Expression of beta-dystroglycan is reduced or absent in many human carcinomas.

PubMed

Cross, S S; Lippitt, J; Mitchell, A; Hollingsbury, F; Balasubramanian, S P; Reed, M W R; Eaton, C; Catto, J W; Hamdy, F; Winder, S J

2008-11-01

Dystroglycan is an important structural and signalling protein that is expressed in most human cells. alpha-Dystroglycan has been investigated and found to be reduced in human cancers, but there is only one published study on the expression of beta-dystroglycan in human cancer and that was only on small numbers of breast and prostatic cancers. The aim was to conduct a comprehensive immunohistochemical survey of the expression of beta-dystroglycan in normal human tissues and common cancers. Triplicate tissue microarrays of 681 samples of normal human tissues and common cancers were stained using an antibody directed against the cytoplasmic component of beta-dystroglycan. beta-Dystroglycan was strongly expressed at the intercellular junctions and basement membranes of all normal human epithelia. Expression of beta-dystroglycan was absent or markedly reduced in 100% of oesophageal adenocarcinomas, 97% of colonic cancers, 100% of transitional cell carcinomas of the urothelium and 94% of breast cancers. In the breast cancers, the only tumours that showed any retention of beta-dystroglycan expression were small low-grade oestrogen receptor-positive tumours. The only cancers that showed retention of beta-dystroglycan expression were cutaneous basal cell carcinomas. There is loss or marked reduction of beta-dystroglycan expression (by immunohistochemistry) in the vast majority of human cancers surveyed. Since beta-dystroglycan is postulated to have a tumour suppressor effect, this loss may have important functional significance.

Assignment of the structural gene for human beta glucuronidase to chromosome 7 and tetrameric association of subunits in the enzyme molecule.

PubMed Central

Chern, C J; Croce, C M

1976-01-01

The structural locus for human beta glucuronidase is assigned to chromosome 7, a localization based upon concordant segregation of the expression of the human enzyme and the presence of human chromosome 7 in somatic cell hybrid clones derived independently from fusions of different human and mouse cells. Hybrid clones containing only human chromosome 7 are included in this study. Electrophoresis of beta glucuronidase also has revealed that human beta glucuronidase has a tetrametric structure. Images Fig. 1 Fig. 2 Fig. 3 PMID:941902

A majority of mice show long-term expression of a human. beta. -globin gene after retrovirus transfer into hematopoietic stem cells

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

Bender, M.A.; Gelinas, R.E.; Miller

1989-04-01

Murine bone marrow was infected with a high-titer retrovirus vector containing the human {beta}-globin and neomycin phosphotransferase genes. Anemic W/W/sup v/ mice were transplanted with infected marrow which in some cases had been exposed to the selective agent G418. Human {beta}-globin expression was monitored in transplanted animals by using a monoclonal antibody specific for human {beta}-globin polypeptide, and hematopoietic reconstitution was monitored by using donor and recipient mice which differed in hemoglobin type. In some experiments all transplanted mice expressed the human {beta}-globin polypeptide for over 4 months, and up to 50% of peripheral erythrocytes contained detectable levels of polypeptide.more » DNA analysis of transplanted animals revealed that virtually every myeloid cell contained a provirus. Integration site analysis and reconstitution of secondary marrow recipients suggested that every mouse was reconstituted with at least one infected stem cell which had extensive repopulation capability. The ability to consistently transfer an active {beta}-globin gene into mouse hematopoietic cells improves the feasibility of using these techniques for somatic cell gene therapy in humans.« less

Clostridium perfringens enterotoxin is a superantigen reactive with human T cell receptors V beta 6.9 and V beta 22

PubMed Central

1992-01-01

Candidate superantigens were screened for their ability to induce lysis of human histocompatibility leukocyte antigen class II-positive targets by human CD8+ influenza-specific cytotoxic T cell (CTL) lines. Clostridium perfringens enterotoxin (CPET) induced major histocompatibility complex unrestricted killing by some but not all CTL lines. Using "anchored" polymerase chain reactions, CPET was shown to selectively stimulate peripheral blood lymphocytes bearing T cell receptor V beta 6.9 and V beta 22 in five healthy donors. V beta 24, V beta 21, V beta 18, V beta 5, and V beta 6.1-5 appeared to be weakly stimulated. Antigen processing was not required for CPET to induce proliferation. Like the staphylococcal enterotoxins, CPET is a major cause of food poisoning. These data suggest that superantigenic and enterotoxigenic properties may be closely linked. PMID:1512551

MicroRNA-29a is up-regulated in beta-cells by glucose and decreases glucose-stimulated insulin secretion

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

Bagge, Annika; Clausen, Trine R.; Larsen, Sylvester

Highlights: Black-Right-Pointing-Pointer MicroRNA-29a (miR-29a) levels are increased by glucose in human and rat islets and INS-1E cells. Black-Right-Pointing-Pointer miR-29a increases proliferation of INS-1E beta-cells. Black-Right-Pointing-Pointer Forced expression of miR-29a decreases glucose-stimulated insulin secretion (GSIS). Black-Right-Pointing-Pointer Depletion of beta-cell miR-29a improves GSIS. Black-Right-Pointing-Pointer miR-29a may be a mediator of glucose toxicity in beta-cells. -- Abstract: Chronically elevated levels of glucose impair pancreatic beta-cell function while inducing beta-cell proliferation. MicroRNA-29a (miR-29a) levels are increased in several tissues in diabetic animals and mediate decreased insulin-stimulated glucose-transport of adipocytes. The aim was to investigate the impact of glucose on miR-29a levels in INS-1E beta-cellsmore » and in human islets of Langerhans and furthermore to evaluate the impact of miR-29a on beta-cell function and proliferation. Increased glucose levels up-regulated miR-29a in beta-cells and human and rat islets of Langerhans. Glucose-stimulated insulin-secretion (GSIS) of INS-1E beta-cells was decreased by forced expression of miR-29a, while depletion of endogenous miR-29a improved GSIS. Over-expression of miR-29a increased INS-1E proliferation. Thus, miR-29a up-regulation is involved in glucose-induced proliferation of beta-cells. Furthermore, as depletion of miR-29a improves beta-cell function, miR-29a is a mediator of glucose-induced beta-cell dysfunction. Glucose-induced up-regulation of miR-29a in beta-cells could be implicated in progression from impaired glucose tolerance to type 2 diabetes.« less

Expression and function of glycogen synthase kinase-3 in human hair follicles.

PubMed

Yamauchi, Koichi; Kurosaka, Akira

2010-05-01

Beta-catenin is involved in the hair follicle morphogenesis and stem cell differentiation, and inhibition of glycogen synthase kinase-3 (GSK-3) increases beta-catenin concentration in the cytoplasm. To examine the effects of GSK-3 inhibition on the hair follicle epithelium, we first examined the expression of GSK-3 in plucked human hair follicles by RT-PCR and found GSK-3 expression in hair follicles. Western blotting with a GSK-3beta-specific antibody, Y174, also demonstrated GSK-3beta expression in the follicles. Moreover, GSK-3beta immunostaining with Y174 showed that GSK-3beta colocalized with hair follicle bulge markers. Contrary to GSK-3beta, GSK-3 alpha was widely expressed throughout the follicles when immunostained with a specific antibody, EP793Y. We then investigated the influence of GSK-3 inhibition. A GSK-3 inhibitor, BIO, promoted the growth of human outer root sheath cells, which could be cultured for up to four passages. The BIO-treated cells exhibited smaller and more undifferentiated morphology than control cells. Moreover, in organ culture of plucked human hair, outer root sheath cells in the middle of a hair follicle proliferated when cultured with BIO. These results indicate that GSK-3beta is expressed in hair bulge stem cells and BIO promotes the growth of ORS cells, possibly by regulating the GSK-3 signaling pathway.

Physical association and functional interaction between beta1 integrin and CD98 on human T lymphocytes

NASA Technical Reports Server (NTRS)

Miyamoto, Yuko J.; Mitchell, Jason S.; McIntyre, Bradley W.

2003-01-01

CD98 is a cell surface protein previously characterized as a cell activation marker, an amino acid transporter, and has recently been implicated in integrin-related functions. Integrins are cell surface proteins, important for homotypic cell aggregation, cell adhesion, and coactivation of T lymphocytes. We have previously shown that the anti-CD98 mAb 80A10, when coimmobilized with anti-CD3 mAb OKT3, is able to mediate human T cell coactivation that is inhibited by anti-beta1 integrin specific mAb 18D3. These results indicated a functional association of CD98 and beta1 integrin signaling but left open the question of a physical association. We now show the induction of homotypic aggregation through CD98 among human T cells and this aggregation was inhibited by anti-beta1 integrin mAb. Therefore, CD98-dependent lymphocyte proliferation and adhesion may involve integrins. Competitive binding assays and fluorescence colocalization analysis suggested that CD98 and beta1 integrin were physically associated. Differential extraction techniques and immunoprecipitations provided the first evidence that the alpha4beta1 integrin and CD98 are specifically associated on human T lymphocytes.

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed Central

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-01-01

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes. Images PMID:1533935

Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines.

PubMed

Tumkosit, Prem; Kuryatov, Alexander; Luo, Jie; Lindstrom, Jon

2006-10-01

Nicotinic acetylcholine receptors (AChRs) containing alpha6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha6* AChRs usually contain beta3 subunits. beta3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha6beta2beta3 and alpha6beta4beta3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha6beta2 or alpha6beta4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a beta3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha6beta2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha6beta2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha6 and beta3 subunits to aid in the characterization of these AChRs. The alpha6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta3 can play important roles in alpha6* AChR expression, stability, and up-regulation by nicotine.

Loss of intra-islet heparan sulfate is a highly sensitive marker of type 1 diabetes progression in humans.

PubMed

Simeonovic, Charmaine J; Popp, Sarah K; Starrs, Lora M; Brown, Debra J; Ziolkowski, Andrew F; Ludwig, Barbara; Bornstein, Stefan R; Wilson, J Dennis; Pugliese, Alberto; Kay, Thomas W H; Thomas, Helen E; Loudovaris, Thomas; Choong, Fui Jiun; Freeman, Craig; Parish, Christopher R

2018-01-01

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells in pancreatic islets are progressively destroyed. Clinical trials of immunotherapies in recently diagnosed T1D patients have only transiently and partially impacted the disease course, suggesting that other approaches are required. Our previous studies have demonstrated that heparan sulfate (HS), a glycosaminoglycan conventionally expressed in extracellular matrix, is present at high levels inside normal mouse beta cells. Intracellular HS was shown to be critical for beta cell survival and protection from oxidative damage. T1D development in Non-Obese Diabetic (NOD) mice correlated with loss of islet HS and was prevented by inhibiting HS degradation by the endoglycosidase, heparanase. In this study we investigated the distribution of HS and heparan sulfate proteoglycan (HSPG) core proteins in normal human islets, a role for HS in human beta cell viability and the clinical relevance of intra-islet HS and HSPG levels, compared to insulin, in human T1D. In normal human islets, HS (identified by 10E4 mAb) co-localized with insulin but not glucagon and correlated with the HSPG core proteins for collagen type XVIII (Col18) and syndecan-1 (Sdc1). Insulin-positive islets of T1D pancreases showed significant loss of HS, Col18 and Sdc1 and heparanase was strongly expressed by islet-infiltrating leukocytes. Human beta cells cultured with HS mimetics showed significantly improved survival and protection against hydrogen peroxide-induced death, suggesting that loss of HS could contribute to beta cell death in T1D. We conclude that HS depletion in beta cells, possibly due to heparanase produced by insulitis leukocytes, may function as an important mechanism in the pathogenesis of human T1D. Our findings raise the possibility that intervention therapy with dual activity HS replacers/heparanase inhibitors could help to protect the residual beta cell mass in patients recently diagnosed with T1D.

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.

The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of. beta. -glucuronidase

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

Watanabe, H.; Grubb, J.H.; Sly, W.S.

1990-10-01

The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functionalmore » receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.« less

Expression profile of senescence-associated beta-galactosidase and activation of telomerase in human ovarian surface epithelial cells undergoing immortalization.

PubMed

Litaker, J R; Pan, J; Cheung, Y; Zhang, D K; Liu, Y; Wong, S C; Wan, T S; Tsao, S W

1998-11-01

Senescence is a specific physiological stage of cells characterized by long population doubling time. It accounts for the inability of normal somatic cells to undergo indefinite cell division. As the number of population doublings increase, cell cycle regulatory mechanisms come into play and signal cells to exit the cell cycle and become senescent. Senescence has been implicated in the aging process and may function as a tumor suppressor mechanism in human cells. The ability to measure the degree of cellular senescence is important in understanding the biological processes regulating cell aging and immortalization. Senescent cells exhibit an enzyme termed senescence-associated histochemical staining. Cells immortalized by viral oncogenes often enter a stage of crisis at the early phase of immortalization. The cells at crisis have a long population doubling time. Cells at the crisis stage resemble senescent cells and the expression of SA- beta-Gal may be used to monitor the process of immortalization. In this study the expression profile of SA-beta-Gal was examined in human ovarian surface epithelial cells (HOSE 6-3) undergoing immortalization by the human papilloma viral oncogene E6 and E7 (HPV E6 and E7). Our results showed a low percentage (12.0%) of HOSE 6-3 cells expressing SA-beta-Gal activity at the pre-crisis stage. The percentage of HOSE 6-3 cells expressing SA-beta-Gal activity was highest (39.2%) at the crisis stage. When HOSE 6-3 cells achieved immortalized status there was a sharp decrease in cells (1. 3%) expressing SA-beta-Gal activity. In addition, an inverse relationship between the expression of SA-beta-Gal activity and telomerase activity was noted in cells undergoing immortalization. The results confirm that the SA-beta-Gal enzyme is a good marker for monitoring the population of cells undergoing senescence at different stages of immortalization and that telomerase activation is a characteristic feature of post-crisis cells.

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.

Human dendritic cells produce TGF-beta 1 under the influence of lung carcinoma cells and prime the differentiation of CD4+CD25+Foxp3+ regulatory T cells.

PubMed

Dumitriu, Ingrid E; Dunbar, Donald R; Howie, Sarah E; Sethi, Tariq; Gregory, Christopher D

2009-03-01

Dendritic cells (DCs) have a central role in the development of adaptive immune responses, including antitumor immunity. Factors present in the tumor milieu can alter the maturation of DCs and inhibit their capacity to activate T cells. Using gene expression analysis, we found that human DCs increased the expression of TGF-beta1 transcripts following culture with human lung carcinoma cells (LCCs). These DCs produced increased amounts of TGF-beta1 protein compared with DCs not exposed to tumor cells. LCCs also decreased the expression of CD86 and HLA-DR by immature DCs. Furthermore, LCCs decreased CD86 expression and the production of TNF-alpha and IL-12 p70 by mature DCs. Moreover, LCCs also converted mature DCs into cells producing TGF-beta1. These TGF-beta1-producing DCs were poor at eliciting the activation of naive CD4(+) T cells and sustaining their proliferation and differentiation into Th1 (IFN-gamma(+)) effectors. Instead, TGF-beta1-producing DCs demonstrated an increased ability to generate CD4(+)CD25(+)Foxp3(+) regulatory T cells that suppress the proliferation of T lymphocytes. These results identify a novel mechanism by which the function of human DCs is altered by tumor cells and contributes to the evasion of the immune response.

Studies on the bioavailability of the provitamin A carotenoid, beta-carotene, using human exfoliated colonic epithelial cells.

PubMed

Gireesh, T; Nair, P P; Sudhakaran, P R

2004-08-01

The possibility of using exfoliated colonic epithelial cells for assessing the bioavailability of beta-carotene was examined. Analysis of exfoliated colonic epithelial cells showed the presence of beta-carotene and vitamin A. The beta-carotene content was significantly lower in cells from stool samples of subjects on a beta-carotene-poor diet than those receiving a single dose of a beta-carotene supplement. Colonic epithelial cells isolated from stool samples collected daily during a wash-out period while the subjects were on a beta-carotene-poor diet showed a steady decrease in beta-carotene content, reaching the lowest value on day 7. Kinetic analysis showed that a single dose of a beta-carotene supplement in the form of spirulina (Spirulina platensis) or agathi (Sesbania grandiflora) after the wash-out period caused an increase in the beta-carotene content after a lag period of 5-7 d, but the vitamin A levels during these periods were not significantly affected. Analysis of plasma beta-carotene concentration also showed similar changes, which correlated with those of exfoliated colonic cells. A relationship between the beta-carotene content of the diet and that of the colonic epithelial cells suggests that analysis of the beta-carotene content in exfoliated human colonic epithelial cells is a useful non-invasive method to assess the bioavailability of provitamin A beta-carotene.

Brain transplantation of immortalized human neural stem cells promotes functional recovery in mouse intracerebral hemorrhage stroke model.

PubMed

Lee, Hong J; Kim, Kwang S; Kim, Eun J; Choi, Hyun B; Lee, Kwang H; Park, In H; Ko, Yong; Jeong, Sang W; Kim, Seung U

2007-05-01

We have generated stable, immortalized cell lines of human NSCs from primary human fetal telencephalon cultures via a retroviral vector encoding v-myc. HB1.F3, one of the human NSC lines, expresses a normal human karyotype of 46, XX, and nestin, a cell type-specific marker for NSCs. F3 has the ability to proliferate continuously and differentiate into cells of neuronal and glial lineage. The HB1.F3 human NSC line was used for cell therapy in a mouse model of intracerebral hemorrhage (ICH) stroke. Experimental ICH was induced in adult mice by intrastriatal administration of bacterial collagenase; 1 week after surgery, the rats were randomly divided into two groups so as to receive intracerebrally either human NSCs labeled with beta-galactosidase (n = 31) or phosphate-buffered saline (PBS) (n = 30). Transplanted NSCs were detected by 5-bromo-4-chloro-3-indolyl-beta-d-galactoside histochemistry or double labeling with beta-galactosidase (beta-gal) and mitogen-activated protein (MAP)2, neurofilaments (both for neurons), or glial fibrillary acidic protein (GFAP) (for astrocytes). Behavior of the animals was evaluated for period up to 8 weeks using modified Rotarod tests and a limb placing test. Transplanted human NSCs were identified in the perihematomal areas and differentiated into neurons (beta-gal/MAP2(+) and beta-gal/NF(+)) or astrocytes (beta-gal/GFAP(+)). The NSC-transplanted group showed markedly improved functional performance on the Rotarod test and limb placing after 2-8 weeks compared with the control PBS group (p < .001). These results indicate that the stable immortalized human NSCs are a valuable source of cells for cell replacement and gene transfer for the treatment of ICH and other human neurological disorders. Disclosure of potential conflicts of interest is found at the end of this article.

Expression of {beta}{sub 1} integrins in human endometrial stromal and decidual cells

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

Shiokawa, Shigetatsu; Yoshimura, Yasunori; Nakamura, Yukio

The present study was undertaken to investigate the expression of {beta}{sub 1} integrins in human endometrium and decidua using flow cytometry, immunohistochemistry, and immunoprecipitation. Fluorescence-activated flow cytometry demonstrated the greater expression of the {beta}{sub 1}, {alpha}{sub 1}, {alpha}{sub 2}, and {alpha}{sub 5} subunits of the {beta}{sub 1} integrin family in cultured stromal cells from the midsecretory phase, than in those of the early proliferative phase. The addition of estradiol (E{sub 2}) and progesterone (P) to cultured stromal cells in the early proliferative phase increased the expression of {beta}{sub 1} integrins in vitro. Flow cytometry also demonstrated the expression of themore » {beta}{sub 1}, {alpha}{sub 1}, {alpha}{sub 2}, {alpha}{sub 3}, {alpha}{sub 5}, and {alpha}{sub 6} subunits of {beta}{sub 1} integrin family in cultured decidual cells, and the enriched-fraction of prolactin (PRL)-producing decidual cells isolated by Percoll gradients showed high levels of {beta}{sub 1} integrins expression. Immunohistochemistry confirmed the {beta}{sub 1} integrin cell surface phenotypes in cultured decidual cells observed by flow cytometry. In summary, the present study demonstrated that endometrial stromal and decidual cells expressed {beta}{sub 1} integrin subunits at their surfaces. The expression exhibited a variability throughout the menstrual cycles, being predominantly detected in the secretory phase, and was maintained highly in the decidua. Thus, {beta}{sub 1} integrins in human endometrium and decidua may be important in mediating the organization of extracellular matrix proteins derived from embryos during the early stage of implantation. 43 refs., 7 figs., 2 tabs.« less

Beta-lactam antibiotics modulate T-cell functions and gene expression via covalent binding to cellular albumin.

PubMed

Mor, Felix; Cohen, Irun R

2013-02-19

Recent work has suggested that beta-lactam antibiotics might directly affect eukaryotic cellular functions. Here, we studied the effects of commonly used beta-lactam antibiotics on rodent and human T cells in vitro and in vivo on T-cell-mediated experimental autoimmune diseases. We now report that experimental autoimmune encephalomyelitis and adjuvant arthritis were significantly more severe in rats treated with cefuroxime and other beta-lactams. T cells appeared to mediate the effect: an anti-myelin basic protein T-cell line treated with cefuroxime or penicillin was more encephalitogenic in adoptive transfer experiments. The beta-lactam ampicillin, in contrast to cefuroxime and penicillin, did not enhance encephalomyelitis, but did inhibit the autoimmune diabetes developing spontaneously in nonobese diabetic mice. Gene expression analysis of human peripheral blood T cells showed that numerous genes associated with T helper 2 (Th2) and T regulatory (Treg) differentiation were down-regulated in T cells stimulated in the presence of cefuroxime; these genes were up-regulated in the presence of ampicillin. The T-cell protein that covalently bound beta-lactam antibiotics was found to be albumin. Human and rodent T cells expressed albumin mRNA and protein, and penicillin-modified albumin was taken up by rat T cells, leading to enhanced encephalitogenicity. Thus, beta-lactam antibiotics in wide clinical use have marked effects on T-cell behavior; beta-lactam antibiotics can function as immunomodulators, apparently through covalent binding to albumin.

Transduction of beta3 integrin subunit cDNA confers on human keratinocytes the ability to adhere to gelatin.

PubMed

Kubo, Miyoko; Clark, Richard A F; Katz, Anne B; Taichman, Lorne B; Jin, Zaishun; Zhao, Ying; Moriguchi, Takahiko

2007-04-01

alphavbeta3 is a multiligand integrin receptor that interacts with fibrinogen (FG), fibrin (FB), fibronectin (FN), vitronectin (VN), and denatured collagen. We previously reported that cultured normal human keratinocytes, like in vivo keratinocytes, do not express alphavbeta3 on the cell surface, and do not adhere to and migrate on FG and FB. Furthermore, we reported that human keratinocytes transduced with beta3 integrin subunit cDNA by a retrovirus-mediated transduction method express alphavbeta3 on the cell surface and adhere to FG, FB, FN, and VN significantly compared with beta-galactosidase (beta-gal) cDNA-transduced keratinocytes (control). In this study, we determined whether these beta3 integrin subunit cDNA-transduced keratinocytes or normal human keratinocytes adhere to denatured collagen (gelatin) using a 1 h cell adhesion assay. beta3 cDNA-transduced keratinocytes adhered to gelatin, whereas no significant adhesion was observed with the control cells (beta-gal cDNA-transduced keratinocytes and normal human keratinocytes). The adhesion to gelatin was inhibited by LM609, a monoclonal antibody to alphavbeta3, and RGD peptides but not by normal mouse IgG1 nor RGE peptides. Thus, transduction of beta3 integrin subunit cDNA confers on human keratinocytes the ability to adhere to denatured collagen (gelatin) as well as to FG, FB, VN, and FN. Otherwise, normal human keratinocytes do not adhere to gelatin. These data support the idea that beta3 cDNA-transduced human keratinocytes can be a good material for cultured epithelium to achieve better take rate with acute or chronic wounds, in which FG, FB, and denatured collagen are abundantly present.

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.

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.

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

Induction of experimental bone metastasis in mice by transfection of integrin alpha 4 beta 1 into tumor cells.

PubMed Central

Matsuura, N.; Puzon-McLaughlin, W.; Irie, A.; Morikawa, Y.; Kakudo, K.; Takada, Y.

1996-01-01

Cell adhesion receptors (eg, integrins and CD44) play an important role in invasion and metastasis during tumor progression. The increase in integrin alpha 4 beta 1 expression on primary melanomas has been reported to significantly correlate with the development of metastases. alpha 4 beta 1 is a cell surface heterodimer that mediates cell-cell and cell-extracellular matrix interactions through adhesion to vascular cell adhesion molecule (VCAM)-1 and to the IIICS region of fibronectin. To test the effects of alpha 4 beta 1 expression on tumor cell metastasis, Chinese hamster ovary cells were transfected with human alpha 4 cDNA. Whereas alpha 4-negative Chinese hamster ovary cells developed only pulmonary metastasis, alpha 4-positive Chinese hamster ovary cells developed bone and pulmonary metastasis in 3 to 4 weeks when injected intravenously into nude mice. Bone metastasis was inhibited by antibody against alpha 4 or VCAM-1. Expression of alpha 3 beta 1, alpha 6 beta 1, or alpha V beta 1 did not induce bone metastasis. Expression of alpha 4 beta 1 also induced bone metastasis in K562 human erythroleukemia cells injected into SCID mice. These results demonstrate that alpha 4 beta 1 can induce tumor cell trafficking to bone, probably via interaction with VCAM-1 that is constitutively expressed on bone marrow stromal cells. Images Figure 1 Figure 3 PMID:8546226

A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug.

PubMed

de Graaf, M; Boven, E; Oosterhoff, D; van der Meulen-Muileman, I H; Huls, G A; Gerritsen, W R; Haisma, H J; Pinedo, H M

2002-03-04

Monoclonal antibodies against tumour-associated antigens could be useful to deliver enzymes selectively to the site of a tumour for activation of a non-toxic prodrug. A completely human fusion protein may be advantageous for repeated administration, as host immune responses may be avoided. We have constructed a fusion protein consisting of a human single chain Fv antibody, C28, against the epithelial cell adhesion molecule and the human enzyme beta-glucuronidase. The sequences encoding C28 and human enzyme beta-glucuronidase were joined by a sequence encoding a flexible linker, and were preceded by the IgGkappa signal sequence for secretion of the fusion protein. A CHO cell line was engineered to secrete C28-beta-glucuronidase fusion protein. Antibody specificity and enzyme activity were retained in the secreted fusion protein that had an apparent molecular mass of 100 kDa under denaturing conditions. The fusion protein was able to convert a non-toxic prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate to doxorubicin, resulting in cytotoxicity. A bystander effect was demonstrated, as doxorubicin was detected in all cells after N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate administration when only 10% of the cells expressed the fusion protein. This is the first fully human and functional fusion protein consisting of an scFv against epithelial cell adhesion molecule and human enzyme beta-glucuronidase for future use in tumour-specific activation of a non-toxic glucuronide prodrug. Copyright 2002 Cancer Research UK

Stimulation of interleukin-1 beta production of human dental pulp cells by Porphyromonas endodontalis lipopolysaccharide.

PubMed

Hosoya, S; Matsushima, K

1997-01-01

IL-1 beta is synthesized as an inactive precursor, which is subsequently processed by IL-1 beta converting enzyme (ICE) and found extracellularly as a mature biologically active polypeptide. Also, IL-1 beta has been detected in necrotic and inflamed dental pulp. We examined the IL-1 beta production in human dental pulp (HDP) cells treated with lipopolysaccharide (LPS) from Porphyromonas endodontalis (P. e.) isolated from root canals and radicular cyst fluids. We demonstrated that P. e. LPS stimulated IL-1 beta release from HDP cells in a time- and dose-dependent manner. However, ICE activity was not increased by P. e. LPS. Northern blot hybridization analysis revealed that the IL-1 beta mRNA level in HDP cells was increased by P. e. LPS. These results suggest that stimulation of IL-1 beta release from HDP cells by P. e. LPS may have an important role in the progression of inflammation in pulpal and periapical disease.

The receptor protein tyrosine phosphatase (RPTP){beta}/{zeta} is expressed in different subtypes of human breast cancer

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

Perez-Pinera, Pablo; Garcia-Suarez, Olivia; Instituto Universitario de Oncologia del Principado de Asturias, Oviedo

2007-10-12

Increasing evidence suggests mutations in human breast cancer cells that induce inappropriate expression of the 18-kDa cytokine pleiotrophin (PTN, Ptn) initiate progression of breast cancers to a more malignant phenotype. Pleiotrophin signals through inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP){beta}/{zeta}, leading to increased tyrosine phosphorylation of different substrate proteins of RPTP{beta}/{zeta}, including {beta}-catenin, {beta}-adducin, Fyn, GIT1/Cat-1, and P190RhoGAP. PTN signaling thus has wide impact on different important cellular systems. Recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTP{beta}/{zeta} signaling pathway; this discovery potentially is very important, since constitutive ALK activity of nucleophosmin (NPM)-ALK fusionmore » protein is causative of anaplastic large cell lymphomas, and, activated ALK is found in other malignant cancers. Recently ALK was identified in each of 63 human breast cancers from 22 subjects. We now demonstrate that RPTP{beta}/{zeta} is expressed in each of these same 63 human breast cancers that previously were found to express ALK and in 10 additional samples of human breast cancer. RPTP{beta}/{zeta} furthermore was localized not only in its normal association with the cell membrane but also scattered in cytoplasm and in nuclei in different breast cancer cells and, in the case of infiltrating ductal carcinomas, the distribution of RPTP{beta}/{zeta} changes as the breast cancer become more malignant. The data suggest that the PTN/RPTP{beta}/{zeta} signaling pathway may be constitutively activated and potentially function to constitutively activate ALK in human breast cancer.« less

Regulation of human nitric oxide synthase 2 expression by Wnt beta-catenin signaling.

PubMed

Du, Qiang; Park, Kyung Soo; Guo, Zhong; He, Peijun; Nagashima, Makoto; Shao, Lifang; Sahai, Rohit; Geller, David A; Hussain, S Perwez

2006-07-15

Nitric oxide (NO.), an important mediator of inflammation, and beta-catenin, a component of the Wnt-adenomatous polyposis coli signaling pathway, contribute to the development of cancer. We have identified two T-cell factor 4 (Tcf-4)-binding elements (TBE1 and TBE2) in the promoter of human inducible NO synthase 2 (NOS2). We tested the hypothesis that beta-catenin regulates human NOS2 gene. Mutation in either of the two TBE sites decreased the basal and cytokine-induced NOS2 promoter activity in different cell lines. The promoter activity was significantly reduced when both TBE1 and TBE2 sites were mutated (P < 0.01). Nuclear extract from HCT116, HepG2, or DLD1 cells bound to NOS2 TBE1 or TBE2 oligonucleotides in electrophoretic mobility shift assays and the specific protein-DNA complexes were supershifted with anti-beta-catenin or anti-Tcf-4 antibody. Overexpression of beta-catenin and Tcf-4 significantly increased both basal and cytokine-induced NOS2 promoter activity (P < 0.01), and the induction was dependent on intact TBE sites. Overexpression of beta-catenin or Tcf-4 increased NOS2 mRNA and protein expression in HCT116 cells. Lithium chloride (LiCl), an inhibitor of glycogen synthase kinase-3beta, increased cytosolic and nuclear beta-catenin level, NOS2 expression, and NO. production in primary human and rat hepatocytes and cancer cell lines. Treatment with Wnt-3A-conditioned medium increased beta-catenin and NOS2 expression in fetal human hepatocytes. When administered in vivo, LiCl increased hepatic beta-catenin level in a dose-dependent manner with simultaneous increase in NOS2 expression. These data are consistent with the hypothesis that beta-catenin up-regulates NOS2 and suggest a novel mechanism by which the Wnt/beta-catenin signaling pathway may contribute to cancer by increasing NO. production.

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

The effect of TGF-beta2 on MMP-2 production and activity in highly metastatic human bladder carcinoma cell line 5637.

PubMed

Dehnavi, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram; Ataei, Zahra; Aryan, Hajar

2009-06-01

Transforming growth factor-beta (TGF-beta) superfamily regulates matrix metalloproteinases (MMP), which intrinsically regulate various cell behaviors leading to metastasis. We investigated the effect of TGF-beta(2) on MMP-2 regulation in human bladder carcinoma cell line 5637. Zymography, ELISA, and real-time polymerase chain reaction revealed that TGF-beta(2) stimulated MMP-2 production, but the transcription of its gene remained unchanged. Wortmannin could not inhibit MMP-2 secretion and activity and conversely the amount of the protein and its enzymatic activity were increased. These data suggest that TGF-beta(2) increased MMP-2 at the posttranscriptional level and this upregulation was independent of phosphatidylinositol 3-kinase signaling pathway.

Biotin uptake by mouse and human pancreatic beta cells/islets: a regulated, lipopolysaccharide-sensitive carrier-mediated process

PubMed Central

Ghosal, Abhisek; Sekar, Thillai V.

2014-01-01

Biotin is essential for the normal function of pancreatic beta cells. These cells obtain biotin from their surroundings via transport across their cell membrane. Little is known about the uptake mechanism involved, how it is regulated, and how it is affected by internal and external factors. We addressed these issues using the mouse-derived pancreatic beta-TC-6 cells and freshly isolated mouse and human primary pancreatic beta cells as models. The results showed biotin uptake by pancreatic beta-TC-6 cells occurs via a Na+-dependent, carrier-mediated process, that is sensitive to desthiobiotin, as well as to pantothenic acid and lipoate; the process is also saturable as a function of concentration (apparent Km = 22.24 ± 5.5 μM). These cells express the sodium-dependent multivitamin transporter (SMVT), whose knockdown (with doxycycline-inducible shRNA) led to a sever inhibition in biotin uptake. Similarly, uptake of biotin by mouse and human primary pancreatic islets is Na+-dependent and carrier-mediated, and both cell types express SMVT. Biotin uptake by pancreatic beta-TC-6 cells is also adaptively regulated (via transcriptional mechanism) by extracellular substrate level. Chronic treatment of pancreatic beta-TC-6 cells with bacterial lipopolysaccharides (LPS) leads to inhibition in biotin uptake. This inhibition is mediated via a Toll-Like receptor 4-mediated process and involves a decrease in membrane expression of SMVT. These findings show, for the first time, that pancreatic beta cells/islets take up biotin via a specific and regulated carrier-mediated process, and that the process is sensitive to the effect of LPS. PMID:24904078

Beta Adrenergic Signaling: A Targetable Regulator of Angiosarcoma and Hemangiosarcoma

PubMed Central

Dickerson, Erin B.; Bryan, Brad A.

2015-01-01

Human angiosarcomas and canine hemangiosarcomas are highly aggressive cancers thought to arise from cells of vascular origin. The pathological features, morphological organization, and clinical behavior of canine hemangiosarcomas are virtually indistinct from those of human angiosarcomas. Overall survival with current standard-of-care approaches remains dismal for both humans and dogs, and each is likely to succumb to their disease within a short duration. While angiosarcomas in humans are extremely rare, limiting their study and treatment options, canine hemangiosarcomas occur frequently. Therefore, studies of these sarcomas in dogs can be used to advance treatment approaches for both patient groups. Emerging data suggest that angiosarcomas and hemangiosarcomas utilize beta adrenergic signaling to drive their progression by regulating the tumor cell niche and fine-tuning cellular responses within the tumor microenvironment. These discoveries indicate that inhibition of beta adrenergic signaling could serve as an Achilles heel for these tumors and emphasize the need to design therapeutic strategies that target tumor cell and stromal cell constituents. In this review, we summarize recent discoveries and present new hypotheses regarding the roles of beta adrenergic signaling in angiosarcomas and hemangiosarcomas. Because the use of beta adrenergic receptor antagonists is well established in human and veterinary medicine, beta blockade could provide an immediate adjunct therapy for treatment along with a tangible opportunity to improve upon the outcomes of both humans and dogs with these diseases. PMID:29061946

The mechanism of cell death in human cultured colon adenocarcinoma cell line COLO 201 induced by beta-D-N-acetylglucosaminyl-p-nitrophenol.

PubMed

Kukidome, J; Kakizaki, I; Takagaki, K; Matsuki, A; Munakata, A; Endo, M

2001-05-01

COLO 201, human colon adenocarcinoma cells were incubated with artificial primers, p-nitrophenyl-glycoside derivatives at 1.0 mmol (mM) in the medium containing 10% fetal bovine serum to detect sugar chain elongation. However, when p-nitrophenyl-beta-N-acetylglucosamine (beta-GlcNAc-PNP) was added, the medium changed color to yellow and the cells were dead. To explain this finding, the cells were incubated with 1.0 mM each of beta-GlcNAc-PNP and 4-methylumbelliferyl-beta-N-acetylglucosamine, then the number of living cells was measured in a time course. In beta-GlcNAc-PNP, the living cells were decreased at 24 hours. The cells were survived with N-acetylglucosamine, whereas in the presence of p-nitrophenol (PNP) the living cells were decreased. It was suggested that PNP released from beta-GlcNAc-PNP induced the cell death. Activity of beta-D-N-acetylglucosaminidase was detected in fetal bovine serum. It was shown that PNP induced the cell death in time-and-dose dependent manner. Genomic DNA from COLO 201 analyzed by agarose gel electrophoresis was fragmentated. PNP analogues were tested for toxicity, and the results suggested that the phenolic OH-group linked to benzene ring and nitro-group linked to the structure in para-form (PNP) was the most effective.

Activation of Beta-Catenin Signaling in Androgen Receptor–Negative Prostate Cancer Cells

PubMed Central

Wan, Xinhai; Liu, Jie; Lu, Jing-Fang; Tzelepi, Vassiliki; Yang, Jun; Starbuck, Michael W.; Diao, Lixia; Wang, Jing; Efstathiou, Eleni; Vazquez, Elba S.; Troncoso, Patricia; Maity, Sankar N.; Navone, Nora M.

2012-01-01

Purpose To study Wnt/beta-catenin in castrate-resistant prostate cancer (CRPC) and understand its function independently of the beta-catenin–androgen receptor (AR) interaction. Experimental Design We performed beta-catenin immunocytochemical analysis, evaluated TOP-flash reporter activity (a reporter of beta-catenin–mediated transcription), and sequenced the beta-catenin gene in MDA PCa 118a, MDA PCa 118b, MDA PCa 2b, and PC-3 prostate cancer (PCa) cells. We knocked down beta-catenin in AR-negative MDA PCa 118b cells and performed comparative gene-array analysis. We also immunohistochemically analyzed beta-catenin and AR in 27 bone metastases of human CRPCs. Results Beta-catenin nuclear accumulation and TOP-flash reporter activity were high in MDA PCa 118b but not in MDA PCa 2b or PC-3 cells. MDA PCa 118a and 118b cells carry a mutated beta-catenin at codon 32 (D32G). Ten genes were expressed differently (false discovery rate, 0.05) in MDA PCa 118b cells with downregulated beta-catenin. One such gene, hyaluronan synthase 2 (HAS2), synthesizes hyaluronan, a core component of the extracellular matrix. We confirmed HAS2 upregulation in PC-3 cells transfected with D32G-mutant beta-catenin. Finally, we found nuclear localization of beta-catenin in 10 of 27 human tissue specimens; this localization was inversely associated with AR expression (P = 0.056, Fisher’s exact test), suggesting that reduced AR expression enables Wnt/beta-catenin signaling. Conclusion We identified a previously unknown downstream target of beta-catenin, HAS2, in PCa, and found that high beta-catenin nuclear localization and low or no AR expression may define a subpopulation of men with bone-metastatic PCa. These findings may guide physicians in managing these patients. PMID:22298898

Prostaglandin E2 suppresses beta1-integrin expression via E-prostanoid receptor in human monocytes/macrophages.

PubMed

Hasegawa, Shunji; Ichiyama, Takashi; Kohno, Fumitaka; Korenaga, Yuno; Ohsaki, Ayami; Hirano, Reiji; Haneda, Yasuhiro; Fukano, Reiji; Furukawa, Susumu

2010-01-01

Beta1-integrins mediate cell attachment to different extracellular matrix proteins, intracellular proteins, and intercellular adhesions. Recently, it has been reported that prostaglandin E2 (PGE2) has anti-inflammatory properties such as inhibition of the expression of adhesion molecules or production of chemokines. However, the effect of PGE2 on the expression of beta1-integrin remains unknown. In this study, we investigated the effects of PGE2 on the expression of beta1-integrin in the human monocytic cell line THP-1 and in CD14+ monocytes/macrophages in human peripheral blood. For this, we examined the role of four subtypes of PGE2 receptors and E-prostanoid (EP) receptors on PGE2-mediated inhibition. We found that PGE2 significantly inhibited the expression of beta1-integrin, mainly through EP4 receptors in THP-1 cells and CD14+ monocytes/macrophages in human peripheral blood. We suggest that PGE2 has anti-inflammatory effects, leading to the inhibited expression of beta1-integrin in human monocytes/macrophages, and that the EP4 receptor may play an important role in PGE2-mediated inhibition. Copyright (c) 2010 Elsevier Inc. All rights reserved.

A cell-based assay to assess the persistence of action of agonists acting at recombinant human beta(2) adrenoceptors.

PubMed

Summerhill, Susan; Stroud, Timothy; Nagendra, Roshini; Perros-Huguet, Christelle; Trevethick, Michael

2008-01-01

The aim was to establish a robust, 96-well, cell-based assay to assess the potency and persistence of action of agonists acting at human recombinant beta(2) adrenoceptors expressed in CHO (Chinese Hamster Ovary) cells and to compare this with published duration of action data in guinea pig isolated trachea and human bronchus. Cells were treated with either: (i) beta-adrenoceptor agonist for 30 min, washed and cyclicAMP (cAMP) measured 30 min later-termed 'washed' cells or, (ii) treated with solvent for 30 min, washed, and then treated with beta-adrenoceptor agonist for 30 min and cAMP measured-termed 'unwashed' cells. The 'washed' EC(50) was divided by the 'unwashed' EC(50) to determine a rightward shift concentration ratio, which was indicative of the persistence of action at the receptor. At the beta(2) adrenoceptor salmeterol, carmoterol and indacaterol were resistant to washing with a concentration ratio of <5, indicating a long persistence of action, whereas formoterol, isoprenaline and salbutamol were washed out with a ratio of 32, >294 and >800 respectively, suggesting a shorter persistence of action. At beta(1) and beta(3) adrenoceptors all compounds washed out. The persistent effects of salmeterol at beta(2) following washing could be reversed by the selective beta(2) antagonist ICI 118551, suggesting continued receptor activation. The data presented agree well with published data assessing duration of action of beta(2) agonists in human isolated bronchus and guinea pig isolated trachea. Key features are: (a) it is a 96-well format which can be used to assess many compounds in a single experiment, (b) both potency and persistence of agonist action are assessed in the same assay, (c) any effects of concentration on the persistence of action can be highlighted, and (d) it allows triage of compounds prior to tissue bath studies thus reducing the use of animal tissue.

Streptococcal modulation of cellular invasion via TGF-beta1 signaling.

PubMed

Wang, Beinan; Li, Shaoying; Southern, Peter J; Cleary, Patrick P

2006-02-14

Group A Streptococcus (GAS) and other bacterial pathogens are known to interact with integrins as an initial step in a complex pathway of bacterial ingestion by host cells. Efficient GAS invasion depends on the interaction of bound fibronectin (Fn) with integrins and activation of integrin signaling. TGF-beta1 regulates expression of integrins, Fn, and other extracellular matrix proteins, and positively controls the integrin signaling pathway. Therefore, we postulated that TGF-beta1 levels could influence streptococcal invasion of mammalian cells. Pretreatment of HEp-2 cells with TGF-beta1 increased their capacity to ingest GAS when the bacteria expressed fibronectin-binding proteins (M1 or PrtF1). Western blots revealed significant induction of alpha5 integrin and Fn expression by HEp-2 cells in response to TGF-beta1. Increased ingestion of streptococci by these cells was blocked by a specific inhibitor of the TGF-beta1 receptor I and antibodies directed against alpha5 integrin and Fn, indicating that increased invasion depends on TGF-beta1 up-regulation of both the alpha5 integrin and Fn. The capacity of TGF-beta1 to up-regulate integrin expression and intracellular invasion by GAS was reproduced in primary human tonsil fibroblasts, which could be a source of TGF-beta1 in chronically infected tonsils. The relationship between TGF-beta1 and GAS invasion was strengthened by the observation that TGF-beta1 production was stimulated in GAS-infected primary human tonsil fibroblasts. These findings suggest a mechanism by which GAS induce a cascade of changes in mammalian tissue leading to elevated expression of the alpha5beta1 receptor, enhanced invasion, and increased opportunity for survival and persistence in their human host.

Ectopic production of beta-HCG by a maxillary squamous cell carcinoma.

PubMed

Scholl, P D; Jurco, S; Austin, J R

1997-12-01

Paraneoplastic syndromes of the head and neck are rare. Hypercalcemia and leukocytosis have been described. The literature was reviewed, and a case of a squamous cell carcinoma of the maxilla producing beta human chorionic gonadotropin (beta-HCG) is presented. A 47-year-old white man with a T4N1M0 squamous cell carcinoma of the left maxilla was treated with a maxillectomy and neck dissection for an N1 positive neck. After completing his planned radiotherapy, he developed distant metastases, which included an axillary node that stained positive for human beta-HCG. Retrospective review of the primary specimen showed beta-HCG positivity in an anaplastic component of the tumor along with vascular invasion. The first case in the literature of a paraneoplastic syndrome with beta-HCG production in association with squamous cell carcinoma of the maxilla is presented. This case history fits the aggressive nature of beta HCG producing tumors elsewhere in the body.

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

Thiazolidinedione, a peroxisome proliferator-activated receptor-gamma ligand, modulates the E-cadherin/beta-catenin system in a human pancreatic cancer cell line, BxPC-3.

PubMed

Ohta, Tetsuo; Elnemr, Ayman; Yamamoto, Miyuki; Ninomiya, Itasu; Fushida, Sachio; Nishimura, Gen-Ichi; Fujimura, Takashi; Kitagawa, Hirohisa; Kayahara, Masato; Shimizu, Koichi; Yi, Shuangqin; Miwa, Koichi

2002-07-01

Activation of peroxisome proliferator-activated receptor (PPAR)-gamma induces terminal differentiation and growth inhibition associated with G1 cell cycle arrest in some cancer cells. The multifunctional molecule beta-catenin performs important roles in intercellular adhesion and signal transduction. However, no report has focused on actions of PPAR-gamma in regulating the E-cadherin/beta-catenin system. We examined whether thiazolidinedione (TZD), a potent PPAR-gamma ligand, could modulate the E-cadherin/beta-catenin system in a human pancreatic cancer cell line, BxPC-3, that has been found to express PPAR-gamma. According to Western blotting, TZD markedly increased differentiation markers including E-cadherin and carcinoembryonic antigen, while beta-catenin did not change significantly. In untreated cells, fluorescence immunostaining demonstrated beta-catenin predominantly in the cytoplasm and/or nucleus; in TZD-treated cells, beta-catenin localization had dramatically shifted to the plasma membrane, in association with increased E-cadherin at this site. Thus, a PPAR-gamma ligand appears to participate not only in induction of differentiation in pancreatic cancer cells, but also in the regulation of the E-cadherin/beta-catenin system. Such ligands may prove clinically useful as cytostatic anticancer agents.

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.

TGF-beta1 inhibits Cx43 expression and formation of functional syncytia in cultured smooth muscle cells from human detrusor.

PubMed

Neuhaus, Jochen; Heinrich, Marco; Schwalenberg, Thilo; Stolzenburg, Jens-Uwe

2009-02-01

Human detrusor smooth muscle cells (hBSMCs) are coupled by connexin 43 (Cx43)-positive gap junctions to form functional syncytia. Gap junctional communication likely is necessary for synchronised detrusor contractions and is supposed to be altered in voiding disturbances. Other authors have shown that the pleiotropic cytokine TGF-beta1 upregulates Cx43 expression in human aortic smooth muscle cells. In this study, we examined the TGF-beta1 effects on Cx43 expression in cultured hBSMCs. hBSMC cultures, established from patients undergoing cystectomy, were treated with recombinant human TGF-beta1. Cx43 expression was then examined by Western blotting, real-time PCR, and immunocytochemistry. Dye-injection experiments were used to study the size of functional syncytia. Dye-coupling experiments revealed stable formation of functional syncytia in passaged cell cultures (P1-P4). Stimulation with TGF-beta1 led to significant reduction of Cx43 immunoreactivity and coupling. Cx43 protein expression was significantly downregulated and Cx43 mRNA was only 30% of the control level. Interestingly, low phosphorylation species of Cx43 were particularly affected. Our experiments demonstrated a significant down regulation of connexin 43 by TGF-beta1 in cultured hBSMCs. These findings support the view that TGF-beta1 is involved in the pathophysiology of urinary bladder dysfunction.

Priming affects the activity of a specific region of the promoter of the human beta interferon gene.

