig. The homology of beluga whale IL-1beta nucleic acid and deduced amino acid sequences with those from these mammalian species ranged from 74.6 to 86.0% and 62.7 to 77.1%, respectively, whereas that of TNF-alpha varied from 79.3 to 90.8% and 75.3 to 87.7%, respectively. Phylogenetic analyses based on deduced amino acid sequences showed that the beluga whale IL-1beta and TNF-alpha were most closely related to those of the ruminant species (cattle, sheep, and deer). The beluga whale IL-1beta- and TNF-alpha-encoding sequences were thereafter successfully expressed in Escherichia coli as fusion proteins by using procaryotic expression vectors. The fusion proteins were used to produce beluga whale IL-1beta- and TNF-alpha-specific rabbit antisera. Images Figure 3. Figure 4. Figure 5. PMID:11768130

  20. The Role of Placental 11-Beta Hydroxysteroid Dehydrogenase Type 1 and Type 2 Methylation on Gene Expression and Infant Birth Weight.

    PubMed

    Green, Benjamin B; Armstrong, David A; Lesseur, Corina; Paquette, Alison G; Guerin, Dylan J; Kwan, Lauren E; Marsit, Carmen J

    2015-06-01

    Maternal stress has been linked to infant birth weight outcomes, which itself may be associated with health later in life. The placenta acts as a master regulator for the fetal environment, mediating intrauterine exposures to stress through the activity of genes regulating glucocorticoids, including the 11beta-hydroxysteroid dehydrogenase (HSD11B) type 1 and 2 genes, and so we hypothesized that variation in these genes will be associated with infant birth weight. We investigated DNA methylation levels at six sites across the two genes, as well as mRNA expression for each, and the relationship to infant birth weight. Logistic regressions correcting for potential confounding factors revealed a significant association between methylation at a single CpG site within HSD11B1 and being born large for gestational age. In addition, our analysis identified correlations between methylation and gene expression, including sex-specific transcriptional regulation of HSD11B2. Our work is one of the first comprehensive views of DNA methylation and expression in the placenta for both HSD11B types 1 and 2, linking epigenetic alterations with the regulation of fetal stress and birth weight outcomes. © 2015 by the Society for the Study of Reproduction, Inc.

  1. Functional properties of an isolated. cap alpha beta. heterodimeric human placenta insulin-like growth factor 1 receptor complex

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Feltz, S.M.; Swanson, M.L.; Wemmie, J.A.

    1988-05-03

    Treatment of human placenta membranes at pH 8.5 in the presence of 2.0 mM dithiothreitol (DTT) for 5 min, followed by the simultaneous removal of the DTT and pH adjustment of pH 7.6, resulted in the formation of a functional ..cap alpha beta.. heterodimeric insulin-like growth factor 1 (IGF-1) receptor complex from the native ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state. The membrane-bound ..cap alpha beta.. heterodimeric complex displayed similar curvilinear /sup 125/I-IGF-1 equilibrium binding compared to the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric complex. /sup 125/I-IGF-1 binding to both the isolated ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta..more » heterodimeric complexes demonstrated a marked straightening of the Scatchard plots, compared to the placenta membrane-bound IGF-1 receptors, with a 2-fold increase in the high-affinity binding component. IGF-1 stimulation of IGF-1 receptor autophosphorylation indicated that the ligand-dependent activation of ..cap alpha beta.. heterodimeric protein kinase activity occurred concomitant with the reassociation into a covalent ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric state. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of human placenta membranes results in the formation of an ..cap alpha beta.. heterodimeric IGF-1 receptor complex, (ii) unlike the insulin receptor, high-affinity homogeneous IGF-1 binding occurs in both the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta.. heterodimeric complexes, and (iii) IGF-1-dependent autophosphorylation of the ..cap alpha beta.. heterodimeric IGF-1 receptor complex correlates wit an IGF-1 dependent covalent reassociation into an ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state.« less

  2. Tumor necrosis factor receptor-1 can function through a G alpha q/11-beta-arrestin-1 signaling complex.

    PubMed

    Kawamata, Yuji; Imamura, Takeshi; Babendure, Jennie L; Lu, Juu-Chin; Yoshizaki, Takeshi; Olefsky, Jerrold M

    2007-09-28

    Tumor necrosis factor-alpha (TNFalpha) is a proinflammatory cytokine secreted from macrophages and adipocytes. It is well known that chronic TNFalpha exposure can lead to insulin resistance both in vitro and in vivo and that elevated blood levels of TNFalpha are observed in obese and/or diabetic individuals. TNFalpha has many acute biologic effects, mediated by a complex intracellular signaling pathway. In these studies we have identified new G-protein signaling components to this pathway in 3T3-L1 adipocytes. We found that beta-arrestin-1 is associated with TRAF2 (TNF receptor-associated factor 2), an adaptor protein of TNF receptors, and that TNFalpha acutely stimulates tyrosine phosphorylation of G alpha(q/11) with an increase in G alpha(q/11) activity. Small interfering RNA-mediated knockdown of beta-arrestin-1 inhibits TNFalpha-induced tyrosine phosphorylation of G alpha(q/11) by interruption of Src kinase activation. TNFalpha stimulates lipolysis in 3T3-L1 adipocytes, and beta-arrestin-1 knockdown blocks the effects of TNFalpha to stimulate ERK activation and glycerol release. TNFalpha also led to activation of JNK with increased expression of the proinflammatory gene, monocyte chemoattractant protein-1 and matrix metalloproteinase 3, and beta-arrestin-1 knockdown inhibited both of these effects. Taken together these results reveal novel elements of TNFalpha action; 1) the trimeric G-protein component G alpha(q/11) and the adapter protein beta-arrestin-1 can function as signaling molecules in the TNFalpha action cascade; 2) beta-arrestin-1 can couple TNFalpha stimulation to ERK activation and lipolysis; 3) beta-arrestin-1 and G alpha(q/11) can mediate TNFalpha-induced phosphatidylinositol 3-kinase activation and inflammatory gene expression.

  3. Values of the phase space factors for double beta decay

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Stoica, Sabin, E-mail: stoica@theory.nipne.ro; Mirea, Mihai; Horia Hulubei National Institute of Physics and Nuclear Engineering, 30 Reactorului street, P.O. Box MG6, Magurele

    2015-10-28

    We report an up-date list of the experimentally most interesting phase space factors for double beta decay (DBD). The electron/positron wave functions are obtained by solving the Dirac equations with a Coulomb potential derived from a realistic proton density distribution in nucleus and with inclusion of the finite nuclear size (FNS) and electron screening (ES) effects. We build up new numerical routines which allow us a good control of the accuracy of calculations. We found several notable differences as compared with previous results reported in literature and possible sources of these discrepancies are discussed.

  4. Macrophage colony-stimulating factor accelerates wound healing and upregulates TGF-beta1 mRNA levels through tissue macrophages.

    PubMed

    Wu, L; Yu, Y L; Galiano, R D; Roth, S I; Mustoe, T A

    1997-10-01

    Macrophage colony-stimulating factor (M-CSF) is produced by many cell types involved in wound repair, yet it acts specifically on monocytes and macrophages. The monocyte-derived cell is thought to be important in wound healing, but the importance of the role of tissue macrophages in wound healing has not been well defined. Dermal ulcers were created in normal and ischemic ears of young rabbits. Either rhM-CSF (17 microg/wound) or buffer was applied to each wound. Wounds were bisected and analyzed histologically at Days 7 and 10 postwounding. The amounts of epithelial growth and granulation tissue deposition were measured in all wounds. The level of increase of TGF-beta1 mRNA level in M-CSF-treated wounds was examined using competitive RT-PCR. M-CSF increased new granulation tissue formation by 37% (N = 21, P < 0.01) and 50% (P < 0.01) after single and multiple treatments, respectively, in nonischemic wounds. TGF-beta1 mRNA levels in rhM-CSF-treated wounds increased 5.01-fold (N = 8) over vehicle-treated wounds under nonischemic conditions. In contrast, no effect could be detected in ischemic wounds treated with rhM-CSF, and these wounds only showed a 1.66-fold increase in TGF-beta1 mRNA levels when compared to ischemic wounds treated with vehicle alone. GAPDH, a housekeeping gene, showed no change. As mesenchymal cells lack receptors for M-CSF, the improved healing of wounds treated with topical rhM-CSF must reflect a generalized enhancement of activation and function of tissue macrophages, as demonstrated by upregulation of TGF-beta. The lack of effect under ischemic conditions suggests that either macrophage activity and/or response to M-CSF is adversely affected under those conditions; this may suggest the pathogenesis of impaired wound healing at the cellular level. Copyright 1997 Academic Press.

  5. Effects of low intensity laser acupoint irradiation on inhibiting islet beta-cell apoptosis in rats with type 2 diabetes

    NASA Astrophysics Data System (ADS)

    Xiong, Guoxin; Xiong, Leilei; Li, Xinzhong

    2016-09-01

    To investigate the effects of low intensity semiconductor laser acupoint irradiation on inhibiting islet beta-cell apoptosis in rats with type 2 diabetes, a method using a high-fat diet and low-dose intraperitoneal injections of streptozotocin established a type 2 diabetes mellitus rat model. Model rats were randomly divided into a laser acupoint irradiation group, rosiglitazone control group, and placebo group; each group had 10 rats. In addition, 10 normal male rats were selected for the normal control group. The Housanli, Neiting and Yishu acupoints of the rats in the laser acupoint irradiation group were irradiated with a 10 mW semiconductor laser; each point was irradiated for 15 min, once every 2 d over 28 d, for a total of 14 episodes of irradiation. The rosiglitazone group rats were given rosiglitazone (0.2 mg kg-1) intragastrically; the placebo group rats were given 0.9% brine (0.2 mg kg-1) intragastrically, once daily, for four consecutive weeks. The change of fasting blood glucose was determined before and after each treatment. The islet beta-cell apoptosis was determined. The islet beta-cell apoptosis rates of the laser acupoint irradiation group and the rosiglitazone group were significantly lower than the rate of the placebo group. Even though the rate was lower in the laser acupoint irradiation group than in the rosiglitazone group, there was no significant difference between them. It is shown that acupoint irradiation with a semiconductor laser can effectively inhibit islet beta-cell apoptosis in rats with type 2 diabetes.

  6. Regulation of adhesion and growth of fibrosarcoma cells by NF-kappa B RelA involves transforming growth factor beta.

    PubMed Central

    Perez, J R; Higgins-Sochaski, K A; Maltese, J Y; Narayanan, R

    1994-01-01

    The NF-kappa B transcription factor is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Antisense oligomer inhibition of the RelA subunit of NF-kappa B results in a block of cellular adhesion and inhibition of tumor cell growth. Investigation of the molecular basis for these effects showed that in vitro inhibition of the growth of transformed fibroblasts by relA antisense oligonucleotides can be reversed by the parental-cell-conditioned medium. Cytokine profile analysis of these cells treated with relA antisense oligonucleotides revealed inhibition of transforming growth factor beta 1 (TGF-beta 1 to the transformed fibroblasts reversed the inhibitory effects of relA antisense oligomers on soft agar colony formation and cell adhesion to the substratum. Direct inhibition of TGF-beta 1 expression by antisense phosphorothioates to TGF-beta 1 mimicked the in vitro effects of blocking cell adhesion that are elicited by antisense relA oligomers. These results may explain the in vitro effects of relA antisense oligomers on fibrosarcoma cell growth and adhesion. Images PMID:8035811

  7. Stability of monomeric Cro variants: Isoenergetic transformation of a type I' to a type II' beta-hairpin by single amino acid replacements.

    PubMed

    Mollah, A K M M; Stennis, Rhonda L; Mossing, Michael C

    2003-05-01

    The thermodynamic stabilities of three monomeric variants of the bacteriophage lambda Cro repressor that differ only in the sequence of two amino acids at the apex of an engineered beta-hairpin have been determined. The sequences of the turns are EVK-XX-EVK, where the two central residues are DG, GG, and GT, respectively. Standard-state unfolding free energies, determined from circular dichroism measurements as a function of urea concentration, range from 2.4 to 2.7 kcal/mole, while those determined from guanidine hydrochloride range from 2.8 to 3.3 kcal/mole for the three proteins. Thermal denaturation yields van't Hoff unfolding enthalpies of 36 to 40 kcal /mole at midpoint temperatures in the range of 53 to 58 degrees C. Extrapolation of the thermal denaturation free energies with heat capacities of 400 to 600 cal/mole deg gives good agreement with the parameters determined in denaturant titrations. As predicted from statistical surveys of amino acid replacements in beta-hairpins, energetic barriers to transformation from a type I' turn (DG) to a type II' turn (GT) can be quite small.

  8. Bone sialoprotein mediates the tumor cell-targeted prometastatic activity of transforming growth factor beta in a mouse model of breast cancer.

    PubMed

    Nam, Jeong-Seok; Suchar, Adam M; Kang, Mi-Jin; Stuelten, Christina H; Tang, Binwu; Michalowska, Aleksandra M; Fisher, Larry W; Fedarko, Neal S; Jain, Alka; Pinkas, Jan; Lonning, Scott; Wakefield, Lalage M

    2006-06-15

    Transforming growth factor betas (TGF-beta) play a dual role in carcinogenesis, functioning as tumor suppressors early in the process, and then switching to act as prometastatic factors in late-stage disease. We have previously shown that high molecular weight TGF-beta antagonists can suppress metastasis without the predicted toxicities. To address the underlying mechanisms, we have used the 4T1 syngeneic mouse model of metastatic breast cancer. Treatment of mice with a monoclonal anti-TGF-beta antibody (1D11) significantly suppressed metastasis of 4T1 cells to the lungs. When metastatic 4T1 cells were recovered from lungs of 1D11-treated and control mice, the most differentially expressed gene was found to be bone sialoprotein (Bsp). Immunostaining confirmed the loss of Bsp protein in 1D11-treated lung metastases, and TGF-beta was shown to regulate and correlate with Bsp expression in vitro. Functionally, knockdown of Bsp in 4T1 cells reduced the ability of TGF-beta to induce local collagen degradation and invasion in vitro, and treatment with recombinant Bsp protected 4T1 cells from complement-mediated lysis. Finally, suppression of Bsp in 4T1 cells reduced metastasis in vivo. We conclude that Bsp is a plausible mediator of at least some of the tumor cell-targeted prometastatic activity of TGF-beta in this model and that Bsp expression in metastases can be successfully suppressed by systemic treatment with anti-TGF-beta antibodies.

  9. Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1{alpha}, suppress amyloid {beta}-induced neurotoxicity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Raman, Dayanidhi; Milatovic, Snjezana-Zaja; Milatovic, Dejan

    2011-11-15

    Alzheimer's disease (AD) is characterized by a progressive cognitive decline and accumulation of neurotoxic oligomeric peptides amyloid-{beta} (A{beta}). Although the molecular events are not entirely known, it has become evident that inflammation, environmental and other risk factors may play a causal, disruptive and/or protective role in the development of AD. The present study investigated the ability of the chemokines, macrophage inflammatory protein-2 (MIP-2) and stromal cell-derived factor-1{alpha} (SDF-1{alpha}), the respective ligands for chemokine receptors CXCR2 and CXCR4, to suppress A{beta}-induced neurotoxicity in vitro and in vivo. Pretreatment with MIP-2 or SDF-1{alpha} significantly protected neurons from A{beta}-induced dendritic regression and apoptosismore » in vitro through activation of Akt, ERK1/2 and maintenance of metalloproteinase ADAM17 especially with SDF-1{alpha}. Intra-cerebroventricular (ICV) injection of A{beta} led to reduction in dendritic length and spine density of pyramidal neurons in the CA1 area of the hippocampus and increased oxidative damage 24 h following the exposure. The A{beta}-induced morphometric changes of neurons and increase in biomarkers of oxidative damage, F{sub 2}-isoprostanes, were significantly inhibited by pretreatment with the chemokines MIP-2 or SDF-1{alpha}. Additionally, MIP-2 or SDF-1{alpha} was able to suppress the aberrant mislocalization of p21-activated kinase (PAK), one of the proteins involved in the maintenance of dendritic spines. Furthermore, MIP-2 also protected neurons against A{beta} neurotoxicity in CXCR2-/- mice, potentially through observed up regulation of CXCR1 mRNA. Understanding the neuroprotective potential of chemokines is crucial in defining the role for their employment during the early stages of neurodegeneration. -- Research highlights: Black-Right-Pointing-Pointer Neuroprotective ability of the chemokines MIP2 and CXCL12 against A{beta} toxicity. Black

  10. SME-type carbapenem-hydrolyzing class A beta-lactamases from geographically diverse Serratia marcescens strains.

    PubMed

    Queenan, A M; Torres-Viera, C; Gold, H S; Carmeli, Y; Eliopoulos, G M; Moellering, R C; Quinn, J P; Hindler, J; Medeiros, A A; Bush, K

    2000-11-01

    Three sets of carbapenem-resistant Serratia marcescens isolates have been identified in the United States: 1 isolate in Minnesota in 1985 (before approval of carbapenems for clinical use), 5 isolates in Los Angeles (University of California at Los Angeles [UCLA]) in 1992, and 19 isolates in Boston from 1994 to 1999. All isolates tested produced two beta-lactamases, an AmpC-type enzyme with pI values of 8.6 to 9.0 and one with a pI value of approximately 9.5. The enzyme with the higher pI in each strain hydrolyzed carbapenems and was not inhibited by EDTA, similar to the chromosomal class A SME-1 beta-lactamase isolated from the 1982 London strain S. marcescens S6. The genes encoding the carbapenem-hydrolyzing enzymes were cloned in Escherichia coli and sequenced. The enzyme from the Minnesota isolate had an amino acid sequence identical to that of SME-1. The isolates from Boston and UCLA produced SME-2, an enzyme with a single amino acid change relative to SME-1, a substitution from valine to glutamine at position 207. Purified SME enzymes from the U. S. isolates had beta-lactam hydrolysis profiles similar to that of the London SME-1 enzyme. Pulsed-field gel electrophoresis analysis revealed that the isolates showed some similarity but differed by at least three genetic events. In conclusion, a family of rare class A carbapenem-hydrolyzing beta-lactamases first described in London has now been identified in S. marcescens isolates across the United States.

  11. Role of estrogen receptors alpha, beta and GPER1/GPR30 in pancreatic beta-cells.

    PubMed

    Nadal, Angel; Alonso-Magdalena, Paloma; Soriano, Sergi; Ripoll, Cristina; Fuentes, Esther; Quesada, Ivan; Ropero, Ana Belen

    2011-01-01

    Estrogen receptors (ER) are emerging as important molecules involved in the adaptation of beta-cells to insulin resistance. The onset of type 2 diabetes is marked by insulin secretory dysfunction and decreased beta-cell mass. During pregnancy, puberty and obesity there is increased metabolic demand and insulin resistance is developed. This metabolic state increases the demand on beta-cells to augment insulin biosynthesis and release. In this respect, ERalpha is directly implicated in the E2-regulation of insulin content and secretion, while ERbeta is in the E2-potentiation of glucose-induced insulin release. Both receptors develop their actions within the physiological range of E2. In addition, the G protein-coupled estrogen receptor (GPER1/GPR30) seems to be implicated in the E2-regulation of stimulus-secretion coupling in the three cell types of the islet. The increased demand of insulin production for long time may lead to beta-cell stress and apoptosis. ERalpha, ERbeta and GPER1/GPR30 are involved in preventing beta-cell apoptosis, impeding the loss of critical beta-cell mass. Therefore, estrogen receptors may play an essential role in the adaptation of the pancreas to insulin resistant periods.

  12. Extracellular heat shock protein HSP90{beta} secreted by MG63 osteosarcoma cells inhibits activation of latent TGF-{beta}1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Suzuki, Shigeki; Kulkarni, Ashok B., E-mail: ak40m@nih.gov

    2010-07-30

    Transforming growth factor-beta 1 (TGF-{beta}1) is secreted as a latent complex, which consists of latency-associated peptide (LAP) and the mature ligand. The release of the mature ligand from LAP usually occurs through conformational change of the latent complex and is therefore considered to be the first step in the activation of the TGF-{beta} signaling pathway. So far, factors such as heat, pH changes, and proteolytic cleavage are reportedly involved in this activation process, but the precise molecular mechanism is still far from clear. Identification and characterization of the cell surface proteins that bind to LAP are important to our understandingmore » of the latent TGF-{beta} activation process. In this study, we have identified heat shock protein 90 {beta} (HSP90{beta}) from the cell surface of the MG63 osteosarcoma cell line as a LAP binding protein. We have also found that MG63 cells secrete HSP90{beta} into extracellular space which inhibits the activation of latent TGF-{beta}1, and that there is a subsequent decrease in cell proliferation. TGF-{beta}1-mediated stimulation of MG63 cells resulted in the increased cell surface expression of HSP90{beta}. Thus, extracellular HSP90{beta} is a negative regulator for the activation of latent TGF-{beta}1 modulating TGF-{beta} signaling in the extracellular domain. -- Research highlights: {yields} Transforming growth factor-beta 1 (TGF-{beta}1) is secreted as a latent complex. {yields} This complex consists of latency-associated peptide (LAP) and the mature ligand. {yields} The release of the mature ligand from LAP is the first step in TGF-{beta} activation. {yields} We identified for the first time a novel mechanism for this activation process. {yields} Heat shock protein 90 {beta} is discovered as a negative regulator for this process.« less

  13. UBV and H. beta. photometry of faint early-type stars in Crux and Centaurus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Muzzio, J.C.; Feinstein, A.; Orsatti, A.M.

    1976-08-01

    UBV and H..beta.. photoelectric observations of faint early-type stars in a small region in Crux near the open cluster Hogg 15 and another in Centaurus are presented. The data suggest large absorption in Crux and small absorption in Centaurus. The spread in the distance moduli of the observed stars seems to be in agreement with the view that a spiral arm is seen tangentially near l = 305/sup 0/.

  14. The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

    PubMed Central

    2011-01-01

    Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-/-, integrin-beta1-/-, focal adhesion kinase (FAK)-/- and Src/Yes/Fyn-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2-/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker molecule between Cdc42 and

  15. Bioinformatics Knowledge Map for Analysis of Beta-Catenin Function in Cancer

    PubMed Central

    Arighi, Cecilia N.; Wu, Cathy H.

    2015-01-01

    Given the wealth of bioinformatics resources and the growing complexity of biological information, it is valuable to integrate data from disparate sources to gain insight into the role of genes/proteins in health and disease. We have developed a bioinformatics framework that combines literature mining with information from biomedical ontologies and curated databases to create knowledge “maps” of genes/proteins of interest. We applied this approach to the study of beta-catenin, a cell adhesion molecule and transcriptional regulator implicated in cancer. The knowledge map includes post-translational modifications (PTMs), protein-protein interactions, disease-associated mutations, and transcription factors co-activated by beta-catenin and their targets and captures the major processes in which beta-catenin is known to participate. Using the map, we generated testable hypotheses about beta-catenin biology in normal and cancer cells. By focusing on proteins participating in multiple relation types, we identified proteins that may participate in feedback loops regulating beta-catenin transcriptional activity. By combining multiple network relations with PTM proteoform-specific functional information, we proposed a mechanism to explain the observation that the cyclin dependent kinase CDK5 positively regulates beta-catenin co-activator activity. Finally, by overlaying cancer-associated mutation data with sequence features, we observed mutation patterns in several beta-catenin PTM sites and PTM enzyme binding sites that varied by tissue type, suggesting multiple mechanisms by which beta-catenin mutations can contribute to cancer. The approach described, which captures rich information for molecular species from genes and proteins to PTM proteoforms, is extensible to other proteins and their involvement in disease. PMID:26509276

  16. The role of peroxisome proliferator-activated receptor-{beta}/{delta} in epidermal growth factor-induced HaCaT cell proliferation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liang Pengfei; Jiang Bimei; Yang Xinghua

    2008-10-15

    Epidermal growth factor (EGF) has been shown to be a potent mitogen for epidermal cells both in vitro and in vivo, thus contributing to the development of an organism. It has recently become clear that peroxisome proliferator-activated receptor-{beta}/{delta} (PPAR{beta}/{delta}) expression and activation is involved in the cell proliferation. However, little is known about the role of PPAR{beta}/{delta} in EGF-induced proliferation of HaCaT keratinocytes. In this study, HaCaT cells were cultured in the presence and absence of EGF and we identified that EGF induced an increase of PPAR{beta}/{delta} mRNA and protein level expression in time-dependent and dose-dependent manner, and AG1487, anmore » EGF receptor (EGFR) special inhibitor, caused attenuation of PPAR{beta}/{delta} protein expression. Electrophoretic mobility shift assay (EMSA) revealed that EGF significantly increased PPAR{beta}/{delta} binding activity in HaCaT keratinocytes. Antisense phosphorothioate oligonucleotides (asODNs) against PPAR{beta}/{delta} caused selectively inhibition of PPAR{beta}/{delta} protein content induced by EGF and significantly attenuated EGF-mediated cell proliferation. Treatment of the cells with L165041, a specific synthetic ligand for PPAR{beta}/{delta}, significantly enhanced EGF-mediated cell proliferation. Finally, c-Jun ablation inhibited PPAR{beta}/{delta} up-regulation induced by EGF, and chromatin immunoprecipitation (ChIP) showed that c-Jun bound to the PPAR{beta}/{delta} promoter and the binding increased in EGF-stimulated cells. These results demonstrate that EGF induces PPAR{beta}/{delta} expression in a c-Jun-dependent manner and PPAR{beta}/{delta} plays a vital role in EGF-stimulated proliferation of HaCaT cells.« less

  17. Exercise and Beta-Glucan Consumption (Saccharomyces cerevisiae) Improve the Metabolic Profile and Reduce the Atherogenic Index in Type 2 Diabetic Rats (HFD/STZ)

    PubMed Central

    Andrade, Eric Francelino; Lima, Andressa Ribeiro Veiga; Nunes, Ingrid Edwiges; Orlando, Débora Ribeiro; Gondim, Paula Novato; Zangeronimo, Márcio Gilberto; Alves, Fernando Henrique Ferrari; Pereira, Luciano José

    2016-01-01

    Physical activity and the ingestion of dietary fiber are non-drug alternatives commonly used as adjuvants to glycemic control in diabetic individuals. Among these fibers, we can highlight beta-glucans. However, few studies have compared isolated and synergic effects of physical exercise and beta-glucan ingestion, especially in type 2 diabetic rats. Therefore, we evaluated the effects beta-glucan (Saccharomyces cerevisiae) consumption, associated or not to exercise, on metabolic parameters of diabetic Wistar rats. The diabetes mellitus (DM) was induced by high-fat diet (HFD) associated with a low dose of streptozotocin (STZ—35 mg/kg). Trained groups were submitted to eight weeks of exercise in aquatic environment. In the last 28 days of experiment, animals received 30 mg/kg/day of beta-glucan by gavage. Isolated use of beta-glucan decreased glucose levels in fasting, Glycated hemoglobin (HbA1c), triglycerides (TAG), total cholesterol (TC), low-density lipoprotein (LDL-C), the atherogenic index of plasma. Exercise alone also decreased blood glucose levels, HbA1c, and renal lesions. An additive effect for reducing the atherogenic index of plasma and renal lesions was observed when both treatments were combined. It was concluded that both beta-glucan and exercise improved metabolic parameters in type 2 (HFD/STZ) diabetic rats. PMID:27999319

  18. Increased expression of mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase type 2 in human atria during atrial fibrillation.

    PubMed

    De-An, Pei; Li, Li; Zhi-Yun, Xu; Jin-Yu, Huang; Zheng-Ming, Xu; Min, Wang; Qiang, Yao; Shi-Eng, Huang

    2010-01-01

    Atrialfibrillation (AF) is associated with the activation of the renin-angiotensin-aldosterone system in the atria. It is not clear whether the expression of a mineralocorticoid receptor (MR), or 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2), conferring aldosterone specificity to the MR, in patients with AF is altered. Patients with AF may be associated with increased expression of MR and 11betaHSD2 in the atria. Atrial tissue samples of 25 patients with rheumatic heart valve disease undergoing a valve replacement operation were examined. A total of 13 patients had chronic persistent AF (>6 mo) and 12 patients had no history of AF. The MR and 11betaHSD2 expression were analyzed at the mRNA and protein level. The localization of MR and 11betaHSD2 in atrial tissue was performed using specific immunohistochemistry staining. The results of real-time quantitative polymerase chain reaction (PCR) showed that AF groups, in comparison with sinus rhythm, had a higher mRNA expression level of MR or 11betaHSD2 (all P < 0.01). Both the MR and 11betaHSD2 protein expression level in atrial tissue were also significantly increased in patients with AF compared with patients with sinus rhythm (P < 0.05 or P < 0.01). The immunohistochemical staining of MR or 11betaHSD2 demonstrated that MR and 11betaHSD2 predominately located in the cytoplasm of myocardial cells in the atrium and the intensity and density of immunostaining appeared to be increased in the atria of patients with AF compared to those without AF. Increasing expression of MR and 11betaHSD2 in the atria during AF is one of the molecular mechanisms for development of atrial interstitial fibrosis in patients with AF. These findings may have an important impact on the treatment of AF with aldosterone antagonists. Copyright 2010 Wiley Periodicals, Inc.

  19. Synthesis and cytotoxic analysis of some disodium 3beta,6beta-dihydroxysterol disulfates.

    PubMed

    Cui, Jianguo; Wang, Hui; Huang, Yanmin; Xin, Yi; Zhou, Aimin

    2009-01-01

    Disodium 3beta,6beta-dihydroxy-5alpha-cholestane disulfate (1) was synthesized in 4 steps with a high overall yield from cholesterol. First, cholesterol (4a) was converted to cholest-4-en-3,6-dione (5a) via oxidation with pyridinium chlorochromate (PCC) and then 5a was reduced by NaBH(4) in the presence of NiCl(2) to produce cholest-3beta,6beta-diol (6a). The reaction of 6a with the triethylamine-sulfur trioxide complex generated diammonium 3beta,6beta-dihydroxy-5alpha-cholestane disulfate (7a) and the treatment of 7a by cation exchange resin 732 (sodium form)(Na(+)) yielded the target steroid 1. Disodium 24-ethyl-3beta,6beta-dihydroxycholest-22-ene disulfate (2) and disodium 24-ethyl-3beta,6beta-dihydroxycholestane disulfate (3) were synthesized using a similar method. The cytotoxicity of these compounds against Sk-Hep-1 (human liver carcinoma cell line), H-292 (human lung carcinoma cell line), PC-3 (human prostate carcinoma cell line) and Hey-1B (human ovarian carcinoma cell line) cells was investigated. Our results indicate that presence of a cholesterol-type side chain at position 17 is necessary for their biological activity.

  20. Angiotensin II increases CTGF expression via MAPKs/TGF-{beta}1/TRAF6 pathway in atrial fibroblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gu, Jun; Liu, Xu, E-mail: xkliuxu@yahoo.cn; Wang, Quan-xing, E-mail: shmywqx@126.com

    2012-10-01

    The activation of transforming growth factor-{beta}1(TGF-{beta}1)/Smad signaling pathway and increased expression of connective tissue growth factor (CTGF) induced by angiotensin II (AngII) have been proposed as a mechanism for atrial fibrosis. However, whether TGF{beta}1/non-Smad signaling pathways involved in AngII-induced fibrogenetic factor expression remained unknown. Recently tumor necrosis factor receptor associated factor 6 (TRAF6)/TGF{beta}-associated kinase 1 (TAK1) has been shown to be crucial for the activation of TGF-{beta}1/non-Smad signaling pathways. In the present study, we explored the role of TGF-{beta}1/TRAF6 pathway in AngII-induced CTGF expression in cultured adult atrial fibroblasts. AngII (1 {mu}M) provoked the activation of P38 mitogen activated proteinmore » kinase (P38 MAPK), extracellular signal-regulated kinase 1/2(ERK1/2) and c-Jun NH(2)-terminal kinase (JNK). AngII (1 {mu}M) also promoted TGF{beta}1, TRAF6, CTGF expression and TAK1 phosphorylation, which were suppressed by angiotensin type I receptor antagonist (Losartan) as well as p38 MAPK inhibitor (SB202190), ERK1/2 inhibitor (PD98059) and JNK inhibitor (SP600125). Meanwhile, both TGF{beta}1 antibody and TRAF6 siRNA decreased the stimulatory effect of AngII on TRAF6, CTGF expression and TAK1 phosphorylation, which also attenuated AngII-induced atrial fibroblasts proliferation. In summary, the MAPKs/TGF{beta}1/TRAF6 pathway is an important signaling pathway in AngII-induced CTGF expression, and inhibition of TRAF6 may therefore represent a new target for reversing Ang II-induced atrial fibrosis. -- Highlights: Black-Right-Pointing-Pointer MAPKs/TGF{beta}1/TRAF6 participates in AngII-induced CTGF expression in atrial fibroblasts. Black-Right-Pointing-Pointer TGF{beta}1/TRAF6 participates in AngII-induced atrial fibroblasts proliferation. Black-Right-Pointing-Pointer TRAF6 may represent a new target for reversing Ang II-induced atrial fibrosis.« less

  1. Increased production of 11beta-hydroxysteroid dehydrogenase type 2 in the kidney microsomes of squirrel monkeys (Saimiri spp.).

    PubMed

    Sadosky, Patti W; Scammell, Jonathan G

    2008-04-01

    In squirrel monkeys (Saimiri spp.), cortisol circulates at levels much higher than those seen in man and other Old World primates, but squirrel monkeys exhibit no physiologic signs of the mineralocorticoid effects of cortisol. These observations suggest that squirrel monkeys have mechanisms for protection of the mineralocorticoid receptor (MR) from these high levels of cortisol. We previously showed that the serum cortisol to cortisone ratio in these animals is low relative to that in human serum, suggesting that production of the MR protective enzyme, 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), is increased in squirrel monkeys. Here, we directly evaluate whether increased production of 11beta-HSD2, which inactivates cortisol to cortisone, is a mechanism for protection of MR. In vitro assays showed that 11beta-HSD2 activity in squirrel monkey kidney microsomes was 3 to 7 times higher than that seen in kidney microsomes from pig or rabbit. 11beta-HSD2 protein detected by Western blot analysis was 4 to 9 times greater in squirrel monkey microsomes than in pig or rabbit microsomes. Comparison of the effect of expression of either human or squirrel monkey 11beta-HSD2 on MR transactivation activity showed similar inhibition of MR response to cortisol by both enzymes, indicating that the intrinsic activities of the human and squirrel monkey enzymes are similar. These findings suggest that one mechanism by which squirrel monkeys protect the MR from activation by high cortisol levels in the kidney is by upregulation of 11beta-HSD2 activity through increased production of the enzyme.

  2. Endogenous interleukin-22 protects against inflammatory bowel disease but not autoimmune cholangitis in dominant negative form of transforming growth factor beta receptor type II mice.

    PubMed

    Yang, G-X; Sun, Y; Tsuneyama, K; Zhang, W; Leung, P S C; He, X-S; Ansari, A A; Bowlus, C; Ridgway, W M; Gershwin, M E

    2016-08-01

    During chronic inflammation, interleukin (IL)-22 expression is up-regulated in both CD4 and CD8 T cells, exerting a protective role in infections. However, in autoimmunity, IL-22 appears to have either a protective or a pathogenic role in a variety of murine models of autoimmunity and, by extrapolation, in humans. It is not clear whether IL-22 itself mediates inflammation or is a by-product of inflammation. We have taken advantage of the dominant negative form of transforming growth factor beta receptor type II (dnTGF-βRII) mice that develop both inflammatory bowel disease and autoimmune cholangitis and studied the role and the biological function of IL-22 by generating IL-22(-/-) dnTGF-βRII mice. Our data suggest that the influence of IL-22 on autoimmunity is determined in part by the local microenvironment. In particular, IL-22 deficiency exacerbates tissue injury in inflammatory bowel disease, but has no influence on either the hepatocytes or cholangiocytes in the same model. These data take on particular significance in the previously defined effects of IL-17A, IL-12p40 and IL-23p19 deficiency and emphasize that, in colitis, there is a dominant role of IL-23/T helper type 17 (Th17) signalling. Furthermore, the levels of IL-22 are IL-23-dependent. The use of cytokine therapy in patients with autoimmune disease has significant potential, but must take into account the overlapping and often promiscuous effects that can theoretically exacerbate inflammation. © 2016 British Society for Immunology.

  3. Endothelial binding of beta toxin to small intestinal mucosal endothelial cells in early stages of experimentally induced Clostridium perfringens type C enteritis in pigs.

    PubMed

    Schumacher, V L; Martel, A; Pasmans, F; Van Immerseel, F; Posthaus, H

    2013-07-01

    Beta toxin (CPB) is known to be an essential virulence factor in the development of lesions of Clostridium perfringens type C enteritis in different animal species. Its target cells and exact mechanism of toxicity have not yet been clearly defined. Here, we evaluate the suitability of a neonatal piglet jejunal loop model to investigate early lesions of C. perfringens type C enteritis. Immunohistochemically, CPB was detected at microvascular endothelial cells in intestinal villi during early and advanced stages of lesions induced by C. perfringens type C. This was first associated with capillary dilatation and subsequently with widespread hemorrhage in affected intestinal segments. CPB was, however, not demonstrated on intestinal epithelial cells. This indicates a tropism of CPB toward endothelial cells and suggests that CPB-induced endothelial damage plays an important role in the early stages of C. perfringens type C enteritis in pigs.

  4. Beta-endorphin and islet hormone release in type-2 diabetes mellitus the effects of normoglycemia, enkephalin, naloxone and somatostatin.

    PubMed

    Giugliano, D; Cozzolino, D; Salvatore, T; Ceriello, A; Giunta, R; Torella, R; D'Onofrio, F

    1987-01-01

    The present study was aimed at characterizing the effects of beta-endorphin on plasma glucose, insulin and glucagon plasma levels in subjects with type-2 diabetes mellitus. Infusion of 0.5 mg/h human beta-endorphin produced significant and simultaneous increments in both insulin and glucagon concentrations and decreased plasma glucose levels (-18 +/- 4 mg/dl, 60 min level, p less than 0.01). When the same diabetics were rendered euglycemic by an insulin infusion (1 mU/kg/min), beta-endorphin did not produce the expected decrease in plasma glucose concentrations nor raise plasma insulin levels; only the response of glucagon was preserved. Normal subjects were rendered hyperglycemic by an intravenous glucose infusion to match the plasma glucose levels of diabetic subjects. In this condition, beta-endorphin produced a significant increase of insulin concentrations, whereas glucagon remained suppressed. The intravenous administration of the long-acting met-enkephalin analogue DAMME (0.25 mg) blunted the hormonal responses to the subsequent beta-endorphin infusion in diabetic patients, although the inhibition was short-lived (30-40 min). Naloxone (5 mg), an opiate antagonist, did not produce any significant change in the insulin and glucagon responses to beta-endorphin, while somatostatin (0.25 mg/h) completely abolished the hormonal responses to the opioid.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Newly proposed hormonal criteria via genotypic proof for type II 3beta-hydroxysteroid dehydrogenase deficiency.

    PubMed

    Lutfallah, Chantal; Wang, Weihua; Mason, J Ian; Chang, Ying Tai; Haider, Anzar; Rich, Barry; Castro-Magana, Mariano; Copeland, Kenneth C; David, Raphael; Pang, Songya

    2002-06-01

    To define the hormonal criteria via genotypic proof for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) deficiency in the adrenals and gonads, we investigated the type II 3beta-HSD genotype in 55 patients with clinical and/or hormonal presentation suggesting compromised adrenal with or without gonadal 3beta-HSD activity. Fourteen patients (11 males and 3 females) had ambiguous genitalia with or without salt wasting and with or without premature pubarche. One female neonate had salt wasting only. Twenty-five children (4 males and 21 females) had premature pubarche only. Fifteen adolescent and adult females had hirsutism with or without menstrual disorder. The type II 3beta-HSD gene, including the promoter region up to -1053 base, all exons I, II, III, IV, and exon and intron boundaries, was sequenced in all subjects. Eight patients had a proven or predictably deleterious mutation in both alleles of the type II 3beta-HSD gene, and 47 patients had no apparent mutation in the gene. ACTH-stimulated (1 h post iv bolus of 250 microg Cortrosyn) serum 17-hydroxypregnenolone (Delta5-17P) levels and basal and ACTH-stimulated ratios of Delta5-17P to cortisol (F) in the genotypic proven patients were unequivocally higher than those of age-matched or pubic hair stage matched genotype-normal patients or control subjects (n = 7-30 for each group). All other baseline and ACTH-stimulated hormone parameters, including dehydroepiandrosterone (DHEA) levels, ratios of Delta5-17P to 17-OHP and DHEA to androstenedione in the genotype-proven patients, overlapped with the genotype-normal patients or control subjects. The hormonal findings in the genotype-proven patients suggest that the following hormonal criteria are compatible with 3beta-HSD deficiency congenital adrenal hyperplasia (numeric and graphic reference standards from infancy to adulthood are provided): ACTH-stimulated Delta5-17P levels in 1) neonatal infants with ambiguous genitalia at or greater than 378 nmol/liter equivalent

  6. Do immunotherapy and beta cell replacement play a synergistic role in the treatment of type 1 diabetes?

    PubMed

    Li, Dong-Sheng; Warnock, Garth L; Tu, Han-Jun; Ao, Ziliang; He, Zehua; Lu, Hong; Dai, Long-Jun

    2009-10-07

    Type 1 diabetes (T1D) is the result of the autoimmune response against pancreatic insulin-producing ss-cells. Its ultimate consequence is beta-cell insufficiency-mediated dysregulation of blood glucose control. In terms of T1D treatment, immunotherapy addresses the cause of T1D, mainly through re-setting the balance between autoimmunity and regulatory mechanisms. Regulatory T cells play an important role in this immune intervention. An alternative T1D treatment is beta-cell replacement, which can reverse the consequence of the disease by replacing destroyed beta-cells in the diabetic pancreas. The applicable insulin-producing cells can be directly obtained from islet transplantation or generated from other cell sources such as autologous adult stem cells, embryonic stem cells, and induced pluripotent stem cells. In this review, we summarize the recent research progress and analyze the possible advantages and disadvantages of these two therapeutic options especially focusing on the potential synergistic effect on T1D treatment. Exploring the optimal combination of immunotherapy and beta-cell replacement will pave the way to the most effective cure for this devastating disease.

  7. Activities of beta-lactam antibiotics against Escherichia coli strains producing extended-spectrum beta-lactamases.

    PubMed Central

    Jacoby, G A; Carreras, I

    1990-01-01

    Seven extended-spectrum beta-lactamases related to TEM and four enzymes derived from SHV-1 were transferred to a common Escherichia coli host so that the activity of a variety of beta-lactams could be tested in a uniform genetic environment. For most derivatives, penicillinase activity was 10% or less than that of strains making TEM-1, TEM-2, or SHV-1 beta-lactamase, suggesting that reduced catalytic efficiency accompanied the broader substrate spectrum. Despite this deficit, resistance to aztreonam, carumonam, cefdinir, cefepime, cefixime, cefmenoxime, cefotaxime, cefotiam, cefpirome, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, and E1040 was enhanced. For strains producing TEM-type enzymes, however, MICs of carumonam, cefepime, cefmenoxime, cefotiam, cefpirome, and ceftibuten were 8 micrograms/ml or less. Susceptibilities of cefmetazole, cefotetan, cefoxitin, flomoxef, imipenem, meropenem, moxalactam, temocillin, FCE 22101, and Sch 34343 were unaffected. FCE 22101, imipenem, meropenem, and Sch 34343 were inhibitory for all strains at 1 microgram/ml or less. In E. coli an OmpF- porin mutation in combination with an extended-spectrum beta-lactamase enhanced resistance to many of these agents, but generally by only fourfold. Hyperproduction of chromosomal AmpC beta-lactamase increased resistance to 7-alpha-methoxy beta-lactams but not that to temocillin. When tested at 8 micrograms/ml, clavulanate was more potent than sulbactam or tazobactam in overcoming resistance to ampicillin, while cefoperazone-sulbactam was more active than ticarcillin-clavulanate or piperacillin-tazobactam, especially against TEM-type extended-spectrum beta-lactamases. PMID:2193623

  8. Binding of TEM-1 beta-lactamase to beta-lactam antibiotics by frontal affinity chromatography.

    PubMed

    Chen, Xiu; Li, Yuhua; Zhang, Yan; Yang, Jianting; Bian, Liujiao

    2017-04-15

    TEM-1 beta-lactamases can accurately catalyze the hydrolysis of the beta-lactam rings in beta-lactam antibiotics, which make beta-lactam antibiotics lose its activity, and the prerequisite for the hydrolysis procedure in the binding interaction of TEM-1 beta-lactamases with beta-lactam antibiotics is the beta-lactam rings in beta-lactam antibiotics. Therefore, the binding of TEM-1 beta-lactamase to three beta-lactam antibiotics including penicillin G, cefalexin as well as cefoxitin was explored here by frontal affinity chromatography in combination with fluorescence spectra, adsorption and thermodynamic data in the temperature range of 278-288K under simulated physiological conditions. The results showed that all the binding of TEM-1 beta-lactamase to the three antibiotics were spontaneously exothermic processes with the binding constants of 8.718×10 3 , 6.624×10 3 and 2.244×10 3 (mol/L), respectively at 288K. All the TEM-1 beta-lactamases were immobilized on the surface of the stationary phase in the mode of monolayer and there existed only one type of binding sites on them. Each TEM-1 beta-lactamase bound with only one beta-lactam antibiotic and hydrogen bond interaction and Van der Waals force were the main forces between them. This work provided an insight into the binding interactions between TEM-1 beta-lactamases and beta-lactam antibiotics, which may be beneficial for the designing and developing of new substrates resistant to TEM-1 beta-lactamases. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Granulocyte-macrophage colony-stimulating factor amplification of interleukin-1beta and tumor necrosis factor alpha production in THP-1 human monocytic cells stimulated with lipopolysaccharide of oral microorganisms.

    PubMed

    Baqui, A A; Meiller, T F; Chon, J J; Turng, B F; Falkler, W A

    1998-05-01

    Cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), are used to assist in bone marrow recovery during cancer chemotherapy. Interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) play important roles in inflammatory processes, including exacerbation of periodontal diseases, one of the most common complications in patients who undergo this therapy. A human monocyte cell line (THP-1) was utilized to investigate IL-1beta and TNF-alpha production following GM-CSF supplementation with lipopolysaccharide (LPS) from two oral microorganisms, Porphyromonas gingivalis and Fusobacterium nucleatum. LPS of P. gingivalis or F. nucleatum was prepared by a phenol-water extraction method and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and determination of total protein and endotoxin contents. Resting THP-1 cells were treated with LPS of P. gingivalis or F. nucleatum and/or GM-CSF (50 IU/ml) by using different concentrations for various time periods. Production of IL-1beta and TNF-alpha in THP-1 cells was measured by solid-phase enzyme-linked immunosorbent assay. Reverse transcription (RT)-PCR was used to evaluate the gene expression of resting and treated THP-1 cells. IL-1beta was not detected in untreated THP-1 cells. IL-1beta production was, however, stimulated sharply at 4 h. GM-CSF amplified IL-1beta production in THP-1 cells treated with LPS from both oral anaerobes. No IL-1beta-specific mRNA transcript was detected in untreated THP-1 cells. However, IL-1beta mRNA was detected by RT-PCR 2 h after stimulation of THP-1 cells with LPS from both organisms. GM-CSF did not shorten the IL-1beta transcriptional activation time. GM-CSF plus F. nucleatum or P. gingivalis LPS activated THP-1 cells to produce a 1.6-fold increase in TNF-alpha production at 4 h over LPS stimulation alone. These investigations with the in vitro THP-1 model indicate that there may be an increase in the cellular immune response to oral

  10. Evaluation of thrombogenicity of beta-propiolactone/ultraviolet (beta-PL/UV) treated PPSB in chimpanzees

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kotitschke, R.; Stephan, W.; Prince, A.M.

    1983-05-01

    The thrombogenicity of beta-PL/UV-treated PPSB (factor IX concentrate) was evaluated in chimpanzees. PPSB isolated from beta-propiolactone-treated and UV-irradiated plasma was injected into chimpanzees at a dose of approximately 100 units/kg body weight. An FDA licensed PPSB preparation served as the negative control, and a preparation containing activated as well as precursor clotting factors served as the positive control. 15 minutes, 1 h, 4 h, and 24 h after the PPSB application the following parameters were determined in the chimpanzee blood: factors II, VII, IX, X, VIII, fibrinogen, AT III, thrombin coagulase, Quick value, APTT and platelet count. Neither the untreatedmore » control preparation, nor the PPSB isolated from beta-propiolactone-treated and UV-irradiated plasma, showed signs of thrombogenicity in the chimpanzee model. The positive control indicated that the chimpanzee is a suitable model for the thrombogenicity testing of activated clotting factors.« less

  11. The antifibrotic effects of TGF-{beta}1 siRNA on hepatic fibrosis in rats

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lang, Qing; Liu, Qi; Xu, Ning

    2011-06-10

    Highlights: {yields} We constructed CCL4 induced liver fibrosis model successfully. {yields} We proofed that the TGF-{beta}1 siRNA had a definite therapy effect to CCL4 induced liver fibrosis. {yields} The therapy effect of TGF-{beta}1 siRNA had dose-dependent. -- Abstract: Background/aims: Hepatic fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs), which proliferate during fibrotic liver injury. Transforming growth factor (TGF)-{beta}1 is the dominant stimulus for extracellular matrix (ECM) production by stellate cells. Our study was designed to investigate the antifibrotic effects of using short interference RNA (siRNA) to target TGF-{beta}1 in hepatic fibrosis and its mechanismmore » in rats exposed to a high-fat diet and carbon tetrachloride (CCL4). Methods: A total of 40 healthy, male SD (Sprague-Dawley) rats were randomly divided into five even groups containing of eight rats each: normal group, model group, TGF-{beta}1 siRNA 0.125 mg/kg treatment group, TGF-{beta}1 siRNA 0.25 mg/kg treatment group and TGF-{beta}1 siRNA negative control group (0.25 mg/kg). CCL4 and a high-fat diet were used for 8 weeks to induce hepatic fibrosis. All the rats were then sacrificed to collect liver tissue samples. A portion of the liver samples were soaked in formalin for Hematoxylin-Eosin staining, classifying the degree of liver fibrosis, and detecting the expression of type I and III collagen and TGF-{beta}1; the remaining liver samples were stored in liquid nitrogen to be used for detecting TGF-{beta}1 by Western blotting and for measuring the mRNA expression of type I and III collagen and TGF-{beta}1 by quantitative real-time polymerase chain reaction. Results: Comparing the TGF-{beta}1 siRNA 0.25 mg/kg treatment group to the model group, the TGF-{beta}1 siRNA negative control group and the TGF-{beta}1 siRNA 0.125 mg/kg treatment group showed significantly reduced levels of pathological changes, protein expression and the m

  12. Targeting Transforming Growth Factor Beta to Enhance the Fracture Resistance of Bone

    DTIC Science & Technology

    2013-01-01

    Transforming Growth Factor Beta to Enhance the Fracture Resistance of Bone is to determine whether the suppression of TGF-β activity improves the fracture...effect primarily occurred in the old rats. Effect of TGF-β suppression on fracture resistance in female mice Since the suppression of TGF-β activity by...treated mice. This suggests that 1D11 treatment depleted the osteoprogenitor pool to some extent as inhibition of TGF-β activity in vivo may favor

  13. Inhibitory effect of transforming growth factor-. beta. (TGF-. beta. ) on insulin-like growth factor 1 (IGF-1)-induced proliferation and differentiation in primary cultures of pig preadipocytes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Richardson, R.L.; Hausman, G.J.; Gaskins, H.R.

    1990-02-26

    The influence of serum, IGF-1 and TGF-{beta} on the differentiation of preadipocytes was examined in primary cultures of porcine adipose tissue cells. In serum-supplemented or serum-free, IGF-1 (1 and 10 nM) had no effect on total cell number. However, IGF-1 (10nM) increased adipocyte number only in serum-supplemented (1% pig serum) cultures, whereas TGF-{beta} (15 pm) reduced the adipocyte number in the presence and absence of IGF-1. Replication of preadipocytes was analyzed with a ({sup 3}H) thymidine assay. Preadipocyte proliferation (cpm in adipocyte fraction) was increased by IGF-1 (10nM) only in cultures containing pig serum. TGF-{beta} had no effect on preadipocytemore » proliferation specifically, but slightly increased total ({sup 3}H) thymidine incorporation in cultures with serum. Glycerol phosphate dehydrogenase (GPDH) specific activity was decreased by adding TGF-{beta} to serum-free cultures but TGF-{beta} had little effect in serum-supplemented cultures. Cellular secretion of IGF-1 was decreased when TGF-{beta} was added to serum-free or serum-supplemented cultures. These studies indicate that TGF-{beta} does not inhibit adipocyte development in the initial growth phase, but may inhibit differentiation and/or hypertrophy at a later stage of development.« less

  14. [Associations of insulin resistance and pancreatic beta-cell function with plasma glucose level in type 2 diabetes].

    PubMed

    Nian, Xiaoping; Sun, Gaisheng; Dou, Chunmei; Hou, Hongbo; Fan, Xiuping; Yu, Hongmei; Ma, Ling; He, Bingxian

    2002-06-10

    To investigate the influence of insulin resistance and pancreatic beta-cell function on plasma glucose level in type 2 diabetes so as to provide theoretical basis for reasonable selection of hypoglycemic agents. The plasma non-specific insulin (NSINS), true insulin (TI) and glucose in eight-one type 2 diabetics, 38 males and 43 females, with a mean age of 53 years, were examined 0, 30, 60 and 120 minutes after they had 75 grams of instant noodles. The patients were divided into two groups according to their fasting plasma glucose (FPG): group A (FPG < 8.89 mmol/L) and group B (FPG> = 8.89 mmol/L). The insulin resistance was evaluated by HOMA-IR, the beta-cell function was evaluated by HOMA-beta formula and the formula deltaI(30)/deltaG(30) = (deltaI(30)-deltaI(0))/(deltaG(30)-deltaG(0)). The insulin area under curve (INSAUC) was evaluated by the formula INSAUC=FINS/2+INS(30)+INS(60)+INS(120)/2. The mean FPG was 6.23 mmol/L in group A and 12.6 mmol/L in group B. PG2H was 11.7 mmol/L in group A and 19.2 mmol/L in group B. The TI levels in group B at 0, 30, 60, 120 min during standard meal test were significantly higher than those in group A: 6.15 +/- 1.06 vs 4.77 +/- 1.06, 9.76 +/- 1.1 vs 5.88 +/- 1.1,14.68 +/- 1.11 vs 6.87 +/- 1.1 and 17.13 +/- 1.12 vs 8.0 +/- 1.1 microU/dl (all P< 0.01). The NSINS showed the same trend. The insulin resistance in group B was 1.5 times that in group A. With the insulin resistance adjusted, the beta cell function in group A was 5 to 6 times that in group B. The INSAUC in group A was 1.66 times larger than that in group B, especially the INSAUC for true insulin (2 times larger). The contribution of insulin resistance and beta cell function to PG2H was half by half in group A and 1:8 in group B. beta cell function calculated by insulin (Homa-beta) explained 41% of the plasma glucose changes in group A and 54% of the plasma glucose changes in group B. The contribution of insulin deficiency to plasma glocose was 3.3.times that of insulin

  15. ROS signaling, oxidative stress and Nrf2 in pancreatic beta-cell function

    PubMed Central

    Pi, Jingbo; Zhang, Qiang; Fu, Jingqi; Woods, Courtney G.; Hou, Yongyong; Corkey, Barbara E; Collins, Sheila; Andersen, Melvin E.

    2009-01-01

    This review focuses on the emerging evidence that reactive oxygen species (ROS) derived from glucose metabolism, such as H2O2, act as metabolic signaling molecules for glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells. Particular emphasis is placed on the potential inhibitory role of endogenous antioxidants, which rise in response to oxidative stress, in glucose-triggered ROS and GSIS. We propose that cellular adaptive response to oxidative stress challenge, such as nuclear factor E2-related factor 2 (Nrf2)-mediated antioxidant induction, plays paradoxical roles in pancreatic beta-cell function. On the one hand, induction of antioxidant enzymes protects beta-cells from oxidative damage and possible cell death, thus minimizing oxidative damage-related impairment of insulin secretion. On the other hand, the induction of antioxidant enzymes by Nrf2 activation blunts glucose-triggered ROS signaling, thus resulting in reduced GSIS. These two premises are potentially relevant to impairment of beta-cells occurring in the late and early stage of Type 2 diabetes, respectively. In addition, we summarized our recent findings that persistent oxidative stress due to absence of uncoupling protein 2 activates cellular adaptive response which is associated with impaired pancreatic beta-cell function. PMID:19501608

  16. Regulation of natriuretic peptide receptor A and B expression by transforming growth factor-beta 1 in cultured aortic smooth muscle cells.

    PubMed

    Fujio, N; Gossard, F; Bayard, F; Tremblay, J

    1994-06-01

    Two types of natriuretic peptide receptors (NPR-A and NPR-B) are membrane guanylate cyclases whose relative expression varies in different tissues. Because natriuretic peptides have been shown to inhibit aortic smooth muscle proliferation, we investigated the regulation of NPR-A and NPR-B in these cells under different proliferative conditions. NPR subtype mRNA levels were measured by our newly developed quantitative reverse transcription-polymerase chain reaction assay using mutated NPR-A and NPR-B cRNA as internal standards. The functional impact of their expression was determined by atrial natriuretic peptide (ANP)- and C-type natriuretic peptide (CNP)-induced stimulation of cyclic GMP production. In the intact aorta, NPR-B mRNA levels were found to be 10-fold higher than those of NPR-A. This dominance was further amplified (1000-fold) in long-term cultures (10 to 15 passages) of aortic smooth muscle cells (ASMC). Higher cyclic GMP production with CNP than with ANP was observed in cultured ASMC from Wistar-Kyoto (WKY) rats. Similar stimulation by the two agonists was noted in spontaneously hypertensive rat (SHR) cells, paralleled by a 10-fold increase in NPR-A mRNA levels and ANP stimulation of cyclic GMP in hypertensive cells. The present study also evaluated NPR-A and NPR-B mRNA control by transforming growth factor-beta 1 (TGF-beta 1), an important regulator of cell proliferation that is overexpressed in SHR ASMC. TGF-beta 1 decreased both NPR-A and NPR-B mRNA levels with a predominant effect in SHR cells at high cell density.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Mutation in transforming growth factor beta induced protein associated with granular corneal dystrophy type 1 reduces the proteolytic susceptibility through local structural stabilization.

    PubMed

    Underhaug, Jarl; Koldsø, Heidi; Runager, Kasper; Nielsen, Jakob Toudahl; Sørensen, Charlotte S; Kristensen, Torsten; Otzen, Daniel E; Karring, Henrik; Malmendal, Anders; Schiøtt, Birgit; Enghild, Jan J; Nielsen, Niels Chr

    2013-12-01

    Hereditary mutations in the transforming growth factor beta induced (TGFBI) gene cause phenotypically distinct corneal dystrophies characterized by protein deposition in cornea. We show here that the Arg555Trp mutant of the fourth fasciclin 1 (FAS1-4) domain of the protein (TGFBIp/keratoepithelin/βig-h3), associated with granular corneal dystrophy type 1, is significantly less susceptible to proteolysis by thermolysin and trypsin than the WT domain. High-resolution liquid-state NMR of the WT and Arg555Trp mutant FAS1-4 domains revealed very similar structures except for the region around position 555. The Arg555Trp substitution causes Trp555 to be buried in an otherwise empty hydrophobic cavity of the FAS1-4 domain. The first thermolysin cleavage in the core of the FAS1-4 domain occurs on the N-terminal side of Leu558 adjacent to the Arg555 mutation. MD simulations indicated that the C-terminal end of helix α3' containing this cleavage site is less flexible in the mutant domain, explaining the observed proteolytic resistance. This structural change also alters the electrostatic properties, which may explain increased propensity of the mutant to aggregate in vitro with 2,2,2-trifluoroethanol. Based on our results we propose that the Arg555Trp mutation disrupts the normal degradation/turnover of corneal TGFBIp, leading to accumulation and increased propensity to aggregate through electrostatic interactions. © 2013.

  18. Mutation in Transforming Growth Factor Beta Induced protein associated with Granular Corneal Dystrophy Type 1 Reduces the Proteolytic Susceptibility through Local Structural Stabilization#

    PubMed Central

    Underhaug, Jarl; Koldsø, Heidi; Runager, Kasper; Nielsen, Jakob Toudahl; Sørensen, Charlotte S.; Kristensen, Torsten; Otzen, Daniel E.; Karring, Henrik; Malmendal, Anders; Schiøtt, Birgit; Enghild, Jan J.; Nielsen, Niels Chr.

    2014-01-01

    Hereditary mutations in the transforming growth factor beta induced (TGFBI) gene cause phenotypically distinct corneal dystrophies characterized by protein deposition in cornea. We show here that the Arg555Trp mutant of the fourth fasciclin 1 (FAS1-4) domain of the protein (TGFBIp/keratoepithelin/βig-h3), associated with granular corneal dystrophy type 1, is significantly less susceptible to proteolysis by thermolysin and trypsin than the WT domain. High-resolution liquid-state NMR of the WT and Arg555Trp mutant FAS1-4 domains revealed very similar structures except for the region around position 555. The Arg555Trp substitution causes Trp555 to be buried in an otherwise empty hydrophobic cavity of the FAS1-4 domain. The first thermolysin cleavage in the core of the FAS1-4 domain occurs on the N-terminal side of Leu558 adjacent to the Arg555 mutation. MD simulations indicated that the C-terminal end of helix α3′ containing this cleavage site is less flexible in the mutant domain, explaining the observed proteolytic resistance. This structural change also alters the electrostatic properties, which may explain increased propensity of the mutant to aggregate in vitro with 2,2,2-trifluoroethanol. Based on our results we propose that the Arg555Trp mutation disrupts the normal degradation/turnover of corneal TGFBIp, leading to accumulation and increased propensity to aggregate through electrostatic interactions. PMID:24129074

  19. Nicergoline, a drug used for age-dependent cognitive impairment, protects cultured neurons against beta-amyloid toxicity.

    PubMed

    Caraci, Filippo; Chisari, Mariangela; Frasca, Giuseppina; Canonico, Pier Luigi; Battaglia, Angelo; Calafiore, Marco; Battaglia, Giuseppe; Bosco, Paolo; Nicoletti, Ferdinando; Copani, Agata; Sortino, Maria Angela

    2005-06-14

    Nicergoline, a drug used for the treatment of Alzheimer's disease and other types of dementia, was tested for its ability to protect neurons against beta-amyloid toxicity. Pure cultures of rat cortical neurons were challenged with a toxic fragment of beta-amyloid peptide (betaAP(25-35)) and toxicity was assessed after 24 h. Micromolar concentrations of nicergoline or its metabolite, MDL, attenuated betaAP(25-35)-induced neuronal death, whereas MMDL (another metabolite of nicergoline), the alpha1-adrenergic receptor antagonist, prazosin, or the serotonin 5HT-2 receptor antagonist, methysergide, were inactive. Nicergoline increased the basal levels of Bcl-2 and reduced the increase in Bax levels induced by beta-amyloid, indicating that the drug inhibits the execution of an apoptotic program in cortical neurons. In mixed cultures of rat cortical cells containing both neurons and astrocytes, nicergoline and MDL were more efficacious than in pure neuronal cultures in reducing beta-amyloid neurotoxicity. Experiments carried out in pure cultures of astrocytes showed that a component of neuroprotection was mediated by a mechanism of glial-neuronal interaction. The conditioned medium of cultured astrocytes treated with nicergoline or MDL for 72-96 h (collected 24 h after drug withdrawal) was neuroprotective when transferred to pure neuronal cultures challenged with beta-amyloid. In cultured astrocytes, nicergoline increased the intracellular levels of transforming-growth factor-beta and glial-derived neurotrophic factor, two trophic factors that are known to protect neurons against beta-amyloid toxicity. These results raise the possibility that nicergoline reduces neurodegeneration in the Alzheimer's brain.

  20. Beta cell function and BMI in ethnically diverse children with newly diagnosed autoimmune type 1 diabetes

    USDA-ARS?s Scientific Manuscript database

    The objective of our study was to examine the relationship between BMI and beta-cell function at diagnosis of autoimmune type 1 diabetes (T1D) in a large group of ethnically diverse children. Cross-sectional analysis of 524 children (60.8% White, 19.5% Hispanic, 14.5% African-American, 5.2% other n...

  1. Hydrological and environmental variables outperform spatial factors in structuring species, trait composition, and beta diversity of pelagic algae.

    PubMed

    Wu, Naicheng; Qu, Yueming; Guse, Björn; Makarevičiūtė, Kristė; To, Szewing; Riis, Tenna; Fohrer, Nicola

    2018-03-01

    There has been increasing interest in algae-based bioassessment, particularly, trait-based approaches are increasingly suggested. However, the main drivers, especially the contribution of hydrological variables, of species composition, trait composition, and beta diversity of algae communities are less studied. To link species and trait composition to multiple factors (i.e., hydrological variables, local environmental variables, and spatial factors) that potentially control species occurrence/abundance and to determine their relative roles in shaping species composition, trait composition, and beta diversities of pelagic algae communities, samples were collected from a German lowland catchment, where a well-proven ecohydrological modeling enabled to predict long-term discharges at each sampling site. Both trait and species composition showed significant correlations with hydrological, environmental, and spatial variables, and variation partitioning revealed that the hydrological and local environmental variables outperformed spatial variables. A higher variation of trait composition (57.0%) than species composition (37.5%) could be explained by abiotic factors. Mantel tests showed that both species and trait-based beta diversities were mostly related to hydrological and environmental heterogeneity with hydrological contributing more than environmental variables, while purely spatial impact was less important. Our findings revealed the relative importance of hydrological variables in shaping pelagic algae community and their spatial patterns of beta diversities, emphasizing the need to include hydrological variables in long-term biomonitoring campaigns and biodiversity conservation or restoration. A key implication for biodiversity conservation was that maintaining the instream flow regime and keeping various habitats among rivers are of vital importance. However, further investigations at multispatial and temporal scales are greatly needed.

  2. Effect of transforming growth factor-beta1 on embryonic and posthatch muscle growth and development in normal and low score normal chicken.

    PubMed

    Li, X; Velleman, S G

    2009-02-01

    During skeletal muscle development, transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of muscle cell proliferation and differentiation. The TGF-beta1 signal is carried by Smad proteins into the cell nucleus, inhibiting the expression of key myogenic regulatory factors including MyoD and myogenin. However, the molecular mechanism by which TGF-beta1 inhibits muscle cell proliferation and differentiation has not been well documented in vivo. The present study investigated the effect of TGF-beta1 on in vivo skeletal muscle growth and development. A chicken line, Low Score Normal (LSN) with reduced muscling and upregulated TGF-beta1 expression, was used and compared to a normal chicken line. The injection of TGF-beta1 at embryonic day (ED) 3 significantly reduced the pectoralis major (p. major) muscle weight in the normal birds at 1 wk posthatch, whereas no significant difference was observed in the LSN birds. The difference between normal and LSN birds in response to TGF-beta1 is likely due to different levels of endogenous TGF-beta1 where the LSN birds have increased TGF-beta1 expression in their p. major muscle at both 17 ED and 6 wk posthatch. Smad3 expression was reduced by TGF-beta1 from 10 ED to 1 wk posthatch in normal p. major muscle. Unlike Smad3, Smad7 expression was not significantly affected by TGF-beta1 until posthatch in both normal and LSN p. major muscle. Expression of MyoD was reduced 35% by TGF-beta1 during embryonic development in normal p. major muscle, whereas LSN p. major muscle showed a delayed decrease at 1 d posthatch in MyoD expression in response to the TGF-beta1 treatment. Myogenin expression was reduced 29% by TGF-beta1 after hatch in normal p. major muscle. In LSN p. major muscle, TGF-beta1 treatment significantly decreased myogenin expression by 43% at 1 d posthatch and 32% at 1 wk posthatch. These data suggested that TGF-beta1 reduced p. major muscle growth by inhibiting MyoD and myogenin expression during both embryonic

  3. Inhibiting platelet-derived growth factor beta reduces Ewing's sarcoma growth and metastasis in a novel orthotopic human xenograft model.

    PubMed

    Wang, Yong Xin; Mandal, Deendayal; Wang, Suizhau; Hughes, Dennis; Pollock, Raphael E; Lev, Dina; Kleinerman, Eugenie; Hayes-Jordan, Andrea

    2009-01-01

    Despite aggressive therapy, Ewing's sarcoma (ES) patients have a poor five-year overall survival of only 20-40%. Pulmonary metastasis is the most common form of demise in these patients. The pathogenesis of pulmonary metastasis is poorly understood and few orthotopic models exist that allow study of spontaneous pulmonary metastasis in ES. We have developed a novel orthotopic xenograft model in which spontaneous pulmonary metastases develop. While the underlying biology of ES is incompletely understood, in addition to the EWS-FLI-1 mutation, it is known that platelet-derived growth factor receptor beta (PDGFR-beta) is highly expressed in ES. Hypothesizing that PDGFR-beta expression is indicative of a specific role for this receptor protein in ES progression, the effect of PDGFR-beta inhibition on ES growth and metastasis was assessed in this novel orthotopic ES model. Silencing PDGFR-beta reduced spontaneous growth and metastasis in ES. Preclinical therapeutically relevant findings such as these may ultimately lead to new treatment initiatives in ES.

  4. Turn stability in beta-hairpin peptides: Investigation of peptides containing 3:5 type I G1 bulge turns.

    PubMed

    Blandl, Tamas; Cochran, Andrea G; Skelton, Nicholas J

    2003-02-01

    The turn-forming ability of a series of three-residue sequences was investigated by substituting them into a well-characterized beta-hairpin peptide. The starting scaffold, bhpW, is a disulfide-cyclized 10-residue peptide that folds into a stable beta-hairpin with two antiparallel strands connected by a two-residue reverse turn. Substitution of the central two residues with the three-residue test sequences leads to less stable hairpins, as judged by thiol-disulfide equilibrium measurements. However, analysis of NMR parameters indicated that each molecule retains a significant folded population, and that the type of turn adopted by the three-residue sequence is the same in all cases. The solution structure of a selected peptide with a PDG turn contained an antiparallel beta-hairpin with a 3:5 type I + G1 bulge turn. Analysis of the energetic contributions of individual turn residues in the series of peptides indicates that substitution effects have significant context dependence, limiting the predictive power of individual amino acid propensities for turn formation. The most stable and least stable sequences were also substituted into a more stable disulfide-cyclized scaffold and a linear beta-hairpin scaffold. The relative stabilities remained the same, suggesting that experimental measurements in the bhpW context are a useful way to evaluate turn stability for use in protein design projects. Moreover, these scaffolds are capable of displaying a diverse set of turns, which can be exploited for the mimicry of protein loops or for generating libraries of reverse turns.

  5. Three-dimensional crystal structure of recombinant murine interferon-beta.

    PubMed Central

    Senda, T; Shimazu, T; Matsuda, S; Kawano, G; Shimizu, H; Nakamura, K T; Mitsui, Y

    1992-01-01

    The crystal structure of recombinant murine interferon-beta (IFN-beta) has been solved by the multiple isomorphous replacement method and refined to an R-factor of 20.5% against 2.6 A X-ray diffraction data. The structure shows a variant of the alpha-helix bundle with a new chain-folding topology, which seems to represent a basic structural framework of all the IFN-alpha and IFN-beta molecules belonging to the type I family. Functionally important segments of the polypeptide chain, as implied through numerous gene manipulation studies carried out so far, are spatially clustered indicating the binding site(s) to the receptor(s). Comparison of the present structure with those of other alpha-helical cytokine proteins, including porcine growth hormone, interleukin 2 and interferon gamma, indicated either a topological similarity in chain folding or a similar spatial arrangement of the alpha-helices. Images PMID:1505514

  6. Assembly of Q{beta} viral RNA polymerase with host translational elongation factors EF-Tu and -Ts.

    PubMed

    Takeshita, Daijiro; Tomita, Kozo

    2010-09-07

    Replication and transcription of viral RNA genomes rely on host-donated proteins. Qbeta virus infects Escherichia coli and replicates and transcribes its own genomic RNA by Qbeta replicase. Qbeta replicase requires the virus-encoded RNA-dependent RNA polymerase (beta-subunit), and the host-donated translational elongation factors EF-Tu and -Ts, as active core subunits for its RNA polymerization activity. Here, we present the crystal structure of the core Qbeta replicase, comprising the beta-subunit, EF-Tu and -Ts. The beta-subunit has a right-handed structure, and the EF-Tu:Ts binary complex maintains the structure of the catalytic core crevasse of the beta-subunit through hydrophobic interactions, between the finger and thumb domains of the beta-subunit and domain-2 of EF-Tu and the coiled-coil motif of EF-Ts, respectively. These hydrophobic interactions are required for the expression and assembly of the Qbeta replicase complex. Thus, EF-Tu and -Ts have chaperone-like functions in the maintenance of the structure of the active Qbeta replicase. Modeling of the template RNA and the growing RNA in the catalytic site of the Qbeta replicase structure also suggests that structural changes of the RNAs and EF-Tu:Ts should accompany processive RNA polymerization and that EF-Tu:Ts in the Qbeta replicase could function to modulate the RNA folding and structure.

  7. A defect in inducible beta-galactosidase of B lymphocytes in the osteopetrotic (mi/mi) mouse.

    PubMed Central

    Yamamoto, N; Naraparaju, V R

    1996-01-01

    Macrophages were activated by administration of an inflammatory lipid metabolite, lysophosphatidylcholine (lyso-Pc), to wild type mice but not murine (microphthalmic) osteopetrotic (mi/mi) mutant mice. In vitro treatment of wild type mouse peritoneal cells with lyso-Pc efficiently activated macrophages whereas lyso-Pc-treatment of mi mutant mouse peritoneal cells resulted in no activation of macrophages. Generation of macrophage activating factor requires a precursor protein, serum vitamin D binding protein (DBP), and participation of lyso-Pc-inducible beta-galactosidase of B lymphocytes. Lyso-Pc-inducible beta-galactosidase of B lymphocytes was found to be defective in mi mutant mice. PMID:8881764

  8. Lymphotoxin beta receptor (Lt betaR): dual roles in demyelination and remyelination and successful therapeutic intervention using Lt betaR-Ig protein.

    PubMed

    Plant, Sheila R; Iocca, Heather A; Wang, Ying; Thrash, J Cameron; O'Connor, Brian P; Arnett, Heather A; Fu, Yang-Xin; Carson, Monica J; Ting, Jenny P-Y

    2007-07-11

    Inflammation mediated by macrophages is increasingly found to play a central role in diseases and disorders that affect a myriad of organs, prominent among these are diseases of the CNS. The neurotoxicant-induced, cuprizone model of demyelination is ideally suited for the analysis of inflammatory events. Demyelination on exposure to cuprizone is accompanied by predictable microglial activation and astrogliosis, and, after cuprizone withdrawal, this activation reproducibly diminishes during remyelination. This study demonstrates enhanced expression of lymphotoxin beta receptor (Lt betaR) during the demyelination phase of this model, and Lt betaR is found in areas enriched with microglial and astroglial cells. Deletion of the Lt betaR gene (Lt betaR-/-) resulted in a significant delay in demyelination but also a slight delay in remyelination. Inhibition of Lt betaR signaling by an Lt betaR-Ig fusion decoy protein successfully delayed demyelination in wild-type mice. Unexpectedly, this Lt betaR-Ig decoy protein dramatically accelerated the rate of remyelination, even after the maximal pathological disease state had been reached. This strongly indicates the beneficial role of Lt betaR-Ig in the delay of demyelination and the acceleration of remyelination. The discrepancy between remyelination rates in these systems could be attributed to developmental abnormalities in the immune systems of Lt betaR-/- mice. These findings bode well for the use of an inhibitory Lt betaR-Ig as a candidate biological therapy in demyelinating disorders, because it is beneficial during both demyelination and remyelination.

  9. Neutrinoless double beta decay in type="smallcap">Gerda

    NASA Astrophysics Data System (ADS)

    Grabmayr, Peter; Gerda Collaboration

    2015-10-01

    The Germanium Detector Array (type="smallcap">Gerda) experiment searches for the neutrinoless double beta decay in 76Ge. This lepton number violating process is predicted by extensions of the standard model. type="smallcap">Gerda follows a staged approach by increasing mass and lowering the background level from phase to phase. type="smallcap">Gerda is setup at the Gran Sasso underground laboratory of INFN, Italy. An array of high-purity germanium detectors is lowered directly in liquid argon for shielding and cooling. Further background reduction is achieved by an instrumented water buffer. In Phase I an exposure of 21.6 kg yr was collected at a background level of 10-2 cts/(keV kg yr). The lower limit on the half-life of 76Ge > 2 . 1 .1025 yr (90% C.L.) has been published. Further analyses search for decay into excited states or the accompanied Majoron decay. Presently, Phase II is in preparation which intends to reach a background level of 10-3 cts/(keV kg yr) and to increase the exposure to 100 kg yr. About 20 kg of novel thick-window BEGe (Broad Energy Germanium) detectors will be added and the liquid argon will be instrumented. The status of Phase II preparation and results from the commissioning runs will be presented as well as some further results from Phase I.

  10. The role of transforming growth factor-beta in PEG-rHuMGDF-induced reversible myelofibrosis in rats.

    PubMed

    Yanagida, M; Ide, Y; Imai, A; Toriyama, M; Aoki, T; Harada, K; Izumi, H; Uzumaki, H; Kusaka, M; Tokiwa, T

    1997-12-01

    Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) injected at a suprapharmacologic dose (100 microg/kg) daily for 5 d in normal rats caused marked increases in marrow megakaryocytes and platelet counts at 6-8 d followed by gradual decreases to control levels at 10-20 d. Interestingly, in addition to the expected thrombopoiesis, PEG-rHuMGDF was associated with myelofibrosis with a predominance of reticulin fibres at day 10 followed by complete normalization by day 20. At 6-8 d, the levels of transforming growth factor-beta1 (TGF-beta1) in the extracellular fluid of the marrow, the platelet poor plasma, and the platelet extract were increased 23-, 7- and 2-fold, respectively. The elevated levels of TGF-beta1 were gradually reduced to baseline levels at 13-20 d in accordance with the normalization of myelofibrosis and thrombopoiesis. An ultrastructural analysis showed that large fragments of megakaryocytes were deposited in the marrow parenchyma of PEG-rHuMGDF-treated rats at day 6. PEG-rHuMGDF administration at pharmacologic doses (1 and 10 microg/kg) did not induce the deposition of reticulin fibres in the marrow. These findings suggest that TGF-beta1 leaked from megakaryocytes is involved in the development of the PEG-rHuMGDF-induced myelofibrosis and that this is a reversible process related to the regulation of the excess production of platelets.

  11. TGF-beta inhibits IL-1beta-activated PAR-2 expression through multiple pathways in human primary synovial cells.

    PubMed

    Tsai, Shin-Han; Sheu, Ming-Thau; Liang, Yu-Chih; Cheng, Hsiu-Tan; Fang, Sheng-Shiung; Chen, Chien-Ho

    2009-10-23

    To investigate the mechanism how Transforming growth factor-beta(TGF-beta) represses Interleukin-1beta (IL-1beta)-induced Proteinase-Activated Receptor-2 (PAR-2) expression in human primary synovial cells (hPSCs). Human chondrocytes and hPSCs isolated from cartilages and synovium of Osteoarthritis (OA) patients were cultured with 10% fetal bovine serum media or serum free media before treatment with IL-1beta, TGF-beta1, or Connective tissue growth factor (CTGF). The expression of PAR-2 was detected using reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting. Collagen zymography was performed to assess the activity of Matrix metalloproteinases-13 (MMP-13). It was demonstrated that IL-1beta induces PAR-2 expression via p38 pathway in hPSCs. This induction can be repressed by TGF-beta and was observed to persist for at least 48 hrs, suggesting that TGF-beta inhibits PAR-2 expression through multiple pathways. First of all, TGF-beta was able to inhibit PAR-2 activity by inhibiting IL-1beta-induced p38 signal transduction and secondly the inhibition was also indirectly due to MMP-13 inactivation. Finally, TGF-beta was able to induce CTGF, and in turn CTGF represses PAR-2 expression by inhibiting IL-1beta-induced phospho-p38 level. TGF-beta could prevent OA from progression with the anabolic ability to induce CTGF production to maintain extracellular matrix (ECM) integrity and to down regulate PAR-2 expression, and the anti-catabolic ability to induce Tissue inhibitors of metalloproteinase-3 (TIMP-3) production to inhibit MMPs leading to avoid PAR-2 over-expression. Because IL-1beta-induced PAR-2 expressed in hPSCs might play a significantly important role in early phase of OA, PAR-2 repression by exogenous TGF-beta or other agents might be an ideal therapeutic target to prevent OA from progression.

  12. Transforming growth factor-beta and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury.

    PubMed

    Yoshida, Katsunori; Matsuzaki, Koichi; Mori, Shigeo; Tahashi, Yoshiya; Yamagata, Hideo; Furukawa, Fukiko; Seki, Toshihito; Nishizawa, Mikio; Fujisawa, Junichi; Okazaki, Kazuichi

    2005-04-01

    After liver injury, transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) regulate the activation of hepatic stellate cells (HSCs) and tissue remodeling. Mechanisms of PDGF signaling in the TGF-beta-triggered cascade are not completely understood. TGF-beta signaling involves phosphorylation of Smad2 and Smad3 at linker and C-terminal regions. Using antibodies to distinguish Smad2/3 phosphorylated at linker regions from those phosphorylated at C-terminal regions, we investigated Smad2/3-mediated signaling in rat liver injured by CCl(4) administration and in cultured HSCs. In acute liver injury, Smad2/3 were transiently phosphorylated at both regions. Although linker-phosphorylated Smad2 remained in the cytoplasm of alpha-smooth muscle actin-immunoreactive mesenchymal cells adjacent to necrotic hepatocytes in centrilobular areas, linker-phosphorylated Smad3 accumulated in the nuclei. c-Jun N-terminal kinase (JNK) in the activated HSCs directly phosphorylated Smad2/3 at linker regions. Co-treatment of primary cultured HSCs with TGF-beta and PDGF activated the JNK pathway, subsequently inducing endogenous linker phosphorylation of Smad2/3. The JNK pathway may be involved in migration of resident HSCs within the space of Disse to the sites of tissue damage because the JNK inhibitor SP600125 inhibited HSC migration induced by TGF-beta and PDGF signals. Moreover, treatment of HSCs with both TGF-beta and PDGF increased transcriptional activity of plasminogen activator inhibitor-1 through linker phosphorylation of Smad3. In conclusion, TGF-beta and PDGF activate HSCs by transmitting their signals through JNK-mediated Smad2/3 phosphorylation at linker regions, both in vivo and in vitro.

  13. Platelet-derived growth factor-DD targeting arrests pathological angiogenesis by modulating glycogen synthase kinase-3beta phosphorylation.

    PubMed

    Kumar, Anil; Hou, Xu; Lee, Chunsik; Li, Yang; Maminishkis, Arvydas; Tang, Zhongshu; Zhang, Fan; Langer, Harald F; Arjunan, Pachiappan; Dong, Lijin; Wu, Zhijian; Zhu, Linda Y; Wang, Lianchun; Min, Wang; Colosi, Peter; Chavakis, Triantafyllos; Li, Xuri

    2010-05-14

    Platelet-derived growth factor-DD (PDGF-DD) is a recently discovered member of the PDGF family. The role of PDGF-DD in pathological angiogenesis and the underlying cellular and molecular mechanisms remain largely unexplored. In this study, using different animal models, we showed that PDGF-DD expression was up-regulated during pathological angiogenesis, and inhibition of PDGF-DD suppressed both choroidal and retinal neovascularization. We also demonstrated a novel mechanism mediating the function of PDGF-DD. PDGF-DD induced glycogen synthase kinase-3beta (GSK3beta) Ser(9) phosphorylation and Tyr(216) dephosphorylation in vitro and in vivo, leading to increased cell survival. Consistently, GSK3beta activity was required for the antiangiogenic effect of PDGF-DD targeting. Moreover, PDGF-DD regulated the expression of GSK3beta and many other genes important for angiogenesis and apoptosis. Thus, we identified PDGF-DD as an important target gene for antiangiogenic therapy due to its pleiotropic effects on vascular and non-vascular cells. PDGF-DD inhibition may offer new therapeutic options to treat neovascular diseases.

  14. Ascending aortic curvature as an independent risk factor for type A dissection, and ascending aortic aneurysm formation: a mathematical model.

    PubMed

    Poullis, Michael P; Warwick, Richard; Oo, Aung; Poole, Robert J

    2008-06-01

    To develop a mathematical model to demonstrate that ascending aortic curvature is an independent risk factor for type A dissections, in addition to hypertension, bicuspid aortic valve, aneurysm of ascending aorta, and intrinsic aortic tissue abnormalities, like Marfan's syndrome. A steady state one-dimensional flow analysis was performed, utilising Newton's third law of motion. Five different clinical scenarios were evaluated: (1) effect of aortic curvature; (2) effect of beta-blockers, (3) effect of patient size, (4) forces on a Marfan's aorta, and (5) site of entry flap in aortic dissection. Aortic curvature increases the forces exerted on the ascending aorta by a factor of over 10-fold. Aortic curvature can cause patients with a systolic blood pressure of 8 0mmHg to have greater forces exerted on their aorta despite smaller diameters and lower cardiac outputs, than patients with systolic blood pressures of 120 mmHg. In normal diameter aortas, beta-blockers have minimal effect compared with aortic curvature. Aortic curvature may help to explain why normal diameter aortas can dissect, and also that the point of the entry tear may be potentially predictable. Aortic curvature has major effects on the forces exerted on the aorta in patients with Marfan's syndrome. Aortic curvature is relatively more important that aortic diameter, blood pressure, cardiac output, beta-blocker use, and patient size with regard to the force acting on the aortic wall. This may explain why some patients with normal diameter ascending aortas with or without Marfan's syndrome develop type A dissections and aneurysms. Aortic curvature may also help to explain the site of entry tear in acute type A dissection. Further clinical study is needed to validate this study's finding.

  15. Effects of beta-lactamases and omp mutation on susceptibility to beta-lactam antibiotics in Escherichia coli.

    PubMed Central

    Hiraoka, M; Okamoto, R; Inoue, M; Mitsuhashi, S

    1989-01-01

    Four types of beta-lactamases consisting of a penicillinase type I (TEM-1), a penicillinase type II (OXA-1), a cephalosporinase of Citrobacter freundii, and a cephalosporinase of Proteus vulgaris were introduced into Escherichia coli MC4100 and its omp mutants, MH1160 (MC4100 ompR1) and MH760 (MC4100 ompR2), by transformation. Effects of the combination of the omp mutations and these beta-lactamases on the susceptibility of E. coli strains were studied with 15 beta-lactam antibiotics including cephalosporins, cephamycins, penicillins, imipenem, and aztreonam. The ompR1 mutant, MH1160, lacks OmpF and OmpC, and it showed reduced susceptibility to 11 of the 15 beta-lactam agents. The reduction in susceptibility to cefoxitin, moxalactam, and flomoxef was much greater than reduction in susceptibility to the other agents. When the ompR1 mutant produced the cephalosporinase of C. freundii, the susceptibility of the mutant to 12 of the 15 beta-lactam antibiotics decreased. The reduction in susceptibility of MH1160 to 10 of the 12 agents affected by the enzyme was two- to fourfold greater than that observed in MC4100. Such a synergistic effect was also observed with the cephalosporinase of P. vulgaris and ompR1 mutation against six cephalosporins, moxalactam, and aztreonam. Images PMID:2658786

  16. Induction of Tyrosine Phosphorylation of UV-Activated EGFR by the Beta-Human Papillomavirus Type 8 E6 Leads to Papillomatosis.

    PubMed

    Taute, Stefanie; Pfister, Herbert J; Steger, Gertrud

    2017-01-01

    Epidemiological evidence is accumulating that beta-human papillomaviruses (HPV) synergize with UV-light in the development of precancerous actinic keratosis, and cutaneous squamous cell carcinomas (cSCC), one of the most common cancers in the Caucasian population. We previously demonstrated the tumorigenic activity of beta-HPV type 8 (HPV8) in the skin of transgenic mice and its cooperation with UV-light. Analysis of underlying mechanisms now showed that in keratinocytes expressing the HPV8E6 protein a transient increase of tyrosine phosphorylated epidermal growth factor receptor (EGFR) in response to UV-irradiation occurred, while EGFR tyrosine phosphorylation, i.e., receptor tyrosine kinase (RTK)-activity was hardly affected in empty vector control cells. FACS and immunofluorescences revealed that the EGFR was internalized into early endosomes in response to UV-exposure in both, HPV8E6 positive and in control cells, yet with a higher rate in the presence of HPV8E6. Moreover, only in HPV8E6 expressing keratinocytes the EGFR was further sorted into CD63+ intraluminal vesicles, indicative for trafficking to late endosomes. The latter requires the ubiquitination of the EGFR, and in correlation, we could show that only in HPV8E6 positive keratinocytes the EGFR was ubiquitinated upon UV-exposure. HPV8E6 and tyrosine phosphorylated EGFR directly interacted which was enhanced by UV-irradiation. The treatment of K14-HPV8E6 transgenic mice with Canertinib, an inhibitor of the RTK-activity of the EGFR, suppressed skin papilloma growth in response to UV-irradiation. This confirms the crucial role of the RTK-activity of the EGFR in HPV8E6 and UV-mediated papillomatosis in transgenic mice. Taken together, our results demonstrate that HPV8E6 alters the signaling of the UV-activated EGFR and this is a critical step in papilloma formation in response to UV-light in transgenic mice. Our results provide a molecular basis how a beta-HPV type may support early steps of skin tumor

  17. Induction of Tyrosine Phosphorylation of UV-Activated EGFR by the Beta-Human Papillomavirus Type 8 E6 Leads to Papillomatosis

    PubMed Central

    Taute, Stefanie; Pfister, Herbert J.; Steger, Gertrud

    2017-01-01

    Epidemiological evidence is accumulating that beta-human papillomaviruses (HPV) synergize with UV-light in the development of precancerous actinic keratosis, and cutaneous squamous cell carcinomas (cSCC), one of the most common cancers in the Caucasian population. We previously demonstrated the tumorigenic activity of beta-HPV type 8 (HPV8) in the skin of transgenic mice and its cooperation with UV-light. Analysis of underlying mechanisms now showed that in keratinocytes expressing the HPV8E6 protein a transient increase of tyrosine phosphorylated epidermal growth factor receptor (EGFR) in response to UV-irradiation occurred, while EGFR tyrosine phosphorylation, i.e., receptor tyrosine kinase (RTK)-activity was hardly affected in empty vector control cells. FACS and immunofluorescences revealed that the EGFR was internalized into early endosomes in response to UV-exposure in both, HPV8E6 positive and in control cells, yet with a higher rate in the presence of HPV8E6. Moreover, only in HPV8E6 expressing keratinocytes the EGFR was further sorted into CD63+ intraluminal vesicles, indicative for trafficking to late endosomes. The latter requires the ubiquitination of the EGFR, and in correlation, we could show that only in HPV8E6 positive keratinocytes the EGFR was ubiquitinated upon UV-exposure. HPV8E6 and tyrosine phosphorylated EGFR directly interacted which was enhanced by UV-irradiation. The treatment of K14-HPV8E6 transgenic mice with Canertinib, an inhibitor of the RTK-activity of the EGFR, suppressed skin papilloma growth in response to UV-irradiation. This confirms the crucial role of the RTK-activity of the EGFR in HPV8E6 and UV-mediated papillomatosis in transgenic mice. Taken together, our results demonstrate that HPV8E6 alters the signaling of the UV-activated EGFR and this is a critical step in papilloma formation in response to UV-light in transgenic mice. Our results provide a molecular basis how a beta-HPV type may support early steps of skin tumor

  18. Simultaneous beta and gamma spectroscopy

    DOEpatents

    Farsoni, Abdollah T.; Hamby, David M.

    2010-03-23

    A phoswich radiation detector for simultaneous spectroscopy of beta rays and gamma rays includes three scintillators with different decay time characteristics. Two of the three scintillators are used for beta detection and the third scintillator is used for gamma detection. A pulse induced by an interaction of radiation with the detector is digitally analyzed to classify the type of event as beta, gamma, or unknown. A pulse is classified as a beta event if the pulse originated from just the first scintillator alone or from just the first and the second scintillator. A pulse from just the third scintillator is recorded as gamma event. Other pulses are rejected as unknown events.

  19. Dexamethasone potently enhances phorbol ester-induced IL-1beta gene expression and nuclear factor NF-kappaB activation.

    PubMed

    Wang, Y; Zhang, J J; Dai, W; Lei, K Y; Pike, J W

    1997-07-15

    The synthetic glucocorticoid dexamethasone, an immunosuppressive and anti-inflammatory agent, was investigated for its effect on PMA-mediated expression of the inflammatory cytokine IL-1beta in the human monocytic leukemic cell line THP-1. PMA alone induced the production of low levels of IL-1beta in THP-1 cells, whereas dexamethasone alone had no effect. However, dexamethasone potently enhanced PMA-mediated IL-1beta production. Using a selective and potent inhibitor of protein kinase C, we found that synergistic interaction between PMA and dexamethasone requires protein kinase C activation. PMA has been known to activate nuclear factor NF-kappaB in THP-1 cells. Using an oligonucleotide probe corresponding to an NF-kappaB DNA-binding motif of the IL-1beta gene promoter in gel electrophoresis mobility shift assays, we demonstrated that PMA-induced NF-kappaB activation was greatly potentiated by dexamethasone. Our results indicate that glucocorticoids can be positive regulators of inflammatory cytokine gene expression during monocytic cell differentiation.

  20. Conformational interconversions in peptide beta-turns: analysis of turns in proteins and computational estimates of barriers.

    PubMed

    Gunasekaran, K; Gomathi, L; Ramakrishnan, C; Chandrasekhar, J; Balaram, P

    1998-12-18

    The two most important beta-turn features in peptides and proteins are the type I and type II turns, which differ mainly in the orientation of the central peptide unit. Facile conformational interconversion is possible, in principle, by a flip of the central peptide unit. Homologous crystal structures afford an opportunity to structurally characterize both possible conformational states, thus allowing identification of sites that are potentially stereochemically mobile. A representative data set of 250 high-resolution (types (type I/type II) in related structures. A total of 55 examples of beta-turns were identified as possible candidates for a stereochemically mobile site. Of the 55 examples, 45 could be classified as a potential site for interconversion between type I and type II beta-turns, while ten correspond to flips from type I' to type II' structures. As a further check, the temperature factors of the central peptide unit carbonyl oxygen atom of the 55 examples were examined. The analysis reveals that the turn assignments are indeed reliable. Examination of the secondary structures at the flanking positions of the flippable beta-turns reveals that seven examples occur in the loop region of beta-hairpins, indicating that the formation of ordered secondary structures on either side of the beta-turn does not preclude local conformational variations. In these beta-turns, Pro (11 examples), Lys (nine examples) and Ser (seven examples) were most often found at the i+1 position. Glycine was found to occur overwhelmingly at position i+2 (28 examples), while Ser (seven examples) and Asn (six examples) were amongst the most frequent residues. Activation energy barriers for the interconversion between type I and type II beta-turns were computed

  1. Ultrasound Stimulation of Insulin Release from Pancreatic Beta Cells as a Potential Novel Treatment for Type 2 Diabetes.

    PubMed

    Suarez Castellanos, Ivan; Jeremic, Aleksandar; Cohen, Joshua; Zderic, Vesna

    2017-06-01

    Type 2 diabetes mellitus is a complex metabolic disease that has reached epidemic proportions in the United States and around the world. This disease is characterized by loss of insulin secretion and, eventually, destruction of insulin-producing pancreatic beta cells. Controlling type 2 diabetes is often difficult as pharmacological management routinely requires complex therapy with multiple medications, and loses its effectiveness over time. The objective of this study was to explore the effectiveness of a novel, non-pharmacological approach that uses the application of ultrasound energy to augment insulin release from rat INS 832/13 beta cells. The cells were exposed to unfocused ultrasound for 5 min at a peak intensity of 1 W/cm 2 and frequencies of 400 kHz, 600 kHz, 800 kHz and 1 MHz. Insulin release was measured with enzyme-linked immunosorbent assay and cell viability was assessed via the trypan blue dye exclusion test. A marked release (approximately 150 ng/10 6  cells, p < 0.05) of insulin was observed when beta cells were exposed to ultrasound at 400 and 600 kHz as compared with their initial control values; however, this release was accompanied by a substantial loss in cell viability. Ultrasound application at frequencies of 800 kHz resulted in 24 ng/10 6  cells released insulin (p < 0.05) as compared with its unstimulated base level, while retaining cell viability. Insulin release from beta cells caused by application of 800-kHz ultrasound was comparable to that reported by the secretagogue glucose, thus operating within physiological secretory capacity of these cells. Ultrasound has potential as a novel and alternative method to current approaches aimed at correcting secretory deficiencies in patients with type 2 diabetes. Copyright © 2017 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

  2. Effect of [6]-gingerol on myofibroblast differentiation in transforming growth factor beta 1-induced nasal polyp-derived fibroblasts.

    PubMed

    Park, Sook A; Park, Il-Ho; Cho, Jung-Sun; Moon, You-Mi; Lee, Seung Hoon; Kim, Tae Hoon; Lee, Sang Hag; Lee, Heung-Man

    2012-01-01

    [6]-Gingerol is one of the major pungent principles of ginger and has diverse effects, including anti-inflammatory, and antioxidative effects. Reactive oxygen species (ROS) are released during the phenotypic transformation of fibroblasts to myofibroblasts, a process that is involved in the growth of nasal polyps by inducing extracellular matrix (ECM) accumulation. The purpose of this study was to determine the effect of [6]-gingerol on myofibroblast differentiation and collagen production of nasal polyp-derived fibroblasts (NPDFs) and to determine if the effect of [6]-gingerol is linked to an antioxidant effect. NPDFs were incubated and treated with transforming growth factor (TGF) beta 1. The ROS generated by NPDFs were determined using 2″,7″-dichlorfluorescein-diacetate. The fluorescence was captured by a fluorescent microscope and measured using a fluorometer. The expression of alpha-smooth muscle actin (SMA) and collagen type IV mRNA was determined by a reverse transcription-polymerase chain reaction, and the expression of α-SMA protein and pSmad2/3 was determined by immunofluorescence microscopy and or Western blotting. The amount of total soluble collagen production was analyzed by the SirCol collagen dye-binding assay. TGF-beta 1 stimulation increased ROS production by NPDFs. [6]-Gingerol decreased the production of ROS in TGF-beta 1-induced NPDFs. Myofibroblast differentiation, collagen production, and phosphorylation of Smad2/3 were prevented by [6]-gingerol and inhibition of ROS generation with antioxidant such as diphenyliodonium, N-acetylcysteine, and ebselen. These results suggest the possibility that [6]-gingerol may play an important role in inhibiting the production of the ECM in the development of nasal polyps through an antioxidant effect.

  3. Increased expression of transforming growth factor beta s after acute oedematous pancreatitis in rats suggests a role in pancreatic repair.

    PubMed Central

    Riesle, E; Friess, H; Zhao, L; Wagner, M; Uhl, W; Baczako, K; Gold, L I; Korc, M; Büchler, M W

    1997-01-01

    BACKGROUND: Transforming growth factor beta isoforms (TGF beta s) belong to a family of multifunctional regulators of cellular growth and differentiation. They are mitogenic and chemotactic for fibroblasts and are potent stimulators of extracellular matrix production (collagen) and deposition. Upregulation of TGF beta transcription has been reported for several in vivo systems during repair after injury. AIMS: To study the expression of the three mammalian isoforms of TGF beta (TGF beta 1-3) and their relation to collagen expression as a marker for fibroblast response in acute oedematous pancreatitis in rats. METHODS: Using northern blot analysis and immunohistochemistry, the expression and localisation of TGF beta isoforms, collagen, and amylase were analysed during the course of acute oedematous pancreatitis in rats, experimentally induced by intravenous caerulein infusion. RESULTS: Induction of acute pancreatitis resulted in a biphasic peak pattern of expression of TGF beta 1, beta 2, and beta 3 mRNA, with a pronounced increase from day 1 to day 3 (sixfold, 2.5-fold, fivefold, respectively) and again from day 5 to day 7 (three-fold, 2.3-fold, 3.5-fold, respectively). The temporal changes in TGF beta mRNA identically paralleled the expression in collagen mRNA. In contrast, amylase mRNA expression, used as a general indicator of acinar cell integrity, was slightly decreased after induction of acute pancreatitis. Immunohistochemical analysis of pancreatitis tissue showed that increased expression of TGF beta s was mainly present in the pancreatic acinar and ductal cells; this was evident within one day after pancreatitis induction. CONCLUSION: Overexpression of TGF beta s after induction of acute pancreatitis suggests a role for these proteins in pancreatic repair and remodelling. The increased levels of TGF beta s may help suppress immune activation, and may contribute to the increase in the extracellular matrix including collagen and to the repair of the

  4. Activin-A, transforming growth factor-beta, and myostatin signaling pathway in experimental dilated cardiomyopathy.

    PubMed

    Mahmoudabady, Maryam; Mathieu, Myrielle; Dewachter, Laurence; Hadad, Ielham; Ray, Lynn; Jespers, Pascale; Brimioulle, Serge; Naeije, Robert; McEntee, Kathleen

    2008-10-01

    The pathogenic mechanisms of dilated cardiomyopathy are still uncertain. A number of cytokines and growth factors participate in the remodeling process of the disease. We investigated the cardiac myostatin, transforming growth factor (TGF)beta, and activin-A/Smad growth inhibitory signaling pathway in experimental dilated cardiomyopathy. Transvenous endomyocardial biopsies of the interventricular septum were taken weekly in 15 beagle dogs during the development of heart failure (HF) induced by rapid pacing over a period of 7 weeks. Genes involved in the myostatin-TGFbeta-activin-A/Smad signaling pathway and the cardiac hypertrophic process were quantified by real-time quantitative polymerase chain reaction. Left ventricular volume, function, and mass were evaluated by echocardiography. Overpacing was associated with increased left ventricular volumes and decreased ejection fraction, whereas the left ventricular mass remained unchanged. TGFbeta was increased in moderate HF. Activin-A mRNA expression was 4-fold higher in overt congestive HF than at baseline. A 2-fold decrease of activin type II receptors and activin receptor interacting protein 2 gene expressions were observed, as well as a transient decrease of follistatin. Activin type I receptors, activin receptor interacting protein 1, follistatin-related gene, and myostatin remained unchanged. The inhibitory Smad 7, a negative feedback loop regulator of the Smad pathway, was overexpressed in severe HF. Gene expression of the cyclin-dependent kinase inhibitor p21, a direct target gene of the Smad pathway, was 8-fold up-regulated in HF, whereas cyclin D1 was down-regulated. We conclude that tachycardia-induced dilated cardiomyopathy is characterized by gene overexpression of the TGFbeta-activin-A/Smad signaling pathway and their target gene p21 and by the absence of ventricular hypertrophy.

  5. Productive interaction between transmembrane mutants of the bovine papillomavirus E5 protein and the platelet-derived growth factor beta receptor.

    PubMed

    Lai, Char-Chang; Edwards, Anne P B; DiMaio, Daniel

    2005-02-01

    The bovine papillomavirus E5 protein is a 44-amino-acid transmembrane protein that transforms cells by binding to the transmembrane region of the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in sustained receptor signaling. However, there are published reports that certain mutants with amino acid substitutions in the membrane-spanning segment of the E5 protein transform cells without activating the PDGF beta receptor. We re-examined several of these transmembrane mutants, and here we present five lines of evidence that these mutants do in fact activate the PDGF beta receptor, resulting in cellular signaling and transformation.

  6. A novel NADP(+)-dependent dehydrogenase activity for 7alpha/beta- and 11beta-hydroxysteroids in human liver nuclei: A third 11beta-hydroxysteroid dehydrogenase.

    PubMed

    Robinzon, B; Prough, R A

    2009-06-15

    Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11betaHSD enzyme activity against corticosterone, dehydrocorticosterone, 7alpha- and 7beta-hydroxy-dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP(+) or NAD(+), but not NADPH and NADH, as pyridine nucleotide cofactor with K(m) values of 12+/-2 and 390+/-2microM, compared to the K(m) for microsomal 11betaHSD1 of 43+/-8 and 264+/-24microM, respectively. The K(m) for corticosterone in the NADP(+)-dependent nuclear oxidation reaction was 102+/-16nM, compared to 4.3+/-0.8microM for 11betaHSD1. The K(cat) values for nuclear activity with NADP(+) was 1687nmol/min/mg/micromol, compared to 755nmol/min/mg/micromol for microsomal 11betaHSD1 activity. Inhibitors of 11betaHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11betaHSD Type 1 and 2.

  7. Identification of beta-Lactamases and beta-Lactam-Related Proteins in Human Pathogenic Bacteria using a Computational Search Approach.

    PubMed

    Brambila-Tapia, Aniel Jessica Leticia; Perez-Rueda, Ernesto; Barrios, Humberto; Dávalos-Rodríguez, Nory Omayra; Dávalos-Rodríguez, Ingrid Patricia; Cardona-Muñoz, Ernesto Germán; Salazar-Páramo, Mario

    2017-08-01

    A systematic analysis of beta-lactamases based on comparative proteomics has not been performed thus far. In this report, we searched for the presence of beta-lactam-related proteins in 591 bacterial proteomes belonging to 52 species that are pathogenic to humans. The amino acid sequences for 19 different types of beta-lactamases (ACT, CARB, CifA, CMY, CTX, FOX, GES, GOB, IMP, IND, KPC, LEN, OKP, OXA, OXY, SHV, TEM, NDM, and VIM) were obtained from the ARG-ANNOT database and were used to construct 19 HMM profiles, which were used to identify potential beta-lactamases in the completely sequenced bacterial proteomes. A total of 2877 matches that included the word "beta-lactamase" and/or "penicillin" in the functional annotation and/or in any of its regions were obtained. These enzymes were mainly described as "penicillin-binding proteins," "beta-lactamases," and "metallo-beta-lactamases" and were observed in 47 of the 52 species studied. In addition, proteins classified as "beta-lactamases" were observed in 39 of the species included. A positive correlation between the number of beta-lactam-related proteins per species and the proteome size was observed (R 0.78, P < 0.00001). This correlation partially explains the high presence of beta-lactam-related proteins in large proteomes, such as Nocardia brasiliensis, Bacillus anthracis, and Mycobacterium tuberculosis, along with their absence in small proteomes, such as Chlamydia spp. and Mycoplasma spp. We detected only five types of beta-lactamases (TEM, SHV, CTX, IMP, and OXA) and other related proteins in particular species that corresponded with those reported in the literature. We additionally detected other potential species-specific beta-lactamases that have not yet been reported. In the future, better results will be achieved due to more accurate sequence annotations and a greater number of sequenced genomes.

  8. Effect of atorvastatin on pancreatic Beta-cell function and insulin resistance in type 2 diabetes mellitus patients: a randomized pilot study.

    PubMed

    Goyal, Aman; Singh, Surender; Tandon, Nikhil; Gupta, Nandita; Gupta, Yogendra Kumar

    2014-12-01

    Statins are commonly used for the management of dyslipidemia in type 2 diabetes mellitus patients. We hypothesized that atorvastatin could modulate the beta-cell function by altering the levels of proapoptotic and antiapoptotic lipoproteins and could also have an effect on insulin resistance. The aim of the present pilot study was to assess the effect of atorvastatin 10 mg on pancreatic beta-cell function and insulin resistance in patients with hyperlipidemia and type 2 diabetes by using the homeostasis model assessment-2 (HOMA2) index. Fifty-one type 2 diabetes patients receiving oral antidiabetes drugs, not taking statins, with baseline low-density lipoprotein cholesterol between 2.6 mmol/L and 4.1 mmol/L were included. Forty-three patients (21 in placebo group and 22 in atorvastatin group) completed the study and were taken up for final analysis. Fasting blood samples were obtained at baseline and at 12 weeks to determine levels of blood glucose, lipid profile, insulin, C-peptide and glycosylated hemoglobin (A1C). Atorvastatin nonsignificantly increased fasting serum insulin (+14.29%, p=0.18), accompanied by marginal nonsignificant increases in fasting plasma glucose and A1C. There was a decrease in HOMA2 percent beta-cell function (-2.9%, p=0.72) and increase in HOMA2 insulin resistance (+14%, p=0.16) in the atorvastatin group as compared with baseline, but the difference was not statistically significant. Atorvastatin in the dose used failed to produce significant change in pancreatic beta-cell function and insulin resistance in type 2 diabetes patients as assessed by the HOMA2 index. The possible explanations include absence of lipotoxicity at prevailing levels of dyslipidemia at baseline or inadequacy of statin dose used in the study. (Clinical Trials Registry-India: CTRI/2008/091/000099). Copyright © 2014 Canadian Diabetes Association. Published by Elsevier Inc. All rights reserved.

  9. Prenatal programming of metabolic syndrome in the common marmoset is associated with increased expression of 11beta-hydroxysteroid dehydrogenase type 1.