PubMed Central

Dron, M; Lacasa, M; Tovey, M G

1990-01-01

Treatment of Daudi or HeLa cells with human interferon (IFN) alpha 8 before induction with either poly(I)-poly(C) or Sendai virus resulted in an 8- to 100-fold increase in IFN production. The extent of priming in Daudi cells paralleled the increase in the intracellular content of IFN-beta mRNA. IFN-alpha mRNA remained undetectable in poly(I)-poly(C)-treated Daudi cells either before or after priming. An IFN-resistant clone of Daudi cells was found to produce 4- to 20-fold more IFN after priming, indicating that priming was unrelated to the phenotype of IFN sensitivity. IFN treatment of either Daudi or HeLa cells transfected with the human IFN-beta promoter (-282 to -37) linked to the chloramphenicol acetyltransferase (CAT) gene resulted in an increase in CAT activity after induction with poly(I)-poly(C) or Sendai virus. A synthetic double-stranded oligonucleotide corresponding to an authentic 30-base-pair (bp) region of the human IFN-beta promoter between positions -91 and -62 was found to confer virus inducibility upon the reporter CAT gene in HeLa cells. IFN treatment of HeLa cells transfected with this 30-bp region of the IFN-beta promoter in either the correct or reversed orientation also increased CAT activity upon subsequent induction. IFN treatment alone had no detectable effect on the activity of either the 30-bp region or the complete human IFN promoter. Images PMID:2153928

Human Beta Defensin 2 Selectively Inhibits HIV-1 in Highly Permissive CCR6⁺CD4⁺ T Cells.

PubMed

Lafferty, Mark K; Sun, Lingling; Christensen-Quick, Aaron; Lu, Wuyuan; Garzino-Demo, Alfredo

2017-05-16

Chemokine receptor type 6 (CCR6)⁺CD4⁺ T cells are preferentially infected and depleted during HIV disease progression, but are preserved in non-progressors. CCR6 is expressed on a heterogeneous population of memory CD4⁺ T cells that are critical to mucosal immunity. Preferential infection of these cells is associated, in part, with high surface expression of CCR5, CXCR4, and α4β7. In addition, CCR6⁺CD4⁺ T cells harbor elevated levels of integrated viral DNA and high levels of proliferation markers. We have previously shown that the CCR6 ligands MIP-3α and human beta defensins inhibit HIV replication. The inhibition required CCR6 and the induction of APOBEC3G. Here, we further characterize the induction of apolipoprotein B mRNA editing enzyme (APOBEC3G) by human beta defensin 2. Human beta defensin 2 rapidly induces transcriptional induction of APOBEC3G that involves extracellular signal-regulated kinases 1/2 (ERK1/2) activation and the transcription factors NFATc2, NFATc1, and IRF4. We demonstrate that human beta defensin 2 selectively protects primary CCR6⁺CD4⁺ T cells infected with HIV-1. The selective protection of CCR6⁺CD4⁺ T cell subsets may be critical in maintaining mucosal immune function and preventing disease progression.

TGF-beta is specifically expressed in human dermal papilla cells and modulates hair folliculogenesis.

PubMed

Inoue, Keita; Aoi, Noriyuki; Yamauchi, Yuji; Sato, Takahiro; Suga, Hirotaka; Eto, Hitomi; Kato, Harunosuke; Tabata, Yasuhiko; Yoshimura, Kotaro

2009-01-01

Dermal papilla cells (DPCs) in the mammalian hair follicle have been shown to develop hair follicles through epithelial-mesenchymal interactions. A cell therapy to regenerate human hair is theoretically possible by expanding autologous human DPCs (hDPCs) and transplanting them into bald skin, though much remains to be overcome before clinical success. In this study, we compared gene signatures of hDPCs at different passages and human dermal fibroblasts, and found transforming growth factor (TGF)-beta(2) to be highly expressed in cultured hDPCs. Keratinocyte conditioned medium, which is known to help preserve the hair-inducing capacity of hDPCs, up-regulated TGF-beta(2) expression of hDPCs and also enhanced their alkaline phosphatase (ALP) activity, a known index for hair-inductive capacity. Through screening of components secreted from keratinocytes, the vitamin D(3) analogue was found to promote TGF-beta(2) expression and ALP activity of hDPCs. In animal hair folliculogenesis models using rat epidermis and expanded hDPCs, inhibition of TGF-beta(2) signalling at the ligand or receptor level significantly impaired hair folliculogenesis and maturation. These results suggest an important role for TGF-beta(2) in hair follicle morphogenesis and provide insights into the establishment of future cell therapies for hair regrowth by transplanting expanded DPCs.

Beta-cell metabolic alterations under chronic nutrient overload in rat and human islets

USDA-ARS?s Scientific Manuscript database

The aim of this study was to assess multifactorial Beta-cell responses to metabolic perturbations in primary rat and human islets. Treatment of dispersed rat islet cells with elevated glucose and free fatty acids (FFAs, oleate:palmitate = 1:1 v/v) resulted in increases in the size and the number of ...

Endoplasmic Reticulum Stress Links Oxidative Stress to Impaired Pancreatic Beta-Cell Function Caused by Human Oxidized LDL.

PubMed

Plaisance, Valérie; Brajkovic, Saška; Tenenbaum, Mathie; Favre, Dimitri; Ezanno, Hélène; Bonnefond, Amélie; Bonner, Caroline; Gmyr, Valéry; Kerr-Conte, Julie; Gauthier, Benoit R; Widmann, Christian; Waeber, Gérard; Pattou, François; Froguel, Philippe; Abderrahmani, Amar

2016-01-01

Elevated plasma concentration of the pro-atherogenic oxidized low density lipoprotein cholesterol (LDL) triggers adverse effects in pancreatic beta-cells and is associated with type 2 diabetes. Here, we investigated whether the endoplasmic reticulum (ER) stress is a key player coupling oxidative stress to beta-cell dysfunction and death elicited by human oxidized LDL. We found that human oxidized LDL activates ER stress as evidenced by the activation of the inositol requiring 1α, and the elevated expression of both DDIT3 (also called CHOP) and DNAJC3 (also called P58IPK) ER stress markers in isolated human islets and the mouse insulin secreting MIN6 cells. Silencing of Chop and inhibition of ER stress markers by the chemical chaperone phenyl butyric acid (PBA) prevented cell death caused by oxidized LDL. Finally, we found that oxidative stress accounts for activation of ER stress markers induced by oxidized LDL. Induction of Chop/CHOP and p58IPK/P58IPK by oxidized LDL was mimicked by hydrogen peroxide and was blocked by co-treatment with the N-acetylcystein antioxidant. As a conclusion, the harmful effects of oxidized LDL in beta-cells requires ER stress activation in a manner that involves oxidative stress. This mechanism may account for impaired beta-cell function in diabetes and can be reversed by antioxidant treatment.

Methylation of insulin DNA in response to proinflammatory cytokines during the progression of autoimmune diabetes in NOD mice.

PubMed

Rui, Jinxiu; Deng, Songyan; Lebastchi, Jasmin; Clark, Pamela L; Usmani-Brown, Sahar; Herold, Kevan C

2016-05-01

Type 1 diabetes is caused by the immunological destruction of pancreatic beta cells. Preclinical and clinical data indicate that there are changes in beta cell function at different stages of the disease, but the fate of beta cells has not been closely studied. We studied how immune factors affect the function and epigenetics of beta cells during disease progression and identified possible triggers of these changes. We studied FACS sorted beta cells and infiltrating lymphocytes from NOD mouse and human islets. Gene expression was measured by quantitative real-time RT-PCR (qRT-PCR) and methylation of the insulin genes was investigated by high-throughput and Sanger sequencing. To understand the role of DNA methyltransferases, Dnmt3a was knocked down with small interfering RNA (siRNA). The effects of cytokines on methylation and expression of the insulin gene were studied in humans and mice. During disease progression in NOD mice, there was an inverse relationship between the proportion of infiltrating lymphocytes and the beta cell mass. In beta cells, methylation marks in the Ins1 and Ins2 genes changed over time. Insulin gene expression appears to be most closely regulated by the methylation of Ins1 exon 2 and Ins2 exon 1. Cytokine transcription increased with age in NOD mice, and these cytokines could induce methylation marks in the insulin DNA by inducing methyltransferases. Similar changes were induced by cytokines in human beta cells in vitro. Epigenetic modification of DNA by methylation in response to immunological stressors may be a mechanism that affects insulin gene expression during the progression of type 1 diabetes.

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.

HES6 reverses nuclear reprogramming of insulin-producing cells following cell fusion

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

Ball, Andrew J.; Abrahamsson, Annelie E.; Tyrberg, Bjoern

2007-04-06

To examine the mechanism by which growth-stimulated pancreatic {beta}-cells dedifferentiate, somatic cell fusions were performed between MIN6, a highly differentiated mouse insulinoma, and {beta}lox5, a cell line derived from human {beta}-cells which progressively dedifferentiated in culture. MIN6/{beta}lox5 somatic cells hybrids underwent silencing of insulin expression and a marked decline in PDX1, NeuroD, and MafA, indicating that {beta}lox5 expresses a dominant transacting factor(s) that represses {beta}-cell differentiation. Expression of Hes1, which inhibits endocrine differentiation was higher in hybrid cells than in parental MIN6 cells. Hes6, a repressor of Hes1, was highly expressed in primary {beta}-cells as well as MIN6, but wasmore » repressed in hybrids. Hes6 overexpression using a retroviral vector led to a decrease in Hes1 levels, an increase in {beta}-cell transcription factors and partial restoration of insulin expression. We conclude that the balance of Notch activators and inhibitors may play an important role in maintaining the {beta}-cell differentiated state.« less

Stimulation of interleukin-1beta-independent interleukin-6 production in human dental pulp cells by lipopolysaccharide.

PubMed

Hosoya, S; Matsushima, K; Ohbayashi, E; Yamazaki, M; Shibata, Y; Abiko, Y

1996-12-01

Dental pulpal infection is most commonly caused by extensive dental caries. A principal driving force behind pulpal disease response appears to lie in the immune system's response to bacteria. However, the production of interleukin (IL)-1beta and IL-6 in human dental pulp (HDP) cells in response to lipopolysaccharide (LPS) has not been well characterized. We examined IL-1beta and IL-6 production in HDP cells by challenging with LPS from Porphyromonas endodontalis, which is a Gram-negative bacteria found in root canals. Our results presented here showed that when HDP cells were stimulated by LPS, the production of IL-6 always preceded that of IL-1beta. Since the IL-6 production was observed even in the presence of the IL-1beta receptor antagonist, we concluded IL-6 production was independent of the IL-1beta molecule in LPS-stimulated HDP cells. This idea was further supported by the results obtained from RT-PCR experiments, in which IL-6 mRNA, but not IL-1beta mRNA, was present in the RNA preparation isolated from the early stage of cells.

Proliferation of Estrogen Receptor alpha Positive Mammary Epithelial Cells is Restrained by TGFbeta1 in Adult Mice

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

Ewan, Kenneth B.R.; Oketch-Rabah, Hellen A.; Ravani, Shraddha A.

2005-03-03

Transforming growth factor {beta}1 (TGF{beta}1) is a potent inhibitor of mammary epithelial proliferation. In human breast, estrogen receptor {alpha} (ER{alpha}) cells rarely co-localize with markers of proliferation, but their increased frequency correlates with breast cancer risk. To determine whether TGF{beta}1 is necessary for the quiescence of ER{alpha}-positive population, we examined mouse mammary epithelial gland at estrus. Approximately 35% of cells showed TGF{beta}1 activation, which co-localized with nuclear receptor-phosphorylated Smad 2/3, indicating that TGF{beta} signaling is autocrine. Furthermore, nuclear Smad co-localized with nuclear ER{alpha}. To test whether TGF{beta} was functional, we examined genetically engineered mice with different levels of TGF{beta}1. ER{alpha}more » co-localization with markers of proliferation (i.e. Ki-67 or BrdU) at estrus was significantly increased in the mammary glands of Tgf{beta}1 C57/bl/129SV heterozygote mice. This relationship was maintained following pregnancy, but was absent at puberty. Conversely, mammary epithelial expression of constitutively active TGF{beta}1 via the MMTV promoter suppressed proliferation of ER{alpha} positive cells. Thus, TGF{beta}1 activation functionally restrains ER{alpha} positive cells from proliferating in adult mammary gland. Accordingly, we propose that TGF{beta}1 dysregulation may promote proliferation of ER{alpha} positive cells associated with breast cancer risk in humans.« less

Expression and in vitro regulation of integrins by normal human urothelial cells.

PubMed

Southgate, J; Kennedy, W; Hutton, K A; Trejdosiewicz, L K

1995-08-01

Integrins are thought to be essential adhesion receptors for the maintenance of tissue histioarchitecture. The purpose of this study was to determine integrin expression patterns in the human stratified transitional epithelium of the urinary tract (urothelium). In situ expression patterns were compared with in vitro expression, using a normal cell culture model system in which the effects of cell stratification can be studied independently of differentiation. By immunohistological criteria, the urothelia of bladder, ureter and renal pelvis expressed alpha 2 beta 1 and alpha 3 beta 1 integrins in all layers at intercellular junctions, and cytoplasmically in the lower strata. By contrast, alpha 6 beta 4 and occasionally alpha v beta 4 were expressed only by basal cells and localised to the basal lamina. These expression patterns were unaltered in specimens where an inflammatory cell infiltrate was present. In long-term cultures of normal urothelial cells maintained in a low-Ca++ serum-free medium, the monolayer cultures expressed alpha 2 beta 1, alpha 3 beta 1 and alpha 5 beta 1 integrins at intercellular junctions and in cytoplasmic inclusions, whereas alpha 6 beta 4 was distributed in a random pattern over the substratum. Increasing exogenous Ca++ concentrations induced cell stratification and desmosome formation, but not cytodifferentiation. Under these conditions, alpha 6 beta 4 became cell-, rather than substratum-associated, localising particularly to filopodia and lamellipodia. Quantitation of integrin expression by flow cytometry confirmed increased surface expression of alpha 6 beta 4 in high Ca++ media, and also of alpha 3 and alpha 5, but not alpha 2, subunits. These results suggest that alpha 2 beta 1 and alpha 3 beta 1 integrins, although differentially regulated, are mainly involved in homotypic cell-cell interactions and the maintenance of a stratified morphology, whereas alpha 6 beta 4 is the principal integrin involved in substratum adhesion.

Tumor-associated antigen human chorionic gonadotropin beta contains numerous antigenic determinants recognized by in vitro-induced CD8+ and CD4+ T lymphocytes.

PubMed

Dangles, Virginie; Halberstam, Ilan; Scardino, Antonio; Choppin, Jeannine; Wertheimer, Mireille; Richon, Sophie; Quelvennec, Erwann; Moirand, Romain; Guillet, Jean-Gérard; Kosmatopoulos, Kostas; Bellet, Dominique; Zeliszewski, Dominique

2002-02-01

The beta subunit of human chorionic gonadotropin (hCG beta) is markedly overexpressed by neoplastic cells of differing histological origin including those present in colon, breast, prostate and bladder tumors. We have previously shown that some patients with hCG beta-producing urothelial tumors have circulating T cells that proliferate in response to hCG beta. To make a comprehensive study of hCG beta as a potential target for cancer immunotherapy, we investigated whether hCG beta peptides could induce CD4+ or CD8+ T-cell responses in vitro. By stimulating peripheral blood mononuclear cells (PBMCs) from three donors with mixtures of overlapping 16-mer synthetic peptides analogous to portions of either the hCG beta 20-71 or the hCG beta 102-129 region, we established six CD4+ T-cell lines that proliferated specifically in response to five distinct determinants located within these two hCG beta regions. Three antigenic determinants (hCG beta 52-67, 106-121 and 114-125) were presented by HLA-DR molecules, while the two other antigenic determinants (hCG beta 48-63 and 56-67) were presented by HLA-DQ molecules. Interestingly, one T-cell line specific for peptide hCG beta 106-121 recognized hCG beta peptides comprising, at position 117, either an alanine or an aspartic acid residue, with the latter residue being present within the protein expressed by some tumor cells. In addition, three other hCG beta-derived peptides that exhibited HLA-A*0201 binding ability were able to stimulate CD8+ cytotoxic T cells from two HLA-A*0201 donors. These three immunogenic peptides corresponded to regions hCG beta 40-48, hCG beta 44-52 and hCG beta 75-84. Our results indicate that the tumor-associated antigen hCG beta possesses numerous antigenic determinants liable to stimulate CD4+ and CD8+ T lymphocytes, and might thus be an effective target antigen for the immunotherapy of hCG beta-producing tumors.

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 interaction between the MR and aldosterone or cortisol is in part a nuclear event.

Characterisation of the insulinotropic activity of an aqueous extract of Gymnema sylvestre in mouse beta-cells and human islets of Langerhans.

PubMed

Liu, Bo; Asare-Anane, Henry; Al-Romaiyan, Altaf; Huang, Guocai; Amiel, Stephanie A; Jones, Peter M; Persaud, Shanta J

2009-01-01

Leaves of the Gymnema sylvestre (GS) plant have been used to treat diabetes mellitus for millennia, but the previously documented insulin secretagogue effects of GS extracts in vitro may be non-physiological through damage to the beta-cells. We have now examined the effects of a novel GS extract (termed OSA) on insulin secretion from the MIN6 beta-cell line and isolated human islets of Langerhans. Insulin secretion from MIN6 cells was stimulated by OSA in a concentration-dependent manner, with low concentrations (0.06-0.25 mg/ml) having no deleterious effects on MIN6 cell viability, while higher concentrations (> or = 0.5 mg/ml) caused increased Trypan blue uptake. OSA increased beta-cell Ca2+ levels, an effect that was mediated by Ca2+ influx through voltage-operated calcium channels. OSA also reversibly stimulated insulin secretion from isolated human islets and its insulin secretagogue effects in MIN6 cells and human islets were partially dependent on the presence of extracellular Ca2+. These data indicate that low concentrations of the GS isolate OSA stimulate insulin secretion in vitro, at least in part as a consequence of Ca2+ influx, without compromising beta-cell viability. Identification of the component of the OSA extract that stimulates regulated insulin exocytosis, and further investigation of its mode(s) of action, may provide promising lead targets for Type 2 diabetes therapy. 2009 S. Karger AG, Basel.

[Effect of ginseng polysaccharide-induced wnt/beta-catenin signal transduction pathway on apoptosis of human nasopharyngeal cancer cells CNE-2].

PubMed

Fan, Jia-Ming; Liu, Ze-Hong; Li, Jing; Wang, Ya-Ping; Yang, Lv-Yuan; Huang, Jiang-Ju

2013-10-01

To observe the effect of ginseng polysaccharide (GPS) on the proliferation and apoptosis of human nasopharyngeal cancer cells CNE-2, and discuss the possible mechanism. The effect of GPS on the growth of CNE-2 cells was observed by CCK8 assay. CNE-2 cells in the logarithmic phase were collected and processed respectively with different concentrations (0, 0. 1, 0. 2, 0. 3. 0. 4 g L-1) of GPS for 48 h. The flow cytometry was used to detect its induction effect on CNE-2 cell apoptosis. Hoechst-33258 cell staining and electron microscope were used to observe the morphological changes of cells. The beta-catenin mRNA expression was detected by Real-time PCR. The protein localizations and expressions of beta-catenin and TCF4 were tested by the immunofluorescence staining. The expressions of beta-catenin, Bcl-2 and Bax proteins were detected by Western blot. CCK8 assay results showed that GPS could remarkably inhibit the proliferation of CNE-2 cells, with dose-time dependence. IC50 of cells induced with GPS for 48 h was 0. 39 g L-1. After being processed with GPS with concentrations of 0.1, 0. 2, 0. 3, 0. 4 g L-1 for 48 h, the cell apoptosis rates of human nasopharyngeal cancer cells CNE-2 were (5. 69 +/- 0. 29)% , (10. 3 +/- 0. 63)% , (15. 4 +/- 0. 74 ) % and (35. 7 +/- 1. 86)% , respectively. Significant difference was observed compared with the control group (2. 08 +/- 0. 11) % (P <0. 05). The results of Hoechst-33258 staining showed the characteristics of cell apoptosis. Under the electron microscope, apoptosis bodies could be observed among CNE-2 cells induced with GPS with the concentration of 0. 4 g L -1 for 48 h. The results of Real-time PCR showed a significant reduction in beta-catenin mRNA expression. The results of laser confocal microscopy revealed notable decrease of beta-catenin and TCF4 expression in nucleus and transfer from nucleus to cell membranes in beta-catenin expression areas after being processed with GPS for 48 h. Western blot showed significant decrease in the expressions of beta-catenin and anti-apoptosis protein Bcl-2, with an increasing expression in apoptosis-promoting protein Bax (P <0. 05). GPS could significantly inhibit the proliferation of CNE-2 cells and promote thier apoptosis. The obstruction of Wnt/beta-catenin signaling pathway may be an important mechanism for GPS to induce the apoptosis of human nasopharyngeal cancer cells CNE-2.

Cytokine regulation on the synthesis of nitric oxide in vivo by chronically infected human polymorphonuclear leucocytes.

PubMed Central

Takeichi, O; Saito, I; Okamoto, Y; Tsurumachi, T; Saito, T

1998-01-01

To determine if nitric oxide (NO) is produced by chronically infected human polymorphonuclear leucocytes (PMNs) in vivo, inflamed exudates (periapical exudates: PE) collected from periapical periodontitis patients were examined. Cell-free supernatants and cells were separated by centrifugation. Significant levels of nitrite concentrations were observed in the supernatants. The production of inducible NO synthase (iNOS) in highly purified PMNs derived from PEs was then immunocytochemically determined using rabbit anti-human iNOS antiserum. In vitro, human peripheral blood PMNs (PB-PMNs) isolated from patients were cultured with a combination of Esherichia coli-lipopolysaccharide (LPS), recombinant human interferon-gamma (rhIFN-gamma) and/or interleukin-1 beta (rhIL-1 beta). The stimulated PB-PMNs showed steady-state levels of nitrite. The stimulation of LPS, rhIFN-gamma and rhIL-1 beta showed more NO induction than that of LPS with either IFN-gamma or IL-1 beta, suggesting the synergistic effects of cytokines. Cryostat sections of surgically removed periapical tissues were also immunohistochemically examined for iNOS, IFN-gamma and IL-1 beta. Two-colour immunohistochemistry revealed the interaction of iNOS-producing PMNs and IFN-gamma- or IL-1 beta-producing mononuclear cells. On the basis of these data, we concluded that with the stimulation of inflammatory cytokines derived from mononuclear cells, PMNs can spontaneously produce NO at the site of chronic infection. The present studies are consistent with a hypothesis suggesting that PMNs could be regulated and delicately balanced to produce NO by mononuclear cell-derived cytokines in vivo. NO-producing cells may play a pivotal role in chronic inflammation. Images Figure 2 Figure 4 Figure 5 Figure 6 PMID:9616379

MicroRNA-320a suppresses human colon cancer cell proliferation by directly targeting {beta}-catenin

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

Sun, Jian-Yong; State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, 710032 Xi'an; Huang, Yi

2012-04-20

Highlights: Black-Right-Pointing-Pointer miR-320a is downregulated in human colorectal carcinoma. Black-Right-Pointing-Pointer Overexpression of miR-320a inhibits colon cancer cell proliferation. Black-Right-Pointing-Pointer {beta}-Catenin is a direct target of miR-320a in colon cancer cells. Black-Right-Pointing-Pointer miR-320a expression inversely correlates with mRNA expression of {beta}-catenin's target genes in human colon carcinoma. -- Abstract: Recent profile studies of microRNA (miRNA) expression have documented a deregulation of miRNA (miR-320a) in human colorectal carcinoma. However, its expression pattern and underlying mechanisms in the development and progression of colorectal carcinoma has not been elucidated clearly. Here, we performed real-time PCR to examine the expression levels of miR-320a in colonmore » cancer cell lines and tumor tissues. And then, we investigated its biological functions in colon cancer cells by a gain of functional strategy. Further more, by the combinational approaches of bioinformatics and experimental validation, we confirmed target associations of miR-320a in colorectal carcinoma. Our results showed that miR-320a was frequently downregulated in cancer cell lines and colon cancer tissues. And we demonstrated that miR-320a restoration inhibited colon cancer cell proliferation and {beta}-catenin, a functionally oncogenic molecule was a direct target gene of miR-320a. Finally, the data of real-time PCR showed the reciprocal relationship between miR-320a and {beta}-catenin's downstream genes in colon cancer tissues. These findings indicate that miR-320a suppresses the growth of colon cancer cells by directly targeting {beta}-catenin, suggesting its application in prognosis prediction and cancer treatment.« less

Transforming growth factor-beta1 transcriptionally activates CD34 and prevents induced differentiation of TF-1 cells in the absence of any cell-cycle effects.

PubMed

Marone, M; Scambia, G; Bonanno, G; Rutella, S; de Ritis, D; Guidi, F; Leone, G; Pierelli, L

2002-01-01

A number of cytokines modulate self-renewal and differentiation of hematopoietic elements. Among these is transforming growth factor beta1 (TGF-beta1), which regulates cell cycle and differentiation of hematopoietic cells, but has pleiotropic activities depending on the state of responsiveness of the target cells. It has been previously shown by us and other authors that TGF-beta1 maintains human CD34(+) hematopoietic progenitors in an undifferentiated state, independently of any cell cycle effects, and that depletion of TGF-beta1 triggers differentiation accompanied by a decrease in CD34 antigen expression. In the present work, we show that exogenous TGF-beta1 upregulates the human CD34 antigen in the CD34(+) cell lines TF-1 and KG-1a, but not in the more differentiated CD34(-) cell lines HL-60 and K-562. We further studied this effect in the pluripotent erythroleukemia cell line TF-1. Here, TGF-beta1 did not effect cell growth, but induced transcriptional activation of full-length CD34 and prevented differentiation induced by differentiating agents. This effect was associated with nuclear translocation of Smad-2, activation of TAK-1, and with a dramatic decrease in p38 phosphorylation. In other systems TGF-beta1 has been shown to activate a TGF-beta-activated kinase 1 (TAK1), which in turn, activates p38. The specific inhibitor of p38 phosphorylation, SB202190, also increased CD34 RNA expression, indicating the existence of a link between p-38 inhibition by TGF-beta1 and CD34 overexpression. Our data demonstrate that TGF-beta1 transcriptionally activates CD34 and prevents differentiation of TF-1 cells by acting independently through the Smad, TAK1 and p38 pathways, and thus provide important clues for the understanding of hematopoietic development and a potential tool to modify response of hematopoietic cells to mitogens or differentiating agents.

MST1 is a key regulator of beta cell apoptosis and dysfunction in diabetes.

PubMed

Ardestani, Amin; Paroni, Federico; Azizi, Zahra; Kaur, Supreet; Khobragade, Vrushali; Yuan, Ting; Frogne, Thomas; Tao, Wufan; Oberholzer, Jose; Pattou, Francois; Conte, Julie Kerr; Maedler, Kathrin

2014-04-01

Apoptotic cell death is a hallmark of the loss of insulin-producing beta cells in all forms of diabetes mellitus. Current treatments fail to halt the decline in functional beta cell mass, and strategies to prevent beta cell apoptosis and dysfunction are urgently needed. Here, we identified mammalian sterile 20-like kinase-1 (MST1) as a critical regulator of apoptotic beta cell death and function. Under diabetogenic conditions, MST1 was strongly activated in beta cells in human and mouse islets and specifically induced the mitochondrial-dependent pathway of apoptosis through upregulation of the BCL-2 homology-3 (BH3)-only protein BIM. MST1 directly phosphorylated the beta cell transcription factor PDX1 at T11, resulting in the latter's ubiquitination and degradation and thus in impaired insulin secretion. MST1 deficiency completely restored normoglycemia, beta cell function and survival in vitro and in vivo. We show MST1 as a proapoptotic kinase and key mediator of apoptotic signaling and beta cell dysfunction and suggest that it may serve as target for the development of new therapies for diabetes.

DNase I hypersensitivity and epsilon-globin transcriptional enhancement are separable in locus control region (LCR) HS1 mutant human beta-globin YAC transgenic mice.

PubMed

Shimotsuma, Motoshi; Okamura, Eiichi; Matsuzaki, Hitomi; Fukamizu, Akiyoshi; Tanimoto, Keiji

2010-05-07

Expression of the five beta-like globin genes (epsilon, Ggamma, Agamma, delta, beta) in the human beta-globin locus depends on enhancement by the locus control region, which consists of five DNase I hypersensitive sites (5'HS1 through 5'HS5). We report here a novel enhancer activity in 5'HS1 that appears to be potent in transfected K562 cells. Deletion analyses identified a core activating element that bound to GATA-1, and a two-nucleotide mutation that disrupted GATA-1 binding in vitro abrogated 5'HS1 enhancer activity in transfection experiments. To determine the in vivo role of this GATA site, we generated multiple lines of human beta-globin YAC transgenic mice bearing the same two-nucleotide mutation. In the mutant mice, epsilon-, but not gamma-globin, gene expression in primitive erythroid cells was severely attenuated, while adult beta-globin gene expression in definitive erythroid cells was unaffected. Interestingly, DNaseI hypersensitivity near the 5'HS1 mutant sequence was eliminated in definitive erythroid cells, whereas it was only mildly affected in primitive erythroid cells. We therefore conclude that, although the GATA site in 5'HS1 is critical for efficient epsilon-globin gene expression, hypersensitive site formation per se is independent of 5'HS1 function, if any, in definitive erythroid cells.

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

Fibronectin regulates the activation of THP-1 cells by TGF-beta1.

PubMed

Wang, A C; Fu, L

2001-03-01

To determine how fibronectin regulates the immunomodulatory effects of transforming growth factor (TGF)-beta on THP-1 cells. THP-1 monocytic cell line. THP-1 cells were primed for 48 h in the presence or absence of 250 pM TGF-beta1. Assays or assessments carried out, together with statistical test applied. We found that adherence to fibronectin dramatically modulates the effects of TGF-beta1 on the human monocytic cell line THP-1. TGF-beta did not significantly affect constitutive interleukin (IL)-8 secretion or IL-1beta-induced IL-8 secretion from suspended cells. In contrast, TGF-beta stimulated IL-8 secretion as well as augmented IL-1beta-induced IL-8 secretion from adherent cells. The differential effects of TGF-beta1 on IL-8 secretion from suspended and adherent cells could not be explained by differences in IL-1 receptor antagonist production. The effects of fibronectin on TGF-beta1 induced IL-8 secretion from THP-1 cells were mimicked by adhesion to immobilized anti-a4beta1 integrin antibody and to a fibronectin fragment containing the CS-1 domain. These results indicate that alpha4beta1-mediated adhesion to fibronectin may play a key role during inflammation by profoundly influencing the effects of TGF-beta1 on monocytes.

Early stages in the development of human T, natural killer and thymic dendritic cells.

PubMed

Spits, H; Blom, B; Jaleco, A C; Weijer, K; Verschuren, M C; van Dongen, J J; Heemskerk, M H; Res, P C

1998-10-01

T-cell development is initiated when CD34+ pluripotent stem cells or their immediate progeny leave the bone marrow to migrate to the thymus. Upon arrival in the thymus the stem cell progeny is not yet committed to the T-cell lineage as it has the capability to develop into T, natural killer (NK) and dendritic cells (DC). Primitive hematopoietic progenitor cells in the human thymus express CD34 and lack CD1a. When these progenitor cells develop into T cells they traverse a number of checkpoints. One early checkpoint is the induction of T-cell commitment, which correlates with appearance of CD1a and involves the loss of capacity to develop into NK cells and DC and the initiation of T-cell receptor (TCR) gene rearrangements. Basic helix-loop-helix transcription factors play a role in induction of T-cell commitment. CD1a+CD34+ cells develop into CD4+CD8 alpha+ beta+ cells by upregulating first CD4, followed by CD8 alpha and then CD8 beta. Selection for productive TCR beta gene rearrangements (beta selection) likely occurs in the CD4+CD8 alpha+ beta- and CD4+CD8 alpha+ beta+ populations. Although the T and NK-cell lineages are closely related to each other, NK cells can develop independently of the thymus. The fetal thymus is most likely one site of NK-cell development.

Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells

NASA Astrophysics Data System (ADS)

Zalzman, Michal; Gupta, Sanjeev; Giri, Ranjit K.; Berkovich, Irina; Sappal, Baljit S.; Karnieli, Ohad; Zern, Mark A.; Fleischer, Norman; Efrat, Shimon

2003-06-01

Beta-cell replacement is considered to be the most promising approach for treatment of type 1 diabetes. Its application on a large scale is hindered by a shortage of cells for transplantation. Activation of insulin expression, storage, and regulated secretion in stem/progenitor cells offers novel ways to overcome this shortage. We explored whether fetal human progenitor liver cells (FH) could be induced to differentiate into insulin-producing cells after expression of the pancreatic duodenal homeobox 1 (Pdx1) gene, which is a key regulator of pancreatic development and insulin expression in beta cells. FH cells possess a considerable replication capacity, and this was further extended by introduction of the gene for the catalytic subunit of human telomerase. Immortalized FH cells expressing Pdx1 activated multiple beta-cell genes, produced and stored considerable amounts of insulin, and released insulin in a regulated manner in response to glucose. When transplanted into hyperglycemic immunodeficient mice, the cells restored and maintained euglycemia for prolonged periods. Quantitation of human C-peptide in the mouse serum confirmed that the glycemia was normalized by the transplanted human cells. This approach offers the potential of a novel source of cells for transplantation into patients with type 1 diabetes.

Preventive effects of heregulin-beta1 on macrophage foam cell formation and atherosclerosis.

PubMed

Xu, Gang; Watanabe, Takuya; Iso, Yoshitaka; Koba, Shinji; Sakai, Tetsuo; Nagashima, Masaharu; Arita, Shigeko; Hongo, Shigeki; Ota, Hidekazu; Kobayashi, Youichi; Miyazaki, Akira; Hirano, Tsutomu

2009-08-28

Human heregulins, neuregulin-1 type I polypeptides that activate proliferation, differentiation, and survival of glial cells, neurons, and myocytes, are expressed in macrophage foam cells within human coronary atherosclerotic lesions. Macrophage foam cell formation, characterized by cholesterol ester accumulation, is modulated by scavenger receptor class A (SR-A), acyl-coenzyme A:cholesterol acyltransferase (ACAT)1, and ATP-binding cassette transporter (ABC)A1. The present study clarified the roles of heregulins in macrophage foam cell formation and atherosclerosis. Plasma heregulin-beta(1) levels were significantly decreased in 31 patients with acute coronary syndrome and 33 patients with effort angina pectoris compared with 34 patients with mild hypertension and 40 healthy volunteers (1.3+/-0.3, 2.0+/-0.4 versus 7.6+/-1.4, 8.2+/-1.2 ng/mL; P<0.01). Among all patients with acute coronary syndrome and effort angina pectoris, plasma heregulin-beta(1) levels were further decreased in accordance with the severity of coronary artery lesions. Expression of heregulin-beta(1) was observed at trace levels in intracoronary atherothrombosis obtained by aspiration thrombectomy from acute coronary syndrome patients. Heregulin-beta(1), but not heregulin-alpha, significantly reduced acetylated low-density lipoprotein-induced cholesterol ester accumulation in primary cultured human monocyte-derived macrophages by reducing SR-A and ACAT1 expression and by increasing ABCA1 expression at both mRNA and protein levels. Heregulin-beta(1) significantly decreased endocytic uptake of [(125)I]acetylated low-density lipoprotein and ACAT activity, and increased cholesterol efflux to apolipoprotein (Apo)A-I from human macrophages. Chronic infusion of heregulin-beta(1) into ApoE(-/-) mice significantly suppressed the development of atherosclerotic lesions. This study provided the first evidence that heregulin-beta(1) inhibits atherogenesis and suppresses macrophage foam cell formation via SR-A and ACAT1 downregulation and ABCA1 upregulation.

Antitubercular cassane furanoditerpenoids from the roots of Caesalpinia pulcherrima.

PubMed

Promsawan, Netnapa; Kittakoop, Prasat; Boonphong, Surat; Nongkunsarn, Pakawan

2003-08-01

Activity-guided fractionation of a root extract of Caesalpinia pulcherrima led to the isolation of two cassane-furanoditerpenoids, 6 beta-benzoyl-7 beta-hydroxyvouacapen-5 alpha-ol (1) and 6 beta-cinnamoyl-7beta-hydroxyvouacapen-5 alpha-ol (2). Compound 2 showed strong antitubercular activity with a minimum inhibitory concentration (MIC) of 6.25 microg/mL, whereas the benzoyl analogue (1) was less active (MIC 25 microg/mL). Both compounds expressed moderate cytotoxic activity towards KB (human oral carcinonoid cancer), BC (human breast cancer) and NCl-H187 (small cell lung cancer) cell lines.

High LET Radiation Can Enhance TGF(Beta) Induced EMT and Cross-Talk with ATM Pathways

NASA Technical Reports Server (NTRS)

Wang, Minli; Hada, Megumi; Huff, Janice; Pluth, Janice M.; Anderson, Janniffer; ONeill, Peter; Cucinotta, Francis A.

2010-01-01

The TGF(Beta) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation in mammary epithelial cells. We investigated possible interactions between the TGF(Beta) and ATM pathways following simulated space radiation using hTERT immortalized human esophageal epithelial cells (EPC-hTERT), mink lung epithelial cells (Mv1lu), and several human fibroblast cell lines. TGF(Beta) is a key modulator of the Epithelial-Mesenchymal Transition (EMT), important in cancer progression and metastasis. The implication of EMT by radiation also has several lines of developing evidence, however is poorly understood. The identification of TGF(Beta) induced EMT can be shown in changes to morphology, related gene over expression or down regulation, which can be detected by RT-PCR, and immunostaining and western blotting. In this study, we have observed morphologic and molecular alternations consistent with EMT after Mv1lu cells were treated with TGF(Beta) High LET radiation enhanced TGF(Beta) mediated EMT with a dose as low as 0.1Gy. In order to consider the TGF(Beta) interaction with ATM we used a potent ATM inhibitor Ku55933 and investigated gene expression changes and Smad signaling kinetics. Ku559933 was observed to reverse TGF(Beta) induced EMT, while this was not observed in dual treated cells (radiation+TGF(Beta)). In EPC-hTERT cells, TGF(Beta) alone was not able to induce EMT after 3 days of application. A combined treatment with high LET, however, significantly caused the alteration of EMT markers. To study the function of p53 in the process of EMT, we knocked down P53 through RNA interference. Morphology changes associated with EMT were observed in epithelial cells with silenced p53. Our study indicates: high LET radiation can enhance TGF(Beta) induced EMT; while ATM is triggering the process of TGF(Beta)-induced EMT, p53 might be an essential repressor for EMT phenotypes.

Smad2 and Smad3 phosphorylated at both linker and COOH-terminal regions transmit malignant TGF-beta signal in later stages of human colorectal cancer.

PubMed

Matsuzaki, Koichi; Kitano, Chiaki; Murata, Miki; Sekimoto, Go; Yoshida, Katsunori; Uemura, Yoshiko; Seki, Toshihito; Taketani, Shigeru; Fujisawa, Jun-ichi; Okazaki, Kazuichi

2009-07-01

Transforming growth factor (TGF)-beta initially inhibits growth of mature epithelial cells. Later, however, autocrine TGF-beta signaling acts in concert with the Ras pathway to induce a proliferative and invasive phenotype. TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also Ras-associated kinases, which differentially phosphorylate the mediators Smad2 and Smad3 to create distinct phosphorylated forms: COOH-terminally phosphorylated Smad2/3 (pSmad2C and pSmad3C) and both linker and COOH-terminally phosphorylated Smad2/3 (pSmad2L/C and pSmad3L/C). In this study, we investigated actions of pSmad2L/C and pSmad3L/C in cancer progression. TGF-beta inhibited cell growth by down-regulating c-Myc oncoprotein through the pSmad2C and pSmad3C pathway; TGF-beta signaling, in turn, enhanced cell growth by up-regulating c-Myc through the cyclin-dependent kinase (CDK) 4-dependent pSmad2L/C and pSmad3L/C pathways in cell nuclei. Alternatively, TbetaRI and c-Jun NH2-terminal kinase (JNK) together created cytoplasmic pSmad2L/C, which entered the nucleus and stimulated cell invasion, partly by up-regulating matrix metalloproteinase-9. In 20 clinical samples, pSmad2L/C and pSmad3L/C showed nuclear localization at invasion fronts of all TGF-beta-producing human metastatic colorectal cancers. In vitro kinase assay confirmed that nuclear CDK4 and cytoplasmic JNK obtained from the tumor tissue could phosphorylate Smad2 or Smad3 at their linker regions. We suggest that CDK4, together with JNK, alters tumor-suppressive TGF-beta signaling to malignant characteristics in later stages of human colorectal cancer. The linker phosphorylation of Smad2 and Smad3 may represent a target for intervention in human metastatic cancer.

Regulation of the mRNA-binding protein AUF1 by activation of the beta-adrenergic receptor signal transduction pathway.

PubMed

Pende, A; Tremmel, K D; DeMaria, C T; Blaxall, B C; Minobe, W A; Sherman, J A; Bisognano, J D; Bristow, M R; Brewer, G; Port, J

1996-04-05

In both cell culture based model systems and in the failing human heart, beta-adrenergic receptors ( beta-AR) undergo agonist-mediated down-regulation. This decrease correlates closely with down-regulation of its mRNA, an effect regulated in part by changes in mRNA stability. Regulation of mRNA stability has been associated with mRNA-binding proteins that recognize A + U-rich elements within the 3'-untranslated regions of many mRNAs encoding proto-oncogene and cytokine mRNAs. We demonstrate here that the mRNA-binding protein, AUF1, is present in both human heart and in hamster DDT1-MF2 smooth muscle cells and that its abundance is regulated by beta-AR agonist stimulation. In human heart, AUF1 mRNA and protein was significantly increased in individuals with myocardial failure, a condition associated with increases in the beta-adrenergic receptor agonist norepinephrine. In the same hearts, there was a significant decrease (approximately 50%) in the abundance of beta1-AR mRNA and protein. In DDT1-MF2 cells, where agonist-mediated destabilization of beta2-AR mRNA was first described, exposure to beta-AR agonist resulted in a significant increase in AUF1 mRNA and protein (approximately 100%). Conversely, agonist exposure significantly decreased (approximately 40%) beta2-adrenergic receptor mRNA abundance. Last, we demonstrate that AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3'-untranslated region of the human beta1-AR mRNA and that purified, recombinant p37AUF1 protein also binds to beta1-AR 3'-untranslated region mRNA.

Pretargeting vs. direct targeting of human betalox5 islet cells subcutaneously implanted in mice using an anti-human islet cell antibody.

PubMed

Liu, Guozheng; Dou, Shuping; Akalin, Ali; Rusckowski, Mary; Streeter, Philip R; Shultz, Leonard D; Greiner, Dale L

2012-07-01

We previously demonstrated MORF/cMORF pretargeting of human islets and betalox 5 cells (a human beta cell line) transplanted subcutaneously in mice with the anti-human islet antibody, HPi1. We now compare pretargeting with direct targeting in the beta cell transplant model to evaluate the degree to which target/non-target (T/NT) ratios may be improved by pretargeting. Specific binding of an anti-human islet antibody HPi1 to the beta cells transplanted subcutaneously in mice was examined against a negative control antibody. We then compared pretargeting by MORF-HPi1 plus 111In-labeled cMORF to direct targeting by 111In-labeled HPi1. HPi1 binding to betalox5 human cells in the transplant was shown by immunofluorescence. Normal organ 111In backgrounds by pretargeting were always lower, although target accumulations were similar. More importantly, the transplant to pancreas and liver ratios was, respectively, 26 and 10 by pretargeting as compared to 9 and 0.6 by direct targeting. Pretargeting greatly improves the T/NT ratios, and based on the estimated endocrine to exocrine ratio within a pancreas, pretargeting may be approaching the sensitivity required for successful imaging of human islets within this organ. Copyright © 2012 Elsevier Inc. All rights reserved.

Difference between beta1-adrenoceptor autoantibodies of human and animal origin-Limitations detecting beta1-adrenoceptor autoantibodies using peptide based ELISA technology.

PubMed

Wenzel, Katrin; Schulze-Rothe, Sarah; Müller, Johannes; Wallukat, Gerd; Haberland, Annekathrin

2018-01-01

Cell-based analytics for the detection of the beta1-adrenoceptor autoantibody (beta1-AAB) are functional, yet difficult to handle, and should be replaced by easily applicable, routine lab methods. Endeavors to develop solid-phase-based assays such as ELISA to exploit epitope moieties for trapping autoantibodies are ongoing. These solid-phase-based assays, however, are often unreliable when used with human patient material, in contrast to animal derived autoantibodies. We therefore tested an immunogen peptide-based ELISA for the detection of beta1-AAB, and compared commercially available goat antibodies against the 2nd extracellular loop of human beta1-adrenoceptor (ADRB1-AB) to autoantibodies enriched from patient material. The functionality of these autoantibodies was tested in a cell based assay for comparison and their structural appearance was investigated using 2D gel electrophoresis. The ELISA showed a limit of detection for ADRB1-AB of about 1.5 nmol antibody/L when spiked in human control serum and only about 25 nmol/L when spiked in species identical (goat) matrix material. When applied to samples of human origin, the ELISA failed to identify the specific beta1-AABs. A low concentration of beta1-AAB, together with structural inconsistency of the patient originated samples as seen from the 2D Gel appearance, might contribute to the failure of the peptide based ELISA technology to detect human beta1-AABs.

Normal T lymphocytes can express two different T cell receptor beta chains: implications for the mechanism of allelic exclusion

PubMed Central

1995-01-01

We have examined the extent of allelic exclusion at the T cell receptor (TCR) beta locus using monoclonal antibodies specific for V beta products. A small proportion (approximately 1%) of human peripheral blood T cells express two V beta as determined by flow cytometric analysis, isolation of representative clones, and sequencing of the corresponding V beta chains. Dual beta T cells are present in both the CD45R0+ and CD45R0- subset. These results indicate that dual beta expression is compatible with both central and peripheral selection. They also suggest that the substantial degree of TCR beta allelic exclusion is dependent only on asynchronous rearrangements at the beta locus, whereas the role of the pre-TCR is limited to signaling the presence of at least one functional beta protein. PMID:7699339

Cytotoxic components of Pereskia bleo (Kunth) DC. (Cactaceae) leaves.

PubMed

Malek, Sri Nurestri Abdul; Shin, Sim Kae; Wahab, Norhanom Abdul; Yaacob, Hashim

2009-05-06

Dihydroactinidiolide (1) and a mixture of sterols [campesterol (2), stigmasterol (3) and beta-sitosterol (4)], together with the previously isolated individual compounds beta-sitosterol (4), 2,4-di-tert-butylphenol (5), alpha-tocopherol (6), phytol (7) were isolated from the active ethyl acetate fraction of Pereskia bleo (Kunth) DC. (Cactaceae) leaves. Cytotoxic activities of the above mentioned compounds against five human carcinoma cell lines, namely the human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), human hormone-dependent breast carcinoma cell line (MCF7) and human lung carcinoma cell line (A549); and non-cancer human fibroblast cell line (MRC-5) were investigated. Compound 5 possessed very remarkable cytotoxic activity against KB cells, with an IC(50 )value of 0.81microg/mL. This is the first report on the cytotoxic activities of the compounds isolated from Pereskia bleo.

Human Beta Cells Produce and Release Serotonin to Inhibit Glucagon Secretion from Alpha Cells.

PubMed

Almaça, Joana; Molina, Judith; Menegaz, Danusa; Pronin, Alexey N; Tamayo, Alejandro; Slepak, Vladlen; Berggren, Per-Olof; Caicedo, Alejandro

2016-12-20

In the pancreatic islet, serotonin is an autocrine signal increasing beta cell mass during metabolic challenges such as those associated with pregnancy or high-fat diet. It is still unclear whether serotonin is relevant for regular islet physiology and hormone secretion. Here, we show that human beta cells produce and secrete serotonin when stimulated with increases in glucose concentration. Serotonin secretion from beta cells decreases cyclic AMP (cAMP) levels in neighboring alpha cells via 5-HT 1F receptors and inhibits glucagon secretion. Without serotonergic input, alpha cells lose their ability to regulate glucagon secretion in response to changes in glucose concentration, suggesting that diminished serotonergic control of alpha cells can cause glucose blindness and the uncontrolled glucagon secretion associated with diabetes. Supporting this model, pharmacological activation of 5-HT 1F receptors reduces glucagon secretion and has hypoglycemic effects in diabetic mice. Thus, modulation of serotonin signaling in the islet represents a drug intervention opportunity. Published by Elsevier Inc.

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 features result in preferential synthesis of type 1 chain structures.

Cross-talk between ERK MAP kinase and Smad signaling pathways enhances TGF-beta-dependent responses in human mesangial cells.

PubMed

Hayashida, Tomoko; Decaestecker, Mark; Schnaper, H William

2003-08-01

Transforming growth factor beta (TGF-beta) stimulates renal cell fibrogenesis by a poorly understood mechanism. Previously, we suggested a synergy between TGF-beta1 activated extracellular signal-regulated kinase (ERK) and Smad signaling in collagen production by human glomerular mesangial cells. In a heterologous DNA binding transcription assay, biochemical or dominant-negative ERK blockade reduced TGF-beta1 induced Smad3 activity. Total serine phosphorylation of Smad2/3, but not phosphorylation of the C-terminal SS(P)XS(P) motif, was decreased by pretreatment with the MEK/ERK inhibitors, PD98059 (10 microM) or U0126 (25 microM). This effect was not seen in the mouse mammary epithelial NMuMG cell line, indicating that ERK-dependent activation of Smad2/3 occurs only in certain cell types. TGF-beta stimulated phosphorylation of an expressed Smad3A construct, with a mutated C-terminal SS(P)XS(P) motif, was reduced by a MEK/ERK inhibitor. In contrast, MEK/ERK inhibition did not affect phosphorylation of a Smad3 construct mutated at consensus phosphorylation sites in the linker region (Smad3EPSM). Constitutively active MEK (caMEK) induced alpha2(I) collagen promoter activity, an effect blocked by co-transfected Smad3EPSM, but not Smad3A. The effects of caMEK and TGF-beta1 on collagen promoter activity were additive. These results indicate that ERK-dependent R-Smad linker region phosphorylation enhances collagen I synthesis and imply positive cross talk between the ERK and Smad pathways in human mesangial cells.