    PubMed

    Nyirenda, Moffat J; Carter, Roderick; Tang, Justin I; de Vries, Annick; Schlumbohm, Christina; Hillier, Stephen G; Streit, Frank; Oellerich, Michael; Armstrong, Victor W; Fuchs, Eberhard; Seckl, Jonathan R

    2009-12-01

    Recent studies in humans and animal models of obesity have shown increased adipose tissue activity of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which amplifies local tissue glucocorticoid concentrations. The reasons for this 11beta-HSD1 dysregulation are unknown. Here, we tested whether 11beta-HSD1 expression, like the metabolic syndrome, is "programmed" by prenatal environmental events in a nonhuman primate model, the common marmoset monkey. We used a "fetal programming" paradigm where brief antenatal exposure to glucocorticoids leads to the metabolic syndrome in the offspring. Pregnant marmosets were given the synthetic glucocorticoid dexamethasone orally for 1 week in either early or late gestation, or they were given vehicle. Tissue 11beta-HSD1 and glucocorticoid receptor mRNA expression were examined in the offspring at 4 and 24 months of age. Prenatal dexamethasone administration, selectively during late gestation, resulted in early and persistent elevations in 11beta-HSD1 mRNA expression and activity in the liver, pancreas, and subcutaneous-but not visceral-fat. The increase in 11beta-HSD1 occurred before animals developed obesity or overt features of the metabolic syndrome. In contrast to rodents, in utero dexamethasone exposure did not alter glucocorticoid receptor expression in metabolic tissues in marmosets. These data suggest that long-term upregulation of 11beta-HSD1 in metabolically active tissues may follow prenatal "stress" hormone exposure and indicates a novel mechanism for fetal origins of adult obesity and the metabolic syndrome.

  10. ICU Acquisition Rate, Risk Factors, and Clinical Significance of Digestive Tract Colonization With Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae: A Systematic Review and Meta-Analysis.

    PubMed

    Detsis, Marios; Karanika, Styliani; Mylonakis, Eleftherios

    2017-04-01

    To evaluate the acquisition rate, identify risk factors, and estimate the risk for subsequent infection, associated with the colonization of the digestive tract with extended-spectrum beta-lactamase-producing Enterobacteriaceae during ICU-hospitalization. PubMed, EMBASE, and reference lists of all eligible articles. Included studies provided data on ICU-acquired colonization with extended-spectrum beta-lactamase-producing Enterobacteriaceae in previously noncolonized and noninfected patients and used the double disk synergy test for extended-spectrum beta-lactamase-producing Enterobacteriaceae phenotypic confirmation. Studies reporting extended-spectrum beta-lactamase-producing Enterobacteriaceae outbreaks or data on pediatric population were excluded. Two authors independently assessed study eligibility and performed data extraction. Thirteen studies (with 15,045 ICUs-patients) were evaluated using a random-effect model and a meta-regression analysis. The acquisition rate of digestive tract colonization during ICU stay was 7% (95% CI, 5-10) and it varies from 3% (95% CI, 2-4) and 4% (95% CI, 2-6) in the Americas and Europe to 21% (95% CI, 9-35) in the Western Pacific region. Previous hospitalization (risk ratio, 1.57 [95% CI, 1.07-2.31]) or antibiotic use (risk ratio, 1.65 [95% CI, 1.15-2.37]) and exposure to beta-lactams/beta-lactamase inhibitors (risk ratio, 1.78 [95% CI, 1.24-2.56]) and carbapenems (risk ratio, 2.13 [95% CI, 1.49-3.06]) during the ICU stay were independent risk factors for ICU-acquired colonization. Importantly, colonized patients were more likely to develop an extended-spectrum beta-lactamase-producing Enterobacteriaceae infection (risk ratio, 49.62 [95% CI, 20.42-120.58]). The sensitivity and specificity of prior colonization to predict subsequent extended-spectrum beta-lactamase-producing Enterobacteriaceae infection were 95.1% (95% CI, 54.7-99.7) and 89.2% (95% CI, 77.2-95.3), respectively. The ICU acquisition rate of extended-spectrum beta

  11. Proteomic profiling of bone marrow mesenchymal stem cells upon TGF-beta stimulation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Daojing; Park, Jennifer S.; Chu, Julia S.F.

    Bone marrow mesenchymal stem cells (MSCs) can differentiate into different types of cells, and have tremendous potential for cell therapy and tissue engineering. Transforming growth factor {beta}1 (TGF-{beta}) plays an important role in cell differentiation and vascular remodeling. We showed that TGF-{beta} induced cell morphology change and an increase in actin fibers in MSCs. To determine the global effects of TGF-{beta} on MSCs, we employed a proteomic strategy to analyze the effect of TGF-{beta} on the human MSC proteome. By using two-dimensional gel electrophoresis and electrospray ionization coupled to Quadrupole/time-of-flight tandem mass spectrometers, we have generated a proteome reference mapmore » of MSCs, and identified {approx}30 proteins with an increase or decrease in expression or phosphorylation in response to TGF-{beta}. The proteins regulated by TGF-{beta} included cytoskeletal proteins, matrix synthesis proteins, membrane proteins, metabolic enzymes, etc. TGF-{beta} increased the expression of smooth muscle (SM) {alpha}-actin and decreased the expression of gelsolin. Over-expression of gelsolin inhibited TGF-{beta}-induced assembly of SM {alpha}-actin; on the other hand, knocking down gelsolin expression enhanced the assembly of {alpha}-actin and actin filaments without significantly affecting {alpha}-actin expression. These results suggest that TGF-{beta} coordinates the increase of {alpha}-actin and the decrease of gelsolin to promote MSC differentiation. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.« less

  12. High-Fluence Light-Emitting Diode-Generated Red Light Modulates the Transforming Growth Factor-Beta Pathway in Human Skin Fibroblasts.

    PubMed

    Mamalis, Andrew; Jagdeo, Jared

    2018-05-24

    Skin fibrosis is a significant medical problem with limited available treatment modalities. The key cellular characteristics include increased fibroblast proliferation, collagen production, and transforming growth factor-beta (TGF-B)/SMAD pathway signaling. The authors have previously shown that high-fluence light-emitting diode red light (HF-LED-RL) decreases cellular proliferation and collagen production. Herein, the authors investigate the ability of HF-LED-RL to modulate the TGF-B/SMAD pathway. Normal human dermal fibroblasts were cultured and irradiated with a commercially available hand-held LED array. After irradiation, cell lysates were collected and levels of pSMAD2, TGF-Beta 1, and TGF-Beta I receptor were measured using Western blot. High-fluence light-emitting diode red light decreased TGF-Beta 1 ligand (TGF-B1) levels after irradiation. 320 J/cm HF-LED-RL resulted in 59% TGF-B1 and 640 J/cm HF-LED-RL resulted in 54% TGF-B1, relative to controls. 640 J/cm HF-LED-RL resulted in 62% pSMAD2 0 hours after irradiation, 65% pSMAD2 2 hours after irradiation, and 95% 4 hours after irradiation, compared with matched controls. High-fluence light-emitting diode red light resulted in no significant difference in transforming growth factor-beta receptor I levels compared with matched controls. Skin fibrosis is a significant medical problem with limited available treatment modalities. Light-emitting diode-generated red light is a safe, economic, and noninvasive modality that has a body of in vitro evidence supporting the reduction of key cellular characteristics associated with skin fibrosis.

  13. Evaluation of the transforming growth factor-beta activity in normal and dry eye human tears by CCL-185 cell bioassay.

    PubMed

    Zheng, Xiaofen; De Paiva, Cintia S; Rao, Kavita; Li, De-Quan; Farley, William J; Stern, Michael; Pflugfelder, Stephen C

    2010-09-01

    To develop a new bioassay method using human lung epithelial cells (CCL-185) to assess activity of transforming growth factor beta (TGF-beta) in human tear fluid from normal subjects and patients with dry eye. Two epithelial cell lines, mink lung cells (CCL-64) and human lung cells (CCL-185), were compared to detect the active form of TGF-beta by BrdU incorporation (quantitation of cell DNA synthesis) and WST assay (metabolic activity of viable cells). The effect of TGF-beta on the growth of CCL-185 cells was observed microscopically. Human tears from normal control subjects and patients with dry eye (DE) with and without Sjögren syndrome were evaluated for TGF-beta concentration by Luminex microbead assay, and TGF-beta activity by the CCL-185 cell growth inhibition bioassay. The metabolic activity of viable CCL-185 cells, measured by WST, was shown to be proportional to the TGF-beta1 concentration (R = 0.919) and confirmed by BrdU assay (R = 0.969). Compared with CCL-185, metabolic activity of viable cells and DNA synthesis, measured by WST and BrdU incorporation assays, were shown to be less proportional to the TGF-beta1 concentration in the CCL-64 line (R = 0.42 and 0.17, respectively). Coincubation with human anti-TGF-beta1 antibody (MAB-240) yielded a dose-dependent inhibition of TGF-beta1 (0.3 ng/mL) activity. CCL-185 cell growth observed microscopically was noted to decrease in response to increasing TGF-beta1 concentrations. Levels of immuodetectable TGF-beta1 and TGF-beta2 were similar in normal and DE tears. TGF-beta bioactivity in DE human tears measured by the CCL-185 cells assay was found to be higher (9777.5 +/- 10481.9 pg/mL) than those in normal controls (4129.3 +/- 1342.9 pg/mL) (P < 0.05). Among patients with DE, TGF-beta bioactivity was highest in those with Sjögren syndrome. Approximately, 79.1% of TGF-beta in DE tears and 37.6% TGF-beta in normal tears were found to be biologically active. The CCL-185 cell assay was found to be a suitable

  14. Ligand-Dependent Disorder of Loop Observed in Extended-Spectrum SHV-Type beta-Lactamase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    J Sampson; W Ke; C Bethel

    2011-12-31

    Among Gram-negative bacteria, resistance to {beta}-lactams is mediated primarily by {beta}-lactamases (EC 3.2.6.5), periplasmic enzymes that inactivate {beta}-lactam antibiotics. Substitutions at critical amino acid positions in the class A {beta}-lactamase families result in enzymes that can hydrolyze extended-spectrum cephalosporins, thus demonstrating an 'extended-spectrum' {beta}-lactamase (ESBL) phenotype. Using SHV ESBLs with substitutions in the {Omega} loop (R164H and R164S) as target enzymes to understand this enhanced biochemical capability and to serve as a basis for novel {beta}-lactamase inhibitor development, we determined the spectra of activity and crystal structures of these variants. We also studied the inactivation of the R164H and R164Smore » mutants with tazobactam and SA2-13, a unique {beta}-lactamase inhibitor that undergoes a distinctive reaction chemistry in the active site. We noted that the reduced K{sub i} values for the R164H and R164S mutants with SA2-13 are comparable to those with tazobactam (submicromolar). The apo enzyme crystal structures of the R164H and R164S SHV variants revealed an ordered {Omega} loop architecture that became disordered when SA2-13 was bound. Important structural alterations that result from the binding of SA2-13 explain the enhanced susceptibility of these ESBL enzymes to this inhibitor and highlight ligand-dependent {Omega} loop flexibility as a mechanism for accommodating and hydrolyzing {beta}-lactam substrates.« less

  15. Quantitative analysis of the synthesis and secretion of type VII collagen in cultured human dermal fibroblasts with a sensitive sandwich enzyme-linked immunoassay.

    PubMed

    Amano, Satoshi; Ogura, Yuki; Akutsu, Nobuko; Nishiyama, Toshio

    2007-02-01

    Type VII collagen is the major component of anchoring fibrils in the epidermal basement membrane. Its expression has been analyzed by immunostaining or Northern blotting, but rarely at the protein level. In this study, we have quantitatively examined the effects of ascorbic acid and various cytokines/growth factors on the protein synthesis and secretion of type VII collagen by human dermal fibroblasts in culture, using a developed, highly sensitive sandwich enzyme-linked immunoassay with two kinds of specific monoclonal antibodies against the non-collagenous domain-1. Ascorbic acid and its derivative induced a twofold increase in type VII collagen synthesis, and markedly increased the secretion of type VII collagen into the medium when compared with the control culture. This effect was not influenced by the presence of transforming growth factor-beta1 (TGF-beta1). The synthesis of type VII collagen was elevated by TGF-beta1, platelet-derived growth factor, tumor necrosis factor-alpha, and interleukin-1beta, but not by TGF-alpha. Thus, our data indicate that the synthesis and secretion of type VII collagen in human dermal fibroblasts are regulated by ascorbate and the enhancement of type VII collagen gene expression by cytokines/growth factors is accompanied with elevated production of type VII collagen at the protein level.

  16. Transforming growth factor beta induced FoxP3+ regulatory T cells suppress Th1 mediated experimental colitis.

    PubMed

    Fantini, M C; Becker, C; Tubbe, I; Nikolaev, A; Lehr, H A; Galle, P; Neurath, M F

    2006-05-01

    The imbalance between effector and regulatory T cells plays a central role in the pathogenesis of inflammatory bowel diseases. In addition to the thymus, CD4+CD25+ regulatory T cells can be induced in the periphery from a population of CD25- T cells by treatment with transforming growth factor beta (TGF-beta). Here, we analysed the in vivo function of TGF-beta induced regulatory T (Ti-Treg) cells in experimental colitis. Ti-Treg cells were generated in cell culture in the presence or absence of TGF-beta and tested for their regulatory potential in experimental colitis using the CD4+CD62L+ T cell transfer model. Ti-Treg cells significantly suppressed Th1 mediated colitis on CD4+CD62L+ T cell transfer in vivo, as shown by high resolution endoscopy, histology, immunohistochemistry, and cytokine analysis. Further analysis of in vivo and in vitro expanded Ti-Treg cells showed that exogenous interleukin 2 (IL-2) was crucial for survival and expansion of these cells. Our data suggest that regulatory Ti-Treg cells expand by TGF-beta and exogenous IL-2 derived from effector T cells at the site of inflammation. In addition to Tr1 and thymic CD4+CD25+ T cells, peripheral Ti-Treg cells emerge as a class of regulatory T cells with therapeutic potential in T cell mediated chronic intestinal inflammation.

  17. Stat5-mediated regulation of the human type II 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene: activation by prolactin.

    PubMed

    Feltus, F A; Groner, B; Melner, M H

    1999-07-01

    Altered PRL levels are associated with infertility in women. Molecular targets at which PRL elicits these effects have yet to be determined. These studies demonstrate transcriptional regulation by PRL of the gene encoding the final enzymatic step in progesterone biosynthesis: 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD). A 9/9 match with the consensus Stat5 response element was identified at -110 to -118 in the human Type II 3beta-HSD promoter. 3beta-HSD chloramphenicol acetyltransferase (CAT) reporter constructs containing either an intact or mutated Stat5 element were tested for PRL activation. Expression vectors for Stat5 and the PRL receptor were cotransfected with a -300 --> +45 3beta-HSD CAT reporter construct into HeLa cells, which resulted in a 21-fold increase in reporter activity in the presence of PRL. Promoter activity showed an increased response with a stepwise elevation of transfected Stat5 expression or by treatment with increasing concentrations of PRL (max, 250 ng/ml). This effect was dramatically reduced when the putative Stat5 response element was removed by 5'-deletion of the promoter or by the introduction of a 3-bp mutation into critical nucleotides in the element. Furthermore, 32P-labeled promoter fragments containing the Stat5 element were shifted in electrophoretic mobility shift assay experiments using nuclear extracts from cells treated with PRL, and this complex was supershifted with antibodies to Stat5. These results demonstrate that PRL has the ability to regulate expression of a key human enzyme gene (type II 3beta-HSD) in the progesterone biosynthetic pathway, which is essential for maintaining pregnancy.

  18. Equine endometrial fibrosis correlates with 11beta-HSD2, TGF-beta1 and ACE activities.

    PubMed

    Ganjam, V K; Evans, T J

    2006-03-27

    Endometrial periglandular fibrosis (EPF) contributes to embryonic and fetal loss in mares. Equine EPF correlates inversely with conception and successful gestation. In the modified Kenney endometrial biopsy classification system, EPF categories I, IIA, IIB, and III correspond to minimal, mild, moderate, and severe fibrosis (+/-inflammation), respectively. Paraffin sections of biopsy specimens were stained with H&E, and picrosirius red (specific for fibrillar collagens types I and III), to determine %EPCVF. Endometrial ACE-binding activity, TGF-beta1 and 11beta-HSD2 activities were also measured. Ultrastructural changes in EPF categories IIB and III endometria strongly suggested myofibroblastic transformation. ACE-binding activity was highest in EPF category IIB; however, endometrial TGF-beta1 and 11beta-HSD2 activities were significantly correlated to the severity of EPF (P<0.05). We conclude that, locally generated angiotensin II initiates the expression of TGF-beta1 resulting in myofibroblastic transformation. 11Beta-HSD2 in concert appears to modulate the severity of endometrial fibrosis.

  19. A comparative analysis reveals weak relationships between ecological factors and beta diversity of stream insect metacommunities at two spatial levels.

    PubMed

    Heino, Jani; Melo, Adriano S; Bini, Luis Mauricio; Altermatt, Florian; Al-Shami, Salman A; Angeler, David G; Bonada, Núria; Brand, Cecilia; Callisto, Marcos; Cottenie, Karl; Dangles, Olivier; Dudgeon, David; Encalada, Andrea; Göthe, Emma; Grönroos, Mira; Hamada, Neusa; Jacobsen, Dean; Landeiro, Victor L; Ligeiro, Raphael; Martins, Renato T; Miserendino, María Laura; Md Rawi, Che Salmah; Rodrigues, Marciel E; Roque, Fabio de Oliveira; Sandin, Leonard; Schmera, Denes; Sgarbi, Luciano F; Simaika, John P; Siqueira, Tadeu; Thompson, Ross M; Townsend, Colin R

    2015-03-01

    The hypotheses that beta diversity should increase with decreasing latitude and increase with spatial extent of a region have rarely been tested based on a comparative analysis of multiple datasets, and no such study has focused on stream insects. We first assessed how well variability in beta diversity of stream insect metacommunities is predicted by insect group, latitude, spatial extent, altitudinal range, and dataset properties across multiple drainage basins throughout the world. Second, we assessed the relative roles of environmental and spatial factors in driving variation in assemblage composition within each drainage basin. Our analyses were based on a dataset of 95 stream insect metacommunities from 31 drainage basins distributed around the world. We used dissimilarity-based indices to quantify beta diversity for each metacommunity and, subsequently, regressed beta diversity on insect group, latitude, spatial extent, altitudinal range, and dataset properties (e.g., number of sites and percentage of presences). Within each metacommunity, we used a combination of spatial eigenfunction analyses and partial redundancy analysis to partition variation in assemblage structure into environmental, shared, spatial, and unexplained fractions. We found that dataset properties were more important predictors of beta diversity than ecological and geographical factors across multiple drainage basins. In the within-basin analyses, environmental and spatial variables were generally poor predictors of variation in assemblage composition. Our results revealed deviation from general biodiversity patterns because beta diversity did not show the expected decreasing trend with latitude. Our results also call for reconsideration of just how predictable stream assemblages are along ecological gradients, with implications for environmental assessment and conservation decisions. Our findings may also be applicable to other dynamic systems where predictability is low.

  20. Optical spectroscopic elucidation of beta-turns in disulfide bridged cyclic tetrapeptides.

    PubMed

    Borics, Attila; Murphy, Richard F; Lovas, Sándor

    2007-01-01

    Vibrational circular dichroism (VCD) spectroscopic features of type II beta-turns were characterized previously, but, criteria for differentiation between beta-turn types had not been established yet. Model tetrapeptides, cyclized through a disulfide bridge, were designed on the basis of previous experimental results and the observed incidence of amino acid residues in the i + 1 and i + 2 positions in beta-turns, to determine the features of VCD spectra of type I and II beta-turns. The results were correlated with electronic circular dichroism (ECD) spectra and VCD spectra calculated from conformational data obtained by molecular dynamics (MD) simulations. All cyclic tetrapeptides yielded VCD signals with a higher frequency negative and a lower frequency positive couplet with negative lobes overlapping. MD simulations confirmed the conformational homogeneity of these peptides in solution. Comparison with ECD spectroscopy, MD, and quantum chemical calculation results suggested that the low frequency component of VCD spectra originating from the tertiary amide vibrations could be used to distinguish between types of beta-turn structures. On the basis of this observation, VCD spectroscopic features of type II and VIII beta-turns and ECD spectroscopic properties of a type VIII beta-turn were suggested. The need for independent experimental as well as theoretical investigations to obtain decisive conformational information was recognized. Copyright 2006 Wiley Periodicals, Inc.

  1. Microvascular pericytes express platelet-derived growth factor-beta receptors in human healing wounds and colorectal adenocarcinoma.

    PubMed Central

    Sundberg, C.; Ljungström, M.; Lindmark, G.; Gerdin, B.; Rubin, K.

    1993-01-01

    The expression of platelet-derived growth factor- beta (PDGF-beta) receptors in the microvasculature of human healing wounds and colorectal adenocarcinoma was investigated. Frozen sections were subjected to double immunofluorescence staining using monoclonal antibodies (MAbs) specific for pericytes (MAb 225.28 recognizing the high-molecular weight-melanoma-associated antigen, expressed by activated pericytes during angiogenesis), endothelial cells (MAb PAL-E), laminin, as well as PDGF-beta receptors (MAb PDGFR-B2) and its ligand PDGF-B chain (MAb PDGF 007). Stained sections were analyzed by computer-aided imaging processing that allowed for a numerical quantification of the degree of colocalization of the investigated antigens. An apparent background colocalization, varying between 23 and 35%, between markers for cells not expected to co-localize was recorded. This background could be due to limitations of camera resolution, to out-of-focus fluorescence, and to interdigitations of the investigated structures. In all six tumor specimens, co-localization of PDGF-beta receptors and PAL-E was not different from the background co-localization, whereas that of PDGF-beta receptors and high-molecular weight-melanoma-associated antigen was significantly higher with mean values between 57 and 71%. Qualitatively, the same pattern was obtained in the two investigated healing wounds. PDGF-B chain did not co-localize with either PAL-E or high-molecular weight-melanoma-associated antigen, but PDGF-B chain-expressing cells were, however, frequently found juxtaposed to the microvasculature. The expression of PDGF-beta receptors on pericytes in activated microvessels and the presence of PDGF-B chain-expressing cells in close proximity to the microvasculature of healing wounds and colorectal adenocarcinoma is compatible with a role for PDGF in the physiology of the microvasculature in these conditions. Images Figure 1 p1381-a Figure 3 Figure 4 PMID:8238254

  2. Stretch and interleukin 1 beta: pro-labour factors with similar mitogen-activated protein kinase effects but differential patterns of transcription factor activation and gene expression.

    PubMed

    Sooranna, S R; Engineer, N; Liang, Z; Bennett, P R; Johnson, M R

    2007-07-01

    IL-1beta and stretch increase uterine smooth muscle cell (USMC) prostaglandin H synthase 2 (PGHS-2) and interleukin (IL)-8 mRNA expression in a mitogen-activated protein kinase (MAPK) dependent mechanism. We have tested our hypothesis that stretch and IL-1beta activate different components of the MAPK cascade in USMC and investigated the effects of specific MAPK inhibitors on these components. Further, we have used a Jun N-terminal kinase (JNK) and p38 activator, anisomycin, to compare the effect of differential MAPK activation on the expression of PGHS-2, IL-8 and oxytocin receptor (OTR) mRNA with that seen in response to stretch and IL-1beta. Stretch, IL-1beta and anisomycin activated similar components of the MAPK cascade and specific inhibitors of MAPK altered phosphorylation of MAPK and downstream cascade components as expected. Expression of OTR mRNA was increased by stretch and anisomycin in a MAPK-independent manner. All three stimuli increased PGHS-2 and IL-8 mRNA expression in a MAPK-dependent manner, but while the MAPK inhibitors reduced the IL-1beta-induced activation of activating transcription factor (ATF)-2, liver activating protein (LAP) and c-jun, the stretch-induced increase in LAP was unaffected by MAPK-inhibition and only JNK inhibition appeared to reduce c-jun activation. These observations show that stretch, IL-1beta and anisomycin activate the same components of the MAPK cascade, but differentially activate LAP and liver inhibitory protein (LIP) perhaps accounting for the increase in OTR by stretch and anisomycin but not IL-1beta observed in this study.

  3. An endocrine pathway in the prostate, ERbeta, AR, 5alpha-androstane-3beta,17beta-diol, and CYP7B1, regulates prostate growth.

    PubMed

    Weihua, Zhang; Lathe, Richard; Warner, Margaret; Gustafsson, Jan-Ake

    2002-10-15

    Epithelial proliferation of the ventral prostate in rodents peaks between 2 and 4 weeks of age, and by week 8, proliferating cells are rare. We have used ERbeta(-/-) and CYP7B1(-/-) mice to investigate the role of ERbeta and one of its ligands, 5alpha-androstane-3beta,17beta-diol (3betaAdiol), in growth of the ventral prostate. Before puberty, ERbeta was found in quiescent but not in proliferating cells, and proliferating cells occurred more frequently in ventral prostates of ERbeta(-/-) mice than in wild-type littermates. Treatment with 3betaAdiol decreased proliferation in wild-type but not in ERbeta(-/-) mice. In rats, treatment with 3betaAdiol from postnatal day 2 to 28 resulted in reduction in growth of ventral prostates. The prostates of CYP7B1(-/-) mice were hypoproliferative before puberty and smaller than those of their wild-type littermates after puberty. Because CYP7B1 represents the major pathway for inactivating 3betaAdiol in the prostate, we suggest that ERbeta, 3betaAdiol, and CYP7B1 are the components of a pathway that regulates growth of the rodent ventral prostate. In this pathway, ERbeta is an antiproliferative receptor, 3betaAdiol is an ERbeta ligand, and CYP7B1 is the enzyme that regulates ERbeta function by regulating the level of 3betaAdiol.

  4. All of the human beta-type globin genes compete for LCR enhancer activity in embryonic erythroid cells of yeast artificial chromosome transgenic mice.

    PubMed

    Okamura, Eiichi; Matsuzaki, Hitomi; Campbell, Andrew D; Engel, James Douglas; Fukamizu, Akiyoshi; Tanimoto, Keiji

    2009-12-01

    In primitive erythroid cells of human beta-globin locus transgenic mice (TgM), the locus control region (LCR)-proximal epsilon- and gamma-globin genes are transcribed, whereas the distal delta- and beta-globin genes are silent. It is generally accepted that the beta-globin gene is competitively suppressed by gamma-globin gene expression at this developmental stage. Previously, however, we observed that epsilon-globin gene expression was severely attenuated when its distance from the LCR was extended, implying that beta-globin gene might also be silenced because of its great distance from the LCR. Here, to clarify the beta-globin gene silencing mechanism, we established TgM lines carrying either gamma- or epsilon- plus gamma-globin promoter deletions, without significantly altering the distance between the beta-globin gene and the LCR. Precocious expression of delta- and beta-globin genes was observed in primitive erythroid cells of mutant, but not wild-type TgM, which was most evident when both the epsilon and gamma promoters were deleted. Thus, we clearly demonstrated that the repression of the delta- and beta-globin genes in primitive erythroid cells is dominated by competitive silencing by the epsilon- and gamma-globin gene promoters, and that epsilon- and the other beta-like globin genes might be activated by two distinct mechanisms by the LCR.

  5. Chemical synthesis of beta-O-4 type artificial lignin.

    PubMed

    Kishimoto, Takao; Uraki, Yasumitsu; Ubukata, Makoto

    2006-04-07

    An artificial lignin polymer containing only the beta-O-4 substructure was synthesized. The procedure consists of two key steps: 1) polycondensation of a brominated monomer by aromatic Williamson reaction; and 2) subsequent reduction of the carbonyl polymer. 13C-NMR and HMQC spectra of the polymer were consistent with beta-O-4 substructures in milled wood lignin isolated from Japanese fir wood. The weight average degree of polymerization (DP(w)) ranged from 19.5 to 30.6, which is comparable to enzymatically synthesized artificial lignin from p-hydroxycinnamyl alcohols (dehydrogenation polymer, DHP) and some isolated lignins. Using this new lignin model polymer, it will now be possible to reinvestigate the properties and reactivity of the main lignin structure in terms of its polymeric character.

  6. Association of hypoxia inducible factor-1 alpha gene polymorphism with both type 1 and type 2 diabetes in a Caucasian (Hungarian) sample

    PubMed Central

    Nagy, Geza; Kovacs-Nagy, Reka; Kereszturi, Eva; Somogyi, Aniko; Szekely, Anna; Nemeth, Nora; Hosszufalusi, Nora; Panczel, Pal; Ronai, Zsolt; Sasvari-Szekely, Maria

    2009-01-01

    Background Hypoxia inducible factor-1 alpha (HIF-1α) is a transcription factor that plays an important role in neo-vascularisation, embryonic pancreas beta-cell mass development, and beta cell protection. Recently a non synonymous single nucleotide polymorphism (g.C45035T SNP, rs11549465) of HIF-1α gene, resulting in the p.P582S amino acid change has been shown to be associated with type 2 diabetes (T2DM) in a Japanese population. Our aim was to replicate these findings on a Caucasian (Hungarian) population, as well as to study whether this genetic effect is restricted to T2DM or can be expanded to diabetes in general. Methods A large Caucasian sample (N = 890) was recruited including 370 T2DM, 166 T1DM and 354 healthy subjects. Genotyping was validated by two independent methods: a restriction fragment analysis (RFLP) and a real time PCR using TaqMan probes. An overestimation of heterozygotes by RFLP was observed as a consequence of a nearby SNP (rs34005929). Therefore genotyping results of the justified TaqMan system were accepted. The measured genotype distribution corresponded to Hardy-Weinberg equilibrium (P = 0.740) Results As the TT genotype was extremely rare in the population (0.6% in clinical sample and 2.5% in controls), the genotypes were grouped as T absent (CC) and T present (CT and TT). Genotype-wise analysis showed a significant increase of T present group in controls (24.0%) as compared to patients (16.8%, P = 0.008). This genetic effect was demonstrated in the separated samples of type 1 (15.1%, P = 0.020), and also in type 2 (17.6%, P = 0.032) diabetes. Allele-wise analysis gave identical results showing a higher frequency of the T allele in the control sample (13.3%) than in the clinical sample (8.7%, P = 0.002) with similar results in type 1 (7.8%, P = 0.010) and type 2 (9.1%, P = 0.011) diabetes. The odds ratio for diabetes (either type 1 or 2) was 1.56 in the presence of the C allele. Conclusion We confirmed the protective effect of a rare

  7. On the role of transforming growth factor-beta in the growth inhibitory effects of retinoic acid in human pancreatic cancer cells.

    PubMed

    Singh, Brahmchetna; Murphy, Richard F; Ding, Xian-Zhong; Roginsky, Alexandra B; Bell, Richard H; Adrian, Thomas E

    2007-12-24

    Retinoids are potent growth inhibitory and differentiating agents in a variety of cancer cell types. We have shown that retinoids induce growth arrest in all pancreatic cancer cell lines studied, regardless of their p53 and differentiation status. However, the mechanism of growth inhibition is not known. Since TGF-beta2 is markedly induced by retinoids in other cancers and mediates MUC4 expression in pancreatic cancer cells, we investigated the role of TGF-beta in retinoic acid-mediated growth inhibition in pancreatic cancer cells. Retinoic acid markedly inhibited proliferation of two cell lines (Capan-2 and Hs766T) in a concentration and time-dependent manner. Retinoic acid increased TGF-beta2 mRNA content and secretion of the active and latent forms of TGF-beta2 (measured by ELISA and bioassay). The concentrations of active and TGF-beta2 secreted in response to 0.1 - 10 muM retinoic acid were between 1-5 pM. TGF-beta2 concentrations within this range also inhibited proliferation. A TGF-beta neutralizing antibody blocked the growth inhibitory effects of retinoic acid in Capan-2 cells and partially inhibitory the effects in Hs766T cells. These findings indicate that TGF-beta can cause growth inhibition of pancreatic cancer cells, in a p53-independent manner. Furthermore, it demonstrates the fundamental role of TGF-beta in growth inhibition in response to retinoic acid treatment is preserved in vitro.

  8. Quantification of beta A4 protein deposition in the medial temporal lobe: a comparison of Alzheimer's disease and senile dementia of the Lewy body type.

    PubMed

    Gentleman, S M; Williams, B; Royston, M C; Jagoe, R; Clinton, J; Perry, R H; Ince, P G; Allsop, D; Polak, J M; Roberts, G W

    1992-08-03

    The distribution of beta-amyloid protein (beta A4) was examined in the medial temporal lobes from cases of Alzheimer's disease (AD) (n = 13), senile dementia of Lewy body type (SDLT) (n = 12) and age matched controls (n = 9). Using a previously described image analysis technique the extent of beta A4 pathology was determined in ten distinct anatomical sites within the medial temporal lobe. AD and SDLT cases contained very similar amounts of beta A4 in the areas sampled and both contained significantly more beta A4 than the age matched controls, particularly in the dentate and parahippocampal gyri. The similarity of the beta A4 load in the two conditions is in contrast to reported differences in the number of neurofibrillary tangles which can be observed. It is suggested that AD and SDLT represent a spectrum of pathology which centres around the aberrant processing of the beta A4 precursor protein.

  9. Chemical factors determine olfactory system beta oscillations in waking rats.

    PubMed

    Lowry, Catherine A; Kay, Leslie M

    2007-07-01

    Recent studies have pointed to olfactory system beta oscillations of the local field potential (15-30 Hz) and their roles both in learning and as specific responses to predator odors. To describe odorant physical properties, resultant behavioral responses and changes in the central olfactory system that may induce these oscillations without associative learning, we tested rats with 26 monomolecular odorants spanning 6 log units of theoretical vapor pressure (estimate of relative vapor phase concentration) and 10 different odor mixtures. We found odorant vapor phase concentration to be inversely correlated with investigation time on the first presentation, after which investigation times were brief and not different across odorants. Analysis of local field potentials from the olfactory bulb and anterior piriform cortex shows that beta oscillations in waking rats occur specifically in response to the class of volatile organic compounds with vapor pressures of 1-120 mmHg. Beta oscillations develop over the first three to four presentations and are weakly present for some odorants in anesthetized rats. Gamma oscillations show a smaller effect that is not restricted to the same range of odorants. Olfactory bulb theta oscillations were also examined as a measure of effective afferent input strength, and the power of these oscillations did not vary systematically with vapor pressure, suggesting that it is not olfactory bulb drive strength that determines the presence of beta oscillations. Theta band coherence analysis shows that coupling strength between the olfactory bulb and piriform cortex increases linearly with vapor phase concentration, which may facilitate beta oscillations above a threshold.

  10. Transforming growth factor-beta inhibits the expression of clock genes.

    PubMed

    Gast, Heidemarie; Gordic, Sonja; Petrzilka, Saskia; Lopez, Martin; Müller, Andreas; Gietl, Anton; Hock, Christoph; Birchler, Thomas; Fontana, Adriano

    2012-07-01

    Disturbances of sleep-wake rhythms are an important problem in Alzheimer's disease (AD). Circadian rhythms are regulated by clock genes. Transforming growth factor-beta (TGF-β) is overexpressed in neurons in AD and is the only cytokine that is increased in cerebrospinal fluid (CSF). Our data show that TGF-β2 inhibits the expression of the clock genes Period (Per)1, Per2, and Rev-erbα, and of the clock-controlled genes D-site albumin promoter binding protein (Dbp) and thyrotroph embryonic factor (Tef). However, our results showed that TGF-β2 did not alter the expression of brain and muscle Arnt-like protein-1 (Bmal1). The concentrations of TGF-β2 in the CSF of 2 of 16 AD patients and of 1 of 7 patients with mild cognitive impairment were in the dose range required to suppress the expression of clock genes. TGF-β2-induced dysregulation of clock genes may alter neuronal pathways, which may be causally related to abnormal sleep-wake rhythms in AD patients. © 2012 New York Academy of Sciences.

  11. Glycosaminoglycan and transforming growth factor beta1 changes in human plasma and urine during the menstrual cycle, in vitro fertilization treatment, and pregnancy.

    PubMed

    De Muro, Pierina; Capobianco, Giampiero; Formato, Marilena; Lepedda, Antonio Junior; Cherchi, Gian Mario; Gordini, Laila; Dessole, Salvatore

    2009-07-01

    To evaluate transforming growth factor beta1 (TGF-beta1) and glycosaminoglycans (GAG) changes in human plasma and urine during the menstrual cycle, IVF-ET, and pregnancy. Prospective clinical study. University hospital. Thirteen women with apparently normal menstrual cycle (group 1); 18 women undergoing IVF-ET (group 2); and 14 low-risk pregnant women (group 3). We assayed plasma and urine concentrations of TGF-beta1, urine content, and distribution of GAG. Blood and urine samples were collected during days 2 to 3, 12 to 13, and 23 to 24 in group 1; in group 2, samples were obtained at menstrual phase, oocyte pick-up day, and 15 days after ET; in group 3, samples were obtained during gestational weeks 10-12, 22-24, and 30-32 and 1 month after delivery. Changes in TGF-beta1 and GAG content. The mean value of total urinary trypsin inhibitor/chondroitin sulfate (UTI/CS) showed a distinct peak at day 12 of the menstrual cycle in the fertile women in whom we monitored the ovulatory period. In the IVF-ET group, GAG distribution and TGF-beta1 levels showed significant differences during the cycle. We observed increased levels of plasma TGF-beta1 15 days after ET. A significant increase of total UTI/CS value with increasing gestation was detected. Transforming growth factor beta1 and GAG levels could represent an additional tool to monitor reproductive events and could be useful, noninvasive markers of ovulation and ongoing pregnancy.

  12. Requirement for the SnoN oncoprotein in transforming growth factor beta-induced oncogenic transformation of fibroblast cells.

    PubMed

    Zhu, Qingwei; Pearson-White, Sonia; Luo, Kunxin

    2005-12-01

    Transforming growth factor beta (TGF-beta) was originally identified by virtue of its ability to induce transformation of the AKR-2B and NRK fibroblasts but was later found to be a potent inhibitor of the growth of epithelial, endothelial, and lymphoid cells. Although the growth-inhibitory pathway of TGF-beta mediated by the Smad proteins is well studied, the signaling pathway leading to the transforming activity of TGF-beta in fibroblasts is not well understood. Here we show that SnoN, a member of the Ski family of oncoproteins, is required for TGF-beta-induced proliferation and transformation of AKR-2B and NRK fibroblasts. TGF-beta induces upregulation of snoN expression in both epithelial cells and fibroblasts through a common Smad-dependent mechanism. However, a strong and prolonged activation of snoN transcription that lasts for 8 to 24 h is detected only in these two fibroblast lines. This prolonged induction is mediated by Smad2 and appears to play an important role in the transformation of both AKR-2B and NRK cells. Reduction of snoN expression by small interfering RNA or shortening of the duration of snoN induction by a pharmacological inhibitor impaired TGF-beta-induced anchorage-independent growth of AKR-2B cells. Interestingly, Smad2 and Smad3 play opposite roles in regulating snoN expression in both fibroblasts and epithelial cells. The Smad2/Smad4 complex activates snoN transcription by direct binding to the TGF-beta-responsive element in the snoN promoter, while the Smad3/Smad4 complex inhibits it through a novel Smad inhibitory site. Mutations of Smad4 that render it defective in heterodimerization with Smad3, which are found in many human cancers, convert the activity of Smad3 on the snoN promoter from inhibitory to stimulatory, resulting in increased snoN expression in cancer cells. Thus, we demonstrate a novel role of SnoN in the transforming activity of TGF-beta in fibroblasts and also uncovered a mechanism for the elevated SnoN expression in

  13. Analysis of the resistance-breaking ability of different beet necrotic yellow vein virus isolates loaded into a single Polymyxa betae population in soil.

    PubMed

    Bornemann, Kathrin; Varrelmann, Mark

    2011-06-01

    The genome of most Beet necrotic yellow vein virus (BNYVV) isolates is comprised of four RNAs. The ability of certain isolates to overcome Rz1-mediated resistance in sugar beet grown in the United States and Europe is associated with point mutations in the pathogenicity factor P25. When the virus is inoculated mechanically into sugar beet roots at high density, the ability depends on an alanine to valine substitution at P25 position 67. Increased aggressiveness is shown by BNYVV P type isolates, which carry an additional RNA species that encodes a second pathogenicity factor, P26. Direct comparison of aggressive isolates transmitted by the vector, Polymyxa betae, has been impossible due to varying population densities of the vector and other soilborne pathogens that interfere with BNYVV infection. Mechanical root inoculation and subsequent cultivation in soil that carried a virus-free P. betae population was used to load P. betae with three BNYVV isolates: a European A type isolate, an American A type isolate, and a P type isolate. Resistance tests demonstrated that changes in viral aggressiveness towards Rz1 cultivars were independent of the vector population. This method can be applied to the study of the synergism of BNYVV with other P. betae-transmitted viruses.

  14. Upregulation of adipose 11-beta-hydroxysteroid dehydrogenase type 1 expression in ovariectomized rats is due to obesity rather than lack of estrogen.

    PubMed

    Paulsen, Søren K; Nielsen, Maria P; Richelsen, Bjørn; Bruun, Jens M; Flyvbjerg, Allan; Pedersen, Steen B

    2008-04-01

    Increased tissue activity of cortisol induced by the activation of inert cortisone to active cortisol through 11-beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) may play a role in the metabolic syndrome. We recently found that 11beta-HSD1 in subcutaneous adipose tissue (AT) was lower in lean women compared with lean men. Estrogen suppresses hepatic and renal 11beta-HSD1 in rats; hence we investigated the in vitro effect of estrogen on human and rat AT, and the in vivo effects on rat AT 11beta-HSD1 expression. Wistar rats were divided into four groups of eight animals. One group was sham-operated (controls) and others were ovariectomized (OVX). One OVX group was left untreated (OVX-E), another (OVX+E) received estrogen treatment, and one received a hypo-caloric diet (OVX-E+D), matching the weight gain of the control group. AT from women undergoing liposuction or surgery and from killed male and female rats were incubated with estrogen alone or in the presence of IL-1beta. Gene expressions were determined by real-time reverse transcriptase PCR. Ovariectomy resulted in a 280% increase in adipose 11beta-HSD1 expression P < 0.05). 11beta-HSD1 expression in the (OVX+E)-group was significantly reduced compared with the nonsubstituted group (P < 0.05). 11beta-HSD1 expression in the (OVX-E+D)-group was reduced significantly (P < 0.05) when compared with the level of the estrogen-substituted group. No significant differences between the control group, the (OVX+E)-group, and the (OVX-E+D)-group were found. In the in vitro studies, no direct effect of estrogen on adipose 11beta-HSD1 was found. The upregulation of 11beta-HSD1 in ovariectomized rats was most likely due to changes in body composition rather than lack of estrogen.

  15. Toward beta cell replacement for diabetes

    PubMed Central

    Johannesson, Bjarki; Sui, Lina; Freytes, Donald O; Creusot, Remi J; Egli, Dieter

    2015-01-01

    The discovery of insulin more than 90 years ago introduced a life-saving treatment for patients with type 1 diabetes, and since then, significant progress has been made in clinical care for all forms of diabetes. However, no method of insulin delivery matches the ability of the human pancreas to reliably and automatically maintain glucose levels within a tight range. Transplantation of human islets or of an intact pancreas can in principle cure diabetes, but this approach is generally reserved for cases with simultaneous transplantation of a kidney, where immunosuppression is already a requirement. Recent advances in cell reprogramming and beta cell differentiation now allow the generation of personalized stem cells, providing an unlimited source of beta cells for research and for developing autologous cell therapies. In this review, we will discuss the utility of stem cell-derived beta cells to investigate the mechanisms of beta cell failure in diabetes, and the challenges to develop beta cell replacement therapies. These challenges include appropriate quality controls of the cells being used, the ability to generate beta cell grafts of stable cellular composition, and in the case of type 1 diabetes, protecting implanted cells from autoimmune destruction without compromising other aspects of the immune system or the functionality of the graft. Such novel treatments will need to match or exceed the relative safety and efficacy of available care for diabetes. PMID:25733347

  16. Liposomal gene transfer of keratinocyte growth factor improves wound healing by altering growth factor and collagen expression.

    PubMed

    Pereira, Clifford T; Herndon, David N; Rocker, Roland; Jeschke, Marc G

    2007-05-15

    Growth factors affect the complex cascade of wound healing; however, interaction between different growth factors during dermal and epidermal regeneration are still not entirely defined. In the present study, we thought to determine the interaction between keratinocyte growth factor (KGF) administered as liposomal cDNA with other dermal and epidermal growth factors and collagen synthesis in an acute wound. Rats received an acute wound and were divided into two groups to receive weekly subcutaneous injections of liposomes plus the Lac-Z gene (0.22 microg, vehicle), or liposomes plus the KGF cDNA (2.2 microg) and Lac-Z gene (0.22 microg). Histological and immunohistochemical techniques were used to determine growth factor, collagen expression, and dermal and epidermal structure. KGF cDNA increased insulin-like growth factor-I (IGF-I), insulin-like growth factor binding protein-3 (IGFBP-3), and fibroblast growth factor (FGF), decreased transforming growth factor-beta (TGF-beta), while it had no effect on platelet-derived growth factor (PDGF) levels in the wound. KGF cDNA significantly increased collagen Type IV at both the wound edge as well as the wound bed, while it had no effect on collagen Type I and III. KGF cDNA increased re-epithelialization, improved dermal regeneration, and increased neovascularization. Exogenous administered KGF cDNA causes increases in IGF-I, IGF-BP3, FGF, and collagen IV and decreases TGF-beta concentration. KGF gene transfer accelerates wound healing without causing an increase in collagen I or III.

  17. Characterization of a novel transcriptionally active domain in the transforming growth factor beta-regulated Smad3 protein.

    PubMed

    Prokova, Vassiliki; Mavridou, Sofia; Papakosta, Paraskevi; Kardassis, Dimitris

    2005-01-01

    Transforming growth factor beta (TGFbeta) regulates transcriptional responses via activation of cytoplasmic effector proteins termed Smads. Following their phosphorylation by the type I TGFbeta receptor, Smads form oligomers and translocate to the nucleus where they activate the transcription of TGFbeta target genes in cooperation with nuclear cofactors and coactivators. In the present study, we have undertaken a deletion analysis of human Smad3 protein in order to characterize domains that are essential for transcriptional activation in mammalian cells. With this analysis, we showed that Smad3 contains two domains with transcriptional activation function: the MH2 domain and a second middle domain that includes the linker region and the first two beta strands of the MH2 domain. Using a protein-protein interaction assay based on biotinylation in vivo, we were able to show that a Smad3 protein bearing an internal deletion in the middle transactivation domain is characterized by normal oligomerization and receptor activation properties. However, this mutant has reduced transactivation capacity on synthetic or natural promoters and is unable to interact physically and functionally with the histone acetyltransferase p/CAF. The loss of interaction with p/CAF or other coactivators could account, at least in part, for the reduced transactivation capacity of this Smad3 mutant. Our data support an essential role of the previously uncharacterized middle region of Smad3 for nuclear functions, such as transcriptional activation and interaction with coactivators.

  18. Chronology of endocrine differentiation and beta-cell neogenesis.

    PubMed

    Miyatsuka, Takeshi

    2016-01-01

    Diabetes is a chronic and incurable disease, which results from absolute or relative insulin insufficiency. Therefore, pancreatic beta cells, which are the only type of cell that expresses insulin, is considered to be a potential target for the cure of diabetes. Although the findings regarding beta-cell neogenesis during pancreas development have been exploited to induce insulin-producing cells from non-beta cells, there are still many hurdles towards generating fully functional beta cells that can produce high levels of insulin and respond to physiological signals. To overcome these problems, a solid understanding of pancreas development and beta-cell formation is required, and several mouse models have been developed to reveal the unique features of each endocrine cell type at distinct developmental time points. Here I review our understanding of pancreas development and endocrine differentiation focusing on recent progresses in improving temporal cell labeling in vivo.

  19. Inequalities of extended beta and extended hypergeometric functions.

    PubMed

    Mondal, Saiful R

    2017-01-01

    We study the log-convexity of the extended beta functions. As a consequence, we establish Turán-type inequalities. The monotonicity, log-convexity, log-concavity of extended hypergeometric functions are deduced by using the inequalities on extended beta functions. The particular cases of those results also give the Turán-type inequalities for extended confluent and extended Gaussian hypergeometric functions. Some reverses of Turán-type inequalities are also derived.

  20. TGF-beta1 inhibits expression and activity of hENT1 in a nitric oxide-dependent manner in human umbilical vein endothelium.

    PubMed

    Vega, José L; Puebla, Carlos; Vásquez, Rodrigo; Farías, Marcelo; Alarcón, Julio; Pastor-Anglada, Marçal; Krause, Bernardo; Casanello, Paola; Sobrevia, Luis

    2009-06-01

    We studied whether transforming growth factor beta1 (TGF-beta1) modulates human equilibrative nucleoside transporters 1 (hENT1) expression and activity in human umbilical vein endothelial cells (HUVECs). hENT1-mediated adenosine transport and expression are reduced in gestational diabetes and hyperglycaemia, conditions associated with increased synthesis and release of nitric oxide (NO) and TGF-beta1 in this cell type. TGF-beta1 increases NO synthesis via activation of TGF-beta receptor type II (TbetaRII), and NO inhibits hENT1 expression and activity in HUVECs. HUVECs (passage 2) were used for experiments. Total and hENT1-mediated adenosine transport was measured in the absence or presence of TGF-beta1, NG-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor), S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor), and/or KT-5823 (protein kinase G inhibitor) in control cells and cells expressing a truncated form of TGF-beta1 receptor type II (TTbetaRII). Western blot and real-time PCR were used to determine hENT1 protein abundance and mRNA expression. SLC29A1 gene promoter and specific protein 1 (Sp1) transcription factor activity was assayed. Vascular reactivity was assayed in endothelium-intact or -denuded umbilical vein rings. TGF-beta1 reduced hENT1-mediated adenosine transport, hENT1 protein abundance, hENT1 mRNA expression, and SLC29A1 gene promoter activity, but increased Sp1 binding to DNA. TGF-beta1 effect was blocked by L-NAME and KT-5823 and mimicked by SNAP in control cells. However, TGF-beta1 was ineffective in cells expressing TTbetaRII or a mutated Sp1 consensus sequence. Vasodilatation in response to TGF-beta1 and S-(4-nitrobenzyl)-6-thio-inosine (an ENT inhibitor) was endothelium-dependent and blocked by KT-5823 and ZM-241385. hENT1 is down-regulated by activation of TbetaRII by TGF-beta1 in HUVECs, a phenomenon where NO and Sp1 play key roles. These findings comprise physiological mechanisms that could be important in diseases where TGF-beta

  1. Dissecting the role of matrix metalloproteinases (MMP) and integrin alpha(v)beta3 in angiogenesis in vitro: absence of hemopexin C domain bioactivity, but membrane-Type 1-MMP and alpha(v)beta3 are critical.

    PubMed

    Nisato, Riccardo E; Hosseini, Ghamartaj; Sirrenberg, Christian; Butler, Georgina S; Crabbe, Thomas; Docherty, Andrew J P; Wiesner, Matthias; Murphy, Gillian; Overall, Christopher M; Goodman, Simon L; Pepper, Michael S

    2005-10-15

    Matrix metalloproteinase (MMP)-2 and its hemopexin C domain autolytic fragment (also called PEX) have been proposed to be crucial for angiogenesis. Here, we have investigated the dependency of in vitro angiogenesis on MMP-mediated extracellular proteolysis and integrin alpha(v)beta3-mediated cell adhesion in a three-dimensional collagen I model. The hydroxamate-based synthetic inhibitors BB94, CT1399, and CT1847 inhibited endothelial cell invasion, as did neutralizing anti-membrane-type 1-MMP (MT1-MMP) antibodies and tissue inhibitor of MMP (TIMP)-2 and TIMP-3 but not TIMP-1. This confirmed the pivotal importance of MT1-MMP over other MMPs in this model. Invasion was also inhibited by a nonpeptidic antagonist of integrin alpha(v)beta3, EMD 361276. Although PEX strongly inhibited pro-MMP-2 activation, when contaminating lipopolysaccharide was neutralized, PEX neither affected angiogenesis nor bound integrin alpha(v)beta(3). Moreover, no specific binding of pro-MMP-2 to integrin alpha(v)beta3 was found, whereas only one out of four independently prepared enzymatically active MMP-2 preparations could bind integrin alpha(v)beta3 , and this in a PEX-independent manner. Likewise, integrin alpha(v)beta3 -expressing cells did not bind MMP-2-coated surfaces. Hence, these findings show that endothelial cell invasion of collagen I gels is MT1-MMP and alpha(v)beta3 - dependent but MMP-2 independent and does not support a role for PEX in alpha(v)beta3 integrin binding or in modulating angiogenesis in this system.

  2. Nonessential role of beta3 and beta5 integrin subunits for efficient clearance of cellular debris after light-induced photoreceptor degeneration.

    PubMed

    Joly, Sandrine; Samardzija, Marijana; Wenzel, Andreas; Thiersch, Markus; Grimm, Christian

    2009-03-01

    During light-induced photoreceptor degeneration, large amounts of cellular debris are formed that must be cleared from the subretinal space. The integrins alphavbeta5 and alphavbeta3 are involved in the normal physiological process of phagocytosis in the retina. This study was conducted to investigate the question of whether the lack of beta5 and/or beta3 integrin subunits might influence the course of retinal degeneration and/or clearance of photoreceptor debris induced by acute exposure to light. Wild-type, beta5(-/-) and beta3(-/-) single-knockout, and beta3(-/-)/beta5(-/-) Ccl2(-/-)/beta5(-/-) double-knockout mice were exposed to 13,000 lux of white light for 2 hours to induce severe photoreceptor degeneration. Real-time PCR and Western blot analysis were used to analyze gene and protein expression, light- and electron microscopy to judge retinal morphology, and immunofluorescence to study retinal distribution of proteins. Individual or combined deletion of beta3 and beta5 integrin subunits did not affect the pattern of photoreceptor cell loss or the clearance of photoreceptor debris in mice compared with that in wild-type mice. Invading macrophages may contribute to efficient phagocytosis. However, ablation of the MCP-1 gene did not prevent macrophage recruitment. Several chemokines in addition to MCP-1 were induced after light-induced damage that may have compensated for the deletion of MCP-1. Acute clearance of a large amount of cellular debris from the subretinal space involves invading macrophages and does not depend on beta3 and beta5 integrins.

  3. Method for protecting bone marrow against chemotherapeutic drugs and radiation therapy using transforming growth factor beta 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Keller, J.R.; Ruscetti, F.W.; Wiltrout, R.

    1989-06-29

    Presented is a method for protecting hematopoietic stem cells from the myelotoxicity of chemotherapeutic drugs or radiation therapy, which comprises administering to a subject a therapeutically effective amount of transforming growth factor beta 1 for protecting bone marrow from the myelotoxicity of chemotherapeutic drugs or radiation therapy.

  4. Inactivation properties of voltage-gated K+ channels altered by presence of beta-subunit.

    PubMed

    Rettig, J; Heinemann, S H; Wunder, F; Lorra, C; Parcej, D N; Dolly, J O; Pongs, O

    1994-05-26

    Structural and functional diversity of voltage-gated Kv1-type potassium channels in rat brain is enhanced by the association of two different types of subunits, the membrane-bound, poreforming alpha-subunits and a peripheral beta-subunit. We have cloned a beta-subunit (Kv beta 1) that is specifically expressed in the rat nervous system. Association of Kv beta 1 with alpha-subunits confers rapid A-type inactivation on non-inactivating Kv1 channels (delayed rectifiers) in expression systems in vitro. This effect is mediated by an inactivating ball domain in the Kv beta 1 amino terminus.

  5. TGF-beta and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions.

    PubMed

    Mori, Shigeo; Matsuzaki, Koichi; Yoshida, Katsunori; Furukawa, Fukiko; Tahashi, Yoshiya; Yamagata, Hideo; Sekimoto, Go; Seki, Toshihito; Matsui, Hirofumi; Nishizawa, Mikio; Fujisawa, Jun-ichi; Okazaki, Kazuichi

    2004-09-23

    Although hepatocyte growth factor (HGF) can act synergistically or antagonistically with transforming growth factor-beta (TGF-beta) signaling, molecular mechanism of their crosstalk remains unknown. Using antibodies which selectively distinguished receptor-regulated Smads (R-Smads) phosphorylated at linker regions from those at C-terminal regions, we herein showed that either HGF or TGF-beta treatment of normal stomach-origin cells activated the JNK pathway, thereafter inducing endogenous R-Smads phosphorylation at linker regions. However, the phosphorylation at their C-terminal regions was not induced by HGF treatment. The activated JNK could directly phosphorylate R-Smads in vitro at the same sites that were phosphorylated in response to TGF-beta or HGF in vivo. Thus, the linker regions of R-Smads were the common phosphorylation sites for HGF and TGF-beta signaling pathways. The phosphorylation induced by simultaneous treatment with HGF and TGF-beta allowed R-Smads to associate with Smad4 and to translocate into the nucleus. JNK pathway involved HGF and TGF-beta-mediated infiltration potency since a JNK inhibitor SP600125 caused the reduction of invasive capacity induced by HGF and TGF-beta signals. Moreover, a combined treatment with HGF and TGF-beta led to a potent increase in plasminogen activator inhibitor type 1 transcriptional activity through Smad3 phosphorylation at the linker region. In contrast, HGF treatment reduced TGF-beta-dependent activation of p15INK4B promoter, in which Smad3 phosphorylation at the C-terminal region was involved. In conclusion, HGF and TGF-beta transmit the signals through JNK-mediated R-Smads phosphorylation at linker regions.

  6. Genotype–phenotype correlation among beta-thalassemia and beta-thalassemia/HbE disease in Thai children: predictable clinical spectrum using genotypic analysis

    PubMed Central

    Traivaree, Chanchai; Monsereenusorn, Chalinee; Rujkijyanont, Piya; Prasertsin, Warakorn; Boonyawat, Boonchai

    2018-01-01

    Introduction Beta-thalassemia is a group of inherited hemolytic anemias and one of the most common genetic disorders in Thailand. The clinical spectrum of beta-thalassemia disease ranges from mild to severe clinical symptoms including mild beta-thalassemia intermedia (TI) and severe beta-thalassemia major (TM). Objective This study aimed to determine the correlation between beta-globin gene (HBB) mutations and their phenotypic manifestations by evaluating patients’ clinical characteristics, transfusion requirements, growth and hematologic parameters, and hemoglobin typing among pediatric patients treated at Phramongkutklao Hospital. Materials and methods Seventy beta-thalassemia patients, including 63 with beta-thalassemia/hemoglobin E (HbE) and 7 with either homozygous or compound heterozygous beta-thalassemia, were enrolled in this study. Their clinical presentation, growth parameters and laboratory findings were reviewed and analyzed. The mean follow-up time was 10.52±5.62 years. Mutation analysis in each individual was performed using multiplex amplification refractory mutation system (M-ARMS), direct DNA sequencing of beta-globin gene and gap PCR for 3.4 kb deletion detection. Results All 7 homozygous and compound heterozygous beta-thalassemia patients were classified in TM. Among 63 patients with beta-thalassemia/HbE, 58 were classified in TM and 4 were classified in TI. Mean age at diagnosis was 0.8±0.49 years for homozygous or compound heterozygous beta-thalassemia and 3.43±3.5 years for beta-thalassemia/HbE. The most common HBB mutation was HBB:c.126_129delCTTT [codon 41/42 (-TCTT)] found in 34 alleles (48.6%). The height for age was also lower in homozygous beta-thalassemia patients (<3rd percentile) compared to compound heterozygous beta-thalassemia patients (25–50th percentile). Conclusion This study revealed a genotype–phenotype correlation of the most prevalent beta-thalassemia in Thai children using diagnostic capacity in genotypic analysis

  7. Beta-Amyloid Deposition and Alzheimer's Type Changes Induced by Borrelia Spirochetes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Miklossy,J.; Kis, A.; Radenovic, A.

    2006-01-01

    The pathological hallmarks of Alzheimer's disease (AD) consist of {beta}-amyloid plaques and neurofibrillary tangles in affected brain areas. The processes, which drive this host reaction are unknown. To determine whether an analogous host reaction to that occurring in AD could be induced by infectious agents, we exposed mammalian glial and neuronal cells in vitro to Borrelia burgdorferi spirochetes and to the inflammatory bacterial lipopolysaccharide (LPS). Morphological changes analogous to the amyloid deposits of AD brain were observed following 2-8 weeks of exposure to the spirochetes. Increased levels of {beta}-amyloid presursor protein (A{beta}PP) and hyperphosphorylated tau were also detected by Westernmore » blots of extracts of cultured cells that had been treated with spirochetes or LPS. These observations indicate that, by exposure to bacteria or to their toxic products, host responses similar in nature to those observed in AD may be induced.« less

  8. ALK and TGF-Beta Resistance in Breast Cancer

    DTIC Science & Technology

    2017-10-01

    and H.F. Lodish, Role of transforming growth factor beta in human disease. N Engl J Med, 2000. 342(18): p. 1350-8. 3. Massague, J., S.W. Blain, and... Transforming growth factor-beta signaling in normal and malignant hematopoiesis. Leukemia, 2003. 17(9): p. 1731-7. 5. Lehman, H.L., et al., Modeling and

  9. Spatial Factors Play a Major Role as Determinants of Endemic Ground Beetle Beta Diversity of Madeira Island Laurisilva

    PubMed Central

    Boieiro, Mário; Carvalho, José C.; Cardoso, Pedro; Aguiar, Carlos A. S.; Rego, Carla; de Faria e Silva, Israel; Amorim, Isabel R.; Pereira, Fernando; Azevedo, Eduardo B.; Borges, Paulo A. V.; Serrano, Artur R. M.

    2013-01-01

    The development in recent years of new beta diversity analytical approaches highlighted valuable information on the different processes structuring ecological communities. A crucial development for the understanding of beta diversity patterns was also its differentiation in two components: species turnover and richness differences. In this study, we evaluate beta diversity patterns of ground beetles from 26 sites in Madeira Island distributed throughout Laurisilva – a relict forest restricted to the Macaronesian archipelagos. We assess how the two components of ground beetle beta diversity (βrepl – species turnover and βrich - species richness differences) relate with differences in climate, geography, landscape composition matrix, woody plant species richness and soil characteristics and the relative importance of the effects of these variables at different spatial scales. We sampled 1025 specimens from 31 species, most of which are endemic to Madeira Island. A spatially explicit analysis was used to evaluate the contribution of pure environmental, pure spatial and environmental spatially structured effects on variation in ground beetle species richness and composition. Variation partitioning showed that 31.9% of species turnover (βrepl) and 40.7% of species richness variation (βrich) could be explained by the environmental and spatial variables. However, different environmental variables controlled the two types of beta diversity: βrepl was influenced by climate, disturbance and soil organic matter content whilst βrich was controlled by altitude and slope. Furthermore, spatial variables, represented through Moran’s eigenvector maps, played a significant role in explaining both βrepl and βrich, suggesting that both dispersal ability and Madeira Island complex orography are crucial for the understanding of beta diversity patterns in this group of beetles. PMID:23724065

  10. Beta-1,4-glucanase-like protein from the cyanobacterium Synechocystis PCC6803 is a beta-1,3-1,4-glucanase and functions in salt stress tolerance.

    PubMed

    Tamoi, Masahiro; Kurotaki, Hideki; Fukamizo, Tamo

    2007-07-01

    In the present study, we characterized the gene (Cyanobase accession number slr0897) designated Ssglc encoding a beta-1,4-glucanase-like protein (SsGlc) from Synechocystis PCC6803. The deduced amino acid sequence for Ssglc showed a high degree of similarity to sequences of GH (glycoside hydrolase) family 9 beta-1,4-glucanases (cellulases) from various sources. Surprisingly, the recombinant protein obtained from the Escherichia coli expression system was able to hydrolyse barley beta-glucan and lichenan (beta-1,3-1,4-glucan), but not cellulose (beta-1,4-glucan), curdlan (beta-1,3-glucan), or laminarin (beta-1,3-1,6-glucan). A 1H-NMR analysis of the enzymatic products revealed that the enzyme hydrolyses the beta-1,4-glycosidic linkage of barley beta-glucan through an inverting mechanism. The data indicated that SsGlc was a novel type of GH9 glucanase which could specifically hydrolyse the beta-1,3-1,4-linkage of glucan. The growth of mutant Synechocystis cells in which the Ssglc gene was disrupted by a kanamycin-resistance cartridge gene was almost the same as that of the wild-type cells under continuous light (40 micromol of photons/m2 per s), a 12 h light (40 micromol of photons/m2 per s)/12 h dark cycle, cold stress (4 degrees C), and high light stress (200 micromol of photons/m2 per s). However, under salt stress (300-450 mM NaCl), growth of the Ssglc-disrupted mutant cells was significantly inhibited as compared with that of the wild-type cells. The Ssglc-disrupted mutant cells showed a decreased rate of O2 consumption and NaHCO3-dependent O2 evolution as compared with the wild-type cells under salt stress. Under osmotic stress (100-400 mM sorbitol), there was no difference in growth between the wild-type and the Ssglc-disrupted mutant cells. These results suggest that SsGlc functions in salt stress tolerance in Synechocystis PCC6803.

  11. Analysis of common transforming growth factor beta-1 gene polymorphisms in gastric and duodenal ulcer disease: pilot study.

    PubMed

    Polonikov, Alexey V; Ivanov, Vladimir P; Belugin, Dmitry A; Khoroshaya, Irina V; Kolchanova, Inessa O; Solodilova, Mariya A; Tutochkina, Margarita P; Stepchenko, Alexander A

    2007-04-01

    Transforming growth factor-beta1 (TGF-beta1) has been shown to be an important cytokine that plays a role in cell proliferation, differentiation, tissue injury repair and ulcer healing. The purpose of this pilot study was to investigate if common polymorphisms Leu10Pro, Arg25Pro and C-509T within the TGF-beta1 gene are associated with susceptibility to gastric and duodenal ulcer disease in Russians. Blood samples from 377 unrelated patients with gastric and duodenal ulcer disease and 226 sex- and age-matched healthy controls were used to determine TGF-beta1 gene polymorphisms by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Leu10Pro substitution in the signal peptide of TGF-beta1 has been found to be associated with susceptibility to gastric ulcer (odds ratio [OR] 1.76, 95% confidence interval [CI] 1.12-2.77). A genotype combination of 10Leu/Leu x 25Arg/Arg x -509C/C was also associated with susceptibility to gastric ulcer disease (OR 1.81, P = 0.01). In addition, the frequency of a combination of genotypes 10Pro/Pro x 25Arg/Pro x -509C/T was statistically lower in patients with duodenal ulcer than in controls (OR 0.42, P = 0.05). A significant difference (P = 0.04) in the distribution of rare haplotypes of the TGF-beta1 gene between patients with duodenal ulcer and healthy controls has been found. Polymorphism Leu10Pro was in positive linkage disequilibrium with C-509T polymorphism (coefficient D = 0.191; P < 0.0001). These findings indicate that the Leu10Pro and C-509T polymorphisms may be involved in the modulation of expression of the TGF-beta1 gene, and therefore a predisposition to peptic ulcer disease could be linked to particular alleles of this gene. In particular, a possible role of TGF-beta1 in the pathogenesis of gastric ulcer disease is discussed.

  12. IFN-beta1b augments glucocorticoid-induced suppression of tumor necrosis factor-alpha production by increasing the number of glucocorticoid receptors on a human monocytic cell line.

    PubMed

    Uitdehaag, B M; Hoekstra, K; Koper, J W; Polman, C H; Dijkstra, C D

    2001-03-01

    We studied the effect of recombinant interferon-beta1b (IFN-beta1b) on the sensitivity to glucocorticoids (GC) and on the number of GC receptors (GCR) in the human monocytic cell line THP-1. We found that IFN-beta1b augments the suppressive effect that dexamethasone has on the stimulated production of tumor necrosis factor-alpha (TNF-alpha), most likely related to the increased number of GCR observed after exposure to IFN-beta1b. This provides a possible clue to the mechanism of action of IFN-beta in multiple sclerosis.

  13. Computerized tomography platform using beta rays

    NASA Astrophysics Data System (ADS)

    Paetkau, Owen; Parsons, Zachary; Paetkau, Mark

    2017-12-01

    A computerized tomography (CT) system using a 0.1 μCi Sr-90 beta source, Geiger counter, and low density foam samples was developed. A simple algorithm was used to construct images from the data collected with the beta CT scanner. The beta CT system is analogous to X-ray CT as both types of radiation are sensitive to density variations. This system offers a platform for learning opportunities in an undergraduate laboratory, covering topics such as image reconstruction algorithms, radiation exposure, and the energy dependence of absorption.

  14. Chromosome mapping of the human arrestin (SAG), {beta}-arrestin 2 (ARRB2), and {beta}-adrenergic receptor kinase 2 (ADRBK2) genes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Calabrese, G.; Sallese, M.; Stornaiuolo, A.

    1994-09-01

    Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, {beta}-arrestin. Both {beta}ARK and {beta}-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, {beta}ARK1, {beta}ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/{beta}-arrestin gene family includes arrestin (also known as S-antigen), {beta}-arrestin 1, and {beta}-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), {beta}arrestin 2 (ARRB2), and {beta}ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment ofmore » the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for {beta}-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for {beta}ARK2 (ADRBK2) to chromosome 22q11. 17 refs., 1 fig.« less

  15. TGF{beta} induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ebi, Masahide; Kataoka, Hiromi, E-mail: hkataoka@med.nagoya-cu.ac.jp; Shimura, Takaya

    2010-11-19

    Research highlights: {yields} TGF{beta} induces EGFR transactivation through proHB-EGF shedding by activated ADAM members in gastric cancer cells. {yields} TGF{beta} induces nuclear translocation of HB-EGF-CTF cleaved by ADAM members. {yields} TGF{beta} enhances cell growth by EGFR transactivation and HB-EGF-CTF nuclear translocation and ADAM inhibitors block these effects. {yields} Silencing of ADAM17 also blocks EGFR transactivation, HB-EGF-CTF nuclear translocation and cancer cell growth by TGF{beta}. {yields} ADAM17 may play a crucial role in this TGF{beta}-HB-EGF signal transduction. -- Abstract: Background and aims: Transforming growth factor-beta (TGF{beta}) is known to potently inhibit cell growth. Loss of responsiveness to TGF{beta} inhibition on cellmore » growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGF{beta} and HB-EGF signal transduction via ADAM activation. Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGF{beta}. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGF{beta} was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGF{beta} was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown. Result: TGF{beta}-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGF{beta} induced shedding of proHB-EGF allowing HB-EGF-CTF to

  16. Development of a new family of conformationally restricted peptides as potent nucleators of beta-turns. Design, synthesis, structure, and biological evaluation of a beta-lactam peptide analogue of melanostatin.

    PubMed

    Palomo, Claudio; Aizpurua, Jesus M; Benito, Ana; Miranda, José Ignacio; Fratila, Raluca M; Matute, Carlos; Domercq, Maria; Gago, Federico; Martin-Santamaria, Sonsoles; Linden, Anthony

    2003-12-31

    Novel enantiopure (i)-(beta-lactam)-(Gly)-(i+3) peptide models, defined by the presence of a central alpha-alkyl-alpha-amino-beta-lactam ring placed as the (i+1) residue, have been synthesized in a totally stereocontrolled way by alpha-alkylation of suitable N-[bis(trimethylsilyl)methyl]-beta-lactams. The structural properties of these beta-lactam pseudopeptides have been studied by X-ray crystallography, Molecular Dynamics simulation, and NOESY-restrained NMR simulated annealing techniques, showing a strong tendency to form stable type II or type II' beta-turns either in the solid state or in highly coordinating DMSO solutions. Tetrapeptide models containing syn- or anti-alpha,beta-dialkyl-alpha-amino-beta-lactam rings have also been synthesized and their conformations analyzed, revealing that alpha-alkyl substitution is essential for beta-turn stabilization. A beta-lactam analogue of melanostatin (PLG amide) has also been prepared, characterized as a type-II beta-turn in DMSO-d6 solution, and tested by competitive binding assay as a dopaminergic D2 modulator in rat neuron cultured cells, displaying moderate agonist activity in the micromolar concentration range. On the basis of these results, a novel peptidomimetic design concept, based on the separation of constraint and recognition elements, is proposed.

  17. Can transforming growth factor-beta1 and retinoids modify the activity of estradiol and antiestrogens in MCF-7 breast cancer cells?.

    PubMed

    Czeczuga-Semeniuk, Ewa; Anchim, Tomasz; Dziecioł, Janusz; Dabrowska, Milena; Wołczyński, Sławomir

    2004-01-01

    Retinoic acid and transforming growth factor-beta (TGF-beta) affect differentiation, proliferation and carcinogenesis of epithelial cells. The effect of both compounds on the proliferation of cells of the hormone sensitive human breast cancer cell line (ER+) MCF-7 was assessed in the presence of estradiol and tamoxifen. The assay was based on [3H]thymidine incorporation and the proliferative activity of PCNA- and Ki 67-positive cells. The apoptotic index and expression of the Bcl-2 and p53 antigens in MCF-7 cells were also determined. Exogenous TGF-beta1 added to the cell culture showed antiproliferative activity within the concentration range of 0.003-30 ng/ml. Irrespective of TGF-beta1 concentrations, a marked reduction in the stimulatory action of estradiol (10(-9) and 10(-8) M) was observed whereas in combination with tamoxifen (10(-7) and 10(-6) M) only 30 ng/ml TGF-beta1 caused a statistically significant reduction to approximately 30% of the proliferative cells. In further experiments we examined the effect of exposure of breast cancer cells to retinoids in combination with TGF-beta1. The incorporation of [3H]thymidine into MCF-7 cells was inhibited to 52 +/- 19% (control =100%) by 3 ng/ml TGF-beta1, and this dose was used throughout. It was found that addition of TGF-beta1 and isotretinoin to the culture did not decrease proliferation, while TGF-beta1 and tretinoin at low concentrations (3 x 10(-8) and 3 x 10(-7) M) reduced the percentage of proliferating cells by approximately 30% (67+/-8% and 67+/-5%, P<0.05 compared to values in the tretinoin group). Both retinoids also led to a statistically significant decrease in the stimulatory effect of 10(-9) M estradiol, attenuated by TGF-beta1. In addition, the retinoids in combination with TGF-beta1 and tamoxifen (10(-6) M) caused a further reduction in the percentage of proliferating cells. Immunocytochemical analysis showed that all the examined compounds gave a statistically significant reduction in the

  18. Tyrosine residues 654 and 670 in {beta}-cat enin are crucial in regulation of Met-{beta}-catenin interactions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zeng, Gang; Apte, Udayan; Micsenyi, Amanda

    2006-11-01

    {beta}-catenin, a key component of the canonical Wnt pathway, is also regulated by tyrosine phosphorylation that regulates its association to E-cadherin. Previously, we reported its association with the hepatocyte growth factor (HGF) receptor Met at the membrane. HGF induced Met-{beta}-catenin dissociation and nuclear translocation of {beta}-catenin, which was tyrosine-phosphorylation-dependent. Here, we further investigate the Met-{beta}-catenin interaction by selectively mutating several tyrosine residues, alone or in combination, in {beta}-catenin. The mutants were subcloned into FLAG-CMV vector and stably transfected into rat hepatoma cells, which were treated with HGF. All single or double-mutant-transfected cells continued to show HGF-induced nuclear translocation of FLAG-{beta}-cateninmore » except the mutations affecting 654 and 670 simultaneously (Y654/670F), which coincided with the lack of formation of {beta}-catenin-TCF complex and DNA synthesis, in response to the HGF treatment. In addition, the Y654/670F-transfected cells also showed no phosphorylation of {beta}-catenin or dissociation from Met in response to HGF. Thus, intact 654 and 670 tyrosine residues in {beta}-catenin are crucial in HGF-mediated {beta}-catenin translocation, activation and mitogenesis.« less

  19. Factors of transforming growth factor beta signalling are co-regulated in human hepatocellular carcinoma.

    PubMed

    Longerich, Thomas; Breuhahn, Kai; Odenthal, Margarete; Petmecky, Katharina; Schirmacher, Peter

    2004-12-01

    Transforming growth factor beta (TGFbeta) is a central mitoinhibitory factor for epithelial cells, and alterations of TGFbeta signalling have been demonstrated in many different human cancers. We have analysed human hepatocellular carcinomas (HCCs) for potential pro-tumourigenic alterations in regard to expression of Smad4 and mutations and expression changes of the pro-oncogenic transcriptional co-repressors Ski and SnoN, as well as mRNA levels of matrix metalloproteinase-2 (MMP2), which is transcriptionally regulated by TGFbeta. Smad4 mRNA was detected in all HCCs; while, using immunohistology, loss of Smad4 expression was found in 10% of HCCs. Neither mutations in the transformation-relevant sequences nor significant pro-tumourigenic expression changes of the Ski and SnoN genes were detected. In HCC cell lines, expression of both genes was regulated, potentially involving phosphorylation. Ski showed a distinct nuclear speckled pattern, indicating recruitment to active transcription complexes. MMP2 mRNA levels were increased in 19% of HCCs, whereas MMP2 mRNA was not detectable in HCC cell lines, suggesting that MMP2 was derived only from tumour stroma cells. Transcript levels of Smad4, Ski, SnoN and MMP2 correlated well. These data argue against a significant role of Ski and SnoN in human hepatocarcinogenesis and suggest that, in the majority of HCCs, the analysed factors are co-regulated by an upstream mechanism, potentially by TGFbeta itself.

  20. Deficiency of bone marrow beta3-integrin enhances non-functional neovascularization.

    PubMed

    Watson, Alan R; Pitchford, Simon C; Reynolds, Louise E; Direkze, Natalie; Brittan, Mairi; Alison, Malcolm R; Rankin, Sara; Wright, Nicholas A; Hodivala-Dilke, Kairbaan M

    2010-03-01

    beta3-Integrin is a cell surface adhesion and signalling molecule important in the regulation of tumour angiogenesis. Mice with a global deficiency in beta3-integrin show increased pathological angiogenesis, most likely due to increased vascular endothelial growth factor receptor 2 expression on beta3-null endothelial cells. Here we transplanted beta3-null bone marrow (BM) into wild-type (WT) mice to dissect the role of BM beta3-integrin deficiency in pathological angiogenesis. Mice transplanted with beta3-null bone marrow show significantly enhanced angiogenesis in subcutaneous B16F0 melanoma and Lewis lung carcinoma (LLC) cell models and in B16F0 melanoma lung metastasis when compared with tumours grown in mice transplanted with WT bone marrow. The effect of bone marrow beta3-integrin deficiency was also assessed in the RIPTAg mouse model of pancreatic tumour growth. Again, angiogenesis in mice lacking BM beta3-integrin was enhanced. However, tumour weight between the groups was not significantly altered, suggesting that the enhanced blood vessel density in the mice transplanted with beta3-null bone marrow was not functional. Indeed, we demonstrate that in mice transplanted with beta3-null bone marrow a significant proportion of tumour blood vessels are non-functional when compared with tumour blood vessels in WT-transplanted controls. Furthermore, beta3-null-transplanted mice showed an increased angiogenic response to VEGF in vivo when compared with WT-transplanted animals. BM beta3-integrin deficiency affects the mobilization of progenitor cells to the peripheral circulation. We show that VEGF-induced mobilization of endothelial progenitor cells is enhanced in mice transplanted with beta3-null bone marrow when compared with WT-transplanted controls, suggesting a possible mechanism underlying the increased blood vessel density seen in beta3-null-transplanted mice. In conclusion, although BM beta3-integrin is not required for pathological angiogenesis, our

  1. Transforming Growth Factor Beta-2 Mutations in Barlow's Disease and Aortic Dilatation.

    PubMed

    Disha, Kushtrim; Schulz, Solveig; Kuntze, Thomas; Girdauskas, Evaldas

    2017-07-01

    We report on a patient operated on for degenerative myxomatous mitral and tricuspid valve disease (Barlow's disease) and aortic root dilatation. A valve repair operation and the postoperative course were uneventful. Multigenerational genetic analyses revealed two different mutations in the transforming growth factor beta-2 gene in the same patient. The two mutations in different exons were inherited from both parents each. None of the parents presented with either valve dysfunction or aortic root dilatation. This rare case illustrates potentially common genetic and signaling pathways of concomitant myxomatous valve disease and aortic root dilatation. Copyright © 2017 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

  2. Small C-terminal domain phosphatases dephosphorylate the regulatory linker regions of Smad2 and Smad3 to enhance transforming growth factor-beta signaling.

    PubMed

    Wrighton, Katharine H; Willis, Danielle; Long, Jianyin; Liu, Fang; Lin, Xia; Feng, Xin-Hua

    2006-12-15

    Transforming growth factor-beta (TGF-beta) controls a diverse set of cellular processes, and its canonical signaling is mediated via TGF-beta-induced phosphorylation of receptor-activated Smads (2 and 3) at the C-terminal SXS motif. We recently discovered that PPM1A can dephosphorylate Smad2/3 at the C-terminal SXS motif, implicating a critical role for phosphatases in regulating TGF-beta signaling. Smad2/3 activity is also regulated by phosphorylation in the linker region (and N terminus) by a variety of intracellular kinases, making it a critical platform for cross-talk between TGF-beta and other signaling pathways. Using a functional genomic approach, we identified the small C-terminal domain phosphatase 1 (SCP1) as a specific phosphatase for Smad2/3 dephosphorylation in the linker and N terminus. A catalytically inactive SCP1 mutant (dnSCP1) had no effect on Smad2/3 phosphorylation in vitro or in vivo. Of the other FCP/SCP family members SCP2 and SCP3, but not FCP1, could also dephosphorylate Smad2/3 in the linker/N terminus. Depletion of SCP1/2/3 enhanced Smad2/3 linker phosphorylation. SCP1 increased TGF-beta-induced transcriptional activity in agreement with the idea that phosphorylation in the Smad2/3 linker must be removed for a full transcriptional response. SCP1 overexpression also counteracts the inhibitory effect of epidermal growth factor on TGF-beta-induced p15 expression. Taken together, this work identifies the first example of a Smad2/3 linker phosphatase(s) and reveals an important new substrate for SCPs.

  3. Effect of doxycycline on transforming growth factor-beta-1-induced matrix metalloproteinase 2 expression, migration, and collagen contraction in nasal polyp-derived fibroblasts.

    PubMed

    Shin, Jae-Min; Park, Joo-Hoo; Kang, Byungjin; Lee, Seoung-Ae; Park, Il-Ho; Lee, Heung-Man

    2016-11-01

    It is well known that doxycycline has antibacterial and anti-inflammatory effects. In this study, we aimed to investigate the effects of doxycycline on the transforming growth factor (TGF) beta 1-induced matrix metalloproteinase (MMP) 2 expression, migration, and collagen contraction, and to determine its molecular mechanism on nasal polyp-derived fibroblasts (NPDF). NPDFs were isolated from the nasal polyps of six patients. Doxycycline was used to pretreat TGF-beta-1-induced NPDFs and ex vivo organ cultures of nasal polyps. Cytotoxicity was evaluated by using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. Smad2/3 is one of the major transcription factors of TGF-beta signaling. The expression levels of MMP2 and Smad2/3 were measured by using Western blotting, reverse transcription-polymerase chain reaction, and immunofluorescence staining. The enzymic activity of MMP2 was analyzed by using gelatin zymography. Fibroblast migration was evaluated by using transwell migration assays. Contractile activity was measured by a collagen gel contraction assay. The expression level of MMP2 in nasal polyp tissues increased in comparison with inferior turbinate tissues. TGF-beta-1-induced NPDFs were not affected by doxycycline (0-40 μg/mL). The expression levels of MMP2 and activation of Smad2/3 in TGF-beta-1-induced NPDFs and in organ cultures of nasal polyps were significantly downregulated with doxycycline pretreatment. Doxycycline also reduced TGF-beta-1-induced fibroblast migration and collagen contraction in NPDFs. Doxycycline inhibited TGF-beta-1-induced MMP2 expression, migration, and collagen contraction via the Smad2/3 signal pathways in NPDFs.

  4. [Study of beta-turns in globular proteins].

    PubMed

    Amirova, S R; Milchevskiĭ, Iu V; Filatov, I V; Esipova, N G; Tumanian, V G

    2005-01-01

    The formation of beta-turns in globular proteins has been studied by the method of molecular mechanics. Statistical method of discriminant analysis was applied to calculate energy components and sequences of oligopeptide segments, and after this prediction of I type beta-turns has been drawn. The accuracy of true positive prediction is 65%. Components of conformational energy considerably affecting beta-turn formation were delineated. There are torsional energy, energy of hydrogen bonds, and van der Waals energy.

  5. beta subunits influence the biophysical and pharmacological differences between P- and Q-type calcium currents expressed in a mammalian cell line.

    PubMed

    Moreno, H; Rudy, B; Llinás, R

    1997-12-09

    Human epithelial kidney cells (HEK) were prepared to coexpress alpha1A, alpha2delta with different beta calcium channel subunits and green fluorescence protein. To compare the calcium currents observed in these cells with the native neuronal currents, electrophysiological and pharmacological tools were used conjointly. Whole-cell current recordings of human epithelial kidney alpha1A-transfected cells showed small inactivating currents in 80 mM Ba2+ that were relatively insensitive to calcium blockers. Coexpression of alpha1A, betaIb, and alpha2delta produced a robust inactivating current detected in 10 mM Ba2+, reversibly blockable with low concentration of omega-agatoxin IVA (omega-Aga IVA) or synthetic funnel-web spider toxin (sFTX). Barium currents were also supported by alpha1A, beta2a, alpha2delta subunits, which demonstrated the slowest inactivation and were relatively insensitive to omega-Aga IVA and sFTX. Coexpression of beta3 with the same combination as above produced inactivating currents also insensitive to low concentration of omega-Aga IVA and sFTX. These data indicate that the combination alpha1A, betaIb, alpha2delta best resembles P-type channels given the rate of inactivation and the high sensitivity to omega-Aga IVA and sFTX. More importantly, the specificity of the channel blocker is highly influenced by the beta subunit associated with the alpha1A subunit.

  6. Selective inhibition of sheep kidney 11 beta-hydroxysteroid dehydrogenase isoform 2 activity by 5 alpha-reduced (but not 5 beta) derivatives of adrenocorticosteroids.

    PubMed

    Latif, S A; Sheff, M F; Ribeiro, C E; Morris, D J

    1997-02-01

    We have previously reported that 5 alpha and 5 beta pathways of steroid metabolism are controlled in vivo by dietary Na+ and glycyrrhetinic acid, see Gorsline et al. 1988; Latif et al. 1990. The present investigations provide evidence supporting the suggestion that endogenous substances may regulate the glucocorticoid inactivating isoenzymes, 11 beta-HSD (hydroxysteroid dehydrogenase) 1 (liver) and 11 beta-HSD2 (kidney). The activity of 11 beta-HSD is impaired in essential hypertension, following licorice ingestion, and in patients with apparent mineralocorticoid excess where 11 beta-HSD2 is particularly affected. In all three conditions, excretion of the less common 5 alpha metabolites is elevated in urine. We now report on the differential abilities of a series of Ring A reduced (5 alpha and 5 beta) adrenocorticosteroid and progesterone metabolites to inhibit these isoenzymes. Using liver microsomes with NADP+ as co-factor (11 beta-HSD1), and sheep kidney microsomes with NAD+ as co-factor (11 beta-HSD2), we have systematically investigated the abilities of a number of adrenocorticosteroids and their derivatives to inhibit the individual isoforms of 11 beta-HSD. A striking feature is the differential sensitivity of the two isoenzymes to inhibition by 5 alpha and 5 beta derivatives. 11 beta-HSD1 is inhibited by both 5 alpha and certain 5 beta derivatives. 11 beta-HSD-2 was selectively inhibited only by 5 alpha derivatives: 5 beta derivatives were without inhibitory activity toward this isoform of 11 beta-HSD. These results indicate the importance of the structural conformation of the A and B Rings in conferring specific inhibitory properties on these compounds. In addition, we discuss the effects of additions or substitutions of other functional groups on the inhibitory potency of these steroid molecules against 11 beta-HSD1 and 11 beta-HSD2.

  7. The H,K-ATPase beta-subunit can act as a surrogate for the beta-subunit of Na,K-pumps.

    PubMed

    Horisberger, J D; Jaunin, P; Reuben, M A; Lasater, L S; Chow, D C; Forte, J G; Sachs, G; Rossier, B C; Geering, K

    1991-10-15

    Na,K-ATPase and H,K-ATPase are the only members of the P-type ATPases in which a glycosylated beta-subunit is part of the purified active enzyme. In this study, we have followed the synthesis and the posttranslational processing of the beta-subunit of H,K-ATPase (beta HK) in Xenopus oocytes injected with beta HK cRNA and have tested whether it can act as a surrogate for the beta-subunit of Na,K-ATPase (beta NaK) to support the functional expression of Na,K-pumps. In Xenopus oocytes, beta HK is processed from an Endo H-sensitive 51-kDa coreglycosylated form to an Endo H-resistant 71-kDa fully glycosylated form. Similar to beta NaK, beta HK can stabilize and increase the trypsin resistance of alpha-subunits of Na,K-ATPase (alpha NaK). Finally, expression of beta HK together with alpha NaK leads to an increased number of ouabain binding sites at the plasma membrane accompanied by an increased Rb+ uptake and Na,K-pump current. Our data suggest that beta HK, similar to beta NaK, can assemble to alpha NaK, support the structural maturation and the intracellular transport of catalytic alpha NaK, and ultimately form active alpha NaK-beta HK complexes with Na,K-pump transport properties.

  8. High power beta electron device - Beyond betavoltaics

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ayers, William M.; Gentile, Charles A.

    Developing watt level power sources with beta emitting radioisotopes has been limited by the inability to utilize high energy (> 100 KeV) beta emitters at high radioisotope loadings without damaging the energy conversion materials. A new type of beta electron power source is described that removes those restrictions. This approach contains the radioisotope in a beta transparent titanium tube and confines beta electrons emitted through the tube wall to spiral trajectories around the tube with an axial magnetic field. The confined beta electrons dissipate energy though multiple interactions with surrounding excimer precursor gas atoms to efficiently generate photons. Photovoltaic cellsmore » convert the photons to electrical power. Since the beta electrons dissipate energy in the excimer precursor gas, the device can be loaded with more than 10 13 Bq of radioisotope to generate 100 milliwatt to watt levels of electrical power without damaging the device materials or degrading its performance. Furthermore, the power source can use a variety of beta radioisotopes and scales by stacking the devices.« less

  9. High power beta electron device - Beyond betavoltaics

    DOE PAGES

    Ayers, William M.; Gentile, Charles A.

    2017-11-10

    Developing watt level power sources with beta emitting radioisotopes has been limited by the inability to utilize high energy (> 100 KeV) beta emitters at high radioisotope loadings without damaging the energy conversion materials. A new type of beta electron power source is described that removes those restrictions. This approach contains the radioisotope in a beta transparent titanium tube and confines beta electrons emitted through the tube wall to spiral trajectories around the tube with an axial magnetic field. The confined beta electrons dissipate energy though multiple interactions with surrounding excimer precursor gas atoms to efficiently generate photons. Photovoltaic cellsmore » convert the photons to electrical power. Since the beta electrons dissipate energy in the excimer precursor gas, the device can be loaded with more than 10 13 Bq of radioisotope to generate 100 milliwatt to watt levels of electrical power without damaging the device materials or degrading its performance. Furthermore, the power source can use a variety of beta radioisotopes and scales by stacking the devices.« less

  10. High power beta electron device - Beyond betavoltaics.

    PubMed

    Ayers, William M; Gentile, Charles A

    2018-01-01

    Developing watt level power sources with beta emitting radioisotopes has been limited by the inability to utilize high energy (> 100KeV) beta emitters at high radioisotope loadings without damaging the energy conversion materials. A new type of beta electron power source is described that removes those restrictions. The approach contains the radioisotope in a beta transparent titanium tube and confines beta electrons emitted through the tube wall to spiral trajectories around the tube with an axial magnetic field. The confined beta electrons dissipate energy though multiple interactions with surrounding excimer precursor gas atoms to efficiently generate photons. Photovoltaic cells convert the photons to electrical power. Since the beta electrons dissipate energy in the excimer precursor gas, the device can be loaded with more than 10 13 Bq of radioisotope to generate 100 milliwatt to watt levels of electrical power without damaging the device materials or degrading its performance. The power source can use a variety of beta radioisotopes and scales by stacking the devices. Copyright © 2017. Published by Elsevier Ltd.

  11. Increasing protein stability by improving beta-turns.

    PubMed

    Fu, Hailong; Grimsley, Gerald R; Razvi, Abbas; Scholtz, J Martin; Pace, C Nick

    2009-11-15

    Our goal was to gain a better understanding of how protein stability can be increased by improving beta-turns. We studied 22 beta-turns in nine proteins with 66-370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some beta-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein, Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase alpha-subunit, and Maltose binding protein. Of the 15 single proline mutations, 11 increased stability (Average = 0.8 +/- 0.3; Range = 0.3-1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. On the basis of this and our previous work, we conclude that proteins can generally be stabilized by replacing nonproline residues with proline residues at the i + 1 position of Type I and II beta-turns and at the i position in Type II beta-turns. Other turn positions can sometimes be used if the phi angle is near -60 degrees for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in beta-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in beta-turns that could be replaced by Gly to increase protein stability. Improving beta-turns by substituting Pro residues is a generally useful way of increasing protein stability. 2009 Wiley-Liss, Inc.

  12. Milk Intolerance, Beta-Casein and Lactose.

    PubMed

    Pal, Sebely; Woodford, Keith; Kukuljan, Sonja; Ho, Suleen

    2015-08-31

    True lactose intolerance (symptoms stemming from lactose malabsorption) is less common than is widely perceived, and should be viewed as just one potential cause of cows' milk intolerance. There is increasing evidence that A1 beta-casein, a protein produced by a major proportion of European-origin cattle but not purebred Asian or African cattle, is also associated with cows' milk intolerance. In humans, digestion of bovine A1 beta-casein, but not the alternative A2 beta-casein, releases beta-casomorphin-7, which activates μ-opioid receptors expressed throughout the gastrointestinal tract and body. Studies in rodents show that milk containing A1 beta-casein significantly increases gastrointestinal transit time, production of dipeptidyl peptidase-4 and the inflammatory marker myeloperoxidase compared with milk containing A2 beta-casein. Co-administration of the opioid receptor antagonist naloxone blocks the myeloperoxidase and gastrointestinal motility effects, indicating opioid signaling pathway involvement. In humans, a double-blind, randomized cross-over study showed that participants consuming A1 beta-casein type cows' milk experienced statistically significantly higher Bristol stool values compared with those receiving A2 beta-casein milk. Additionally, a statistically significant positive association between abdominal pain and stool consistency was observed when participants consumed the A1 but not the A2 diet. Further studies of the role of A1 beta-casein in milk intolerance are needed.

  13. Milk Intolerance, Beta-Casein and Lactose

    PubMed Central

    Pal, Sebely; Woodford, Keith; Kukuljan, Sonja; Ho, Suleen

    2015-01-01

    True lactose intolerance (symptoms stemming from lactose malabsorption) is less common than is widely perceived, and should be viewed as just one potential cause of cows’ milk intolerance. There is increasing evidence that A1 beta-casein, a protein produced by a major proportion of European-origin cattle but not purebred Asian or African cattle, is also associated with cows’ milk intolerance. In humans, digestion of bovine A1 beta-casein, but not the alternative A2 beta-casein, releases beta-casomorphin-7, which activates μ-opioid receptors expressed throughout the gastrointestinal tract and body. Studies in rodents show that milk containing A1 beta-casein significantly increases gastrointestinal transit time, production of dipeptidyl peptidase-4 and the inflammatory marker myeloperoxidase compared with milk containing A2 beta-casein. Co-administration of the opioid receptor antagonist naloxone blocks the myeloperoxidase and gastrointestinal motility effects, indicating opioid signaling pathway involvement. In humans, a double-blind, randomized cross-over study showed that participants consuming A1 beta-casein type cows’ milk experienced statistically significantly higher Bristol stool values compared with those receiving A2 beta-casein milk. Additionally, a statistically significant positive association between abdominal pain and stool consistency was observed when participants consumed the A1 but not the A2 diet. Further studies of the role of A1 beta-casein in milk intolerance are needed. PMID:26404362

  14. An elevated serum beta-2-microglobulin level is an adverse prognostic factor for overall survival in patients with early-stage Hodgkin disease.

    PubMed

    Chronowski, Gregory M; Wilder, Richard B; Tucker, Susan L; Ha, Chul S; Sarris, Andreas H; Hagemeister, Fredrick B; Barista, Ibrahim; Hess, Mark A; Cabanillas, Fernando; Cox, James D

    2002-12-15

    The relative importance of prognostic factors in patients with early-stage Hodgkin disease remains controversial. The purpose of this study was to evaluate prognostic factors among patients who received chemotherapy before radiotherapy. From 1987 to 1995, 217 consecutive patients ranging in age from 16 to 88 years (median, 28 years) with Ann Arbor Stage I (n = 55) or II (n = 162) Hodgkin disease underwent chemotherapy before radiotherapy at a single center. Most were treated on prospective studies. Patients received a median of three cycles of induction chemotherapy. Mitoxantrone, vincristine, vinblastine, and prednisone (NOVP), doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD), mechlorethamine, vincristine, procarbazine, and prednisone (MOPP), cyclophosphamide, vinblastine, procarbazine, prednisone, doxorubicin, bleomycin, dacarbazine, and CCNU (CVPP/ABDIC), or other chemotherapeutic regimens were given to 160, 18, 15, 10, and 14 patients, respectively. The median radiotherapy dose was 40 Gy. Serum beta-2-microglobulin (beta-2M) levels ranged from 1.0 to 4.1 mg/L (median, 1.7 mg/L; upper limit of normal, 2.0 mg/L). We studied univariate and multivariate associations between survival and the following clinical features: serum beta-2M level above 1.25 times the upper limit of normal (n = 12), male gender (n = 113), hypoalbuminemia (n = 11), and bulky mediastinal disease (n = 94). Follow-up of surviving patients ranged from 0.9 to 13.4 years (median, 6.6 years) and 92% were observed for 3.0 or more years. Nineteen patients have died. Only elevation of the serum beta-2M level was an independent adverse prognostic factor for overall survival (P = 0.0009). The prognostic significance of a simple, widely available, and inexpensive blood test, beta-2M, has not been studied routinely in patients with Hodgkin disease and should be tested prospectively in large, cooperative group trials. Copyright 2002 American Cancer Society.DOI 10.1002/cncr.10998

  15. TGF-beta3 is expressed in taste buds and inhibits proliferation of primary cultured taste epithelial cells.

    PubMed

    Nakamura, Shin-ichi; Kawai, Takayuki; Kamakura, Takashi; Ookura, Tetsuya

    2010-01-01

    Transforming growth factor-betas (TGF-betas), expressed in various tissues, play important roles in embryonic development and adult tissue homeostasis through their effects on cell proliferation, cell differentiation, cell death, and cell motility. However, expression of TGF-beta signaling components and their biological effect on taste epithelia has not been elucidated. We performed expression analysis of TGF-beta signaling components in taste epithelia and found that the TGF-beta3 mRNA was specifically expressed in taste buds. Type II TGF-betas receptor (TbetaR-II) mRNA was specifically expressed in the tongue epithelia including the taste epithelia. To elucidate the biological function of TGF-beta3 in taste epithelia, we performed proliferation assay with primary cultured taste epithelial cells. In the presence of TGF-beta3, percentage of BrdU-labeled cells decreased significantly, suggesting that the TGF-beta3 inhibited the proliferation of cultured taste epithelial cells through inhibiting cell-cycle entry into S phase. By quantitative reverse transcription-polymerase chain reaction assay, we found that the TGF-beta3 resulted in an increased level of expression of p15Ink4b and p21Cip1, suggesting that the TGF-beta3 inhibited the taste epithelial cell proliferation through inhibiting G1cyclin-Cdk complexes. Taken together, these results suggested that the TGF-beta3 may regulate taste epithelial cell homeostasis through controlling cell proliferation.

  16. Targeting expression of a transforming growth factor beta 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation.

    PubMed Central

    Jhappan, C; Geiser, A G; Kordon, E C; Bagheri, D; Hennighausen, L; Roberts, A B; Smith, G H; Merlino, G

    1993-01-01

    Transforming growth factor-beta 1 (TGF-beta 1) possesses highly potent, diverse and often opposing cell-specific activities, and has been implicated in the regulation of a variety of physiologic and developmental processes. To determine the effects of in vivo overexpression of TGF-beta 1 on mammary gland function, transgenic mice were generated harboring a fusion gene consisting of the porcine TGF-beta 1 cDNA placed under the control of regulatory elements of the pregnancy-responsive mouse whey-acidic protein (WAP) gene. Females from two of four transgenic lines were unable to lactate due to inhibition of the formation of lobuloalveolar structures and suppression of production of endogenous milk protein. In contrast, ductal development of the mammary glands was not overtly impaired. There was a complete concordance in transgenic mice between manifestation of the lactation-deficient phenotype and expression of RNA from the WAP/TGF-beta 1 transgene, which was present at low levels in the virgin gland, but was greatly induced at mid-pregnancy. TGF-beta 1 was localized to numerous alveoli and to the periductal extracellular matrix in the mammary gland of transgenic females late in pregnancy by immunohistochemical analysis. Glands reconstituted from cultured transgenic mammary epithelial cells duplicated the inhibition of lobuloalveolar development observed in situ in the mammary glands of pregnant transgenic mice. Results from this transgenic model strongly support the hypothesis that TGF-beta 1 plays an important in vivo role in regulating the development and function of the mammary gland. Images PMID:8491177

  17. Mesenchymal stem cells derived from human exocrine pancreas express transcription factors implicated in beta-cell development.

    PubMed

    Baertschiger, Reto M; Bosco, Domenico; Morel, Philippe; Serre-Beinier, Veronique; Berney, Thierry; Buhler, Leo H; Gonelle-Gispert, Carmen

    2008-07-01

    Transplantation of in vitro generated islets or insulin-producing cells represents an attractive option to overcome organ shortage. The aim of this study was to isolate, expand, and characterize cells from human exocrine pancreas and analyze their potential to differentiate into beta cells. Fibroblast-like cells growing out of human exocrine tissue were characterized by flow cytometry and by their capacity to differentiate into mesenchymal cell lineages. During cell expansion and after differentiation toward beta cells, expression of transcription factors of endocrine pancreatic progenitors was analyzed by reverse transcription polymerase chain reaction. Cells emerged from 14/18 human pancreatic exocrine fractions and were expanded up to 40 population doublings. These cells displayed surface antigens similar to mesenchymal stem cells from bone marrow. A culture of these cells in adipogenic and chondrogenic differentiation media allowed differentiation into adipocyte- and chondrocyte-like cells. During expansion, cells expressed transcription factors implicated in islet development such as Isl1, Nkx2.2, Nkx6.1, nestin, Ngn3, Pdx1, and NeuroD. Activin A and hepatocyte growth factor induced an expression of insulin, glucagon, and glucokinase. Proliferating cells with characteristics of mesenchymal stem cells and endocrine progenitors were isolated from exocrine tissue. Under specific conditions, these cells expressed little insulin. Human pancreatic exocrine tissue might thus be a source of endocrine cell progenitors.

  18. Production and Precipitation Hardening of Beta-Type Ti-35Nb-10Cu Alloy Foam for Implant Applications

    NASA Astrophysics Data System (ADS)

    Mutlu, Ilven; Yeniyol, Sinem; Oktay, Enver

    2016-04-01

    In this study, beta-type Ti-35Nb-10Cu alloy foams were produced by powder metallurgy method for dental implant applications. 35% Nb was added to stabilize the beta-Ti phase with low Young's modulus. Cu addition enhanced sinterability and gave precipitation hardening capacity to the alloy. Sintered specimens were precipitation hardened in order to enhance the mechanical properties. Electrochemical corrosion behavior of the specimens was examined by electrochemical impedance spectroscopy in artificial saliva. Electrochemical impedance spectroscopy results indicated that the oxide film on the surface of foam is a bi-layer structure consisting of outer porous layer and inner barrier layer. Impedance values of barrier layer were higher than porous layer. Corrosion resistance of specimens decreased at high fluoride concentrations and at low pH of artificial saliva. Corrosion resistance of alloys was slightly decreased with aging. Mechanical properties, microstructure, and surface roughness of the specimens were also examined.

  19. A Multifunctional Bread Rich in Beta Glucans and Low in Starch Improves Metabolic Control in Type 2 Diabetes: A Controlled Trial

    PubMed Central

    Tessari, Paolo; Lante, Anna

    2017-01-01

    Design: Functional foods may be useful for people with diabetes. The soluble fibers beta glucans can modify starch digestion and improve postprandial glucose response. We analyzed the metabolic effects of a specifically designed ‘functional’ bread, low in starch, rich in fibers (7 g/100 g), with a beta glucan/starch ratio of (7.6:100, g/g), in people with type 2 diabetes mellitus. Methods: Clinical and metabolic data from two groups of age-, sex- and glycated hemoglobin-matched diabetic subjects, taking either the functional bread or regular white bread, over a roughly six-month observation period, were retrieved. Results: Bread intake did not change during the trial. The functional bread reduced glycated hemoglobin by ~0.5% (absolute units) vs. pre-treatment values (p = 0.028), and by ~0.6% vs. the control group (p = 0.027). Post-prandial and mean plasma glucose was decreased in the treatment group too. Body weight, blood pressure and plasma lipids did not change. The acceptance of the functional bread was good in the majority of subjects, except for taste. Conclusions: A starch-restricted, fiber-rich functional bread, with an increased beta glucan/starch ratio, improved long term metabolic control, and may be indicated in the dietary treatment of type 2 diabetes. PMID:28304350

  20. Betaglycan expression is transcriptionally up-regulated during skeletal muscle differentiation. Cloning of murine betaglycan gene promoter and its modulation by MyoD, retinoic acid, and transforming growth factor-beta.

    PubMed

    Lopez-Casillas, Fernando; Riquelme, Cecilia; Perez-Kato, Yoshiaki; Ponce-Castaneda, M Veronica; Osses, Nelson; Esparza-Lopez, Jose; Gonzalez-Nunez, Gerardo; Cabello-Verrugio, Claudio; Mendoza, Valentin; Troncoso, Victor; Brandan, Enrique

    2003-01-03

    Betaglycan is a membrane-anchored proteoglycan co-receptor that binds transforming growth factor beta (TGF-beta) via its core protein and basic fibroblast growth factor through its glycosaminoglycan chains. In this study we evaluated the expression of betaglycan during the C(2)C(12) skeletal muscle differentiation. Betaglycan expression, as determined by Northern and Western blot, was up-regulated during the conversion of myoblasts to myotubes. The mouse betaglycan gene promoter was cloned, and its sequence showed putative binding sites for SP1, Smad3, Smad4, muscle regulatory factor elements such as MyoD and MEF2, and retinoic acid receptor. Transcriptional activity of the mouse betaglycan promoter reporter was also up-regulated in differentiating C(2)C(12) cells. We found that MyoD, but not myogenin, stimulated this transcriptional activity even in the presence of high serum. Betaglycan promoter activity was increased by RA and inhibited by the three isoforms of TGF-beta. On the other hand, basic fibroblast growth factor, BMP-2, and hepatocyte growth factor/scatter factor, which are inhibitors of myogenesis, had little effect. In myotubes, up-regulated betaglycan was also detectable by TGF-beta affinity labeling and immunofluorescence microscopy studies. The latter indicated that betaglycan was localized both on the cell surface and in the ECM. Forced expression of betaglycan in C(2)C(12) myoblasts increases their responsiveness to TGF-beta2, suggesting that it performs a TGF-beta presentation function in this cell lineage. These results indicate that betaglycan expression is up-regulated during myogenesis and that MyoD and RA modulate its expression by a mechanism that is independent of myogenin.

  1. Functional overload increases beta-MHC promoter activity in rodent fast muscle via the proximal MCAT (betae3) site.

    PubMed

    Giger, Julia M; Haddad, Fadia; Qin, Anqi X; Baldwin, Kenneth M

    2002-03-01

    Functional overload (OL) of the rat plantaris muscle by the removal of synergistic muscles induces a shift in the myosin heavy chain (MHC) isoform expression profile from the fast isoforms toward the slow type I, or, beta-MHC isoform. Different length rat beta-MHC promoters were linked to a firefly luciferase reporter gene and injected in control and OL plantaris muscles. Reporter activities of -3,500, -914, -408, and -215 bp promoters increased in response to 1 wk of OL. The smallest -171 bp promoter was not responsive to OL. Mutation analyses of putative regulatory elements within the -171 and -408 bp region were performed. The -408 bp promoters containing mutations of the betae1, distal muscle CAT (MCAT; betae2), CACC, or A/T-rich (GATA), were still responsive to OL. Only the proximal MCAT (betae3) mutation abolished the OL response. Gel mobility shift assays revealed a significantly higher level of complex formation of the betae3 probe with nuclear protein from OL plantaris compared with control plantaris. These results suggest that the betae3 site functions as a putative OL-responsive element in the rat beta-MHC gene promoter.