Advances in pancreatic islet monolayer culture on glass surfaces enable super-resolution microscopy and insights into beta cell ciliogenesis and proliferation

PubMed Central

Phelps, Edward A.; Cianciaruso, Chiara; Santo-Domingo, Jaime; Pasquier, Miriella; Galliverti, Gabriele; Piemonti, Lorenzo; Berishvili, Ekaterine; Burri, Olivier; Wiederkehr, Andreas; Hubbell, Jeffrey A.; Baekkeskov, Steinunn

2017-01-01

A robust and reproducible method for culturing monolayers of adherent and well-spread primary islet cells on glass coverslips is required for detailed imaging studies by super-resolution and live-cell microscopy. Guided by an observation that dispersed islet cells spread and adhere well on glass surfaces in neuronal co-culture and form a monolayer of connected cells, we demonstrate that in the absence of neurons, well-defined surface coatings combined with components of neuronal culture media collectively support robust attachment and growth of primary human or rat islet cells as monolayers on glass surfaces. The islet cell monolayer cultures on glass stably maintain distinct mono-hormonal insulin+, glucagon+, somatostatin+ and PP+ cells and glucose-responsive synchronized calcium signaling as well as expression of the transcription factors Pdx-1 and NKX-6.1 in beta cells. This technical advance enabled detailed observation of sub-cellular processes in primary human and rat beta cells by super-resolution microscopy. The protocol is envisaged to have broad applicability to sophisticated analyses of pancreatic islet cells that reveal new biological insights, as demonstrated by the identification of an in vitro protocol that markedly increases proliferation of primary beta cells and is associated with a reduction in ciliated, ostensibly proliferation-suppressed beta cells. PMID:28401888

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

Developmental regulation of DNA replication timing at the human beta globin locus.

PubMed

Simon, I; Tenzen, T; Mostoslavsky, R; Fibach, E; Lande, L; Milot, E; Gribnau, J; Grosveld, F; Fraser, P; Cedar, H

2001-11-01

The human beta globin locus replicates late in most cell types, but becomes early replicating in erythroid cells. Using FISH to map DNA replication timing around the endogenous beta globin locus and by applying a genetic approach in transgenic mice, we have demonstrated that both the late and early replication states are controlled by regulatory elements within the locus control region. These results also show that the pattern of replication timing is set up by mechanisms that work independently of gene transcription.

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.

Activation of antigen-specific cytotoxic T lymphocytes by beta 2-microglobulin or TAP1 gene disruption and the introduction of recipient-matched MHC class I gene in allogeneic embryonic stem cell-derived dendritic cells.

PubMed

Matsunaga, Yusuke; Fukuma, Daiki; Hirata, Shinya; Fukushima, Satoshi; Haruta, Miwa; Ikeda, Tokunori; Negishi, Izumi; Nishimura, Yasuharu; Senju, Satoru

2008-11-01

A method for the genetic modification of dendritic cells (DC) was previously established based on the in vitro differentiation of embryonic stem (ES) cells to DC (ES-DC). The unavailability of human ES cells genetically identical to the patients will be a problem in the future clinical application of this technology. This study attempted to establish a strategy to overcome this issue. The TAP1 or beta(2)-microglobulin (beta(2)m) gene was disrupted in 129 (H-2(b))-derived ES cells and then expression vectors for the H-2K(d) or beta(2)m-linked form of K(d) (beta2m-K(d)) were introduced, thus resulting in two types of genetically engineered ES-DC, TAP1(-/-)/K(d) ES-DC and beta(2)m(-/-)/beta(2)m-K(d) ES-DC. As intended, both of the transfectant ES-DC expressed K(d) but not the intrinsic H-2(b) haplotype-derived MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) and TAP1(-/-)/K(d) ES-DC were not recognized by pre-activated H-2(b)-reactive CTL and did not prime H-2(b) reactive CTL in vitro or in vivo. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC and TAP1(-/-)/K(d) ES-DC had a survival advantage in comparison to beta(2)m(+/-)/beta(2)m-K(d) ES-DC and TAP1(+/+)/K(d) ES-DC, when transferred into BALB/c mice. K(d)-restricted RSV-M2-derived peptide-loaded ES-DC could prime the epitope-specific CTL upon injection into the BALB/c mice, irrespective of the cell surface expression of intrinsic H-2(b) haplotype-encoded MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC were significantly more efficient in eliciting immunity against RSV M2 protein-expressing tumor cells than beta(2)m(+/-)/beta(2)m-K(d) ES-DC. The modification of the beta(2)m or TAP gene may therefore be an effective strategy to resolve the problem of HLA class I allele mismatch between human ES or induced pluripotent stem cells and the recipients to be treated.

Activation of Wnt signaling pathway by human papillomavirus E6 and E7 oncogenes in HPV16-positive oropharyngeal squamous carcinoma cells.

PubMed

Rampias, Theodore; Boutati, Eleni; Pectasides, Eirini; Sasaki, Clarence; Kountourakis, Panteleimon; Weinberger, Paul; Psyrri, Amanda

2010-03-01

We sought to determine the role of human papillomavirus (HPV) E6 and E7 oncogenes in nuclear beta-catenin accumulation, a hallmark of activated canonical Wnt signaling pathway. We used HPV16-positive oropharyngeal cancer cell lines 147T and 090, HPV-negative cell line 040T, and cervical cell lines SiHa (bearing integrated HPV16) and HeLa (bearing integrated HPV18) to measure the cytoplasmic and nuclear beta-catenin levels and the beta-catenin/Tcf transcriptional activity before and after E6/E7 gene silencing. Repression of HPV E6 and E7 genes induced a substantial reduction in nuclear beta-catenin levels. Luciferase assay showed that transcriptional activation of Tcf promoter by beta-catenin was lower after silencing. The protein levels of beta-catenin are tightly regulated by the ubiquitin/proteasome system. We therefore performed expression analysis of regulators of beta-catenin degradation and nuclear transport and showed that seven in absentia homologue (Siah-1) mRNA and protein levels were substantially upregulated after E6/E7 repression. Siah-1 protein promotes the degradation of beta-catenin through the ubiquitin/proteasome system. To determine whether Siah-1 is important for the proteasomal degradation of beta-catenin in HPV16-positive oropharyngeal cancer cells, we introduced a Siah-1 expression vector into 147T and 090 cells and found substantial reduction of endogenous beta-catenin in these cells. Thus, E6 and E7 are involved in beta-catenin nuclear accumulation and activation of Wnt signaling in HPV-induced cancers. In addition, we show the significance of the endogenous Siah-1-dependent ubiquitin/proteasome pathway for beta-catenin degradation and its regulation by E6/E7 viral oncoproteins in HPV16-positive oropharyngeal cancer cells.

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.

Effects of chondroitin sulfate and interleukin-1beta on human chondrocyte cultures exposed to pressurization: a biochemical and morphological study.

PubMed

Nerucci, F; Fioravanti, A; Cicero, M R; Collodel, G; Marcolongo, R

2000-07-01

Objective This study investigated the in vitro effects of chondroitin sulfate (CS) on human articular chondrocytes cultivated in the presence or in the absence of interleukin-1beta (IL-1beta) during 10 days of culture with and without pressurization cycles. Design The effects of CS (10 and 100 microg/ml) with and without IL-1beta were assessed in the culture medium of cells exposed to pressurization cycles in the form of synusoidal waves (minimum pressure 1 Mpa, maximum pressure 5 Mpa) and a frequency of 0.25 Hz for 3 h by immunoenzymatic method on microplates for the quantitative measurement of human proteoglycans (PG). On the 4th and 10th day of culture the cells were used for morphological analysis by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Results The presence of IL-1beta determines a significant decrease in PG concentration measured in the culture medium. When the cells are cultured in the presence of IL-1beta and CS, a statistically significant restoration of PG levels is observed. Under pressurization conditions, we observed that PG concentration in the medium of cells presents a significant increase at baseline conditions, in the presence of IL-1beta+CS10 and IL-1beta+CS100, but not with IL-1beta alone. The results concerning metabolic evaluation are confirmed by the morphologic findings obtained by TEM and SEM. Conclusions These in vitro studies confirm the protective role of CS, which counteracts the IL-1beta induced effects and they confirm the importance of pressure on chondrocyte metabolism and morphology.

Nr-CAM is a target gene of the beta-catenin/LEF-1 pathway in melanoma and colon cancer and its expression enhances motility and confers tumorigenesis.

PubMed

Conacci-Sorrell, Maralice E; Ben-Yedidia, Tamar; Shtutman, Michael; Feinstein, Elena; Einat, Paz; Ben-Ze'ev, Avri

2002-08-15

beta-catenin and plakoglobin (gamma-catenin) are homologous molecules involved in cell adhesion, linking cadherin receptors to the cytoskeleton. beta-catenin is also a key component of the Wnt pathway by being a coactivator of LEF/TCF transcription factors. To identify novel target genes induced by beta-catenin and/or plakoglobin, DNA microarray analysis was carried out with RNA from cells overexpressing either protein. This analysis revealed that Nr-CAM is the gene most extensively induced by both catenins. Overexpression of either beta-catenin or plakoglobin induced Nr-CAM in a variety of cell types and the LEF/TCF binding sites in the Nr-CAM promoter were required for its activation by catenins. Retroviral transduction of Nr-CAM into NIH3T3 cells stimulated cell growth, enhanced motility, induced transformation, and produced rapidly growing tumors in nude mice. Nr-CAM and LEF-1 expression was elevated in human colon cancer tissue and cell lines and in human malignant melanoma cell lines but not in melanocytes or normal colon tissue. Dominant negative LEF-1 decreased Nr-CAM expression and antibodies to Nr-CAM inhibited the motility of B16 melanoma cells. The results indicate that induction of Nr-CAM transcription by beta-catenin or plakoglobin plays a role in melanoma and colon cancer tumorigenesis, probably by promoting cell growth and motility.

Induction of TGF-beta1 and TGF-beta1-dependent predominant Th17 differentiation by group A streptococcal infection.

PubMed

Wang, Beinan; Dileepan, Thamotharampillai; Briscoe, Sarah; Hyland, Kendra A; Kang, Johnthomas; Khoruts, Alexander; Cleary, P Patrick

2010-03-30

Recurrent group A Streptococcus (GAS) tonsillitis and associated autoimmune diseases indicate that the immune response to this organism can be ineffective and pathological. TGF-beta1 is recognized as an essential signal for generation of regulatory T cells (Tregs) and T helper (Th) 17 cells. Here, the impact of TGF-beta1 induction on the T-cell response in mouse nasal-associated lymphoid tissue (NALT) following intranasal (i.n.) infections is investigated. ELISA and TGF-beta1-luciferase reporter assays indicated that persistent infection of mouse NALT with GAS sets the stage for TGF-beta1 and IL-6 production, signals required for promotion of a Th17 immune response. As predicted, IL-17, the Th17 signature cytokine, was induced in a TGF-beta1 signaling-dependent manner in single-cell suspensions of both human tonsils and NALT. Intracellular cytokine staining and flow cytometry demonstrated that CD4(+) IL-17(+) T cells are the dominant T cells induced in NALT by i.n. infections. Moreover, naive mice acquired the potential to clear GAS by adoptive transfer of CD4(+) T cells from immunized IL-17A(+)/(+) mice but not cells from IL-17A(-)/(-) mice. These experiments link specific induction of TGF-beta1 by a bacterial infection to an in vivo Th17 immune response and show that this cellular response is sufficient for protection against GAS. The association of a Th17 response with GAS infection reveals a potential mechanism for destructive autoimmune responses in humans.

Integrin alpha 3 beta 1 participates in the phagocytosis of extracellular matrix molecules by human breast cancer cells.

PubMed

Coopman, P J; Thomas, D M; Gehlsen, K R; Mueller, S C

1996-11-01

The mechanisms and receptors involved in phagocytosis by nonhematopoietic cells are not well understood. The involvement of the alpha 3 beta 1 integrin in phagocytosis of the extracellular matrix by human breast cancer cells was studied. The possible role of this integrin was suggested since alpha 3 and beta 1 but not alpha 2 subunits are concentrated at membrane sites where local degradation of fluorescently labeled gelatin occurs. Strikingly, anti-alpha 3 integrin monoclonal antibodies (mAbs) stimulate the phagocytosis of fluorescently labeled gelatin films, gelatin beads, and Matrigel films in a quantitative phagocytosis assay. Stimulation of the gelatin uptake by the anti-alpha 3 mAb is dose responsive, saturable, and time dependent. Antibodies against other integrin subunits have a lower stimulatory effect (anti-beta 1) or no significant effect (anti-alpha 2, -alpha 5, -alpha 6, and -alpha v) on gelatin phagocytosis. The synthetic HGD-6 human laminin peptide that binds specifically the alpha 3 beta 1 integrin, but not the scrambled HSGD-6 control peptide, also markedly stimulates gelatin uptake in a dose-responsive way. Furthermore, the stimulatory effects of the HGD-6 peptide and the anti-alpha 3 mAb are additive, suggesting that they might promote phagocytosis in different ways. Other laminin (YIGSR, IKVAV) and fibronectin (GRGDS) peptides have no effect on gelatin phagocytosis. Immunofluorescence shows that the alpha 3 and the beta 1, but not the alpha 2 integrin subunit, concentrate into patches on the cell surface after treatment with their respective mAbs. And, both gelatin and the alpha 3 beta 1 but not the alpha 2 beta 1 integrin are cointernalized and routed to acidic vesicles such as lysosomes. In conclusion, we demonstrate that human breast cancer cells locally degrade and phagocytose the extracellular matrix and show for the first time that the alpha 3 beta 1 integrin participates in this phagocytosis. We hypothesize that the anti-alpha 3 antibodies and the laminin peptide HGD-6 activate the alpha 3 beta 1 integrin, which results in a downstream signaling cascade stimulating phagocytosis.

hCG, the wonder of today's science

PubMed Central

2012-01-01

Background hCG is a wonder. Firstly, because hCG is such an extreme molecule. hCG is the most acidic glycoprotein containing the highest proportion of sugars. Secondly, hCG exists in 5 common forms. Finally, it has so many functions ranging from control of human pregnancy to human cancer. This review examines these molecules in detail. Content These 5 molecules, hCG, sulfated hCG, hyperglycosylated hCG, hCG free beta and hyperglycosylated free beta are produced by placental syncytiotrophoblast cells and pituitary gonadotrope cells (group 1), and by placental cytotrophoblast cells and human malignancies (group 2). Group 1 molecules are both hormones that act on the hCG/LH receptor. These molecules are central to human menstrual cycle and human pregnancy. Group 2 molecules are autocrines, that act by antagonizing a TGF beta receptor. These molecules are critical to all advanced malignancies. Conclusions The hCG groups are molecules critical to both the molecules of pregnancy or human life, and to the advancement of cancer, or human death. PMID:22455390

Smad7 induces tumorigenicity by blocking TGF-beta-induced growth inhibition and apoptosis.

PubMed

Halder, Sunil K; Beauchamp, R Daniel; Datta, Pran K

2005-07-01

Smad proteins play a key role in the intracellular signaling of the transforming growth factor beta (TGF-beta) superfamily of extracellular polypeptides that initiate signaling to regulate a wide variety of biological processes. The inhibitory Smad, Smad7, has been shown to function as intracellular antagonists of TGF-beta family signaling and is upregulated in several cancers. To determine the effect of Smad7-mediated blockade of TGF-beta signaling, we have stably expressed Smad7 in a TGF-beta-sensitive, well-differentiated, and non-tumorigenic cell line, FET, that was derived from human colon adenocarcinoma. Smad7 inhibits TGF-beta-induced transcriptional responses by blocking complex formation between Smad 2/3 and Smad4. While Smad7 has no effect on TGF-beta-induced activation of p38 MAPK and ERK, it blocks the phosphorylation of Akt by TGF-beta and enhances TGF-beta-induced phosphorylation of c-Jun. FET cells expressing Smad7 show anchorage-independent growth and enhance tumorigenicity in athymic nude mice. Smad7 blocks TGF-beta-induced growth inhibition by preventing TGF-beta-induced G1 arrest. Smad7 inhibits TGF-beta-mediated downregulation of c-Myc, CDK4, and Cyclin D1, and suppresses the expression of p21(Cip1). As a result, Smad7 inhibits TGF-beta-mediated downregulation of Rb phosphorylation. Furthermore, Smad7 inhibits the apoptosis of these cells. Together, Smad7 may increase the tumorigenicity of FET cells by blocking TGF-beta-induced growth inhibition and by inhibiting apoptosis. Thus, this study provides a mechanism by which a portion of human colorectal tumors may become refractory to tumor-suppressive actions of TGF-beta that might result in increased tumorigenicity.

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-beta1 plasma level is increased as in gestational diabetic mothers or patients with diabetes mellitus.

Generation of Beta Cells from Human Pluripotent Stem Cells: Potential for Regenerative Medicine

PubMed Central

Nostro, Maria Cristina; Keller, Gordon

2015-01-01

The loss of beta cells in Type I Diabetes ultimately leads to insulin dependence and major complications that are difficult to manage by insulin injections. Given the complications associated with long-term administration of insulin, cell-replacement therapy is now under consideration as an alternative treatment that may someday provide a cure for this disease. Over the past 10 years, islet transplantation trials have demonstrated that it is possible to replenish beta cell function in Type I Diabetes patients and, at least temporarily, eliminate their dependency on insulin. While not yet optimal, the success of these trials has provided proof-of-principle that cell replacement therapy is a viable option for treating diabetes. Limited access to donor islets has launched a search for alternative source of beta cells for cell therapy purposes and focused the efforts of many investigators on the challenge of deriving such cells from human embryonic and induced pluripotent stem cells. Over the past five years, significant advances have been made in understanding the signaling pathways that control lineage development from hPSCs and as a consequence, it is now possible to routinely generate human insulin producing cells from both hESCs and hiPSCs. While these achievements are impressive, significant challenges do still exist, as the majority of insulin producing cells generated under these conditions are polyhormonal and non functional, likely reflecting the emergence of the polyhormonal population that is known to arise in the early embryo during the phase of pancreatic development known as the ‘first transition’. Functional beta cells, which arise during the second phase or transition of pancreatic development have been generated from hPSCs, however they are detected only following transplantation of progenitor stage cells into immunocompromised mice. With this success, our challenge now is to define the pathways that control the development and maturation of this second transition population from hPSCs, and establish conditions for the generation of functional beta cells in vitro. PMID:22750147

Decreased hepatocyte membrane potential differences and GABAA-beta3 expression in human hepatocellular carcinoma.

PubMed

Minuk, Gerald Y; Zhang, Manna; Gong, Yuewen; Minuk, Leonard; Dienes, Hans; Pettigrew, Norman; Kew, Michael; Lipschitz, Jeremy; Sun, Dongfeng

2007-03-01

To determine whether hepatocyte membrane potential differences (PDs) are depolarized in human HCC and whether depolarization is associated with changes in GABAA receptor expression, hepatocyte PDs and gamma-aminobutyric acid (GABA)A receptor messenger RNA (mRNA) and protein expression were documented in HCC tissues via microelectrode impalement, real-time reverse-transcriptase polymerase chain reaction, and Western blot analysis, respectively. HCC tissues were significantly depolarized (-19.8+/-1.3 versus -25.9+/-3.2 mV, respectively [P<0.05]), and GABAA-beta3 expression was down-regulated (GABAA-beta3 mRNA and protein expression in HCC; 5,693+/-1,385 and 0.29+/-0.11 versus 11,046+/-4,979 copies/100 mg RNA and 0.62+/-0.16 optical density in adjacent tumor tissues, respectively [P=0.002 and P<0.0001, respectively]) when compared with adjacent nontumor tissues. To determine the physiological relevance of the down-regulation, human malignant hepatocytes deficient in GABAA-beta3 receptor expression (Huh-7 cells) were transfected with GABAA-beta3 complementary DNA (cDNA) or vector alone and injected into nu/nu nude mice (n=16-17 group). Tumors developed after a mean (+/-SD) of 51+/-6 days (range: 41-60 days) in 7/16 (44%) mice injected with vector-transfected cells and 70+/-12 days (range: 59-86 days) in 4/17 (24%) mice injected with GABAA-beta3 cDNA-transfected cells (P<0.005). The results of this study indicate that (1) human HCC tissues are depolarized compared with adjacent nontumor tissues, (2) hepatic GABAA-beta3 receptor expression is down-regulated in human HCC, and (3) restoration of GABAA-beta3 receptor expression results in attenuated in vivo tumor growth in nude mice.

Characterization of human DHRS4: an inducible short-chain dehydrogenase/reductase enzyme with 3beta-hydroxysteroid dehydrogenase activity.

PubMed

Matsunaga, Toshiyuki; Endo, Satoshi; Maeda, Satoshi; Ishikura, Shuhei; Tajima, Kazuo; Tanaka, Nobutada; Nakamura, Kazuo T; Imamura, Yorishige; Hara, Akira

2008-09-15

Human DHRS4 is a peroxisomal member of the short-chain dehydrogenase/reductase superfamily, but its enzymatic properties, except for displaying NADP(H)-dependent retinol dehydrogenase/reductase activity, are unknown. We show that the human enzyme, a tetramer composed of 27kDa subunits, is inactivated at low temperature without dissociation into subunits. The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Human DHRS4 reduced various aromatic ketones and alpha-dicarbonyl compounds including cytotoxic 9,10-phenanthrenequinone. The overexpression of the peroxisomal enzyme in cultured cells did not increase the cytotoxicity of 9,10-phenanthrenequinone. While its activity towards all-trans-retinal was low, human DHRS4 efficiently reduced 3-keto-C(19)/C(21)-steroids into 3beta-hydroxysteroids. The stereospecific conversion to 3beta-hydroxysteroids was observed in endothelial cells transfected with vectors expressing the enzyme. The mRNA for the enzyme was ubiquitously expressed in human tissues and several cancer cells, and the enzyme in HepG2 cells was induced by peroxisome-proliferator-activated receptor alpha ligands. The results suggest a novel mechanism of cold inactivation and role of the inducible human DHRS4 in 3beta-hydroxysteroid synthesis and xenobiotic carbonyl metabolism.

Immunogenicity of an interferon-beta1a product.

PubMed

Kauffman, M A; Sterin-Prync, A; Papouchado, M; González, E; Vidal, A J; Grossberg, S E; Chuppa, S; Odoriz, B; Vrech, C; Diez, R A; Ferro, H H

2011-01-01

In order to determine whether Blastoferon®, a biosimilar interferon (IFN)- beta 1a formulation, shares epitopes with other known IFN-beta products, a series of neutralization bioassays were performed with a set of well-characterized anti-IFN- beta monoclonal antibodies and human sera (World Health Organization Reference Reagents). The bioassay was the interferon-induced inhibition of virus cytopathic effect on human cells in culture (EMC virus and A-549 cells). Computer-calculated results were reported as Tenfold Reduction Units (TRU)/ml. To further assess Blastoferon® immunogenicity, in vivo production of anti-IFN beta antibodies was determined in sera of patients included in the pharmacovigilance plan of Blastoferon® by the level of IFN- beta 1a binding antibodies (by enzyme immunoassay -EIA) and neutralizing antibodies (in the Wish-VSV system). The highly characterized neutralizing monoclonal antibodies A1 and A5 that bind to specific regions of the IFN- beta molecule reacted positively with the three beta 1a IFNs: Blastoferon®, Rebif®, and the IFN- beta WHO Second International Standard 00/572. As expected, the non-neutralizing monoclonal antibodies B4 and B7 did not neutralize any of the IFN- beta preparations. The commercially available monoclonal antibody B-02 reacted essentially equally with Rebif® and Blastoferon®. The WHO Reference Reagent human serum anti-IFN- beta polyclonal antibody neutralized all the IFN- beta products, whereas the WHO Reference Reagent human serum anti-IFN-alpha polyclonal antibody G037-501-572 appropriately failed to react with any of the IFN- beta products. On the basis of in vitro reactivity with known, well-characterized monoclonal and polyclonal antibody preparations, Blastoferon® shares immunological determinants with other human interferon- beta products, especially IFN- beta 1a. In vivo antibodies were detected by EIA in 72.9% of 37 chronically treated multiple sclerosis patients, whereas neutralizing antibodies were found in 8.1% of them. Blastoferon® appears to have immunological characteristics comparable to other IFN- beta 1a products.

Nuclear import of glucokinase in pancreatic beta-cells is mediated by a nuclear localization signal and modulated by SUMOylation.

PubMed

Johansson, Bente Berg; Fjeld, Karianne; Solheim, Marie Holm; Shirakawa, Jun; Zhang, Enming; Keindl, Magdalena; Hu, Jiang; Lindqvist, Andreas; Døskeland, Anne; Mellgren, Gunnar; Flatmark, Torgeir; Njølstad, Pål Rasmus; Kulkarni, Rohit N; Wierup, Nils; Aukrust, Ingvild; Bjørkhaug, Lise

2017-10-15

The localization of glucokinase in pancreatic beta-cell nuclei is a controversial issue. Although previous reports suggest such a localization, the mechanism for its import has so far not been identified. Using immunofluorescence, subcellular fractionation and mass spectrometry, we present evidence in support of glucokinase localization in beta-cell nuclei of human and mouse pancreatic sections, as well as in human and mouse isolated islets, and murine MIN6 cells. We have identified a conserved, seven-residue nuclear localization signal ( 30 LKKVMRR 36 ) in the human enzyme. Substituting the residues KK 31,32 and RR 35,36 with AA led to a loss of its nuclear localization in transfected cells. Furthermore, our data indicates that SUMOylation of glucokinase modulates its nuclear import, while high glucose concentrations do not significantly alter the enzyme nuclear/cytosolic ratio. Thus, for the first time, we provide data in support of a nuclear import of glucokinase mediated by a redundant mechanism, involving a nuclear localization signal, and which is modulated by its SUMOylation. These findings add new knowledge to the functional role of glucokinase in the pancreatic beta-cell. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Popp, R.A.; Popp, D.M.; Johnson, F.M.

Mice homozygous for a spontaneous mutation, in which the ..beta..-major globin gene is deleted, have clinical symptoms of ..beta..-thalassemia. These mice have a hypocellular, hypochromic, microcytic anemia that becomes more severe with increasing age. The defective red cell morphology, decreased osmotic fragility of erythrocytes and shortened red cell life span found in ..beta..-thalassemic mice are similar to those observed in human ..beta..-thalassemia. Synthesis of ..beta..-globin is depressed but not as much as might be expected because the expression of the..beta..-minor globin gene is enhanced to encode two to three times more globin than in normal mice. Splenomegaly, an enlarged poolmore » of stem cells for erythropoiesis, and iron overloading occur in older mice. The fact that these mice remain moderately healthy makes them a very suitable animal model in which to develop and test alternative techniques of gene therapy that could be successfully applied to the treatment of human thalassemia. Homozygous ..beta..-thalassemic mice have large deposits of iron in their tissues, which might make these mice also useful for in vivo tests of the effectiveness and possible long-term side effects for newly developed iron chelators.« less

Generation of insulin-producing human mesenchymal stem cells using recombinant adeno-associated virus.

PubMed

Kim, Jeong Hwan; Park, Si-Nae; Suh, Hwal

2007-02-28

The purpose of current experiment is the generation of insulin-producing human mesenchymal stem cells as therapeutic source for the cure of type 1 diabetes. Type 1 diabetes is generally caused by insulin deficiency accompanied by the destruction of islet beta-cells. In various trials for the treatment of type 1 diabetes, cell-based gene therapy using stem cells is considered as one of the most useful candidate for the treatment. In this experiment, human mesenchymal stem cells were transduced with AAV which is containing furin-cleavable human preproinsulin gene to generate insulin-producing cells as surrogate beta-cells for the type 1 diabetes therapy. In the rAAV production procedure, rAAV was generated by transfection of AD293 cells. Human mesenchymal stems cells were transduced using rAAV with a various multiplicity of infection. Transduction of recombinant AAV was also tested using beta-galactosidse expression. Cell viability was determined by using MTT assay to evaluate the toxicity of the transduction procedure. Expression and production of Insulin were tested using reverse transcriptase-polymerase chain reaction and immunocytochemistry. Secretion of human insulin and C-peptide from the cells was assayed using enzyme-linked immunosorbent assay. Production of insulin and C-peptide from the test group represented a higher increase compared to the control group. In this study, we examined generation of insulin-producing cells from mesenchymal stem cells by genetic engineering for diabetes therapy. This work might be valuable to the field of tissue engineering for diabetes treatment.

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.

A Novel Di-Leucine Motif at the N-Terminus of Human Organic Solute Transporter Beta Is Essential for Protein Association and Membrane Localization.

PubMed

Xu, Shuhua; Soroka, Carol J; Sun, An-Qiang; Backos, Donald S; Mennone, Albert; Suchy, Frederick J; Boyer, James L

2016-01-01

The heteromeric membrane protein Organic Solute Transporter alpha/beta is the major bile acid efflux transporter in the intestine. Physical association of its alpha and beta subunits is essential for their polarized basolateral membrane localization and function in the transport of bile acids and other organic solutes. We identified a highly conserved acidic dileucine motif (-EL20L21EE) at the extracellular amino-tail of organic solute transporter beta from multiple species. To characterize the role of this protein interacting domain in the association of the human beta and alpha subunits and in membrane localization of the transporter, Leu20 and Leu21 on the amino-tail of human organic solute transporter beta were replaced with alanines by site-directed mutagenesis. Co-immunoprecipitation study in HEK293 cells demonstrated that substitution of the leucine residues with alanines prevented the interaction of the human beta mutant with the alpha subunit. Membrane biotinylation demonstrated that the LL/AA mutant eliminated membrane expression of both subunits. Computational-based modelling of human organic solute transporter beta suggested that the LL/AA mutation substantially alters both the structure and lipophilicity of the surface, thereby not only affecting the interaction with the alpha subunit but also possibly impacting the capacity of the beta subunit to traffick through the cell and interact with the membrane. In summary, our findings indicate that the dileucine motif in the extracellular N-terminal region of human organic solute transporter beta subunit plays a critical role in the association with the alpha subunit and in its polarized plasma membrane localization.

(+/-)-3-[4-(2-dimethylamino-1-methylethoxy)-phenyl]-1H-pyrazolo[3,4- B]pyridine-1-acetic acid (Y-25510) stimulates production of IL-1 beta and IL-6 at the level of messenger RNA expression in cultured human monocytes.

PubMed

Kusuhara, H; Komatsu, H; Hisadome, M; Ikeda, Y

1996-12-01

(+/-)-3-[4-(2-Dimethylamino-1-methylethoxy)phenyl]-1H-pyrazolo[3, 4-b]pyridine-1-acetic acid (Y-25510) stimulated the mRNA expression for interleukin-1 beta (IL-1 beta), and enhanced the expression induced by lipopolysaccharide (LPS) in cultured human peripheral blood mononuclear cells (PBMC) and THP-1 cells, a cell-line derived from human monocytic leukemia. Y-25510 also stimulated the mRNA expression for IL-6 in both types of the cells, however, the stimulation required the presence of LPS. In THP-1 cells, the stimulation of IL-1 beta mRNA expression by Y-25510 was suppressed by cycloheximide, an inhibitor of protein synthesis. This phenomenon indicates that the stimulation requires de norv protein synthesis. In contrast, the stimulation of mRNA expression for IL-6 by Y-25510 was not suppressed by cycloheximide but suppressed by N alpha-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of nuclear transcription factor-kappa B (NF-kappa B) activation, in the presence of LPS, suggesting that the stimulation requires NF-kappa activation. These results demonstrate that Y-25510 stimulates the mRNA expression for IL-1 beta and IL-6 by different mechanisms. Dexamethasone suppressed the LPS-induced expression of mRNA for IL-1 beta and IL-6 in THP-1 cells, whereas the drug never suppressed the mRNA expression for these cytokines in the presence of Y-25510. The result indicates that Y-25510 stimulates the mRNA expression for IL-1 beta and IL-6 by different mechanisms from those of LPS.

Smad3 phosphoisoform-mediated signaling during sporadic human colorectal carcinogenesis.

PubMed

Matsuzaki, K

2006-06-01

Transforming growth factor-beta (TGF-beta) signaling occurring during human colorectal carcinogenesis involves a shift in TGF-beta function, reducing the cytokine's antiproliferative effect, while increasing actions that promote invasion and metastasis. TGF-beta signaling involves phosphorylation of Smad3 at serine residues 208 and 213 in the linker region and serine residues 423 and 425 in the C-terminal region. Exogenous TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), changing unphosphorylated Smad3 to its phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker phosphorylated Smad3 (pSmad3L). Either pSmad3C or pSmad3L oligomerizes with Smad4, and translocates into nuclei. While the TbetaRI/pSmad3C pathway inhibits growth of normal epithelial cells in vivo, JNK/pSmad3L-mediated signaling promotes tumor cell invasion and extracellular matrix synthesis by activated mesenchymal cells. Furthermore, hepatocyte growth factor signaling interacts with TGF-beta to activate the JNK/pSmad3L pathway, accelerating nuclear transport of cytoplasmic pSmad3L. This reduces accessibility of unphosphorylated Smad3 to membrane-anchored TbetaRI, preventing Smad3C phosphorylation, pSmad3C-mediated transcription, and antiproliferative effects of TGF-beta on epithelial cells. As neoplasia progresses from normal colorectal epithelium through adenoma to invasive adenocarcinoma with distant metastasis, nuclear pSmad3L gradually increases while pSmad3C decreases. The shift from TbetaRI/pSmad3C-mediated to JNK/pSmad3L-mediated signaling is a major mechanism orchestrating a complex transition of TGF-beta signaling during sporadic human colorectal carcinogenesis. This review summarizes the recent understanding of Smad3 phosphoisoform-mediated signaling, particularly 'cross-talk' between Smad3 and JNK pathways that cooperatively promote oncogenic activities. Understanding of these actions should help to develop more effective therapy against human colorectal cancer, involving inhibition of JNK/pSmad3L pathway.

SKI knockdown inhibits human melanoma tumor growth in vivo.

PubMed

Chen, Dahu; Lin, Qiushi; Box, Neil; Roop, Dennis; Ishii, Shunsuke; Matsuzaki, Koichi; Fan, Tao; Hornyak, Thomas J; Reed, Jon A; Stavnezer, Ed; Timchenko, Nikolai A; Medrano, Estela E

2009-12-01

The SKI protein represses the TGF-beta tumor suppressor pathway by associating with the Smad transcription factors. SKI is upregulated in human malignant melanoma tumors in a disease-progression manner and its overexpression promotes proliferation and migration of melanoma cells in vitro. The mechanisms by which SKI antagonizes TGF-beta signaling in vivo have not been fully elucidated. Here we show that human melanoma cells in which endogenous SKI expression was knocked down by RNAi produced minimal orthotopic tumor xenograft nodules that displayed low mitotic rate and prominent apoptosis. These minute tumors exhibited critical signatures of active TGF-beta signaling including high levels of nuclear Smad3 and p21(Waf-1), which are not found in the parental melanomas. To understand how SKI promotes tumor growth we used gain- and loss-of-function approaches and found that simultaneously to blocking the TGF-beta-growth inhibitory pathway, SKI promotes the switch of Smad3 from tumor suppression to oncogenesis by favoring phosphorylations of the Smad3 linker region in melanoma cells but not in normal human melanocytes. In this context, SKI is required for preventing TGF-beta-mediated downregulation of the oncogenic protein c-MYC, and for inducing the plasminogen activator inhibitor-1, a mediator of tumor growth and angiogenesis. Together, the results indicate that SKI exploits multiple regulatory levels of the TGF-beta pathway and its deficiency restores TGF-beta tumor suppressor and apoptotic activities in spite of the likely presence of oncogenic mutations in melanoma tumors.

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

Kang, Kyungsu; Lee, Kyung-Mi; Yoo, Ji-Hye

Graphical abstract: Schematic diagram of the possible molecular mechanism underlying the inhibition of the Wnt/{beta}-catenin signaling pathway and the induction of G0/G1-phase arrest by gomisins J and N, derived from the fruits of S. chinensis, in HCT116 human colon cancer cells. Highlights: Black-Right-Pointing-Pointer Gomisins J and N inhibited Wnt/{beta}-catenin signaling pathway in HCT116 cells. Black-Right-Pointing-Pointer Gomisins J and N disrupted the binding of {beta}-catenin to specific DNA sequences, TBE. Black-Right-Pointing-Pointer Gomisins J and N inhibited the HCT116 cell proliferation through G0/G1 phase arrest. Black-Right-Pointing-Pointer Gomisins J and N inhibited the expression of Cyc D1, a Wnt/{beta}-catenin target gene. -- Abstract:more » Here, we report that gomisin J and gomisin N, dibenzocyclooctadiene type lignans isolated from Schisandra chinensis, inhibit Wnt/{beta}-catenin signaling in HCT116 cells. Gomisins J and N appear to inhibit Wnt/{beta}-catenin signaling by disrupting the interaction between {beta}-catenin and its specific target DNA sequences (TCF binding elements, TBE) rather than by altering the expression of the {beta}-catenin protein. Gomisins J and N inhibit HCT116 cell proliferation by arresting the cell cycle at the G0/G1 phase. The G0/G1 phase arrest induced by gomisins J and N appears to be caused by a decrease in the expression of Cyclin D1, a representative target gene of the Wnt/{beta}-catenin signaling pathway, as well as Cdk2, Cdk4, and E2F-1. Therefore, gomisins J and N, the novel Wnt/{beta}-catenin inhibitors discovered in this study, may serve as potential agents for the prevention and treatment of human colorectal cancers.« less

Visualization of astaxanthin localization in HT29 human colon adenocarcinoma cells by combined confocal resonance Raman and fluorescence microspectroscopy.

PubMed

Briviba, Karlis; Bornemann, Rainer; Lemmer, Ulrich

2006-11-01

Astaxanthin, a carotenoid found in plants and seafood, exhibits antiproliferative, antioxidant and anticarcinogenic properties. We show that astaxanthin delivered with tetrahydrofuran is effectively taken up by cultured colon adenocarcinoma cells and is localized mostly in the cytoplasm as detected by confocal resonance Raman and broad-band fluorescence microspectroscopy image analysis. Cells incubated with beta-carotene at the same concentration as astaxanthin (10 microM) showed about a 50-fold lower cellular amount of beta-carotene, as detected by HPLC. No detectable Raman signal of beta-carotene was found in cells, but a weak broad-band fluorescence signal of beta-carotene was observed. beta-Carotene, like astaxanthin, was localized mostly in the cytoplasm. The heterogeneity of astaxanthin and beta-carotene cellular distribution in cells of intestinal origin suggests that the possible defense against reactive molecules by carotenoids in these cells may also be heterogeneous.

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 information towards understanding the pathogenesis of proliferative vitreoretinal diseases.« less

Diverse roles of integrin receptors in articular cartilage.

PubMed

Shakibaei, M; Csaki, C; Mobasheri, A

2008-01-01

Integrins are heterodimeric integral membrane proteins made up of alpha and beta subunits. At least eighteen alpha and eight beta subunit genes have been described in mammals. Integrin family members are plasma membrane receptors involved in cell adhesion and active as intra- and extracellular signalling molecules in a variety of processes including embryogenesis, hemostasis, tissue repair, immune response and metastatic spread of tumour cells. Integrin beta 1 (beta1-integrin), the protein encoded by the ITGB1 gene (also known as CD29 and VLAB), is a multi-functional protein involved in cell-matrix adhesion, cell signalling, cellular defense, cell adhesion, protein binding, protein heterodimerisation and receptor-mediated activity. It is highly expressed in the human body (17.4 times higher than the average gene in the last updated revision of the human genome). The extracellular matrix (ECM) of articular cartilage is a unique environment. Interactions between chondrocytes and the ECM regulate many biological processes important to homeostasis and repair of articular cartilage, including cell attachment, growth, differentiation and survival. The beta1-integrin family of cell surface receptors appears to play a major role in mediating cell-matrix interactions that are important in regulating these fundamental processes. Chondrocyte mechanoreceptors have been proposed to incorporate beta1-integrins and mechanosensitive ion channels which link with key ECM, cytoskeletal and signalling proteins to maintain the chondrocyte phenotype, prevent chondrocyte apoptosis and regulate chondrocyte-specific gene expression. This review focuses on the expression and function of beta1-integrins in articular chondrocytes, its role in the unique biology of these cells and its distribution in cartilage.

Reduction of the immunostainable length of the hippocampal dentate granule cells' primary cilia in 3xAD-transgenic mice producing human A{beta}{sub 1-42} and tau

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

Chakravarthy, Balu, E-mail: Balu.Chakravarthy@nrc-cnrc.gc.ca; Gaudet, Chantal; Menard, Michel

2012-10-12

Highlights: Black-Right-Pointing-Pointer A{beta} and tau-induced neurofibrillary tangles play a key role in Alzheimer's disease. Black-Right-Pointing-Pointer A{beta}{sub 1-42} and mutant tau protein together reduce the primary cilium length. Black-Right-Pointing-Pointer This shortening likely reduces cilium-dependent neurogenesis and memory function. Black-Right-Pointing-Pointer This provides a model of an A{beta}/tau targeting of a neuronal signaling organelle. -- Abstract: The hippocampal dentate gyrus is one of the two sites of continuous neurogenesis in adult rodents and humans. Virtually all dentate granule cells have a single immobile cilium with a microtubule spine or axoneme covered with a specialized cell membrane loaded with receptors such as the somatostatinmore » receptor 3 (SSTR3), and the p75 neurotrophin receptor (p75{sup NTR}). The signals from these receptors have been reported to stimulate neuroprogenitor proliferation and the post-mitotic maturation of newborn granule cells into functioning granule cells. We have found that in 6-24-months-old triple transgenic Alzheimer's disease model mice (3xTg-AD) producing both A{beta}{sub 1-42} and the mutant human tau protein tau{sub P301L,} the dentate granule cells still had immunostainable SSTR3- and p75{sup NTR}-bearing cilia but they were only half the length of the immunostained cilia in the corresponding wild-type mice. However, the immunostainable length of the granule cell cilia was not reduced either in 2xTg-AD mice accumulating large amounts of A{beta}{sub 1-42} or in mice accumulating only a mutant human tau protein. Thus it appears that a combination of A{beta}{sub 1-42} and tau protein accumulation affects the levels of functionally important receptors in 3xTg-AD mice. These observations raise the important possibility that structural and functional changes in granule cell cilia might have a role in AD.« less

Beta cells transfer vesicles containing insulin to phagocytes for presentation to T cells.

PubMed

Vomund, Anthony N; Zinselmeyer, Bernd H; Hughes, Jing; Calderon, Boris; Valderrama, Carolina; Ferris, Stephen T; Wan, Xiaoxiao; Kanekura, Kohsuke; Carrero, Javier A; Urano, Fumihiko; Unanue, Emil R

2015-10-06

Beta cells from nondiabetic mice transfer secretory vesicles to phagocytic cells. The passage was shown in culture studies where the transfer was probed with CD4 T cells reactive to insulin peptides. Two sets of vesicles were transferred, one containing insulin and another containing catabolites of insulin. The passage required live beta cells in a close cell contact interaction with the phagocytes. It was increased by high glucose concentration and required mobilization of intracellular Ca2+. Live images of beta cell-phagocyte interactions documented the intimacy of the membrane contact and the passage of the granules. The passage was found in beta cells isolated from islets of young nonobese diabetic (NOD) mice and nondiabetic mice as well as from nondiabetic humans. Ultrastructural analysis showed intraislet phagocytes containing vesicles having the distinct morphology of dense-core granules. These findings document a process whereby the contents of secretory granules become available to the immune system.

Generation of glucose-sensitive insulin-secreting beta-like cells from human embryonic stem cells by incorporating a synthetic lineage-control network.

PubMed

Saxena, Pratik; Bojar, Daniel; Zulewski, Henryk; Fussenegger, Martin

2017-10-10

We previously reported novel technology to differentiate induced pluripotent stem cells (IPSCs) into glucose-sensitive insulin-secreting beta-like cells by engineering a synthetic lineage-control network regulated by the licensed food additive vanillic acid. This genetic network was able to program intricate expression dynamics of the key transcription factors Ngn3 (neurogenin 3, OFF-ON-OFF), Pdx1 (pancreatic and duodenal homeobox 1, ON-OFF-ON) and MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A, OFF-ON) to guide the differentiation of IPSC-derived pancreatic progenitor cells to beta-like cells. In the present study, we show for the first time that this network can also program the expression dynamics of Ngn3, Pdx1 and MafA in human embryonic stem cell (hESC)-derived pancreatic progenitor cells and drive differentiation of these cells into glucose-sensitive insulin-secreting beta-like cells. Therefore, synthetic lineage-control networks appear to be a robust methodology for differentiating pluripotent stem cells into somatic cell types for basic research and regenerative medicine. Copyright © 2017 Elsevier B.V. All rights reserved.

The species origin of the cellular microenvironment influences markers of beta cell fate and function in EndoC-βH1 cells.

PubMed

Jeffery, N; Richardson, S; Beall, C; Harries, L W

2017-12-15

Interaction between islet cell subtypes and the extracellular matrix influences beta-cell function in mammals. The tissue architecture of rodent islets is very different to that of human islets; cell-to-cell communication and interaction with the extracellular matrix may vary between species. In this work, we have compared the responses of the human EndoC-βH1 cell line to non-human and human-derived growth matrices in terms of growth morphology, gene expression and glucose-stimulated insulin secretion (GSIS). EndoC-βH1 cells demonstrated a greater tendency to form cell clusters when cultured in a human microenvironment and exhibited reduced alpha cell markers at the mRNA level; mean expression difference - 0.23 and - 0.51; p = 0.009 and 0.002 for the Aristaless-related homeobox (ARX) and Glucagon (GCG) genes respectively. No differences were noted in the protein expression of mature beta cell markers such as Pdx1 and NeuroD1 were noted in EndoC-βH1 cells grown in a human microenvironment but cells were however more sensitive to glucose (4.3-fold increase in insulin secretion following glucose challenge compared with a 1.9-fold increase in cells grown in a non-human microenvironment; p = 0.0003). Our data suggests that the tissue origin of the cellular microenvironment has effects on the function of EndoC-βH1 cells in vitro, and the use of a more human-like culture microenvironment may bring benefits in terms of increased physiological relevance. Copyright © 2017 Elsevier Inc. All rights reserved.

Id-1 promotes TGF-{beta}1-induced cell motility through HSP27 activation and disassembly of adherens junction in prostate epithelial cells

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

Di Kaijun; Wong, Y.C.; Wang Xianghong

Id-1 (inhibitor of differentiation or DNA binding-1) has been positively associated with cell proliferation, cell cycle progression, and invasiveness during tumorigenesis. In addition, Id-1 has been shown to modulate cellular sensitivity to TGF-{beta}1 (transforming growth factor {beta}1). Here we demonstrate a novel role of Id-1 in promoting TGF-{beta}1-induced cell motility in a non-malignant prostate epithelial cell line, NPTX. We found that Id-1 promoted F-actin stress fiber formation in response to TGF-{beta}1, which was associated with increased cell-substrate adhesion and cell migration in NPTX cells. In addition, this positive effect of Id-1 on TGF-{beta}1-induced cell motility was mediated through activation ofmore » MEK-ERK signaling pathway and subsequent phosphorylation of HSP27 (heat shock protein 27). Furthermore, Id-1 disrupted the adherens junction complex in TGF-{beta}1-treated cells through down-regulation of E-cadherin, redistribution of {beta}-catenin, along with up-regulation of N-cadherin. These lines of evidence reveal a novel tumorigenic role of Id-1 through reorganization of actin cytoskeleton and disassembly of cell-cell adhesion in response to TGF-{beta}1 in human prostate epithelial cells, and suggest that intracellular Id-1 levels might be a determining factor for switching TGF-{beta}1 from a growth inhibitor to a tumor promoter during prostate carcinogenesis.« less

Tangeretin suppresses IL-1beta-induced cyclooxygenase (COX)-2 expression through inhibition of p38 MAPK, JNK, and AKT activation in human lung carcinoma cells.

PubMed

Chen, Kuan-Hung; Weng, Meng-Shih; Lin, Jen-Kun

2007-01-15

Tangeretin (5,6,7,8,4'-pentamethoxyflavone) is a polymethoxylated flavonoid concentrated in the peel of citrus fruits. Recent studies have shown that tangeretin exhibits anti-proliferative, anti-invasive, anti-metastatic, and antioxidant activities. However, the anti-inflammatory properties of tangeretin are unclear. In this study, we examine the effects of tangeretin and its structure-related compound, nobiletin, on the expression of cyclooxygenases-2 (COX-2) in human lung epithelial carcinoma cells, A549, and human non-small cell lung carcinoma cells, H1299. Tangeretin exerts a much better inhibitory activity than nobiletin against IL-1beta-induced production of COX-2 in A549 cells, and it effectively represses the constitutively expressed COX-2 in H1299. RT-PCR was used to investigate the transcriptional inhibition of COX-2 by tangeretin. COX-2 mRNA was rapidly induced by IL-1beta in 3h and markedly suppressed by tangeretin. IL-1beta-induced the activation of ERK, p38 MAPK, JNK, and AKT in A549 cells. COX-2 expression in response to IL-1beta was attenuated by pretreatment with SB203580, SP600125, and LY294002, but not with PD98059, suggesting the involvement of p38 MAPK, JNK, and PI3K in this response. Pretreatment of cells with tangeretin inhibited IL-1beta-induced p38 MAPK, JNK, and AKT phosphorylation and the downstream activation of NF-kappaB. These results may reveal that the tangeretin inhibition of IL-1beta-induced COX-2 expression in A549 cells is, at least in part, mediated through suppression of NF-kappaB transcription factor as well as through suppression of the signaling proteins of p38 MAPK, JNK, and PI3K, but not of ERK.

Expression of HSP27, HSP72 and MRP proteins in in vitro co-culture of colon tumour cell spheroids with normal cells after incubation with rhTGF- beta1 and/or CPT-11.

PubMed

Paduch, Roman; Jakubowicz-Gil, Joanna; Kandefer-Szerszen, Martyna

2009-12-01

We studied the expression of inducible heat shock protein (HSP27, HSP72) and multidrug-resistance protein (MRP) in co-cultures of human colon carcinoma cell spheroids obtained from different grades of tumour with normal human colon epithelium, myofibroblast and endothelial cell monolayers. We also measured the influence of recombinant human transforming growth factor beta1 (rhTGF-beta1) and camptothecin (CPT-11), added as single agents or in combination, on the levels of the HSPs, MRP, interleukin (IL)-6 and nitric oxide (NO). An immunoblotting analysis with densitometry showed that rhTGF-beta1 and/or CPT-11 increased HSP27, HSP72 and MRP expression in tumour cells and myofibroblasts, as well as in co-cultures compared with appropriate controls. By contrast, in colonic epithelium, inhibition of HSPs and MRP was comparable with that of the control. In endothelial cells, HSP72 was undetectable. Direct interaction of colon tumour spheroids with normal myofibroblasts caused a significant, tumour-grade dependent increase in IL-6 production. Production of IL-6 was significantly lowered by rhTGF-beta1 and/or CPT-11. Tumour cell spheroids cultivated alone produced larger amounts of NO than normal cells. In co-culture, the level of the radical decreased compared with the sum of NO produced by the monocultures of the two types of cells. rhTGF-beta1 and/or CPT-11 decreased NO production both in tumour and normal cell monocultures and their co-cultures. In conclusion, direct interactions between tumour and normal cells influence the expression of HSP27, HSP72 and MRP, and alter IL-6 and NO production. rhTGF-beta1 and/or CPT-11 may potentate resistance to chemotherapy by increasing HSP and MRP expression but, on the other hand, they may limit tumour cell spread by decreasing the level of some soluble mediators of inflammation (IL-6 and NO).

Preliminary in vivo efficacy studies of a recombinant rhesus anti-alpha(4)beta(7) monoclonal antibody.

PubMed

Pereira, L E; Onlamoon, N; Wang, X; Wang, R; Li, J; Reimann, K A; Villinger, F; Pattanapanyasat, K; Mori, K; Ansari, A A

2009-01-01

Recent findings established that primary targets of HIV/SIV are lymphoid cells within the gastrointestinal (GI) tract. Focus has therefore shifted to T-cells expressing alpha(4)beta(7) integrin which facilitates trafficking to the GI tract via binding to MAdCAM-1. Approaches to better understand the role of alpha(4)beta(7)+ T-cells in HIV/SIV pathogenesis include their depletion or blockade of their synthesis, binding and/or homing capabilities in vivo. Such studies can ideally be conducted in rhesus macaques (RM), the non-human primate model of AIDS. Characterization of alpha(4)beta(7) expression on cell lineages in RM blood and GI tissues reveal low densities of expression by NK cells, B-cells, naïve and TEM (effector memory) T-cells. High densities were observed on TCM (central memory) T-cells. Intravenous administration of a single 50mg/kg dose of recombinant rhesus alpha(4)beta(7) antibody resulted in significant initial decline of alpha(4)beta(7)+ lymphocytes and sustained coating of the alpha(4)beta(7) receptor in both the periphery and GI tissues.

Generating hypoimmunogenic human embryonic stem cells by the disruption of beta 2-microglobulin.

PubMed

Lu, Pengfei; Chen, Jijun; He, Lixiazi; Ren, Jiangtao; Chen, Haide; Rao, Lingjun; Zhuang, Qinggang; Li, Hui; Li, Lei; Bao, Lei; He, Ji; Zhang, Wei; Zhu, Faming; Cui, Chun; Xiao, Lei

2013-12-01

Immune rejection hinders the application of human embryonic stem cells (hESCs) in transplantation therapy. Human leukocyte antigens (HLAs) on the cell surface are the major cause of graft rejection. In this study, we generated HLA class I-deficient hESCs via disruption of beta 2-microglobulin (β2m), the light chain of HLA Class I. We found that HLA class I proteins were not present on the cell surface of β2m-null hESCs. These cells showed the same pluripotency as wildtype hESCs and demonstrated hypoimmunogenicity. Thus, HLA class I-deficient hESCs might serve as an unlimited cell source for the generation of universally compatible "off-the-shelf" cell grafts, tissues or organs in the future.

Studies of insulin secretory responses and of arachidonic acid incorporation into phospholipids of stably transfected insulinoma cells that overexpress group VIA phospholipase A2 (iPLA2beta ) indicate a signaling rather than a housekeeping role for iPLA2beta.

PubMed

Ma, Z; Ramanadham, S; Wohltmann, M; Bohrer, A; Hsu, F F; Turk, J

2001-04-20

A cytosolic 84-kDa group VIA phospholipase A(2) (iPLA(2)beta) that does not require Ca(2+) for catalysis has been cloned from several sources, including rat and human pancreatic islet beta-cells and murine P388D1 cells. Many potential iPLA(2)beta functions have been proposed, including a signaling role in beta-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA(2)beta function rest in part on effects of inhibiting iPLA(2)beta activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A(2). Manipulation of iPLA(2)beta expression by molecular biologic means is an alternative approach to study iPLA(2)beta functions, and we have used a retroviral construct containing iPLA(2)beta cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA(2)beta. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA(2)beta-overexpressing lines exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents, and BEL substantially attenuates stimulated secretion. Electrospray ionization mass spectrometric analyses of arachidonic acid incorporation into INS-1 cell PC indicate that neither overexpression nor inhibition of iPLA(2)beta affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA(2)beta indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA(2)beta associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA(2)beta in insulin-secreting beta-cells.

TGF-beta1 stimulates expression of the aromatase (CYP19) gene in human osteoblast-like cells and THP-1 cells.

PubMed

Shozu, M; Zhao, Y; Simpson, E R

2000-02-25

Recent evidence has shown that bone is not only a target of estrogen action but also a source of local estrogen production. Bone cells such as osteoblasts express aromatase (P450arom) and the expression of P450arom in osteoblasts is positively regulated in a tissue specific fashion, as in the case of other tissues which express P450arom. To clarify the physiological factors regulating expression of P450arom in bone, we tested TGF-beta1 using osteoblast-like cells obtained from human fetuses as well as THP-1 cells. TGF-beta1 increased IL-1beta+DEX- induced aromatase activity in osteoblast-like cells, while it inhibited activity in skin fibroblasts. Similar enhancement of aromatase activity by TGF-beta1 was found in DEX-stimulated THP-1 cells and this cell line was used for further experiments. In THP-1 cells, TGF-beta1 enhanced DEX-induced aromatase activity almost linearly by 12 h and thereafter. Increased levels of P450arom transcripts were also demonstrated by RT-PCR at 3 h of TGF-beta1 treatment and thereafter. Cyclohexamide abolished enhancement of activity but did not inhibit the accumulation of P450arom transcripts induced by TGF-beta1. Increase in P450arom expression by TGF-beta1 was attributable to expression driven by promoter I.4. TGF-beta1 did not change the half life of P450arom transcripts. To identify the cis-acting elements responsible for TGF-beta1 action on aromatase expression, transient transfection assays were performed using a series of deletion constructs for promoter I.4 (P450-I.4/Luc). Two constructs (-410/+14 and-340/+14) that contain a functional glucocorticoid response element (GRE) and downstream sequence showed significant increase of luciferase activity in response to TGF-beta1. Deletion and mutation of the GRE in P450-I.4/Luc (-340/+14) abolished the TGF-beta1. The luciferase activity of a (GRE)(1)-SV40/Luc construct was also stimulated by TGF-beta1. These results indicate that TGF-beta1 increases the expression of P450arom at the level of transcription through promoter I.4, at least in part via an enhancement of transactivation activity of the GR in THP-1 cells. TGF-beta1 is suggested to be one of the physiological up-regulatory factors of bone aromatase.

Differentiation stage-specific regulation of primitive human hematopoietic progenitor cycling by exogenous and endogenous inhibitors in an in vivo model.

PubMed

Cashman, J D; Clark-Lewis, I; Eaves, A C; Eaves, C J

1999-12-01

Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice transplanted with human cord blood or adult marrow cells and injected 6 weeks posttransplant with 2 daily doses of transforming growth factor-beta(1) (TGF-beta(1)), monocyte chemoattractant protein-1 (MCP-1), or a nonaggregating form of macrophage inflammatory protein-1alpha (MIP-1alpha) showed unique patterns of inhibition of human progenitor proliferation 1 day later. TGF-beta(1) was active on long-term culture initiating cells (LTC-IC) and on primitive erythroid and granulopoietic colony-forming cells (HPP-CFC), but had no effect on mature CFC. MCP-1 inhibited the cycling of both types of HPP-CFC but not LTC-IC. MIP-1alpha did not inhibit either LTC-IC or granulopoietic HPP-CFC but was active on erythroid HPP-CFC and mature granulopoietic CFC. All of these responses were independent of the source of human cells transplanted. LTC-IC of either human cord blood or adult marrow origin continue to proliferate in NOD/SCID mice for many weeks, although the turnover of all types of human CFC in mice transplanted with adult human marrow (but not cord blood) is downregulated after 6 weeks. Interestingly, administration of either MIP-1beta, an antagonist of both MIP-1alpha and MCP-1 or MCP-1(9-76), an antagonist of MCP-1 (and MCP-2 and MCP-3), into mice in which human marrow-derived CFC had become quiescent, caused the rapid reactivation of these progenitors in vivo. These results provide the first definition of stage-specific inhibitors of human hematopoietic progenitor cell cycling in vivo. In addition they show that endogenous chemokines can contribute to late graft failure, which can be reversed by the administration of specific antagonists.

An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells

PubMed Central

Massumi, Mohammad; Pourasgari, Farzaneh; Nalla, Amarnadh; Batchuluun, Battsetseg; Nagy, Kristina; Neely, Eric; Gull, Rida; Nagy, Andras; Wheeler, Michael B.

2016-01-01

The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25–30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL, and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion, targeting selected signaling pathways for 25–30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs, small molecules or genes that may have potential to influence beta-cell function. PMID:27755557

Steroids from the leaves of Chinese Melia azedarach and their cytotoxic effects on human cancer cell lines.

PubMed

Wu, Shi-Biao; Ji, Yan-Ping; Zhu, Jing-Jing; Zhao, Yun; Xia, Gang; Hu, Ying-He; Hu, Jin-Feng

2009-09-01

Three new (1-3) and several known (4-6) steroids were isolated from the leaves of Chinese Melia azedarach. The structures of the new compounds were elucidated by means of spectroscopic methods including 2D NMR techniques and mass spectrometry to be (20S)-5,24(28)-ergostadiene-3beta,7alpha,16beta,20-tetrol (1), (20S)-5-ergostene-3beta,7alpha,16beta,20-tetrol (2), and 2alpha,3beta-dihydro-5-pregnen-16-one (3). The cytotoxicities of the isolated compounds against three human cancer cell lines (A549, H460, U251) were evaluated; only compounds 1, 2, and (20S)-5-stigmastene-3beta,7alpha,20-triol (4) were found to show significant cyctotoxic effects with IC(50)s from 12.0 to 30.1 microg/mL.

Inefficiency in GM2 ganglioside elimination by human lysosomal beta-hexosaminidase beta-subunit gene transfer to fibroblastic cell line derived from Sandhoff disease model mice.

PubMed

Itakura, Tomohiro; Kuroki, Aya; Ishibashi, Yasuhiro; Tsuji, Daisuke; Kawashita, Eri; Higashine, Yukari; Sakuraba, Hitoshi; Yamanaka, Shoji; Itoh, Kohji

2006-08-01

Sandhoff disease (SD) is an autosomal recessive GM2 gangliosidosis caused by the defect of lysosomal beta-hexosaminidase (Hex) beta-subunit gene associated with neurosomatic manifestations. Therapeutic effects of Hex subunit gene transduction have been examined on Sandhoff disease model mice (SD mice) produced by the allelic disruption of Hexb gene encoding the murine beta-subunit. We demonstrate here that elimination of GM2 ganglioside (GM2) accumulated in the fibroblastic cell line derived from SD mice (FSD) did not occur when the HEXB gene only was transfected. In contrast, a significant increase in the HexB (betabeta homodimer) activity toward neutral substrates, including GA2 (asialo-GM2) and oligosaccharides carrying the terminal N-acetylglucosamine residues at their non-reducing ends (GlcNAc-oligosaccharides) was observed. Immunoblotting with anti-human HexA (alphabeta heterodimer) serum after native polyacrylamide gel electrophoresis (Native-PAGE) revealed that the human HEXB gene product could hardly form the chimeric HexA through associating with the murine alpha-subunit. However, co-introduction of the HEXA encoding the human alpha-subunit and HEXB genes caused significant corrective effect on the GM2 degradation by producing the human HexA. These results indicate that the recombinant human HexA could interspeciesly associate with the murine GM2 activator protein to degrade GM2 accumulated in the FSD cells. Thus, therapeutic effects of the recombinant human HexA isozyme but not human HEXB gene product could be evaluated by using the SD mice.

Improved human islet preparations using Glucocorticoid and Exendin-4

PubMed Central

Miki, Atsushi.; Ricordi, Camillo.; Yamamoto, Toshiyuki.; Sakuma, Yasunaru.; Misawa, Ryosuke.; Mita, Atsuyoshi.; Inverardi, Luca.; Alejandro, Rodolfo; Ichii, Hirohito.

2014-01-01

Objectives The effects of Glucocorticoid during culture on human islet cells have been controversial. Exendin-4 (EX) enhances the insulin secretion and significantly improves clinical outcomes in islet cell transplantation. In this study, we examined the effects of Glucocorticoids and exendin-4 on human islet cells during pre-transplant culture. Methods Methylprednisolone (MP) and/or EX were added to the standard culture medium for clinical islet cell transplantation. Islets were cultured for 24 hours with three different conditions (Control: no additives, MP alone, MP+EX). Beta cell fractional viability, cellular composition, multiple cytokine/chemokine production, multiple phosphorylation proteins and glucose induced insulin secretion were evaluated. Results Viable beta cell survival in MP and MP+EX group was significantly higher than in the control group. EX prevented MP induced reduction of insulin secretion. MP supplementation to the culture medium decreased cytokine and chemokine production. Moreover, Erk1/2 phosphorylation was significantly increased by MP and MP+EX. Conclusions Glucocorticoid supplementation into culture media significantly decreased the cytokine/chemokine production and increased the Erk1/2 phosphorylation, resulting in the improvement of human beta cell survival. In addition, EX maintained the insulin secretion suppressed by MP. The supplementation of MP and EX together could be a useful strategy to create suitable human islets for transplantation. PMID:25036907

Translation and assembly of HLA-DR antigens in Xenopus oocytes injected with mRNA from a human B-cell line.

PubMed Central

Long, E O; Gross, N; Wake, C T; Mach, J P; Carrel, S; Accolla, R; Mach, B

1982-01-01

HLA-DR antigens are polymorphic cell surface glycoproteins, expressed primarily in B lymphocytes and macrophages, which are thought to play an important role in the immune response. Two polypeptide chains, alpha and beta, are associated at the cell surface, and a third chain associates with alpha and beta intracellularly. RNA isolated from the human B-cell line Raji was injected in Xenopus laevis oocytes. Immunoprecipitates of translation products with several monoclonal antibodies revealed the presence of HLA-DR antigens similar to those synthesized in Raji cells. One monoclonal antibody was able to bind the beta chain after dissociation of the three polypeptide chains with detergent. The presence of all three chains was confirmed by two-dimensional gel electrophoresis. The glycosylation pattern of the three chains was identical to that observed in vivo, as evidenced in studies using tunicamycin, an inhibitor of N-linked glycosylation. The presence of alpha chains assembled with beta chains in equimolar ratio was further demonstrated by amino-terminal sequencing. An RNA fraction enriched for the three mRNAs, encoding alpha, beta, and intracellular chains, was isolated. This translation-assembly system and the availability of monoclonal antibodies make it possible to assay for mRNA encoding specific molecules among the multiple human Ia-like antigens. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6821356

Effects of preventing O-glycosylation on the secretion of human chorionic gonadotropin in Chinese hamster ovary cells

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

Matzuk, M.M.; Krieger, M.; Corless, C.L.

1987-09-01

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common ..cap alpha.. subunit but differ in their hormone-specific ..beta..-subunits. The ..beta.. subunit of hCG (hCG..beta..) is unique among the ..beta.. subunits in that it contains four mucin-like O-linked oligosaccharides attached to a carboxyl-terminal extension. To study the effects of O-glycosylation on the secretion and assembly of hCG, expression vectors containing either hCG..beta.. gene alone or together with the hCG..cap alpha.. gene were transfected into a mutant Chinese hamster ovary cell line, 1d1D, which exhibits a reversible defect in O-glycosylation. The results revealmore » that hCG..beta.. can be secreted normally in the absence of its O-linked oligosaccharides. hCG..beta.. devoid of O-linked carbohydrate can also combine efficiently with hCG..cap alpha.. and be secreted as an intact dimer. The authors conclude that in Chinese hamster ovary cells, the hCG..beta.. O-linked chains play no role in the assembly and secretion of hCG. The normal and O-linked oligosaccharide-deficient forms of hCG secreted by these cells should prove useful in examining the role of O-linked chains on the biological function of hCG.« less

Specific knockdown of Oct4 and beta2-microglobulin expression by RNA interference in human embryonic stem cells and embryonic carcinoma cells.

PubMed

Matin, Maryam M; Walsh, James R; Gokhale, Paul J; Draper, Jonathan S; Bahrami, Ahmad R; Morton, Ian; Moore, Harry D; Andrews, Peter W

2004-01-01

We have used RNA interference (RNAi) to downregulate beta2-microglobulin and Oct4 in human embryonal carcinoma (hEC) cells and embryonic stem (hES) cells, demonstrating that RNAi is an effective tool for regulating specific gene activity in these human stem cells. The knockdown of Oct4 but not beta2-microglobulin expression in both EC and ES cells resulted in their differentiation, as indicated by a marked change in morphology, growth rate, and surface antigen phenotype, with respect to SSEA1, SSEA3, and TRA-1-60 expression. Expression of hCG and Gcm1 was also induced following knockdown of Oct4 expression, in both 2102Ep hEC cells and in H7 and H14 hES cells, consistent with the conclusion that, as in the mouse, Oct4 is required to maintain the undifferentiated stem cell state, and that differentiation to trophectoderm occurs in its absence. NTERA2 hEC cells also differentiated, but not to trophectoderm, suggesting their equivalence to a later stage of embryogenesis than other hEC and hES cells.

Novel function of STAT1beta in B cells: induction of cell death by a mechanism different from that of STAT1alpha.

PubMed

Najjar, Imen; Schischmanoff, Pierre Olivier; Baran-Marszak, Fanny; Deglesne, Pierre-Antoine; Youlyouz-Marfak, Ibtissam; Pampin, Mathieu; Feuillard, Jean; Bornkamm, Georg W; Chelbi-Alix, Mounira K; Fagard, Remi

2008-12-01

Alternate splicing of STAT1 produces two isoforms: alpha, known as the active form, and beta, previously shown to act as a dominant-negative factor. Most studies have dealt with STAT1alpha, showing its involvement in cell growth control and cell death. To examine the specific function of either isoform in cell death, a naturally STAT1-deficient human B cell line was transfected to express STAT1alpha or STAT1beta. STAT1alpha, expressed alone, enhanced cell death, potentiated the fludarabine-induced apoptosis, and enhanced the nuclear location, the phosphorylation, and the transcriptional activity of p53. Unexpectedly, STAT1beta, expressed alone, induced cell death through a mechanism that was independent of the nuclear function of p53. Indeed, in STAT1beta-expressing B cells, p53 was strictly cytoplasmic where it formed clusters, and there was no induction of the transcriptional activity of p53. These data reveal a novel role of STAT1beta in programmed cell death, which is independent of p53.

Role for granulocyte colony-stimulating factor in the generation of human T regulatory type 1 cells.

PubMed

Rutella, Sergio; Pierelli, Luca; Bonanno, Giuseppina; Sica, Simona; Ameglio, Franco; Capoluongo, Ettore; Mariotti, Andrea; Scambia, Giovanni; d'Onofrio, Giuseppe; Leone, Giuseppe

2002-10-01

Granulocyte colony-stimulating factor (G-CSF) may affect T-cell homeostasis by multiple mechanisms, inducing polarization of cytokine secretion, inhibition of T-cell proliferation, and enhancement of T-cell apoptosis. We analyzed the production of interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-beta1) by T cells from healthy volunteer donors treated with recombinant human G-CSF. Highly purified CD4(+) T cells obtained before and after G-CSF administration (pre-G and post-G, respectively) were activated using the allogeneic mixed leukocyte reaction. Post-G CD4(+) T cells produced high levels of IL-10 but undetectable levels of IL-2 and IL-4, whereas the level of TGF-beta1 release was comparable to that of pre-G CD4(+) T cells. Notably, post-G CD4(+) T cells proliferated poorly in response to alloantigens and to recall antigens and suppressed the proliferation of autologous CD4(+) T cells in a cell contact-independent and an antigen-nonspecific manner. TGF-beta1 and IL-10 were not dispensable for post-G CD4(+) T cells to mediate suppression, as shown by neutralization studies. Compared with pre-G CD4(+) T cells, alloantigen-activated post-G CD4(+) T cells preferentially expressed markers associated with memory T cells, in conjunction with reduced levels of CD28 and CD62L. Collectively, these data demonstrate that CD4(+) T cells exposed to G-CSF in vivo acquire the properties of T regulatory (Tr) cells once triggered in vitro through the T-cell receptor, including a peculiar cytokine production profile (IL-10(++)TGF-beta1(+)IL-2(low/-)IL-4(low/-)), an intrinsic low proliferative capacity, and a contact-independent suppression of antigen-driven proliferation. Tr cells generated ex vivo after exposure to G-CSF might be clinically relevant for transplantation medicine and for the treatment of human immune-mediated diseases.

Effect of mitomycin C on IL-1R expression, IL-1-related hepatocyte growth factor secretion and corneal epithelial cell migration.

PubMed

Chen, Tsan-Chi; Chang, Shu-Wen

2010-03-01

To investigate how mitomycin C (MMC) modulates hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) secretions in human corneal fibroblasts and regulates human corneal epithelial (HCE) cell migration. Primary human corneal fibroblasts were treated with MMC (0.05, 0.1, or 0.2 mg/mL for 5 minutes) and were cultivated with or without interleukin (IL)-1beta. Transcript and secretion of HGF and KGF were determined by quantitative real-time RT-PCR and Western blot analysis, respectively. The effect of MMC-treated fibroblasts on HCE cell migration was evaluated using a transwell migration assay. The influence of MMC on HGF expression/secretion and HCE cell migration was further confirmed by RNA interference. The number of IL-1 receptors (IL-1R) on the fibroblast surface was analyzed by flow cytometry. MMC alone did not affect endogenous HGF expression, whereas IL-1beta alone significantly upregulated HGF transcripts and secretion. By modifying IL-1R numbers, MMC further upregulated IL-1beta-related HGF expression at a concentration of 0.05 mg/mL but to a lesser extent at 0.1 and 0.2 mg/mL. KGF transcripts and intracellular expression were suppressed by MMC dose dependently in the presence or absence of IL-1beta, whereas KGF secretion was not affected. Conditioned medium from MMC-treated fibroblasts exerted a similar concentration-dependent effect on HCE cell migration, enhancing migration most significantly at 0.05 mg/mL MMC in the presence of IL-1beta. The MMC dose-dependent modulation of HCE cell migration was abolished in HGF-silenced fibroblasts. MMC differentially modulated IL-1R expression at various concentrations and regulated HGF and KGF differently. MMC alone did not alter HGF expression. In the presence of IL-1beta, MMC-treated corneal fibroblasts modified HCE cell migration through IL-1beta-induced HGF secretion.

PPAR{gamma} activates ABCA1 gene transcription but reduces the level of ABCA1 protein in HepG2 cells

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

Mogilenko, Denis A., E-mail: denis@iem.sp.ru; Department of Embryology, St. Petersburg State University, 199034 St. Petersburg; Shavva, Vladimir S.

Research highlights: {yields} PPAR{gamma} activates ABCA1 gene expression but decreases ABCA1 protein content in human hepatoma cell line HepG2. {yields} Treatment of HepG2 cells with PPAR{gamma} agonist GW1929 leads to dissociation of LXR{beta} from ABCA1-LXR{beta} complex. {yields} Inhibition of protein kinases MEK1/2 abolishes PPAR{gamma}-mediated dissociation of LXR{beta} from ABCA1/LXR{beta} complex. {yields} Activation of PPAR{gamma} leads to increasing of the level of LXR{beta} associated with LXRE within ABCA1 gene promoter. -- Abstract: Synthesis of ABCA1 protein in liver is necessary for high-density lipoproteins (HDL) formation in mammals. Nuclear receptor PPAR{gamma} is known as activator of ABCA1 expression, but details of PPAR{gamma}-mediatedmore » regulation of ABCA1 at both transcriptional and post-transcriptional levels in hepatocytes have not still been well elucidated. In this study we have shown, that PPAR{gamma} activates ABCA1 gene transcription in human hepatoma cells HepG2 through increasing of LXR{beta} binding with promoter region of ABCA1 gene. Treatment of HepG2 cells with PPAR{gamma} agonist GW1929 leads to dissociation of LXR{beta} from ABCA1/LXR{beta} complex and to nuclear translocation of this nuclear receptor resulting in reduction of ABCA1 protein level 24 h after treatment. Inhibition of protein kinases MEK1/2 abolishes PPAR{gamma}-mediated dissociation of LXR{beta} from ABCA1/LXR{beta} complex, but does not block PPAR{gamma}-dependent down-regulation of ABCA1 protein in HepG2 cells. These data suggest that PPAR{gamma} may be important for regulation of the level of hepatic ABCA1 protein and indicate the new interplays between PPAR{gamma}, LXR{beta} and MEK1/2 in regulation of ABCA1 mRNA and protein expression.« less

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 endotoxin following GM-CSF therapy, as evidenced by production of the tissue-reactive cytokines IL-1beta and TNF-alpha.

Integrins beta 5, beta 3 and alpha v are apically distributed in endometrial epithelium.

PubMed

Aplin, J D; Spanswick, C; Behzad, F; Kimber, S J; Vićovac, L

1996-07-01

Several adhesion molecules have been shown to occur at the surface of endometrial cells. One of these is the integrin alpha v subunit which associates with various beta chains including beta 5. We demonstrate the presence of integrin beta 5 polypeptide in human endometrial epithelial cells throughout the menstrual cycle using immunocytochemistry with monospecific antibodies, and at the mRNA level by thermal amplification from endometrial cDNA. Integrin beta 5 is also found in a population of bone marrow-derived cells. A notable feature of the distribution of the beta 5 subunit in the glandular and luminal epithelium is its apical localization, which may suggest an involvement in implantation. However, no evidence was found for regulated expression of epithelial beta 5. In mouse, the beta 5 subunit is found at both the apical and basal surface of epithelial cells and expression is essentially oestrous cycle-independent. Comparisons are made in both species with the distribution of the alpha v and beta 3 subunits which also localize to the apical epithelium.

Impairment of glucose-induced insulin secretion in human pancreatic islets transplanted to diabetic nude mice.

PubMed

Jansson, L; Eizirik, D L; Pipeleers, D G; Borg, L A; Hellerström, C; Andersson, A

1995-08-01

Hyperglycemia-induced beta-cell dysfunction may be an important component in the pathogenesis of non-insulin-dependent diabetes mellitus. However, most available data in this field were obtained from rodent islets. To investigate the relevance of this hypothesis for human beta-cells in vivo, human pancreatic islets were transplanted under the renal capsule of nude mice. Experimental groups were chosen so that grafted islets were exposed to either hyper- or normoglycemia or combinations of these for 4 or 6 wk. Grafts of normoglycemic recipients responded with an increased insulin release to a glucose stimulus during perfusion, whereas grafts of hyperglycemic recipients failed to respond to glucose. The insulin content of the grafts in the latter groups was only 10% of those observed in controls. Recipients initially hyperglycemic (4 wk), followed by 2 wk of normoglycemia regained a normal graft insulin content, but a decreased insulin response to glucose remained. No ultrastructural signs of beta-cell damage were observed, with the exception of increased glycogen deposits in animals hyperglycemic at the time of killing. It is concluded that prolonged exposure to a diabetic environment induces a long-term secretory defect in human beta-cells, which is not dependent on the size of the islet insulin stores.

Impairment of glucose-induced insulin secretion in human pancreatic islets transplanted to diabetic nude mice.

PubMed Central

Jansson, L; Eizirik, D L; Pipeleers, D G; Borg, L A; Hellerström, C; Andersson, A

1995-01-01

Hyperglycemia-induced beta-cell dysfunction may be an important component in the pathogenesis of non-insulin-dependent diabetes mellitus. However, most available data in this field were obtained from rodent islets. To investigate the relevance of this hypothesis for human beta-cells in vivo, human pancreatic islets were transplanted under the renal capsule of nude mice. Experimental groups were chosen so that grafted islets were exposed to either hyper- or normoglycemia or combinations of these for 4 or 6 wk. Grafts of normoglycemic recipients responded with an increased insulin release to a glucose stimulus during perfusion, whereas grafts of hyperglycemic recipients failed to respond to glucose. The insulin content of the grafts in the latter groups was only 10% of those observed in controls. Recipients initially hyperglycemic (4 wk), followed by 2 wk of normoglycemia regained a normal graft insulin content, but a decreased insulin response to glucose remained. No ultrastructural signs of beta-cell damage were observed, with the exception of increased glycogen deposits in animals hyperglycemic at the time of killing. It is concluded that prolonged exposure to a diabetic environment induces a long-term secretory defect in human beta-cells, which is not dependent on the size of the islet insulin stores. Images PMID:7635965

IL-9 expression by human eosinophils: regulation by IL-1beta and TNF-alpha.

PubMed

Gounni, A S; Nutku, E; Koussih, L; Aris, F; Louahed, J; Levitt, R C; Nicolaides, N C; Hamid, Q

2000-09-01

IL-9 is a pleiotropic cytokine that exhibits biologic activity on cells of diverse hemopoietic lineage. IL-9 stimulates the proliferation of activated T cells, enhances the production of IgE from B cells, and promotes the proliferation and differentiation of mast cells and hematopoietic progenitors. In this study we evaluated the expression of IL-9 messenger (m)RNA and protein by human peripheral blood eosinophils. We also investigated the role of IL-1beta and TNF-alpha in the release of IL-9 from human peripheral blood eosinophils. RT-PCR, in situ hybridization, and immunocytochemistry were used to investigate the presence of IL-9 mRNA and protein in human peripheral blood eosinophils from asthmatic patients and normal control subjects. Furthermore, biologic assay was used to investigate the release of IL-9 protein from IL-1beta- or TNF-alpha-stimulated eosinophils in vitro. RT-PCR analysis showed the presence of IL-9 mRNA in human peripheral blood eosinophil RNA preparations from subjects with atopic asthma, as well as in the eosinophil-differentiated HL-60 cell line. By using in situ hybridization, a significant difference (P <.01) in IL-9 mRNA expression was detected in human peripheral blood eosinophils freshly isolated from asthmatic subjects compared with those isolated from normal control subjects. Furthermore, the percentage of IL-9 immunoreactive eosinophils from asthmatic patients was increased compared with that found in normal control subjects (P <.01). We also demonstrate that cultured human peripheral blood eosinophils from asthmatic subjects synthesize and release IL-9 protein, which is upregulated on stimulation with TNF-alpha and IL-1beta. Human eosinophils express biologically active IL-9, which suggests that these cells may influence the recruitment and activation of effector cells linked to the pathogenesis of allergic disease. These observations provide further evidence for the role of eosinophils in regulating airway immune responses.

Effect of prolonged exposure to sublethal concentrations of DDT and DDE on protein expression in human pancreatic beta cells.

PubMed

Pavlikova, Nela; Smetana, Pavel; Halada, Petr; Kovar, Jan

2015-10-01

Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cell line exposed to three sublethal concentrations (0.1 μM, 1 μM, 10 μM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10 μM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10 μM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE. Copyright © 2015 Elsevier Inc. All rights reserved.

Decursin and PDBu: two PKC activators distinctively acting in the megakaryocytic differentiation of K562 human erythroleukemia cells.

PubMed

Kim, Hyeon Ho; Ahn, Kyung Seop; Han, Hogyu; Choung, Se Young; Choi, Sang-Yun; Kim, Ik-Hwan

2005-12-01

Protein kinase C (PKC) plays an important role in the proliferation and differentiation of various cell types including normal and leukemic hematopoietic cells. Phorbol 12,13-dibutyrate (PDBu) induces the megakaryocytic differentiation of K562 human erythroleukemia cells through PKC activation. Decursin, a pyranocoumarin from Angelica gigas, exhibits the cytotoxic effects on various human cancer cell lines and in vitro PKC activation. We report here the differences between two PKC activators, tumor-suppressing decursin and tumor-promoting PDBu, in their actions on the megakaryocytic differentiation of K562 cells. First of all, decursin inhibited PDBu-induced bleb formation in K562 cells. Decursin also inhibited the PDBu-induced megakaryocytic differentiation of K562 cells that is characterized by an increase in substrate adhesion, the secretion of granulocyte/macrophage colony stimulating factor (GM-CSF) and interleukin-6 (IL-6), and the surface expression of integrin beta3. The binding of PDBu to PKC was competitively inhibited by decursin. Decursin induced the more rapid down-regulation of PKC alpha and betaII isozymes than that induced by PDBu in K562 cells. Unlike PDBu, decursin promoted the translocation of PKC alpha and betaII to the nuclear membrane. Decursin-induced faster down-regulation and nuclear translocation of PKC alpha and betaII were not affected by the presence of PDBu. All these results indicate that decursin and phorbol ester are PKC activators distinctively acting in megakaryocytic differentiation and PKC modulation in K562 leukemia cells.

Fibronectin-mediation cell adhesion is required for induction of 92-kDa type IV collagenase/gelatinase (MMP-9) gene expression during macrophage differentiation : the signaling role of protein kinase C-{beta}.

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

Xie, B.; Laouar, A.; Huberman, E.

1998-05-08

Induction of the 92-kDa gelatinase (MMP-9) gene expression is associated with macrophage differentiation. In this study, we explored the regulatory mechanisms underlying this differentiation-associated MMP-9 gene expression in human HL-60 myeloid leukemia cells and human peripheral blood monocytes. Phorbol 12-myristate 13-acetate (PMA) markedly induced MMP-9 gene expression in HL-60 cells; the induction closely paralleled the timing and extent of PMA-induced cell adhesion and spreading, a hallmark of macrophage differentiation. Similarly, treatment with PMA or macrophage-colony stimulating factor stimulated adherence and spreading of blood monocytes with a concurrent 7- or 5-fold increase in MMP-9 production, respectively. In protein kinase C (PKC)-betamore » -deficient HL-60 variant cells (HL-525), PMA failed to induce cell adhesion and MMP-9 gene expression. Transfecting HL-525 cells with a PKC-beta expression plasmid restored PKC-beta levels and PMA inducibility of cell adhesion and spreading as well as MMP-9 gene expression. Induction of cell adhesion and MMP-9 gene expression in HL-60 cells and blood monocytes was strongly inhibited by neutralizing monoclonal antibodies to fibronectin (FN) and its receptor {alpha}5{beta}1 integrin. HL-525 cells, which constitutively display high levels of surface {alpha}5{beta}1 integrin, adhered and spread on immobilized FN with concomitant induction of MMP-9 gene expression. Cytochalasins B and D were each a potent inhibitor of MMP-9 production. Our results suggest that {alpha}5{beta}1 integrin-mediated interaction of immature hematopoietic cells with FN plays a critical role in modulating matrix-degrading activities during macrophage differentiation.« less

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.

Effects of combined treatment with interferon and mezerein on melanogenesis and growth in human melanoma cells.

PubMed

Fisher, P B; Prignoli, D R; Hermo, H; Weinstein, I B; Pestka, S

1985-01-01

We have analyzed the effects of various human interferons produced in bacteria and the antileukemic compound mezerein (MEZ) on growth and melanogenesis in human melanoma cells. In four human melanoma cell lines, recombinant human fibroblast interferon (IFN-beta) was more active than recombinant human leukocyte interferons (IFN-alpha A, IFN-alpha D, or IFN-alpha A/D (Bgl] in inhibiting cellular proliferation. When monolayer cultures were exposed to 1000 IU/ml IFN-beta for four days the degree of growth inhibition in the different melanoma cell lines varied between 94 and 26%. Similarly, four days growth in medium containing 10 ng/ml MEZ resulted in either no inhibition of growth or as much as 53% inhibition of growth, depending on the specific melanoma cell line tested. MEZ induced dendrite-like processes, cytoplasmic projections morphologically similar to those normally found in neurons and melanocytes, in all four melanoma cell lines, whereas none of the interferons tested had this effect. The combination of interferon and MEZ resulted in a dramatic inhibition in cellular proliferation in all four melanoma cell lines. When cell extracts were assayed for melanin content, a marker of melanoma cell differentiation, the combination of IFN-beta and MEZ resulted in higher levels of melanin than with either agent alone. Dendrite-like formation was also prominent in the cultures treated with this combination. These results indicate that the antiproliferative effect of interferon toward human melanoma dells can be enhanced by treatment with MEZ and that this effect is associated with an enhancement of terminal differentiation.

Control of yeast mating signal transduction by a mammalian. beta. sub 2 -adrenergic receptor and G sub s. alpha. subunit

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

King, K.; Caron, M.G.; Lefkowitz, R.J.

1990-10-05

To facilitate functional and mechanistic studies of receptor-G protein interactions by expression of the human {beta}{sub 2}-adrenergic receptor (h{beta}-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h{beta}-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h{beta}-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h{beta}-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by {beta}-adrenergic receptor agonists was achieved in cells coexpressing h{beta}-AR andmore » a mammalian G protein (G{sub s}) {alpha} subunit - demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors.« less

TGF-beta-induced apoptosis in human thyrocytes is mediated by p27kip1 reduction and is overridden in neoplastic thyrocytes by NF-kappaB activation.

PubMed

Bravo, Susana B; Pampín, Sandra; Cameselle-Teijeiro, José; Carneiro, Carmen; Domínguez, Fernando; Barreiro, Francisco; Alvarez, Clara V

2003-10-30

Millions of people worldwide suffer goiter, a proliferative disease of the follicular cells of the thyroid that may become neoplastic. Thyroid neoplasms have low proliferative index, low apoptotic index and a high incidence of metastasis. TGF-beta is overexpressed in thyroid follicular tumor cells. To investigate the role of TGF-beta in thyroid tumor progression, we established cultures of human thyrocytes from different proliferative pathologies (Grave's disease, multinodular goiter, follicular adenoma, papillary carcinoma), lymph node metastasis, and a normal thyroid sample. All cultures maintained the thyrocyte phenotype. TGF-beta induced cell-cycle arrest in all cultures, in contrast with results reported for other epithelial tumors. In deprived medium, TGF-beta induced apoptosis in normal thyrocyte cultures and all neoplastic cultures except the metastatic cultures. This apoptosis was mediated by a reduction in p27kip1 levels, inducing cell-cycle initiation. Antisense p27 expression induced apoptosis in the absence of TGF-beta. By contrast, in cells in which p27 was overexpressed, TGF-beta had a survival effect. In growth medium, a net survival effect occurs in neoplastic thyrocytes only, not normal thyrocytes, due to activation of the NF-kappaB survival program. Together, these findings suggest that (a) thyroid neoplasms are due to reduced apoptosis, not increased division, in line with the low proliferative index of these pathologies, and (b) TGF-beta induces apoptosis in normal thyrocytes via p27 reduction, but that in neoplastic thyrocytes this effect is overridden by activation of the NF-kappaB program.

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.

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.

The insulin and islet amyloid polypeptide genes contain similar cell-specific promoter elements that bind identical beta-cell nuclear complexes.

PubMed Central

German, M S; Moss, L G; Wang, J; Rutter, W J

1992-01-01

The pancreatic beta cell makes several unique gene products, including insulin, islet amyloid polypeptide (IAPP), and beta-cell-specific glucokinase (beta GK). The functions of isolated portions of the insulin, IAPP, and beta GK promoters were studied by using transient expression and DNA binding assays. A short portion (-247 to -197 bp) of the rat insulin I gene, the FF minienhancer, contains three interacting transcriptional regulatory elements. The FF minienhancer binds at least two nuclear complexes with limited tissue distribution. Sequences similar to that of the FF minienhancer are present in the 5' flanking DNA of the human IAPP and rat beta GK genes and also the rat insulin II and mouse insulin I and II genes. Similar minienhancer constructs from the insulin and IAPP genes function as cell-specific transcriptional regulatory elements and compete for binding of the same nuclear factors, while the beta GK construct competes for protein binding but functions poorly as a minienhancer. These observations suggest that the patterns of expression of the beta-cell-specific genes result in part from sharing the same transcriptional regulators. Images PMID:1549125

Molecular cloning of a novel widely expressed human 80 kDa 17 beta-hydroxysteroid dehydrogenase IV.

PubMed Central

Adamski, J; Normand, T; Leenders, F; Monté, D; Begue, A; Stéhelin, D; Jungblut, P W; de Launoit, Y

1995-01-01

Reactions of oestrogens and androgens at position C-17 are catalysed by 17 beta-hydroxysteroid dehydrogenases (17 beta-HSDs). Cloning of the cDNA of a novel human 17 beta-HSD IV and expression of its mRNA are described. A probe derived from the recently discovered porcine 17 beta-oestradiol dehydrogenase (17 beta-EDH) was used to isolate a 2.6 kb human cDNA encoding a continuous protein of 736 amino acids of high (84%) similarity to the porcine 17 beta-EDH. The calculated molecular mass of the human enzyme is 79,595 Da. Other sequence similarities shared by the two enzymes are: an N-terminal sequence which is similar to that of members of the short-chain alcohol dehydrogenase family; amino acids 343-607 which are similar to the C-terminal domains of a trifunctional Candida tropicalis enzyme and the FOX2 gene product of Saccharomyces cerevisiae; amino acids 596-736 which are similar to human sterol carrier protein 2. The previously cloned human 17 beta-HSD I, II and III are less than 25% identical with 17 beta-HSD IV. mRNA for HSD IV is a single species of 3.0 kb, present in many tissues with highest concentrations in liver, heart, prostate and testes. When over-expressed in mammalian cells, the human 17 beta-HSD IV enzyme displays a specific unidirectional oxidative 17 beta-HSD activity. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7487879

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

Interleukin-2-dependent long-term cultures of low-density lymphocytes allow the proliferation of lymphokine-activated killer cells with natural killer, Ti gamma/delta or TNK phenotype.

PubMed

Testa, U; Care, A; Montesoro, E; Fossati, C; Giannella, G; Masciulli, R; Fagioli, M; Bulgarini, D; Habetswallner, D; Isacchi, G

1990-01-01

We have developed a culture system for "long-term" growth of human lymphokine-activated killer (LAK) cells exhibiting an elevated, wide-spectrum antitumor cytotoxicity. The system allows the exponential growth of monocyte-depleted low-density lymphocytes in the presence of human serum and recombinant human interleukin-2 (10(3) U/ml), alone or in combination with interleukin-1 alpha or beta (both at 10 U/ml). Eighteen cultures were established from 18 normal adult donors. The membrane phenotypes of the final LAK cell population, assessed by a panel of monoclonal antibodies (mAb), consist of three main types: (a) NKH-1+, Ti alpha/beta-, Ti gamma/delta-, and CD3- lymphocytes; (b) NKH-1+, Ti alpha/beta-, Ti gamma/delta+, and CD3+ lymphocytes and (c) NKH-1+, Ti alpha/beta+, Ti gamma/delta- and CD3+ lymphocytes. Northern blot analysis showed that all these cell populations express relatively high levels of perforin RNA, particularly cells exhibiting the first phenotype. This culture system may provide a tool for cellular and molecular studies on the mechanisms of antitumor cytotoxicity, as well as the basis for new adoptive immunotherapy protocols in advanced center.

Anaplastic lymphoma kinase is expressed in different subtypes of human breast cancer

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

Perez-Pinera, Pablo; Chang, Y.; Astudillo, A.

2007-06-29

Pleiotrophin (PTN, Ptn) is an 18 kDa cytokine expressed in human breast cancers. Since inappropriate expression of Ptn stimulates progression of breast cancer in transgenic mice and a dominant negative PTN reverses the transformed phenotype of human breast cancer cells that inappropriately express Ptn, it is suggested that constitutive PTN signaling in breast cancer cells that inappropriately express Ptn activates pathways that promote a more aggressive breast cancer phenotype. Pleiotrophin signals by inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP){beta}/{zeta}, and, recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTP{beta}/{zeta} signaling pathway in PTN-stimulated cells,more » not through a direct interaction of PTN with ALK and thus not through the PTN-enforced dimerization of ALK. Since full-length ALK is activated in different malignant cancers and activated ALK is a potent oncogenic protein, we examined human breast cancers to test the possibility that ALK may be expressed in breast cancers and potentially activated through the PTN/RPTP{beta}/{zeta} signaling pathway; we now demonstrate that ALK is strongly expressed in different histological subtypes of human breast cancer; furthermore, ALK is expressed in both nuclei and cytoplasm and, in the 'dotted' pattern characteristic of ALK fusion proteins in anaplastic large cell lymphoma. This study thus supports the possibility that activated ALK may be important in human breast cancers and potentially activated either through the PTN/RPTP{beta}/{zeta} signaling pathway, or, alternatively, as an activated fusion protein to stimulate progression of breast cancer in humans.« less

Inhibition of human megakaryocytopoiesis in vitro by platelet factor 4 (PF4) and a synthetic COOH-terminal PF4 peptide.

PubMed Central

Gewirtz, A M; Calabretta, B; Rucinski, B; Niewiarowski, S; Xu, W Y

1989-01-01

We report that highly purified human platelet factor 4 (PF4) inhibits human megakaryocytopoiesis in vitro. At greater than or equal to 25 micrograms/ml, PF4 inhibited megakaryocyte colony formation approximately 80% in unstimulated cultures, and approximately 58% in cultures containing recombinant human IL 3 and granulocyte-macrophage colony-stimulating factor. Because PF4 (25 micrograms/ml) had no effect on either myeloid or erythroid colony formation lineage specificity of this effect was suggested. A synthetic COOH-terminal PF4 peptide of 24, but not 13 residues, also inhibited megakaryocyte colony formation, whereas a synthetic 18-residue beta-thromboglobulin (beta-TG) peptide and native beta-TG had no such effect when assayed at similar concentrations. The mechanism of PF4-mediated inhibition was investigated. First, we enumerated total cell number, and examined cell maturation in control colonies (n = 200) and colonies (n = 100) that arose in PF4-containing cultures. Total cells per colony did not differ dramatically in the two groups (6.1 +/- 3.0 vs. 4.2 +/- 1.6, respectively), but the numbers of mature large cells per colony was significantly decreased in the presence of PF4 when compared with controls (1.6 +/- 1.5 vs. 3.9 +/- 2.3; P less than 0.001). Second, by using the human leukemia cell line HEL as a model for primitive megakaryocytic cells, we studied the effect of PF4 on cell doubling time, on the expression of both growth-regulated (H3, p53, c-myc,and c-myb), and non-growth-regulated (beta 2-microglobulin) genes. At high concentrations of native PF4 (50 micrograms/ml), no effect on cell doubling time, or H3 or p53 expression was discerned. In contrast, c-myc and c-myb were both upregulated. These results suggested the PF4 inhibited colony formation by impeding cell maturation, as opposed to cell proliferation, perhaps by inducing expression of c-myc and c-myb. The ability of PF4 to inhibit a normal cell maturation function was then tested. Megakaryocytes were incubated in synthetic PF4, or beta-TG peptides for 18 h and effect on Factor V steady-state mRNA levels was determined in 600 individual cells by in situ hybridization. beta-TG peptide had no effect on FV mRNA levels, whereas a approximately 60% decrease in expression of Factor V mRNA was found in megakaryocytes exposed to greater than or equal 100 ng/ml synthetic COOH-terminal PF4 peptide. Accordingly, PF4 modulates megakaryocyte maturation in vitro, and may function as a negative autocrine regulator of human megakaryocytopoiesis. Images PMID:2523411

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.

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 some human cancer cells.

The human interleukin-1 alpha gene is located on the long arm of chromosome 2 at band q13.

PubMed

Lafage, M; Maroc, N; Dubreuil, P; de Waal Malefijt, R; Pébusque, M J; Carcassonne, Y; Mannoni, P

1989-01-01

Interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) are two biochemically distinct, but distantly related, polypeptidic cytokines that play a key role in inflammation, immunologic reactions, and tissue repair. Recently, it has been shown that IL-1 alpha is identical to hematopoietin 1, which was described as a hematopoietic growth factor acting on early progenitor cells in synergy with other hematopoietic growth factors. In this report we discuss our use of in situ hybridization on human prometaphase cells with a human IL-1 alpha cDNA probe to localize the human IL-1 alpha gene on the proximal part of the long arm of chromosome 2 at band q13, in the same chromosomal region as the IL-1 beta gene.