  2. Involvement of H- and N-Ras isoforms in transforming growth factor-{beta}1-induced proliferation and in collagen and fibronectin synthesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Martinez-Salgado, Carlos; Fuentes-Calvo, Isabel; Instituto 'Reina Sofia' de Investigacion Nefrologica, Universidad de Salamanca, 37007 Salamanca

    2006-07-01

    Transforming growth factor {beta}1 (TGF-{beta}1) has a relevant role in the origin and maintenance of glomerulosclerosis and tubule-interstitial fibrosis. TGF-{beta} and Ras signaling pathways are closely related: TGF-{beta}1 overcomes Ras mitogenic effects and Ras counteracts TGF-{beta} signaling. Tubule-interstitial fibrosis is associated to increases in Ras, Erk, and Akt activation in a renal fibrosis model. We study the role of N- and H-Ras isoforms, and the involvement of the Ras effectors Erk and Akt, in TGF-{beta}1-mediated extracellular matrix (ECM) synthesis and proliferation, using embrionary fibroblasts from double knockout (KO) mice for H- and N-Ras (H-ras {sup -/-}/N-ras {sup -/-}) isoforms andmore » from heterozygote mice (H-ras {sup +/-}/N-ras {sup +/-}). ECM synthesis is increased in basal conditions in H-ras {sup -/-}/N-ras {sup -/-} fibroblasts, this increase being higher after stimulation with TGF-{beta}1. TGF-{beta}1-induced fibroblast proliferation is smaller in H-ras {sup -/-}/N-ras {sup -/-} than in H-ras {sup +/-}/N-ras {sup +/-} fibroblasts. Erk activation is decreased in H-ras {sup -/-}/N-ras {sup -/-} fibroblasts; inhibition of Erk activation reduces fibroblast proliferation. Akt activation is higher in double KO fibroblasts than in heterozygotes; inhibition of Akt activation also inhibits ECM synthesis. We suggest that H- and N-Ras isoforms downregulate ECM synthesis, and mediate proliferation, in part through MEK/Erk activation. PI3K-Akt pathway activation may be involved in the increase in ECM synthesis observed in the absence of H- and N-Ras.« less

  3. Stage-specific control of connective tissue growth factor (CTGF/CCN2) expression in chondrocytes by Sox9 and beta-catenin.

    PubMed

    Huang, Bau-Lin; Brugger, Sean M; Lyons, Karen M

    2010-09-03

    CCN2/connective tissue growth factor is highly expressed in hypertrophic chondrocytes and is required for chondrogenesis. However, the transcriptional mechanisms controlling its expression in cartilage are largely unknown. The activity of the Ccn2 promoter was, therefore, investigated in osteochondro-progenitor cells and hypertrophic chondrocytes to ascertain these mechanisms. Sox9 and T-cell factor (TCF) x lymphoid enhancer factor (LEF) factors contain HMG domains and bind to related consensus sites. TCF x LEF factors are normally repressive but when bound to DNA in a complex with beta-catenin become activators of gene expression. In silico analysis of the Ccn2 proximal promoter identified multiple consensus TCF x LEF elements, one of which was also a consensus binding site for Sox9. Using luciferase reporter constructs, the TCF x LEF x Sox9 site was found to be involved in stage-specific expression of Ccn2. Luciferase, electrophoretic mobility shift assay (EMSA), and ChIP analysis revealed that Sox9 represses Ccn2 expression by binding to the consensus TCF x LEF x Sox9 site. On the other hand, the same assays showed that in hypertrophic chondrocytes, TCF x LEF x beta-catenin complexes occupy the consensus TCF x LEF x Sox9 site and activate Ccn2 expression. Furthermore, transgenic mice in which lacZ expression is driven under the control of the proximal Ccn2 promoter revealed that the proximal Ccn2 promoter responded to Wnt signaling in cartilage. Hence, we propose that differential occupancy of the TCF x LEF x Sox9 site by Sox9 versus beta-catenin restricts high levels of Ccn2 expression to hypertrophic chondrocytes.

  4. Orphan nuclear receptor small heterodimer partner inhibits transforming growth factor-beta signaling by repressing SMAD3 transactivation.

    PubMed

    Suh, Ji Ho; Huang, Jiansheng; Park, Yun-Yong; Seong, Hyun-A; Kim, Dongwook; Shong, Minho; Ha, Hyunjung; Lee, In-Kyu; Lee, Keesook; Wang, Li; Choi, Hueng-Sik

    2006-12-22

    Orphan nuclear receptor small heterodimer partner (SHP) is an atypical member of the nuclear receptor superfamily; SHP regulates the nuclear receptor-mediated transcription of target genes but lacks a conventional DNA binding domain. In this study, we demonstrate that SHP represses transforming growth factor-beta (TGF-beta)-induced gene expression through a direct interaction with Smad, a transducer of TGF-beta signaling. Transient transfection studies demonstrate that SHP represses Smad3-induced transcription. In vivo and in vitro protein interaction assays revealed that SHP directly interacts with Smad2 and Smad3 but not with Smad4. Mapping of domains mediating the interaction between SHP and Smad3 showed that the entire N-terminal domain (1-159 amino acids) of SHP and the linker domain of Smad3 are involved in this interaction. In vitro glutathione S-transferase pulldown competition experiments revealed the SHP-mediated repression of Smad3 transactivation through competition with its co-activator p300. SHP also inhibits the activation of endogenous TGF-beta-responsive gene promoters, the p21, Smad7, and plasminogen activator inhibitor-1 (PAI-1) promoters. Moreover, adenovirus-mediated overexpression of SHP decreases PAI-1 mRNA levels, and down-regulation of SHP by a small interfering RNA increases both the transactivation of Smad3 and the PAI-1 mRNA levels. Finally, the PAI-1 gene is expressed in SHP(-/-) mouse hepatocytes at a higher level than in normal hepatocytes. Taken together, these data indicate that SHP is a novel co-regulator of Smad3, and this study provides new insights into regulation of TGF-beta signaling.

  5. Transforming growth factor beta-3 and environmental factors and cleft lip with/without cleft palate.

    PubMed

    Guo, Zeqiang; Huang, Chengle; Ding, Kaihong; Lin, Jianyan; Gong, Binzhong

    2010-07-01

    To identify the interactions among two loci (C641A and G15572-) of transforming growth factor beta 3 (TGFbeta3), and exposures in pregnancy with cleft lip with/without cleft palate (CL/P), a hospital-based case-control study was conducted. Associations among offspring polymorphisms of TGFbeta3 C641A and G15572-, paternal smoking, paternal high-risk drinking, maternal passive smoking, and maternal multivitamin supplement with CL/P were analyzed by logistic regression analysis, and the results showed that maternal passive smoking exposures and maternal multivitamin use were associated with the risk of CL/P but offspring polymorphisms of TGFbeta3 C641A and G15572-, paternal smoking, and paternal high-risk drinking were not. Interactions among these variables were analyzed using the multifactor dimensionality reduction method, and the results showed that the two-factor model, including maternal passive smoking and TGFbeta3 C641A, among all models evaluated had the best ability to predict CL/P risk with a maximum cross-validation consistency (9/10) and a maximum average testing accuracy (0.5892; p = 0.0010). These findings suggested that maternal passive smoking exposure is a risk factor for CL/P, whereas maternal multivitamin supplement is a protective factor. The polymorphism of TGFbeta3 C641A participates in interaction effect for CL/P with environmental exposures, although the polymorphism was not associated with CL/P in single-locus analysis, and synergistic effect of TGFbeta3 C641A and maternal passive smoking could provide a new tool for identifying high-risk individuals of CL/P and also an additional evidence that CL/P is determined by both genetic and environmental factors.

  6. Incorporating beta-turns and a turn mimetic out of context in loop 1 of the WW domain affords cooperatively folded beta-sheets.

    PubMed

    Kaul, R; Angeles, A R; Jäger, M; Powers, E T; Kelly, J W

    2001-06-06

    To probe the conformational requirements of loop 1 in the Pin1 WW domain, the residues at the i + 2 and i + 3 positions of a beta-turn within this loop were replaced by dPro-Gly and Asn-Gly, which are known to prefer the conformations required at the i + 1 and i + 2 positions of type II' and type I' beta-turns. Conformational specificity or lack thereof was further examined by incorporating into the i + 2 and i + 3 positions a non-alpha-amino acid-based beta-turn mimetic (4-(2'-aminoethyl)-6-dibenzofuran propionic acid residue, 1), which was designed to replace the i + 1 and i + 2 positions of beta-turns. All these Pin WW variants are monomeric and folded as discerned by analytical ultracentrifugation, NMR, and CD. They exhibit cooperative two-state transitions and display thermodynamic stability within 0.5 kcal/mol of the wild-type WW domain, demonstrating that the acquisition of native structure and stability does not require a specific sequence and, by extension, conformation within loop 1. However, it could be that these loop 1 mutations alter the kinetics of antiparallel beta-sheet folding, which will be addressed by subsequent kinetic studies.

  7. Smad signaling pathway is a pivotal component of tissue inhibitor of metalloproteinases-3 regulation by transforming growth factor beta in human chondrocytes.

    PubMed

    Qureshi, Hamid Yaqoob; Ricci, Gemma; Zafarullah, Muhammad

    2008-09-01

    Transforming growth factor beta (TGF-beta1) promotes cartilage matrix synthesis and induces tissue inhibitor of metalloproteinases-3 (TIMP-3), which inhibits matrix metalloproteinases, aggrecanases and TNF-alpha-converting enzyme implicated in articular cartilage degradation and joint inflammation. TGF-beta1 activates Akt, ERK and Smad2 pathways in chondrocytes. Here we investigated previously unexplored roles of specific Smads in TGF-beta1 induction of TIMP-3 gene by pharmacological and genetic knockdown approaches. TGF-beta1-induced Smad2 phosphorylation and TIMP-3 protein expression could be inhibited by the Smad2/3 phosphorylation inhibitors, PD169316 and SB203580 and by Smad2-specific siRNA. Specific inhibitor of Smad3 (SIS3) and Smad3 siRNA abolished TGF-beta induction of TIMP-3. Smad2/3 siRNAs also down regulated TIMP-3 promoter-driven luciferase activities, suggesting transcriptional regulation. SiRNA-driven co-Smad4 knockdown abrogated TIMP-3 augmentation by TGF-beta. TIMP-3 promoter deletion analysis revealed that -828 deletion retains the original promoter activity while -333 and -167 deletions display somewhat reduced activity suggesting that most of the TGF-beta-responsive, cis-acting elements are found in the -333 fragment. Chromatin Immunoprecipitation (ChIP) analysis confirmed binding of Smad2 and Smad4 with the -940 and -333 promoter sequences. These results suggest that receptor-activated Smad2 and Smad3 and co-Smad4 critically mediate TGF-beta-stimulated TIMP-3 expression in human chondrocytes and TIMP-3 gene is a target of Smad signaling pathway.

  8. Significance of the interleukin-1 receptor antagonist/interleukin-1 beta ratio as a prognostic factor in patients with pulmonary sarcoidosis.

    PubMed

    Mikuniya, T; Nagai, S; Takeuchi, M; Mio, T; Hoshino, Y; Miki, H; Shigematsu, M; Hamada, K; Izumi, T

    2000-01-01

    Various factors such as serum angiotensin-converting enzyme (sACE) activity, bronchoalveolar lavage (BAL) fluid lymphocyte percent, CD4/CD8 ratio, and shadows on chest radiograph have been identified as indexes of disease activity in patients with sarcoidosis. However, it remains to be confirmed whether these factors can predict clinical outcomes. To examine whether the interleukin-1 receptor antagonist (IL-1ra)/IL-1 beta ratio can predict the clinical course, we prospectively followed the clinical courses of 30 patients with pulmonary sarcoidosis 4 years after measurement of immunoreactive amounts of IL-1ra or IL-1 beta in the culture supernatants obtained from BAL fluid macrophages. Immunoreactive amounts of IL-1ra or IL-1 beta were measured using ELISA. Changes in pulmonary function, sACE activity, and shadows on chest radiographs during observation periods were evaluated as markers of changes in disease activity. We found that the patients whose shadows on chest radiographs showed improvement had a higher molar IL-1ra/IL-1 beta ratio than the patients whose shadows persistently remained 4 years after BAL examination (p < 0.05). The molar ratio was found to be positively correlated with improvement of percent vital capacity (p < 0.05) and negatively correlated with the ratio of sACE activity at the time of the last observation to sACE activity at the time of BAL (sACE(LAST)/sACE(BAL), p < 0.01). The sACE(LAST)/sACE(BAL) ratio was significantly lower in patients whose shadows on chest radiographs decreased than in those whose shadows remained unchanged (p < 0.005). The IL-1ra/IL-1 beta ratio in the BAL fluid macrophage culture supernatants in patients with pulmonary sarcoidosis could be a useful marker in predicting the persistence of granulomatous lesions (chronicity). Copyright 2000 S. Karger AG, Basel

  9. Protective effects of St. John's wort extract and its component hyperforin against cytokine-induced cytotoxicity in a pancreatic beta-cell line.

    PubMed

    Menegazzi, Marta; Novelli, Michela; Beffy, Pascale; D'Aleo, Valentina; Tedeschi, Elisa; Lupi, Roberto; Zoratti, Elisa; Marchetti, Piero; Suzuki, Hisanori; Masiello, Pellegrino

    2008-01-01

    In both type 1 and type 2 diabetes, increased production of cytokines on autoimmune or metabolic basis is supposed to trigger an inflammatory process leading to dysfunction and death of pancreatic beta-cells. Therefore, anti-inflammatory pharmacological approaches aimed at blocking cytokine signalling pathways and consequent cytotoxicity in beta-cells are highly advisable. Based on previous evidence of cytokine antagonistic effects in other cell types, we explored the protective action of Hypericum perforatum (St-John's-wort) extract and its component hyperforin against cytokine-induced functional impairment and apoptosis in the INS-1E beta-cell line, searching for the underlying mechanisms. The results showed that either St-John's-wort extract or hyperforin (at 1-3 microM) prevented cytokine-induced impairment in glucose-stimulated insulin secretion and protected cells against apoptosis in a dose-dependent fashion. Inducible-NO-synthase expression was also potently hindered by the vegetal compounds. Interestingly, cytokine-induced activations of the signal-transducer-and-activator-of-transcription-1 (STAT-1) and the nuclear-factor-kappaB (NF-kappaB) were both down-regulated by SJW extract or HPF (range 0.5-5 microM) when evaluated by electrophoretic-mobility-shift-assay. Other transcription factors (CBF-1, SP-1) were unaffected. Components of SJW extract other than HPF were much less effective in down-regulating cytokine signalling. Significantly, inhibition of cytokine-elicited STAT-1 and NF-kappaB activation was confirmed in isolated rat and human islets incubated in the presence of these vegetal compounds. In conclusion, St-John's-wort extract and hyperforin are non-peptidyl compounds which, at low concentrations, target key mechanisms of cytokine-induced beta-cell injury, thereby improving beta-cell function and survival. Thus, they are potentially valuable for the prevention or limitation of beta-cell loss in diabetes.

  10. Interferon-gamma interferes with transforming growth factor-beta signaling through direct interaction of YB-1 with Smad3.

    PubMed

    Higashi, Kiyoshi; Inagaki, Yutaka; Fujimori, Ko; Nakao, Atsuhito; Kaneko, Hideo; Nakatsuka, Iwao

    2003-10-31

    Transforming growth factor-beta (TGF-beta) and interferon-gamma (IFN-gamma) exert antagonistic effects on collagen synthesis in human dermal fibroblasts. We have recently shown that Y box-binding protein YB-1 mediates the inhibitory effects of IFN-gamma on alpha2(I) procollagen gene (COL1A2) transcription through the IFN-gamma response element located between -161 and -150. Here we report that YB-1 counter-represses TGF-beta-stimulated COL1A2 transcription by interfering with Smad3 bound to the upstream sequence around -265 and subsequently by interrupting the Smad3-p300 interaction. Western blot and immunofluorescence analyses using inhibitors for Janus kinases or casein kinase II suggested that the casein kinase II-dependent signaling pathway mediates IFN-gamma-induced nuclear translocation of YB-1. Down-regulation of endogenous YB-1 expression by double-stranded YB-1-specific RNA abrogated the transcriptional repression of COL1A2 by IFN-gamma in the absence and presence of TGF-beta. In transient transfection assays, overexpression of YB-1 in human dermal fibroblasts exhibited antagonistic actions against TGF-beta and Smad3. Physical interaction between Smad3 and YB-1 was demonstrated by immunoprecipitation-Western blot analyses, and electrophoretic mobility shift assays using the recombinant Smad3 and YB-1 proteins indicated that YB-1 forms a complex with Smad3 bound to the Smad-binding element. Glutathione S-transferase pull-down assays showed that YB-1 binds to the MH1 domain of Smad3, whereas the central and carboxyl-terminal regions of YB-1 were required for its interaction with Smad3. YB-1 also interferes with the Smad3-p300 interaction by its preferential binding to p300. Altogether, the results provide a novel insight into the mechanism by which IFN-gamma/YB-1 counteracts TGF-beta/Smad3. They also indicate that IFN-gamma/YB-1 inhibits COL1A2 transcription by dual actions: via the IFN-gamma response element and through a cross-talk with the TGF-beta

  11. Immunocytochemistry suggests that the prevalence of a sub-type of beta-proteins determines the hardness in the epidermis of the hard-shelled turtle.

    PubMed

    Alibardi, Lorenzo

    2014-01-01

    The corneous layer of the epidermis in hard-shelled turtles largely derives from the accumulation of beta-proteins as indicated by microscopic, in situ hybridization, and immunocytochemical and Western blotting analysis. The expression of mRNAs of one of the most common type of beta-proteins shows higher expression in upper spinosus and pre-corneous keratinocytes of growing scutes. Two beta-proteins of 14-16 kDa, indicated as Tu2 and Tu17 and representing two subtypes of beta-proteins co-accumulate in the thick corneous layer of the epidermis in hard-shelled turtle. The two beta-proteins apparently mix in differentiating and mature corneocytes although Tu2 appears more prevalent than Tu17. The specific role of the different subtypes in the formation of the hard corneous material of the carapace and plastron is not clear. It is hypothesized that the relative amount of beta-proteins belonging to the two subclasses in relation to the alpha-keratin meshwork present in keratinocytes contributes to the formation of a variably resistant and inflexible corneous layer. Tu17 may have a more globular structure than Tu2 and is likely present in denser areas of the corneous layer containing also alpha-keratin. The increase of cysteine-glycine-rich beta-proteins in the matrix located among alpha-keratin filaments may allow the formation of a hard corneous material, probably through increase of cross-bridge formation and hydrophobicity. © 2013 Wiley Periodicals, Inc.

  12. SIRT1 inhibits proliferation of pancreatic cancer cells expressing pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, by suppression of {beta}-catenin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cho, Il-Rae; Koh, Sang Seok; Department of Functional Genomics, University of Science and Technology, Daejeon 305-333

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer SIRT1 inhibits protein levels of {beta}-catenin and its transcriptional activity. Black-Right-Pointing-Pointer Nuclear localization of SIRT1 is not required for the decrease of {beta}-catenin expression. Black-Right-Pointing-Pointer SIRT1-mediated degradation of {beta}-catenin is not required for GSK-3{beta} and Siah-1 but for proteosome. Black-Right-Pointing-Pointer SIRT1 activation inhibits proliferation of pancreatic cancer cells expressing PAUF. -- Abstract: Because we found in a recent study that pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, induces a rapid proliferation of pancreatic cells by up-regulation of {beta}-catenin, we postulated that {beta}-catenin might be a target molecule for pancreatic cancer treatment. We thus speculated whether SIRT1, knownmore » to target {beta}-catenin in a colon cancer model, suppresses {beta}-catenin in those pancreatic cancer cells that express PAUF (Panc-PAUF). We further evaluated whether such suppression would lead to inhibition of the proliferation of these cells. The ectopic expression of either SIRT1 or resveratrol (an activator of SIRT1) suppressed levels of {beta}-catenin protein and its transcriptional activity in Panc-PAUF cells. Conversely, suppression of SIRT1 expression by siRNA enhanced {beta}-catenin expression and transcriptional activity. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for reduction of {beta}-catenin. Treatment with MG132, a proteasomal inhibitor, restored {beta}-catenin protein levels, suggesting that SIRT1-mediated degradation of {beta}-catenin requires proteasomal activity. It was reported that inhibition of GSK-3{beta} or Siah-1 stabilizes {beta}-catenin in colon cancer cells, but suppression of GSK-3{beta} or Siah-1 using siRNA in the presence of resveratrol instead diminished {beta}-catenin protein levels in Panc-PAUF cells. This suggests that GSK-3{beta} and Siah-1 are not involved

  13. Synthesis, Characterization, In Vitro Evaluation, and Preclinical Profiling of beta-Cyclodextrin Polyrotaxane Families for Use As Potential Niemann-Pick Type C Therapeutics

    NASA Astrophysics Data System (ADS)

    Collins, Christopher J.

    Niemann-Pick Disease Type C (NPC) is a rare, autosomal recessive genetic disorder featuring a loss of proteins responsible for unesterified cholesterol (UC) trafficking through the late endosomes/lysosomes (LE/LY) of every cell of the body. Disruption of this pathway leads to abnormal accumulation and storage of UC and other lipids. A broad range of visceral and neurological symptoms result from this accumulation exhibiting a variable age of onset and a disease progression that is ultimately fatal. The disease has an incidence of approximately 1 in 120,000 live births and has no known effective treatment. beta-Cyclodextrin (beta-CD) are natural small molecules macrocycles composed of glucose units with a hydrophobic inner cavity and hydrophilic outer rims. beta-CD derivatives have recently been shown to be effective therapeutics for NPC in cellular and animal models. In the mouse model of the disease, beta-CD therapy increases overall lifetime by as much as 50% and slows the progression of neurodegeneration. The progress has led to the initiation of a National Institutes of Health phase I clinical trial. A main drawback of beta-CD administration is the poor pharmacokinetic profile characterized by rapid renal clearance of the drug through the urine. Libraries of beta-CD derivative carrying high molecular weight polyrotaxane (PR) systems have been designed to prevent glomerular filtration of the injected beta-CD dose. An initial family of unmodified beta-CD PRs was synthesized, characterized, and their therapeutic efficacy was tested in NPC fibroblasts. This was followed by screening of PRs consisting of mixed beta-CD derivative threading featuring charged sulfobutylether beta-CD. Finally, we sought to define PR structure-property effects on in vivo pharmacokinetics, biodistribution, toxicity, immunogenicity, and protein hard corona composition. This was accomplished using a family of gadolinium carrying PRs composed of triblock Pluronic co-polymers of varying

  14. Both conditional ablation and overexpression of E2 SUMO-conjugating enzyme (UBC9) in mouse pancreatic beta cells result in impaired beta cell function.

    PubMed

    He, Xiaoyu; Lai, Qiaohong; Chen, Cai; Li, Na; Sun, Fei; Huang, Wenting; Zhang, Shu; Yu, Qilin; Yang, Ping; Xiong, Fei; Chen, Zhishui; Gong, Quan; Ren, Boxu; Weng, Jianping; Eizirik, Décio L; Zhou, Zhiguang; Wang, Cong-Yi

    2018-04-01

    Post-translational attachment of a small ubiquitin-like modifier (SUMO) to the lysine (K) residue(s) of target proteins (SUMOylation) is an evolutionary conserved regulatory mechanism. This modification has previously been demonstrated to be implicated in the control of a remarkably versatile regulatory mechanism of cellular processes. However, the exact regulatory role and biological actions of the E2 SUMO-conjugating enzyme (UBC9)-mediated SUMOylation function in pancreatic beta cells has remained elusive. Inducible beta cell-specific Ubc9 (also known as Ube2i) knockout (KO; Ubc9 Δbeta ) and transgenic (Ubc9 Tg ) mice were employed to address the impact of SUMOylation on beta cell viability and functionality. Ubc9 deficiency or overexpression was induced at 8 weeks of age using tamoxifen. To study the mechanism involved, we closely examined the regulation of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) through SUMOylation in beta cells. Upon induction of Ubc9 deficiency, Ubc9 Δbeta islets exhibited a 3.5-fold higher accumulation of reactive oxygen species (ROS) than Ubc9 f/f control islets. Islets from Ubc9 Δbeta mice also had decreased insulin content and loss of beta cell mass after tamoxifen treatment. Specifically, at day 45 after Ubc9 deletion only 40% of beta cell mass remained in Ubc9 Δbeta mice, while 90% of beta cell mass was lost by day 75. Diabetes onset was noted in some Ubc9 Δbeta mice 8 weeks after induction of Ubc9 deficiency and all mice developed diabetes by 10 weeks following tamoxifen treatment. In contrast, Ubc9 Tg beta cells displayed an increased antioxidant ability but impaired insulin secretion. Unlike Ubc9 Δbeta mice, which spontaneously developed diabetes, Ubc9 Tg mice preserved normal non-fasting blood glucose levels without developing diabetes. It was noted that SUMOylation of NRF2 promoted its nuclear expression along with enhanced transcriptional activity, thereby preventing ROS accumulation in

  15. Neutrophil chemotaxis in response to TGF-beta isoforms (TGF-beta 1, TGF-beta 2, TGF-beta 3) is mediated by fibronectin.

    PubMed

    Parekh, T; Saxena, B; Reibman, J; Cronstein, B N; Gold, L I

    1994-03-01

    TGF-beta isoforms regulate numerous cellular functions including cell growth and differentiation, the cellular synthesis and secretion of extracellular matrix proteins, such as fibronectin (Fn), and the immune response. We have previously shown that TGF-beta 1 is the most potent chemoattractant described for human peripheral blood neutrophils (PMNs), suggesting that TGF-beta s may play a role in the recruitment of PMNs during the initial phase of the inflammatory response. In our current studies, we demonstrate that the maximal chemotactic response was attained near 40 fM for all mammalian TGF-beta isoforms. However, there was a statistically significant difference in migratory distance of the PMNs: TGF-beta 2 (556 microM) > TGF-beta 3 (463 microM) > TGF-beta 1 (380 microM) (beta 2: beta 3, p < or = 0.010; beta 3: beta 1, p < or = 0.04; beta 2: beta 1, p < or = 0.0012). A mAb to the cell binding domain (CBD) of Fn inhibited the chemotactic response to TGF-beta 1 and TGF-beta 3 by 63% and to TGF-beta 2 by 70%, whereas the response to FMLP, a classic chemoattractant, was only inhibited by 18%. In contrast, a mAb to a C-terminal epitope of Fn did not retard migration (< 1.5%). The Arg-gly-Asp-ser tetrapeptide inhibited chemotaxis by approximately the same extent as the anti-CBD (52 to 83%). Furthermore, a mAb against the VLA-5 integrin (VLA-5; Fn receptor) also inhibited TGF-beta-induced chemotaxis. These results indicate that chemotaxis of PMNs in response to TGF-beta isoforms is mediated by the interaction of the Arg-gly-Asp-ser sequence in the CBD of Fn with an integrin on the PMN cell surface, primarily the VLA-5 integrin. TGF-beta isoforms also elicited the release of cellular Fn from PMNs; we observed a 2.3-fold increase in Fn (389 to 401 ng/ml) in the supernatants of TGF-beta-stimulated PMNs compared with unstimulated cells (173.6 ng/ml). The concentration of TGF-beta required to cause maximal release of Fn from PMNs (4000 fM) is a concentration at which TGF-beta

  16. Lipopolysaccharide-induced IL-18 secretion from murine Kupffer cells independently of myeloid differentiation factor 88 that is critically involved in induction of production of IL-12 and IL-1beta.

    PubMed

    Seki, E; Tsutsui, H; Nakano, H; Tsuji, N; Hoshino, K; Adachi, O; Adachi, K; Futatsugi, S; Kuida, K; Takeuchi, O; Okamura, H; Fujimoto, J; Akira, S; Nakanishi, K

    2001-02-15

    IL-18, produced as biologically inactive precursor, is secreted from LPS-stimulated macrophages after cleavage by caspase-1. In this study, we investigated the mechanism underlying caspase-1-mediated IL-18 secretion. Kupffer cells constantly stored IL-18 and constitutively expressed caspase-1. Inhibition of new protein synthesis only slightly reduced IL-18 secretion, while it decreased and abrogated their IL-1beta and IL-12 secretion, respectively. Kupffer cells deficient in Toll-like receptor (TLR) 4, an LPS-signaling receptor, did not secrete IL-18, IL-1beta, and IL-12 upon LPS stimulation. In contrast, Kupffer cells lacking myeloid differentiation factor 88 (MyD88), an adaptor molecule for TLR-mediated-signaling, secreted IL-18 without IL-1beta and IL-12 production in a caspase-1-dependent and de novo synthesis-independent manner. These results indicate that MyD88 is essential for IL-12 and IL-1beta production from Kupffer cells while their IL-18 secretion is mediated via activation of endogenous caspase-1 without de novo protein synthesis in a MyD88-independent fashion after stimulation with LPS. In addition, infection with Listeria monocytogenes, products of which have the capacity to activate TLR, increased serum levels of IL-18 in wild-type and MyD88-deficient mice but not in caspase-1-deficient mice, whereas it induced elevation of serum levels of IL-12 in both wild-type and caspase-1-deficient mice but not in MyD88-deficient mice. Taken together, these results suggested caspase-1-dependent, MyD88-independent IL-18 release in bacterial infection.

  17. Core-binding factor beta interacts with Runx2 and is required for skeletal development.

    PubMed

    Yoshida, Carolina A; Furuichi, Tatsuya; Fujita, Takashi; Fukuyama, Ryo; Kanatani, Naoko; Kobayashi, Shinji; Satake, Masanobu; Takada, Kenji; Komori, Toshihisa

    2002-12-01

    Core-binding factor beta (CBFbeta, also called polyomavirus enhancer binding protein 2beta (PEBP2B)) is associated with an inversion of chromosome 16 and is associated with acute myeloid leukemia in humans. CBFbeta forms a heterodimer with RUNX1 (runt-related transcription factor 1), which has a DNA binding domain homologous to the pair-rule protein runt in Drosophila melanogaster. Both RUNX1 and CBFbeta are essential for hematopoiesis. Haploinsufficiency of another runt-related protein, RUNX2 (also called CBFA1), causes cleidocranial dysplasia in humans and is essential in skeletal development by regulating osteoblast differentiation and chondrocyte maturation. Mice deficient in Cbfb (Cbfb(-/-)) die at midgestation, so the function of Cbfbeta in skeletal development has yet to be ascertained. To investigate this issue, we rescued hematopoiesis of Cbfb(-/-) mice by introducing Cbfb using the Gata1 promoter. The rescued Cbfb(-/-) mice recapitulated fetal liver hematopoiesis in erythroid and megakaryocytic lineages and survived until birth, but showed severely delayed bone formation. Although mesenchymal cells differentiated into immature osteoblasts, intramembranous bones were poorly formed. The maturation of chondrocytes into hypertrophic cells was markedly delayed, and no endochondral bones were formed. Electrophoretic mobility shift assays and reporter assays showed that Cbfbeta was necessary for the efficient DNA binding of Runx2 and for Runx2-dependent transcriptional activation. These findings indicate that Cbfbeta is required for the function of Runx2 in skeletal development.

  18. Beta-structures in fibrous proteins.

    PubMed

    Kajava, Andrey V; Squire, John M; Parry, David A D

    2006-01-01

    The beta-form of protein folding, one of the earliest protein structures to be defined, was originally observed in studies of silks. It was then seen in early studies of synthetic polypeptides and, of course, is now known to be present in a variety of guises as an essential component of globular protein structures. However, in the last decade or so it has become clear that the beta-conformation of chains is present not only in many of the amyloid structures associated with, for example, Alzheimer's Disease, but also in the prion structures associated with the spongiform encephalopathies. Furthermore, X-ray crystallography studies have revealed the high incidence of the beta-fibrous proteins among virulence factors of pathogenic bacteria and viruses. Here we describe the basic forms of the beta-fold, summarize the many different new forms of beta-structural fibrous arrangements that have been discovered, and review advances in structural studies of amyloid and prion fibrils. These and other issues are described in detail in later chapters.

  19. The new generation dihydropyridine type calcium blockers, bearing 4-phenyl oxypropanolamine, display alpha-/beta-adrenoceptor antagonist and long-acting antihypertensive activities.

    PubMed

    Liang, Jhy-Chong; Yeh, Jwu-Lai; Wang, Chia-Sui; Liou, Shwu-Fen; Tsai, Chieh-Ho; Chen, Ing-Jun

    2002-03-01

    A new series of dihydropyridine derivatives, bearing oxypropanolamine moiety on phenyl ring at the 4-position of the dihydropyridine base, were prepared. Oxypropanolamine was synthesized by replacing the phenolic OH of vanillin or other compounds, having a phenyl aldehyde group, with epichlorohydrin, followed by cleavaging the obtained epoxide compounds with tert-butylamine, n-butylamine or 2-methoxy-1-oxyethylamino benzene (guaiacoxyethylamine), respectively. Obtained various oxypropanolamine compounds, still remaining a phenyl aldehyde moiety, were then performed by Hantzsch condensation reaction with methylacetoacetate or ethylacetoacetate, respectively, to give our new series of dihydropyridine linked with the 4-phenyl ring. These compounds were evaluated for inotropic, chronotropic, and aorta contractility that associated with calcium channel and adrenoceptor antagonist activities. Dihydropyridine derivatives that with oxypropanolamine side chain on their 4-phenyl ring associated alpha-/beta-adrenoceptor blocking activities created a new family of calcium entry and the third generation beta-adrenoceptor blockers. Optimizing this research to obtain more potent alpha-/beta-adrenoceptor blocking and long-acting antihypertensive oxypropanolamine on the 4-phenyl ring of dihydropyridine series compounds was thus accomplished and classified as third generation dihydropyridine type calcium channel blockers, in comparison with previous short-acting type nifedipine and long-acting type amlodipine. We concluded that compounds 1a, 1b and 1g showed not only markedly high calcium-antagonistic activity but also the highest antihypertensive effect; compounds 1b, 1c, 1f, 1g, 1i and 1j induced sustained antihypertensive effects are major and attributed to their calcium entry and alpha-adrenoceptor blocking activities in the blood vessel due to their introduction of 2-methoxy, 1-oxyethylamino benzene moiety in the side chain on the 4-phenyl ring of dihydropyridine. Bradycardiac

  20. TGF-beta antisense oligonucleotides reduce mRNA expression of matrix metalloproteinases in cultured wound-healing-related cells.

    PubMed

    Philipp, Katrin; Riedel, Frank; Germann, Günter; Hörmann, Karl; Sauerbier, Michael

    2005-02-01

    The pathology of chronic dermal ulcers is characterized by excessive proteolytic activity which degrades extracellular matrix. The transforming growth factor-beta (TGF-beta) has been identified as an important component of wound healing. Recent developments in molecular therapy offer exciting prospects for the modulation of wound healing, specifically those targeting TGF-beta. We investigated the effect of TGF-beta antisense oligonucleotides on the mRNA expression of matrix metalloproteinases in cultured human keratinocytes, fibroblasts and endothelial cells using multiplex RT-PCR. The treatment of keratinocytes and fibroblasts with TGF-beta antisense oligonucleotides resulted in a significant decrease of expression of mRNA of MMP-1 and MMP-9 compared to controls. Accordingly, a decreased expression of MMP-1 mRNA in endothelial cells was detectable. Other MMPs were not affected. Affecting all dermal wound-healing-related cell types, TGF-beta antisense oligonucleotide technology may be a potential therapeutic option for the inhibition of proteolytic tissue destruction in chronic wounds. Pharmaceutical intervention in this area ultimately may help clinicians to proactively intervene in an effort to prevent normal wounds from becoming chronic.

  1. Adverse CNS-effects of beta-adrenoceptor blockers.

    PubMed

    Gleiter, C H; Deckert, J

    1996-11-01

    In 1962 propranolol, the first beta adrenoceptor antagonist (beta blocker), was brought on to the market. There is now a host of different beta blockers available, and these compounds are among the most commonly prescribed groups of drugs. The efficacy of beta blockers has been proven predominantly for the treatment of cardiovascular diseases. Beta blockers are also used for certain types of CNS disorders, such as anxiety disorders, essential tremor and migraine. While low toxicity means that they have a favorable risk-benefit ratio, given the high intensity of use, it is essential to have a comprehensive knowledge of adverse events. Adverse events of beta blockers that can be related to the CNS are quite often neglected, even in textbooks of clinical pharmacology or review articles, and thus often misdiagnosed. The following article, therefore, after summarizing the use of beta blockers for CNS indications, critically reviews the literature on centrally mediated adverse events. General pharmacological features of beta blockers and their molecular basis of action will briefly be addressed to the extent that they are or may become relevant for central nervous pharmacotherapy and side-effects.

  2. VISA is an adapter protein required for virus-triggered IFN-beta signaling.

    PubMed

    Xu, Liang-Guo; Wang, Yan-Yi; Han, Ke-Jun; Li, Lian-Yun; Zhai, Zhonghe; Shu, Hong-Bing

    2005-09-16

    Viral infection or stimulation of TLR3 triggers signaling cascades, leading to activation of the transcription factors IRF-3 and NF-kappaB, which collaborate to induce transcription of type I interferon (IFN) genes. In this study, we identified a protein termed VISA (for virus-induced signaling adaptor) as a critical component in the IFN-beta signaling pathways. VISA recruits IRF-3 to the cytoplasmic viral dsRNA sensor RIG-I. Depletion of VISA inhibits virus-triggered and RIG-I-mediated activation of IRF-3, NF-kappaB, and the IFN-beta promoter, suggesting that VISA plays a central role in virus-triggered TLR3-independent IFN-beta signaling. Our data also indicate that VISA interacts with TRIF and TRAF6 and mediates bifurcation of the TLR3-triggered NF-kappaB and IRF-3 activation pathways. These findings suggest that VISA is critically involved in both virus-triggered TLR3-independent and TLR3-mediated antiviral IFN signaling.

  3. Phospholipase C-{delta}{sub 1} regulates interleukin-1{beta} and tumor necrosis factor-{alpha} mRNA expression

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chung, Eric; Jakinovich, Paul; Bae, Aekyung

    Phospholipase C-{delta}{sub 1} (PLC{delta}{sub 1}) is a widely expressed highly active PLC isoform, modulated by Ca{sup 2+} that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLC{delta}{sub 1} modulated expression of the pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}), tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLC{delta}{sub 1} was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLC{delta}{sub 1}more » knockdown enhanced expression IL-1{beta} and tumor necrosis factor-{alpha} (TNF-{alpha}) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLC{delta}{sub 1} knock down caused persistently high Nf{kappa}b levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLC{delta}{sub 1} knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1{beta} and TNF-{alpha} mRNA's induced by PLC{delta}{sub 1} knockdown. Our results show that loss of PLC{delta}{sub 1} enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nf{kappa}b pathway. Our findings are consistent with the idea that PLC{delta}{sub 1} is

  4. [S632A3 promotes LPS-induced IFN-beta production through inhibiting the activation of GSK-3beta].

    PubMed

    Zhang, Na; Yang, Xin; Xu, Rong; Wang, Zhen; Song, Dan-Qing; Li, Dian-Dong; Deng, Hong-Bin

    2013-07-01

    LPS stimulation of macrophages production of IFN-beta plays a key role in innate immunity defending the microbial invasion. In this study, the effect of S632A3 promoting LPS-induced IFN-beta production and the underlying mechanism were investigated, mRNA level was measured by real-time PCR, cytokine production was determined by ELISA, GSK-3beta activity was investigated by kinase assay, protein phosphorylation and expression were evaluated by Western blotting. The results revealed that S632A3 significantly augmented IFN-beta production by LPS-stimulated macrophages. S632A3 inhibition of the activation of GSK-3beta, reduced the threonine 239 phosphorylation of transcription factor c-Jun but increased the total level of c-Jun in LPS-stimulated macrophages. Moreover, small interfering RNA-mediated knockdown of c-Jun level abrogated the ability of S632A3 to augment IFN-beta. The study thus demonstrates S632A3 being a new anti-inflammation lead compound and provides a molecular mechanism by which S632A3 promoted LPS-induced IFN-beta production in macrophages through inhibiting the activation of GSK-3beta.

  5. The aCORN backscatter-suppressed beta spectrometer

    DOE PAGES

    Hassan, M. T.; Bateman, F.; Collett, B.; ...

    2017-06-16

    Backscatter of electrons from a beta detector, with incomplete energy deposition, can lead to undesirable effects in many types of experiments. We present and discuss the design and operation of a backscatter-suppressed beta spectrometer that was developed as part of a program to measure the electron–antineutrino correlation coefficient in neutron beta decay (aCORN). An array of backscatter veto detectors surrounds a plastic scintillator beta energy detector. The spectrometer contains an axial magnetic field gradient, so electrons are efficiently admitted but have a low probability for escaping back through the entrance after backscattering. Lastly, the design, construction, calibration, and performance ofmore » the spectrometer are discussed.« less

  6. Analysis of betaS and betaA genes in a Mexican population with African roots.

    PubMed

    Magaña, María Teresa; Ongay, Zoyla; Tagle, Juan; Bentura, Gilberto; Cobián, José G; Perea, F Javier; Casas-Castañeda, Maricela; Sánchez-López, Yoaly J; Ibarra, Bertha

    2002-01-01

    To investigate the origin of the beta(A) and beta(S) genes in a Mexican population with African roots and a high frequency of hemoglobin S, we analyzed 467 individuals (288 unrelated) from different towns in the states of Guerrero and Oaxaca in the Costa Chica region. The frequency of the sickle-cell trait was 12.8%, which may represent a public health problem. The frequencies of the beta-haplotypes were determined from 350 nonrelated chromosomes (313 beta(A) and 37 beta(S)). We observed 15 different beta(A) haplotypes, the most common of which were haplotypes 1 (48.9%), 2 (13.4%), and 3 (13.4%). The calculation of pairwise distributions and Nei's genetic distance analysis using 32 worldwide populations showed that the beta(A) genes are more closely related to those of Mexican Mestizos and North Africans. Bantu and Benin haplotypes and haplotype 9 were related to the beta(S) genes, with frequencies of 78.8, 18.2, and 3.0%, respectively. Comparison of these haplotypes with 17 other populations revealed a high similitude with the population of the Central African Republic. These data suggest distinct origins for the beta(A) and beta(S) genes in Mexican individuals from the Costa Chica region.

  7. Genetic cathepsin B deficiency reduces beta-amyloid in transgenic mice expressing human wild-type amyloid precursor protein.

    PubMed

    Hook, Vivian Y H; Kindy, Mark; Reinheckel, Thomas; Peters, Christoph; Hook, Gregory

    2009-08-21

    Neurotoxic beta-amyloid (Abeta) peptides participate in Alzheimer's disease (AD); therefore, reduction of Abeta generated from APP may provide a therapeutic approach for AD. Gene knockout studies in transgenic mice producing human Abeta may identify targets for reducing Abeta. This study shows that knockout of the cathepsin B gene in mice expressing human wild-type APP (hAPPwt) results in substantial decreases in brain Abeta40 and Abeta42 by 67% and decreases in levels of the C-terminal beta-secretase fragment (CTFbeta) derived from APP. In contrast, knockout of cathepsin B in mice expressing hAPP with the rare Swedish (Swe) and Indiana (Ind) mutations had no effect on Abeta. The difference in reduction of Abeta in hAPPwt mice, but not in hAPPSwe/Ind mice, shows that the transgenic model can affect cathepsin B gene knockout results. Since most AD patients express hAPPwt, these data validate cathepsin B as a target for development of inhibitors to lower Abeta in AD.

  8. Evidence for novel beta-sheet structures in Iowa mutant beta-amyloid fibrils.

    PubMed

    Tycko, Robert; Sciarretta, Kimberly L; Orgel, Joseph P R O; Meredith, Stephen C

    2009-07-07

    Asp23-to-Asn mutation within the coding sequence of beta-amyloid, called the Iowa mutation, is associated with early onset, familial Alzheimer's disease and cerebral amyloid angiopathy, in which patients develop neuritic plaques and massive vascular deposition predominantly of the mutant peptide. We examined the mutant peptide, D23N-Abeta40, by electron microscopy, X-ray diffraction, and solid-state NMR spectroscopy. D23N-Abeta40 forms fibrils considerably faster than the wild-type peptide (k = 3.77 x 10(-3) min(-1) and 1.07 x 10(-4) min(-1) for D23N-Abeta40 and the wild-type peptide WT-Abeta40, respectively) and without a lag phase. Electron microscopy shows that D23N-Abeta40 forms fibrils with multiple morphologies. X-ray fiber diffraction shows a cross-beta pattern, with a sharp reflection at 4.7 A and a broad reflection at 9.4 A, which is notably smaller than the value for WT-Abeta40 fibrils (10.4 A). Solid-state NMR measurements indicate molecular level polymorphism of the fibrils, with only a minority of D23N-Abeta40 fibrils containing the in-register, parallel beta-sheet structure commonly found in WT-Abeta40 fibrils and most other amyloid fibrils. Antiparallel beta-sheet structures in the majority of fibrils are indicated by measurements of intermolecular distances through (13)C-(13)C and (15)N-(13)C dipole-dipole couplings. An intriguing possibility exists that there is a relationship between the aberrant structure of D23N-Abeta40 fibrils and the unusual vasculotropic clinical picture in these patients.

  9. Interaction with beta-arrestin determines the difference in internalization behavor between beta1- and beta2-adrenergic receptors.

    PubMed

    Shiina, T; Kawasaki, A; Nagao, T; Kurose, H

    2000-09-15

    The beta(1)-adrenergic receptor (beta(1)AR) shows the resistance to agonist-induced internalization. As beta-arrestin is important for internalization, we examine the interaction of beta-arrestin with beta(1)AR with three different methods: intracellular trafficking of beta-arrestin, binding of in vitro translated beta-arrestin to intracellular domains of beta(1)- and beta(2)ARs, and inhibition of betaAR-stimulated adenylyl cyclase activities by beta-arrestin. The green fluorescent protein-tagged beta-arrestin 2 translocates to and stays at the plasma membrane by beta(2)AR stimulation. Although green fluorescent protein-tagged beta-arrestin 2 also translocates to the plasma membrane, it returns to the cytoplasm 10-30 min after beta(1)AR stimulation. The binding of in vitro translated beta-arrestin 1 and beta-arrestin 2 to the third intracellular loop and the carboxyl tail of beta(1)AR is lower than that of beta(2)AR. The fusion protein of beta-arrestin 1 with glutathione S-transferase inhibits the beta(1)- and beta(2)AR-stimulated adenylyl cyclase activities, although inhibition of the beta(1)AR-stimulated activity requires a higher concentration of the fusion protein than that of the beta(2)AR-stimulated activity. These results suggest that weak interaction of beta(1)AR with beta-arrestins explains the resistance to agonist-induced internalization. This is further supported by the finding that beta-arrestin can induce internalization of beta(1)AR when beta-arrestin 1 does not dissociate from beta(1)AR by fusing to the carboxyl tail of beta(1)AR.

  10. Hepatitis B virus X protein shifts human hepatic transforming growth factor (TGF)-beta signaling from tumor suppression to oncogenesis in early chronic hepatitis B.

    PubMed

    Murata, Miki; Matsuzaki, Koichi; Yoshida, Katsunori; Sekimoto, Go; Tahashi, Yoshiya; Mori, Shigeo; Uemura, Yoshiko; Sakaida, Noriko; Fujisawa, Junichi; Seki, Toshihito; Kobayashi, Kazuki; Yokote, Koutaro; Koike, Kazuhiko; Okazaki, Kazuichi

    2009-04-01

    Hepatitis B virus X (HBx) protein is suspected to participate in oncogenesis during chronic hepatitis B progression. Transforming growth factor beta (TGF-beta) signaling involves both tumor suppression and oncogenesis. TGF-beta activates TGF-beta type I receptor (TbetaRI) and c-Jun N-terminal kinase (JNK), which differentially phosphorylate the mediator Smad3 to become C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). Reversible shifting of Smad3-mediated signaling between tumor suppression and oncogenesis in HBx-expressing hepatocytes indicated that TbetaRI-dependent pSmad3C transmitted a tumor-suppressive TGF-beta signal, while JNK-dependent pSmad3L promoted cell growth. We used immunostaining, immunoblotting, and in vitro kinase assay to compare pSmad3L- and pSmad3C-mediated signaling in biopsy specimens representing chronic hepatitis, cirrhosis, or hepatocellular carcinoma (HCC) from 90 patients chronically infected with hepatitis B virus (HBV) with signaling in liver specimens from HBx transgenic mice. In proportion to plasma HBV DNA levels, early chronic hepatitis B specimens showed prominence of pSmad3L in hepatocytic nuclei. HBx-activated JNK/pSmad3L/c-Myc oncogenic pathway was enhanced, while the TbetaRI/pSmad3C/p21(WAF1) tumor-suppressive pathway was impaired as human and mouse HBx-associated hepatocarcinogenesis progressed. Of 28 patients with chronic hepatitis B who showed strong oncogenic pSmad3L signaling, six developed HCC within 12 years; only one of 32 patients showing little pSmad3L developed HCC. In contrast, seven of 30 patients with little Smad3C phosphorylation developed HCC, while no patient who retained hepatocytic tumor-suppressive pSmad3C developed HCC within 12 years. HBx shifts hepatocytic TGF-beta signaling from the tumor-suppressive pSmad3C pathway to the oncogenic pSmad3L pathway in early carcinogenic process. Hepatocytic pSmad3L and pSmad3C assessment in HBV-infected liver specimens should prove

  11. Aberrant hypertrophy in Smad3-deficient murine chondrocytes is rescued by restoring transforming growth factor beta-activated kinase 1/activating transcription factor 2 signaling: a potential clinical implication for osteoarthritis.

    PubMed

    Li, Tian-Fang; Gao, Lin; Sheu, Tzong-Jen; Sampson, Erik R; Flick, Lisa M; Konttinen, Yrjö T; Chen, Di; Schwarz, Edward M; Zuscik, Michael J; Jonason, Jennifer H; O'Keefe, Regis J

    2010-08-01

    To investigate the biologic significance of Smad3 in the progression of osteoarthritis (OA), the crosstalk between Smad3 and activating transcription factor 2 (ATF-2) in the transforming growth factor beta (TGFbeta) signaling pathway, and the effects of ATF-2 overexpression and p38 activation in chondrocyte differentiation. Joint disease in Smad3-knockout (Smad3(-/-)) mice was examined by microfocal computed tomography and histologic analysis. Numerous in vitro methods including immunostaining, real-time polymerase chain reaction, Western blotting, an ATF-2 DNA-binding assay, and a p38 kinase activity assay were used to study the various signaling responses and protein interactions underlying the altered chondrocyte phenotype in Smad3(-/-) mice. In Smad3(-/-) mice, an end-stage OA phenotype gradually developed. TGFbeta-activated kinase 1 (TAK1)/ATF-2 signaling was disrupted in Smad3(-/-) mouse chondrocytes at the level of p38 MAP kinase (MAPK) activation, resulting in reduced ATF-2 phosphorylation and transcriptional activity. Reintroduction of Smad3 into Smad3(-/-) cells restored the normal p38 response to TGFbeta. Phosphorylated p38 formed a complex with Smad3 by binding to a portion of Smad3 containing both the MAD homology 1 and linker domains. Additionally, Smad3 inhibited the dephosphorylation of p38 by MAPK phosphatase 1 (MKP-1). Both ATF-2 overexpression and p38 activation repressed type X collagen expression in wild-type and Smad3(-/-) chondrocytes. P38 was detected in articular cartilage and perichondrium; articular and sternal chondrocytes expressed p38 isoforms alpha, beta, and gamma, but not delta. Smad3 is involved in both the onset and progression of OA. Loss of Smad3 abrogates TAK1/ATF-2 signaling, most likely by disrupting the Smad3-phosphorylated p38 complex, thereby promoting p38 dephosphorylation and inactivation by MKP-1. ATF-2 and p38 activation inhibit chondrocyte hypertrophy. Modulation of p38 isoform activity may provide a new therapeutic

  12. Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kouchi, Zen, E-mail: zkouchi@toyaku.ac.jp; Fujiwara, Yuki; Yamaguchi, Hideki

    2011-05-20

    Highlights: {yields} We analyzed Phosphatidylinositol 5-phosphate kinase II{beta} (PIPKII{beta}) function in cancer. {yields} PIPKII{beta} is required for vitamin D receptor-mediated E-cadherin upregulation in SW480. {yields} PIPKII{beta} suppresses cellular motility through E-cadherin induction in SW480 cells. {yields} Nuclear PIP{sub 2} but not plasma membrane-localized PIP{sub 2} mediates E-cadherin upregulation. -- Abstract: Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1{alpha},25-dihydroxyvitamin D{sub 3} (1{alpha},25(OH){sub 2}D{sub 3}) has anti-cancer activity in several colon cancers. 1{alpha},25(OH){sub 2}D{sub 3} induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however,more » its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKII{beta}) but not PIPKII{alpha} is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLC{delta}1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P{sub 2}) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLC{delta}1 PHD inhibited 1{alpha},25(OH){sub 2}D{sub 3}-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P{sub 2} production mediates E-cadherin expression through PIPKII{beta} in a VDR-dependent manner. PIPKII{beta} is also involved in the suppression of the cell motility induced by 1{alpha},25(OH){sub 2}D{sub 3}. These results indicate that PIPKII{beta}-mediated PI(4,5)P{sub 2} signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.« less

  13. Procollagen C-proteinase enhancer-1 (PCPE-1) interacts with beta2-microglobulin (beta2-m) and may help initiate beta2-m amyloid fibril formation in connective tissues.

    PubMed

    Morimoto, Hisanori; Wada, Jun; Font, Bernard; Mott, Joni D; Hulmes, David J S; Ookoshi, Tadakazu; Naiki, Hironobu; Yasuhara, Akihiro; Nakatsuka, Atsuko; Fukuoka, Kousuke; Takatori, Yuji; Ichikawa, Haruo; Akagi, Shigeru; Nakao, Kazushi; Makino, Hirofumi

    2008-04-01

    Dialysis related amyloidosis (DRA) is a progressive and serious complication in patients under long-term hemodialysis and mainly leads to osteo-articular diseases. Although beta(2)-microglobulin (beta2-m) is the major structural component of beta2-m amyloid fibrils, the initiation of amyloid formation is not clearly understood. Here, we have identified procollagen C-proteinase enhancer-1 (PCPE-1) as a new interacting protein with beta2-m by screening a human synovium cDNA library. The interaction of beta2-m with full-length PCPE-1 was confirmed by immunoprecipitation, solid-phase binding and pull-down assays. By yeast two-hybrid analysis and pull-down assay, beta2-m appeared to interact with PCPE-1 via the NTR (netrin-like) domain and not via the CUB (C1r/C1s, Uegf and BMP-1) domain region. In synovial tissues derived from hemodialysis patients with DRA, beta2-m co-localized and formed a complex with PCPE-1. beta2-m did not alter the basal activity of bone morphogenetic protein-1/procollagen C-proteinase (BMP-1/PCP) nor BMP-1/PCP activity enhanced by PCPE-1. PCPE-1 did not stimulate beta2-m amyloid fibril formation from monomeric beta2-m in vitro under acidic and neutral conditions as revealed by thioflavin T fluorescence spectroscopy and electron microscopy. Since PCPE-1 is abundantly expressed in connective tissues rich in type I collagen, it may be involved in the initial accumulation of beta2-m in selected tissues such as tendon, synovium and bone. Furthermore, since such preferential deposition of beta2-m may be linked to subsequent beta2-m amyloid fibril formation, the disruption of the interaction between beta2-m and PCPE-1 may prevent beta2-m amyloid fibril formation and therefore PCPE-1 could be a new target for the treatment of DRA.