Characterization of resident lymphocytes in human pancreatic islets

PubMed Central

Radenkovic, M.; Uvebrant, K.; Skog, O.; Sarmiento, L.; Avartsson, J.; Storm, P.; Vickman, P.; Bertilsson, P.‐A.; Fex, M.; Korgsgren, O.

2016-01-01

Summary The current view of type 1 diabetes (T1D) is that it is an immune‐mediated disease where lymphocytes infiltrate the pancreatic islets, promote killing of beta cells and cause overt diabetes. Although tissue resident immune cells have been demonstrated in several organs, the composition of lymphocytes in human healthy pancreatic islets have been scarcely studied. Here we aimed to investigate the phenotype of immune cells associated with human islets of non‐diabetic organ donors. A flow cytometry analysis of isolated islets from perfused pancreases (n = 38) was employed to identify alpha, beta, T, natural killer (NK) and B cells. Moreover, the expression of insulin and glucagon transcripts was evaluated by RNA sequencing. Up to 80% of the lymphocytes were CD3+ T cells with a remarkable bias towards CD8+ cells. Central memory and effector memory phenotypes dominated within the CD8+ and CD4+ T cells and most CD8+ T cells were positive for CD69 and up to 50–70% for CD103, both markers of resident memory cells. The frequency of B and NK cells was low in most islet preparations (12 and 3% of CD45+ cells, respectively), and the frequency of alpha and beta cells varied between donors and correlated clearly with insulin and glucagon mRNA expression. In conclusion, we demonstrated the predominance of canonical tissue resident memory CD8+ T cells associated with human islets. We believe that these results are important to understand more clearly the immunobiology of human islets and the disease‐related phenotypes observed in diabetes. PMID:27783386

Lysophospholipid presentation by CD1d and recognition by a human Natural Killer T-cell receptor

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

López-Sagaseta, Jacinto; Sibener, Leah V.; Kung, Jennifer E.

2014-10-02

Invariant Natural Killer T (iNKT) cells use highly restricted {alpha}{beta} T cell receptors (TCRs) to probe the repertoire of lipids presented by CD1d molecules. Here, we describe our studies of lysophosphatidylcholine (LPC) presentation by human CD1d and its recognition by a native, LPC-specific iNKT TCR. Human CD1d presenting LPC adopts an altered conformation from that of CD1d presenting glycolipid antigens, with a shifted {alpha}1 helix resulting in an open A pocket. Binding of the iNKT TCR requires a 7-{angstrom} displacement of the LPC headgroup but stabilizes the CD1d-LPC complex in a closed conformation. The iNKT TCR CDR loop footprint onmore » CD1d-LPC is anchored by the conserved positioning of the CDR3{alpha} loop, whereas the remaining CDR loops are shifted, due in part to amino-acid differences in the CDR3{beta} and J{beta} segment used by this iNKT TCR. These findings provide insight into how lysophospholipids are presented by human CD1d molecules and how this complex is recognized by some, but not all, human iNKT cells.« less

Unraveling the role of the ghrelin gene peptides in the endocrine pancreas.

PubMed

Granata, Riccarda; Baragli, Alessandra; Settanni, Fabio; Scarlatti, Francesca; Ghigo, Ezio

2010-09-01

The ghrelin gene peptides include acylated ghrelin (AG), unacylated ghrelin (UAG), and obestatin (Ob). AG, mainly produced by the stomach, exerts its central and peripheral effects through the GH secretagogue receptor type 1a (GHS-R1a). UAG, although devoid of GHS-R1a-binding affinity, is an active peptide, sharing with AG many effects through an unknown receptor. Ob was discovered as the G-protein-coupled receptor 39 (GPR39) ligand; however, its physiological actions remain unclear. The endocrine pancreas is necessary for glucose homeostasis maintenance. AG, UAG, and Ob are expressed in both human and rodent pancreatic islets from fetal to adult life, and the pancreas is the major source of ghrelin in the perinatal period. GHS-R1a and GPR39 expression has been shown in beta-cells and islets, as well as specific binding sites for AG, UAG, and Ob. Ghrelin colocalizes with glucagon in alpha-islet cells, but is also uniquely expressed in epsilon-islet cells, suggesting a role in islet function and development. Indeed, AG, UAG, and Ob regulate insulin secretion in beta-cells and isolated islets, promote beta-cell proliferation and survival, inhibit beta-cell and human islet cell apoptosis, and modulate the expression of genes that are essential in pancreatic islet cell biology. They even induce beta-cell regeneration and prevent diabetes in streptozotocin-treated neonatal rats. The receptor(s) mediating their effects are not fully characterized, and a signaling crosstalk has been suggested. The present review summarizes the newest findings on AG, UAG, and Ob expression in pancreatic islets and the role of these peptides on beta-cell development, survival, and function.

Antiproliferative properties of toremifene on AIDS-related Kaposi's sarcoma cells.

PubMed

Hong, Angela; Leigh, Bryan R

2002-12-01

Kaposi's sarcoma (KS) is the most common neoplastic apoptosis manifestation of acquired immunodeficiency syndrome. Toremifene is known to upregulate transforming growth factor beta-1 (TGF-beta1), which is a growth-inhibitory factor for KS. We investigated the in vitro effect of toremifene on KS cells. MTT assay was used to measure the growth of four KS cell lines and a human umbilical vein endothelial (HUVE) cell line after incubation with toremifene. Reverse transcription polymerase chain reaction and ELISA were used to measure the level of TGF-beta1. The IC(50) for the KS cells ranged from 2.2 to 3.2 microM, and 80% of the growth inhibition occurred within 24 h. Toremifene enhanced TGF-beta1 mRNA expression, and the level of TGF-beta1 increased from 103 to 473 pg/ml after 48 h of incubation. Toremifene had no effect on the growth of HUVE cells. Toremifene has a specific antiproliferative effect on KS cells. The stimulation of TGF-beta1 production may play a role in the antiproliferative process. Copyright 2002 S. Karger AG, Basel

Characterization of beta-cell mass and insulin resistance in diet-induced obese and diet-resistant rats.

PubMed

Paulsen, Sarah J; Jelsing, Jacob; Madsen, Andreas N; Hansen, Gitte; Lykkegaard, Kirsten; Larsen, Leif K; Larsen, Philip J; Levin, Barry E; Vrang, Niels

2010-02-01

The selectively bred diet-induced obese (DIO) and diet-resistant (DR) rats represent a polygenetic animal model mimicking most clinical variables characterizing the human metabolic syndrome. When fed a high-energy (HE) diet DIO rats develop visceral obesity, dyslipidemia, hyperinsulinemia, and insulin resistance but never frank diabetes. To improve our understanding of the underlying cause for the deteriorating glucose and insulin parameters, we have investigated possible adaptive responses in DIO and DR rats at the level of the insulin-producing beta-cells. At the time of weaning, DR rats were found to have a higher body weight and beta-cell mass compared to DIO rats, and elevated insulin and glucose responses to an oral glucose load. However, at 2.5 months of age, and for the remaining study period, the effect of genotype became evident: the chow-fed DIO rats steadily increased their body weight and beta-cell mass, as well as insulin and glucose levels compared to the DR rats. HE feeding affected both DIO and DR rats leading to an increased body weight and an increased beta-cell mass. Interestingly, although the beta-cell mass in DR rats and chow-fed DIO rats appeared to constantly increase with age, the beta-cell mass in the HE-fed DIO rats did not continue to do so. This might constitute part of an explanation for their reduced glucose tolerance. Collectively, the data support the use of HE-fed DIO rats as a model of human obesity and insulin resistance, and accentuate its relevance for studies examining the benefit of pharmaceutical compounds targeting this disease complex.

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

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

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.

Neurotensin receptor 1 gene activation by the Tcf/beta-catenin pathway is an early event in human colonic adenomas.

PubMed

Souazé, Frédérique; Viardot-Foucault, Véronique; Roullet, Nicolas; Toy-Miou-Leong, Mireille; Gompel, Anne; Bruyneel, Erik; Comperat, Eva; Faux, Maree C; Mareel, Marc; Rostène, William; Fléjou, Jean-François; Gespach, Christian; Forgez, Patricia

2006-04-01

Alterations in the Wnt/APC (adenomatous polyposis coli) signalling pathway, resulting in beta-catenin/T cell factor (Tcf)-dependent transcriptional gene activation, are frequently detected in familial and sporadic colon cancers. The neuropeptide neurotensin (NT) is widely distributed in the gastrointestinal tract. Its proliferative and survival effects are mediated by a G-protein coupled receptor, the NT1 receptor. NT1 receptor is not expressed in normal colon epithelial cells, but is over expressed in a number of cancer cells and tissues suggesting a link to the outgrowth of human colon cancer. Our results demonstrate that the upregulation of NT1 receptor occurring in colon cancer is the result of Wnt/APC signalling pathway activation. We first established the functionality of the Tcf response element within the NT1 receptor promoter. Consequently, we observed the activation of NT1 receptor gene by agents causing beta-catenin cytosolic accumulation, as well as a strong decline of endogenous receptor when wt-APC was restored. At the cellular level, the re-establishment of wt-APC phenotype resulted in the impaired functionality of NT1 receptor, like the breakdown in NT-induced intracellular calcium mobilization and the loss of NT pro-invasive effect. We corroborated the Wnt/APC signalling pathway on the NT1 receptor promoter activation with human colon carcinogenesis, and showed that NT1 receptor gene activation was perfectly correlated with nuclear or cytoplasmic beta-catenin localization while NT1 receptor was absent when beta-catenin was localized at the cell-cell junction in early adenomas of patients with familial adenomatous polyposis, hereditary non-polyposis colorectal cancer and loss of heterozygosity tumours. In this report we establish a novel link in vitro between the Tcf/beta-catenin pathway and NT1 receptor promoter activation.

Dose-related influence of sodium selenite on apoptosis in human thyroid follicles in vitro induced by iodine, EGF, TGF-beta, and H2O2.

PubMed

Lehmann, Petra; Rank, Petra; Hallfeldt, Klaus L J; Krebs, Bjarne; Gärtner, Roland

2006-08-01

Apoptosis of thyroid follicular cells is induced by high doses of iodide, epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta), as well as H2O2 and might be attenuated by antioxidants. Therefore, we examined the apoptotic index induced by these substances in selenium-treated vs untreated human thyroid follicular cells. Reconstituted human thyroid follicles were incubated with sodium selenite (10 or 100 nM) for 72 h; controls received none. The follicles were then distributed to 24-well plates and incubated with potassium iodide (5, 10, or 20 nM), EGF (5 ng/mL), TGF-beta (5 ng/mL), or H2O2 (100 muM). Apoptosis was determined by a mitochondrial potential assay and the number of apoptotic cells counted by two independent, experienced technicians and the glutathione peroxidase (GPx) activity was determined. Asignificant increase of apoptic cells was obtained in control thyroid follicles treated with iodine (5, 10, or 20 microM), thyroidstimulating hormone (TSH) 1, or 10 mU/mL in combination with 5 and 10 microM iodine, EGF (5 ng/mL) and TGF-beta (5 ng/mL), or H2O2 (100 microM) (p < 0.001). In contrast, in thyroid follicles preincubated with 10 or 100 nM sodium selenite, the apoptototic index was identical to the basal rate. In H2O2-treated follicles, the apoptotic index was still significantly elevated but 50% lower compared to control cells. The GPx activity increased from 1.4 +/- 0.2 to 2.25 +/- 0.4 mU/microg DNA with 10 nMselenite and 2.6 + 0.4 mU/microg DNA with 100 nM selenite. Sodium selenite might increase the antioxidative potential in human thyroid follicles in vitro and therefore diminish the apoptosis induced by TGF-beta, EGF, iodide, and even H2O2.

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. We next studied the effect of TGF-beta 1 and TGF-beta 2 on the proliferation of early to late passage HKc/HPVa6, HKc/GFI and HKc/DR. While early passage HKc/HPV16 were as sensitive as normal HKc to growth inhibition by TGF-beta 1 and TGF-beta 2, the cells became increasingly resistant to TGF-beta during in vitro progression, with the proliferation of HKc/DR being virtually unaffected by TGF-beta 1 or TGF-beta 2 treatment. Overall, loss of growth inhibition by RA parallels loss of TGF-beta sensitivity.(ABSTRACT TRUNCATED AT 400 WORDS)

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

PubMed Central

De Groef, Sofie; Leuckx, Gunter; Van Gassen, Naomi; Staels, Willem; Cai, Ying; Yuchi, Yixing; Coppens, Violette; De Leu, Nico; Heremans, Yves; Baeyens, Luc; Van de Casteele, Mark; Heimberg, Harry

2015-01-01

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories. PMID:26273954

Spatiotemporal Dynamics of Insulitis in Human Type 1 Diabetes

PubMed Central

Wedgwood, Kyle C. A.; Richardson, Sarah J.; Morgan, Noel G.; Tsaneva-Atanasova, Krasimira

2016-01-01

Type 1 diabetes (T1D) is an auto-immune disease characterized by the selective destruction of the insulin secreting beta cells in the pancreas during an inflammatory phase known as insulitis. Patients with T1D are typically dependent on the administration of externally provided insulin in order to manage blood glucose levels. Whilst technological developments have significantly improved both the life expectancy and quality of life of these patients, an understanding of the mechanisms of the disease remains elusive. Animal models, such as the NOD mouse model, have been widely used to probe the process of insulitis, but there exist very few data from humans studied at disease onset. In this manuscript, we employ data from human pancreases collected close to the onset of T1D and propose a spatio-temporal computational model for the progression of insulitis in human T1D, with particular focus on the mechanisms underlying the development of insulitis in pancreatic islets. This framework allows us to investigate how the time-course of insulitis progression is affected by altering key parameters, such as the number of the CD20+ B cells present in the inflammatory infiltrate, which has recently been proposed to influence the aggressiveness of the disease. Through the analysis of repeated simulations of our stochastic model, which track the number of beta cells within an islet, we find that increased numbers of B cells in the peri-islet space lead to faster destruction of the beta cells. We also find that the balance between the degradation and repair of the basement membrane surrounding the islet is a critical component in governing the overall destruction rate of the beta cells and their remaining number. Our model provides a framework for continued and improved spatio-temporal modeling of human T1D. PMID:28082906

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.

A kinetic comparison of the processing and secretion of the alpha beta dimer and the uncombined alpha and beta subunits of chorionic gonadotropin synthesized by human choriocarcinoma cells.

PubMed

Peters, B P; Krzesicki, R F; Hartle, R J; Perini, F; Ruddon, R W

1984-12-25

Human choriocarcinoma cells (JAR) synthesize the alpha and beta subunits of the glycoprotein hormone chorionic gonadotropin (hCG) (R.W. Ruddon, C.A. Hanson, A. H. Bryan, G.J. Putterman, E.L. White, F. Perini, K. S. Meade, and P.H. Aldenderfer (1980) J. Biol. Chem. 255, 1000-1007). In addition to the hCG dimer (alpha beta), JAR cells secrete uncombined alpha and beta subunits into the culture medium (L.A. Cole, R.J. Hartle, J.A. Laferla, and R.W. Ruddon (1983) Endocrinology 113, 1176-1178). Pulse-chase studies with [35S]methionine or [3H]mannose were carried out in order to compare free alpha, free beta, and the alpha beta dimer with regard to the kinetics of synthesis, N-linked oligosaccharide processing, and secretion and to determine the kinetics of alpha-beta subunit combination. A panel of three antisera was used to immunoprecipitate directly the free subunits and the alpha beta dimer sequentially from the same cell lysates and culture media. The alpha subunit of hCG was synthesized in a slight molar excess (1.2-1.5-fold) over the beta subunit, and alpha beta dimer was rapidly formed by combination of the intracellular alpha and beta precursors. Dimer formation was already apparent in JAR cells following a 10-min biosynthetic labeling incubation with [35S]methionine. The combination of subunits ceased by 30 min of chase even though 51% of alpha and 44% of beta remained free within the cells. Combination of the alpha and beta precursors had occurred before their N-linked oligosaccharides were processed beyond the Man8GlcNAc2 structure. The initial trimming of glucosyl and mannosyl units from the high-mannose oligosaccharides of the hCG precursors occurred more rapidly for free alpha and CG-alpha than for free beta and CG-beta. JAR cells accumulated alpha precursors bearing mostly Man8GlcNAc2 units and beta precursors bearing Man8GlcNAc2 units that represent the substrates of the rate-limiting step in the secretory pathway. In spite of the fact that their N-linked oligosaccharides were trimmed at different rates, free alpha, free beta, and alpha beta dimer were all secreted into the medium at the same rate, with a half-time of 35 min. The secreted hCG forms were stable in the chase medium between 4 and 8h, indicating that extracellular degradation, combination of free subunits to form dimer, or dissociation of dimer to form free subunits did not occur.(ABSTRACT TRUNCATED AT 400 WORDS)

Paradigm Shift in Thyroid Hormone Mechanism of Action | Center for Cancer Research

Cancer.gov

Thyroid hormone (TH) is one of the primary endocrine regulators of human metabolism and homeostasis. Acting through three forms of the thyroid hormone receptor (THR; alpha-1, beta-1, and beta-2), TH regulates target gene expression in nearly every cell in the body, modulating fundamental processes, such as basal metabolic rate, long bone growth, and neural maturation. TH is also essential for proper development and differentiation of all cells of the human body.

Effect of beta-carotene-rich tomato lycopene beta-cyclase ( tlcy-b) on cell growth inhibition in HT-29 colon adenocarcinoma cells.

PubMed

Palozza, Paola; Bellovino, Diana; Simone, Rossella; Boninsegna, Alma; Cellini, Francesco; Monastra, Giovanni; Gaetani, Sancia

2009-07-01

Lycopene beta-cyclase (tlcy-b) tomatoes, obtained by modulating carotenogenesis via genetic engineering, contain a large amount of beta-carotene, as clearly visible by their intense orange colour. In the present study we have subjected tlcy-b tomatoes to an in vitro simulated digestion and analysed the effects of digestate on cell proliferation. To this aim we used HT-29 human colon adenocarcinoma cells, grown in monolayers, as a model. Digested tomatoes were diluted (20 ml, 50 ml and 100 ml/l) in culture medium and added to the cells for different incubation times (24 h, 48 h and 72 h). Inhibition of cell growth by tomato digestate was dose-dependent and resulted from an arrest of cell cycle progression at the G0/G1 and G2/M phase and by apoptosis induction. A down-regulation of cyclin D1, Bcl-2 and Bcl-xl expression was observed. We also found that heat treatment of samples before digestion enhanced beta-carotene release and therefore cell growth inhibition. To induce with purified beta-carotene solubilised in tetrahydrofuran the same cell growth inhibition obtained with the tomato digestate, a higher amount of the carotenoid was necessary, suggesting that beta-carotene micellarised during digestion is utilised more efficiently by the cells, but also that other tomato molecules, reasonably made available during digestion, may be present and cooperate with beta-carotene in promoting cell growth arrest.

Irradiation With Carbon Ion Beams Induces Apoptosis, Autophagy, and Cellular Senescence in a Human Glioma-Derived Cell Line

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

Jinno-Oue, Atsushi; Shimizu, Nobuaki; 21st Century Center of Excellence Program for Biomedical Research Using Accelerator Technology, Maebashi, Gunma

2010-01-15

Purpose: We examined biological responses of human glioma cells to irradiation with carbon ion beams (C-ions). Methods and Materials: A human glioma-derived cell line, NP-2, was irradiated with C-ions. Apoptotic cell nuclei were stained with Hoechst 33342. Induction of autophagy was examined either by staining cells with monodansylcadaverine (MDC) or by Western blotting to detect conversion of microtuble-associated protein light chain 3 (MAP-LC3) (LC3-I) to the membrane-bound form (LC3-II). Cellular senescence markers including induction of senescence-associated beta-galactosidase (SA-beta-gal) were examined. The mean telomere length of irradiated cells was determined by Southern blot hybridization. Expression of tumor suppressor p53 and cyclin/cyclin-dependentmore » kinase inhibitor p21{sup WAF1/CIP1} in the irradiated cells was analyzed by Western blotting. Results: When NP-2 cells were irradiated with C-ions at 6 Gy, the major population of the cells died of apoptosis and autophagy. The residual fraction of attached cells (<1% of initially irradiated cells) could not form a colony: however, they showed a morphological phenotype consistent with cellular senescence, that is, enlarged and flattened appearance. The senescent nature of these attached cells was further indicated by staining for SA-beta-gal. The mean telomere length was not changed after irradiation with C-ions. Phosphorylation of p53 at serine 15 as well as the expression of p21{sup WAF1/CIP1} was induced in NP-2 cells after irradiation. Furthermore, we found that irradiation with C-ions induced cellular senescence in a human glioma cell line lacking functional p53. Conclusions: Irradiation with C-ions induced apoptosis, autophagy, and cellular senescence in human glioma cells.« less

The group B streptococcal alpha C protein binds alpha1beta1-integrin through a novel KTD motif that promotes internalization of GBS within human epithelial cells.

PubMed

Bolduc, Gilles R; Madoff, Lawrence C

2007-12-01

Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis and meningitis among neonates and a cause of morbidity among pregnant women and immunocompromised adults. GBS epithelial cell invasion is associated with expression of alpha C protein (ACP). Loss of ACP expression results in a decrease in GBS internalization and translocation across human cervical epithelial cells (ME180). Soluble ACP and its 170 amino acid N-terminal region (NtACP), but not the repeat protein RR', bind to ME180 cells and reduce internalization of wild-type GBS to levels obtained with an ACP-deficient isogenic mutant. In the current study, ACP colocalized with alpha(1)beta(1)-integrin, resulting in integrin clustering as determined by laser scanning confocal microscopy. NtACP contains two structural domains, D1 and D2. D1 is structurally similar to fibronectin's integrin-binding region (FnIII10). D1's (KT)D146 motif is structurally similar to the FnIII10 (RG)D1495 integrin-binding motif, suggesting that ACP binds alpha(1)beta(1)-integrin via the D1 domain. The (KT)D146A mutation within soluble NtACP reduced its ability to bind alpha(1)beta(1)-integrin and inhibit GBS internalization within ME180 cells. Thus ACP binding to human epithelial cell integrins appears to contribute to GBS internalization within epithelial cells.

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.

beta. -Amyloid precursor protein of Alzheimer disease occurs as 110- to 135-kilodalton membrane-associated proteins in neural and nonneural tissues

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

Selkoe, D.J.; Podlisny, M.B.; Joachim, C.L.

1988-10-01

Progressive cerebral deposition of extracellular filaments composed of the {beta}-amyloid protein ({beta}AP) is a constant feature of Alzheimer disease (AD). Since the gene on chromosome 21 encoding the {beta}AP precursor ({beta}APP) is not known to be altered in AD, transcriptional or posttranslational changes may underlie accelerated {beta}AP deposition. Using two antibodies to the predicted carboxyl terminus of {beta}APP, the authors have identified the native {beta}APP in brain and nonneural human tissues as a 110- to 135-kDa protein complex that is insoluble in buffer and found in various membrane-rich subcellular fractions. These proteins are relatively uniformly distributed in adult brain, abundantmore » in fetal brain, and detected in nonneural tissues that contain {beta}APP mRNA. Similarly sized proteins occur in rat, cow, and monkey brain and in cultured human HL-60 and HeLa cells; the precise patterns in the 110- to 135-kDa range are heterogeneous among various tissues and cell lines. They conclude that the highly conserved {beta}APP molecule occurs in mammalian tissues as a heterogeneous group of membrane-associated proteins of {approx} 120 kDa. Detection of the nonamyloidogenic carboxyl terminus within plaques suggests that proteolytic processing of the {beta}APP into insoluble filaments occurs locally in cortical regions that develop {beta}-amyloid deposits with age.« less

In vitro biological evaluation of beta-TCP/HDPE--A novel orthopedic composite: a survey using human osteoblast and fibroblast bone cells.

PubMed

Homaeigohar, S Sh; Shokrgozar, M A; Khavandi, A; Sadi, A Yari

2008-02-01

Beta-tricalcium phosphate reinforced high density polyethylene (beta-TCP/HDPE) was prepared to simulate bone composition and to study its capacity to act as bone tissue. This material was produced by replacing the mineral component and collagen soft tissue of the bone with beta-TCP and HDPE, respectively. The biocompatibility of the composite samples with different volume fractions of TCP (20, 30 and 40 vol %) was examined in vitro using two osteoblast cell lines G-292 and Saos-2, and also a type of fibroblast cell isolated from bone tissue, namely human bone fibroblast (HBF) by proliferation, and cell adhesion assays. Cell-material interaction with the surface of the composite samples was examined by scanning electron microscopy (SEM). The effect of beta-TCP/HDPE on the behavior of osteoblast and fibroblast cells was compared with those of composite and negative control samples; polyethylene (PE) and tissue culture polystyrene (TPS), respectively. In general, the results showed that the composite samples containing beta-TCP as reinforcement supported a higher rate of proliferation by various bone cells after 3, 7, and 14 days of incubation compared to the composite control sample. Furthermore, more osteoblast cells were attached to the surface of the composite samples when compared to the composite control samples after the above incubation periods (p < 0.05), while in the case of HBF an equal or even higher number of cells adhered to PE was observed. The number of adhered osteoblast cells was almost equal and in some days even higher than the number of adhered cells on negative control sample, while in the case of fibroblast this difference was significantly higher than TPS (p < 0.05). Adhered cells presented a normal morphology by SEM and many of the cells were observed to be undergoing cell division. These findings indicate that beta-TCP/HDPE composites are biocompatible, nontoxic, and act to stimulate proliferation and adhesion of the cells, whether osteoblast or fibroblast. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2008.

4beta-Hydroxywithanolide E from Physalis peruviana (golden berry) inhibits growth of human lung cancer cells through DNA damage, apoptosis and G2/M arrest.

PubMed

Yen, Ching-Yu; Chiu, Chien-Chih; Chang, Fang-Rong; Chen, Jeff Yi-Fu; Hwang, Chi-Ching; Hseu, You-Cheng; Yang, Hsin-Ling; Lee, Alan Yueh-Luen; Tsai, Ming-Tz; Guo, Zong-Lun; Cheng, Yu-Shan; Liu, Yin-Chang; Lan, Yu-Hsuan; Chang, Yu-Ching; Ko, Ying-Chin; Chang, Hsueh-Wei; Wu, Yang-Chang

2010-02-18

The crude extract of the fruit bearing plant, Physalis peruviana (golden berry), demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown. Herein, we isolated the main pure compound, 4beta-Hydroxywithanolide (4betaHWE) derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299) using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE) assay was used to evaluate the DNA damage due to the drug. It was shown that DNA damage was significantly induced by 1, 5, and 10 microg/mL 4betaHWE for 2 h in a dose-dependent manner (p < 0.005). A trypan blue exclusion assay showed that the proliferation of cells was inhibited by 4betaHWE in both dose- and time-dependent manners (p < 0.05 and 0.001 for 24 and 48 h, respectively). The half maximal inhibitory concentrations (IC50) of 4betaHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 microg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4betaHWE produced cell cycle perturbation in the form of sub-G1 accumulation and slight arrest at the G2/M phase with 1 microg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G2/M arrest for H1299 cells treated with 5 microg/mL for 24 h. In this study, we demonstrated that golden berry-derived 4betaHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer.

Bmi-1 extends the life span of normal human oral keratinocytes by inhibiting the TGF-{beta} signaling

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

Kim, Reuben H., E-mail: rkim@dentistry.ucla.edu; UCLA Dental Research Institute, Los Angeles, CA 90095; UCLA Jonsson Comprehensive Cancer Center, Los Angeles, CA 90095

2010-10-01

We previously demonstrated that Bmi-1 extended the in vitro life span of normal human oral keratinocytes (NHOK). We now report that the prolonged life span of NHOK by Bmi-1 is, in part, due to inhibition of the TGF-{beta} signaling pathway. Serial subculture of NHOK resulted in replicative senescence and terminal differentiation and activation of TGF-{beta} signaling pathway. This was accompanied with enhanced intracellular and secreted TGF-{beta}1 levels, phosphorylation of Smad2/3, and increased expression of p15{sup INK4B} and p57{sup KIP2}. An ectopic expression of Bmi-1 in NHOK (HOK/Bmi-1) decreased the level of intracellular and secreted TGF-{beta}1 induced dephosphorylation of Smad2/3, andmore » diminished the level of p15{sup INK4B} and p57{sup KIP2}. Moreover, Bmi-1 expression led to the inhibition of TGF-{beta}-responsive promoter activity in a dose-specific manner. Knockdown of Bmi-1 in rapidly proliferating HOK/Bmi-1 and cancer cells increased the level of phosphorylated Smad2/3, p15{sup INK4B}, and p57{sup KIP2}. In addition, an exposure of senescent NHOK to TGF-{beta} receptor I kinase inhibitor or anti-TGF-{beta} antibody resulted in enhanced replicative potential of cells. Taken together, these data suggest that Bmi-1 suppresses senescence of cells by inhibiting the TGF-{beta} signaling pathway in NHOK.« less

Tumor suppression function of the Big-h3 gene in radiation carcinogenesis

NASA Astrophysics Data System (ADS)

Zhao, Y.; Piao, C.; Hei, T.

Interaction between cell and extracellular matrix (ECM) plays a crucial role in tumor invasiveness and metastasis. Using an immortalized human bronchial epithelial (BEP2D) cell model, we show here that expression of Big-h3 gene, a secreted adhesion molecule induced by transforming growth factor- beta (TGF-beta ), is markedly decreased in independently generated, high LET radiation-induced tumor cell lines (TL1-TL5) relative to parental BEP2D cells. Expression of this gene was restored to control level in fusion cell lines between the tumorigenic and parental BEP2D cells that were no longer tumorigenic in nude mice. Transfection of Big-h3 gene into tumor cells resulted in a significant reduction of tumor growth. While integrin receptor alpha 5/beta 1 was overexpressed in tumor cells, its expression was corrected to the level of control BEP2D cells after Big-h3 transfection. These data suggest that Big-h3 is involved in tumor progression by regulating integrin receptor alpha 5/beta 1. . WWee We further show that down regulation of Big-h3 results from loss of expression of TGFbeta1 in tumor cells. The findings provide strong evidence that the Big-h3 gene has tumor suppressor function in radiation induced tumorigenic human bronchial epithelial cells and suggest a potential target for interventional therapy.

Arginine supplementation of sickle transgenic mice reduces red cell density and Gardos channel activity.

PubMed

Romero, José R; Suzuka, Sandra M; Nagel, Ronald L; Fabry, Mary E

2002-02-15

Nitric oxide (NO), essential for maintaining vascular tone, is produced from arginine by nitric oxide synthase. Plasma arginine levels are low in sickle cell anemia, and it is reported here that low plasma arginine is also found in our sickle transgenic mouse model that expresses human alpha, human beta(S), and human beta(S-Antilles) and is homozygous for the mouse beta(major) deletion (S+S-Antilles). S+S-Antilles mice were supplemented with a 4-fold increase in arginine that was maintained for several months. Mean corpuscular hemoglobin concentration (MCHC) decreased and the percent high-density red cells was reduced. Deoxy K(+) efflux is characteristic of red cells in sickle cell disease and contributes to the disease process by increasing the MCHC and rendering the cells more susceptible to polymer formation. This flux versus the room air flux was reduced in S+S-Antilles red cells from an average value of 1.6 +/- 0.3 mmol per liter of red cells x minute (FU) in nonsupplemented mice to 0.9 +/- 0.3 FU (n = 4, P < .02, paired t test) in supplemented mice. In room air, V(max) of the Ca(++)-activated K(+) channel (Gardos) was reduced from 4.1 +/- 0.6 FU (off diet) to 2.6 +/- 0.4 FU (n = 7 and 8, P < .04, t test) in arginine-supplemented mice versus clotrimazole. In conclusion, the major mechanism by which arginine supplementation reduces red cell density (MCHC) in S+S-Antilles mice is by inhibiting the Ca(++)-activated K(+) channel.

IL-1beta suppresses the formation of osteoclasts by increasing OPG production via an autocrine mechanism involving celecoxib-related prostaglandins in chondrocytes.

PubMed

Watanabe, Yusuke; Namba, Aki; Aida, Yukiko; Honda, Kazuhiro; Tanaka, Hideki; Suzuki, Naoto; Matsumura, Hideo; Maeno, Masao

2009-01-01

Elevated interleukin (IL)-1 concentrations in synovial fluid have been implicated in joint bone and cartilage destruction. Previously, we showed that IL-1beta stimulated the expression of prostaglandin (PG) receptor EP4 via increased PGE(2) production. However, the effect of IL-1beta on osteoclast formation via chondrocytes is unclear. Therefore, we examined the effect of IL-1beta and/or celecoxib on the expression of macrophage colony-stimulating factor (M-CSF), receptor activator of NF-kappaB ligand (RANKL), and osteoprotegerin (OPG) in human chondrocytes, and the indirect effect of IL-1beta on osteoclast-like cell formation using RAW264.7 cells. OPG and RANKL expression increased with IL-1beta; whereas M-CSF expression decreased. Celecoxib blocked the stimulatory effect of IL-1beta. Conditioned medium from IL-1beta-treated chondrocytes decreased TRAP staining in RAW264.7 cells. These results suggest that IL-1beta suppresses the formation of osteoclast-like cells via increased OPG production and decreased M-CSF production in chondrocytes, and OPG production may increase through an autocrine mechanism involving celecoxib-related PGs.

Control of growth and squamous differentiation in normal human bronchial epithelial cells by chemical and biological modifiers and transferred genes.

PubMed Central

Pfeifer, A M; Lechner, J F; Masui, T; Reddel, R R; Mark, G E; Harris, C C

1989-01-01

The majority of human lung cancers arise from bronchial epithelial cells. The normal pseudostratified bronchial epithelium is composed of basal, mucous, and ciliated cells. This multi-differentiated epithelium usually responds to xenobiotics and physical injury by undergoing basal cell hyperplasia, mucous cell hyperplasia, and squamous metaplasia. One step of the multistage process of carcinogenesis is thought to involve aberrations in control of the squamous metaplastic processes. Decreased responsiveness to regulators of terminal squamous differentiation may confer a selective clonal expansion advantage to an initiated cell. We studied the effects of endogenous [e.g., transforming growth factor beta 1 (TGF-beta 1) and serum] and exogenous [e.g., 12-O-tetradecanoyl-13-phorbol-acetate (TPA), tobacco smoke condensate, and aldehydes] modifiers of normal human bronchial epithelial (NHBE) cell in a serum-free culture system. NHBE cells are growth inhibited by all of these compounds and induced to undergo squamous differentiation by TGF-beta 1 or TPA. In contrast, lung carcinoma cell lines are relatively resistant to inducers of terminal squamous differentiation which may provide them with a selective growth advantage. Chemical agents and activated protooncogenes (ras,raf,myc) altered the response to endogenous and exogenous inducers of squamous differentiation and caused extended cellular lifespan, aneuploidy, and/or tumorigenicity. The data suggest a close relationship between dysregulation of terminal differentiation pathways and neoplastic transformation of human bronchial epithelial cells. PMID:2538323

Coordinate regulation of estrogen-mediated fibronectin matrix assembly and epidermal growth factor receptor transactivation by the G protein-coupled receptor, GPR30.

PubMed

Quinn, Jeffrey A; Graeber, C Thomas; Frackelton, A Raymond; Kim, Minsoo; Schwarzbauer, Jean E; Filardo, Edward J

2009-07-01

Estrogen promotes changes in cytoskeletal architecture not easily attributed to the biological action of estrogen receptors, ERalpha and ERbeta. The Gs protein-coupled transmembrane receptor, GPR30, is linked to specific estrogen binding and rapid estrogen-mediated release of heparin-bound epidermal growth factor. Using marker rescue and dominant interfering mutant strategies, we show that estrogen action via GPR30 promotes fibronectin (FN) matrix assembly by human breast cancer cells. Stimulation with 17beta-estradiol or the ER antagonist, ICI 182, 780, results in the recruitment of FN-engaged integrin alpha5beta1 conformers to fibrillar adhesions and the synthesis of FN fibrils. Concurrent with this cellular response, GPR30 promotes the formation of Src-dependent, Shc-integrin alpha5beta1 complexes. Function-blocking antibodies directed against integrin alpha5beta1 or soluble Arg-Gly-Asp peptide fragments derived from FN specifically inhibited GPR30-mediated epidermal growth factor receptor transactivation. Estrogen-mediated FN matrix assembly and epidermal growth factor receptor transactivation were similarly disrupted in integrin beta1-deficient GE11 cells, whereas reintroduction of integrin beta1 into GE11 cells restored these responses. Mutant Shc (317Y/F) blocked GPR30-induced FN matrix assembly and tyrosyl phosphorylation of erbB1. Interestingly, relative to recombinant wild-type Shc, 317Y/F Shc was more readily retained in GPR30-induced integrin alpha5beta1 complexes, yet this mutant did not prevent endogenous Shc-integrin alpha5beta1 complex formation. Our results suggest that GPR30 coordinates estrogen-mediated FN matrix assembly and growth factor release in human breast cancer cells via a Shc-dependent signaling mechanism that activates integrin alpha5beta1.

Human beta-defensin 3 has immunosuppressive activity in vitro and in vivo.

PubMed

Semple, Fiona; Webb, Sheila; Li, Hsin-Ni; Patel, Hetal B; Perretti, Mauro; Jackson, Ian J; Gray, Mohini; Davidson, Donald J; Dorin, Julia R

2010-04-01

Beta-defensins are antimicrobial peptides with an essential role in the innate immune response. In addition beta-defensins can also chemoattract cells involved in adaptive immunity. Until now, based on evidence from dendritic cell stimulation, human beta defensin-3 (hBD3) was considered pro-inflammatory. We present evidence here that hBD3 lacks pro-inflammatory activity in human and mouse primary Mphi. In addition, in the presence of LPS, hBD3 and the murine orthologue Defb14 (but not hBD2), effectively inhibit TNF-alpha and IL-6 accumulation implying an anti-inflammatory function. hBD3 also inhibits CD40/IFN-gamma stimulation of Mphi and in vivo, hBD3 significantly reduces the LPS-induced TNF-alpha level in serum. Recent work has revealed that hBD3 binds melanocortin receptors but we provide evidence that these are not involved in hBD3 immunomodulatory activity. This implies a dual role for hBD3 in antimicrobial activity and resolution of inflammation.

Amelioration of murine beta-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both gamma-globin and the MGMT drug-resistance gene.

PubMed

Zhao, Huifen; Pestina, Tamara I; Nasimuzzaman, Md; Mehta, Perdeep; Hargrove, Phillip W; Persons, Derek A

2009-06-04

Correction of murine models of beta-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human gamma-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT), driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs, which can be administered to kill residual untransduced, diseased HSCs, whereas transduced cells are protected. Mice transplanted with beta-thalassemic HSCs transduced with a gamma-globin/MGMT vector initially had subtherapeutic levels of red cells expressing gamma-globin. To enrich gamma-globin-expressing cells, transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of gamma-globin-expressing red cells and the amount of fetal hemoglobin, leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of gamma-globin-expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for beta-thalassemia.

A chitosan/beta-glycerophosphate thermo-sensitive gel for the delivery of ellagic acid for the treatment of brain cancer.

PubMed

Kim, Sungwoo; Nishimoto, Satoru K; Bumgardner, Joel D; Haggard, Warren O; Gaber, M Waleed; Yang, Yunzhi

2010-05-01

We report here the development of a chitosan/beta-glycerophosphate(Ch/beta-GP) thermo-sensitive gel to deliver ellagic acid (EA) for cancer treatment. The properties of the Ch/beta-GP gels were characterized regarding chemical structure, surface morphology, and viscoelasticity. In vitro EA release rate from the EA loaded Ch/beta-GP gel and chitosan degradation rate were investigated. The anti-tumor effect of the EA loaded Ch/beta-GP gel on brain cancer cells (human U87 glioblastomas and rat C6 glioma cells) was evaluated by examining cell viability. Cell number and activity were monitored by the MTS assay. The Ch/beta-GP solution formed a heat-induced gel at body temperature, and the gelation temperature and time were affected by the final pH of the Ch/beta-GP solution. The lysozyme increased the EA release rate by 2.5 times higher than that in the absence of lysozyme. Dialyzed chitosan solution with final pH 6.3 greatly reduced the beta-GP needed for gelation, thereby significantly improving the biocompatibility of gel (p < 0.001). The chitosan gels containing 1% (w/v) of ellagic acid significantly reduced viability of U87 cells and C6 cells compared with the chitosan gels at 3 days incubation (p < 0.01, and p < 0.001, respectively). Copyright 2010 Elsevier Ltd. All rights reserved.

Hyperoxia reduces insulin release and induces mitochondrial dysfunction with possible implications for hyperoxic treatment of neonates.

PubMed

Hals, Ingrid; Ohki, Tsuyoshi; Singh, Rinku; Ma, Zuheng; Björklund, Anneli; Balasuriya, Chandima; Scholz, Hanne; Grill, Valdemar

2017-10-01

We previously showed that hyperoxia in vitro negatively affects beta cells of the rat. Here, we tested for possible clinical significance as well as mitochondrial interactions by hyperoxia, using human islets (function and viability), INS-1 832/13 cells (mitochondrial metabolism), and mouse neonates (effects in vivo). Lastly, we assessed relevant parameters in a cohort of individuals born preterm and then exposed to hyperoxia. Human islets and INS-1 832/13 cells were exposed to 24 h of hyperoxia (90-92% oxygen). Mouse neonates were subjected to 5 days of continuous hyperoxia. Individuals born preterm were evaluated in terms of glucose homeostasis and beta cell function by HbA1c and the HOMA2 formula. In human islets, hyperoxia significantly reduced glucose-stimulated insulin secretion by 42.2 ± 5.3% and viability assessed by MTT by 22.5 ± 5.4%. Hyperoxia down-regulated mitochondrial complex II by 21 ± 5% and upregulated complex III by 26 ± 10.1% and complex IV by 37 ± 10.6%. Partly similar effects on mitochondrial complexes were found in hyperoxia-exposed INS-1 832/13 cells. Exposure to hyperoxia swiftly reduced oxygen consumption in these cells and increased mitochondrial uncoupling. Hyperoxia transiently but significantly reduced insulin release in mouse neonates. Individuals born preterm displayed higher HbA1c versus controls, as well as insulin resistance. Thus, hyperoxia exerts negative effects in vitro on human beta cells and results indicate inhibitory effects on insulin secretion in vivo in mouse neonates. Negative effects may be lessened by the demonstrated swift and profound mitochondrial adaptability. Our findings open the possibility that hyperoxia could negatively affect beta cells of preterm human neonates. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Imaging of alpha(v)beta(3) expression by a bifunctional chimeric RGD peptide not cross-reacting with alpha(v)beta(5).

PubMed

Zannetti, Antonella; Del Vecchio, Silvana; Iommelli, Francesca; Del Gatto, Annarita; De Luca, Stefania; Zaccaro, Laura; Papaccioli, Angela; Sommella, Jvana; Panico, Mariarosaria; Speranza, Antonio; Grieco, Paolo; Novellino, Ettore; Saviano, Michele; Pedone, Carlo; Salvatore, Marco

2009-08-15

To test whether a novel bifunctional chimeric peptide comprising a cyclic Arg-Gly-Asp pentapeptide covalently bound to an echistatin domain can discriminate alpha(v)beta(3) from alpha(v)beta(5) integrin, thus allowing the in vivo selective visualization of alpha(v)beta(3) expression by single-photon and positron emission tomography (PET) imaging. The chimeric peptide was preliminarily tested for inhibition of alpha(v)beta(3)-dependent cell adhesion and competition of 125I-echistatin binding to membrane of stably transfected K562 cells expressing alpha(v)beta(3) (Kalpha(v)beta(3)) or alpha(v)beta(5) (Kalpha(v)beta(5)) integrin. The chimeric peptide was then conjugated with diethylenetriaminepentaacetic acid and labeled with 111In for single-photon imaging, whereas a one-step procedure was used for labeling the full-length peptide and a truncated derivative, lacking the last five C-terminal amino acids, with 18F for PET imaging. Nude mice bearing tumors from Kalpha(v)beta(3), Kalpha(v)beta(5), U87MG human glioblastoma, and A431 human epidermoid cells were subjected to single-photon and PET imaging. Adhesion and competitive binding assays showed that the novel chimeric peptide selectively binds to alpha(v)beta(3) integrin and does not cross-react with alpha(v)beta(5). In agreement with in vitro findings, single-photon and PET imaging studies showed that the radiolabeled chimeric peptide selectively localizes in tumor xenografts expressing alphavbeta3 and fails to accumulate in those expressing alpha(v)beta(5) integrin. When 18F-labeled truncated derivative was used for PET imaging, alphavbeta3- and alpha(v)beta(5)-expressing tumors were visualized, indicating that the five C-terminal amino acids are required to differentially bind the two integrins. Our findings indicate that the novel chimeric Arg-Gly-Asp peptide, having no cross-reaction with alphavbeta5 integrin, allows highly selective alphavbeta3 expression imaging and monitoring.

Accurate assessment and identification of naturally occurring cellular cobalamins.

PubMed

Hannibal, Luciana; Axhemi, Armend; Glushchenko, Alla V; Moreira, Edward S; Brasch, Nicola E; Jacobsen, Donald W

2008-01-01

Accurate assessment of cobalamin profiles in human serum, cells, and tissues may have clinical diagnostic value. However, non-alkyl forms of cobalamin undergo beta-axial ligand exchange reactions during extraction, which leads to inaccurate profiles having little or no diagnostic value. Experiments were designed to: 1) assess beta-axial ligand exchange chemistry during the extraction and isolation of cobalamins from cultured bovine aortic endothelial cells, human foreskin fibroblasts, and human hepatoma HepG2 cells, and 2) to establish extraction conditions that would provide a more accurate assessment of endogenous forms containing both exchangeable and non-exchangeable beta-axial ligands. The cobalamin profile of cells grown in the presence of [ 57Co]-cyanocobalamin as a source of vitamin B12 shows that the following derivatives are present: [ 57Co]-aquacobalamin, [ 57Co]-glutathionylcobalamin, [ 57Co]-sulfitocobalamin, [ 57Co]-cyanocobalamin, [ 57Co]-adenosylcobalamin, [ 57Co]-methylcobalamin, as well as other yet unidentified corrinoids. When the extraction is performed in the presence of excess cold aquacobalaminacting as a scavenger cobalamin (i.e. "cold trapping"), the recovery of both [ 57Co]-glutathionylcobalamin and [ 57Co]-sulfitocobalamin decreases to low but consistent levels. In contrasts, the [ 57Co]-nitrocobalamin observed in the extracts prepared without excess aquacobalamin is undetected in extracts prepared with cold trapping. This demonstrates that beta-ligand exchange occur with non-covalently bound beta-ligands. The exception to this observation is cyanocobalamin with a non-exchangeable CN- group. It is now possible to obtain accurate profiles of cellular cobalamin.

Type I interferon (IFN-alpha/beta) rescues B-lymphocytes from apoptosis via PI3Kdelta/Akt, Rho-A, NFkappaB and Bcl-2/Bcl(XL).

PubMed

Badr, Gamal; Saad, Heba; Waly, Hanan; Hassan, Khadega; Abdel-Tawab, Hanem; Alhazza, Ibrahim M; Ahmed, Emad A

2010-01-01

Although IFN-alpha was reported to promote the survival of peripheral B-lymphocytes via the PI3-kinase-Akt pathway, the triggered signalling pathways involved in the protection of B cell from apoptosis need to be clarified. Using flow cytometry and western blot analysis, we have found that type 1 IFNs (IFN-alpha/beta) protect human B cells in culture from spontaneous apoptosis and from apoptosis mediated by anti-CD95 agonist, in a dose- and time-dependant manner. IFN-alpha/beta-mediated anti-apoptotic effect on human B cells was totally abrogated by blockade of IFNR1 chain. Our data indicate that PI3Kdelta, Rho-A, NFkappaB and Bcl-2/Bcl(XL) are active downstream of IFN receptors and are the major effectors of IFN-alpha/beta-rescued B cells from apoptosis. Furthermore, immunohistochemical results show marked reduction in numbers of CD20 positive B cell in both spleen and Peyer's patches from mice treated with anti-IFNR1 blocking antibody compared with control group. Moreover, ultrastructural observations of these organs show an obvious increase in apoptotic cells from mice treated with anti-IFNR1 blocking antibody. Our results provide more details about the triggered signalling pathways and the phosphorylation cascade which are involved in the protection of B cell from apoptosis after treatment with IFN-alpha/beta. Copyright 2010 Elsevier Inc. All rights reserved.

Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells.

PubMed

Alper, O; De Santis, M L; Stromberg, K; Hacker, N F; Cho-Chung, Y S; Salomon, D S

2000-11-15

Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness. Copyright 2000 Wiley-Liss, Inc.

Structural domains required for channel function of the mouse transient receptor potential protein homologue TRP1beta.

PubMed

Engelke, Michael; Friedrich, Olaf; Budde, Petra; Schäfer, Christina; Niemann, Ursula; Zitt, Christof; Jüngling, Eberhard; Rocks, Oliver; Lückhoff, Andreas; Frey, Jürgen

2002-07-17

Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca(2+) influx channels by co-assembly. However, little is known which domains facilitate the interaction of subunits. Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1beta (mTRP1beta) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1beta expressing cells exhibited enhanced Ca(2+) influx and enhanced whole-cell membrane currents compared to mTRP1beta deletion mutants. Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus. These results suggest that the N-terminus of mTRP1beta is required for structural organization thus forming functional channels.

Inhibition of in vitro growth and arrest in the G0/G1 phase of HCT8 line human colon cancer cells by kaempferide triglycoside from Dianthus caryophyllus.

PubMed

Martineti, Valentina; Tognarini, Isabella; Azzari, Chiara; Carbonell Sala, Silvia; Clematis, Francesca; Dolci, Marcello; Lanzotti, Virginia; Tonelli, Francesco; Brandi, Maria Luisa; Curir, Paolo

2010-09-01

The effects of phytoestrogens have been studied in the hypothalamic-pituitary-gonadal axis and in various non-gonadal targets. Epidemiologic and experimental evidence indicates a protective effect of phytoestrogens also in colorectal cancer. The mechanism through which estrogenic molecules control colorectal cancer tumorigenesis could possibly involve estrogen receptor beta, the predominantly expressed estrogen receptor subtype in colon mucosa.To validate this hypothesis, we therefore used an engineered human colon cancer cell line induced to overexpress estrogen receptor beta, beside its native cell line, expressing very low levels of ERbeta and not expressing ERalpha; as a phytoestrogenic molecule, we used kaempferide triglycoside, a glycosylated flavonol from a Dianthus caryophyllus cultivar. The inhibitory properties of this molecule toward vegetal cell growth have been previously demonstrated: however, no data on its activity on animal cell or information about the mechanism of this activity are available. Kaempferide triglycoside proved to inhibit the proliferation of native and estrogen receptor beta overexpressing colon cancer cells through a mechanism not mediated by ligand binding dependent estrogen receptor activation. It affected HCT8 cell cycle progression by increasing the G(0)/G(1) cell fraction and in estrogen receptor beta overexpressing cells increased two antioxidant enzymes. Interestingly, the biological effects of this kaempferide triglycoside were strengthened by the presence of high levels of estrogen receptor beta.Pleiotropic molecular effects of phytoestrogens may explain their protective activity against colorectal cancer and may represent an interesting area for future investigation with potential clinical applications. Copyright 2010 John Wiley & Sons, Ltd.

Geraniol and beta-ionone inhibit proliferation, cell cycle progression, and cyclin-dependent kinase 2 activity in MCF-7 breast cancer cells independent of effects on HMG-CoA reductase activity.

PubMed

Duncan, Robin E; Lau, Dominic; El-Sohemy, Ahmed; Archer, Michael C

2004-11-01

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the formation of mevalonate, a precursor of cholesterol that is also required for cell proliferation. Mevalonate depletion results in a G1 phase cell cycle arrest that is mediated in part by impaired activity of cyclin-dependent kinase (CDK) 2, and decreased expression of positive regulators of G1 to S phase progression. Inhibition of mevalonate synthesis may, therefore, be a useful strategy to impair the growth of malignant cells. Plant isoprenoids, including beta-ionone and geraniol, have previously been shown to inhibit rodent mammary tumor development, and rodent and avian hepatic HMG-CoA reductase activity. We hypothesized that the putative anti-proliferative and cell cycle inhibitory effects of beta-ionone and geraniol on MCF-7 human breast cancer cells in culture are mediated by mevalonate depletion resulting from inhibition of HMG-CoA reductase activity. Flow cytometric analysis showed a G1 arrest in isoprenoid-treated MCF-7 cells, and also a G2/M arrest at higher concentrations of isoprenoids. These compounds minimally affected the growth of MCF-10F normal breast epithelial cells. Both beta-ionone and geraniol inhibited CDK 2 activity and dose-dependently decreased the expression of cyclins D1, E, and A, and CDK 2 and 4, without changing the expression of p21cip1 or p27kip1. Although both beta-ionone and geraniol also inhibited MCF-7 proliferation, only geraniol inhibited HMG-CoA reductase activity. While these effects were significantly correlated (r2=0.89, P <0.01), they were not causally related, since exogenous mevalonate did not restore growth in geraniol-inhibited cells. These findings indicate that mechanisms other than impaired mevalonate synthesis mediate the anti-proliferative and cell cycle regulatory effects of beta-ionone and geraniol in human breast cancer cells.

Mechanisms and regulation of polymorphonuclear leukocyte and eosinophil adherence to human airway epithelial cells.

PubMed

Jagels, M A; Daffern, P J; Zuraw, B L; Hugli, T E

1999-09-01

Polymorphonuclear leukocytes (PMN) and eosinophils (Eos) are important cellular participants in a variety of acute and chronic inflammatory reactions in the airway. Histologic evidence has implicated direct interactions between these two subsets of leukocytes and airway epithelial cells during inflammation. A comprehensive characterization and comparison of physiologic stimuli and adhesion molecule involvement in granulocyte-epithelial-cell interactions done with nontransformed human airway epithelial cells has not been reported. We therefore examined the regulation and biochemical mechanisms governing granulocyte-epithelial-cell adhesion, using either purified PMN or Eos and primary cultures of human bronchial epithelial cells (HBECs). We investigated the involvement of a number of proinflammatory signals associated with allergic and nonallergic airway inflammation, as well as the contribution of several epithelial and leukocyte adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and members of the beta(1), beta(2), and beta(7) integrin families. ICAM-1 was expressed at low levels on cultured HBECs and was markedly upregulated after stimulation with interferon (IFN)-gamma or, to a lesser extent, with tumor necrosis factor (TNF)-alpha or interleukin (IL)-1. VCAM-1 was not present on resting HBECs, and was not upregulated after stimulation with IFN-gamma, IL-1, IL-4, or TNF-alpha. PMN adhesion to HBECs could be induced either through activation of PMN with IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), or C5a, but not with IL-5 or by preactivation of HBECs with TNF-alpha or IFN-gamma. Blocking antibody studies indicated that PMN-HBEC adherence depended on beta(2) integrins, primarily alpha(M)beta(2) (Mac-1). Adherence of Eos to HBECs could be induced through activation of Eos with IL-5, GM-CSF, or C5a, but not with IL-8 or by prior activation of HBECs with TNF-alpha of IFN-gamma. Maximal adhesion of Eos and PMN required pretreatment of HBECs with either TNF-alpha or IFN-gamma in addition to leukocyte activation. Adherence of Eos to unstimulated HBECs was mediated through both beta(1) and beta(2) integrins, whereas adhesion of Eos to activated HBECs was dominated by beta(2) integrins. Adhesion of both Eos and PMN was inhibited by treatment of HBECs with blocking antibodies to ICAM-1. Differential utilization of beta(1) and beta(2) integrins by Eos, depending on the activation state of the epithelium, is a novel finding and may affect activation and/or recruitment of Eos in airway tissue. Mechanisms of adhesion of HBECs to Eos and PMN, as evidenced by the different responsiveness of the two latter types of cells to IL-8 and IL-5, may account for a prevalence of Eos over PMN in certain airway diseases.

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.

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

Seomun, Young; Joo, Choun-Ki

Lumican is a major proteoglycans of the human cornea. Lumican knock-out mice have been shown to lose corneal transparency and to display delayed wound healing. The purpose of this study was to define the role of lumican in corneal epithelial cell migration. Over-expression of lumican in human corneal epithelial (HCE-T) cells increased both cell migration and proliferation, and increased levels of integrins {alpha}2 and {beta}1. ERK 1/2 was also activated in lumican over-expressed cells. When we treated HCE-T cells with the ERK-specific inhibitor U0126, cell migration and the expression of integrin {beta}1 were completely blocked. These data provide evidence thatmore » lumican stimulates cell migration in the corneal epithelium by activating ERK 1/2, and point to a novel signaling pathway implicated in corneal epithelial cell migration.« less

A facile and efficient synthesis of some (6E)-hydroximino-4-en-3-one steroids, steroidal oximes from Cinachyrella spp. sponges.

PubMed

Cui, Jianguo; Huang, Liliang; Fan, Lei; Zhou, Aimin

2008-03-01

Using beta-sitosterol as a starting material, (6E)-hydroximino-24-ethylcholest-4-en-3-one (1), a natural steroidal oxime from Cinachyrella alloclada and C. apion, was synthesized in four steps with a high overall yield. First, beta-sitosterol (5a) is transformed into the corresponding 24-ethylcholest-4-en-3,6-dione (6a) via oxidation with pyridinium chlorochromate (PCC). Selective reduction of 6a by NaBH(4) in the presence of CoCl(2) gives 24-ethylcholest- 4-en-3beta-ol-6-one (7a). The reaction of 7a with hydroxylamine hydrochloride offers the oxime 8a and the oxidation of 8a by Jones reagent gives the target steroid 1. (6E)-Hydroximinocholest-4-en-3-one (2) and (6E)-hydroximino-24-ethylcholest-4,22-dien-3-one (4) were synthesized by a similar method. The cytotoxicity of the synthesized 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 were investigated. The presence of a cholesterol-type side chain appears to be necessary for the biological activity.

Crystal Structures of T Cell Receptor (Beta) Chains Related to Rheumatoid Arthritis

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

Li,H.; van Vranken, S.; Zhao, Y.

The crystal structures of the V{beta}17+ {beta} chains of two human T cell receptors (TCRs), originally derived from the synovial fluid (SF4) and tissue (C5-1) of a patient with rheumatoid arthritis (RA), have been determined in native (SF4) and mutant (C5-1{sub F104{yields}Y/C187{yields}S}) forms, respectively. These TCR {beta} chains form homo-dimers in solution and in crystals. Structural comparison reveals that the main-chain conformations in the CDR regions of the C5-1 and SF4 V{beta}17 closely resemble those of a V{beta}17 JM22 in a bound form; however, the CDR3 region shows different conformations among these three V{beta}17 structures. At the side-chain level, conformationalmore » differences were observed at the CDR2 regions between our two ligand-free forms and the bound JM22 form. Other significant differences were observed at the V{beta} regions 8-12, 40-44, and 82-88 between C5-1/SF4 and JM22 V{beta}17, implying that there is considerable variability in the structures of very similar {beta} chains. Structural alignments also reveal a considerable variation in the V{beta}-C{beta} associations, and this may affect ligand recognition. The crystal structures also provide insights into the structure basis of T cell recognition of Mycoplasma arthritidis mitogen (MAM), a superantigen that may be implicated in the development of human RA. Structural comparisons of the V{beta} domains of known TCR structures indicate that there are significant similarities among V{beta} regions that are MAM-reactive, whereas there appear to be significant structural differences among those V{beta} regions that lack MAM-reactivity. It further reveals that CDR2 and framework region (FR) 3 are likely to account for the binding of TCR to MAM.« less

An in vitro bioassay for xenobiotics using the SXR-driven human CYP3A4/lacZ reporter gene.

PubMed

Lee, Mi R; Kim, Yeon J; Hwang, Dae Y; Kang, Tae S; Hwang, Jin H; Lim, Chae H; Kang, Hyung K; Goo, Jun S; Lim, Hwa J; Ahn, Kwang S; Cho, Jung S; Chae, Kap R; Kim, Yong K

2003-01-01

The dose and time effect of nine xenobiotics, including 17beta-estradiol, corticosterone, dexamethasone, progesterone, nifedipine, bisphenol A, rifampicin, methamphetamine, and nicotine were investigated, in vitro, using human steroid and xenobiotics receptor (SXR)-binding sites on the human CYP3A4 promoter, which can enhance the linked lacZ reporter gene transcription. To test this, liver-specific SAP (human serum amyloid P component)-SXR (SAP/SXR) and human CYP3A4 promoter-regulated lacZ (hCYP3A4/lacZ) constructs were transiently transfected into HepG2 and NIH3T3 cells to compare the xenobiotic responsiveness between human and nonhuman cell lines. In the HepG2 cells, rifampicin, followed by corticosterone, nicotine, methamphetamine, and dexamethasone, exhibited enhanced levels of the lacZ transcript, whereas those of bisphenol A and nifedipine were found to be reduced. No significant responses were observed with 17beta-estradiol or progesterone. In addition, 17beta-estradiol and progesterone did not change the levels of the lacZ transcripts in the HepG2 cells, but did induce significant increases in the transcripts of the NIH3T3 cells. Treatment with corticosterone and dexamethasone, which were highly expressed in the HepG2 cells, did not affect the levels of the lacZ transcript in NIH3T3 cells. These results show that lacZ transcripts can be measured, rapidly and reproducibly, using reverse transcriptase-polymerase chain reaction (RT-PCR) based on the expression of the hCYP3A4/lacZ reporter gene, and was mediated by the SXR. Thus, this in vitro reporter gene bioassay is useful for measuring xenobiotic activities, and is a means to a better relevant bioassay, using human cells, human genes and human promoters, in order to get a closer look at actual human exposure.

Molecular basis for the regulation of islet beta cell mass in mice: the role of E-cadherin

PubMed Central

Wakae-Takada, N.; Xuan, S.; Watanabe, K.; Meda, P.; Leibel, R. L.

2014-01-01

Aims/hypothesis In rodents and humans, the rate of beta cell proliferation declines rapidly after birth; formation of the islets of Langerhans begins perinatally and continues after birth. Here, we tested the hypothesis that increasing levels of E-cadherin during islet formation mediate the decline in beta cell proliferation rate by contributing to a reduction of nuclear β-catenin and D-cyclins. Methods We examined E-cadherin, nuclear β-catenin, and D-cyclin levels, as well as cell proliferation during in vitro and in vivo formation of islet cell aggregates, using β-TC6 cells and transgenic mice with green fluorescent protein (GFP)-labelled beta cells, respectively. We tested the role of E-cadherin using antisense-mediated reductions of E-cadherin in β-TC6 cells, and mice segregating for a beta cell-specific E-cadherin knockout (Ecad [also known as Cdh1] βKO). Results In vitro, pseudo-islets of β-TC6 cells displayed increased E-cadherin but decreased nuclear β-catenin and cyclin D2, and reduced rates of cell proliferation, compared with monolayers. Antisense knockdown of E-cadherin increased cell proliferation and levels of cyclins D1 and D2. After birth, beta cells showed increased levels of E-cadherin, but decreased levels of D-cyclin, whereas islets of Ecad βKO mice showed increased levels of D-cyclins and nuclear β-catenin, as well as increased beta cell proliferation. These islets were significantly larger than those of control mice and displayed reduced levels of connexin 36. These changes correlated with reduced insulin response to ambient glucose, both in vitro and in vivo. Conclusions/interpretation The findings support our hypothesis by indicating an important role of E-cadherin in the control of beta cell mass and function. PMID:23354125

Molecular basis for the regulation of islet beta cell mass in mice: the role of E-cadherin.

PubMed

Wakae-Takada, N; Xuan, S; Watanabe, K; Meda, P; Leibel, R L

2013-04-01

In rodents and humans, the rate of beta cell proliferation declines rapidly after birth; formation of the islets of Langerhans begins perinatally and continues after birth. Here, we tested the hypothesis that increasing levels of E-cadherin during islet formation mediate the decline in beta cell proliferation rate by contributing to a reduction of nuclear β-catenin and D-cyclins. We examined E-cadherin, nuclear β-catenin, and D-cyclin levels, as well as cell proliferation during in vitro and in vivo formation of islet cell aggregates, using β-TC6 cells and transgenic mice with green fluorescent protein (GFP)-labelled beta cells, respectively. We tested the role of E-cadherin using antisense-mediated reductions of E-cadherin in β-TC6 cells, and mice segregating for a beta cell-specific E-cadherin knockout (Ecad [also known as Cdh1] βKO). In vitro, pseudo-islets of β-TC6 cells displayed increased E-cadherin but decreased nuclear β-catenin and cyclin D2, and reduced rates of cell proliferation, compared with monolayers. Antisense knockdown of E-cadherin increased cell proliferation and levels of cyclins D1 and D2. After birth, beta cells showed increased levels of E-cadherin, but decreased levels of D-cyclin, whereas islets of Ecad βKO mice showed increased levels of D-cyclins and nuclear β-catenin, as well as increased beta cell proliferation. These islets were significantly larger than those of control mice and displayed reduced levels of connexin 36. These changes correlated with reduced insulin response to ambient glucose, both in vitro and in vivo. The findings support our hypothesis by indicating an important role of E-cadherin in the control of beta cell mass and function.

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 myeloid cells. The expression of steroid metabolizing enzymes in myeloid cells is able to create a microenvironment which is uncoupled from dominating systemic estrogens. These findings may be relevant in the autocrine, paracrine or iuxtacrine cellular crosstalk of myeloid cells in their respective states of terminal differentiation, e.g. in bone metabolism and inflammation.

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.

Effect of prolonged exposure to sublethal concentrations of DDT and DDE on protein expression in human pancreatic beta cells

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

Pavlikova, Nela, E-mail: nela.pavlikova@lf3.cuni.cz; Smetana, Pavel; Halada, Petr

Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cellmore » line exposed to three sublethal concentrations (0.1 μM, 1 μM, 10 μM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10 μM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10 μM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE. - Highlights: • Epidemiologic studies connect pollution with incidence of diabetes mellitus. • We explored the effect of DDT and DDE on protein expression in the NES2Y pancreatic beta cell line. • One month exposure to three sublethal concentrations of DDT and DDE was employed. • Expression of alpha-enolase, actin, cytokeratin 8 and 18 was reduced in NES2Y/DDT. • Expression of HNRH1 and cytokeratin 18 was reduced in NES2Y/DDE.« less

Extensive esterification of adrenal C19-delta 5-sex steroids to long-chain fatty acids in the ZR-75-1 human breast cancer cell line

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

Poulin, R.; Poirier, D.; Merand, Y.

1989-06-05

Estrogen-sensitive human breast cancer cells (ZR-75-1) were incubated with the 3H-labeled adrenal C19-delta 5-steroids dehydroepiandrosterone (DHEA) and its fully estrogenic derivative, androst-5-ene-3 beta,17 beta-diol (delta 5-diol) for various time intervals. When fractionated by solvent partition, Sephadex LH-20 column chromatography and silica gel TLC, the labeled cell components were largely present (40-75%) in three highly nonpolar, lipoidal fractions. Mild alkaline hydrolysis of these lipoidal derivatives yielded either free 3H-labeled DHEA or delta 5-diol. The three lipoidal fractions cochromatographed with the synthetic DHEA 3 beta-esters, delta 5-diol 3 beta (or 17 beta)-monoesters and delta 5-diol 3 beta,17 beta-diesters of long-chain fatty acids.more » DHEA and delta 5-diol were mainly esterified to saturated and mono-unsaturated fatty acids. For delta 5-diol, the preferred site of esterification of the fatty acids is the 3 beta-position while some esterification also takes place at the 17 beta-position. Time course studies show that ZR-75-1 cells accumulate delta 5-diol mostly (greater than 95%) as fatty acid mono- and diesters while DHEA is converted to delta 5-diol essentially as the esterified form. Furthermore, while free C19-delta 5-steroids rapidly diffuse out of the cells after removal of the precursor (3H)delta 5-diol, the fatty acid ester derivatives are progressively hydrolyzed, and DHEA and delta 5-diol thus formed are then sulfurylated prior to their release into the culture medium. The latter process however is rate-limited, since new steady-state levels of free steroids and fatty acid esters are rapidly reached and maintained for extended periods of time after removal of precursor, thus maintaining minimal concentrations of intracellular steroids.« less

Increased neuronal beta-amyloid precursor protein expression in human temporal lobe epilepsy: association with interleukin-1 alpha immunoreactivity.

PubMed

Sheng, J G; Boop, F A; Mrak, R E; Griffin, W S

1994-11-01

Levels of immunoreactive beta-amyloid precursor protein and interleukin-1 alpha were found to be elevated in surgically resected human temporal lobe tissue from patients with intractable epilepsy compared with postmortem tissue from neurologically unaffected patients (controls). In tissue from epileptics, the levels of the 135-kDa beta-amyloid precursor protein isoform were elevated to fourfold (p < 0.05) those of controls and those of the 130-kDa isoform to threefold (p < 0.05), whereas those of the 120-kDa isoform (p > 0.05) were not different from control values. beta-Amyloid precursor protein-immunoreactive neurons were 16 times more numerous, and their cytoplasm and proximal processes were more intensely immunoreactive in tissue sections from epileptics than controls (133 +/- 12 vs. 8 +/- 3/mm2; p < 0.001). However, neither beta-amyloid precursor protein-immunoreactive dystrophic neurites nor beta-amyloid deposits were found in this tissue. Interleukin-1 alpha-immunoreactive cells (microglia) were three times more numerous in epileptics than in controls (80 +/- 8 vs. 25 +/- 5/mm2; p < 0.001), and these cells were often found adjacent to beta-amyloid precursor protein-immunoreactive neuronal cell bodies. Our findings, together with functions established in vitro for interleukin-1, suggest that increased expression of this protein contributes to the increased levels of beta-amyloid precursor protein in epileptics, thus indicating a potential role for both of these proteins in the neuronal dysfunctions, e.g., hyperexcitability, characteristic of epilepsy.

Regulation of glutamate in cultures of human monocytic THP-1 and astrocytoma U-373 MG cells.

PubMed

Klegeris, A; Walker, D G; McGeer, P L

1997-09-01

Glutamate, an excitatory neurotransmitter, is neurotoxic at high concentrations. Neuroglial cells, including astrocytes and microglia, play an important role in regulating its extracellular levels. Cultured human monocytic THP-1 cells increased their glutamate secretion following 18 and 68 h exposure to the inflammatory mediators zymosan, phorbol myristate acetate (PMA), lipopolysaccharide, interferon-gamma, tumor-necrosis factor-alpha and interleukin-1beta. Cultured astrocytoma U-373 MG cells increased their glutamate secretion following similar exposure to zymosan and PMA. DL-Alpha-aminopimelic acid, an inhibitor of the glutamate secretion system, reduced extracellular glutamate in both cell culture systems, while the high-affinity glutamate uptake inhibitors D-Aspartic acid, DL-threo-beta-hydroxyaspartic acid and L-trans-pyrrolidine-2,4-dicarboxylic acid increased extracellular glutamate in U-373 MG, but not THP-1 cell cultures. In co-cultures of THP-1 and U-373 MG cells, extracellular glutamate levels were increased significantly by the Alzheimer beta-amyloid peptide (1-40) and were decreased significantly by the anti-inflammatory drug dexamethasone. These data indicate that inflammatory stimuli may increase extracellular glutamate while antiinflammatory drugs decrease it.

Nicotine promotes cell proliferation via {alpha}7-nicotinic acetylcholine receptor and catecholamine-synthesizing enzymes-mediated pathway in human colon adenocarcinoma HT-29 cells

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

Wong, Helen Pui Shan; Yu Le; Lam, Emily Kai Yee

Cigarette smoking has been implicated in colon cancer. Nicotine is a major alkaloid in cigarette smoke. In the present study, we showed that nicotine stimulated HT-29 cell proliferation and adrenaline production in a dose-dependent manner. The stimulatory action of nicotine was reversed by atenolol and ICI 118,551, a {beta}{sub 1}- and {beta}{sub 2}-selective antagonist, respectively, suggesting the role of {beta}-adrenoceptors in mediating the action. Nicotine also significantly upregulated the expression of the catecholamine-synthesizing enzymes [tyrosine hydroxylase (TH), dopamine-{beta}-hydroxylase (D{beta}H) and phenylethanolamine N-methyltransferase]. Inhibitor of TH, a rate-limiting enzyme in the catecholamine-biosynthesis pathway, reduced the actions of nicotine on cell proliferationmore » and adrenaline production. Expression of {alpha}7-nicotinic acetylcholine receptor ({alpha}7-nAChR) was demonstrated in HT-29 cells. Methyllycaconitine, an {alpha}7-nAChR antagonist, reversed the stimulatory actions of nicotine on cell proliferation, TH and D{beta}H expression as well as adrenaline production. Taken together, through the action on {alpha}7-nAChR nicotine stimulates HT-29 cell proliferation via the upregulation of the catecholamine-synthesis pathway and ultimately adrenaline production and {beta}-adrenergic activation. These data reveal the contributory role {alpha}7-nAChR and {beta}-adrenoceptors in the tumorigenesis of colon cancer cells and partly elucidate the carcinogenic action of cigarette smoke on colon cancer.« less

Targeted overexpression of EZH2 in the mammary gland disrupts ductal morphogenesis and causes epithelial hyperplasia.

PubMed

Li, Xin; Gonzalez, Maria E; Toy, Katherine; Filzen, Tracey; Merajver, Sofia D; Kleer, Celina G

2009-09-01

The Polycomb group protein enhancer of zeste homolog 2 (EZH2), which has roles during development of numerous tissues, is a critical regulator of cell type identity. Overexpression of EZH2 has been detected in invasive breast carcinoma tissue samples and is observed in human breast tissue samples of morphologically normal lobules up to 12 years before the development of breast cancer. The function of EZH2 during preneoplastic progression in the mammary gland is unknown. To investigate the role of EZH2 in the mammary gland, we targeted the expression of EZH2 to mammary epithelial cells using the mouse mammary tumor virus long terminal repeat. EZH2 overexpression resulted in aberrant terminal end bud architecture. By the age of 4 months, 100% of female mouse mammary tumor virus-EZH2 virgin mice developed intraductal epithelial hyperplasia resembling the human counterpart accompanied by premature differentiation of ductal epithelial cells and up-regulation of the luminal marker GATA-3. In addition, remodeling of the mammary gland after parturition was impaired and EZH2 overexpression caused delayed involution. Mechanistically, we found that EZH2 physically interacts with beta-catenin, inducing beta-catenin nuclear accumulation in mammary epithelial cells and activating Wnt/beta-catenin signaling. The biological significance of these data to human hyperplasias is demonstrated by EZH2 up-regulation and colocalization with beta-catenin in human intraductal epithelial hyperplasia, the earliest histologically identifiable precursor of breast carcinoma.

Mesangial cell Fas ligand: upregulation in human lupus nephritis and NF-kappaB-mediated expression in cultured human mesangial cells.

PubMed

Tsukinoki, Tomoko; Sugiyama, Hitoshi; Sunami, Reiko; Kobayashi, Mizuho; Onoda, Tetsuya; Maeshima, Yohei; Yamasaki, Yasushi; Makino, Hirofumi

2004-09-01

Fas ligand (FasL) is a well-known death factor; however, the role of FasL in the regulation of human glomerulonephritis remains unclear. We investigated the renal expression and localization of FasL in various forms of human glomerulonephritis by immunohistochemistry, utilizing confocal laser scanning microscopy. We further evaluated cytokine-induced FasL expression via nuclear factor (NF)kappaB in cultured human mesangial cells (HMC). The level of soluble FasL was measured by a specific enzyme-linked immunosorbent assay (ELISA). The frequency of glomerular FasL-positive cases was higher in lupus nephritis (37.9%) as compared with other forms of glomerulonephritis (8.7%). The glomerular FasL score in proliferative lupus nephritis was significantly higher than that in nonproliferative forms. Patients with a high apoptosis score, severe microhematuria, proteinuria, or decreased renal function had a high FasL score. Double immunolabelling demonstrated that the most prevalent phenotypes of FasL-positive cells were mesangial cells. In cultured HMC, interleukin (IL)1beta, lipopolysaccharide (LPS), or gamma interferon (IFN) upregulated membrane-bound FasL. IL1beta significantly, and LPS or gammaIFN weakly activated NFkappaB, but none of these agents activated NFkappaB/Rel-related nuclear factor of activated T cells (NFATc) or IFN regulatory factor-1. IL1beta-mediated NFkappaB was completely inhibited in the presence of lactacystin, a potent inhibitor of NFkappaB. Lactacystin-mediated inhibition of NFkappaB reduced FasL protein levels. Matrix metalloproteinase (MMP)-7, but not other MMPs (1, 2, 3, 8, or 9), significantly sensitized HMC to release soluble FasL after IL1beta stimulation. The results suggest that: (1) upregulation of mesangial FasL may contribute to the glomerular inflammation in proliferative lupus nephritis in vivo; (2) proinflammatory cytokines, in particular IL1beta, produced in nephritis can upregulate FasL via the transcription factor NFkappaB in HMC; and (3) MMP-7-mediated release of soluble FasL could control the mesangial inflammation.

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.

Effects of alpha/beta-androstenediol immune regulating hormones on bone remodeling and apoptosis in osteoblasts.

PubMed

Urban, Nicole H; Chamberlin, Brett; Ramage, Samuel; Roberts, Zachary; Loria, Roger M; Beckman, Matthew J

2008-06-01

A large body of evidence suggests that the immune system directly impacts bone physiology. We tested whether immune regulating hormones (IRH), 17beta-androstenediol (beta-AED), 7beta,17beta-androstenetriol (beta-AET) or the 17alpha-androstenediol (alpha-AED), and 7alpha,17beta-androstenetriol (alpha-AET) metabolites could directly influence bone remodeling in vitro using human fetal osteoblasts (FOB-9). The impact on bone remodeling was examined by comparing the ratio of RANKL/OPG gene expression in response to AED and AET compounds. The alpha-AED was found to significantly increase in the ratio of RANKL/OPG gene expression and altering the morphology of RANKL stained FOB-9 cells. Cell viability was assessed using a Live/Dead assay. Again alpha-AED was unique in its ability to reduce the proportion of viable cells, and to induce mild apoptosis of FOB-9 cells. Treatment of FOB-9 cells with WY14643, an activator of PPAR-alpha and -gamma, also significantly elevated the percentage of dead cells. This increase was abolished by co-treatment with GW9962, a specific inhibitor of PPAR-gamma. Analysis of PPAR-gamma mRNA by Quantitative RT-PCR and its activation by DNA binding demonstrated that alpha-AED increased PPAR-gamma activation by 19%, while beta-AED conferred a 37% decrease in PPAR-gamma activation. In conclusion, alpha-AED opposed beta-AED by elevating a bone resorption scenario in osteoblast cells. The increase in RANKL/OPG is modulated by an activation of PPAR-gamma that in turn caused mild apoptosis of FOB-9 cells.

Comparison of potentials between stem cells isolated from human anterior cruciate ligament and bone marrow for ligament tissue engineering.

PubMed

Cheng, Ming-Te; Liu, Chien-Lin; Chen, Tain-Hsiung; Lee, Oscar K

2010-07-01

We have previously isolated and identified stem cells from human anterior cruciate ligament (ACL). The purpose of this study was to evaluate the differences in proliferation, differentiation, and extracellular matrix (ECM) formation abilities between bone marrow stem cells (BMSCs) and ACL-derived stem cells (LSCs) from the same donors when cultured with different growth factors, including basic fibroblast growth factor (bFGF), epidermal growth factor, and transforming growth factor-beta 1 (TGF-beta1). Ligament tissues and bone marrow aspirate were obtained from patients undergoing total knee arthroplasty and ACL reconstruction surgeries. Proliferation, colony formation, and population doubling capacity as well as multilineage differentiation potentials of LSCs and BMSCs were compared. Gene expression and ECM production for ligament engineering were also evaluated. It was found that BMSCs possessed better osteogenic differentiation potential than LSCs, while similar adipogenic and chondrogenic differentiation abilities were observed. Proliferation rates of both LSCs and BMSCs were enhanced by bFGF and TGF-beta1. TGF-beta1 treatment significantly increased the expression of type I collagen, type III collagen, fibronectin, and alpha-smooth muscle actin in LSCs, but TGF-beta1 only upregulated type I collagen and tenascin-c in BMSCs. Protein quantification further confirmed the results of differential gene expression and suggested that LSCs and BMSCs increase ECM production upon TGF-beta1 treatment. In summary, in comparison with BMSCs, LSCs proliferate faster and maintain an undifferentiated state with bFGF treatment, whereas under TGF-beta1 treatment, LSCs upregulate major tendinous gene expression and produce a robust amount of ligament ECM protein, making LSCs a potential cell source in future applications of ACL tissue engineering.

Ultraviolet-induced movement of the human DNA repair protein, xeroderma pigmentosum type G, in the nucleus

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

Park, M.S.; Knauf, J.A.; Pendergrass, S.H.

1996-08-06

Xeroderma pigmentosum type G (XPG) is a human genetic disease exhibiting extreme sensitivity to sunlight. XPG patients are defective XPG endonuclease, which is an enzyme essential for DNA repair of the major kinds of solar ultraviolet (UV)-induced DNA damages. Here we describe a novel dynamics of this protein within the cell nucleus after UV irradiation of human cells. USing confocal microscopy, we have localized the immunofluorescent, antigenic signal of XPG protein to foci throughout the cell nucleus. Our biochemical studies also established that XPG protein forms a tight association with nuclear structure(s). In human skin fibroblast cells, the number ofmore » XPG foci decreased within 2 h after UV irradiation, whereas total nuclear XPG fluorescence intensity remained constant, suggesting redistribution of XPG from a limited number of nuclear foci to the nucleus overall. Within 8 h after UV, most XPG antigenic signal was found as foci. Using {beta}-galactosidase-XPG fusion constructs ({beta}-gal-XPG) transfected into HeLa cells, we have identified a single region of XPG that is evidently responsible both for foci formation and for the UV dynamic response. The fusion protein carrying the C terminus of XPG (amino acids 1146-1185) localized {beta}-gal specific antigenic signal to foci and to the nucleolus regions. After UV irradiation, antigenic {beta}-gal translocated reversibly from the subnuclear structures to the whole nucleus with kinetics very similar to the movements of XPG protein. These findings lead us to propose a model in which distribution of XPG protein may regulate the rate of DNA repair within transcriptionally active and inactive compartments of the cell nucleus. 50 refs., 5 figs., 1 tab.« less

Expansion of mesenchymal stem cells from human pancreatic ductal epithelium.

PubMed

Seeberger, Karen L; Dufour, Jannette M; Shapiro, Andrew M James; Lakey, Jonathan R T; Rajotte, Ray V; Korbutt, Gregory S

2006-02-01

Fibroblast-like cells emerging from cultured human pancreatic endocrine and exocrine tissue have been reported. Although a thorough phenotypic characterization of these cells has not yet been carried out, these cells have been hypothesized to be contaminating fibroblasts, mesenchyme and/or possibly beta-cell progenitors. In this study, we expanded fibroblast-like cells from adult human exocrine pancreas following islet isolation and characterized these cells as mesenchymal stem cells (MSCs) based on their cell surface antigen expression and ability to differentiate into mesoderm. Analysis by flow cytometry demonstrated that pancreatic MSCs express cell surface antigens used to define MSCs isolated from bone marrow such as CD13, CD29, CD44, CD49b, CD54, CD90 and CD105. In addition, utilizing protocols used to differentiate MSCs isolated from other somatic tissues, we successfully differentiated pancreatic MSCs into: (1) osteocytes that stained positive for alkaline phosphatase, collagen, mineralization (calcification) and expressed osteocalcin, (2) adipocytes that contained lipid inclusions and expressed fatty acid binding protein 4 and (3) chondrocytes that expressed aggrecan. We also demonstrated that pancreatic MSCs are multipotent and capable of deriving cells of endodermal origin. Pancreatic MSCs were differentiated into hepatocytes that stained positive for human serum albumin and expressed endoderm and liver-specific genes such as GATA 4 and tyrosine aminotransferase. In addition, preliminary protocols used to differentiate these cells into insulin-producing cells resulted in the expression of genes necessary for islet and beta-cell development such as Pax4 and neurogenin 3. Therefore, multipotent MSCs residing within the adult exocrine pancreas could represent a progenitor cell, which when further manipulated could result in the production of functional islet beta-cells.

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

Lu Jiawei; Division of Molecular Medicine, Harbor-UCLA Medical Center, David Geffen School of Medicine, University of California Los Angeles, Torrance, CA 90502; Lu Zhenyu

The corneal endothelial cells form a boundary layer between anterior chamber and cornea. This single cell layer is important to maintain cornea transparency by eliciting net fluid transport into the anterior chamber. Injuries of the corneal endothelial layer in humans lead to corneal swelling and translucence. This hindrance is thought to be due to limited proliferative capacity of the endothelial layer. Fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 2 (TGF-{beta}2) are both found in aqueous humor, and these two cytokines promote and inhibit cell growth, respectively. The intracellular signaling mechanisms by which TGF-{beta}2 suppresses the mitogenic response tomore » FGF-2, however, remain unclear. We have addressed this question by investigating potential crosstalk between FGF-2-induced and TGF-{beta}2-regulated intracellular signaling events in cultured bovine corneal endothelial (BCE) cells. We found that TGF-{beta}2 and FGF-2 oppositely affect BCE cell proliferation and TGF-{beta}2 can override the stimulating effects of FGF-2 by increasing COX-2 expression in these cells. Consistent with these findings, overexpression of COX-2 significantly reduced FGF-2-induced cell proliferation whereas a COX-2 specific inhibitor NS398 reversed the effect of TGF-{beta}2 on FGF-2-induced cell proliferation. The COX-2 product prostaglandin E2 (PGE-2) blocks FGF-2-induced cell proliferation. Whereas FGF-2 stimulates cell proliferation by activating the AKT pathway, TGF-{beta}2 and PGE-2 both inhibit this pathway. In accordance with the effect of PGE-2, cAMP also inhibits FGF-2-induced AKT activation. These findings suggest that the mitogenic response to FGF-2 in vivo in the corneal endothelial layer may be inhibited by TGF-{beta}2-induced suppression of the PI3-kinase/AKT signaling pathway.« less

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans.

PubMed

MacDonald, Patrick E; De Marinis, Yang Zhang; Ramracheya, Reshma; Salehi, Albert; Ma, Xiaosong; Johnson, Paul R V; Cox, Roger; Eliasson, Lena; Rorsman, Patrik

2007-06-01

Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 muM) stimulated glucagon secretion, whereas high concentrations (>10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

Method of increasing radiation sensitivity by inhibition of beta one integrin

DOEpatents

Park, Catherine [San Francisco, CA; Bissell, Mina J [Berkeley, CA

2009-11-17

A method for increasing or monitoring apoptosis in tumor cells by the co-administration of ionizing radiation and an anti-integrin antibody. Increasing apoptosis reduces tumor growth in vivo and in a cell culture model. The antibody is directed against the beta-1 integrin subunit and is inhibitory of beta-1 integrin signaling. Other molecules having an inhibitory effect on beta-1 integrin, either in signaling or in binding to its cognate extracellular receptors may also be used. The present method is particularly of interest in treatment of tumor cells associated with breast cancer, wherein radiation is currently used alone. The present method further contemplates a monoclonal antibody suitable for human administration that may further comprise a radioisotope attached thereto.

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.

Localization of the human {beta}-catenin gene (CTNNB1) to 3p21: A region implicated in tumor development

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

Kraus, C.; Liehr, T.; Ballhausen, G.

1994-09-01

The human {beta}-catenin locus (CTNNB1) was mapped by in situ fluorescence analysis to band p21 on the short arm of chromosome 3, a region frequently affected by somatic alterations in a variety of tumors. PCR primers for the genomic amplification of {beta}-catenin sequences were selected on the basis of homology to exon 4 of the Drosophila armadillo gene. Analysis of a panel of somatic cell hybrids confirmed the localization of {beta}-catenin on human chromosome 3. Furthermore, exclusion mapping of three hybrids carrying defined fragments of the short arm of human chromosome 3 allowed us to determine the position of themore » CTNNB1 locus close to the marker D3S2 in 3p21. 22 refs., 3 figs.« less

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 and Ski interact in these cells. Ski levels dramatically decreased upon TGF-beta treatment of HKc/HPV16, and overexpression of Ski eliminated the ability of TGF-beta to inhibit the URR. Based on these studies, we propose that TGF-beta inhibition of HPV16 early gene expression is mediated by a decrease in Ski levels, which in turn dramatically reduces NFI activity.

The role of IL-1beta in reduced IL-7 production by stromal and epithelial cells: a model for impaired T-cell numbers in the gut during HIV-1 infection.

PubMed

Thang, P H; Ruffin, N; Brodin, D; Rethi, B; Cam, P D; Hien, N T; Lopalco, L; Vivar, N; Chiodi, F

2010-08-01

Interleukin (IL)-7 is a key cytokine in T-cell homeostasis. Stromal cells, intestinal epithelial cells and keratinocytes are known to produce this cytokine. The mechanisms and cellular factors regulating IL-7 production are still unclear. We assessed whether IL-1beta and interferon (IFN)-gamma, cytokines produced during inflammatory conditions, may impact on IL-7 production. We used human intestinal epithelial cells (DLD-1 cell line) and bone marrow stromal cells (HS27 cell line), known to produce IL-7; IL-7 production was evaluated at the mRNA and protein levels. To assess whether treatment of HS27 cells with IL-1beta and/or IFN-gamma leads to changes in the gene expression of cytokines, Toll-like receptors (TLRs) and chemokines, we analysed gene expression profiles using the whole-genome microarray Human Gene 1.0 ST. We found that IFN-gamma enhanced the expression of IL-7 mRNA (P < 0.001) in both cell lines. IL-1beta treatment led to a significant down-regulation (P < 0.001) of IL-7 mRNA expression in both cell lines. The IL-7 concentration in supernatants collected from treated DLD-1 and HS27 cell cultures reflected the trend of IL-7 mRNA levels. The gene profiles revealed dramatic changes in expression of cytokines and their receptors (IL-7/IL-7R alpha; IL-1alpha,IL-1beta/IL-1R1; IFN-gamma/IFN-gammaR1), of IFN regulatory factors (IRF-1 and 2), of TLRs and of important chemo-attractants for T cells. The microarray results were verified by additional methods. Our results are discussed in the setting of inflammation and T-cell survival in the gut compartment during HIV-1 infection where stromal and epithelial cells may produce factors that contribute to impaired IL-7 homeostasis and homing of T cells.

Separation of cell survival, growth, migration, and mesenchymal transdifferentiation effects of fibroblast secretome on tumor cells of head and neck squamous cell carcinoma.

PubMed

Metzler, Veronika Maria; Pritz, Christian; Riml, Anna; Romani, Angela; Tuertscher, Raphaela; Steinbichler, Teresa; Dejaco, Daniel; Riechelmann, Herbert; Dudás, József

2017-11-01

Fibroblasts play a central role in tumor invasion, recurrence, and metastasis in head and neck squamous cell carcinoma. The aim of this study was to investigate the influence of tumor cell self-produced factors and paracrine fibroblast-secreted factors in comparison to indirect co-culture on cancer cell survival, growth, migration, and epithelial-mesenchymal transition using the cell lines SCC-25 and human gingival fibroblasts. Thereby, we particularly focused on the participation of the fibroblast-secreted transforming growth factor beta-1.Tumor cell self-produced factors were sufficient to ensure tumor cell survival and basic cell growth, but fibroblast-secreted paracrine factors significantly increased cell proliferation, migration, and epithelial-mesenchymal transition-related phenotype changes in tumor cells. Transforming growth factor beta-1 generated individually migrating disseminating tumor cell groups or single cells separated from the tumor cell nest, which were characterized by reduced E-cadherin expression. At the same time, transforming growth factor beta-1 inhibited tumor cell proliferation under serum-starved conditions. Neutralizing transforming growth factor beta antibody reduced the cell migration support of fibroblast-conditioned medium. Transforming growth factor beta-1 as a single factor was sufficient for generation of disseminating tumor cells from epithelial tumor cell nests, while other fibroblast paracrine factors supported tumor nest outgrowth. Different fibroblast-released factors might support tumor cell proliferation and invasion, as two separate effects.

Barley and Oat beta-Glucan content measured by Calcofluor fluorescence in a microplate assay

USDA-ARS?s Scientific Manuscript database

Beta-glucans, linear glucan polymers of mixed linkage, are important constituents of cereal cell walls. They have important health benefits in the human diet, but also can negatively affect the use of barley grain as an animal feed. High beta-glucans in barley malt can also cause problems in brewi...

Suppression of proliferation and neurite extension of human neuroblastoma SH-SY5Y cells on immobilized Psathyrella velutina lectin.

PubMed

Kitamura, Noriaki; Ikekita, Masahiko; Hayakawa, Satoru; Funahashi, Hisayuki; Furukawa, Kiyoshi

2004-02-01

Glycoproteins from mammalian brain tissues contain unique N-linked oligosaccharides terminating with beta-N-acetylglucosamine residues. Lectin blot analysis of membrane glycoprotein samples from human neuroblastoma SH-SY5Y cells showed that several protein bands bind to Psathylera velutina lectin (PVL), which interacts with beta-N-acetylglucosamine-terminating oligosaccharides. No lectin positive bands were detected by digestion with jack bean beta-N-acetyl-hexosaminidase or N-glycanase before incubation with the lectin, indicating that the cells contain beta-N-acetylglucosamine-terminating N-linked oligosaccharides. When cells were cultured in dishes with different concentrations of PVL, the cell proliferation was inhibited in a dose-dependent manner. Similarly, the neurite extension, which was stimulated with nerve growth factor, was also inhibited in a manner dependent on the lectin dose. Cell proliferation and neurite extension were recovered by the addition of 10 mM N-acetylglucosamine into the medium. Immunoblot analysis of the activation of mitogen-activated protein (MAP) kinases and protein kinase C revealed that phosphorylation of 42-kDa and 44-kDa MAP kinases and 80-kDa protein kinase C are inhibited when SH-SY5Y cells are cultured in PVL-coated dishes, but are restored by the addition of the haptenic sugar into the medium, indicating that MAP kinase and protein kinase C pathways are inhibited by interaction with immobilized PVL. These results indicate that beta-N-acetylglucosamine-terminating N-linked oligosaccharides expressed on neural cells can induce intracellular signals upon binding to extracellular receptors, and are important for growth regulation of neural cells. Copyright 2003 Wiley-Liss, Inc.

Role of {alpha}{sub v}{beta}{sub 5} integrin receptor in endocytosis of crocidolite and its effect on intracellular glutathione levels in human lung epithelial (A549) cells

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

Pande, Priyadarshini; Mosleh, Tariq A.; Aust, Ann E.

Crocidolite, containing 27% iron by weight, is the most carcinogenic form of asbestos. Crocidolite fibers are endocytized by {alpha}{sub v}{beta}{sub 5} integrin receptors in rabbit pleural mesothelial cells. We show here that crocidolite fibers are endocytized in human lung epithelial (A549) cells and in primary small airway epithelial (SAEC) cells. Presence of the integrin {alpha}{sub v}{beta}{sub 5} blocking antibody, P1F6, significantly reduced the uptake of crocidolite fibers in A549 cells. Thus, the integrin {alpha}{sub v}{beta}{sub 5} receptor is involved in endocytosis of crocidolite fibers in A549 cells as well. Previously, it has been observed that asbestos fibers lead to changesmore » in the intracellular redox environment, i.e. a marked decrease in intracellular glutathione concentrations and an increase in the extracellular glutathione in A549 cells. In addition, the decrease in intracellular glutathione was found to be largely independent of iron present on the surface of the fiber. A549 cells were treated with crocidolite in the presence of endocytosis inhibitor cytochalasin D. Our data indicate that, upon preventing endocytosis, we were able to reverse the decrease in total intracellular glutathione. The decrease in total intracellular glutathione could also be prevented in the presence of the monoclonal antibody P1F6. Thus, we observed that endocytosis of crocidolite fibers via integrin {alpha}{sub v}{beta}{sub 5} receptor is linked to the marked decrease in total intracellular glutathione in A549 cells.« less

Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

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

Soeda, Junpei; Morgan, Maelle; McKee, Chad

Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells inmore » the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by mecamylamine. {alpha}1 and {alpha}3-nAChR mRNA expression was significantly upregulated in NASH fibrosis compared to normal livers. Conclusion: Nicotine at levels in smokers' blood is pro-fibrogenic, through actions on hHSCs expressed nAChRs. Therefore, CS, via its nicotine content, may worsen liver fibrosis. Moreover, nicotinic receptor antagonists may have utility as novel anti-fibrotic agents.« less

Proteolytic processing of endogenous and recombinant beta 4 integrin subunit

PubMed Central

1992-01-01

The alpha 6 beta 4 integrin is a receptor involved in the interaction of epithelial cells with basement membranes. This integrin is unique among the known integrins in that its beta 4 subunit has a large cytoplasmic domain. The function of this cytoplasmic domain is not known. In this paper we show that the beta 4 subunit undergoes proteolytic processing in cultured cells and provide evidence that this also happens in tissues. Immunoprecipitation experiments indicated that the cytoplasmic domain of beta 4 is susceptible to a calcium-dependent protease present in cellular extracts. In vitro assays with purified calpain showed that this enzyme can cleave beta 4 at two distinct sites in the cytoplasmic domain, generating truncated molecules of 165 and 130 kD. Immunoblotting experiments performed on cultured epithelial cells using an antibody to a peptide modeled after the COOH-terminus of the beta 4 subunit showed 70-kD fragments and several fragments of molecular masses between 185 and 115 kD. Similar fragments were detected in CHO cells transfected with the full-length beta 4 cDNA, but not in control transfected cells or in cells transfected with a mutant cDNA lacking the epitope of the cytoplasmic peptide antibody. The sizes of the fragments indicated that both the intracellular and extracellular domains of beta 4 are proteolytically processed. To examine the processing of the beta 4 subunit in epithelial tissues in vivo, human skin frozen sections were stained with antibodies to the ectodomain or the cytoplasmic domain of beta 4. The distinct staining patterns obtained with the two types of antibodies provided evidence that beta 4 is proteolytically processed in vivo in skin. Analogous experiments performed on sections of the cornea suggested that beta 4 is not proteolytically processed at a detectable level in this tissue. Thus, cleavage of the beta 4 subunit occurs in a tissue-specific fashion. These results suggest a potential mechanism of modulating the activities of the alpha 6 beta 4 integrin. PMID:1500432

RANTES release by human airway smooth muscle: effects of prostaglandin E(2) and fenoterol.