  14. Aggregation and lack of secretion of most newly synthesized proinsulin in non-beta-cell lines.

    PubMed

    Zhu, Yong Lian; Abdo, Alexander; Gesmonde, Joan F; Zawalich, Kathleen C; Zawalich, Walter; Dannies, Priscilla S

    2004-08-01

    Myoblasts transfected with HB10D insulin secrete more hormone than those transfected with wild-type insulin, as published previously, indicating that production of wild-type insulin is not efficient in these cells. The ability of non-beta-cells to produce insulin was examined in several cell lines. In clones of neuroendocrine GH(4)C(1) cells stably transfected with proinsulin, two thirds of (35)S-proinsulin was degraded within 3 h of synthesis, whereas (35)S-prolactin was stable. In transiently transfected neuroendocrine AtT20 cells, half of (35)S-proinsulin was degraded within 3 h after synthesis, whereas (35)S-GH was stable. In transiently transfected fibroblast COS cells, (35)S-proinsulin was stable for longer, but less than 10% was secreted 8 h after synthesis. Proinsulin formed a concentrated patch detected by immunofluorescence in transfected cells that did not colocalize with calreticulin or BiP, markers for the endoplasmic reticulum, but did colocalize with membrin, a marker for the cis-medial Golgi complex. Proinsulin formed a Lubrol-insoluble aggregate within 30 min after synthesis in non-beta-cells but not in INS-1E cells, a beta-cell line that normally produces insulin. More than 45% of (35)S-HB10D proinsulin was secreted from COS cells 3 h after synthesis, and this mutant formed less Lubrol-insoluble aggregate in the cells than did wild-type hormone. These results indicate that proinsulin production from these non-beta-cells is not efficient and that proinsulin aggregates in their secretory pathways. Factors in the environment of the secretory pathway of beta-cells may prevent aggregation of proinsulin to allow efficient production.

  15. Orphan nuclear receptor TLX activates Wnt/beta-catenin signalling to stimulate neural stem cell proliferation and self-renewal.

    PubMed

    Qu, Qiuhao; Sun, Guoqiang; Li, Wenwu; Yang, Su; Ye, Peng; Zhao, Chunnian; Yu, Ruth T; Gage, Fred H; Evans, Ronald M; Shi, Yanhong

    2010-01-01

    The nuclear receptor TLX (also known as NR2E1) is essential for adult neural stem cell self-renewal; however, the molecular mechanisms involved remain elusive. Here we show that TLX activates the canonical Wnt/beta-catenin pathway in adult mouse neural stem cells. Furthermore, we demonstrate that Wnt/beta-catenin signalling is important in the proliferation and self-renewal of adult neural stem cells in the presence of epidermal growth factor and fibroblast growth factor. Wnt7a and active beta-catenin promote neural stem cell self-renewal, whereas the deletion of Wnt7a or the lentiviral transduction of axin, a beta-catenin inhibitor, led to decreased cell proliferation in adult neurogenic areas. Lentiviral transduction of active beta-catenin led to increased numbers of type B neural stem cells in the subventricular zone of adult brains, whereas deletion of Wnt7a or TLX resulted in decreased numbers of neural stem cells retaining bromodeoxyuridine label in the adult brain. Both Wnt7a and active beta-catenin significantly rescued a TLX (also known as Nr2e1) short interfering RNA-induced deficiency in neural stem cell proliferation. Lentiviral transduction of an active beta-catenin increased cell proliferation in neurogenic areas of TLX-null adult brains markedly. These results strongly support the hypothesis that TLX acts through the Wnt/beta-catenin pathway to regulate neural stem cell proliferation and self-renewal. Moreover, this study suggests that neural stem cells can promote their own self-renewal by secreting signalling molecules that act in an autocrine/paracrine mode.

  16. Estrogen receptor-beta expression in invasive breast cancer in relation to molecular phenotype: results from the Nurses' Health Study.

    PubMed

    Marotti, Jonathan D; Collins, Laura C; Hu, Rong; Tamimi, Rulla M

    2010-02-01

    The expression of estrogen receptor-alpha (ER-alpha) and related genes has emerged as one of the major determinants of molecular classification of invasive breast cancers. Expression of a second ER, estrogen receptor-beta (ER-beta), has not been previously evaluated in a large population-based study. Therefore, we examined ER-beta expression in a large population of women with breast cancer to assess its relationship to molecular categories of invasive breast cancer. We constructed tissue microarrays from paraffin blocks of 3093 breast cancers that developed in women enrolled in the Nurses' Health Study. Tissue microarray sections were immunostained for ER-alpha, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), cytokeratin 5/6, epidermal growth factor receptor (EGFR) and with a monoclonal antibody to ER-beta. Cancers were categorized as luminal A (ER-alpha+ and/or PR+ and HER2-); luminal B (ER-alpha+ and/or PR+ and HER2+); HER2 (ER-alpha- and PR- and HER2+); and basal-like (ER-alpha-, PR-, HER2- and EGFR or cytokeratin 5/6+). The relationship between expression of ER-beta and molecular class of invasive breast cancer was analyzed. Overall, 68% of breast carcinomas were ER-beta+. Expression of ER-beta was significantly associated with expression of ER-alpha (P<0.0001) and PR (P<0.0001), and was inversely related to expression of HER2 (P=0.004), CK5/6 (P=0.02) and EGFR (P=0.006). Among 2170 invasive cancers with complete immunophenotypic data, 73% were luminal A, 5% luminal B, 6 % HER2 and 11% basal-like. ER-beta expression was significantly related to molecular category (P<0.0001) and was more common in luminal A (72% of cases) and B (68% of cases) than in HER2 or basal-like types. However, despite their being defined by the absence of ER-alpha expression, 55% of HER2-type and 60% of basal-like cancers showed expression of ER-beta. The role of ER-beta in the development and progression of breast cancers defined by lack of expression of ER

  17. GASDERMIN, suppressed frequently in gastric cancer, is a target of LMO1 in TGF-beta-dependent apoptotic signalling.

    PubMed

    Saeki, N; Kim, D H; Usui, T; Aoyagi, K; Tatsuta, T; Aoki, K; Yanagihara, K; Tamura, M; Mizushima, H; Sakamoto, H; Ogawa, K; Ohki, M; Shiroishi, T; Yoshida, T; Sasaki, H

    2007-10-04

    Defining apoptosis-regulatory cascades of the epithelium is important for understanding carcinogenesis, since cancer cells are considered to arise as a result of the collapse of the cascades. We previously reported that a novel gene GASDERMIN (GSDM) is expressed in the stomach but suppressed in gastric cancer cell lines. Furthermore, in this study, we demonstrated that GSDM is expressed in the mucus-secreting pit cells of the gastric epithelium and frequently silenced in primary gastric cancers. We found that GSDM has a highly apoptotic activity and its expression is regulated by a transcription factor LIM domain only 1 (LMO1) through a sequence to which Runt-related transcription factor 3 (RUNX3) binds, in a GSDM promoter region. We observed coexpression of GSDM with LMO1, RUNX3 and type II transforming growth factor-beta receptor (TGF-betaRII) in the pit cells, and found that TGF-beta upregulates the LMO1- and GSDM-expression in the gastric epithelial cell line and induces apoptosis, which was confirmed by the finding that the apoptosis induction is inhibited by suppression of each LMO1-, RUNX3- and GSDM expression, respectively. The present data suggest that TGF-beta, LMO1, possibly RUNX3, and GSDM form a regulatory pathway for directing the pit cells to apoptosis.

  18. The islet beta-cell: fuel responsive and vulnerable.

    PubMed

    Nolan, Christopher J; Prentki, Marc

    2008-10-01

    The pancreatic beta-cell senses blood nutrient levels and is modulated by neurohormonal signals so that it secretes insulin according to the need of the organism. Nutrient sensing involves marked metabolic activation, resulting in the production of coupling signals that promote insulin biosynthesis and secretion. The beta-cell's high capacity for nutrient sensing, however, necessitates reduced protection to nutrient toxicity. This potentially explains why in susceptible individuals, chronic fuel surfeit results in beta-cell failure and type 2 diabetes. Here we discuss recent insights into first, the biochemical basis of beta-cell signaling in response to glucose, amino acids and fatty acids, and second, beta-cell nutrient detoxification. We emphasize the emerging role of glycerolipid/fatty acid cycling in these processes.

  19. Role of Flightless-I (Drosophila) homolog in the transcription activation of type I collagen gene mediated by transforming growth factor beta

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lim, Mi-Sun; Jeong, Kwang Won, E-mail: kwjeong@gachon.ac.kr

    Highlights: • FLII activates TGFβ-mediated expression of COL1A2 gene. • TGFβ induces the association of FLII with SMAD3 and BRG1 in A549 cells. • FLII is required for the recruitment of SWI/SNF complex and chromatin accessibility to COL1A2 promoter. - Abstract: Flightless-I (Drosophila) homolog (FLII) is a nuclear receptor coactivator that is known to interact with other transcriptional regulators such as the SWI/SNF complex, an ATP-dependent chromatin-remodeling complex, at the promoter or enhancer region of estrogen receptor (ER)-α target genes. However, little is known about the role of FLII during transcription initiation in the transforming growth factor beta (TGFβ)/SMAD-dependent signalingmore » pathway. Here, we demonstrate that FLII functions as a coactivator in the expression of type I collagen gene induced by TGFβ in A549 cells. FLII activates the reporter gene driven by COL1A2 promoter in a dose-dependent manner. Co-expression of GRIP1, CARM1, or p300 did not show any synergistic activation of transcription. Furthermore, the level of COL1A2 expression correlated with the endogenous level of FLII mRNA level. Depletion of FLII resulted in a reduction of TGFβ-induced expression of COL1A2 gene. In contrast, over-expression of FLII caused an increase in the endogenous expression of COL1A2. We also showed that FLII is associated with Brahma-related gene 1 (BRG1) as well as SMAD in A549 cells. Notably, the recruitment of BRG1 to the COL1A2 promoter region was decreased in FLII-depleted A549 cells, suggesting that FLII is required for TGFβ-induced chromatin remodeling, which is carried out by the SWI/SNF complex. Furthermore, formaldehyde-assisted isolation of regulatory elements (FAIRE)-quantitative polymerase chain reaction (qPCR) experiments revealed that depletion of FLII caused a reduction in chromatin accessibility at the COL1A2 promoter. These results suggest that FLII plays a critical role in TGFβ/SMAD-mediated transcription of the COL1A2

  20. Expression and function of CD8 alpha/beta chains on rat and human mast cells.

    PubMed

    Kim, Mi-Sun; Kim, Sung-Hoon; Lee, Hye-Jung; Kim, Hyung-Min

    2004-03-01

    The expression and functional role of CD8 glycoprotein, a marker of cytotoxic/suppressor T lymphocytes and NK cells, were not studied on freshly isolated connective tissue type rat peritoneal mast cells, a rat mucosal type mast cell line (RBL 2H3), or human mast cell line (HMC-1). We used the reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, immunohistochemistry and enzyme-linked immunosorbent assay. RT-PCR and Western blot analysis identified the presence of CD8 alpha/beta chains on the mast cells, and immunohistochemistry confirmed CD8alpha expression on rat or human mast cells. Functional studies demonstrated that stimulation of CD8 alpha/beta chains on rat mast cells induced the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), which are regarded as important mediators during infection. However, co-stimulation with stem cell factor had no effect on CD8-induced mediator secretion. Our findings demonstrate novel biological roles of CD8 molecules in mast cells.

  1. Opposite effects of dihydrosphingosine 1-phosphate and sphingosine 1-phosphate on transforming growth factor-beta/Smad signaling are mediated through the PTEN/PPM1A-dependent pathway.

    PubMed

    Bu, Shizhong; Kapanadze, Bagrat; Hsu, Tien; Trojanowska, Maria

    2008-07-11

    Transforming growth factor-beta (TGF-beta) is an important regulator of physiological connective tissue biosynthesis and plays a central role in pathological tissue fibrosis. Previous studies have established that a biologically active lipid mediator, sphingosine 1-phosphate (S1P), mimics some of the profibrotic functions of TGF-beta through cross-activation of Smad signaling. Here we report that another product of sphingosine kinase, dihydrosphingosine 1-phosphate (dhS1P), has an opposite role in the regulation of TGF-beta signaling. In contrast to S1P, dhS1P inhibits TGF-beta-induced Smad2/3 phosphorylation and up-regulation of collagen synthesis. The effects of dhS1P require a lipid phosphatase, PTEN, a key modulator of cell growth and survival. dhS1P stimulates phosphorylation of the C-terminal domain of PTEN and its subsequent translocation into the nucleus. We demonstrate a novel function of nuclear PTEN as a co-factor of the Smad2/3 phosphatase, PPM1A. Complex formation of PTEN with PPM1A does not require the lipid phosphatase activity but depends on phosphorylation of the serine/threonine residues located in the C-terminal domain of PTEN. Upon complex formation with PTEN, PPM1A is protected from degradation induced by the TGF-beta signaling. Consequently, overexpression of PTEN abrogates TGF-beta-induced Smad2/3 phosphorylation. This study establishes a novel role for nuclear PTEN in the stabilization of PPM1A. PTEN-mediated cross-talk between the sphingolipid and TGF-beta signaling pathways may play an important role in physiological and pathological TGF-beta signaling.

  2. Novel Computational Protocols for Functionally Classifying and Characterising Serine Beta-Lactamases

    PubMed Central

    Das, Sayoni; Dawson, Natalie L.; Dobrijevic, Dragana; Orengo, Christine

    2016-01-01

    Beta-lactamases represent the main bacterial mechanism of resistance to beta-lactam antibiotics and are a significant challenge to modern medicine. We have developed an automated classification and analysis protocol that exploits structure- and sequence-based approaches and which allows us to propose a grouping of serine beta-lactamases that more consistently captures and rationalizes the existing three classification schemes: Classes, (A, C and D, which vary in their implementation of the mechanism of action); Types (that largely reflect evolutionary distance measured by sequence similarity); and Variant groups (which largely correspond with the Bush-Jacoby clinical groups). Our analysis platform exploits a suite of in-house and public tools to identify Functional Determinants (FDs), i.e. residue sites, responsible for conferring different phenotypes between different classes, different types and different variants. We focused on Class A beta-lactamases, the most highly populated and clinically relevant class, to identify FDs implicated in the distinct phenotypes associated with different Class A Types and Variants. We show that our FunFHMMer method can separate the known beta-lactamase classes and identify those positions likely to be responsible for the different implementations of the mechanism of action in these enzymes. Two novel algorithms, ASSP and SSPA, allow detection of FD sites likely to contribute to the broadening of the substrate profiles. Using our approaches, we recognise 151 Class A types in UniProt. Finally, we used our beta-lactamase FunFams and ASSP profiles to detect 4 novel Class A types in microbiome samples. Our platforms have been validated by literature studies, in silico analysis and some targeted experimental verification. Although developed for the serine beta-lactamases they could be used to classify and analyse any diverse protein superfamily where sub-families have diverged over both long and short evolutionary timescales. PMID

  3. Amyloid-beta aggregation: selective inhibition of aggregation in mixtures of amyloid with different chain lengths.

    PubMed Central

    Snyder, S W; Ladror, U S; Wade, W S; Wang, G T; Barrett, L W; Matayoshi, E D; Huffaker, H J; Krafft, G A; Holzman, T F

    1994-01-01

    One of the clinical manifestations of Alzheimer's disease is the deposition of the 39-43 residue amyloid-beta (A beta) peptide in aggregated fibrils in senile plaques. Characterization of the aggregation behavior of A beta is one of the critical issues in understanding the role of A beta in the disease process. Using solution hydrodynamics, A beta was observed to form three types of species in phosphate-buffered saline: insoluble aggregates with sedimentation coefficients of approximately 50,000 S and molecular masses of approximately 10(9) Da, "soluble aggregates" with sedimentation coefficients of approximately 30 S and masses of approximately 10(6) Da, and monomer. When starting from monomer, the aggregation kinetics of A beta 1-40 (A beta 40) and A beta 1-42 (A beta 42), alone and in combination, reveal large differences in the tendency of these peptides to aggregate as a function of pH and other solution conditions. At pH 4.1 and 7.0-7.4, aggregation is significantly slower than at pH 5 and 6. Under all conditions, aggregation of the longer A beta 42 was more rapid than A beta 40. Oxidation of Met-35 to the sulfoxide in A beta 40 enhances the aggregation rate over that of the nonoxidized peptide. Aggregation was found to be dependent upon temperature and to be strongly dependent on peptide concentration and ionic strength, indicating that aggregation is driven by a hydrophobic effect. When A beta 40 and A beta 42 are mixed together, A beta 40 retards the aggregation of A beta 42 in a concentration-dependent manner. Shorter fragments have a decreasing ability to interfere with A beta 42 aggregation. Conversely, the rate of aggregation of A beta 40 can be significantly enhanced by seeding slow aggregating solutions with preformed aggregates of A beta 42. Taken together, the inhibition of A beta 42 aggregation by A beta 40, the seeding of A beta 40 aggregation by A beta 42 aggregates, and the chemical oxidation of A beta 40 suggest that the relative abundance and

  4. In vitro effects of beta-lactams combined with beta-lactamase inhibitors against methicillin-resistant Staphylococcus aureus.

    PubMed Central

    Kobayashi, S; Arai, S; Hayashi, S; Sakaguchi, T

    1989-01-01

    The effects of combinations of beta-lactams with two beta-lactamase inhibitors, sulbactam and clavulanic acid, were determined in vitro against 22 clinical isolates of methicillin-resistant Staphylococcus aureus. Combinations of cefpirome, cefotaxime, and cefazolin with sulbactam (10 micrograms/ml) showed synergistic effects against more than 70% of the strains. Combinations of methicillin and penicillin G with sulbactam also showed synergistic effects against 50 and 68% of the strains, respectively, while cefotiam, moxalactam, flomoxef, and cefmetazole in combination with sulbactam showed such effects against only 40% or fewer. Clavulanic acid was synergistic only when combined with penicillin G, the effect probably being due to the beta-lactamase inhibition by the inhibitor. Sulbactam did not improve the antimicrobial activities of the beta-lactams against methicillin-susceptible S. aureus strains. At 42 degrees C the MICs of cefotaxime, methicillin, and flomoxef alone were markedly decreased from the values at 35 degrees C, and no synergy between these beta-lactams and sulbactam appeared. The resistance to penicillin G was not inhibited by incubation at 42 degrees C, and combinations of penicillin G with sulbactam and clavulanic acid showed synergy. The amounts of beta-lactamase produced were not related to the decreases in the MICs of the beta-lactams, except for penicillin G combined with sulbactam. Clavulanic acid showed slightly stronger beta-lactamase-inhibiting activity than sulbactam did. These results suggest that the synergy between sulbactam and the beta-lactams, except for penicillin G, may not be due to beta-lactamase inhibition but to suppression of the methicillin-resistant S. aureus-specific resistance based on other factors. PMID:2786369

  5. [Influence of physiologic 17 beta-estradiol concentrations on gene E6 expression in HVP type 18 in vitro].

    PubMed

    Dziubińska-Parol, Izabella; Gasowska, Urszula; Rzymowska, Jolanta; Kwaśniewska, Anna

    2003-09-01

    Many recent studies indicate that long term use of contraceptives is a strong risk factor in the development of cervical cancer. Steroid hormones, in persistent papilloma virus infection act on various levels, one of them is enhancing transforming activity of the virus. The aim of the study was to estimate if physiological concentrations of 17 beta-estradiol could influence expression of viral transforming genes. HeLa cell lines were incubated with three different physiological concentrations and and on the third day of incubation the level of E6 gene expression was determined. Results show no differences in expression between the control culter, and cultures incubated with physiological concentrations. It indicates that normal levels of 17 beta-estradiol don't play role in transforming process but it also shows need to analyse higher levels of hormones by quantitative analyses in prospective studies.

  6. Polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Morant, Marc

    The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  7. A segmented, enriched N-type germanium detector for neutrinoless double beta-decay experiments

    NASA Astrophysics Data System (ADS)

    Leviner, L. E.; Aalseth, C. E.; Ahmed, M. W.; Avignone, F. T.; Back, H. O.; Barabash, A. S.; Boswell, M.; De Braeckeleer, L.; Brudanin, V. B.; Chan, Y.-D.; Egorov, V. G.; Elliott, S. R.; Gehman, V. M.; Hossbach, T. W.; Kephart, J. D.; Kidd, M. F.; Konovalov, S. I.; Lesko, K. T.; Li, Jingyi; Mei, D.-M.; Mikhailov, S.; Miley, H.; Radford, D. C.; Reeves, J.; Sandukovsky, V. G.; Umatov, V. I.; Underwood, T. A.; Tornow, W.; Wu, Y. K.; Young, A. R.

    2014-01-01

    We present data characterizing the performance of the first segmented, N-type Ge detector, isotopically enriched to 85% 76Ge. This detector, based on the Ortec PT6×2 design and referred to as SEGA (Segmented, Enriched Germanium Assembly), was developed as a possible prototype for neutrinoless double beta-decay measurements by the MAJORANA collaboration. We present some of the general characteristics (including bias potential, efficiency, leakage current, and integral cross-talk) for this detector in its temporary cryostat. We also present an analysis of the resolution of the detector, and demonstrate that for all but two segments there is at least one channel that reaches the MAJORANA resolution goal below 4 keV FWHM at 2039 keV, and all channels are below 4.5 keV FWHM.

  8. Defining the carbohydrate specificities of Abrus precatorius agglutinin as T (Gal beta 1----3GalNAc) greater than I/II (Gal beta 1----3/4GlcNAc).

    PubMed

    Wu, A M; Lin, S R; Chin, L K; Chow, L P; Lin, J Y

    1992-09-25

    The combining site of the nontoxic carbohydrate binding protein (Abrus precatorius agglutinin, APA) purified from the needs of Abrus precatorius (Jequirity bean), was studied by quantitative precipitin and precipitin-inhibition assays. Of 26 glycoproteins and polysaccharides tested, all, except sialic acid-containing glycoproteins and desialized ovine salivary glycoproteins, reacted strongly with the lectin, and precipitated over 70% of the lectin added, indicating that APA has a broad range of affinity and recognizes (internal) Gal beta 1----sequences of carbohydrate chains. The strong reaction with desialized porcine and rat salivary glycoproteins as well as pneumococcus type XIV polysaccharide suggests that APA has affinity for one or more of the following carbohydrate sequences: Thomsen-Friedenreich (T, Gal beta 1----3GalNAc), blood group precursor type I and/or type II (Gal beta 1----3/4GlcNAc) disaccharide determinants of complex carbohydrates. Among the oligosaccharides tested, the T structure was the best inhibitor; it was 2.4 and 3.2 times more active than type II and type I sequences, respectively. The blood group I Ma-active trisaccharide, Gal beta 1----4GlcNAc beta 1----6Gal, was about as active as the corresponding disaccharide (II). From the above results, we conclude that the size of the combining site of the A. precatorius agglutinin is probably as large as a disaccharide and most strongly complementary to the Gal beta 1----3GalNAc (T determinant) sequence. The carbohydrate specificities of this lectin will be further investigated once the related oligosaccharide structures become available.

  9. Inhibition of transforming growth factor-beta1-induced signaling and epithelial-to-mesenchymal transition by the Smad-binding peptide aptamer Trx-SARA.

    PubMed

    Zhao, Bryan M; Hoffmann, F Michael

    2006-09-01

    Overexpression of the inhibitory Smad, Smad7, is used frequently to implicate the Smad pathway in cellular responses to transforming growth factor beta (TGF-beta) signaling; however, Smad7 regulates several other proteins, including Cdc42, p38MAPK, and beta-catenin. We report an alternative approach for more specifically disrupting Smad-dependent signaling using a peptide aptamer, Trx-SARA, which comprises a rigid scaffold, the Escherichia coli thioredoxin A protein (Trx), displaying a constrained 56-amino acid Smad-binding motif from the Smad anchor for receptor activation (SARA) protein. Trx-SARA bound specifically to Smad2 and Smad3 and inhibited both TGF-beta-induced reporter gene expression and epithelial-to-mesenchymal transition in NMuMG murine mammary epithelial cells. In contrast to Smad7, Trx-SARA had no effect on the Smad2 or 3 phosphorylation levels induced by TGF-beta1. Trx-SARA was primarily localized to the nucleus and perturbed the normal cytoplasmic localization of Smad2 and 3 to a nuclear localization in the absence of TGF-beta1, consistent with reduced Smad nuclear export. The key mode of action of Trx-SARA was to reduce the level of Smad2 and Smad3 in complex with Smad4 after TGF-beta1 stimulation, a mechanism of action consistent with the preferential binding of SARA to monomeric Smad protein and Trx-SARA-mediated disruption of active Smad complexes.

  10. Relative stability of major types of beta-turns as a function of amino acid composition: a study based on Ab initio energetic and natural abundance data.

    PubMed

    Perczel, András; Jákli, Imre; McAllister, Michael A; Csizmadia, Imre G

    2003-06-06

    Folding properties of small globular proteins are determined by their amino acid sequence (primary structure). This holds both for local (secondary structure) and for global conformational features of linear polypeptides and proteins composed from natural amino acid derivatives. It thus provides the rational basis of structure prediction algorithms. The shortest secondary structure element, the beta-turn, most typically adopts either a type I or a type II form, depending on the amino acid composition. Herein we investigate the sequence-dependent folding stability of both major types of beta-turns using simple dipeptide models (-Xxx-Yyy-). Gas-phase ab initio properties of 16 carefully selected and suitably protected dipeptide models (for example Val-Ser, Ala-Gly, Ser-Ser) were studied. For each backbone fold most probable side-chain conformers were considered. Fully optimized 321G RHF molecular structures were employed in medium level [B3LYP/6-311++G(d,p)//RHF/3-21G] energy calculations to estimate relative populations of the different backbone conformers. Our results show that the preference for beta-turn forms as calculated by quantum mechanics and observed in Xray determined proteins correlates significantly.

  11. Residual beta cell function in newly diagnosed type 1 diabetes after treatment with atorvastatin: the Randomized DIATOR Trial.

    PubMed

    Martin, Stephan; Herder, Christian; Schloot, Nanette C; Koenig, Wolfgang; Heise, Tim; Heinemann, Lutz; Kolb, Hubert

    2011-03-11

    Recent evidence suggests that the lipid-lowering agent atorvastatin is also a potent immunomodulator. The aim of this study was to investigate the possible effect of atorvastatin on the decline of residual beta cell function in recent-onset type 1 diabetes. The randomised placebo-controlled Diabetes and Atorvastatin (DIATOR) Trial included 89 patients with newly diagnosed type 1 diabetes and islet autoantibodies (mean age 30 years, 40% females), in 12 centres in Germany. Patients received placebo or 80 mg/d atorvastatin for 18 months. As primary outcome stimulated serum C-peptide levels were determined 90 min after a standardized liquid mixed meal. An intent-to-treat analysis was performed. Fasting and stimulated C-peptide levels were not significantly different between groups at 18 months. However, median fasting serum C-peptide levels dropped from baseline to 12 and 18 months in the placebo group (from 0. 34 to 0.23 and 0.20 nmol/l, p<0.001) versus a nonsignificant decline in the atorvastatin group (from 0.34 to 0.27 and 0.30 nmol/l, ns). Median stimulated C-peptide concentrations declined between baseline and 12 months (placebo from 0.89 to 0.71 nmol/l, atorvastatin from 0.88 to 0.73 nmol/l, p<0.01 each) followed by a major loss by month 18 in the placebo group (to 0.48 nmol/l, p = 0.047) but not in the atorvastatin group (to 0.71 nmol/l, ns). Median levels of total cholesterol and C-reactive protein decreased in the atorvastatin group only (p<0.001 and p = 0.04). Metabolic control was similar between groups. Atorvastatin treatment did not significantly preserve beta cell function although there may have been a slower decline of beta-cell function which merits further study. ClinicalTrials.gov NCT00974740.

  12. Competition between Ski and CREB-binding protein for binding to Smad proteins in transforming growth factor-beta signaling.

    PubMed

    Chen, Weijun; Lam, Suvana S; Srinath, Hema; Schiffer, Celia A; Royer, William E; Lin, Kai

    2007-04-13

    The family of Smad proteins mediates transforming growth factor-beta (TGF-beta) signaling in cell growth and differentiation. Smads repress or activate TGF-beta signaling by interacting with corepressors (e.g. Ski) or coactivators (e.g. CREB-binding protein (CBP)), respectively. Specifically, Ski has been shown to interfere with the interaction between Smad3 and CBP. However, it is unclear whether Ski competes with CBP for binding to Smads and whether they can interact with Smad3 at the same binding surface on Smad3. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, we show that Ski-(16-192) interacted directly with a homotrimer of receptor-regulated Smad protein (R-Smad), e.g. Smad2 or Smad3, to form a hexamer; Ski-(16-192) interacted with an R-Smad.Smad4 heterotrimer to form a pentamer. CBP-(1941-1992) was also found to interact directly with an R-Smad homotrimer to form a hexamer and with an R-Smad.Smad4 heterotrimer to form a pentamer. Moreover, these domains of Ski and CBP competed with each other for binding to Smad3. Our mutational studies revealed that domains of Ski and CBP interacted with Smad3 at a portion of the binding surface of the Smad anchor for receptor activation. Our results suggest that Ski negatively regulates TGF-beta signaling by replacing CBP in R-Smad complexes. Our working model suggests that Smad protein activity is delicately balanced by Ski and CBP in the TGF-beta pathway.

  13. Norrin mediates neuroprotective effects on retinal ganglion cells via activation of the Wnt/beta-catenin signaling pathway and the induction of neuroprotective growth factors in Muller cells.

    PubMed

    Seitz, Roswitha; Hackl, Simon; Seibuchner, Thomas; Tamm, Ernst R; Ohlmann, Andreas

    2010-04-28

    Norrin is a secreted protein that binds to frizzled 4 and controls development of capillaries in retina and inner ear. We provide evidence that Norrin has distinct neuroprotective properties that are independent from its effects on vascular development. The function of Norrin was investigated in a mouse model of excitotoxic retinal ganglion cell (RGC) damage after intravitreal injection of NMDA, and in cultured Müller glia or immortalized RGC-5 cells. Intravitreal injection of Norrin significantly increased the number of surviving RGC axons in the optic nerve and decreased apoptotic death of retinal neurons following NMDA-mediated damage. This effect could be blocked by adding dickkopf (DKK)-1, an inhibitor of the Wnt/beta-catenin signaling pathway. Treatment of eyes with combined Norrin/NMDA activated Wnt/beta-catenin signaling and increased the retinal expression of leukemia inhibitory factor and endothelin-2, as well as that of neurotrophic growth factors such as fibroblast growth factor-2, brain-derived neurotrophic factor, lens epithelium-derived growth factor, and ciliary neurotrophic factor. A similar activation of Wnt/beta-catenin signaling and an increased expression of neurotrophic factors was observed in cultured Müller cells after treatment with Norrin, effects that again could be blocked by adding DKK-1. In addition, conditioned cell culture medium of Norrin-treated Müller cells increased survival of differentiated RGC-5 cells. We conclude that Norrin has pronounced neuroprotective properties on retinal neurons with the distinct potential to decrease the damaging effects of NMDA-induced RGC loss. The effects of Norrin involve activation of Wnt/beta-catenin signaling and subsequent induction of neurotrophic growth factors in Müller cells.

  14. Beta Factors for Collinear Asymmetrical Cracks Emanating from an Offset Circular Hole in a Rectangular Plate

    DTIC Science & Technology

    2016-05-01

    Lorimer Street Fishermans Bend, Victoria 3207, Australia Telephone: 1300 333 362 © Commonwealth of Australia 2016 AR-016-732 May 2016...multiple through cracks in plates with and without holes (Final Report for 25 August 1997 – 31 July 2002). AFRL-VA-WP-TR-2004-3112, October 2004. 11. J...remote tension stress. UNCLASSIFIED DST-Group-RR-0437 25 UNCLASSIFIED Figure 15: Beta factors FcR for the right-hand crack of two collinear

  15. Foreign travel is a major risk factor for colonization with Escherichia coli producing CTX-M-type extended-spectrum beta-lactamases: a prospective study with Swedish volunteers.

    PubMed

    Tängdén, Thomas; Cars, Otto; Melhus, Asa; Löwdin, Elisabeth

    2010-09-01

    Foreign travel has been suggested to be a risk factor for the acquisition of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae. To our knowledge, this has not previously been demonstrated in a prospective study. Healthy volunteers traveling outside Northern Europe were enrolled. Rectal swabs and data on potential travel-associated risk factors were collected before and after traveling. A total of 105 volunteers were enrolled. Four of them did not complete the study, and one participant carried ESBL-producing Escherichia coli before travel. Twenty-four of 100 participants with negative pretravel samples were colonized with ESBL-producing Escherichia coli after the trip. All strains produced CTX-M enzymes, mostly CTX-M-15, and some coproduced TEM or SHV enzymes. Coresistance to several antibiotic subclasses was common. Travel to India was associated with the highest risk for the acquisition of ESBLs (88%; n = 7). Gastroenteritis during the trip was an additional risk factor (P = 0.003). Five of 21 volunteers who completed the follow-up after 6 months had persistent colonization with ESBLs. This is the first prospective study demonstrating that international travel is a major risk factor for colonization with ESBL-producing Enterobacteriaceae. Considering the high acquisition rate of 24%, it is obvious that global efforts are needed to meet the emergence and spread of CTX-M enzymes and other antimicrobial resistances.

  16. Cleavage of beta,beta-carotene to flavor compounds by fungi.

    PubMed

    Zorn, H; Langhoff, S; Scheibner, M; Berger, R G

    2003-09-01

    More than 50 filamentous fungi and yeasts, known for de novo synthesis or biotransformation of mono-, sesqui-, tri-, or tetraterpenes, were screened for their ability to cleave beta,beta-carotene to flavor compounds. Ten strains discolored a beta,beta-carotene-containing growth agar, indicating efficient degradation of beta,beta-carotene. Dihydroactinidiolide was formed as the sole conversion product of beta,beta-carotene in submerged cultures of Ganoderma applanatum, Hypomyces odoratus, Kuehneromyces mutabilis, and Trametes suaveolens. When mycelium-free culture supernatants from five species were applied for the conversions, nearly complete degradation of beta,beta-carotene was observed after 12 h. Carotenoid-derived volatile products were detected in the media of Ischnoderma benzoinum, Marasmius scorodonius, and Trametes versicolor. beta-Ionone proved to be the main metabolite in each case, whereas beta-cyclocitral, dihydroactinidiolide, and 2-hydroxy-2,6,6-trimethylcyclohexanone were formed in minor quantities. Using a photometric bleaching test, the beta,beta-carotene cleaving enzyme activities of M. scorodonius were partially characterized.

  17. Inhibition of liver fibrosis by solubilized coenzyme Q10: Role of Nrf2 activation in inhibiting transforming growth factor-beta1 expression

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Choi, Hoo-Kyun; Pokharel, Yuba Raj; Lim, Sung Chul

    2009-11-01

    Coenzyme Q10 (CoQ10), an endogenous antioxidant, is important in oxidative phosphorylation in mitochondria. It has anti-diabetic and anti-cardiovascular disease effects, but its ability to protect against liver fibrosis has not been studied. Here, we assessed the ability of solubilized CoQ10 to improve dimethylnitrosamine (DMN)-induced liver fibrogenesis in mice. DMN treatments for 3 weeks produced a marked liver fibrosis as assessed by histopathological examination and tissue 4-hydroxyproline content. Solubilized CoQ10 (10 and 30 mg/kg) significantly inhibited both the increases in fibrosis score and 4-hydroxyproline content induced by DMN. Reverse transcription-polymerase chain reaction and Western blot analyses revealed that solubilized CoQ10 inhibitedmore » increases in the transforming growth factor-beta1 (TGF-beta1) mRNA and alpha-smooth muscle actin (alpha-SMA) protein by DMN. Interestingly, hepatic glutamate-cysteine ligase (GCL) and glutathione S-transferase A2 (GSTA2) were up-regulated in mice treated with CoQ10. Solubilized CoQ10 also up-regulated antioxidant enzymes such as catalytic subunits of GCL and GSTA2 via activating NF-E2 related factor2 (Nrf2)/antioxidant response element (ARE) in H4IIE hepatoma cells. Moreover, CoQ10's inhibition of alpha-SMA and TGF-beta1 expressions disappeared in Nrf2-null MEF cells. In contrast, Nrf2 overexpression significantly decreased the basal expression levels of alpha-SMA and TGF-beta1 in Nrf2-null MEF cells. These results demonstrated that solubilized CoQ10 inhibited DMN-induced liver fibrosis through suppression of TGF-beta1 expression via Nrf2/ARE activation.« less

  18. Regulatory T cells protect mice against coxsackievirus-induced myocarditis through the transforming growth factor beta-coxsackie-adenovirus receptor pathway.

    PubMed

    Shi, Yu; Fukuoka, Masahiro; Li, Guohua; Liu, Youan; Chen, Manyin; Konviser, Michael; Chen, Xin; Opavsky, Mary Anne; Liu, Peter P

    2010-06-22

    Coxsackievirus B3 infection is an excellent model of human myocarditis and dilated cardiomyopathy. Cardiac injury is caused either by a direct cytopathic effect of the virus or through immune-mediated mechanisms. Regulatory T cells (Tregs) play an important role in the negative modulation of host immune responses and set the threshold of autoimmune activation. This study was designed to test the protective effects of Tregs and to determine the underlying mechanisms. Carboxyfluorescein diacetate succinimidyl ester-labeled Tregs or naïve CD4(+) T cells were injected intravenously once every 2 weeks 3 times into mice. The mice were then challenged with intraperitoneal coxsackievirus B3 immediately after the last cell transfer. Transfer of Tregs showed higher survival rates than transfer of CD4(+) T cells (P=0.0136) but not compared with the PBS injection group (P=0.0589). Interestingly, Tregs also significantly decreased virus titers and inflammatory scores in the heart. Transforming growth factor-beta and phosphorylated AKT were upregulated in Tregs-transferred mice and coxsackie-adenovirus receptor expression was decreased in the heart compared with control groups. Transforming growth factor-beta decreased coxsackie-adenovirus receptor expression and inhibited coxsackievirus B3 infection in HL-1 cells and neonatal cardiac myocytes. Splenocytes collected from Treg-, CD4(+) T-cell-, and PBS-treated mice proliferated equally when stimulated with heat-inactivated virus, whereas in the Treg group, the proliferation rate was reduced significantly when stimulated with noninfected heart tissue homogenate. Adoptive transfer of Tregs protected mice from coxsackievirus B3-induced myocarditis through the transforming growth factor beta-coxsackie-adenovirus receptor pathway and thus suppresses the immune response to cardiac tissue, maintaining the antiviral immune response.

  19. Existence of a regulatory loop between MCP-1 and TGF-beta in glomerular immune injury.

    PubMed

    Wolf, Gunter; Jocks, Thomas; Zahner, Gunther; Panzer, Ulf; Stahl, Rolf A K

    2002-11-01

    Glomerular upregulation of monocyte chemotactic protein-1 (MCP-1), followed by an influx of monocytes resulting eventually in extracellular matrix deposition is a common sequel of many types of glomerulonephritis. However, it is not entirely clear how early expression of MCP-1 is linked to the later development of glomerulosclerosis. Because transforming growth factor-beta (TGF-beta) is a key regulator of extracellular matrix proteins, we hypothesized that there might be a regulatory loop between early glomerular MCP-1 induction and subsequent TGF-beta expression. To avoid interference with other cytokines that may be released from infiltrating monocytes, isolated rat kidneys were perfused with a polyclonal anti-thymocyte-1 antiserum (ATS) and rat serum (RS) as a complement source to induce glomerular injury. Renal TGF-beta protein and mRNA expressions were strongly stimulated after perfusion with ATS-RS. This effect was attenuated by coperfusion with a neutralizing anti-MCP-1 but was partly mimicked by perfusion with recombinant MCP-1 protein. On the other hand, renal MCP-1 expression and production were stimulated by administration of ATS-RS. Additional perfusion with an anti-TGF-beta antibody further aggravated this increase, whereas application of recombinant TGF-beta protein reduced MCP-1 formation. Our data demonstrate an intrinsic regulatory loop in which increased MCP-1 levels stimulate TGF-beta formation in resident glomerular cells in the absence of infiltrating immune competent cells.

  20. beta-Hexachlorocyclohexane (beta-HCH)

    Integrated Risk Information System (IRIS)

    beta - Hexachlorocyclohexane ( beta - HCH ) ; CASRN 319 - 85 - 7 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Asses

  1. TGF-{beta}-stimulated aberrant expression of class III {beta}-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chung, Eun Jee; Chun, Ji Na; Jung, Sun-Ah

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer TGF-{beta} induces aberrant expression of {beta}III in RPE cells via the ERK pathway. Black-Right-Pointing-Pointer TGF-{beta} increases O-GlcNAc modification of {beta}III in RPE cells. Black-Right-Pointing-Pointer Mature RPE cells have the capacity to express a neuron-associated gene by TGF-{beta}. -- Abstract: The class III {beta}-tubulin isotype ({beta}{sub III}) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III {beta}-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology inmore » pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-{beta} (TGF-{beta}) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-{beta} on the aberrant expression of class III {beta}-tubulin and the intracellular signaling pathway mediating these changes. TGF-{beta}-induced aberrant expression and O-linked-{beta}-N-acetylglucosamine (O-GlcNac) modification of class III {beta}-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-{beta} also stimulated phosphorylation of ERK. TGF-{beta}-induced aberrant expression of class III {beta}-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-{beta} stimulated aberrant expression of class III {beta}-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-{beta} stimulation and provide useful

  2. The 76Ge Program to Search for Neutrinoless Double-Beta Decay

    NASA Astrophysics Data System (ADS)

    Guiseppe, Vincente

    2017-09-01

    Neutrinoless double-beta decay searches play a major role in determining the nature of neutrinos, the existence of a lepton violating process, and the effective Majorana neutrino mass. The type="smallcap">Majorana and type="smallcap">Gerda Collaborations are operating arrays of high purity Ge detectors to search for neutrinoless double-beta decay in 76Ge. The type="smallcap">Majorana type="smallcap">Demonstrator is operating at the Sanford Underground Research Facility in South Dakota while the type="smallcap">Gerda experiment is operating at LNGS in Italy. The type="smallcap">Gerda and type="smallcap">Majorana type="smallcap">Demonstrator experiments have achieved the lowest backgrounds in the neutrinoless double-beta decay region of interest. These results, coupled with the superior energy resolution (0.1%) of Ge detectors demonstrate that 76Ge is an ideal isotope for a large next generation experiment. The LEGEND collaboration, with 220 members from 47 institutions around the world, has been formed to pursue a ton scale 76Ge experiment. Building on the successes of type="smallcap">Gerda and type="smallcap">Majorana, the LEGEND collaboration aims to develop a phased neutrinoless double-beta decay experimental program with discovery potential at a half-life significantly longer than 1027 years. This talk will present the initial results from the type="smallcap">Majorana type="smallcap">Demonstrator and type="smallcap">Gerda experiments and the plan for the LEGEND program.

  3. Role of activator protein-1 on the effect of arginine-glycine-aspartic acid containing peptides on transforming growth factor-beta1 promoter activity.

    PubMed

    Ruiz-Torres, M P; Perez-Rivero, G; Diez-Marques, M L; Griera, M; Ortega, R; Rodriguez-Puyol, M; Rodríguez-Puyol, D

    2007-01-01

    While arginine-glycine-aspartic acid-based peptidomimetics have been employed for the treatment of cardiovascular disorders and cancer, their use in other contexts remains to be explored. Arginine-glycine-aspartic acid-serine induces Transforming growth factor-beta1 transcription in human mesangial cells, but the molecular mechanisms involved have not been studied extensively. We explored whether this effect could be due to Activator protein-1 activation and studied the potential pathways involved. Addition of arginine-glycine-aspartic acid-serine promoted Activator protein-1 binding to its cognate sequence within the Transforming growth factor-beta1 promoter as well as c-jun and c-fos protein abundance. Moreover, this effect was suppressed by curcumin, a c-Jun N terminal kinase inhibitor, and was absent when the Activator protein-1 cis-regulatory element was deleted. Activator protein-1 binding was dependent on the activity of integrin linked kinase, as transfection with a dominant negative mutant suppressed both Activator protein-1 binding and c-jun and c-fos protein increment. Integrin linked kinase was, in turn, dependent on Phosphoinositol-3 kinase activity. Arginine-glycine-aspartic acid-serine stimulated Phosphoinositol-3 kinase activity, and Transforming growth factor-beta1 promoter activation was abrogated by the use of Phosphoinositol-3 kinase specific inhibitors. In summary, we propose that arginine-glycine-aspartic acid-serine activates Integrin linked kinase via the Phosphoinositol-3 kinase pathway and this leads to activation of c-jun and c-fos and increased Activator protein-1 binding and Transforming growth factor-beta1 promoter activity. These data may contribute to understand the molecular mechanisms involved in the cellular actions of arginine-glycine-aspartic acid-related peptides and enhance their relevance as these products evolve into clinical therapeutic use.

  4. The role of growth factors in embryonic induction in Xenopus laevis.

    PubMed

    Dawid, I B; Taira, M; Good, P J; Rebagliati, M R

    1992-06-01

    Establishment of the body pattern in all animals, and especially in vertebrate embryos, depends on cell interactions. During the cleavage and blastula stages in amphibians, signal(s) from the vegetal region induce the equatorial region to become mesoderm. Two types of peptide growth factors have been shown by explant culture experiments to be active in mesoderm induction. First, there are several isoforms of fibroblast growth factor (FGF), including aFGF, bFGF, and hst/kFGF. FGF induces ventral, but not the most dorsal, levels of mesodermal tissue; bFGF and its mRNA, and an FGF receptor and its mRNA, are present in the embryo. Thus, FGF probably has a role in mesoderm induction, but is unlikely to be the sole inducing agent in vivo. Second, members of the transforming growth factor-beta (TGF-beta) family. TGF-beta 2 and TGF-beta 3 are active in induction, but the most powerful inducing factors are the distant relatives of TGF-beta named activin A and activin B, which are capable of inducing all types of mesoderm. An important question relates to the establishment of polarity during the induction of mesoderm. While all regions of the animal hemisphere of frog embryos are competent to respond to activins by mesoderm differentiation, only explants that include cells close to the equator form structures with some organization along dorsoventral and anteroposterior axes. These observations suggest that cells in the blastula animal hemisphere are already polarized to some extent, although inducers are required to make this polarity explicit.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Beyond Type D personality: reduced positive affect (anhedonia) predicts impaired health status in chronic heart failure.

    PubMed

    Pelle, Aline J; Pedersen, Susanne S; Szabó, Balázs M; Denollet, Johan

    2009-08-01

    Type D personality has been associated with impaired health status in chronic heart failure (CHF), but other psychological factors may also be important. To determine whether non-Type D patients with low positive affect and Type D patients report lower health status, compared with non-Type D patients with high positive affect at 12-month follow-up in chronic heart failure. Consecutive CHF outpatients (n = 276) filled out the Short Form-12 (health status) and Health Complaints Scale (disease-specific complaints) at inclusion and 12-month follow-up, and the DS14 (Type D personality) and positive affect (Global Mood Scale) at inclusion. Three groups were composed: non-Type D patients without anhedonia, non-Type D patients with anhedonia, and Type D patients. After controlling for demographic and clinical confounders, and scores at inclusion, anhedonic non-Type D patients reported lower mental health status (beta = -.19, P < .004), and more feelings of disability (beta = .10, P = .04), marginally lower physical health status (beta = -.11, P = .07), and equal levels of cardiac symptoms (beta = .04, P = .43), when compared with non-Type D's without anhedonia. Type D patients reported lower levels of impaired mental health status, more cardiac symptoms and feelings of disability (-.31 < beta < .17, all Ps < .05). A trend was shown for physical health status (beta = -.11, P = .09). Non-Type D patients low on positive affect and Type D patients report lower levels of health status in CHF, compared with non-Type D patients with high positive affect. Future studies need to determine whether lack of positive affect is associated with impaired clinical outcome.

  6. [Changes of interlukin-1beta and tumor necrosis factor-alpha levels in gingival crevicular fluid during orthodontic tooth movement].

    PubMed

    Tian, Yu-Lou; Xie, Jiang-Chun; Zhao, Zhen-Jin; Zhang, Yang

    2006-06-01

    To investigate the dynamic changes of interlukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in gingival crevicular fluid (GCF) during orthodontic tooth movement, and to discuss the biological significance. Fifteen patients were chosen as subjects. For each patient, upper and lower canines at one side having one treatment for distal movement by elastic chain served as the experimental teeth, whereas the contralateral ones were used as controls. The GCF were taken before activation and at 1, 24, 48, 72, 168 hours respectively after initiation of the experiment. The levels of IL-1beta and TNF-alpha in GCF were determined by radioimmunoassay. The levels of IL-1beta and TNF-alpha in experimental group began to increase at 24 hours and reached to its peak value at 72 hours after initiation of the experiment, but their levels returned to baseline at 168 hours. Both of them, however, remained at the baseline level in control group. The changes of the two cytokines level were found statistically significant at 48 and 72 hours (P<0.05) between experimental and control group. No statistically significant were observed before activation and at 1, 168 hours after application of orthodontic forces (P>0.05) between experimental and control group. The levels of IL-1beta and TNF-alpha in gingival crevicular fluid experience dynamic changes during the early phase of orthodontic treatment, indicate that they might play an important role in the process of alveolar regeneration and tooth movement.

  7. Effect of prenatal vitamin supplementation on lower-genital levels of HIV type 1 and interleukin type 1 beta at 36 weeks of gestation.

    PubMed

    Fawzi, Wafaie; Msamanga, Gernard; Antelman, Gretchen; Xu, Chong; Hertzmark, Ellen; Spiegelman, Donna; Hunter, David; Anderson, Deborah

    2004-03-01

    Micronutrient status has been associated with shedding of human immunodeficiency virus type 1 (HIV-1) in the lower-genital tract in observational studies. We examined the effect of vitamin supplements on genital HIV-1 shedding and interleukin-1 beta (IL-1 beta ), a cytokine marker of vaginal inflammation and promotion of HIV-1 infection. Consenting HIV-1-infected pregnant women were randomized to receive daily supplementation with vitamin A and/or multivitamins B-complex, C, and E with use of a factorial design. Cervicovaginal lavage (CVL) specimens were obtained shortly before delivery. Significantly more women who received vitamin A had detectable levels of HIV-1 in CVL (74.8%), compared with those who did not receive vitamin A (65.1%) (P=.04, by multivariate analysis). Multivitamin B-complex, C, and E had no effect on the risk of viral shedding. Our results raise concern about the use of vitamin A supplements by HIV-1-infected women. Use of prenatal multivitamin supplements (including vitamins B-complex, C, and E) should be continued despite the lack of effect on HIV-1 transmission because of previously reported positive effects on maternal health and pregnancy outcomes.

  8. Metabolic factors and genetic risk mediate familial type 2 diabetes risk in the Framingham Heart Study

    PubMed Central

    Raghavan, Sridharan; Porneala, Bianca; McKeown, Nicola; Fox, Caroline S.; Dupuis, Josée; Meigs, James B.

    2015-01-01

    Aims/hypothesis Type 2 diabetes mellitus in parents is a strong determinant of diabetes risk in their offspring. We hypothesise that offspring diabetes risk associated with parental diabetes is mediated by metabolic risk factors. Methods We studied initially non-diabetic participants of the Framingham Offspring Study. Metabolic risk was estimated using beta cell corrected insulin response (CIR), HOMA-IR or a count of metabolic syndrome components (metabolic syndrome score [MSS]). Dietary risk and physical activity were estimated using questionnaire responses. Genetic risk score (GRS) was estimated as the count of 62 type 2 diabetes risk alleles. The outcome of incident diabetes in offspring was examined across levels of parental diabetes exposure, accounting for sibling correlation and adjusting for age, sex and putative mediators. The proportion mediated was estimated by comparing regression coefficients for parental diabetes with (βadj) and without (βunadj) adjustments for CIR, HOMA-IR, MSS and GRS (percentage mediated = 1 – βadj / βunadj). Results Metabolic factors mediated 11% of offspring diabetes risk associated with parental diabetes, corresponding to a reduction in OR per diabetic parent from 2.13 to 1.96. GRS mediated 9% of risk, corresponding to a reduction in OR per diabetic parent from 2.13 to 1.99. Conclusions/interpretation Metabolic risk factors partially mediated offspring type 2 diabetes risk conferred by parental diabetes to a similar magnitude as genetic risk. However, a substantial proportion of offspring diabetes risk associated with parental diabetes remains unexplained by metabolic factors, genetic risk, diet and physical activity, suggesting that important familial influences on diabetes risk remain undiscovered. PMID:25619168

  9. Polymorphisms of the IL-1beta and IL-1beta-inducible genes in ulcerative colitis.

    PubMed

    Nohara, Hiroaki; Saito, Yuki; Higaki, Singo; Okayama, Naoko; Hamanaka, Yuichiro; Okita, Kiwamu; Hinoda, Yuji

    2002-11-01

    Ulcerative colitis (UC) is a chronic disorder of undetermined etiology, but a genetic predisposition to UC is well recognized. Among cytokines induced in UC, interleukin 1 (IL-1) appears to have a central role because of its immunological upregulatory and proinflammatory activities. The aim of this study was to assess whether UC is associated with polymorphisms of the IL-1beta gene and three additional genes inducible with IL-1beta in Japanese subjects. A total of 96 patients with UC and 106 ethnically matched controls were genotyped at polymorphic sites in IL-1beta, matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 3 (MMP-3), and inducible nitric oxide synthase (iNOS) genes, using polymerase chain reaction (PCR)-based methods. There was no significant difference in genotype distributions of IL-1beta, MMP-1, MMP-3, and iNOS genes between controls and UC patients in a Japanese population. Also, no significant association of those polymorphisms with various clinical parameters of the patients was found. However, concerning association of age at onset with clinical factors in UC, the frequency of pancolitis was significantly higher in UC patients with age at onset being less than 30 years than in those more than 30 years of age (P = 0.049). No association of the IL-1beta and three IL-1beta-inducible gene polymorphisms with UC was observed in a Japanese population.

  10. Mixed-meal tolerance test versus glucagon stimulation test for the assessment of beta-cell function in therapeutic trials in type 1 diabetes.

    PubMed

    Greenbaum, Carla J; Mandrup-Poulsen, Thomas; McGee, Paula Friedenberg; Battelino, Tadej; Haastert, Burkhard; Ludvigsson, Johnny; Pozzilli, Paolo; Lachin, John M; Kolb, Hubert

    2008-10-01

    Beta-cell function in type 1 diabetes clinical trials is commonly measured by C-peptide response to a secretagogue in either a mixed-meal tolerance test (MMTT) or a glucagon stimulation test (GST). The Type 1 Diabetes TrialNet Research Group and the European C-peptide Trial (ECPT) Study Group conducted parallel randomized studies to compare the sensitivity, reproducibility, and tolerability of these procedures. In randomized sequences, 148 TrialNet subjects completed 549 tests with up to 2 MMTT and 2 GST tests on separate days, and 118 ECPT subjects completed 348 tests (up to 3 each) with either two MMTTs or two GSTs. Among individuals with up to 4 years' duration of type 1 diabetes, >85% had measurable stimulated C-peptide values. The MMTT stimulus produced significantly higher concentrations of C-peptide than the GST. Whereas both tests were highly reproducible, the MMTT was significantly more so (R(2) = 0.96 for peak C-peptide response). Overall, the majority of subjects preferred the MMTT, and there were few adverse events. Some older subjects preferred the shorter duration of the GST. Nausea was reported in the majority of GST studies, particularly in the young age-group. The MMTT is preferred for the assessment of beta-cell function in therapeutic trials in type 1 diabetes.

  11. Risk Factor Analysis of Ciprofloxacin-Resistant and Extended Spectrum Beta-Lactamases Pathogen-Induced Acute Bacterial Prostatitis in Korea.

    PubMed

    Lee, Young; Lee, Dong Gi; Lee, Sang Hyub; Yoo, Koo Han

    2016-11-01

    The objectives of this study were to investigate risk factors and the incidence of ciprofloxacin resistance and extended-spectrum beta-lactamases (ESBL) in patients with acute bacterial prostatitis (ABP). We reviewed the medical records of 307 patients who were diagnosed with ABP between January 2006 and December 2015. The etiologic pathogens and risk factors for ciprofloxacin-resistant E. coli and ESBL-producing microbes, susceptibility to ciprofloxacin, and the incidence of ESBL in patients with ABP were described. History of prior urologic manipulation was an independent risk factor for ciprofloxacin-resistant (P = 0.005) and ESBL-producing microbes (P = 0.005). Advanced age (over 60 years) was an independent risk factor for ciprofloxacin-resistant microbes (P = 0.022). The ciprofloxacin susceptibility for Escherichia coli in groups without prior manipulation was documented 85.7%. For groups with prior manipulation, the susceptibility was 10.0%. Incidence of ESBL-producing microbes by pathogen was 3.8% for E. coli and 1.0% for Klebsiella pneumonia in the absence of manipulation group, and 20% and 33.3% in the presence of manipulation group, respectively. Initial treatment of ABP must consider patient's age and the possibility of prior manipulation to optimize patient treatment. With the high rate of resistance to fluoroquinolone, cephalosporins with amikacin, or carbapenems, or extended-spectrum penicillin with beta lactamase inhibitor should be considered as the preferred empirical ABP treatment in the patients with history of prior urologic manipulation.

  12. Nucleotide sequence of the beta-lactamase gene from Enterococcus faecalis HH22 and its similarity to staphylococcal beta-lactamase genes.

    PubMed Central

    Zscheck, K K; Murray, B E

    1991-01-01

    The nucleotide sequence of the constitutively produced beta-lactamase (Bla) gene from Enterococcus faecalis HH22 was shown to be identical to the published sequences of three of four staphylococcal type A beta-lactamase genes; more differences were seen with the genes for staphylococcal type C and D enzymes. One hundred forty nucleotides upstream of the beta-lactamase start codon were determined for an inducible staphylococcal beta-lactamase and were identical to those of the constitutively expressed enterococcal gene, indicating that the changes resulting in constitutive expression are not due to changes in the promoter or operator region. Moreover, complementation studies indicated that production of the enterococcal enzyme could be repressed. The genes for the enterococcal Bla and an inducible staphylococcal Bla were each cloned into a shuttle vector and transformed into enterococcal and staphylococcal recipients. The major difference between the backgrounds of the two hosts was that more enzyme was produced by the staphylococcal host, regardless of the source of the gene. The location of the enzyme was found to be host dependent, since each cloned gene generated extracellular (free) enzyme in the staphylococcus and cell-bound enzyme in the enterococcus. On the basis of the identities of the enterococcal Bla and several staphylococcal Bla sequences, these data suggest the recent spread of beta-lactamase to enterococci and also suggest the loss of a functional repressor. PMID:1952840

  13. The lethal effects of cytokine-induced nitric oxide on cardiac myocytes are blocked by nitric oxide synthase antagonism or transforming growth factor beta.

    PubMed Central

    Pinsky, D J; Cai, B; Yang, X; Rodriguez, C; Sciacca, R R; Cannon, P J

    1995-01-01

    Inducible nitric oxide (NO) produced by macrophages is cytotoxic to invading organisms and has an important role in host defense. Recent studies have demonstrated inducible NO production within the heart, and that cytokine-induced NO mediates alterations in cardiac contractility, but the cytotoxic potential of nitric oxide with respect to the heart has not been defined. To evaluate the role of inducible nitric oxide synthase (iNOS) on cardiac myocyte cytotoxicity, we exposed adult rat cardiac myocytes to either cytokines alone or to activated J774 macrophages in coculture. Increased expression of both iNOS message and protein was seen in J774 macrophages treated with IFN gamma and LPS and cardiac myocytes treated with TNF-alpha, IL-1 beta, and IFN gamma. Increased NO synthesis was confirmed in both the coculture and isolated myocyte preparations by increased nitrite production. Increased NO synthesis was associated with a parallel increase in myocyte death as measured by CPK release into the culture medium as well as by loss of membrane integrity, visualized by trypan blue staining. Addition of the competitive NO synthase inhibitor L-NMMA to the culture medium prevented both the increased nitrite production and the cytotoxicity observed after cytokine treatment in both the isolated myocyte and the coculture experiments. Because transforming growth-factor beta modulates iNOS expression in other cell types, we evaluated its effects on cardiac myocyte iNOS expression and NO-mediated myocyte cytotoxicity. TGF-beta reduced expression of cardiac myocyte iNOS message and protein, reduced nitrite production, and reduced NO-mediated cytotoxicity in parallel. Taken together, these experiments show the cytotoxic potential of endogenous NO production within the heart, and suggest a role for TGF-beta or NO synthase antagonists to mute these lethal effects. These findings may help explain the cardiac response to sepsis or allograft rejection, as well as the progression of

  14. Detection of VIM- and IMP-type Metallo-Beta-Lactamase Genes in Acinetobacter baumannii Isolates from Patients in Two Hospitals in Tehran

    PubMed Central

    Davoodi, Saba; Boroumand, Mohammad Ali; Sepehriseresht, Saeed; Pourgholi, Leila

    2015-01-01

    Background Acinetobacter baumannii, is an opportunistic pathogen and is responsible for numerous nosocomial infections. In recent years, this microorganism has been resistant to a wide range of antibiotics. One of the most important mechanisms of resistance in this microorganism is production of metallo-beta-lactamases (MBLs). Objectives The aim of this study was to detect VIM- and IMP-type metallo-beta-lactamase genes in Acinetobacter baumanniiisolates from patients in two Hospitals in Tehran. Materials and Methods 104 isolates were tested using the PCR method for the identification of VIM- and IMP-type genes. Results vim1, vim2, imp1 and imp2 genes were detected in 6.7%, 41.7%, 50% and 1.7% of the isolates from Tehran Heart Center, and in 29.5%, 38.6%, 4.5% and 4.5% of the isolates from Shahid Mutahhari Hospital respectively. Discussions Our analysis revealed that the majority of the isolates had at least one of these genes, indicating that MBLs production is an important resistance mechanism in Acinetobacter baumannii. PMID:28959283

  15. Increased mature interleukin-1beta (IL-1beta) secretion from THP-1 cells induced by nigericin is a result of activation of p45 IL-1beta-converting enzyme processing.

    PubMed

    Cheneval, D; Ramage, P; Kastelic, T; Szelestenyi, T; Niggli, H; Hemmig, R; Bachmann, M; MacKenzie, A

    1998-07-10

    Perregaux and Gabel (Perregaux, D., and Gabel, C. A. (1994) J. Biol. Chem. 269, 15195-15203) reported that potassium depletion of lipopolysaccharide-stimulated mouse macrophages induced by the potassium ionophore, nigericin, leads to the rapid release of mature interleukin-1beta (IL-1beta). We have now shown a similar phenomenon in lipopolysaccharide-stimulated human monocytic leukemia THP-1 cells. Rapid secretion of mature, 17-kDa IL-1beta occurred, in the presence of nigericin (4-16 microM). No effects on the release of tumor necrosis factor-alpha, IL-6, or proIL-1beta were seen. Addition of the irreversible interleukin-1beta-converting enzyme (ICE) inhibitor, Z-Val-Ala-Asp-dichlorobenzoate, or a radicicol analog, inhibited nigericin-induced mature IL-1beta release and activation of p45 ICE precursor. The radicicol analog itself did not inhibit ICE, but markedly, and very rapidly depleted intracellular levels of 31-kDa proIL-1beta. By contrast, dexamethasone, cycloheximide, and the Na+/H+ antiporter inhibitor, 5-(N-ethyl-N-isopropyl)amiloride, had no effect on nigericin-induced release of IL-1beta. We have therefore shown conclusively, for the first time, that nigericin-induced release of IL-1beta is dependent upon activation of p45 ICE processing. So far, the mechanism by which reduced intracellular potassium ion concentration triggers p45 ICE processing is not known, but further investigation in this area could lead to the discovery of novel molecular targets whereby control of IL-1beta production might be effected.

  16. Prolonged survival of dendritic cell-vaccinated melanoma patients correlates with tumor-specific delayed type IV hypersensitivity response and reduction of tumor growth factor beta-expressing T cells.

    PubMed

    López, Mercedes N; Pereda, Cristian; Segal, Gabriela; Muñoz, Leonel; Aguilera, Raquel; González, Fermín E; Escobar, Alejandro; Ginesta, Alexandra; Reyes, Diego; González, Rodrigo; Mendoza-Naranjo, Ariadna; Larrondo, Milton; Compán, Alvaro; Ferrada, Carlos; Salazar-Onfray, Flavio

    2009-02-20

    The aim of this work was to assess immunologic response, disease progression, and post-treatment survival of melanoma patients vaccinated with autologous dendritic cells (DCs) pulsed with a novel allogeneic cell lysate (TRIMEL) derived from three melanoma cell lines. Forty-three stage IV and seven stage III patients were vaccinated four times with TRIMEL/DC vaccine. Specific delayed type IV hypersensitivity (DTH) reaction, ex vivo cytokine production, and regulatory T-cell populations were determined. Overall survival and disease progression rates were analyzed using Kaplan-Meier curves and compared with historical records. The overall survival for stage IV patients was 15 months. More than 60% of patients showed DTH-positive reaction against the TRIMEL. Stage IV/DTH-positive patients displayed a median survival of 33 months compared with 11 months observed for DTH-negative patients (P = .0014). All stage III treated patients were DTH positive and remained alive and tumor free for a median follow-up period of 48 months (range, 33 to 64 months). DTH-positive patients showed a marked reduction in the proportion of CD4+ transforming growth factor (TGF) beta+ regulatory T cells compared to DTH-negative patients (1.54% v 5.78%; P < .0001). Our findings strongly suggest that TRIMEL-pulsed DCs provide a standardized and widely applicable source of melanoma antigens, very effective in evoking antimelanoma immune response. To our knowledge, this is the first report describing a correlation between vaccine-induced reduction of CD4+TGFbeta+ regulatory T cells and in vivo antimelanoma immune response associated to improved patient survival and disease stability.

  17. 48 CFR 16.104 - Factors in selecting contract types.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 1 2014-10-01 2014-10-01 false Factors in selecting... CONTRACTING METHODS AND CONTRACT TYPES TYPES OF CONTRACTS Selecting Contract Types 16.104 Factors in selecting contract types. There are many factors that the contracting officer should consider in selecting and...

  18. 48 CFR 16.104 - Factors in selecting contract types.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 1 2013-10-01 2013-10-01 false Factors in selecting... CONTRACTING METHODS AND CONTRACT TYPES TYPES OF CONTRACTS Selecting Contract Types 16.104 Factors in selecting contract types. There are many factors that the contracting officer should consider in selecting and...

  19. Beta-hairpin formation in aqueous solution and in the presence of trifluoroethanol: a (1)H and (13)C nuclear magnetic resonance conformational study of designed peptides.

    PubMed

    Santiveri, Clara M; Pantoja-Uceda, David; Rico, Manuel; Jiménez, M Angeles

    2005-10-15

    In order to check our current knowledge on the principles involved in beta-hairpin formation, we have modified the sequence of a 3:5 beta-hairpin forming peptide with two different purposes, first to increase the stability of the formed 3:5 beta-hairpin, and second to convert the 3:5 beta-hairpin into a 2:2 beta-hairpin. The conformational behavior of the designed peptides was investigated in aqueous solution and in 30% trifluoroethanol (TFE) by analysis of the following nuclear magnetic resonance (NMR) parameters: nuclear Overhauser effect (NOE) data, and C(alpha)H, (13)C(alpha), and (13)C(beta) conformational shifts. From the differences in the ability to adopt beta-hairpin structures in these peptides, we have arrived to the following conclusions: (i) beta-Hairpin population increases with the statistical propensity of residues to occupy each turn position. (ii) The loop length, and in turn, the beta-hairpin type, can be modified as a function of the type of turn favored by the loop sequence. These two conclusions reinforce previous results about the importance of beta-turn sequence in beta-hairpin folding. (iii) Side-chain packing on each face of the beta-sheet may play a major role in beta-hairpin stability; hence simplified analysis in terms of isolated pair interactions and intrinsic beta-sheet propensities is insufficient. (iv) Contributions to beta-hairpin stability of turn and strand sequences are not completely independent. (v) The burial of hydrophobic surface upon beta-hairpin formation that, in turn, depends on side-chain packing also contributes to beta-hairpin stability. (vi) As previously observed, TFE stabilizes beta-hairpin structures, but the extent of the contribution of different factors to beta-hairpin formation is sometimes different in aqueous solution and in 30% TFE. (c) 2005 Wiley Periodicals, Inc. Biopolymers 79: 150-162, 2005.

  20. Corticotropin-releasing factor receptor-1 antagonism mitigates beta amyloid pathology and cognitive and synaptic deficits in a mouse model of Alzheimer's disease.

    PubMed

    Zhang, Cheng; Kuo, Ching-Chang; Moghadam, Setareh H; Monte, Louise; Campbell, Shannon N; Rice, Kenner C; Sawchenko, Paul E; Masliah, Eliezer; Rissman, Robert A

    2016-05-01

    Stress and corticotropin-releasing factor (CRF) have been implicated as mechanistically involved in Alzheimer's disease (AD), but agents that impact CRF signaling have not been carefully tested for therapeutic efficacy or long-term safety in animal models. To test whether antagonism of the type-1 corticotropin-releasing factor receptor (CRFR1) could be used as a disease-modifying treatment for AD, we used a preclinical prevention paradigm and treated 30-day-old AD transgenic mice with the small-molecule, CRFR1-selective antagonist, R121919, for 5 months, and examined AD pathologic and behavioral end points. R121919 significantly prevented the onset of cognitive impairment in female mice and reduced cellular and synaptic deficits and beta amyloid and C-terminal fragment-β levels in both genders. We observed no tolerability or toxicity issues in mice treated with R121919. CRFR1 antagonism presents a viable disease-modifying therapy for AD, recommending its advancement to early-phase human safety trials. Copyright © 2015 Alzheimer's Association. All rights reserved.

  1. Gaucher disease: Physical, kinetic and immunologic investigations of human and canine acid. beta. -glucosidase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fabbro, D.E.

    1988-01-01

    Kinetic and immunologic techniques were developed to investigate the nature of the acid {beta}-glucosidase ({beta}-Glc) defects which results in human and canine Gaucher disease (GD). Two new affinity columns, using the potent inhibitors of {beta}-Glc (N-alkyl-deoxynojirimycins) as affinity ligands, were synthesized and methods were developed to obtain homogeneous {beta}-Glc from normal human placenta. Polyclonal and monoclonal (representing 14 different epitopes from 18 clones) antibodies were produced to the pure normal {beta}-Glc. Monospecific polyclonal IgG and tritiated-bromo-conduritol B epoxide (({sup 3}H)Br-CBE), a specific covalent active site directed inhibitor of {beta}-Glc, were used to quantitate the functional catalytic sites in normal andmore » Type 1 Ashkenazi Jewish GD (AJGD) enzyme preparations: The k{sub cat} values for several new substrates with the mutant enzymes from spleen were about 1.5-fold less than the respective normal enzyme, indicating a nearly normal catalytic capacity of the mutant enzymes. Immunoblotting studies with polyclonal or several monoclonal antibodies indicated three molecular forms of {beta}-Glc (M{sub r} = 67,000, 62,000 to 65,000 and 58,000) in fibroblast extracts from normals and Type 1 AJGD patients. In comparison, only one form of cross-reacting immunologic material (CRIM) was detected in fibroblast extracts from Types 2 and 3 or several non-Jewish Type 1 GD patients.« less

  2. Immunodetection of 11 beta-hydroxysteroid dehydrogenase type 2 in human mineralocorticoid target tissues: evidence for nuclear localization.

    PubMed

    Shimojo, M; Ricketts, M L; Petrelli, M D; Moradi, P; Johnson, G D; Bradwell, A R; Hewison, M; Howie, A J; Stewart, P M

    1997-03-01

    11 beta-Hydroxysteroid dehydrogenase (11 beta HSI) is an enzyme complex responsible for the conversion of hormonally active cortisol to inactive cortisone; two isoforms of the enzyme have been cloned and characterized. Clinical observations from patients with the hypertensive syndrome apparent mineralocorticoid excess, recently explained on the basis of mutations in the human 11 beta HSD2 gene, suggest that it is the 11 beta HSD2 isoform that serves a vital role in dictating specificity upon the mineralocorticoid receptor (MR). We have raised a novel antibody in sheep against human 11 beta HSD2 using synthetic multiantigenic peptides and have examined the localization and subcellular distribution of 11 beta HSD2 in mineralocorticoid target tissues. The immunopurified antibody recognized a single band of approximately 44 kDa in placenta, trophoblast, and distal colon. In kidney tissue, two bands of approximately 44 and 48 kDa were consistently observed. No signal was seen in decidua, adrenal, or liver. Immunoperoxidase studies on the mineralocorticoid target tissues, kidney, colon, and parotid gland indicated positive staining in epithelial cells known to express the MR: respectively, renal collecting ducts, surface and crypt colonic epithelial cells, and parotid duct epithelial cells. No staining was seen in these tissues in other sites. The intracellular localization of 11 beta HSD2 in kidney and colon epithelial cells was addressed using confocal laser microscopy. Parallel measurements of 11 beta HSD2 and nuclear propidium iodide fluorescence on sections scanned through an optical section of approximately 0.1 micron indicated significant 11 beta HSD2 immunofluorescence in the nucleus. In human kidney, colon, and salivary gland, 11 beta HSD2 protects the MR from glucocorticoid excess in an autocrine fashion. Furthermore, within these tissues, 11 beta HSD2, which had been considered to be a microsomal enzyme, is also found in the nucleus, suggesting that the

  3. Inhibin/activin-betaC and -betaE subunits in the Ishikawa human endometrial adenocarcinoma cell line.

    PubMed

    Kimmich, Tanja; Brüning, Ansgar; Käufl, Stephanie D; Makovitzky, Josef; Kuhn, Christina; Jeschke, Udo; Friese, Klaus; Mylonas, Ioannis

    2010-08-01

    Inhibins and activins are important regulators of the female reproductive system. Recently, two novel inhibin subunits, named betaC and betaE, have been identified and shown to be expressed in several human tissues. However, only limited data on the expression of these novel inhibin subunits in normal human endometrial tissue and endometrial adenocarcinoma cell lines exist. Samples of proliferative and secretory human endometrium were obtained from five premenopausal, non-pregnant patients undergoing gynecological surgery for benign diseases. Normal endometrial tissue and Ishikawa endometrial adenocarcinoma cell lines were analyzed by immunohistochemistry, immunofluorescence and RT-PCR. Expression of the inhibin betaC and betaE subunits could be demonstrated at the protein level by means of immunohistochemical evaluation and at the transcriptional level by establishing a betaC- and betaE-specific RT-PCR analysis in normal human endometrial tissue and the parental Ishikawa cell line. Interestingly, in a highly de-differentiated subclone of the Ishikawa cell line lacking estrogen receptor expression, the expression of the inhibin-betaC subunit appeared strongly reduced. Here, we show for the first time that the novel inhibin/activin-betaC and -betaE subunits are expressed in normal human endometrium and the estrogen receptor positive human endometrial carcinoma cell line Ishikawa using RT-PCR and immunohistochemical detection methods. Interestingly, the Ishikawa minus cell line (lacking estrogen receptor expression) demonstrated no to minimal expression of the betaC subunit as observed with immunofluorescence and RT-PCR, suggesting a possible hormone- dependency of this subunit in human endometrial cancer cells. Moreover, because the Ishikawa cell line minus is thought to be a more malignant endometrial cell line than its estrogen receptor positive counterpart, inhibin-betaC subunit might be substantially involved in the pathogenesis and malignant transformation in

  4. The p110beta isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with p110gamma.

    PubMed

    Guillermet-Guibert, Julie; Bjorklof, Katja; Salpekar, Ashreena; Gonella, Cristiano; Ramadani, Faruk; Bilancio, Antonio; Meek, Stephen; Smith, Andrew J H; Okkenhaug, Klaus; Vanhaesebroeck, Bart

    2008-06-17

    The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110alpha, p110beta, and p110delta) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110alpha and p110delta to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110gamma class IB PI3K lack SH2 domains and instead couple p110gamma to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110beta and cells derived from a p110beta-deficient mouse line, that p110beta is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110beta and p110gamma contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110beta but not p110gamma, p110beta mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110gamma in these cells reduced the contribution of p110beta to GPCR signaling. Taken together, these data show that p110beta and p110gamma can couple redundantly to the same GPCR agonists. p110beta, which shows a much broader tissue distribution than the leukocyte-restricted p110gamma, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110gamma expression is low or absent.

  5. Trials in the prevention of type 1 diabetes: current and future.

    PubMed

    Wherrett, Diane K

    2014-08-01

    A major thrust in type 1 diabetes research is stopping the destruction of beta cells that leads to type 1 diabetes. Research over the past 30 years has defined genetic factors and evidence of autoimmunity that have led to the development of robust prediction models in those at high risk for type 1 diabetes. The ability to identify those at risk and the development of new agents and of collaborative research networks has led to multiple trials aimed at preventing beta cell loss. Trials at all stages of beta cell loss have been conducted: primary prevention (prior to the development of autoimmunity); secondary prevention (after autoantibodies are found) and tertiary prevention (intervening after diagnosis to maintain remaining beta cells). Studies have shown mixed results; evidence of maintained insulin secretion after the time of diagnosis has been described in a number of studies, and primary and secondary prevention is proving to be elusive. Much has been learned from the increasing number of studies in the field in terms of network creation, study design and choice of intervention that will facilitate new avenues of investigation. Copyright © 2014 Canadian Diabetes Association. Published by Elsevier Inc. All rights reserved.

  6. [Enterobacteriaceae producing extended spectrum beta-lactamase: epidemiology, risk factors, and prevention].

    PubMed

    Vodovar, D; Marcadé, G; Raskine, L; Malissin, I; Mégarbane, B

    2013-11-01

    Multidrug-resistant bacteria are a major worldwide health public concern. It results from the growing increase in antibiotic prescriptions, which are responsible for selection pressure on bacteria. In France like in other countries, enterobacteriaceae producing extended spectrum beta-lactamase (EESBL) are the predominant multidrug-resistant bacteria. EESBL may be responsible for severe infections and require prescription of broad-spectrum antibacterial agents. The current EESBL outbreak is different from methicillin-resistant Staphylococcus aureus outbreak that occurred in the early 1980. Consistently, EESBL are isolated both in hospital and community. Moreover, standard hygiene measures appear ineffective since EESBL prevalence is still increasing. The current inability to contain EESBL outbreak is due to several factors, including the existence of a wide community- and hospital-acquired tank of EESBL, failure to follow strict rules for hygiene, and the current irrational prescription of antibiotics. Copyright © 2012 Société nationale française de médecine interne (SNFMI). Published by Elsevier SAS. All rights reserved.

  7. Treatment with unsaponifiable extracts of avocado and soybean increases TGF-beta1 and TGF-beta2 levels in canine joint fluid.

    PubMed

    Altinel, Levent; Saritas, Z Kadir; Kose, Kamil Cagri; Pamuk, Kamuran; Aksoy, Yusuf; Serteser, Mustafa

    2007-02-01

    Avocado and soya unsaponifiables (ASU) are plant extracts used as a slow-acting antiarthritic agent. ASU stimulate the synthesis of matrix components by chondrocytes, probably by increasing the production of transforming growth factor-beta (TGF-beta). TGF-beta is expressed by chondrocytes and osteoblasts and is present in cartilage matrix. This study investigates the effect of ASU treatment on the levels of two isoforms of TGFbeta, TGF-beta1 and TGF-beta2, in the knee joint fluid using a canine model. Twenty-four outbred dogs were divided into three groups. The control animals were given a normal diet, while the treated animals were given 300 mg ASU every three days or every day. Joint fluid samples were obtained prior to treatment, and at the end of every month (up to three months). TGF-beta1 and TGF-beta2 levels were measured using a quantitative sandwich enzyme immunoassay technique. ASU treatment caused an increase in TGF-beta1 and TGF-beta2 levels in the joint fluid when compared to controls. The different doses did not cause a significant difference in joint fluid TGF levels. TGF-beta1 levels in the treated animals reached maximum values at the end of the second month and then decreased after the third month, while TGF-beta2 levels showed a marginal increase during the first two months, followed by a marked increase at the end of the third month. In conclusion, ASU increased both TGF-beta1 and TGF-beta2 levels in knee joint fluid.

  8. Impact of transforming growth factor-beta1 on atrioventricular node conduction modification by injected autologous fibroblasts in the canine heart.

    PubMed

    Bunch, T Jared; Mahapatra, Srijoy; Bruce, G Keith; Johnson, Susan B; Miller, Dylan V; Horne, Benjamin D; Wang, Xiao-Li; Lee, Hon-Chi; Caplice, Noel M; Packer, Douglas L

    2006-05-30

    Atrioventricular (AV) nodal ablation for management of atrial fibrillation (AF) is irreversible and requires permanent pacemaker implantation. We hypothesized that as an alternative, implantation of autologous fibroblasts in the perinodal region would focally modify AV nodal conduction and that this modulation would be enhanced by pretreatment with transforming growth factor-beta1 (TGF-beta1), a stimulant of fibroblasts. Skin biopsies were taken from 12 mongrel dogs, and derived fibroblasts were dissociated and grown in culture for 2 weeks. Multiple injections (0.25 mL) were made through an 8F NOGA catheter along the fast/slow AV nodal pathways as guided by an electroanatomic mapping system. Seven dogs received fibroblasts alone (1x10(6) cells/mL), 7 dogs received TGF-beta1 (5 microg), 4 dogs received fibroblasts and TGF-beta1 (1x10(6) cells/mL+5 microg), and 4 dogs received saline only. AV node function was assessed at baseline and after 4 weeks. Saline (80 mL) with assigned therapy (0.25 mL per injection) was injected into the peri-AV nodal region in each dog. At baseline, the AH interval (66+/-3 ms) and the average RR interval (331+/-17 ms) in pacing-induced AF were similar in each cohort. The increase in AH interval in normal sinus rhythm was longer after fibroblast (23+/-4 versus 5+/-5 ms; P=0.05) and fibroblast plus TGF-beta1 (50+/-5 versus 5+/-5 ms; P<0.001) injections than with saline alone, with similar findings during high right atrium and distal coronary sinus pacing. The AH interval was not significantly increased after TGF-beta1 injections. The AH interval was significantly longer after fibroblast plus TGF-beta1 injections than with either therapy (TGF-beta1 or fibroblasts) alone. The RR interval during AF was increased in dogs that received fibroblasts alone (110+/-36 versus -41+/-34 ms) and to a greater extent with the addition of TGF-beta1 (294+/-108 versus -41+/-34 ms). No AV block was seen in any cohort at 4 weeks. Labeled fibroblasts that

  9. Selective regulation of beta 1- and beta 2-adrenoceptors in the human heart by chronic beta-adrenoceptor antagonist treatment.

    PubMed Central

    Michel, M. C.; Pingsmann, A.; Beckeringh, J. J.; Zerkowski, H. R.; Doetsch, N.; Brodde, O. E.

    1988-01-01

    1. In 44 patients undergoing coronary artery bypass grafting, the effect of chronic administration of the beta-adrenoceptor antagonists sotalol, propranolol, pindolol, metoprolol and atenolol on beta-adrenoceptor density in right atria (containing 70% beta 1- and 30% beta 2-adrenoceptors) and in lymphocytes (having only beta 2-adrenoceptors) was studied. 2. beta-Adrenoceptor density in right atrial membranes and in intact lymphocytes was assessed by (-)-[125I]-iodocyanopindolol (ICYP) binding; the relative amount of right atrial beta 1- and beta 2-adrenoceptors was determined by inhibition of ICYP binding by the selective beta 2-adrenoceptor antagonist ICI 118,551 and analysis of the resulting competition curves by the iterative curve fitting programme LIGAND. 3. With the exception of pindolol, all beta-adrenoceptor antagonists increased right atrial beta-adrenoceptor density compared to that observed in atria from patients not treated with beta-adrenoceptor antagonists. 4. All beta-adrenoceptor antagonists increased right atrial beta 1-adrenoceptor density; on the other hand, only sotalol and propranolol also increased right atrial beta 2-adrenoceptor density, whereas metoprolol and atenolol did not affect it and pindolol decreased it. 5. Similarly, in corresponding lymphocytes, only sotalol or propranolol increased beta 2-adrenoceptor density, while metoprolol and atenolol did not affect it and pindolol decreased it. 6. It is concluded that beta-adrenoceptor antagonists subtype-selectively regulate cardiac and lymphocyte beta-adrenoceptor subtypes. The selective increase in cardiac beta 1-adrenoceptor density evoked by metoprolol and atenolol may be one of the reasons for the beneficial effects observed in patients with end-stage congestive cardiomyopathy following intermittent treatment with low doses of selective beta 1-adrenoceptor antagonists. PMID:2902891

  10. Angiotensin II Receptor Antagonism Reduces Transforming Growth Factor Beta and Smad Signaling in Thoracic Aortic Aneurysm

    PubMed Central

    Nataatmadja, Maria; West, Jennifer; Prabowo, Sulistiana; West, Malcolm

    2013-01-01

    ABSTRACT Background The expression of transforming growth factor beta (TGF-β) and Smad3 regulates extracellular matrix homeostasis and inflammation in aortic aneurysms. The expression of Smad3 depends on signaling by angiotensin II (AngII) receptor pathways through TGF-β receptor–dependent and –independent pathways. Methods To determine the expression of AngII type 1 (AT1R) and type 2 receptors (AT2R), TGF-β, and Smad3 in thoracic aortic aneurysms, we performed immunohistochemistry testing on tissue and cultured cells derived from subjects with Marfan syndrome (MFS) and bicuspid aortic valve (BAV) malformation and from normal aortas of subjects who were organ donors. Results MFS and BAV aneurysm tissue showed enhanced accumulation of TGF-β and Smad3 in vascular smooth muscle cells (VSMCs) and in inflammatory cells in the subintimal layer and tunica media. The normal aortic wall exhibited minimal TGF-β and Smad3 staining. Cultured VSMCs from MFS and BAV samples showed nuclear Smad3 and strong cytoplasmic TGF-β expression in the cytoplasmic vesicles. In control cells, Smad3 was located mainly in the cytoplasm, and weak cytoplasmic TGF-β was distributed with a pattern similar to that of the aneurysm-derived cells. Compared to normal aorta cells, AT1R and AT2R expression was increased in both aneurysm types. Treatment of cultured VSMCs with the AT1R antagonist losartan caused both reduced TGF-β vesicle localization and nuclear expression of Smad3. Conclusions Increased TGF-β and Smad3 expression in aneurysm tissue and cultured VSMCs is consistent with aberrant TGF-β expression and the activation of Smad3 signaling. Losartan-mediated reduction in TGF-β expression and the cytoplasmic localization of Smad3 support a role for AT1R antagonism in the inhibition of aneurysm progression. PMID:23532685

  11. Increased expression of c-Ski as a co-repressor in transforming growth factor-beta signaling correlates with progression of esophageal squamous cell carcinoma.

    PubMed

    Fukuchi, Minoru; Nakajima, Masanobu; Fukai, Yasuyuki; Miyazaki, Tatsuya; Masuda, Norihiro; Sohda, Makoto; Manda, Ryokuhei; Tsukada, Katsuhiko; Kato, Hiroyuki; Kuwano, Hiroyuki

    2004-03-01

    Transforming growth factor-beta (TGF-beta) regulates cell growth inhibition, and inactivation of the TGF-beta signaling pathway contributes to tumor development. In our previous study, altered expression of TGF-beta, TGF-beta-specific receptors and Smad4 was shown to correlate with tumor progression in esophageal squamous cell carcinoma (SCC). These components, however, were maintained normally in some patients with esophageal SCC. In our study, the mechanism by which aggressive esophageal SCC maintains these components was investigated, with particular emphasis on the participation of c-Ski and SnoN as transcriptional co-repressors in TGF-beta signaling. Immunohistochemistry for c-Ski and SnoN was carried out on surgical specimens obtained from 80 patients with esophageal SCC. The expression of c-Ski and SnoN was also studied in 6 established cell lines derived from esophageal SCC and compared to an immortalized human esophageal cell line by Western blotting. High levels of expression of c-Ski, detected immunohistologically, were found to correlate with depth of invasion (p = 0.0080) and pathologic stage (p = 0.0447). There was, however, no significant correlation between expression of SnoN and clinicopathologic characteristics. A significant correlation between c-Ski and TGF-beta expression was observed. Moreover, in patients with TGF-beta negative expression, the survival rates of patients with c-Ski positive expression were significantly lower than those of patients with c-Ski negative expression (p = 0.0486). c-Ski was expressed at a high level in 5 of 6 cell lines derived from esophageal SCC compared to immortalized esophageal keratinocytes. Furthermore, the cyclin-dependent kinase (CDK) inhibitor, p21 that was up-regulated by TGF-beta signaling was expressed at a low level in the 5 cell lines. The expression of c-Ski protein as a transcriptional co-repressor in TGF-beta signaling seems to be correlated with tumor progression of esophageal SCC. Copyright 2003

  12. Bombesin-dependent pro-MMP-9 activation in prostatic cancer cells requires beta1 integrin engagement.

    PubMed

    Festuccia, Claudio; Angelucci, Adriano; Gravina, Giovanni; Eleuterio, Enrica; Vicentini, Carlo; Bologna, Mauro

    2002-10-15

    Bombesin-like peptides, including the mammalian homologue gastrin-releasing peptide, are highly expressed and secreted by neuroendocrine cells in prostate carcinoma tissues and are likely to be related to the progression of this neoplastic disease. Previously, we demonstrated that bombesin increased migration and protease expression in androgen-independent cells. In this work we show that bombesin is able to activate pro-MMP-9 through a mechanism involving the beta1 integrin subunit. In fact, MMP-9 processing was evident only when beta1 integrin was engaged with specific adhesive substrates, such as type I collagen, or when cells were seeded on dishes coated with antibodies against beta1 integrin, resulting in activation of the surface ligand. When exogenous pro-MMP-9 was added to PC3 cells, MMP-9 active forms were produced within 30 min by bombesin-treated cultures while control cultures expressed activated forms only after a longer time and at lower levels. MMP-9 activation required cytoskeleton integrity since this effect was abolished by cytochalasin D. Engagement of beta1 integrin caused an increased membrane-linked uPA activity which was required for MMP-9 activation. The cross talk between bombesin- and beta1-integrin-engaged signals seems to be crucial for the modulation of both membrane-linked uPA activity and MMP-9 activation and triggers complex intracellular signaling pathways requiring activation of tyrosine kinase activity, including that of src and PI3K. The beta1 integrin may be considered an important mechanism by which bombesin induces MMP-9 activation. This finding supports the idea that cellular responses to growth factors may be driven by cell-matrix interactions and stresses the role of neuroendocrine factors in prostate carcinoma progression.

  13. Relationships of pancreatic beta-cell function with microalbuminuria and glomerular filtration rate in middle-aged and elderly population without type 2 diabetes mellitus: a Chinese community-based analysis.

    PubMed

    Fu, Shihui; Zhou, Shanjing; Luo, Leiming; Ye, Ping

    2017-01-01

    Relationships of pancreatic beta-cell function abnormality with microalbuminuria (MA) and glomerular filtration rate (GFR) may differ by age, ethnicity and accompanied diseases. Previous studies were generally conducted in Western adult patients with type 2 diabetes mellitus (T2DM), and it is uncertain whether pancreatic beta-cell function is associated with MA and GFR in Chinese community-dwelling middle-aged and elderly population without T2DM. We therefore examined the relationships of pancreatic beta-cell function with two indices of renal damage, MA and GFR, in Chinese community-dwelling middle-aged and elderly population without T2DM. This analysis focused on 380 Beijing residents older than 45 years who were free of T2DM and completed the evaluation of pancreatic beta-cell function. Median age was 67 (49-80) years. Levels of triglyceride, diastolic blood pressure and homeostasis model assessment-beta (HOMA-beta) index were positively related to urine microalbumin ( P <0.05 for all). Age, low-density lipoprotein cholesterol levels and HOMA-beta index were inversely correlated with GFR, while high-density lipoprotein cholesterol levels were positively correlated with GFR ( P <0.05 for all). In all three adjustment models, there was a significant positive association between HOMA-beta index and MA; subjects with higher beta-cell function had higher odds of MA ( P <0.05 for all). There was no association between HOMA-beta index and GFR <60 mL/min/1.73 m 2 in any model ( P >0.05 for all). Modeling the pancreatic beta-cell function with different adjusted variables provided the same conclusion of association with MA; beta-cell function was positively associated with MA. Additionally, there was a specific difference in the adjusted associations of pancreatic beta-cell function with MA and GFR <60 mL/min/1.73 m 2 ; beta-cell function was not independently associated with GFR <60 mL/min/1.73 m 2 . This result indicated that abnormal pancreatic beta-cell function

  14. Clinical characteristics and beta cell function in Chinese patients with newly diagnosed type 2 diabetes mellitus with different levels of serum triglyceride.

    PubMed

    Zheng, Shuang; Zhou, Huan; Han, Tingting; Li, Yangxue; Zhang, Yao; Liu, Wei; Hu, Yaomin

    2015-04-29

    To explore clinical characteristics and beta cell function in Chinese patients with newly diagnosed drug naive type 2 diabetes mellitus (T2DM) with different levels of serum triglyceride (TG). Patients with newly diagnosed T2DM (n = 624) were enrolled and divided into different groups according to levels of serum TG. All patients underwent oral glucose tolerance tests and insulin releasing tests. Demographic data, lipid profiles, glucose levels, and insulin profiles were compared between different groups. Basic insulin secretion function index (homeostasis model assessment for beta cell function index, HOMA-β), modified beta cell function index (MBCI), glucose disposition indices (DI), and early insulin secretion function index (insulinogenic index, IGI) were used to evaluate the beta cell function. Patients of newly diagnosed T2DM with hypertriglyceridemia were younger, fatter and had worse lipid profiles, glucose profiles, and high insulin levels than those with normal TG. There is no difference in early phase insulin secretion among groups of newly diagnosed T2DM patients with different TG levels. The basal beta cell function (HOMA-β and MBCI) initially increased along rising TG levels and then decreased as the TG levels rose further. The insulin sensitivity was relatively high in patients with a low level of TG and low with a high level of TG. Hypertriglyceridemia influences clinical characteristics and β cell function of Chinese patients with newly diagnosed T2DM. A better management of dyslipidemia may, to some extent, reduce the effect of lipotoxicity, thereby improving glucose homeostasis in patients with newly diagnosed T2DM.

  15. Cloning, sequencing, and expression of cDNA for human. beta. -glucuronidase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Oshima, A.; Kyle, J.W.; Miller, R.D.

    1987-02-01

    The authors report here the cDNA sequence for human placental ..beta..-glucuronidase (..beta..-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. They also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH/sub 2/-terminal amino acid sequence determined for human spleen ..beta..-glucuronidase agreed with that inferred from the DNAmore » sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human ..beta..-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human ..beta..-glucuronidase, demonstrate the existence of two populations of mRNA for ..beta..-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length.« less

  16. The role of estrogen receptor {beta} (ER{beta}) in malignant diseases-A new potential target for antiproliferative drugs in prevention and treatment of cancer

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Warner, Margaret; Center for Nuclear Receptors and Cell Signaling, Department of Biochemistry and Cell Biology, University of Houston, Houston, TX; Gustafsson, Jan-Ake, E-mail: jan-ake.gustafsson@mednut.ki.se

    2010-05-21

    The discovery of ER{beta} in the middle of the 1990s represents a paradigm shift in our understanding of estrogen signaling. It has turned out that estrogen action is not mediated by one receptor, ER{alpha}, but by two balancing factors, ER{alpha} and ER{beta}, which are often antagonistic to one another. Excitingly, ER{beta} has been shown to be widespread in the body and to be involved in a multitude of physiological and pathophysiological events. This has led to a strong interest of the pharmaceutical industry to target ER{beta} by drugs against various diseases. In this review, focus is on the role ofmore » ER{beta} in malignant diseases where the anti proliferative activity of ER{beta} gives hope of new therapeutic approaches.« less

  17. Identification of HLA-DP polymorphism with DP alpha and DP beta probes and monoclonal antibodies: correlation with primed lymphocyte typing.

    PubMed Central

    Bodmer, J; Bodmer, W; Heyes, J; So, A; Tonks, S; Trowsdale, J; Young, J

    1987-01-01

    Thirty-four lymphoblastoid cell lines that had been previously typed for HLA-DP antigens by primed lymphocyte typing (PLT) were tested by Southern blotting and by ELISA. Using two DP beta probes and a DP alpha probe with a series of enzymes, it is possible to identify restriction fragment length polymorphism (RFLP) patterns characteristic of DPw1, -2, -3, -4, and possibly -5. ELISA typing results, based on two polymorphic DP antibodies DP11.1 and ILR1, were compared with PLT-defined and RFLP-defined types. Thus, using a range of probes and enzymes it is possible to identify DP polymorphism. The value of monoclonal antibodies for such studies is demonstrated, and the molecular data can, in some cases, pinpoint the amino acids responsible for the specificity of the monoclonal antibodies. Images PMID:2885841

  18. c-Ski, Smurf2, and Arkadia as regulators of TGF-beta signaling: new targets for managing myofibroblast function and cardiac fibrosis.

    PubMed

    Cunnington, Ryan H; Nazari, Mansoreh; Dixon, Ian M C

    2009-10-01

    Recent studies demonstrate the critical role of the extracellular matrix in the organization of parenchymal cells in the heart. Thus, an understanding of the modes of regulation of matrix production by cardiac myofibroblasts is essential. Transforming growth factor beta (TGF-beta) signaling is transduced through the canonical Smad pathway, and the involvement of this pathway in matrix synthesis and other processes requires precise control. Inhibition of Smad signaling may be achieved at the receptor level through the targeting of the TGF-beta type I receptors with an inhibitory Smad7/Smurf2 complex, or at the transcriptional level through c-Ski/receptor-Smad/co-mediator Smad4 interactions. Conversely, Arkadia protein intensifies TGF-beta-induced effects by marking c-Ski and inhibitory Smad7 for destruction. The study of these TGF-beta mediators is essential for future treatment of fibrotic disease, and this review highlights recent relevant findings that may impact our understanding of cardiac fibrosis.

  19. Permeation and gating properties of the L-type calcium channel in mouse pancreatic beta cells

    PubMed Central

    1993-01-01

    Ba2+ currents through L-type Ca2+ channels were recorded from cell- attached patches on mouse pancreatic beta cells. In 10 mM Ba2+, single- channel currents were recorded at -70 mV, the beta cell resting membrane potential. This suggests that Ca2+ influx at negative membrane potentials may contribute to the resting intracellular Ca2+ concentration and thus to basal insulin release. Increasing external Ba2+ increased the single-channel current amplitude and shifted the current-voltage relation to more positive potentials. This voltage shift could be modeled by assuming that divalent cations both screen and bind to surface charges located at the channel mouth. The single- channel conductance was related to the bulk Ba2+ concentration by a Langmuir isotherm with a dissociation constant (Kd(gamma)) of 5.5 mM and a maximum single-channel conductance (gamma max) of 22 pS. A closer fit to the data was obtained when the barium concentration at the membrane surface was used (Kd(gamma) = 200 mM and gamma max = 47 pS), which suggests that saturation of the concentration-conductance curve may be due to saturation of the surface Ba2+ concentration. Increasing external Ba2+ also shifted the voltage dependence of ensemble currents to positive potentials, consistent with Ba2+ screening and binding to membrane surface charge associated with gating. Ensemble currents recorded with 10 mM Ca2+ activated at more positive potentials than in 10 mM Ba2+, suggesting that external Ca2+ binds more tightly to membrane surface charge associated with gating. The perforated-patch technique was used to record whole-cell currents flowing through L-type Ca2+ channels. Inward currents in 10 mM Ba2+ had a similar voltage dependence to those recorded at a physiological Ca2+ concentration (2.6 mM). BAY-K 8644 (1 microM) increased the amplitude of the ensemble and whole-cell currents but did not alter their voltage dependence. Our results suggest that the high divalent cation solutions usually used to

  20. 17betaE2 promotes cell proliferation in endometriosis by decreasing PTEN via NFkappaB-dependent pathway.

    PubMed

    Zhang, Hui; Zhao, Xingbo; Liu, Shu; Li, Jijun; Wen, Zeqing; Li, Mingjiang

    2010-04-12

    The objective of this study was to explore the mechanism of phosphatase and tensin homolog (PTEN) loss in endometriosis. We found that aberrant PTEN expression and mitogen-activated protein kinases (MAPK)/ERK, phosphoinositide 3-kinase (PI3K)/AKt, and nuclear factor-kappaB (NFkappaB) signaling overactivities coexisted in endometriosis. In vitro, 17beta-estradiol rapidly activated the 3 pathways in endometriotic cells and specific inhibitions on the 3 pathways respectively blocked 17beta-estradiol-induced cell proliferation. 17beta-estradiol suppressed PTEN transcription and expression in endometriotic cells which was abolished by specific NFkappaB inhibition. Total/nuclear PTEN-loss and MAPK/ERK, PI3K/AKt, and NFkappaB signal overactivities coexist in endometriosis. In vitro, 17beta-estradiol can promotes cell proliferation in endometriosis by activating PI3K/AKt pathway via an NFkappaB/PTEN-dependent pathway. For the first time we propose the possibility of the presence of a positive feedback-loop: 17beta-estradiol-->high NFkappaB-->low PTEN-->high PI3K-->high NFkappaB, in endometriosis, which may finally promote the proliferation of ectopic endometrial epithelial cells and in turn contributes to the progression of the disease.

  1. Regressive changes in finasteride-treated human hyperplastic prostates correlate with an upregulation of TGF-beta receptor expression.

    PubMed

    Sáez, C; González-Baena, A C; Japón, M A; Giráldez, J; Segura, D I; Miranda, G; Rodríguez-Vallejo, J M; González-Esteban, J; Torrubia, F

    1998-10-01

    Prostatic atrophy has been documented histologically as a consequence of finasteride action on human hyperplastic prostates. An increase in apoptotic rates has also been reported in androgen-deprived hyperplastic prostates. Transforming growth factor beta (TGF-beta) signaling is implicated in apoptotic cell death. TGF-betas have been detected in normal and diseased human prostate. In the normal prostate, TGF-beta acts as a predominantly negative growth regulator. TGF-beta signaling receptors TbetaRI and TbetaRII have been shown to be negatively regulated by androgens. We studied the histological changes in 9 selected finasteride-treated patients with benign prostatic hyperplasia (BPH), and analyzed the levels of expression and localization of TGF-beta receptor types TbetaRI and TbetaRII in these patients as compared to selected BPH controls. The prostatic epithelial compartment seemed to be a primary target site for finasteride action, since we observed moderate to severe glandular atrophy after 4-6 months of treatment. TGF-beta receptors were upregulated in treated cases. We assessed a twofold increase in TbetaRII mRNA levels in treated cases as compared to controls. An increase in both TbetaRI and TbetaRII at the protein level by immunostaining was observed, which also provided a helpful means for detecting glands undergoing regression. We conclude that finasteride may modulate the TGF-beta signaling system to promote changes leading to apoptosis of epithelial cells and prostatic glandular atrophy.

  2. Absence of post-translational aspartyl beta-hydroxylation of epidermal growth factor domains in mice leads to developmental defects and an increased incidence of intestinal neoplasia.

    PubMed

    Dinchuk, Joseph E; Focht, Richard J; Kelley, Jennifer A; Henderson, Nancy L; Zolotarjova, Nina I; Wynn, Richard; Neff, Nicola T; Link, John; Huber, Reid M; Burn, Timothy C; Rupar, Mark J; Cunningham, Mark R; Selling, Bernard H; Ma, Jianhong; Stern, Andrew A; Hollis, Gregory F; Stein, Robert B; Friedman, Paul A

    2002-04-12

    The BAH genomic locus encodes three distinct proteins: junctin, humbug, and BAH. All three proteins share common exons, but differ significantly based upon the use of alternative terminal exons. The biological roles of BAH and humbug and their functional relationship to junctin remain unclear. To evaluate the role of BAH in vivo, the catalytic domain of BAH was specifically targeted such that the coding regions of junctin and humbug remained undisturbed. BAH null mice lack measurable BAH protein in several tissues, lack aspartyl beta-hydroxylase activity in liver preparations, and exhibit no hydroxylation of the epidermal growth factor (EGF) domain of clotting Factor X. In addition to reduced fertility in females, BAH null mice display several developmental defects including syndactyly, facial dysmorphology, and a mild defect in hard palate formation. The developmental defects present in BAH null mice are similar to defects observed in knock-outs and hypomorphs of the Notch ligand Serrate-2. In this work, beta-hydroxylation of Asp residues in EGF domains is demonstrated for a soluble form of a Notch ligand, human Jagged-1. These results along with recent reports that another post-translational modification of EGF domains in Notch gene family members (glycosylation by Fringe) alters Notch pathway signaling, lends credence to the suggestion that aspartyl beta-hydroxylation may represent another post-translational modification of EGF domains that can modulate Notch pathway signaling. Previous work has demonstrated increased levels of BAH in certain tumor tissues and a role for BAH in tumorigenesis has been proposed. The role of hydroxylase in tumor formation was tested directly by crossing BAH KO mice with an intestinal tumor model, APCmin mice. Surprisingly, BAH null/APCmin mice show a statistically significant increase in both intestinal polyp size and number when compared with BAH wild-type/APCmin controls. These results suggest that, in contrast to expectations

  3. Intracellular mediators of transforming growth factor beta superfamily signaling localize to endosomes in chicken embryo and mouse lenses in vivo.

    PubMed

    Rajagopal, Ramya; Ishii, Shunsuke; Beebe, David C

    2007-06-25

    Endocytosis is a key regulator of growth factor signaling pathways. Recent studies showed that the localization to endosomes of intracellular mediators of growth factor signaling may be required for their function. Although there is substantial evidence linking endocytosis and growth factor signaling in cultured cells, there has been little study of the endosomal localization of signaling components in intact tissues or organs. Proteins that are downstream of the transforming growth factor-beta superfamily signaling pathway were found on endosomes in chicken embryo and postnatal mouse lenses, which depend on signaling by members of the TGFbeta superfamily for their normal development. Phosphorylated Smad1 (pSmad1), pSmad2, Smad4, Smad7, the transcriptional repressors c-Ski and TGIF and the adapter molecules Smad anchor for receptor activation (SARA) and C184M, localized to EEA-1- and Rab5-positive vesicles in chicken embryo and/or postnatal mouse lenses. pSmad1 and pSmad2 also localized to Rab7-positive late endosomes. Smad7 was found associated with endosomes, but not caveolae. Bmpr1a conditional knock-out lenses showed decreased nuclear and endosomal localization of pSmad1. Many of the effectors in this pathway were distributed differently in vivo from their reported distribution in cultured cells. Based on the findings reported here and data from other signaling systems, we suggest that the localization of activated intracellular mediators of the transforming growth factor-beta superfamily to endosomes is important for the regulation of growth factor signaling.

  4. Alpha- and beta-keratins of the snake epidermis.

    PubMed

    Toni, Mattia; Alibardi, Lorenzo

    2007-01-01

    Snake scales contain specialized hard keratins (beta-keratins) and alpha- or cyto-keratins in their epidermis. The number, isoelectric point, and the evolution of these proteins in snakes and their similarity with those of other vertebrates are not known. In the present study, alpha- and beta-keratins of snake molts and of the whole epidermis have been studied by using two-dimensional electrophoresis and immunocytochemistry. Specific keratins in snake epidermis have been identified by using antibodies that recognize acidic and basic cytokeratins and avian or lizard scale beta-keratin. Alpha keratins of 40-70 kDa and isoelectric point (pI) at 4.5-7.0 are present in molts. The study suggests that cytokeratins in snakes are acidic or neutral, in contrast to mammals and birds where basic keratins are also present. Beta keratins of 10-15 kDa and a pI of 6.5-8.5 are found in molts. Some beta-keratins appear as basic proteins (pI 8.2) comparable to those present in the epidermis of other reptiles. Some basic "beta-keratins" associate with cytokeratins as matrix proteins and replace cytokeratins forming the corneous material of the mature beta-layer of snake scales, as in other reptiles. The study also suggests that more forms of beta-keratins (more than three different types) are present in the epidermis of snakes.

  5. Glycation induces formation of amyloid cross-beta structure in albumin.

    PubMed

    Bouma, Barend; Kroon-Batenburg, Loes M J; Wu, Ya-Ping; Brünjes, Bettina; Posthuma, George; Kranenburg, Onno; de Groot, Philip G; Voest, Emile E; Gebbink, Martijn F B G

    2003-10-24

    Amyloid fibrils are components of proteinaceous plaques that are associated with conformational diseases such as Alzheimer's disease, transmissible spongiform encephalopathies, and familial amyloidosis. Amyloid polypeptides share a specific quarternary structure element known as cross-beta structure. Commonly, fibrillar aggregates are modified by advanced glycation end products (AGE). In addition, AGE formation itself induces protein aggregation. Both amyloid proteins and protein-AGE adducts bind multiligand receptors, such as receptor for AGE, CD36, and scavenger receptors A and B type I, and the serine protease tissue-type plasminogen activator (tPA). Based on these observations, we hypothesized that glycation induces refolding of globular proteins, accompanied by formation of cross-beta structure. Using transmission electron microscopy, we demonstrate here that glycated albumin condensates into fibrous or amorphous aggregates. These aggregates bind to amyloid-specific dyes Congo red and thioflavin T and to tPA. In contrast to globular albumin, glycated albumin contains amino acid residues in beta-sheet conformation, as measured with circular dichroism spectropolarimetry. Moreover, it displays cross-beta structure, as determined with x-ray fiber diffraction. We conclude that glycation induces refolding of initially globular albumin into amyloid fibrils comprising cross-beta structure. This would explain how glycated ligands and amyloid ligands can bind to the same multiligand "cross-beta structure" receptors and to tPA.