PubMed

Lazzeri, N; Belvisi, M G; Patel, H J; Chung, K F; Yacoub, M H; Mitchell, J A

2001-12-21

In human airway smooth muscle cells, the levels of RANTES were increased upon stimulation with interleukin-1beta together with tumour necrosis factor-alpha (TNF-alpha) (10 ng ml(-1) for each). In this study, we have assessed the effects of prostaglandin E(2) and the beta(2)-adrenoceptor agonist, fenoterol on RANTES (regulated upon activation, normal T cell expressed and secreted) release by these cells. The levels of RANTES released by human airway smooth muscle cells were measured after 24 h of treatment. Prostaglandin E(2) and fenoterol, only in presence of a cyclo-oxygenase inhibitor indomethacin (10(-6) M), provoked a concentration-dependent reduction in RANTES release. These data suggest that, in settings where cyclo-oxygenase activity is low, both drugs may relieve the symptoms of airway diseases by reducing RANTES production.

Suppression of IL-1beta-induced COX-2 expression by trichostatin A (TSA) in human endometrial stromal cells.

PubMed

Wu, Yan; Guo, Sun-Wei

2007-11-01

Over-production of cyclooxygenase-2 (COX-2) plays an important role in the positive feedback loop that leads to proliferation and inflammation in endometriosis. Following our observation that histone deacetylase inhibitors (HDACIs) trichostatin A (TSA) and valproic acid (VPA) can suppress proliferation of endometrial stromal cells, we sought to determine whether TSA suppresses IL-1beta-induced COX-2 expression in endometrial stromal cells. In vitro study using a recently established immortalized endometrial stromal cell line. The stromal cells were pretreated with TSA before stimulation with IL-1beta, and COX-2 gene and protein expression was measured by real-time quantitative RT-PCR and Western blot analysis, respectively. IL-1beta stimulated COX-2 expression in a concentration-dependent manner in endometrial stromal cells. The induced COX-2 gene and protein expression were suppressed by TSA pretreatment. TSA suppresses IL-1beta-induced COX-2 gene and protein expression in endometrial stromal cells. This finding, coupled with the findings that TSA and another HDACI, valproic acid, suppress proliferation and induce cell cycle arrest, suggests that HDACIs are a promising class of compound that has therapeutic potential for endometriosis.

Transcription of Epstein-Barr virus-encoded nuclear antigen 1 promoter Qp is repressed by transforming growth factor-beta via Smad4 binding element in human BL cells.

PubMed

Liang, C L; Tsai, C N; Chung, P J; Chen, J L; Sun, C M; Chen, R H; Hong, J H; Chang, Y S

2000-11-10

In Epstein-Barr virus (EBV)-infected BL cells, the oncogenic EBV-encoded nuclear antigen 1 (EBNA 1) gene is directed from the latent promoter Qp. Yeast one-hybrid screen analysis using the -50 to -37 sequence of Qp as the bait was carried out to identify transcriptional factors that may control Qp activity. Results showed that Smad4 binds the -50 to -37 sequence of Qp, indicating that this promoter is potentially regulated by TGF-beta. The association of Smad4 with Qp was further confirmed by supershift of EMSA complexes using Smad4-specific antibody. The transfection of a Qp reporter construct in two EBV(+) BL cell lines, Rael and WW2, showed that Qp activity is repressed in response to the TGF-beta treatment. This repression involves the interaction of a Smad3/Smad4 complex and the transcriptional repressor TGIF, as determined by cotransfection assay and coimmunoprecipitation analysis. Results suggest that TGF-beta may transcriptionally repress Qp through the Smad4-binding site in human BL cells. Copyright 2000 Academic Press.

Identification of Human Islet Amyloid Polypeptide as a BACE2 Substrate

PubMed Central

Rulifson, Ingrid C.; Cao, Ping; Miao, Li; Kopecky, David; Huang, Linda; White, Ryan D.; Samayoa, Kim; Gardner, Jonitha; Wu, Xiaosu; Chen, Kui; Tsuruda, Trace; Homann, Oliver; Baribault, Helene; Yamane, Harvey; Carlson, Tim; Wiltzius, Jed; Li, Yang

2016-01-01

Pancreatic amyloid formation by islet amyloid polypeptide (IAPP) is a hallmark pathological feature of type 2 diabetes. IAPP is stored in the secretory granules of pancreatic beta-cells and co-secreted with insulin to maintain glucose homeostasis. IAPP is innocuous under homeostatic conditions but imbalances in production or processing of IAPP may result in homodimer formation leading to the rapid production of cytotoxic oligomers and amyloid fibrils. The consequence is beta-cell dysfunction and the accumulation of proteinaceous plaques in and around pancreatic islets. Beta-site APP-cleaving enzyme 2, BACE2, is an aspartyl protease commonly associated with BACE1, a related homolog responsible for amyloid processing in the brain and strongly implicated in Alzheimer’s disease. Herein, we identify two distinct sites of the mature human IAPP sequence that are susceptible to BACE2-mediated proteolytic activity. The result of proteolysis is modulation of human IAPP fibrillation and human IAPP protein degradation. These results suggest a potential therapeutic role for BACE2 in type 2 diabetes-associated hyperamylinaemia. PMID:26840340

Inhibition of estrogen-responsive gene activation by the retinoid X receptor beta: evidence for multiple inhibitory pathways.

PubMed

Segars, J H; Marks, M S; Hirschfeld, S; Driggers, P H; Martinez, E; Grippo, J F; Brown, M; Wahli, W; Ozato, K

1993-04-01

The retinoid X receptor beta (RXR beta; H-2RIIBP) forms heterodimers with various nuclear hormone receptors and binds multiple hormone response elements, including the estrogen response element (ERE). In this report, we show that endogenous RXR beta contributes to ERE binding activity in nuclear extracts of the human breast cancer cell line MCF-7. To define a possible regulatory role of RXR beta regarding estrogen-responsive transcription in breast cancer cells, RXR beta and a reporter gene driven by the vitellogenin A2 ERE were transfected into estrogen-treated MCF-7 cells. RXR beta inhibited ERE-driven reporter activity in a dose-dependent and element-specific fashion. This inhibition occurred in the absence of the RXR ligand 9-cis retinoic acid. The RXR beta-induced inhibition was specific for estrogen receptor (ER)-mediated ERE activation because inhibition was observed in ER-negative MDA-MB-231 cells only following transfection of the estrogen-activated ER. No inhibition of the basal reporter activity was observed. The inhibition was not caused by simple competition of RXR beta with the ER for ERE binding, since deletion mutants retaining DNA binding activity but lacking the N-terminal or C-terminal domain failed to inhibit reporter activity. In addition, cross-linking studies indicated the presence of an auxiliary nuclear factor present in MCF-7 cells that contributed to RXR beta binding of the ERE. Studies using known heterodimerization partners of RXR beta confirmed that RXR beta/triiodothyronine receptor alpha heterodimers avidly bind the ERE but revealed the existence of another triiodothyronine-independent pathway of ERE inhibition. These results indicate that estrogen-responsive genes may be negatively regulated by RXR beta through two distinct pathways.

Characterization of Acyl-CoA Synthetase Isoforms In Pancreatic Beta Cells: Gene Silencing Shows Participation of ACSL3 and ACSL4 In Insulin Secretion

PubMed Central

Ansari, Israr-ul H.; Longacre, Melissa J.; Stoker, Scott W.; Kendrick, Mindy A.; O’Neill, Lucas M.; Zitur, Laura J.; Fernandez, Luis A.; Ntambi, James M.; MacDonald, Michael J.

2017-01-01

Long-chain acyl-CoA synthetases (ACSLs) convert fatty acids to fatty acyl-CoAs to regulate various physiologic processes. We characterized the ACSL isoforms in a cell line of homogeneous rat beta cells (INS-1 832/13 cells) and human pancreatic islets. ACSL4 and ACSL3 proteins were present in the beta cells and human and rat pancreatic islets and concentrated in insulin secretory granules and less in mitochondria and negligible in other intracellular organelles. ACSL1 and ACSL6 proteins were not seen in INS-1 832/13 cells or pancreatic islets. ACSL5 protein was seen only in INS-1 832/13 cells. With shRNA-mediated gene silencing we developed stable ACSL knockdown cell lines from INS-1 832/13 cells. Glucose-stimulated insulin release was inhibited ~ 50% with ACSL4 and ACSL3 knockdown and unaffected in cell lines with knockdown of ACSL5, ACLS6 and ACSL1. Lentivirus shRNA-mediated gene silencing of ACSL4 and ACSL3 in human pancreatic islets inhibited glucose-stimulated insulin release. ACSL4 and ACSL3 knockdown cells showed inhibition of ACSL enzyme activity more with arachidonate than with palmitate as a substrate, consistent with their preference for unsaturated fatty acids as substrates. ACSL4 knockdown changed the patterns of fatty acids in phosphatidylserines and phosphatidylethanolamines. The results show the involvement of ACLS4 and ACLS3 in insulin secretion. PMID:28193492

Induction of insulin-producing cells from human pancreatic progenitor cells.

PubMed

Noguchi, H; Naziruddin, B; Shimoda, M; Fujita, Y; Chujo, D; Takita, M; Peng, H; Sugimoto, K; Itoh, T; Tamura, Y; Olsen, G S; Kobayashi, N; Onaca, N; Hayashi, S; Levy, M F; Matsumoto, S

2010-01-01

We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we sought to identify and isolate human pancreatic stem/progenitor cells. We also tested whether growth factors and protein transduction of pancreatic and duodenal homeobox factor-1 (PDX-1) and BETA2/NeuroD into human pancreatic stem/progenitor cells induced insulin or pancreas-related gene expressions. Human pancreata from brain-dead donors were used for islet isolation with the standard Ricordi technique modified by the Edmonton protocol. The cells from a duct-rich population were cultured in several media, based on those designed for mouse pancreatic or for human embryonic stem cells. To induce cell differentiation, cells were cultured for 2 weeks with exendin-4, nicotinamide, keratinocyte growth factor, PDX-1 protein, or BETA2/NeuroD protein. The cells in serum-free media showed morphologies similar to a mouse pancreatic stem cell line, while the cells in the medium for human embryonic stem cells formed fibroblast-like morphologies. The nucleus/cytoplasm ratios of the cells in each culture medium decreased during the culture. The cells stopped dividing after 30 days, suggesting that they had entered senescence. The cells treated with induction medium differentiated into insulin-producing cells, expressing pancreas-related genes. Duplications of cells from a duct-rich population were limited. Induction therapy with several growth factors and transduction proteins might provide a potential new strategy for induction of transplantable insulin-producing cells. Copyright 2010 Elsevier Inc. All rights reserved.

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.

Applied Developmental Biology: Making Human Pancreatic Beta Cells for Diabetics.

PubMed

Melton, Douglas A

2016-01-01

Understanding the genes and signaling pathways that determine the differentiation and fate of a cell is a central goal of developmental biology. Using that information to gain mastery over the fates of cells presents new approaches to cell transplantation and drug discovery for human diseases including diabetes. © 2016 Elsevier Inc. All rights reserved.

Putative signaling action of amelogenin utilizes the Wnt/beta-catenin pathway.

PubMed

Matsuzawa, M; Sheu, T-J; Lee, Y-J; Chen, M; Li, T-F; Huang, C T; Holz, J D; Puzas, J E

2009-06-01

While it has long been known that amelogenin is essential for the proper development of enamel, its role has generally been seen as structural in nature. However, our new data implicate this protein in the regulation of cell signaling pathways in periodontal ligament cells and osteoblasts. In this article we report the successful purification of a recombinant mouse amelogenin protein and demonstrate that it has signaling activity in isolated mouse calvarial cells and human periodontal ligament cells. To determine the regulatory function of canonical Wnt signaling by amelogenin, we used TOPGAL transgenic mice. These mice express a beta-galactosidase transgene under the control of a LEF/TCF and beta-catenin-inducible promoter. To investigate in greater detail the molecular mechanisms involved in the beta-catenin signaling pathway, isolated osteoblasts and periodontal ligament cells were exposed to full-length recombinant mouse amelogenin and were evaluated for phenotypic changes and beta-catenin signaling using a TOPFLASH construct and the LacZ reporter gene. In these in vitro models, we showed that amelogenin can activate beta-catenin signaling. Using the TOPGAL transgenic mouse we showed that amelogenin expression in vivo is localized mainly around the root, the periodontal ligament and the alveolar bone.

Pancreas Volume and Fat Deposition in Diabetes and Normal Physiology: Consideration of the Interplay Between Endocrine and Exocrine Pancreas.

PubMed

Saisho, Yoshifumi

2016-01-01

The pancreas is comprised of exocrine and endocrine components. Despite the fact that they are derived from a common origin in utero, these two compartments are often studied individually because of the different roles and functions of the exocrine and endocrine pancreas. Recent studies have shown that not only type 1 diabetes (T1D), but also type 2 diabetes (T2D), is characterized by a deficit in beta-cell mass, suggesting that pathological changes in the pancreas are critical events in the natural history of diabetes. In both patients with T1D and those with T2D, pancreas mass and exocrine function have been reported to be reduced. On the other hand, pancreas volume and pancreatic fat increase with obesity. Increased beta-cell mass with increasing obesity has also been observed in humans, and ectopic fat deposits in the pancreas have been reported to cause beta-cell dysfunction. Moreover, neogenesis and transdifferentiation from the exocrine to the endocrine compartment in the postnatal period are regarded as a source of newly formed beta-cells. These findings suggest that there is important interplay between the endocrine and exocrine pancreas throughout life. This review summarizes the current knowledge on physiological and pathological changes in the exocrine and endocrine pancreas (i.e., beta-cell mass), and discusses the potential mechanisms of the interplay between the two compartments in humans to understand the pathophysiology of diabetes better.

Pancreas Volume and Fat Deposition in Diabetes and Normal Physiology: Consideration of the Interplay Between Endocrine and Exocrine Pancreas

PubMed Central

Saisho, Yoshifumi

2016-01-01

The pancreas is comprised of exocrine and endocrine components. Despite the fact that they are derived from a common origin in utero, these two compartments are often studied individually because of the different roles and functions of the exocrine and endocrine pancreas. Recent studies have shown that not only type 1 diabetes (T1D), but also type 2 diabetes (T2D), is characterized by a deficit in beta-cell mass, suggesting that pathological changes in the pancreas are critical events in the natural history of diabetes. In both patients with T1D and those with T2D, pancreas mass and exocrine function have been reported to be reduced. On the other hand, pancreas volume and pancreatic fat increase with obesity. Increased beta-cell mass with increasing obesity has also been observed in humans, and ectopic fat deposits in the pancreas have been reported to cause beta-cell dysfunction. Moreover, neogenesis and transdifferentiation from the exocrine to the endocrine compartment in the postnatal period are regarded as a source of newly formed beta-cells. These findings suggest that there is important interplay between the endocrine and exocrine pancreas throughout life. This review summarizes the current knowledge on physiological and pathological changes in the exocrine and endocrine pancreas (i.e., beta-cell mass), and discusses the potential mechanisms of the interplay between the two compartments in humans to understand the pathophysiology of diabetes better. PMID:28012279

Amyloid precursor protein and Presenilin 1 interaction studied by FRET in human H4 cells.

PubMed

Nizzari, Mario; Venezia, Valentina; Bianchini, Paolo; Caorsi, Valentina; Diaspro, Alberto; Repetto, Emanuela; Thellung, Stefano; Corsaro, Alessandro; Carlo, Pia; Schettini, Gennaro; Florio, Tullio; Russo, Claudio

2007-01-01

The mayor pathologic hallmarks of Alzheimer's disease (AD) are senile plaque and neurofibrillary tangles. Senile plaque are primarily made up of deposits of amyloid-beta protein, a proteolytic product derived from the amyloid precursor protein (APP). APP is a transmembrane protein detected into the endoplasmic reticulum, in the Golgi apparatus, at the cell surface, recycled by endocytosis to endosomes, whose physiological function is unclear. Presenilins (PS), are a component of gamma-secretase complex that cleave alpha-CTFs (carboxy-terminal fragment), or beta-CTFs, leaving 40 or 42 amino acids amyloid-beta peptides and 58 or 56 amino acids intracellular domains (AICD). Where the amyloid-beta peptides is generated is not clear. The study of APP-PS interaction in specific cell compartments provides a good opportunity to light upon the molecular mechanisms regulating the activity of the "gamma-secretase complex," and where beta-amyloid is generated. In our study we used a biophysical assay of protein proximity: fluorescence resonance energy transfer (FRET), that can provide information about molecular interactions when two proteins are in the close proximity (<10 nm), to examine the subcellular localization and interaction between APP and PS1 in human neuroglioma cells (H4). Confocal microscopic analysis reveals extensive colocalization in different cells' compartment, and centrosomal or microtubule organizing center (MTOC) localization of APP and PS1, but not necessarily a close molecular interaction. We used FRET to determine if APP and PS1 interact at the cell centrosome. FRET data suggest a close interaction between APP and PS1 in subcellular compartments and at the centrosome of H4 cells. Using this approach we show that APP and PS1 are closely associated in the centrosomes of the H4 cell.

Bidirectional enhancing activities between human T cell leukemia-lymphoma virus type I and human cytomegalovirus in human term syncytiotrophoblast cells cultured in vitro.

PubMed

Tóth, F D; Aboagye-Mathiesen, G; Szabó, J; Liu, X; Mosborg-Petersen, P; Kiss, J; Hager, H; Zdravkovic, M; Andirkó, I; Aranyosi, J

1995-12-01

The syncytiotrophoblast layer of the human placenta has an important role in limiting transplacental viral spread from mother to fetus. Human cytomegalovirus (HCMV) is capable of establishing a latent infection in syncytiotrophoblast cells, with restriction of gene expression to immediate-early and early proteins. We analyzed the extent of replication of human T cell leukemia-lymphoma virus type I (HTLV-I) in human term syncytiotrophoblasts infected with HTLV-I alone or coinfected with HTLV-I and HCMV. Although syncytiotrophoblasts could be infected with cell-free HTLV-I, no viral protein expression was found in the singly infected cells. On the contrary, coinfection of the cells with HTLV-I and HCMV resulted in simultaneous replication of both viruses. Bidirectional enhancing activities between HTLV-I and HCMV were mediated primarily by the Tax and immediate-early proteins, respectively. The stimulatory effect of HTLV-I Tax on HCMV replication appeared to be mediated partly by tumor necrosis factor beta and transforming growth factor beta-1. We observed formation of pseudotypes with HTLV-I nucleocapsids within HCMV envelopes, whereas HCMV was not pseudotyped by HTLV-I envelopes in dually infected syncytiotrophoblast cells. Our data suggest that in vivo dual infection of syncytiotrophoblast cells with HTLV-I and HCMV may facilitate the transplacental transmission of both viruses.

Biotransformation of glycyrrhizin by human intestinal bacteria and its relation to biological activities.

PubMed

Kim, D H; Hong, S W; Kim, B T; Bae, E A; Park, H Y; Han, M J

2000-04-01

The relationship between the metabolites of glycyrrhizin (18beta-glycyrrhetinic acid-3-O-beta-D-glucuronopyranosyl-(1-->2)-beta-D-glucuronide, GL) and their biological activities was investigated. By human intestinal microflora, GL was metabolized to 18beta-glycyrrhetinic acid (GA) as a main product and to 18beta-glycyrrhetinic acid-3-O-beta-D-glucuronide (GAMG) as a minor product. The former reaction was catalyzed by Eubacterium L-8 and the latter was by Streptococcus LJ-22. Among GL and its metabolites, GA and GAMG had more potent in vitro anti-platelet aggregation activity than GL. GA also showed the most potent cytotoxicity against tumor cell lines and the potent inhibitory activity on rotavirus infection as well as growth of Helicobacter pylori. GAMG, the minor metabolite of GL, was the sweetest.

Regulation of the human SLC25A20 expression by peroxisome proliferator-activated receptor alpha in human hepatoblastoma cells

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

Tachibana, Keisuke, E-mail: nya@phs.osaka-u.ac.jp; Takeuchi, Kentaro; Inada, Hirohiko

2009-11-20

Solute carrier family 25, member 20 (SLC25A20) is a key molecule that transfers acylcarnitine esters in exchange for free carnitine across the mitochondrial membrane in the mitochondrial {beta}-oxidation. The peroxisome proliferator-activated receptor alpha (PPAR{alpha}) is a ligand-activated transcription factor that plays an important role in the regulation of {beta}-oxidation. We previously established tetracycline-regulated human cell line that can be induced to express PPAR{alpha} and found that PPAR{alpha} induces the SLC25A20 expression. In this study, we analyzed the promoter region of the human slc25a20 gene and showed that PPAR{alpha} regulates the expression of human SLC25A20 via the peroxisome proliferator responsive element.

3D heterogeneous islet organoid generation from human embryonic stem cells using a novel engineered hydrogel platform.

PubMed

Candiello, Joseph; Grandhi, Taraka Sai Pavan; Goh, Saik Kia; Vaidya, Vimal; Lemmon-Kishi, Maya; Eliato, Kiarash Rahmani; Ros, Robert; Kumta, Prashant N; Rege, Kaushal; Banerjee, Ipsita

2018-05-25

Organoids, which exhibit spontaneous organ specific organization, function, and multi-cellular complexity, are in essence the in vitro reproduction of specific in vivo organ systems. Recent work has demonstrated human pluripotent stem cells (hPSCs) as a viable regenerative cell source for tissue-specific organoid engineering. This is especially relevant for engineering islet organoids, due to the recent advances in generating functional beta-like cells from human pluripotent stem cells. In this study, we report specific engineering of regenerative islet organoids of precise size and cellular heterogeneity, using a novel hydrogel system, Amikagel. Amikagel facilitated controlled and spontaneous aggregation of human embryonic stem cell derived pancreatic progenitor cells (hESC-PP) into robust homogeneous spheroids. This platform further allowed fine control over the integration of multiple cell populations to produce heterogeneous spheroids, which is a necessity for complex organoid engineering. Amikagel induced hESC-PP spheroid formation enhanced pancreatic islet-specific Pdx-1 and NKX6.1 gene and protein expression, while also increasing the percentage of committed population. hESC-PP spheroids were further induced towards mature beta-like cells which demonstrated increased Beta-cell specific INS1 gene and C-peptide protein expression along with functional insulin production in response to in vitro glucose challenge. Further integration of hESC-PP with biologically relevant supporting endothelial cells resulted in multicellular organoids which demonstrated spontaneous maturation towards islet-specific INS1 gene and C-peptide protein expression along with a significantly developed extracellular matrix support system. These findings establish Amikagel -facilitated platform ideal for islet organoid engineering. Copyright © 2018. Published by Elsevier Ltd.

Age-related decline in mitochondrial DNA copy number in isolated human pancreatic islets.

PubMed

Cree, L M; Patel, S K; Pyle, A; Lynn, S; Turnbull, D M; Chinnery, P F; Walker, M

2008-08-01

Pancreatic beta cell function has been shown to decline with age in man. Depletion of mitochondrial DNA (mtDNA) copy number is associated with impaired insulin secretion in pancreatic beta cell lines, and decreased mtDNA copy number has been observed with age in skeletal muscle in man. We investigated whether mtDNA copy number decreases with age in human pancreatic beta cells, which might in turn contribute to the age-related decline in insulin secretory capacity. We quantified mtDNA copy number in isolated human islet preparations from 15 pancreas donors aged between 17 and 75 years. Islets (n = 20) were individually hand-picked and pooled from each donor isolate for the quantification of mtDNA copy number and deleted mtDNA (%), which were determined using real-time PCR methods. There was a significant negative correlation between mtDNA copy number and islet donor age (r = -0.53, p = 0.044). mtDNA copy number was significantly decreased in islet preparations from donors aged > or =50 years (n = 8) compared with those aged <50 years (n = 7) (median [interquartile range]: 418 [236-503] vs 596 [554-729] mtDNA copy number/diploid genome; p = 0.032). None of the islet preparations harboured high levels of deleted mtDNA affecting the major arc. Given the correlation between mtDNA content and respiratory chain activity, the age-related decrease in mtDNA copy number that we observed in human pancreatic islet preparations may contribute to the age-dependent decline in pancreatic beta cell insulin secretory capacity.

Selective inhibition of prostaglandin E2 receptors EP2 and EP4 inhibits adhesion of human endometriotic epithelial and stromal cells through suppression of integrin-mediated mechanisms.

PubMed

Lee, JeHoon; Banu, Sakhila K; Burghardt, Robert C; Starzinski-Powitz, Anna; Arosh, Joe A

2013-03-01

Endometriosis is a chronic gynecological disease of reproductive age women characterized by the presence of functional endometrial tissues outside the uterine cavity. Interactions between the endometriotic cells and the peritoneal extracellular matrix proteins (ECM) are crucial mechanisms that allow adhesion of the endometriotic cells into peritoneal mesothelia. Prostaglandin E2 (PGE2) plays an important role in the pathogenesis of endometriosis. In previous studies, we have reported that selective inhibition of PGE2 receptors PTGER2 and PTGER4 decreases survival and invasion of human endometriotic epithelial and stromal cells through multiple mechanisms. Results of the present study indicates that selective inhibition of PTGER2- and PTGER4-mediated PGE2 signaling 1) decreases the expression and/or activity of specific integrin receptor subunits Itgb1 (beta1) and Itgb3 (beta3) but not Itgb5 (beta5), Itga1 (alpha1), Itga2 (alpha2), Itga5 (alpha5), and Itgav (alphav); 2) decreases integrin-signaling components focal adhesion kinase or protein kinase 2 (PTK2) and talin proteins; 3) inhibits interactions between Itgb1/Itgb3 subunits, PTK2, and talin and PTGER2/PTGER4 proteins through beta-arrestin-1 and Src kinase protein complex in human endometriotic epithelial cells 12Z and stromal cells 22B; and 4) decreases adhesion of 12Z and 22B cells to ECM collagen I, collagen IV, fibronectin, and vitronectin in a substrate-specific manner. These novel findings provide an important molecular framework for further evaluation of selective inhibition of PTGER2 and PTGER4 as potential nonsteroidal therapy to expand the spectrum of currently available treatment options for endometriosis in child-bearing age women.

Expression of a mutation causing hypertrophic cardiomyopathy disrupts sarcomere assembly in adult feline cardiac myocytes.

PubMed

Marian, A J; Yu, Q T; Mann, D L; Graham, F L; Roberts, R

1995-07-01

Mutations in the beta-myosin heavy chain (beta MyHC) induce hypertrophic cardiomyopathy (HCM), cardiac hypertrophy, and sarcomere disarray, with the latter being the characteristic hallmark. Thus, we sought to determine whether expression of mutant beta MyHC in adult feline cardiac myocytes, a species known to develop HCM with a phenotype identical to that in humans, induces sarcomere disarray. A full-length beta MyHC cDNA was cloned from a human heart cDNA library, and an HCM-causing mutation (Arg403Gln) was induced in the beta MyHC cDNA by site-directed mutagenesis using polymerase chain reaction (PCR). The normal and mutant beta MyHC cDNAs were cloned into p delta E1spIB shuttle vector, downstream from a cytomegalovirus (CMV) promoter. Replication-deficient recombinant adenoviral constructs (Ad5/CMV/beta MyHC-N and Ad5/CMV/beta MyHC-403) were generated through homologous recombination of p delta E1spIB/CMV/beta MyHC-N or Ad5/CMV/beta MyHC-403 and pBHG10 after cotransfection in 293 host cells. Infection of COS-1 cells with the beta MyHC construct resulted in the expression of a full-length myosin protein. Efficiency of infection of isolated adult cardiac myocytes was > 95%. Expression of the beta MyHC constructs into mRNA at 48 hours after infection of feline cardiac myocytes was confirmed by reverse transcription-PCR. The net total protein and beta-myosin synthesis were determined by using the amount of incorporation of [3H]phenylalanine into total protein and beta-myosin, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of (R)-2-(2-aminothiazol-4-yl)-4'-{2-[(2-hydroxy-2-phenylethyl)amino]ethyl} acetanilide (YM178), a novel selective beta3-adrenoceptor agonist, on bladder function.

PubMed

Takasu, Toshiyuki; Ukai, Masashi; Sato, Shuichi; Matsui, Tetsuo; Nagase, Itsuro; Maruyama, Tatsuya; Sasamata, Masao; Miyata, Keiji; Uchida, Hisashi; Yamaguchi, Osamu

2007-05-01

We evaluated the pharmacological characteristics of (R)-2-(2-aminothiazol-4-yl)-4'-{2-[(2-hydroxy-2-phenylethyl)amino]-ethyl} acetanilide (YM178). YM178 increased cyclic AMP accumulation in Chinese hamster ovary (CHO) cells expressing human beta3-adrenoceptor (AR). The half-maximal effective concentration (EC50) value was 22.4 nM. EC50 values of YM178 for human beta1- and beta2-ARs were 10,000 nM or more, respectively. The ratio of intrinsic activities of YM178 versus maximal response induced by isoproterenol (nonselective beta-AR agonist) was 0.8 for human beta3-ARs, 0.1 for human beta1-ARs, and 0.1 for human beta2-ARs. The relaxant effects of YM178 were evaluated in rats and humans bladder strips precontracted with carbachol (CCh) and compared with those of isoproterenol and 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride (CGP-12177A) (beta3-AR agonist). EC50 values of YM178 and isoproterenol in rat bladder strips precontracted with 10(-6) M CCh were 5.1 and 1.4 microM, respectively, whereas those in human bladder strips precontracted with 10(-7) M CCh were 0.78 and 0.28 microM, respectively. In in vivo study, YM178 at a dose of 3 mg/kg i.v. decreased the frequency of rhythmic bladder contraction induced by intravesical filling with saline without suppressing its amplitude in anesthetized rats. These findings suggest the suitability of YM178 as a therapeutic drug for the treatment of symptoms of overactive bladder such as urinary frequency, urgency, and urge incontinence.

Restoring Mitochondrial Function: A Small Molecule-mediated Approach to Enhance Glucose Stimulated Insulin Secretion in Cholesterol Accumulated Pancreatic beta cells

NASA Astrophysics Data System (ADS)

Asalla, Suman; Girada, Shravan Babu; Kuna, Ramya S.; Chowdhury, Debabrata; Kandagatla, Bhaskar; Oruganti, Srinivas; Bhadra, Utpal; Bhadra, Manika Pal; Kalivendi, Shasi Vardhan; Rao, Swetha Pavani; Row, Anupama; Ibrahim, A.; Ghosh, Partha Pratim; Mitra, Prasenjit

2016-06-01

Dyslipidemia, particularly the elevated serum cholesterol levels, aggravate the pathophysiology of type 2 diabetes. In the present study we explored the relationship between fasting blood sugar and serum lipid parameters in human volunteers which revealed a significant linear effect of serum cholesterol on fasting blood glucose. Short term feeding of cholesterol enriched diet to rodent model resulted in elevated serum cholesterol levels, cholesterol accumulation in pancreatic islets and hyperinsulinemia with modest increase in plasma glucose level. To explore the mechanism, we treated cultured BRIN-BD11 pancreatic beta cells with soluble cholesterol. Our data shows that cholesterol treatment of cultured pancreatic beta cells enhances total cellular cholesterol. While one hour cholesterol exposure enhances insulin exocytosis, overnight cholesterol accumulation in cultured pancreatic beta cells affects cellular respiration, and inhibits Glucose stimulated insulin secretion. We further report that (E)-4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl) hydrazono) ethyl) phenol (small molecule M1) prevents the cholesterol mediated blunting of cellular respiration and potentiates Glucose stimulated insulin secretion which was abolished in pancreatic beta cells on cholesterol accumulation.

Restoring Mitochondrial Function: A Small Molecule-mediated Approach to Enhance Glucose Stimulated Insulin Secretion in Cholesterol Accumulated Pancreatic beta cells

PubMed Central

Asalla, Suman; Girada, Shravan Babu; Kuna, Ramya S.; Chowdhury, Debabrata; Kandagatla, Bhaskar; Oruganti, Srinivas; Bhadra, Utpal; Bhadra, Manika Pal; Kalivendi, Shasi Vardhan; Rao, Swetha Pavani; Row, Anupama; Ibrahim, A; Ghosh, Partha Pratim; Mitra, Prasenjit

2016-01-01

Dyslipidemia, particularly the elevated serum cholesterol levels, aggravate the pathophysiology of type 2 diabetes. In the present study we explored the relationship between fasting blood sugar and serum lipid parameters in human volunteers which revealed a significant linear effect of serum cholesterol on fasting blood glucose. Short term feeding of cholesterol enriched diet to rodent model resulted in elevated serum cholesterol levels, cholesterol accumulation in pancreatic islets and hyperinsulinemia with modest increase in plasma glucose level. To explore the mechanism, we treated cultured BRIN-BD11 pancreatic beta cells with soluble cholesterol. Our data shows that cholesterol treatment of cultured pancreatic beta cells enhances total cellular cholesterol. While one hour cholesterol exposure enhances insulin exocytosis, overnight cholesterol accumulation in cultured pancreatic beta cells affects cellular respiration, and inhibits Glucose stimulated insulin secretion. We further report that (E)-4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl) hydrazono) ethyl) phenol (small molecule M1) prevents the cholesterol mediated blunting of cellular respiration and potentiates Glucose stimulated insulin secretion which was abolished in pancreatic beta cells on cholesterol accumulation. PMID:27282931

TGF-beta induces connexin43 gene expression in normal murine mammary gland epithelial cells via activation of p38 and PI3K/AKT signaling pathways.

PubMed

Tacheau, Charlotte; Fontaine, Juliette; Loy, Jennifer; Mauviel, Alain; Verrecchia, Franck

2008-12-01

One of the shared physiological roles between TGF-beta and connexin family members is to inhibit epithelial cell cycle progression and consequently, to provide protection against malignant transformation. Herein, we demonstrated that TGF-beta1 induces the expression of connexin43 (Cx43) in normal murine mammary gland (NMuMG) cell lines at the protein and mRNA levels, and transcriptionally. Using overexpression of a truncated dominant-negative form of Cx43, we determined that the modulation of gap junctional communication by TGF-beta1 plays a key role in the control of NMuMG cells proliferation by TGF-beta1. In addition, using overexpression of truncated dominant-negative forms of either Smad2 or Smad3, and MDA-MB-468 human breast carcinoma cells deficient for Smad4, we determined that the Smad cascade is not implicated in TGF-beta1 effect on Cx43 expression. Using specific pharmacologic inhibitors for JNK, ERK, p38, and PI3K/AKT signaling pathways, we demonstrated the cooperative role of p38 and PI3K/AKT signaling in TGF-beta1-induced Cx43 expression and gap junctional communication. Furthermore, transfection of a c-jun antisense expression vector significantly prevented TGF-beta1-induced Cx43 gene expression demonstrating the involvement of c-Jun/AP-1 pathway together with p38 and PI3K/AKT pathways in mediating TGF-beta1-induced Cx43 gene expression.

Effect of heat treatment and enzymatic digestion on the B cell epitopes of cow's milk proteins.

PubMed

Morisawa, Y; Kitamura, A; Ujihara, T; Zushi, N; Kuzume, K; Shimanouchi, Y; Tamura, S; Wakiguchi, H; Saito, H; Matsumoto, K

2009-06-01

Processing milk leads to changes in clinical allergenicity. However, the mechanism by which heat treatment affects the allergenicity of milk proteins is not fully understood. We investigated the effect of heat treatment and enzymatic digestion on the allergenicity of B cell epitopes of milk proteins using a histamine release assay. Human basophils were passively sensitized using sera from 10 patients with allergies to cow's milk. All the patients experienced symptoms immediately after ingesting milk. The human basophils were obtained from umbilical cord blood mononuclear cells after culturing the mononuclear cells for 3-4 weeks in the presence of IL-3. After sensitization with 10% patient sera for 48 h, the cells were stimulated with untreated, heat-treated, or heat-treated and pepsin-and-trypsin-digested beta-lactoglobulin or alpha-casein for 1 h. The histamine concentrations in the supernatants were then measured by radioimmunoassay. Heat treatment alone did not alter the molecular weight of beta-lactoglobulin or alpha-casein. Heat treatment of beta-lactoglobulin significantly increased its susceptibility to enzymatic digestion in a time- and temperature-dependent manner and reduced its ability to induce histamine release from sensitized basophils. Similar findings were not observed for alpha-casein. The combination of heat treatment and enzymatic digestion reduced the abilities of both beta-lactoglobulin and alpha-casein to induce histamine release from passively sensitized basophils. Heat treatment reduced the allergenicity of beta-lactoglobulin by inducing conformational changes and by increasing its susceptibility to enzymatic digestion, both of which disrupted B cell epitopes, whereas heat treatment alone did not alter the allergenicity of alpha-casein.

Growth regulation of simian and human AIDS-related non-Hodgkin's lymphoma cell lines by TGF-β1 and IL-6

PubMed Central

Ruff, Kristin R; Puetter, Adriane; Levy, Laura S

2007-01-01

Background AIDS-related non-Hodgkin's lymphoma (AIDS-NHL) is the second most frequent cancer associated with AIDS, and is a frequent cause of death in HIV-infected individuals. Experimental analysis of AIDS-NHL has been facilitated by the availability of an excellent animal model, i.e., simian Acquired Immunodeficiency Syndrome (SAIDS) in the rhesus macaque consequent to infection with simian immunodeficiency virus. A recent study of SAIDS-NHL demonstrated a lymphoma-derived cell line to be sensitive to the growth inhibitory effects of the ubiquitous cytokine, transforming growth factor-beta (TGF-beta). The authors concluded that TGF-beta acts as a negative growth regulator of the lymphoma-derived cell line and, potentially, as an inhibitory factor in the regulatory network of AIDS-related lymphomagenesis. The present study was conducted to assess whether other SAIDS-NHL and AIDS-NHL cell lines are similarly sensitive to the growth inhibitory effects of TGF-beta, and to test the hypothesis that interleukin-6 (IL-6) may represent a counteracting positive influence in their growth regulation. Methods Growth stimulation or inhibition in response to cytokine treatment was quantified using trypan blue exclusion or colorimetric MTT assay. Intracellular flow cytometry was used to analyze the activation of signaling pathways and to examine the expression of anti-apoptotic proteins and distinguishing hallmarks of AIDS-NHL subclass. Apoptosis was quantified by flow cytometric analysis of cell populations with sub-G1 DNA content and by measuring activated caspase-3. Results Results confirmed the sensitivity of LCL8664, an immunoblastic SAIDS-NHL cell line, to TGF-beta1-mediated growth inhibition, and further demonstrated the partial rescue by simultaneous treatment with IL-6. IL-6 was shown to activate STAT3, even in the presence of TGF-beta1, and thereby to activate proliferative and anti-apoptotic pathways. By comparison, human AIDS-NHL cell lines differed in their responsiveness to TGF-beta1 and IL-6. Analysis of a recently derived AIDS-NHL cell line, UMCL01-101, indicated that it represents immunoblastic AIDS-DLCBL. Like LCL-8664, UMCL01-101 was sensitive to TGF-beta1-mediated inhibition, rescued partially by IL-6, and demonstrated rapid STAT3 activation following IL-6 treatment even in the presence of TGF-beta1. Conclusion These studies indicate that the sensitivity of immunoblastic AIDS- or SAIDS-DLBCL to TGF-beta1-mediated growth inhibition may be overcome through the stimulation of proliferative and anti-apoptotic signals by IL-6, particularly through the rapid activation of STAT3. PMID:17324269

In vitro immunomodulatory effects of cuphiin D1 on human mononuclear cells.

PubMed

Wang, Ching-Chiung; Chen, Lih-Geeng; Yang, Ling-Ling

2002-01-01

Cuphiin D1 (CD1), a macrocyclic hydrolyzable tannin isolated from Cuphea hyssopifolia, has been shown to exert antitumor activity both in vitro and in vivo. Moreover, the antitumor effects of CD1 are not only related to its cytotoxicity to carcinoma cell lines, but also depend on host-mediated mechanisms. In the present study, CD1 was investigated for its effects on the proliferation and cytokine secretion of human peripheral blood mononuclear cells (PBMCs). At concentrations of from 6.25 to 50 micrograms/ml, it enhanced the 3H-thymidine incorporation of concanavalin A (Con A)-stimulated PBMCs in a dose-dependent manner. Excretion of IL-1 beta, IL-2 and TNF-alpha by CD1-stimulated PBMCs was markedly increased in a dose-dependent manner. The results show that CD1 could stimulate PBMCs release of IL-1 beta, IL-2 and TNF-alpha and then activate T cells. Therefore, CD1-activated T cells via IL-1 beta in vitro might account for the host-mediated CD1 mechanism of action.

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

Heat shock protein-90 beta is expressed at the surface of multipotential mesenchymal precursor cells: generation of a novel monoclonal antibody, STRO-4, with specificity for mesenchymal precursor cells from human and ovine tissues.

PubMed

Gronthos, Stan; McCarty, Rosa; Mrozik, Krzysztof; Fitter, Stephen; Paton, Sharon; Menicanin, Danijela; Itescu, Silviu; Bartold, P Mark; Xian, Cory; Zannettino, Andrew C W

2009-11-01

Mesenchymal stromal cells (MSCs) and their precursor cells (MPCs) can proliferate and differentiate into multiple mesodermal and some ectodermal and endodermal tissues. Culture-expanded MSCs are currently being evaluated as a possible cell therapy to replace/repair injured or diseased tissues. While a number of mAb reagents with specificity to human MSCs, including STRO-1, STRO-3 (BLK ALP), CD71 (SH2, SH3), CD106 (VCAM-1), CD166, and CD271, have facilitated the isolation of purified populations of human MSCs from primary tissues, few if any mAb reagents have been described that can be used to isolate equivalent cells from other species. This is of particular relevance when assessing the tissue regenerative efficacy of MSCs in large immunocompetent, preclinical animal models of disease. In light of this, we sought to generate novel monoclonal antibodies (mAb) with specific reactivity against a cell surface molecule that is expressed at high levels by MSCs from different species. Using CD106 (VCAM-1)-selected ovine MSCs as an immunogen, mAb-producing hybridomas were selected for their reactivity to both human and ovine MSCs. One such hybridoma, termed STRO-4, produced an IgG mAb that reacted with <5% of human and ovine bone marrow (BM) mononuclear cells. As a single selection reagent, STRO-4 mAb was able to enrich colony-forming fibroblasts (CFU-F) in both human and ovine BM by 16- and 8-folds, respectively. Cells isolated with STRO-4 exhibited reactivity with markers commonly associated with MSCs isolated by plastic adherence including CD29, CD44, and CD166. Moreover, when placed in inductive culture conditions in vitro, STRO-4(+) MSCs exhibited multilineage differentiation potential and were capable of forming a mineralized matrix, lipid-filled adipocytes, and chondrocytes capable of forming a glycosaminoglycan-rich matrix. Biochemical analysis revealed that STRO-4 identified the beta isoform of heat shock protein-90 (Hsp90beta). In addition to identifying an antibody reagent that identifies a highly conserved epitope expressed by MSCs from different species, our study also points to a potential role for Hsp90beta in MSC biology.

Beta-Catenin and Epithelial Tumors: A Study Based on 374 Oropharyngeal Cancers

PubMed Central

Santoro, Angela; Pannone, Giuseppe; Papagerakis, Silvana; McGuff, H. Stan; Cafarelli, Barbara; Lepore, Silvia; De Maria, Salvatore; Rubini, Corrado; Mattoni, Marilena; Staibano, Stefania; Mezza, Ernesto; De Rosa, Gaetano; Aquino, Gabriella; Losito, Simona; Loreto, Carla; Crimi, Salvatore; Bufo, Pantaleo

2014-01-01

Introduction. Although altered regulation of the Wnt pathway via beta-catenin is a frequent event in several human cancers, its potential implications in oral/oropharyngeal squamous cell carcinomas (OSCC/OPSCC) are largely unexplored. Work purpose was to define association between beta-catenin expression and clinical-pathological parameters in 374 OSCCs/OP-SCCs by immunohistochemistry (IHC). Materials and Methods. Association between IHC detected patterns of protein expression and clinical-pathological parameters was assessed by statistical analysis and survival rates by Kaplan-Meier curves. Beta-catenin expression was also investigated in OSCC cell lines by Real-Time PCR. An additional analysis of the DNA content was performed on 22 representative OSCCs/OPSCCs by DNA-image-cytometric analysis. Results and Discussion. All carcinomas exhibited significant alterations of beta-catenin expression (P < 0.05). Beta-catenin protein was mainly detected in the cytoplasm of cancerous cells and only focal nuclear positivity was observed. Higher cytoplasmic expression correlated significantly with poor histological differentiation, advanced stage, and worst patient outcome (P < 0.05). By Real-Time PCR significant increase of beta-catenin mRNA was detected in OSCC cell lines and in 45% of surgical specimens. DNA ploidy study demonstrated high levels of aneuploidy in beta-catenin overexpressing carcinomas. Conclusions. This is the largest study reporting significant association between beta-catenin expression and clinical-pathological factors in patients with OSCCs/OPSCCs. PMID:24511551

Pit-1/growth hormone factor 1 splice variant expression in the rhesus monkey pituitary gland and the rhesus and human placenta.

PubMed

Schanke, J T; Conwell, C M; Durning, M; Fisher, J M; Golos, T G

1997-03-01

We have examined the expression of Pit-1 messenger RNA (mRNA) splice variants in the nonhuman primate pituitary and in rhesus and human placenta. Full-length complementary DNAs (cDNAs) representing Pit-1 and the Pit-1 beta splice variants were cloned from a rhesus monkey pituitary cDNA library and were readily detectable by RT-PCR with rhesus pituitary gland RNA. The Pit-1T variant previously reported in mouse pituitary tumor cell lines was not detectable in normal rhesus pituitary tissue, although two novel splice variants were detected. A cDNA approximating the rat Pit-1 delta 4 variant was cloned but coded for a truncated and presumably nonfunctional protein. Only by using a nested RT-PCR approach were Pit-1 and Pit-1 beta variants consistently detectable in both human and rhesus placental tissue. The Pit-1 beta variant mRNA was not detectable in JEG-3 choriocarcinoma cells unless the cells were stimulated with 8-Br-cAMP. Immunoblot studies with nuclear extracts from primary rhesus syncytiotrophoblast cultures or JEG-3 choriocarcinoma cells indicated that although mRNA levels were very low, Pit-1 protein was detectable in differentiated cytotrophoblasts, and levels increased after treatment with 8-Br-cAMP. Two major species of Pit-1 protein were detected that corresponded to the two major bands in rat pituitary GH3 cell nuclear extracts. Low levels of slightly larger bands also were seen, which may represent Pit-1 beta protein or phosphorylated species. We conclude that Pit-1 splice variants expressed in the primate pituitary gland differ from those in the rodent gland and that the Pit-1 and Pit-1 beta mRNAs expressed in the placenta give rise to a pattern of protein expression similar to that seen in pituitary cells, which is inducible by treatment with 8-Br-cAMP.

Antibody Response to Serpin B13 Induces Adaptive Changes in Mouse Pancreatic Islets and Slows Down the Decline in the Residual Beta Cell Function in Children with Recent Onset of Type 1 Diabetes Mellitus.

PubMed

Kryvalap, Yury; Lo, Chi-Wen; Manuylova, Ekaterina; Baldzizhar, Raman; Jospe, Nicholas; Czyzyk, Jan

2016-01-01

Type 1 diabetes mellitus (T1D) is characterized by a heightened antibody (Ab) response to pancreatic islet self-antigens, which is a biomarker of progressive islet pathology. We recently identified a novel antibody to clade B serpin that reduces islet-associated T cell accumulation and is linked to the delayed onset of T1D. As natural immunity to clade B arises early in life, we hypothesized that it may influence islet development during that time. To test this possibility healthy young Balb/c male mice were injected with serpin B13 mAb or IgG control and examined for the number and cellularity of pancreatic islets by immunofluorescence and FACS. Beta cell proliferation was assessed by measuring nucleotide analog 5-ethynyl-2'-deoxyuridine (5-EdU) incorporation into the DNA and islet Reg gene expression was measured by real time PCR. Human studies involved measuring anti-serpin B13 autoantibodies by Luminex. We found that injecting anti-serpin B13 monoclonal Ab enhanced beta cell proliferation and Reg gene expression, induced the generation of ∼80 pancreatic islets per animal, and ultimately led to increase in the beta cell mass. These findings are relevant to human T1D because our analysis of subjects just diagnosed with T1D revealed an association between baseline anti-serpin activity and slower residual beta cell function decline in the first year after the onset of diabetes. Our findings reveal a new role for the anti-serpin immunological response in promoting adaptive changes in the endocrine pancreas and suggests that enhancement of this response could potentially help impede the progression of T1D in humans. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

An Epstein-Barr virus-associated superantigen

PubMed Central

1996-01-01

More than 90% of adults are latently infected with Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, a self-limiting lymphoproliferative disease characterized by extensive T cell activation. Reactivation of this herpesvirus during immunosuppression is often associated with oncogenesis. These considerations led us to analyze the early events that occur after exposure of the immune system to EBV. Strong major histocompatibility complex (MHC) class II- dependent but not MHC-restricted, T cell proliferation was observed in vitro in response to autologous, lytically infected EBV-transformed B cells. By measuring the appearance of the early activation marker CD69 on individual T cell V beta subsets, we could demonstrate selective activation of human V beta 13- T cells. This was confirmed with murine T cell hybridomas expressing various human BV genes. While EBV- Burkitt's lymphoma cells were nonstimulatory, they induced V beta- restricted T cell activation after EBV infection. EBV specific activation was also demonstrated in cord blood cells, excluding a recall-antigen response. Thus, all of the characteristics of a superantigen-stimulated response are seen, indicating that induction of the EBV lytic cycle is associated with the expression of a superantigen in B cells. A model is presented proposing a role for the superantigen in infection, latency, and oncogenesis. PMID:9064357

beta 2-glycoprotein I (apolipoprotein H) modulates uptake and endocytosis associated chemiluminescence in rat Kupffer cells.