  6. Effect of Hypertriglyceridemia on Beta Cell Mass and Function in ApoC3 Transgenic Mice*

    PubMed Central

    Liu, Yun-Zi; Cheng, Xiaoyun; Zhang, Ting; Lee, Sojin; Yamauchi, Jun; Xiao, Xiangwei; Gittes, George; Qu, Shen; Jiang, Chun-Lei; Dong, H. Henry

    2016-01-01

    Hypertriglyceridemia results from increased production and decreased clearance of triglyceride-rich very low-density lipoproteins, a pathological condition that accounts for heightened risk of ischemic vascular diseases in obesity and type 2 diabetes. Despite its intimate association with insulin resistance, whether hypertriglyceridemia constitutes an independent risk for beta cell dysfunction in diabetes is unknown. Answering this fundamental question is stymied by the fact that hypertriglyceridemia is intertwined with hyperglycemia and insulin resistance in obese and diabetic subjects. To circumvent this limitation, we took advantage of apolipoprotein C3 (ApoC3)-transgenic mice, a model with genetic predisposition to hypertriglyceridemia. We showed that ApoC3-transgenic mice, as opposed to age/sex-matched wild-type littermates, develop hypertriglyceridemia with concomitant elevations in plasma cholesterol and non-esterified fatty acid levels. Anti-insulin and anti-glucagon dual immunohistochemistry in combination with morphometric analysis revealed that ApoC3-transgenic and wild-type littermates had similar beta cell and alpha cell masses as well as islet size and architecture. These effects correlated with similar amplitudes of glucose-stimulated insulin secretion and similar degrees of postprandial glucose excursion in ApoC3-transgenic versus wild-type littermates. Oil Red O histology did not visualize lipid infiltration into islets, correlating with the lack of ectopic triglyceride and cholesterol depositions in the pancreata of ApoC3-transgenic versus wild-type littermates. ApoC3-transgenic mice, despite persistent hypertriglyceridemia, maintained euglycemia under both fed and fasting conditions without manifestation of insulin resistance and fasting hyperinsulinemia. Thus, hypertriglyceridemia per se is not an independent risk factor for beta cell dysfunction in ApoC3 transgenic mice. PMID:27226540

  7. Effects of sequential artificial tear and cyclosporine emulsion therapy on conjunctival goblet cell density and transforming growth factor-beta2 production.

    PubMed

    Pflugfelder, Stephen C; De Paiva, Cintia S; Villarreal, Arturo L; Stern, Michael E

    2008-01-01

    To evaluate the effects of sequential treatment with artificial tears and cyclosporine emulsion on conjunctival goblet cell density and production of transforming growth factor (TGF)-beta2 in patients with dry eye disease. Patients with dry eye disease (N = 6) defined by an Ocular Surface Disease Index symptom score >or=25, Schirmer test 1 <10 mm, and corneal fluorescein and conjunctival lissamine green staining scores >or=3 were treated with artificial tears (Refresh Plus; Allergan, Irvine, CA) 4 times a day for 4 weeks, followed by 0.05% cyclosporine emulsion (Restasis; Allergan) twice a day for 12 weeks. Impression cytology was performed on the bulbar conjunctiva of both eyes at baseline, after artificial tear therapy, and after 6 and 12 weeks of cyclosporine therapy. Goblet cells were counted in 5 representative microscopic fields per membrane in those taken from the temporal and inferior bulbar conjunctiva of the worse eye, and membranes taken from the fellow eye were immunostained for TGF-beta2. There were no differences in mean goblet cell density between baseline and 4 weeks of artificial tears in the temporal and inferior bulbar specimens. After 6 weeks of cyclosporine emulsion, goblet cell density was significantly greater than baseline and artificial tears in the inferior bulbar conjunctiva (P < 0.01). After 12 weeks of cyclosporine emulsion, goblet cell density was significantly greater than baseline and artificial tears in both temporal and inferior bulbar sites (P < 0.01). The number of TGF-beta2-positive goblet cells was also noted to increase after 6 and 12 weeks of cyclosporine therapy (P < 0.001). Cyclosporine emulsion, but not artificial tears, increases goblet cell density and production of the immunoregulatory factor TGF-beta2 in the bulbar conjunctiva in patients with dry eye.

  8. Triiodothyronine, beta-adrenergic receptors, agonist responses, and exercise capacity.

    PubMed

    Martin, W H

    1993-07-01

    Although thyroid hormone excess results in increased beta-adrenergic receptor density or agonist responses in some cells of experimental animals, the role of these effects in contributing to clinical manifestations of hyperthyroidism in human subjects is unclear. To shed further light on this issue, we characterized the effect of 2 weeks of excess triiodothyronine administration on cardiac and metabolic responses to graded-dose isoproterenol infusion, skeletal muscle beta-adrenergic receptor density, and physiologic determinants of exercise capacity in young healthy subjects. The slope of the heart rate response to isoproterenol was 36% greater (p < 0.05) after triiodothyronine administration. In addition, beta-adrenergic receptor density was increased (p < 0.01) in all types of skeletal muscle fibers. Maximal oxygen uptake during treadmill exercise declined 5% (p < 0.001) after triiodothyronine administration because of a decrease in the arteriovenous oxygen difference (p < 0.05). The plasma lactate response to submaximal exercise was 25% greater (p < 0.01) in the hyperthyroid state. These effects were paralleled by a decrement in skeletal muscle oxidative capacity and a decrease in cross-sectional area of type 2A skeletal myocytes. Thus, thyroid hormone excess enhances cardiac beta-adrenergic sensitivity under in vivo conditions in human subjects. Nevertheless, exercise capacity is diminished in the hyperthyroid state, an effect that may be related to reduced skeletal muscle oxidative capacity and type 2A fiber atrophy.

  9. Role of glutamine 17 of the bovine papillomavirus E5 protein in platelet-derived growth factor beta receptor activation and cell transformation.

    PubMed

    Klein, O; Polack, G W; Surti, T; Kegler-Ebo, D; Smith, S O; DiMaio, D

    1998-11-01

    The bovine papillomavirus E5 protein is a small, homodimeric transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor through transmembrane and juxtamembrane interactions, resulting in receptor activation and cell transformation. Glutamine 17 in the transmembrane domain of the 44-amino-acid E5 protein is critical for complex formation and receptor activation, and we previously proposed that glutamine 17 forms a hydrogen bond with threonine 513 of the PDGF beta receptor. We have constructed and analyzed mutant E5 proteins containing all possible amino acids at position 17 and examined the ability of these proteins to transform C127 fibroblasts, which express endogenous PDGF beta receptor. Although several position 17 mutants were able to transform cells, mutants containing amino acids with side groups that were unable to participate in hydrogen bonding interactions did not form a stable complex with the PDGF beta receptor or transform cells, in agreement with the proposed interaction between position 17 of the E5 protein and threonine 513 of the receptor. The nature of the residue at position 17 also affected the ability of the E5 proteins to dimerize. Overall, there was an excellent correlation between the ability of the various E5 mutant proteins to bind the PDGF beta receptor, lead to receptor tyrosine phosphorylation, and transform cells. Similar results were obtained in Ba/F3 hematopoietic cells expressing exogenous PDGF beta receptor. In addition, treatment of E5-transformed cells with a specific inhibitor of the PDGF receptor tyrosine kinase reversed the transformed phenotype. These results confirm the central importance of the PDGF beta receptor in mediating E5 transformation and highlight the critical role of the residue at position 17 of the E5 protein in the productive interaction with the PDGF beta receptor. On the basis of molecular modeling analysis and the known chemical properties of the

  10. Lower risk of postinfarct rupture in mouse heart overexpressing beta 2-adrenergic receptors: importance of collagen content.

    PubMed

    Gao, Xiao-Ming; Dilley, Rodney J; Samuel, Chrishan S; Percy, Elodie; Fullerton, Meryl J; Dart, Anthony M; Du, Xiao-Jun

    2002-10-01

    This paper addresses whether the enhanced left ventricular (LV) contractility and heart rate, seen in transgenic mice overexpressing beta -adrenergic receptor in the heart, might raise the incidence of LV rupture after myocardial infarct. Transgenic and wild-type mice underwent left coronary artery occlusion. Postinfarct deaths that occurred 1-7 days after surgery were analyzed. Hemodynamics, morphologic parameters, and collagen content in the LV were determined. A significantly lower incidence of LV rupture was observed in transgenic than in wild-type mice 3-5 days after myocardial infarct (2.5 versus 19.7%, p < 0.05), despite a similar infarct size between the two groups and better hemodynamic function in transgenic mouse hearts. Morphologic analysis showed a more severe infarct expansion in wild-type versus transgenic mice or in mice dying of rupture versus those that died of acute heart failure. Collagen content was higher in the LV of sham-operated transgenic than wild-type mice (p < 0.01) with both type I and type III collagen elevated. Such difference in collagen content between transgenic and wild-type mice was maintained in noninfarcted and infarcted LV. In conclusion, transgenic mice overexpressing beta -adrenergic receptor had a lower risk of cardiac rupture during the acute phase after infarction despite the markedly enhanced LV contractility and heart rate. As a hyperdynamic function due to beta-adrenergic activation would likely increase the risk of cardiac rupture and infarct expansion, the lack of rupture in this transgenic mouse model suggests that the interstitial collagen level is a more important factor than functional status in the pathogenesis of rupture and infarct expansion.

  11. Negative feedback regulation of TGF-beta signaling by the SnoN oncoprotein.

    PubMed

    Stroschein, S L; Wang, W; Zhou, S; Zhou, Q; Luo, K

    1999-10-22

    Smad proteins mediate transforming growth factor-beta (TGF-beta) signaling to regulate cell growth and differentiation. The SnoN oncoprotein was found to interact with Smad2 and Smad4 and to repress their abilities to activate transcription through recruitment of the transcriptional corepressor N-CoR. Immediately after TGF-beta stimulation, SnoN is rapidly degraded by the nuclear accumulation of Smad3, allowing the activation of TGF-beta target genes. By 2 hours, TGF-beta induces a marked increase in SnoN expression, resulting in termination of Smad-mediated transactivation. Thus, SnoN maintains the repressed state of TGF-beta-responsive genes in the absence of ligand and participates in negative feedback regulation of TGF-beta signaling.

  12. A novel mechanism by which hepatocyte growth factor blocks tubular epithelial to mesenchymal transition.

    PubMed

    Yang, Junwei; Dai, Chunsun; Liu, Youhua

    2005-01-01

    Hepatocyte growth factor (HGF) is a potent antifibrotic cytokine that blocks tubular epithelial to mesenchymal transition (EMT) induced by TGF-beta1. However, the underlying mechanism remains largely unknown. This study investigated the signaling events that lead to HGF blockade of the TGF-beta1-initiated EMT. Incubation of human kidney epithelial cells HKC with HGF only marginally affected the expression of TGF-beta1 and its type I and type II receptors, suggesting that disruption of TGF-beta1 signaling likely plays a critical role in mediating HGF inhibition of TGF-beta1 action. However, HGF neither affected TGF-beta1-induced Smad-2 phosphorylation and its subsequent nuclear translocation nor influenced the expression of inhibitory Smad-6 and -7 in tubular epithelial cells. HGF specifically induced the expression of Smad transcriptional co-repressor SnoN but not Ski and TG-interacting factor at both mRNA and protein levels in HKC cells. SnoN physically interacted with activated Smad-2 by forming transcriptionally inactive complex and overrode the profibrotic action of TGF-beta1. In vivo, HGF did not affect Smad-2 activation and its nuclear accumulation in tubular epithelium, but it restored SnoN protein abundance in the fibrotic kidney in obstructive nephropathy. Hence, HGF blocks EMT by antagonizing TGF-beta1's action via upregulating Smad transcriptional co-repressor SnoN expression. These findings not only identify a novel mode of interaction between the signals activated by HGF receptor tyrosine kinase and TGF-beta receptor serine/threonine kinases but also illustrate the feasibility of confining Smad activity as an effective strategy for blocking renal fibrosis.

  13. The genus Rhodosporidium: a potential source of beta-carotene.

    PubMed

    de Miguel, T; Calo, P; Díaz, A; Villa, T G

    1997-03-01

    Four wild-type species of the genus Rhodosporidium have been studied as as possible sources for the industrial production of beta-carotene. HPLC-based studies showed that their carotenoid composition consisted of almost pure beta-carotene at concentrations ranging from 226 to 685 micrograms/g of dried yeast biomass. These results are consistent with those obtained by spectrophotometry at 480 nm.

  14. {beta}-Catenin regulates airway smooth muscle contraction.

    PubMed

    Jansen, Sepp R; Van Ziel, Anna M; Baarsma, Hoeke A; Gosens, Reinoud

    2010-08-01

    beta-Catenin is an 88-kDa member of the armadillo family of proteins that is associated with the cadherin-catenin complex in the plasma membrane. This complex interacts dynamically with the actin cytoskeleton to stabilize adherens junctions, which play a central role in force transmission by smooth muscle cells. Therefore, in the present study, we hypothesized a role for beta-catenin in the regulation of smooth muscle force production. beta-Catenin colocalized with smooth muscle alpha-actin (sm-alpha-actin) and N-cadherin in plasma membrane fractions and coimmunoprecipitated with sm-alpha-actin and N-cadherin in lysates of bovine tracheal smooth muscle (BTSM) strips. Moreover, immunocytochemistry of cultured BTSM cells revealed clear and specific colocalization of sm-alpha-actin and beta-catenin at the sites of cell-cell contact. Treatment of BTSM strips with the pharmacological beta-catenin/T cell factor-4 (TCF4) inhibitor PKF115-584 (100 nM) reduced beta-catenin expression in BTSM whole tissue lysates and in plasma membrane fractions and reduced maximal KCl- and methacholine-induced force production. These changes in force production were not accompanied by changes in the expression of sm-alpha-actin or sm-myosin heavy chain (MHC). Likewise, small interfering RNA (siRNA) knockdown of beta-catenin in BTSM strips reduced beta-catenin expression and attenuated maximal KCl- and methacholine-induced contractions without affecting sm-alpha-actin or sm-MHC expression. Conversely, pharmacological (SB-216763, LiCl) or insulin-induced inhibition of glycogen synthase kinase-3 (GSK-3) enhanced the expression of beta-catenin and augmented maximal KCl- and methacholine-induced contractions. We conclude that beta-catenin is a plasma membrane-associated protein in airway smooth muscle that regulates active tension development, presumably by stabilizing cell-cell contacts and thereby supporting force transmission between neighboring cells.

  15. Fragrant dioxane derivatives identify beta1-subunit-containing GABAA receptors.

    PubMed

    Sergeeva, Olga A; Kletke, Olaf; Kragler, Andrea; Poppek, Anja; Fleischer, Wiebke; Schubring, Stephan R; Görg, Boris; Haas, Helmut L; Zhu, Xin-Ran; Lübbert, Hermann; Gisselmann, Günter; Hatt, Hanns

    2010-07-30

    Nineteen GABA(A) receptor (GABA(A)R) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of beta1-subunit-containing GABA(A)Rs is unknown. Here we report the discovery of a new structural class of GABA(A)R positive modulators with unique beta1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed alpha1betaxgamma2L (x-for 1,2,3) GABA(A)R FDD were 6 times more potent at beta1- versus beta2- and beta3-containing receptors. Serine at position 265 was essential for the high sensitivity of the beta1-subunit to FDD and the beta1N286W mutation nearly abolished modulation; vice versa the mutation beta3N265S shifted FDD sensitivity toward the beta1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to beta1-negative cerebellar Purkinje neurons. Immunostaining for the beta1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by beta1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of beta1-containing GABA(A)Rs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABA(A)Rs.

  16. Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death.

    PubMed

    Clark, Amy L; Kanekura, Kohsuke; Lavagnino, Zeno; Spears, Larry D; Abreu, Damien; Mahadevan, Jana; Yagi, Takuya; Semenkovich, Clay F; Piston, David W; Urano, Fumihiko

    2017-07-17

    Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

  17. 5Beta,6beta-epoxy-17-oxoandrostan-3beta-yl acetate and 5beta,6beta-epoxy-20-oxopregnan-3beta-yl acetate.

    PubMed

    Pinto, Rui M A; Salvador, Jorge A R; Paixão, José A

    2008-05-01

    In the title compounds, C(21)H(30)O(4), (I), and C(23)H(34)O(4), (II), respectively, which are valuable intermediates in the synthesis of important steroid derivatives, rings A and B are cis-(5beta,10beta)-fused. The two molecules have similar conformations of rings A, B and C. The presence of the 5beta,6beta-epoxide group induces a significant twist of the steroid nucleus and a strong flattening of the B ring. The different C17 substituents result in different conformations for ring D. Cohesion of the molecular packing is achieved in both compounds only by weak intermolecular interactions. The geometries of the molecules in the crystalline environment are compared with those of the free molecules as given by ab initio Roothan Hartree-Fock calculations. We show in this work that quantum mechanical ab initio methods reproduce well the details of the conformation of these molecules, including a large twist of the steroid nucleus. The calculated twist values are comparable, but are larger than the observed values, indicating a possible small effect of the crystal packing on the twist angles.

  18. Effect of centrifugation time on growth factor and MMP release of an experimental platelet-rich fibrin-type product.

    PubMed

    Eren, Gülnihal; Gürkan, Ali; Atmaca, Harika; Dönmez, Ayhan; Atilla, Gül

    2016-07-01

    Platelet-rich fibrin (PRF) has a controlled release of growth factors due to the fibrin matrix structure. Different centrifugation protocols were suggested for PRF preparation. Since the derivation method of PRF can alter its contents, in the present study it is aimed to investigate the cell contents and transforming growth factor beta-1 (TGF-β1), platelet-derived growth factor (PDGF-AB), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1 and-8 release from experimental PRF-type membranes obtained with different centrifugation times at 400 gravity. Three blood samples were collected from 20 healthy non-smoker volunteers. One tube was used for whole blood analyses. The other two tubes were centrifuged at 400 g for 10 minutes (group A) or 12 minutes (group B). Each experimental PRF-type membrane was placed in Dulbecco's Modified Eagle's Medium (DMEM)and at 1, 24 and 72 hours, TGF-β1, PDGF-AB, VEGF, MMP-1 and -8 release amounts were analysed by enzyme-linked immunosorbent assay (ELISA). The blood cell count of membranes was determined by subtracting plasma supernatant and red blood cell (RBC) mixture from the whole blood cell counts. At 72 hours, the VEGF level of group B was statistically higher than that of group A (p = 0.040). The centrifugation time was not found to influence the release of other growth factors, enzymes and cell counts. Within the limits of the present study, it might be suggested that centrifugation time at a constant gravity has a significant effect on the VEGF levels released from experimental PRF-type membrane. It can be concluded that due to the importance of VEGF in the tissue healing process, membranes obtained at 12-minute centrifugation time may show a superior potential in wound healing.

  19. Pregnancy outcome in dairy and beef cattle after artificial insemination and treatment with seminal plasma or transforming growth factor beta-1.

    PubMed

    Odhiambo, J F; Poole, D H; Hughes, L; Dejarnette, J M; Inskeep, E K; Dailey, R A

    2009-09-01

    Reduced capability of the uterus to support pregnancy in the absence of its interaction with secretions from male accessory glands has been demonstrated in rodents and to some extent in pigs. However, in cattle, the role of postmating inflammatory response on pregnancy success has not been studied. The current study examined the influence of uterine presensitization with seminal antigens at breeding on pregnancy outcome in cows. Lactating beef (n=1090) and dairy (n=800) cows received 0.5 mL seminal plasma (SP), 40 ng recombinant human transforming growth factor-beta1 (rhTGF-beta1), or 0.5 mL bovine serum albumin (BSA), or were left untreated before or at insemination. Semen was deposited into the anterior cervix using a second insemination gun. Pregnancy was diagnosed at 35 to 40 d postinsemination by transrectal ultrasonography or from records of calves born the subsequent calving season. Pregnancy rates in beef cows did not differ among treatments but differed among trials (69.8%, 52.5% vs. 40.3%; P<0.05). In trials where average pregnancy rates were below 50%, treatments with TGF-beta1 but not SP tended (P<0.07) to increase pregnancy rates in beef cows. In dairy cows, SP and TGF-beta1 improved pregnancy outcome by 10 percentage points, but these increments did not achieve statistical significance. In conclusion, this study did not find any conclusive evidence for the effect of TGF-beta1 or seminal plasma on pregnancy outcome in lactating dairy or beef cows but realized marginal improvements when pregnancy rates were below 50% (compromised fertility).

  20. Local Interstellar Medium Properties and Deuterium Abundances for the Lines of Sight Toward HR 1099, 31 Comae, beta Ceti, and beta Cassiopeiae

    NASA Technical Reports Server (NTRS)

    Piskunov, Nikolai; Wood, Brian E.; Linsky, Jeffrey L.; Dempsey, Robert C.; Ayres, Thomas R.

    1997-01-01

    We analyze Goddard High-Resolution Spectrograph data to infer the properties of local interstellar gas and the Deuterium/Hydrogen (D/H) ratio for lines of sight toward four nearby late-type stars-HR 1099, 31 Comae, beta Ceti, and beta Cassiopeiae. The data consist of spectra of the hydrogen and deuterium Lyman-(alpha) lines, and echelle spectra of the Mg IIh and k lines toward all stars except beta Cas. Spectra of the RS CVn-type spectroscopic binary system HR 1099 were obtained near opposite quadratures to determine the intrinsic stellar emission line profile and the interstellar absorption separately. Multiple-velocity components were found toward HR 1099 and beta Cet. The spectra of 31 Com and beta Cet are particularly interesting because they sample lines of sight toward the north and south Galactic poles, respectively, for which H I and D I column densities were not previously available. The north Galactic pole appears to be a region of low hydrogen density like the 'interstellar tunnel' toward epsilon CMa. The temperature and turbulent velocities of the Local InterStellar Medium (LISM) that we measure for the lines of sight toward HR 1099, 31 Com, beta Cet, and beta Cas are similar to previously measured values (T approx.7000 K and xi = 1.0-1.6 km/s). The deuterium/hydrogen ratios found for these lines of sight are also consistent with previous measurements of other short lines of sight, which suggest D/H approx. 1.6 x 10(sup -5). In contrast, the Mg abundance measured for the beta Cet line of sight (implying a logarithmic depletion of D(Mg) = +0.30 +/- 0.15) is about 5 times larger than the Mg abundance previously observed toward alpha Cen, and about 20 times larger than all other previous measurements for the LISM. These results demonstrate that metal abundances in the LISM vary greatly over distances of only a few parsecs.

  1. Spleen tyrosine kinase mediates high glucose-induced transforming growth factor-{beta}1 up-regulation in proximal tubular epithelial cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yang, Won Seok; Chang, Jai Won; Han, Nam Jeong

    The role of spleen tyrosine kinase (Syk) in high glucose-induced intracellular signal transduction has yet to be elucidated. We investigated whether Syk is implicated in high glucose-induced transforming growth factor-{beta}1 (TGF-{beta}1) up-regulation in cultured human proximal tubular epithelial cells (HK-2 cell). High glucose increased TGF-{beta}1 gene expression through Syk, extracellular signal-regulated kinase (ERK), AP-1 and NF-{kappa}B. High glucose-induced AP-1 DNA binding activity was decreased by Syk inhibitors and U0126 (an ERK inhibitor). Syk inhibitors suppressed high glucose-induced ERK activation, whereas U0126 had no effect on Syk activation. High glucose-induced NF-{kappa}B DNA binding activity was also decreased by Syk inhibitors. Highmore » glucose increased nuclear translocation of p65 without serine phosphorylation of I{kappa}B{alpha} and without degradation of I{kappa}B{alpha}, but with an increase in tyrosine phosphorylation of I{kappa}B{alpha} that may account for the activation of NF-{kappa}B. Both Syk inhibitors and Syk-siRNA attenuated high glucose-induced I{kappa}B{alpha} tyrosine phosphorylation and p65 nuclear translocation. Depletion of p21-activated kinase 2 (Pak2) by transfection of Pak2-siRNA abolished high glucose-induced Syk activation. In summary, high glucose-induced TGF-{beta}1 gene transcription occurred through Pak2, Syk and subsequent ERK/AP-1 and NF-{kappa}B pathways. This suggests that Syk might be implicated in the diabetic kidney disease.« less

  2. Agglucetin, a tetrameric C-type lectin-like venom protein, regulates endothelial cell survival and promotes angiogenesis by activating integrin {alpha}v{beta}3 signaling

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, W.-J.

    2008-05-02

    Agglucetin, a platelet glycoprotein (GP)Ib binding protein from Formosan Agkistrodon acutus (A. acutus) venom, could sustain human umbilical vein endothelial cell (HUVEC) proliferation and HUVEC adhering to immobilized agglucetin showed extensive spreading, which was strongly abrogated by integrin antagonists 7E3 and triflavin. Flow cytometric analyses confirmed the expression of GPIb complex on HUVEC is absent and fluorescein isothiocyanate (FITC)-agglucetin binds to HUVEC in a dose-dependent and saturable manner. Furthermore, native agglucetin specifically and dose-dependently inhibited the binding of FITC-23C6, an anti-{alpha}v{beta}3 monoclonal antibody (mAb), but not antibodies against {alpha}2 and {alpha}5, toward HUVEC and purified {alpha}v{beta}3 also bound to immobilizedmore » agglucetin-{beta} in a dose-dependent manner. Moreover, agglucetin exhibited a pro-angiogenic effect in vitro, as well as the focal adhesion kinase (FAK)-associated signaling molecules responsible for HUVEC activation were initiated by agglucetin. In conclusion, agglucetin, acting as a survival factor, promotes endothelial adhesion and angiogenesis by triggering {alpha}v{beta}3 signaling through FAK/phosphatidylinositol 3-kinase (PI3K)/Akt pathway.« less

  3. Milk composition and lactation of beta-casein-deficient mice.

    PubMed Central

    Kumar, S; Clarke, A R; Hooper, M L; Horne, D S; Law, A J; Leaver, J; Springbett, A; Stevenson, E; Simons, J P

    1994-01-01

    beta-Casein is a major protein component of milk and, in conjunction with the other caseins, it is assembled into micelles. The casein micelles determine many of the physical characteristics of milk, which are important for stability during storage and for milk-processing properties. There is evidence that suggests that beta-casein may also possess other, nonnutritional functions. To address the function of beta-casein, the mouse beta-casein gene was disrupted by gene targeting in embryonic stem cells. Homozygous beta-casein mutant mice are viable and fertile; females can lactate and successfully rear young. beta-Casein was expressed at a reduced level in heterozygotes and was completely absent from the milk of homozygous mutant mice. Despite the deficiency of beta-casein, casein micelles were assembled in heterozygous and homozygous mutants, albeit with reduced diameters. The absence of beta-casein expression was reflected in a reduced total protein concentration in milk, although this was partially compensated for by an increased concentration of other proteins. The growth of pups feeding on the milk of homozygous mutants was reduced relative to those feeding on the milk of wild-type mice. Various genetic manipulations of caseins have been proposed for the qualitative improvement of cow's milk composition. The results presented here demonstrate that beta-casein has no essential function and that the casein micelle is remarkably tolerant of changes in composition. Images PMID:8016126

  4. Epidemiology of type 2 diabetes: risk factors.

    PubMed

    Haffner, S M

    1998-12-01

    A number of cross-sectional and prospective studies that compared the insulin sensitivity of various national and ethnic populations within the U.S. to the total U.S. population were analyzed to find possible risk factors for the development of type 2 diabetes. It was found that the risks for diabetes in African-Americans, Hispanics, and Native Americans are approximately 2, 2.5, and 5 times greater, respectively, than in Caucasians. Studies of the prevalence of type 2 diabetes in Mexican Americans and non-Hispanic whites in San Antonio showed that there is an inverse relationship between socioeconomic status and the prevalence of diabetes. It also appears that cultural effects lead to an increased incidence of obesity in these populations, which may lead to insulin resistance. Genetic factors may also be a contributing factor. A 5-year, prospective study of insulin resistance in Pima Indians showed a relationship between impaired glucose tolerance and subsequent development of type 2 diabetes. In a 7-year study in Mexican Americans, those subjects who had both high insulin secretion and impaired insulin sensitivity had a 14-fold increased risk of developing type 2 diabetes. Regardless of cultural and ethnic factors, the San Antonio Heart Study, which compared Mexican Americans and non-Hispanic whites, showed that in both groups, the strongest predictors of developing type 2 diabetes are elevated fasting insulin concentrations and low insulin secretion.

  5. Beta-blockers for hypertension.

    PubMed

    Wiysonge, C S; Bradley, H; Mayosi, B M; Maroney, R; Mbewu, A; Opie, L H; Volmink, J

    2007-01-24

    )=78.2%, ARI=6.4% NNH=16) and RAS inhibitors (RR 1.41, 95%CI 1.29 to 1.54, I(2)=12.1%; ARI=5.5%, NNH=18), but there was no significant difference with CCBs. The available evidence does not support the use of beta-blockers as first-line drugs in the treatment of hypertension. This conclusion is based on the relatively weak effect of beta-blockers to reduce stroke and the absence of an effect on coronary heart disease when compared to placebo or no treatment. More importantly, it is based on the trend towards worse outcomes in comparison with calcium-channel blockers, renin-angiotensin system inhibitors, and thiazide diuretics. Most of the evidence for these conclusions comes from trials where atenolol was the beta-blocker used (75% of beta-blocker participants in this review). However, it is not known at present whether beta-blockers have differential effects on younger and elderly patients or whether there are differences between the different sub-types of beta-blockers.

  6. Structure of a group A streptococcal phage-encoded virulence factor reveals a catalytically active triple-stranded beta-helix.

    PubMed

    Smith, Nicola L; Taylor, Edward J; Lindsay, Anna-Marie; Charnock, Simon J; Turkenburg, Johan P; Dodson, Eleanor J; Davies, Gideon J; Black, Gary W

    2005-12-06

    Streptococcus pyogenes (group A Streptococcus) causes severe invasive infections including scarlet fever, pharyngitis (streptococcal sore throat), skin infections, necrotizing fasciitis (flesh-eating disease), septicemia, erysipelas, cellulitis, acute rheumatic fever, and toxic shock. The conversion from nonpathogenic to toxigenic strains of S. pyogenes is frequently mediated by bacteriophage infection. One of the key bacteriophage-encoded virulence factors is a putative "hyaluronidase," HylP1, a phage tail-fiber protein responsible for the digestion of the S. pyogenes hyaluronan capsule during phage infection. Here we demonstrate that HylP1 is a hyaluronate lyase. The 3D structure, at 1.8-angstroms resolution, reveals an unusual triple-stranded beta-helical structure and provides insight into the structural basis for phage tail assembly and the role of phage tail proteins in virulence. Unlike the triple-stranded beta-helix assemblies of the bacteriophage T4 injection machinery and the tailspike endosialidase of the Escherichia coli K1 bacteriophage K1F, HylP1 possesses three copies of the active center on the triple-helical fiber itself without the need for an accessory catalytic domain. The triple-stranded beta-helix is not simply a structural scaffold, as previously envisaged; it is harnessed to provide a 200-angstroms-long substrate-binding groove for the optimal reduction in hyaluronan viscosity to aid phage penetration of the capsule.

  7. Identical plasmid AmpC beta-lactamase genes and plasmid types in E. coli isolates from patients and poultry meat in the Netherlands.

    PubMed

    Voets, Guido M; Fluit, Ad C; Scharringa, Jelle; Schapendonk, Claudia; van den Munckhof, Thijs; Leverstein-van Hall, Maurine A; Stuart, James Cohen

    2013-11-01

    The increasing prevalence of third-generation cephalosporin-resistant Enterobacteriaceae is a worldwide problem. Recent studies showed that poultry meat and humans share identical Extended-Spectrum Beta-Lactamase genes, plasmid types, and Escherichia coli strain types, suggesting that transmission from poultry meat to humans may occur. The aim of this study was to compare plasmid-encoded Ambler class C beta-lactamase (pAmpC) genes, their plasmids, and bacterial strain types between E. coli isolates from retail chicken meat and clinical isolates in the Netherlands. In total, 98 Dutch retail chicken meat samples and 479 third-generation cephalosporin non-susceptible human clinical E. coli isolates from the same period were screened for pAmpC production. Plasmid typing was performed using PCR-based replicon typing (PBRT). E coli strains were compared using Multi-Locus-Sequence-Typing (MLST). In 12 of 98 chicken meat samples (12%), pAmpC producing E. coli were detected (all blaCMY-2). Of the 479 human E. coli, 25 (5.2%) harboured pAmpC genes (blaCMY-2 n = 22, blaACT n = 2, blaMIR n = 1). PBRT showed that 91% of poultry meat isolates harboured blaCMY-2 on an IncK plasmid, and 9% on an IncI1 plasmid. Of the human blaCMY-2 producing isolates, 42% also harboured blaCMY-2 on an IncK plasmid, and 47% on an IncI1 plasmid. Thus, 68% of human pAmpC producing E. coli have the same AmpC gene (blaCMY-2) and plasmid type (IncI1 or IncK) as found in poultry meat. MLST showed one cluster containing one human isolate and three meat isolates, with an IncK plasmid. These findings imply that a foodborne transmission route of blaCMY-2 harbouring plasmids cannot be excluded and that further evaluation is required. © 2013.

  8. Cytokine-induced (interleukins-3, -6 and -8 and tumour necrosis factor-beta) activation and deactivation of human neutrophils.

    PubMed Central

    Brom, J; König, W

    1992-01-01

    The effect of various cytokines [interleukin-3(IL-3), IL-6, IL-8, tumour necrosis factor-beta (TNF-beta)] on human neutrophils (PMN) was analysed with regard to the generation of leukotrienes and the involvement of guanosine triphosphate (GTP)-binding proteins (G proteins). Incubation of cytochalasin B-pretreated PMN with cytokines alone did not lead to a generation of leukotrienes. However, the cytokines affected the formyl-methionyl-leucyl-phenylalanine-(FMLP)-induced formation of leukotrienes in a time-dependent manner. Preincubation of the cells with the different cytokines for short periods (15 seconds at 37 degrees) enhanced the subsequent FMLP-induced leukotriene generation, whereas preincubation for prolonged times resulted in a reduced formation of leukotrienes. These results correlated with the respective G protein-associated guanosine triphosphatase (GTPase) activities within isolated membrane fractions. The present study indicates a modulation of the FMLP-induced leukotriene formation by diverse cytokines via interaction with the GTP-binding proteins. PMID:1312995

  9. Geomorphology Drives Amphibian Beta Diversity in Atlantic Forest Lowlands of Southeastern Brazil

    PubMed Central

    Luiz, Amom Mendes; Leão-Pires, Thiago Augusto; Sawaya, Ricardo J.

    2016-01-01

    Beta diversity patterns are the outcome of multiple processes operating at different scales. Amphibian assemblages seem to be affected by contemporary climate and dispersal-based processes. However, historical processes involved in present patterns of beta diversity remain poorly understood. We assess and disentangle geomorphological, climatic and spatial drivers of amphibian beta diversity in coastal lowlands of the Atlantic Forest, southeastern Brazil. We tested the hypothesis that geomorphological factors are more important in structuring anuran beta diversity than climatic and spatial factors. We obtained species composition via field survey (N = 766 individuals), museum specimens (N = 9,730) and literature records (N = 4,763). Sampling area was divided in four spatially explicit geomorphological units, representing historical predictors. Climatic descriptors were represented by the first two axis of a Principal Component Analysis. Spatial predictors in different spatial scales were described by Moran Eigenvector Maps. Redundancy Analysis was implemented to partition the explained variation of species composition by geomorphological, climatic and spatial predictors. Moreover, spatial autocorrelation analyses were used to test neutral theory predictions. Beta diversity was spatially structured in broader scales. Shared fraction between climatic and geomorphological variables was an important predictor of species composition (13%), as well as broad scale spatial predictors (13%). However, geomorphological variables alone were the most important predictor of beta diversity (42%). Historical factors related to geomorphology must have played a crucial role in structuring amphibian beta diversity. The complex relationships between geomorphological history and climatic gradients generated by the Serra do Mar Precambrian basements were also important. We highlight the importance of combining spatially explicit historical and contemporary predictors for understanding

  10. Geomorphology Drives Amphibian Beta Diversity in Atlantic Forest Lowlands of Southeastern Brazil.

    PubMed

    Luiz, Amom Mendes; Leão-Pires, Thiago Augusto; Sawaya, Ricardo J

    2016-01-01

    Beta diversity patterns are the outcome of multiple processes operating at different scales. Amphibian assemblages seem to be affected by contemporary climate and dispersal-based processes. However, historical processes involved in present patterns of beta diversity remain poorly understood. We assess and disentangle geomorphological, climatic and spatial drivers of amphibian beta diversity in coastal lowlands of the Atlantic Forest, southeastern Brazil. We tested the hypothesis that geomorphological factors are more important in structuring anuran beta diversity than climatic and spatial factors. We obtained species composition via field survey (N = 766 individuals), museum specimens (N = 9,730) and literature records (N = 4,763). Sampling area was divided in four spatially explicit geomorphological units, representing historical predictors. Climatic descriptors were represented by the first two axis of a Principal Component Analysis. Spatial predictors in different spatial scales were described by Moran Eigenvector Maps. Redundancy Analysis was implemented to partition the explained variation of species composition by geomorphological, climatic and spatial predictors. Moreover, spatial autocorrelation analyses were used to test neutral theory predictions. Beta diversity was spatially structured in broader scales. Shared fraction between climatic and geomorphological variables was an important predictor of species composition (13%), as well as broad scale spatial predictors (13%). However, geomorphological variables alone were the most important predictor of beta diversity (42%). Historical factors related to geomorphology must have played a crucial role in structuring amphibian beta diversity. The complex relationships between geomorphological history and climatic gradients generated by the Serra do Mar Precambrian basements were also important. We highlight the importance of combining spatially explicit historical and contemporary predictors for understanding

  11. Wild-type cells rescue genotypically Math1-null hair cells in the inner ears of chimeric mice.

    PubMed

    Du, Xiaoping; Jensen, Patricia; Goldowitz, Daniel; Hamre, Kristin M

    2007-05-15

    The transcription factor Math1 has been shown to be critical in the formation of hair cells (HCs) in the inner ear. However, the influence of environmental factors in HC specification suggests that cell extrinsic factors are also crucial to their development. To test whether extrinsic factors impact development of Math1-null (Math1(beta-Gal/beta-Gal)) HCs, we examined neonatal (postnatal ages P0-P4.5) Math1-null chimeric mice in which genotypically mutant and wild-type cells intermingle to form the inner ear. We provide the first direct evidence that Math1-null HCs are able to be generated and survive in the conducive chimeric environment. beta-Galactosidase expression was used to identify genetically mutant cells while cells were phenotypically defined as HCs by morphological characteristics notably the expression of HC-specific markers. Genotypically mutant HCs were found in all sensory epithelia of the inner ear at all ages examined. Comparable results were obtained irrespective of the wild-type component of the chimeric mice. Thus, genotypically mutant cells retain the competence to differentiate into HCs. The implication is that the lack of the Math1 gene in HC precursors can be overcome by environmental influences, such as cell-cell interactions with wild-type cells, to ultimately result in the formation of HCs.

  12. The emergence of non-cytolytic NK1.1+ T cells in the long-term culture of murine tumour-infiltrating lymphocytes: a possible role of transforming growth factor-beta.

    PubMed

    Tamada, K; Harada, M; Ito, O; Takenoyama, M; Mori, T; Matsuzaki, G; Nomoto, K

    1996-12-01

    The mechanism by which murine tumour-infiltrating lymphocytes (TIL) decreased their anti-tumour activity during an in vitro culture with interleukin-2 (IL-2) was investigated. A phenotype analysis revealed that the TIL cultured for 7 days (TIL-d7) were exclusively NKI.1- CD4- CD8+ CD3+ cells and that this population was replaced by natural killer (NK)1.1+ CD4- CD8 CD3+ cells by day 27 (TIL-d27) during the culture of TIL. The TIL-d7 cells showed a cytolytic activity against B16 melanoma, whereas the TIL-d27 cells had lost this activity, suggesting that the decrease in the anti tumour effect of TIL during the culture with IL-2 was due to their populational change. Analysis on the characteristics of the TIL-d27 cells revealed that they expressed skewed T-cell receptor (TCR) V beta 5 and increased mRNA expression of V alpha 14. In addition, they expressed transforming growth factor beta (TGF-beta) mRNA. Interestingly, TGF-beta augmented the proliferation of TIL-d27 cells under the presence of IL-2, but suppressed that of TIL-d7 cells. Moreover, the proliferation of TIL-d27 cells was suppressed by anti-TGF-beta monoclonal antibody. Collectively, these results suggest that, in contrast to its suppressive effect on anti-tumour effector T cells. TGF-beta could be an autocrine growth factor for NKL1.1+ T cells and thereby induce non-cytolytic NK1.1+ T cells in the long-term culture of TIL.

  13. Observations of H-beta and He II lambda 4686 lines in the spectra of flares of UV Cet-type stars

    NASA Astrophysics Data System (ADS)

    Chugainov, P. F.; Petrov, P. P.; Scherbakov, A. G.

    The main results of 45.4 hours of continuous spectroscopic and photoelectric B-band observations of AD Leo, DT Virgo, and YZ CMi are discussed. In two AD Leo flares and two YZ CMi flares, an increase of the central intensity of H-beta was observed 10-20 min before the maximum B-band brightness. The spectra of one AD Leo flare and one YZ CMi flare definitely indicate the formation of broad wings of H-beta occurring mainly during flare maximum. These flares surpass the other four in total optical energy. The Stark effect seems to be the most appropriate explanation for the origin of the wings. The upper limit of the equivalent widths of the He II wavelength 4686 line was higher than that in the quiet state. The equivalent width values cannot be explained by the cascade recombination mechanism if the ratio of optical and X-ray luminosities is nearly the same for all flares of UV Cet-type stars.

  14. Inhibition of AmpC beta-lactamase through a destabilizing interaction in the active site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Trehan, I.; Beadle, B.M.; Shoichet, B.K.

    2010-03-08

    {beta}-Lactamases hydrolyze {beta}-lactam antibiotics, including penicillins and cephalosporins; these enzymes are the most widespread resistance mechanism to these drugs and pose a growing threat to public health. {beta}-Lactams that contain a bulky 6(7){alpha} substituent, such as imipenem and moxalactam, actually inhibit serine {beta}-lactamases and are widely used for this reason. Although mutant serine {beta}-lactamases have arisen that hydrolyze {beta}-lactamase resistant {beta}-lactams (e.g., ceftazidime) or avoid mechanism-based inhibitors (e.g., clavulanate), mutant serine {beta}-lactamases have not yet arisen in the clinic with imipenemase or moxalactamase activity. Structural and thermodynamic studies suggest that the 6(7){alpha} substituents of these inhibitors form destabilizing contacts withinmore » the covalent adduct with the conserved Asn152 in class C {beta}-lactamases (Asn132 in class A {beta}-lactamases). This unfavorable interaction may be crucial to inhibition. To test this destabilization hypothesis, we replaced Asn152 with Ala in the class C {beta}-lactamase AmpC from Escherichia coli and examined the mutant enzyme's thermodynamic stability in complex with imipenem and moxalactam. Consistent with the hypothesis, the Asn152 {yields} Ala substitution relieved 0.44 and 1.10 kcal/mol of strain introduced by imipenem and moxalactam, respectively, relative to the wild-type complexes. However, the kinetic efficiency of AmpC N152A was reduced by 6300-fold relative to that of the wild-type enzyme. To further investigate the inhibitor's interaction with the mutant enzyme, the X-ray crystal structure of moxalactam in complex with N152A was determined to a resolution of 1.83 {angstrom}. Moxalactam in the mutant complex is significantly displaced from its orientation in the wild-type complex; however, moxalactam does not adopt an orientation that would restore competence for hydrolysis. Although Asn152 forces {beta}-lactams with 6(7){alpha

  15. Islet immunity and beta cell reserve of indigenous Black South Africans with ketoacidosis at initial diagnosis of diabetes.

    PubMed

    Ekpebegh, Chukwuma; Longo-Mbenza, Benjamin; Blanco-Blanco, Ernesto

    2013-01-01

    Islet immunity and beta cell reserve status were utilized to classify persons with ketoacidosis as the initial manifestation of diabetes. The clinical features of the various diabetes classes were also characterized. Prospective cross sectional study. Nelson Mandela Academic Hospital, Mthatha, Eastern Cape Province, South Africa. Indigenous Black South Africans with ketoacidosis as the initial manifestation of diabetes. Islet immunity and beta cell reserve were respectively assessed using serum anti-glutamic acid decarboxylase 65 (GAD) antibody and serum C-peptide after 1 mg of intravenous glucagon. Serum anti-GAD 65 antibody > or = 5 units/L and < 5 units/L, respectively defined anti-GAD 65 positive (A+) and negative (A-). Replete (beta+) and deplete (beta-) beta cell reserve were serum C-peptide after glucagon injection of > or = 0.5 ng/mL and < 0.5 ng/mL, respectively. The proportions of patients with A+beta-, A+beta+, A-beta- and A-beta+ and their clinical characteristics were determined. Of the 38 males and 33 females who participated in the study, patients were categorized in various classes: A-beta+, 46.5% (n=33/ 71); A-beta-, 26.8% (n=19/71); A+beta-, 22.5% (n=16/71); and A+beta+, 4.2% (n=3/71). The ages of the various classes were: 41.8 +/- 13.8 years for A-beta+ (n=33); 36.5 +/- 14.6 years for A-beta- (n=19); and 20.6 +/- 7.1 years for the combination of A+beta- with A+beta+ (n=19) (P<.0001, P<.0001 for the combination of A+beta- and A+beta+ vs A-beta+, P=.001 for the combination of A+beta- and A+beta+ vs A-beta-and P=.2 for A-beta- vs A-beta+. The clinical features of type 2 diabetes were most prevalent in A-beta+ class while the A+beta- and A+beta+ groups had the clinical profile of type 1A diabetes. Most of the indigenous Black South African patients with ketoacidosis as the initial manifestation of diabetes had islet immunity, beta cell reserve status and clinical profiles of type 2 diabetes.

  16. GLP-1 mediates antiapoptotic effect by phosphorylating Bad through a beta-arrestin 1-mediated ERK1/2 activation in pancreatic beta-cells.

    PubMed

    Quoyer, Julie; Longuet, Christine; Broca, Christophe; Linck, Nathalie; Costes, Safia; Varin, Elodie; Bockaert, Joël; Bertrand, Gyslaine; Dalle, Stéphane

    2010-01-15

    Strategies based on activating GLP-1 receptor (GLP-1R) are intensively developed for the treatment of type 2 diabetes. The exhaustive knowledge of the signaling pathways linked to activated GLP-1R within the beta-cells is of major importance. In beta-cells, GLP-1 activates the ERK1/2 cascade by diverse pathways dependent on either Galpha(s)/cAMP/cAMP-dependent protein kinase (PKA) or beta-arrestin 1, a scaffold protein. Using pharmacological inhibitors, beta-arrestin 1 small interfering RNA, and islets isolated from beta-arrestin 1 knock-out mice, we demonstrate that GLP-1 stimulates ERK1/2 by two temporally distinct pathways. The PKA-dependent pathway mediates rapid and transient ERK1/2 phosphorylation that leads to nuclear translocation of the activated kinases. In contrast, the beta-arrestin 1-dependent pathway produces a late ERK1/2 activity that is restricted to the beta-cell cytoplasm. We further observe that GLP-1 phosphorylates the cytoplasmic proapoptotic protein Bad at Ser-112 but not at Ser-155. We find that the beta-arrestin 1-dependent ERK1/2 activation engaged by GLP-1 mediates the Ser-112 phosphorylation of Bad, through p90RSK activation, allowing the association of Bad with the scaffold protein 14-3-3, leading to its inactivation. beta-Arrestin 1 is further found to mediate the antiapoptotic effect of GLP-1 in beta-cells through the ERK1/2-p90RSK-phosphorylation of Bad. This new regulatory mechanism engaged by activated GLP-1R involving a beta-arrestin 1-dependent spatiotemporal regulation of the ERK1/2-p90RSK activity is now suspected to participate in the protection of beta-cells against apoptosis. Such signaling mechanism may serve as a prototype to generate new therapeutic GLP-1R ligands.

  17. mRNA Expression of Platelet-Derived Growth Factor Receptor-{beta} and C-KIT: Correlation With Pathologic Response to Cetuximab-Based Chemoradiotherapy in Patients With Rectal Cancer

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Erben, Philipp; Horisberger, Karoline; Muessle, Benjamin

    2008-12-01

    Purpose: Deviant expression of platelet-derived growth factor receptor-{beta} (PDGFR{beta}) and c-kit was shown in patients with colorectal cancer. In the present study, mRNA expression of PDGFR{beta} and c-kit in 33 patients with locally advanced rectal cancer undergoing preoperative chemoradiotherapy with cetuximab/capecitabine/irinotecan in correlation with the tumor regression rate was investigated. Methods and Materials: Pretherapeutic biopsy cores and tumor material from the resected specimens were collected in parallel with normal rectal mucosa. The expression levels of PDGFR{beta} and c-kit were measured by quantitative polymerase chain reaction. Tumors were classified as good responders (tumor regression grade [TRG], 2-3) or poor responders (TRG,more » 0-1). Results: The TRG evaluation of the resected specimen was TRG 0-1 in 11 and TRG 2-3 in 22. The median normalized ratios in the pretreatment mucosa vs. tumor biopsy cores was as follows: PDGFR{beta} ratio of 15.2 vs. 49.5 (p <0.0001) and c-kit ratio of 0.94 vs. 0.67 (p = 0.014). The same tendency was observed for the median PDGFR{beta} ratios after chemoradiotherapy completion: 34.2 vs. 170.0 (p <0.0001). The PDGFR{beta} and c-kit mRNA expression values in the pretreatment tumor biopsy cores were lower than the values in the resected specimens: PDGFR{beta} ratio 49.5 vs. 170.0 (p = 0.0002) and c-kit ratio 0.67 vs. 1.1 (p = 0.0003). Nevertheless, no correlation was seen between the pretherapeutic PDGFR{beta} and c-kit mRNA expression and the pathologic regression rate. Conclusion: Cetuximab-based chemoradiotherapy increased PDGFR{beta} levels even further compared with the pretreatment samples and deserves further investigation.« less

  18. Evaluation of serum levels of C3 and C4 complement factors in patients with beta thalassemia major in Khuzestan Province, Southwest Iran.

    PubMed

    Ghafourian, Mehri; Esmaeili, Mehrnosh; Dashti-Gerdabi, Nader; Sadeghi, Alireza; Malekei Naseri, Ali; Kazemi, Akhtar

    2017-01-01

    Thalassemia syndrome is the most common genetic disorder in the world and infection is the second cause of death in these patients. Measurement of serum C3 and C4 complement factors in serum was done in 60 patients with beta thalassemia major in comparison with 30 healthy subjects as control group. The serum level of C3 and C4 complement factors in 60 patients with beta thalassemia major who were randomly selected from among the patients referred to Shafa Hospital of Ahvaz was evaluated and compared with 30 samples from healthy individuals with no history of recent infectious or autoimmune diseases. It should be noted that single-radial-immunodiffusion assay was used in this study. This study has shown a significant reduction in serum levels of C3 and C4 in patients compared to controls (P value < 0.05). Decreased synthesis or increased consumption of complement factors in patients receiving multiple blood transfusions might lead to continuous contact between the immune system and various antigens, causing nonstop use of complement factors, recurrent infections, changes in parameters of the immune system due to iron overload as well as exposure to infectious factors such as HBV, HCV, HIV, and HTLV through blood transfusion.

  19. Pancreatic Beta Cell Death: Novel Potential Mechanisms in Diabetes Therapy

    PubMed Central

    Palmar, Jim; Nava, Manuel; Tomey, Daniel; Garicano, Carlos

    2018-01-01

    Purpose of Review Describing the diverse molecular mechanisms (particularly immunological) involved in the death of the pancreatic beta cell in type 1 and type 2 diabetes mellitus. Recent Findings Beta cell death is the final event in a series of mechanisms that, up to date, have not been entirely clarified; it represents the pathophysiological mechanism in the natural history of diabetes mellitus. These mechanisms are not limited to an apoptotic process only, which is characteristic of the immune-mediated insulitis in type 1 diabetes mellitus. They also include the action of proinflammatory cytokines, the production of reactive oxygen species, DNA fragmentation (typical of necroptosis in type 1 diabetic patients), excessive production of islet amyloid polypeptide with the consequent endoplasmic reticulum stress, disruption in autophagy mechanisms, and protein complex formation, such as the inflammasome, capable of increasing oxidative stress produced by mitochondrial damage. Summary Necroptosis, autophagy, and pyroptosis are molecular mechanisms that modulate the survival of the pancreatic beta cell, demonstrating the importance of the immune system in glucolipotoxicity processes and the potential role for immunometabolism as another component of what once known as the “ominous octet.” PMID:29670917

  20. A membrane-anchored E-type endo-1,4-beta-glucanase is localized on Golgi and plasma membranes of higher plants.

    PubMed

    Brummell, D A; Catala, C; Lashbrook, C C; Bennett, A B

    1997-04-29

    Endo-1,4-beta-D-glucanases (EGases, EC 3.2.1.4) are enzymes produced in bacteria, fungi, and plants that hydrolyze polysaccharides possessing a 1,4-beta-D-glucan backbone. All previously identified plant EGases are E-type endoglucanases that possess signal sequences for endoplasmic reticulum entry and are secreted to the cell wall. Here we report the characterization of a novel E-type plant EGase (tomato Cel3) with a hydrophobic transmembrane domain and structure typical of type II integral membrane proteins. The predicted protein is composed of 617 amino acids and possesses seven potential sites for N-glycosylation. Cel3 mRNA accumulates in young vegetative tissues with highest abundance during periods of rapid cell expansion, but is not hormonally regulated. Antibodies raised to a recombinant Cel3 protein specifically recognized three proteins, with apparent molecular masses of 93, 88, and 53 kDa, in tomato root microsomal membranes separated by sucrose density centrifugation. The 53-kDa protein comigrated in the gradient with plasma membrane markers, the 88-kDa protein with Golgi membrane markers, and the 93-kDa protein with markers for both Golgi and plasma membranes. EGase enzyme activity was also found in regions of the density gradient corresponding to both Golgi and plasma membranes, suggesting that Cel3 EGase resides in both membrane systems, the sites of cell wall polymer biosynthesis. The in vivo function of Cel3 is not known, but the only other known membrane-anchored EGase is present in Agrobacterium tumefaciens where it is required for cellulose biosynthesis.