PubMed

Gomes, L F; Gonçalves, L M; Fonseca, F L A; Celli, C M; Videla, L A; Chaimovich, H; Junqueira, V B C

2002-07-01

beta 2-Glycoprotein I (beta 2 GPI) is known to influence macrophage uptake of particles with phosphatidylserine containing surfaces, as apoptotic thymocytes and unilamellar vesicles in vitro. Nevertheless, effects upon macrophage activation induced by this interaction are still unknown. beta 2 GPI influence upon the reactive species production by Kupffer cells was evaluated in order to investigate whether beta 2 GPI modulates the macrophage response to negatively charged surfaces. Chemiluminescence of isolated non-parenchymal rat liver cells was measured after phagocytosis of opsonized zymosan or phorbolymristate acetate (PMA) stimulation, in the presence and absence of large unilamellar vesicles (LUVs) containing 25 mol% phosphatidylserine (PS) or 50 mol% cardiolipin (CL) and complementary molar ratio of phosphatidylcholine (PC). beta 2 GPI decreased by 50% the chemiluminescence response induced by opsonized zymosan, with a 66% reduction of the initial light emission rate. PMA stimulated Kupffer cell chemiluminescence was insensitive to human or rat beta 2 GPI. Albumin (500 micrograms/ml) showed no effect upon chemiluminescence. beta 2 GPI increased PS/PC LUV uptake and degradation by Kupffer cells in a concentration-dependent manner, without leakage of the internal contents of the LUVs, as shown by fluorescence intensity enhancement. LUVs opsonized with antiphospholipid antibodies (aPL) from syphilitic patients increased light emission by Kupffer cells. Addition of beta 2 GPI to the assay reduced chemiluminescence due to opsonization with purified IgG antibodies from systemic lupus erythematosus (SLE or syphilis (Sy) patient sera. A marked net increase in chemiluminescence is observed in the presence of Sy aPL antibodies, whereas a decrease was found when SLE aPL were added to the assay, in the presence or absence of beta 2 GPI. At a concentration of 125 micrograms/ml, beta 2 GPI significantly reduced Kupffer cell Candida albicans phagocytosis index and killing score by 50 and 10%, respectively. The present data strongly suggest that particle uptake in the presence of beta 2 GPI is coupled to an inhibition of reactive species production by liver macrophages during the respiratory burst, supporting the role of beta 2 GPI as a mediator of senescent cell removal.

A severe combined immunodeficient-hu in vivo mouse model of human primary mantle cell lymphoma.

PubMed

Wang, Michael; Zhang, Liang; Han, Xiaohong; Yang, Jing; Qian, Jianfei; Hong, Sungyoul; Lin, Pei; Shi, Yuankai; Romaguera, Jorge; Kwak, Larry W; Yi, Qing

2008-04-01

To establish a severe combined immunodeficient (SCID)-hu in vivo mouse model of human primary mantle cell lymphoma (MCL) for the study of the biology and novel therapy of human MCL. Primary MCL cells were isolated from spleen, lymph node, bone marrow aspirates, or peripheral blood of six different patients and injected respectively into human bone chips, which had been s.c. implanted in SCID-hu. Circulating human beta(2)-microglobulin in mouse serum was used to monitor the engraftment and growth of patient's MCL cells. H&E staining and immunohistochemical staining with anti-human CD20 and cyclin D1 antibodies were used to confirm the tumor growth and migration. Increasing levels of circulating human beta(2)-microglobulin in mouse serum indicated that the patient's MCL cells were engrafted successfully into human bone chip of SCID-hu mice. The engraftment and growth of patient's MCL cells were dependent on human bone marrow microenvironment. Immunohistochemical staining with anti-human CD20 and cyclin D1 antibodies confirmed that patient's MCL cells were able to not only survive and propagate in the bone marrow microenvironment of the human fetal bone chips, but also similar to the human disease, migrate to lymph nodes, spleen, bone marrow, and gastrointestinal tract of host mice. Treatment of MCL-bearing SCID-hu mice with atiprimod, a novel antitumor compound against the protection of bone marrow stromal cells, induced tumor regression. This is the first human primary MCL animal model that should be useful for the biological and therapeutic research on MCL.

Arachidonic Acid-Induced Expression of the Organic Solute and Steroid Transporter-beta (Ost-beta) in a Cartilaginous Fish Cell Line

PubMed Central

Hwang, Jae-Ho; Parton, Angela; Czechanski, Anne; Ballatori, Nazzareno; Barnes, David

2008-01-01

The organic solute and steroid transporter (OST/Ost) is a unique membrane transport protein heterodimer composed of subunits designated alpha and beta, that transports conjugated steroids and prostaglandin E2 across the plasma membrane. Ost was first identified in the liver of the cartilaginous fish Leucoraja erinacea, the little skate, and subsequently was found in many other species, including humans and rodents. The present study describes the isolation of a new cell line, LEE-1, derived from an early embryo of L. erinacea, and characterizes the expression of Ost in these cells. The mRNA size and amino acid sequence of Ost-beta in LEE-1 was identical to that previously reported for Ost-beta from skate liver, and the primary structure was identical to that of the spiny dogfish shark (Squalus acanthias) with the exception of a single amino acid. Ost-beta was found both on the plasma membrane and intracellularly in LEE-1 cells, consistent with its localization in other cell types. Interestingly, arachidonic acid, the precursor to eiconsanoids, strongly induced Ost-beta expression in LEE-1 cells and a lipid mixture containing arachidonic acid also induced Ost-alpha. Overall, the present study describes the isolation of a novel marine cell line, and shows that this cell line expresses relatively high levels of Ost when cultured in the presence of arachidonic acid. Although the function of this transport protein in embryo-derived cells is unknown, it may play a role in the disposition of eicosanoids or steroid-derived molecules. PMID:18407792

Contributions of arginines-43 and -94 of human choriogonadotropin. beta. to receptor binding and activation as determined by oligonucleotide-based mutagenesis

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

Fang Chen; Puett, D.

1991-10-22

Members of the glycoprotein hormone family contain a common {alpha} subunit and a hormone-specific {beta} subunit. Human choriogonadotropin (hCG) {beta} is a 145 amino acid residue protein glycosylated at 6 positions (2 N-linked and 4 O-linked oligosaccharides). In an effort to elucidate receptor determinants on hCG{beta}, the authors have used site-directed mutagenesis to prepare and express several mutant cDNAs with replacements at arginines-43 and -94. Arg-43 is invariant in all known mammalian CG/lutropin {beta} amino acid sequences, and Arg-94 is conserved in 10 of the 12 sequences. Moreover, various studies involving synthetic peptides and enzymatic digestions of intact {beta} chainsmore » suggest that these residues may be important in hCG receptor binding. Point mutants were made in which these two arginines were replaced with the corresponding residues in human follitropin {beta}, Leu-43 and Asp-94. The wild-type and mutant {beta} chains were expressed in CHO cells containing a stably integrated gene for bovine {alpha}, and heterodimer formation occurred. These heterologous gonadotropins were active in assays using transformed Leydig cells, competitive binding with standard {sup 125}I-hCG, and cAMP and progesterone production, but the potency was considerably less than that associated with the hCG{beta} wild-type-containing gonadotropin. The double-mutant protein Arg-43 to Leu/Arg-94 to Asp also associated with bovine {alpha}, but the resultant heterodimer exhibited only low activity. Replacement but the Lys-43-containing {beta} chain appeared to exhibit a low degree of subunit association or reduced stability relative to the expressed hCG{beta} wild type. These results demonstrate that arginines-43 and -94 contribute to receptor binding through a positive charge.« less

Novel Confocal Microscopic and Flow Cytometric Based Assays to Visualize and Detect the (Beta)2-Adrenergic Receptor in Human Lymphocyte and Mononuclear Cell Populations

NASA Technical Reports Server (NTRS)

Salicru, A. N.; Crucian, B. E.; Nelman, M. A.; Sams, C. F.; Actor, J. K.; Marshall, G. D.

2006-01-01

The data show that immunophenotyping of leukocyte populations with (beta)2AR is possible with the commercially available Ab, although the FC assay is limited to the IST as a result of the Ab binding site to the intracellular C-terminus of the 2AR. The FC assay has applications for measuring alterations in total (beta)2AR in human leukocyte populations as changes in fluorescence. In addition, CM confirms that both surface and intracellular compartments stain positively for the (beta)2AR and can be used for qualitative assays that screen for changes in receptor compartmentalization and localization.

The effects of topically applied glycolic acid and salicylic acid on ultraviolet radiation-induced erythema, DNA damage and sunburn cell formation in human skin.

PubMed

Kornhauser, Andrija; Wei, Rong-Rong; Yamaguchi, Yuji; Coelho, Sergio G; Kaidbey, Kays; Barton, Curtis; Takahashi, Kaoruko; Beer, Janusz Z; Miller, Sharon A; Hearing, Vincent J

2009-07-01

alpha-Hydroxy acids (alphaHAs) are reported to reduce signs of aging in the skin and are widely used cosmetic ingredients. Several studies suggest that alphaHA can increase the sensitivity of skin to ultraviolet radiation. More recently, beta-hydroxy acids (betaHAs), or combinations of alphaHA and betaHA have also been incorporated into antiaging skin care products. Concerns have also arisen about increased sensitivity to ultraviolet radiation following use of skin care products containing beta-HA. To determine whether topical treatment with glycolic acid, a representative alphaHA, or with salicylic acid, a betaHA, modifies the short-term effects of solar simulated radiation (SSR) in human skin. Fourteen subjects participated in this study. Three of the four test sites on the mid-back of each subject were treated daily Monday-Friday, for a total of 3.5 weeks, with glycolic acid (10%), salicylic acid (2%), or vehicle (control). The fourth site received no treatment. After the last treatment, each site was exposed to SSR, and shave biopsies from all four sites were obtained. The endpoints evaluated in this study were erythema (assessed visually and instrumentally), DNA damage and sunburn cell formation. Treatment with glycolic acid resulted in increased sensitivity of human skin to SSR, measured as an increase in erythema, DNA damage and sunburn cell formation. Salicylic acid did not produce significant changes in any of these biomarkers. Short-term topical application of glycolic acid in a cosmetic formulation increased the sensitivity of human skin to SSR, while a comparable treatment with salicylic acid did not.

Studies on novel 4beta-[(4-substituted)-1,2,3-triazol-1-yl] podophyllotoxins as potential anticancer agents.

PubMed

Bhat, Bilal A; Reddy, P Bhaskar; Agrawal, Satyam Kumar; Saxena, A K; Kumar, H M Sampath; Qazi, G N

2008-10-01

A series of 4beta-[(4-substituted)-1,2,3-triazol-1-yl] podophyllotoxin congeners have been designed and synthesized with significant regioselectivity by employing Cu(I) catalyzed 1,3-dipolar cycloaddition reaction of C4beta-azido podophyllotoxin and C4beta-azido-4'-O-demethyl podophyllotoxin with N-prop-2-yn-1-ylanilines. These compounds were evaluated for anticancer activity against a panel of seven human cancer cell lines. It was interesting to note that all the compounds exhibited promising activity especially against SF-295 (CNS), HCT-15 (colon) and 502713 (colon) cell lines. Compound 11e was found to be the most promising in this study.

Involvement of PKC and ROS in the cytotoxic mechanism of anti-leukemic decursin and its derivatives and their structure-activity relationship in human K562 erythroleukemia and U937 myeloleukemia cells.

PubMed

Kim, Hyeon Ho; Sik Bang, Sung; Seok Choi, Jin; Han, Hogyu; Kim, Ik-Hwan

2005-06-08

Protein kinase C (PKC) plays an important role in the proliferation and differentiation of various cell types including normal and leukemic hematopoietic cells. Recently, various PKC modulators were used as a chemotherapeutic agent of leukemia. Decursin (1), a pyranocoumarin from Angelica gigas, exhibits the cytotoxic effects on various human cancer cell lines and in vitro PKC activation. For the development of more effective anticancer agents with PKC modulation activity, 11 decursin derivatives 2-12 were chemically synthesized and evaluated for their ability to act as a tumor-suppressing PKC activator and as an antagonist to phorbol 12-myristate 13-acetate (PMA), a tumor-promoting PKC activator. In the presence of phosphatidylserine (PS), all of 12 compounds 1-12 activated PKC (mainly alpha, beta, and gamma isozymes) but only three compounds 1-3 activated PKC even in the absence of PS. Six compounds 1-6 containing the coumarin structure were cytotoxic to human K562 erythroleukemia and U937 myeloleukemia cells. A cytotoxic mechanism of decursin and its derivatives was investigated using TUR cells, a PKC betaII-deficient variant of U937 cells. Among six compounds 1-6 with cytotoxicity to K562 and U937 leukemia cells, only three compounds 1-3 were cytotoxic to TUR cells. Therefore, compounds 1-3 and 4-6 inhibit the proliferation of leukemia cells in a PKC betaII-independent and dependent manner, respectively, indicating that the side chain of compounds determines the dependency of their cytotoxicity on PKC betaII. To further elucidate the cytotoxic mechanism of compounds 1 and 2, levels of PKC isozymes and generation of reactive oxygen species (ROS) were investigated. Compounds 1-2 induced the down-regulation of PKC alpha and betaII in K562 cells and the production of ROS in U937 cells. Thus, PKC and ROS are probably important factors in the cytotoxic mechanism of compounds 1-2. From these results, the structure-activity relationship of decursin and its derivatives is as follows: (i) the coumarin structure is required for anti-leukemic activity and (ii) the side chain is a determinant of PKC activation and the cytotoxic mechanism in leukemia cells.

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 Wiley-Liss, Inc.

Integrin expression by human osteoblasts cultured on degradable polymeric materials applicable for tissue engineered bone.

PubMed

El-Amin, Saadiq F; Attawia, Mohamed; Lu, Helen H; Shah, Asist K; Chang, Richard; Hickok, Noreen J; Tuan, Rocky S; Laurencin, Cato T

2002-01-01

The use of biodegradable polymers in the field of orthopaedic surgery has gained increased popularity, as surgical pins and screws, and as potential biological scaffolds for repairing cartilage and bone defects. One such group of polymers that has gained considerable attention are the polyesters, poly(lactide-co-glycolide) (PLAGA) and polylactic acid (PLA), because of their minimal tissue inflammatory response, favorable biocompatibility and degradation characteristics. The objective of this study was to evaluate human osteoblastic cell adherence and growth on PLAGA and PLA scaffolds by examining integrin receptor (alpha2, alpha3, alpha4, alpha5, alpha6 and beta1) expression. Primary human osteoblastic cells isolated from trabecular bone adhered efficiently to both PLAGA and PLA, with the rate of adherence on PLAGA comparable to that of control tissue culture polystyrene (TCPS), and significantly higher than on PLA polymers at 3, 6 and 12 h. Human osteoblastic phenotypic expression, alkaline phosphatase (ALP) activity was positive on both degradable matrices, whereas osteocalcin levels were significantly higher on cells grown on PLAGA than on PLA composites. Interestingly, the integrin subunits, alpha2, alpha3, alpha4, alpha5, alpha6 and beta1 were all expressed at higher levels by osteoblasts cultured on PLAGA than those on PLA as analyzed by westerns blots and by flow cytometry. Among the integrins, alpha2, beta5 and beta1 showed the greatest difference in levels between the two surfaces. Thus, both PLA and PLAGA support osteoblastic adhesion and its accompanying engagement of integrin receptor and expression of osteocalcin and ALP. However PLAGA consistently appeared to be a better substrate for osteoblastic cells based on these parameters. This study is one of the first to investigate the ability of primary human osteoblastic cells isolated from trabecular bone to adhere to the biodegradable polymers PLAGA and PLA, and to examine the expression of their key adhesion receptors (integrins) on these substrates.

SNOM and AFM microscopy techniques to study the effect of non-ionizing radiation on the morphological and biochemical properties of human keratinocytes cell line (HaCaT).

PubMed

Rieti, S; Manni, V; Lisi, A; Giuliani, L; Sacco, D; D'Emilia, E; Cricenti, A; Generosi, R; Luce, M; Grimaldi, S

2004-01-01

In this study we have employed atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM) techniques to study the effect of the interaction between human keratinocytes (HaCaT) and electromagnetic fields at low frequency. HaCaT cells were exposed to a sinusoidal magnetic field at a density of 50 Hz, 1 mT. AFM analysis revealed modification in shape and morphology in exposed cells with an increase in the areas of adhesion between cells. This latter finding was confirmed by SNOM indirect immunofluorescence analysis performed with a fluorescent antibody against the adhesion marker beta4 integrin, which revealed an increase of beta4 integrin segregation in the cell membrane of 50-Hz exposed cells, suggesting that a higher percentage of these cells shows a modified pattern of this adhesion marker.

Differentiation of human-induced pluripotent stem cells into insulin-producing clusters.

PubMed

Shaer, Anahita; Azarpira, Negar; Vahdati, Akbar; Karimi, Mohammad Hosein; Shariati, Mehrdad

2015-02-01

In diabetes mellitus type 1, beta cells are mostly destroyed; while in diabetes mellitus type 2, beta cells are reduced by 40% to 60%. We hope that soon, stem cells can be used in diabetes therapy via pancreatic beta cell replacement. Induced pluripotent stem cells are a kind of stem cell taken from an adult somatic cell by "stimulating" certain genes. These induced pluripotent stem cells may be a promising source of cell therapy. This study sought to produce isletlike clusters of insulin-producing cells taken from induced pluripotent stem cells. A human-induced pluripotent stem cell line was induced into isletlike clusters via a 4-step protocol, by adding insulin, transferrin, and selenium (ITS), N2, B27, fibroblast growth factor, and nicotinamide. During differentiation, expression of pancreatic β-cell genes was evaluated by reverse transcriptase-polymerase chain reaction; the morphologic changes of induced pluripotent stem cells toward isletlike clusters were observed by a light microscope. Dithizone staining was used to stain these isletlike clusters. Insulin produced by these clusters was evaluated by radio immunosorbent assay, and the secretion capacity was analyzed with a glucose challenge test. Differentiation was evaluated by analyzing the morphology, dithizone staining, real-time quantitative polymerase chain reaction, and immunocytochemistry. Gene expression of insulin, glucagon, PDX1, NGN3, PAX4, PAX6, NKX6.1, KIR6.2, and GLUT2 were documented by analyzing real-time quantitative polymerase chain reaction. Dithizone-stained cellular clusters were observed after 23 days. The isletlike clusters significantly produced insulin. The isletlike clusters could increase insulin secretion after a glucose challenge test. This work provides a model for studying the differentiation of human-induced pluripotent stem cells to insulin-producing cells.

Dual role of Ski in pancreatic cancer cells: tumor-promoting versus metastasis-suppressive function.

PubMed

Wang, Peng; Chen, Zhen; Meng, Zhi-Qiang; Fan, Jie; Luo, Jian-Min; Liang, Wang; Lin, Jun-Hua; Zhou, Zhen-Hua; Chen, Hao; Wang, Kun; Shen, Ye-Hua; Xu, Zu-De; Liu, Lu-Ming

2009-09-01

Ski used to be defined as an oncogene that contributes to the resistance of tumor cells to transforming growth factor-beta (TGF-beta)-induced growth arrest. As TGF-beta has a dual effect on tumor growth with both tumor-suppressing and -promoting activity depending on the stage of carcinogenesis and the cell type, the precise role of Ski in carcinogenesis remains unclear. In this study, we show that downregulation of Ski through lentivirus-mediated RNA interference decreases tumor growth both in vitro and in vivo, yet promotes cell invasiveness in vitro, and lung metastasis in vivo in the pancreatic cancer cell line SW1990, which contain wild-type Smad4 expression, and the BxPC3 cell line, which is Smad4 deficient. We also show that the downregulation of Ski increases TGF-beta-induced transcriptional activity, which is associated with increased TGF-beta-dependent Smad2/3 phosphorylation, and results in an altered expression profile of TGF-beta-inducible genes involved in metastasis, angiogenesis and cell proliferation and epithelial-mesenchymal transition. Immunohistochemical analysis of specimens from 71 patients with pancreatic adenocarcinoma showed a significant association between overexpression of Ski and decreased patient survival time (P = 0.0024). Our results suggest that Ski may act as a tumor proliferation-promoting factor or as a metastatic suppressor in human pancreatic cancer.

BetaPIX and GIT1 regulate HGF-induced lamellipodia formation and WAVE2 transport.

PubMed

Morimura, Shigeru; Suzuki, Katsuo; Takahashi, Kazuhide

2009-05-08

Formation of lamellipodia is the first step during cell migration, and involves actin reassembly at the leading edge of migrating cells through the membrane transport of WAVE2. However, the factors that regulate WAVE2 transport to the cell periphery for initiating lamellipodia formation have not been elucidated. We report here that in human breast cancer MDA-MB-231 cells, the hepatocyte growth factor (HGF) induced the association between the constitutive complex of betaPIX and GIT1 with WAVE2, which was concomitant with the induction of lamellipodia formation and WAVE2 transport. Although depletion of betaPIX by RNA interference abrogated the HGF-induced WAVE2 transport and lamellipodia formation, GIT1 depletion caused HGF-independent WAVE2 transport and lamellipodia formation. Collectively, we suggest that betaPIX releases cells from the GIT1-mediated suppression of HGF-independent responses and recruits GIT1 to WAVE2, thereby facilitating HGF-induced WAVE2 transport and lamellipodia formation.

Molecular Mechanisms of Toxicity and Cell Damage by Chemicals in a Human Pancreatic Beta Cell Line, 1.1B4.

PubMed

Vasu, Srividya; McClenaghan, Neville H; Flatt, Peter R

2016-10-01

Mechanisms of toxicity and cell damage were investigated in novel clonal human pancreatic beta cell line, 1.1B4, after exposure to streptozotocin, alloxan, ninhydrin, and hydrogen peroxide. Viability, DNA damage, insulin secretion/content, [Ca]i, and glucokinase/hexokinase, mRNA expression were measured by MTT assay, comet assay, radioimmunoassay, fluorometric imaging plate reader, enzyme-coupled photometry, and real-time polymerase chain reaction, respectively. Chemicals significantly reduced 1.1B4 cell viability in a time/concentration-dependent manner. Chronic 18-hour exposure decreased cellular insulin, glucokinase, and hexokinase activities. Chemicals decreased transcription of INS, GCK, PCSK1, PCSK2, and GJA1 (involved in secretory function). Insulin release and [Ca]i responses to nutrients and membrane-depolarizing agents were impaired. Streptozotocin and alloxan up-regulated transcription of genes, SOD1 and SOD2 (antioxidant enzymes). Ninhydrin and hydrogen peroxide up-regulated SOD2 transcription, whereas alloxan and hydrogen peroxide increased CAT transcription. Chemicals induced DNA damage, apoptosis, and increased caspase 3/7 activity. Streptozotocin and alloxan decreased transcription of BCL2 while increasing transcription of BAX. Chemicals did not affect transcription of HSPA4 and HSPA5 and nitrite production. 1.1B4 cells represent a useful model of human beta cells. Chemicals impaired 1.1B4 cell secretory function and activated antioxidant defense and apoptotic pathways without activating endoplasmic reticulum stress response/nitrosative stress.

Inflammatory stress of pancreatic beta cells drives release of extracellular heat-shock protein 90α.

PubMed

Ocaña, Gail J; Pérez, Liliana; Guindon, Lynette; Deffit, Sarah N; Evans-Molina, Carmella; Thurmond, Debbie C; Blum, Janice S

2017-06-01

A major obstacle in predicting and preventing the development of autoimmune type 1 diabetes (T1D) in at-risk individuals is the lack of well-established early biomarkers indicative of ongoing beta cell stress during the pre-clinical phase of disease. Recently, serum levels of the α cytoplasmic isoform of heat-shock protein 90 (hsp90) were shown to be elevated in individuals with new-onset T1D. We therefore hypothesized that hsp90α could be released from beta cells in response to cellular stress and inflammation associated with the earliest stages of T1D. Here, human beta cell lines and cadaveric islets released hsp90α in response to stress induced by treatment with a combination of pro-inflammatory cytokines including interleukin-1β, tumour necrosis factor-α and interferon-γ. Mechanistically, hsp90α release was found to be driven by cytokine-induced endoplasmic reticulum stress mediated by c-Jun N-terminal kinase (JNK), a pathway that can eventually lead to beta cell apoptosis. Cytokine-induced beta cell hsp90α release and JNK activation were significantly reduced by pre-treating cells with the endoplasmic reticulum stress-mitigating chemical chaperone tauroursodeoxycholic acid. The hsp90α release by cells may therefore be a sensitive indicator of stress during inflammation and a useful tool in assessing therapeutic mitigation of cytokine-induced cell damage linked to autoimmunity. © 2017 John Wiley & Sons Ltd.

CDK2 phosphorylation of Smad2 disrupts TGF-beta transcriptional regulation in resistant primary bone marrow myeloma cells.

PubMed

Baughn, Linda B; Di Liberto, Maurizio; Niesvizky, Ruben; Cho, Hearn J; Jayabalan, David; Lane, Joseph; Liu, Fang; Chen-Kiang, Selina

2009-02-15

Resistance to growth suppression by TGF-beta1 is common in cancer; however, mutations in this pathway are rare in hematopoietic malignancies. In multiple myeloma, a fatal cancer of plasma cells, malignant cells accumulate in the TGF-beta-rich bone marrow due to loss of both cell cycle and apoptotic controls. Herein we show that TGF-beta activates Smad2 but fails to induce cell cycle arrest or apoptosis in primary bone marrow myeloma and human myeloma cell lines due to its inability to activate G(1) cyclin-dependent kinase (CDK) inhibitors (p15(INK4b), p21(CIP1/WAF1), p27(KIP1), p57(KIP2)) or to repress c-myc and Bcl-2 transcription. Correlating with aberrant activation of CDKs, CDK-dependent phosphorylation of Smad2 on Thr(8) (pT8), a modification linked to impaired Smad activity, is elevated in primary bone marrow myeloma cells, even in asymptomatic monoclonal gammopathy of undetermined significance. Moreover, CDK2 is the predominant CDK that phosphorylates Smad2 on T8 in myeloma cells, leading to inhibition of Smad2-Smad4 association that precludes transcriptional regulation by Smad2. Our findings provide the first direct evidence that pT8 Smad2 couples dysregulation of CDK2 to TGF-beta resistance in primary cancer cells, and they suggest that disruption of Smad2 function by CDK2 phosphorylation acts as a mechanism for TGF-beta resistance in multiple myeloma.

Complementary deoxyribonucleic acid cloning of spermatogonial stem cell renewal factor.

PubMed

Miura, Takeshi; Ohta, Takashi; Miura, Chiemi I; Yamauchi, Kohei

2003-12-01

Spermatogonial mitosis can be subdivided into two processes: spermatogonial stem cell renewal and spermatogonial proliferation toward meiosis. Recently it has been indicated that estrogen, estradiol-17beta, is involved in regulating the renewal of spermatogonial stem cells in eel. To determine the genes that directly regulate this process, we used expression screening to identify genes whose expression is regulated by estradiol-17beta in testes. We detected a previously unidentified cDNA clone that is up-regulated by estradiol-17beta stimulation and named it eel spermatogenesis-related substances 34 (eSRS34) cDNA. Homology searching showed that eSRS34 shares amino acid sequence similarity with human platelet-derived endothelial cell growth factor. We examined the function of eSRS34 using several in vitro systems. Recombinant eSRS34 produced by a baculovirus system induced spermatogonial mitosis in testicular organ culture. Furthermore, the addition of an antibody specific for eSRS34 prevented spermatogonial mitosis induced by estradiol-17beta stimulation in a germ cell/somatic cell coculture system. We therefore conclude that eSRS34 is a "spermatogonial stem cell renewal factor."

Trefoil factor 3 isolated from human breast milk downregulates cytokines (IL8 and IL6) and promotes human beta defensin (hBD2 and hBD4) expression in intestinal epithelial cells HT-29

PubMed Central

Barrera, Girolamo Jose; Sanchez, Gabriela; Gonzalez, Jose Emanuele

2012-01-01

Trefoil factors (TFF) are secretory products of mucin producing cells. They play a key role in the maintenance of the surface integrity of oral mucosa and enhance healing of the gastrointestinal mucosa by a process called restitution. TFF comprises the gastric peptides (TFF1), spasmolytic peptide (TFF2), and the intestinal trefoil factor (TFF3). They have an important and necessary role in epithelial restitution within the gastrointestinal tract. Significant amounts of TFF are present in human milk. This study aimed to determine a possible correlation between TFF3 isolated from human breast milk and levels of cytokines (IL8 and IL6) and defensins (hBD2 and hBD4) in intestinal epithelial cells HT-29 treated with trefoil. Samples of human milk were collected within 2-4 weeks postpartum from healthy human mothers (18-30-years-old) by manual breast massage, and TFF3 was purified by ammonium sulfate precipitation, isoelectric precipitation, DEAE-chromatography, and gel filtration. In this work we measured the concentrations and mRNA levels of cytokines and defensins by immunoassay (ELISA) and semiquantitative RT-PCR technique, respectively. Also we measured the peroxidase activity. We present the first evidence of human milk TFF3 purification. Here we show that the presence of TFF3 isolated from milk strongly correlates with downregulation of IL8 and IL6 in human intestinal epithelial cells. On the other hand, TFF3 activated the epithelial cells in culture to produce beta defensins 2 (hBD2) and beta defensins 4 (hBD4). These findings suggest that TFF can activate intestinal epithelial cells and could actively participate in the immune system of breastfed babies by inducing the production of peptides related to innate defence, such as defensins. PMID:23198942

Targeting Alpha5 Beta1 Integrin to Prevent Metastatic Breast Cancer Cell Invasion: PhScN Target Site Definition and Plasma Stability

DTIC Science & Technology

2014-09-01

Staszewski, et al., The PHSCN dendrimer as a more potent inhibitor of human breast cancer cell invasion, extravasation, and lung colony formation...the PHSCN dendrimer as an inhibitor of human prostate cancer cell invasion, extravasation, and lung colony formation. Clin Exp Metastasis, 2010. 27(3

Large Gliadin Peptides Detected in the Pancreas of NOD and Healthy Mice following Oral Administration

PubMed Central

Sidenius, Ulrik; Heegaard, Niels H.

2016-01-01

Gluten promotes type 1 diabetes in nonobese diabetic (NOD) mice and likely also in humans. In NOD mice and in non-diabetes-prone mice, it induces inflammation in the pancreatic lymph nodes, suggesting that gluten can initiate inflammation locally. Further, gliadin fragments stimulate insulin secretion from beta cells directly. We hypothesized that gluten fragments may cross the intestinal barrier to be distributed to organs other than the gut. If present in pancreas, gliadin could interact directly with the immune system and the beta cells to initiate diabetes development. We orally and intravenously administered 33-mer and 19-mer gliadin peptide to NOD, BALB/c, and C57BL/6 mice and found that the peptides readily crossed the intestinal barrier in all strains. Several degradation products were found in the pancreas by mass spectroscopy. Notably, the exocrine pancreas incorporated large amounts of radioactive label shortly after administration of the peptides. The study demonstrates that, even in normal animals, large gliadin fragments can reach the pancreas. If applicable to humans, the increased gut permeability in prediabetes and type 1 diabetes patients could expose beta cells directly to gliadin fragments. Here they could initiate inflammation and induce beta cell stress and thus contribute to the development of type 1 diabetes. PMID:27795959

Induction of an angiogenic phenotype in endometriotic stromal cell cultures by interleukin-1beta.

PubMed

Lebovic, D I; Bentzien, F; Chao, V A; Garrett, E N; Meng, Y G; Taylor, R N

2000-03-01

Activated peritoneal macrophages are associated with endometriosis and may play a central role in its aetiology by releasing interleukin-1beta (IL-1beta) in response to refluxed endometrium. Pari passu with the establishment of endometriotic implants is the development of a vascular supply. In this study we investigated the angiogenic properties of two endometrial proteins, vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6), and assessed their production in response to IL-1beta stimulation in human stromal cells isolated from normal endometrium (NE) and endometriotic lesions (EI). Proliferation of bovine brain capillary endothelial cells (BBCE) with a [(3)H]-thymidine incorporation assay was observed when VEGF (2.1 +/- 0.2-fold; P < 0.05) or VEGF and IL-6 (1.8 +/- 0.1-fold; P < 0.05) were added in vitro, relative to saline-treated control cultures. Northern blot analysis showed induction of VEGF mRNA (2.6-fold; P < 0.05) and IL-6 mRNA (6.3-fold; P < 0.05) transcripts in EI cells, but not NE cells, exposed to IL-1beta. A similar induction was seen with VEGF and IL-6 protein secretion in the responsive EI cells. Reverse transcription-polymerase chain reaction (RT-PCR) for the IL-1 receptor type I (IL-1 RI) indicated that the differential effects of IL-1beta on NE and EI cells was associated with 2.4 +/- 0.1-fold more receptor mRNA in EI versus NE cells. We propose that the ability of IL-1beta to activate an angiogenic phenotype in EI stromal cells but not in NE cells, is mediated by the IL-1 RI.

Modulation of TGF-beta signaling during progression of chronic liver diseases.

PubMed

Matsuzaki, Koichi

2009-01-01

A large body of work has established roles for epithelial cells as important mediators of progressive fibrosis and carcinogenesis. Transforming growth factor-beta (TGF-beta) and pro-inflammatory cytokines are important inducers of fibro-carcinogenesis. TGF-beta signaling involves phosphorylation of Smad3 at middle linker and/or C-terminal regions. Reversible shifting of Smad3-dependent signaling between tumor-suppression and oncogenesis in hyperactive Ras-expressing epithelial cells indicates that Smad3 phosphorylated at the C-terminal region (pSmad3C) transmits a tumor-suppressive TGF-beta signal, while oncogenic activities such as cell proliferation and invasion are promoted by Smad3 phosphorylated at the linker region (pSmad3L). Notably, pSmad3L-mediated signaling promotes extracellular matrix deposition by activated mesenchymal cells. During progression of chronic liver diseases, hepatic epithelial hepatocytes undergo transition from the tumor-suppressive pSmad3C pathway to the fibrogenic/oncogenic pSmad3L pathway, accelerating liver fibrosis and increasing risk of hepatocellular carcinoma. c-Jun N-terminal kinase activated by pro-inflammatory cytokines is mediating this perturbed hepatocytic TGF-beta signaling. Thus, TGF-beta signaling of hepatocytes affected by chronic inflammation offers a general framework for understanding the molecular mechanisms of human fibro-carcinogenesis during progression of chronic liver diseases.

Hematopoietic Stem Cell Therapy as a Counter-Measure for Human Exploration of Deep Space

NASA Technical Reports Server (NTRS)

Ohi, S.; Roach, A.-N.; Ramsahai, S.; Kim, B. C.; Fitzgerald, W.; Riley, D. A.; Gonda, S. R.

2004-01-01

Human exploration of deep space depends, in part, on our ability to counter severe/invasive disorders that astronauts experience in space environments. The known symptoms include hematological/cardiac abnormalities,bone and muscle losses, immunodeficiency, neurological disorders, and cancer. Exploiting the extraordinary plasticity of hematopoietic stem cells (HSCs), which differentiate not only to all types of blood cells, but also to various tissues, we have advanced a hypothesis that ome of the space-caused disorders maybe amenable to hematopoietis stem cell therapy(HSCT) so as to maintain promote human exploration of deep space. Using mouse models of human anemia beta-thaiassemia) as well as spaceflight (hindlimb unloading system), we have obtained feasibility results of HSCT for space anemia, muscle loss, and immunodeficiency. For example, in the case of HSCT for muscle loss, the beta-galactosidese marked HSCs were detected in the hindlimbs of unloaded mouse following transplantation by -X-gal wholemaunt staining procedure. Histochemicaland physical analyses indicated structural contribution of HSCs to the muscle. HSCT for immunodeficiency was investigated ising beta-galactosidese gene-tagged Escherichia coli as the infectious agent. Results of the X-gal staining procedure indicated the rapeutic role of the HSCT. To facilitate the HSCT in space, growth of HSCs were optimized in the NASA Rotating Wall Vessel (RWV) culture systems, including Hydrodynamic Focusing Bioreactor (HFB).

Disruption of alpha beta but not of gamma delta T cell development by overexpression of the helix-loop-helix protein Id3 in committed T cell progenitors.

PubMed Central

Blom, B; Heemskerk, M H; Verschuren, M C; van Dongen, J J; Stegmann, A P; Bakker, A Q; Couwenberg, F; Res, P C; Spits, H

1999-01-01

Enforced expression of Id3, which has the capacity to inhibit many basic helix-loop-helix (bHLH) transcription factors, in human CD34(+) hematopoietic progenitor cells that have not undergone T cell receptor (TCR) gene rearrangements inhibits development of the transduced cells into TCRalpha beta and gamma delta cells in a fetal thymic organ culture (FTOC). Here we document that overexpression of Id3, in progenitors that have initiated TCR gene rearrangements (pre-T cells), inhibits development into TCRalpha beta but not into TCRgamma delta T cells. Furthermore, Id3 impedes expression of recombination activating genes and downregulates pre-Talpha mRNA. These observations suggest possible mechanisms by which Id3 overexpression can differentially affect development of pre-T cells into TCRalpha beta and gamma delta cells. We also observed that cell surface CD4(-)CD8(-)CD3(-) cells with rearranged TCR genes developed from Id3-transduced but not from control-transduced pre-T cells in an FTOC. These cells had properties of both natural killer (NK) and pre-T cells. These findings suggest that bHLH factors are required to control T cell development after the T/NK developmental checkpoint. PMID:10329625

Differential effect of 1{alpha},25-dihydroxyvitamin D{sub 3} on Hsp28 and PKC{beta} gene expression in the phorbol ester-resistant human myeloid HL-525 leukemic cells

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

Lee, Yong J.; Galoforo, S.S.; Berns, C.M.

We investigated the effect of 1{alpha},25-dihydroxyvitamin D{sub 3} [1,25-(OH){sub 2}D{sub 3}] on the expression of the 28-kDa heat shock protein gene (hsp28) and the protein kinase C beta gene (PKC{beta}) in the human myeloid HL-60 leukemic cell variant HL-525, which is resistance to phorbol ester-induced macrophage differentiation. Northern and Western blot analysis showed little or no hsp28 gene expression in the HL-60 cell variant, HL-205, which is susceptible to such differentiation, while a relatively high basal level of hps28 gene expression was observed in the HL-525 cells. However, both cell lines demonstrated heat shock-induced expression of this gene. During treatmentmore » with 50-300 nM 1,25-(OH){sub 2}D{sub 3}, a marked reduction of hsp28 gene expression was not associated with heat shock transcription factor-heat shock element (HSF-HSE) binding activity. Our results suggest that the differential effect of 1,25-(OH){sub 2}D{sub 3} on hsp28 and PKC{beta} gene expression is due to the different sequence composition of the vitamin D response element in the in the promoter region as well as an accessory factor for each gene or that 1,25-(OH){sub 2}D{sub 3} increases PKC{beta} gene expression, which in turn negatively regulates the expression of the hsp28 gene, or vice versa.« less

Expression of hemidesmosomal and extracellular matrix proteins by normal and malignant human prostate tissue.

PubMed Central

Nagle, R. B.; Hao, J.; Knox, J. D.; Dalkin, B. L.; Clark, V.; Cress, A. E.

1995-01-01

The progression of prostate carcinoma may be influenced by the biochemical nature of the basal lamina surrounding the primary carcinoma cells. As a first step toward understanding this process, the composition and structure of the basal lamina in normal prostate, prostatic intraepithelial neoplasia, and human carcinoma were determined. In addition, a comparison was made between the attachments of the normal basal cell to its underlying basal lamina and those made by primary prostate carcinoma. The normal basal cells form both focal adhesions and hemidesmosomal-like structures as observed by transmission electron microscopy. The normal basal cells exhibited a polarized distribution of hemidesmosomal associated proteins including BP180, BP230, HD1, plectin, laminin-gamma 2(B2t), collagen VII, and the corresponding integrin laminin receptors alpha 6 beta 1 and alpha 6 beta 4. The expression and distribution pattern of these proteins were retained in the prostate intraepithelial neoplasia lesions. In contrast, the carcinoma cells uniformly lacked hemidesmosomal structures, the integrin alpha 6 beta 4, BP180, laminin-gamma 2 (B2t), and collagen VII but did express BP230 (30%), plectin, HD1 (15%), and the integrin laminin receptors alpha 3 beta 1 and alpha 6 beta 1. These results suggest that, although a detectable basal lamina structure is present in carcinoma, its composition and cellular attachments are abnormal. The loss of critical cellular attachments may play a role in influencing the progression potential of prostate carcinoma. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7778688

Novel porphyrin conjugates with a potent photodynamic antitumor effect: differential efficacy of mono- and bis-beta-cyclodextrin derivatives in vitro and in vivo.

PubMed

Kralova, Jarmila; Synytsya, Alla; Pouckova, Pavla; Koc, Michal; Dvorak, Michal; Kral, Vladimir

2006-01-01

In the present study we investigated the photosensitizing properties of two novel mono- and bis-cyclodextrin tetrakis (pentafluorophenyl) porphyrin derivatives in several tumor cell lines and in BALB/c mice bearing subcutaneously transplanted syngeneic mouse mammary carcinoma 4T1. Both studied sensitizers were localized mainly in lysosomes and were found to induce cell death by triggering apoptosis in human leukemic cells HL-60. In 4T1 and other cell lines both apoptotic and necrotic modes of cell death occurred depending on drug and light doses. Mono-cyclodextrin porphyrin derivative P(beta-CD)1 exhibited stronger in vitro phototoxic effect than bis-cyclodextrin derivative P(beta-CD)2. However, in vivo P(beta-CD)2 displayed faster tumor uptake with maximal accumulation 6 h after application, leading to complete and prolonged elimination of subcutaneous tumors within 3 days after irradiation (100 J cm(-2)). In contrast, P(beta-CD)1 uptake was slower (48 h) and the reduction of tumor mass was only transient, reaching the maximum at the 12 h interval when a favorable tumor-to-skin ratio appeared. Thus, P(beta-CD)2 represents a new photosensitizing drug displaying fast and selective tumor uptake, strong antitumor activity and fast elimination from the body.

Functional relationship between cationic amino acid transporters and beta-defensins: implications for dry skin diseases and the dry eye.

PubMed

Jäger, Kristin; Garreis, Fabian; Posa, Andreas; Dunse, Matthias; Paulsen, Friedrich P

2010-04-20

The ocular surface, constantly exposed to environmental pathogens, is particularly vulnerable to infection. Hence an advanced immune defence system is essential to protect the eye from microbial attack. Antimicrobial peptides, such as beta-defensins, are essential components of the innate immune system and are the first line of defence against invaders of the eye. High concentrations of L-arginine and L-lysine are necessary for the expression of beta-defensins. These are supplied by epithelial cells in inflammatory processes. The limiting factor for initiation of beta-defensin production is the transport of L-arginine and L-lysine into the cell. This transport is performed to 80% by only one transporter system in the human, the y(+)-transporter. This group of proteins exclusively transports the cationic amino acids L-arginine, L-lysine and L-ornithine and is also known under the term cationic amino acid transporter proteins (CAT-proteins). Various infections associated with L-arginine deficiency (for example psoriasis, keratoconjuctivitis sicca) are also associated with an increase in beta-defensin production. For the first time, preliminary work has shown the expression of human CATs in ocular surface epithelia and tissues of the lacrimal apparatus indicating their relevance for diseases of the ocular surface. In this review, we summarize current knowledge on the human CATs that appear to be integrated in causal regulation cascades of beta-defensins, thereby offering novel concepts for therapeutic perspectives. Copyright 2010 Elsevier GmbH. All rights reserved.

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)

Comparative study on cytogenetic damage induced by homo-aza-steroidal esters in human lymphocytes.

PubMed

Mourelatos, D; Papageorgiou, A; Boutis, L; Catsoulacos, P

1995-02-01

The effect of P[N,N-bis(2-chloroethyl)amino]phenylacetate esters of 3 beta-hydroxy-N-methyl-17 alpha-aza-D-homo-5 alpha-androstan-17-one (compound 3) and 3 beta-hydroxy-17 alpha-aza-D-homo-5 alpha-androstane (compound 2) on sister-chromatid exchange (SCE) frequencies and on human lymphocytes proliferation kinetics was studied. The results are compared with those of the P[N,N-bis(2-chloroethyl)amino]phenylacetate esters of 3 beta-hydroxy-17 alpha-aza-D-homo-5 alpha-androstan-17-one (compound 1). All compounds were found to be active in inducing markedly increased SCE rates and cell division delays. A correlation between potency for SCE induction, effectiveness in cell division delay and previously established antitumour activity of these compounds was observed.

A novel route of transplantation of human cord blood stem cells in preimmune fetal sheep: the intracelomic cavity.

PubMed

Noia, Giuseppe; Pierelli, Luca; Bonanno, Giuseppina; Monego, Giovanni; Perillo, Alessandro; Rutella, Sergio; Cavaliere, Anna Franca; De Santis, Marco; Ligato, Maria Serena; Fortunato, Giuseppe; Scambia, Giovanni; Terzano, Giuseppina Maria; Iannace, Enrico; Zelano, Giovanni; Michetti, Fabrizio; Leone, Giuseppe; Mancuso, Salvatore; Terzano, Marinela; Fotunato, Giuseppe

2003-01-01

The intracelomic route for in utero hematopoietic stem cell transplantation was evaluated in preimmune fetal sheep and the engraftment characteristics were defined. Twelve twin ovine fetuses (gestational age: 40-45 days) received intracelomic transplants of human CD3-depleted (50 x 10(6) per lamb) or CD34-selected (1-2 x 10(5) per lamb) cord blood hematopoietic stem cells. Engraftment was evaluated from cell suspensions of the liver, spleen, bone marrow, and thymus by flow cytometry, cloning assays, and polymerase chain reaction (PCR) analyses of human beta2-microglobulin. Four fetuses (33%) aborted shortly after intracelomic transplantation and were not evaluable for engraftment. Engraftment was detected in four fetuses obtained from cesarean delivery on day 70 after transplantation of CD3-depleted cord blood cells. The degrees of engraftment in these four fetuses ranged from 6%-22% in the different organs (as revealed by antigenic analysis of human CD45 with flow cytometry). Three fetuses obtained after cesarean section at 102 (no. 435184) and 105 (no. 915293, no. 037568) days and one fetus delivered at term that received CD34-selected cord blood cells had human engraftment with 10%, 32%, 20%, and 10% CD45(+) cells in bone marrow, respectively. In six of eight fetuses evaluable for human engraftment, chimerism was confirmed by PCR analysis for human beta2-microglobulin, which also identified human cells in brain, spinal cord, heart, lung, and skeletal muscle. This preliminary study indicates that intracelomic transplantation of human hematopoietic stem cells in fetal lambs is feasible and effective in terms of hematopoietic engraftment.

Neuropilin-1 and neuropilin-2 are differentially expressed in human proteinuric nephropathies and cytokine-stimulated proximal tubular cells.

PubMed

Schramek, Herbert; Sarközi, Rita; Lauterberg, Christina; Kronbichler, Andreas; Pirklbauer, Markus; Albrecht, Rudolf; Noppert, Susie-Jane; Perco, Paul; Rudnicki, Michael; Strutz, Frank M; Mayer, Gert

2009-11-01

Neuropilin-1 (NRP1) and neuropilin-2 (NRP2) are transmembrane glycoproteins with large extracellular domains that interact with class 3 semaphorins, vascular endothelial growth factor (VEGF) family members, and ligands, such as hepatocyte growth factor, platelet-derived growth factor BB, transforming growth factor-beta1 (TGF-beta1), and fibroblast growth factor2 (FGF2). Neuropilins (NRPs) have been implicated in tumor growth and vascularization, as novel mediators of the primary immune response and in regeneration and repair; however, their role in renal pathophysiology is largely unknown. Here, we report upregulation of tubular and interstitial NRP2 protein expression in patients with focal segmental glomerulosclerosis (FSGS). In an additional cohort of patients with minimal change disease (MCD), membranous nephropathy (MN), and FSGS, elevated NRP2 mRNA expression in kidney biopsies inversely correlated with estimated glomerular filtration rate (eGFR) at the time of biopsy. Furthermore, upregulation of NRP2 mRNA correlated with post-bioptic decline of kidney function. Expression of NRP1 and NRP2 in human proximal tubular cells (PTCs) was differentially affected after stimulation with TGF-beta1, interleukin-1beta (IL-1beta), and oncostatin M (OSM). Although the pro-fibrotic mediators, TGF-beta1 and IL-1beta, induced upregulation of NRP2 expression but downregulation of NRP1 expression, OSM stimulated the expression of both NRP1 and NRP2. Basal and OSM-induced NRP1 mRNA expression, as well as TGF-beta1-induced NRP2 mRNA and protein expression were partially mediated by MEK1/2-ERK1/2 signaling. This is the first report suggesting a differential role of NRP1 and NRP2 in renal fibrogenesis, and TGF-beta1, IL-1beta, and OSM represent the first ligands known to stimulate NRP2 expression in mammalian cells.

Autoradiographic localization of beta-adrenoceptors in asthmatic human lung

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

Spina, D.; Rigby, P.J.; Paterson, J.W.

1989-11-01

The autoradiographic distribution and density of beta-adrenoceptors in human non-diseased and asthmatic bronchi were investigated using (125I)iodocyanopindolol (I-CYP). Analysis of the effects of the beta-adrenoceptor antagonists on I-CYP binding demonstrated that betaxolol (20 nM, beta 1-selective) had no significant effect on specific grain density in either nonasthmatic or asthmatic human bronchus, whereas ICI-118551 (20 nM, beta 2-selective) inhibited I-CYP binding by 85 +/- 9% and 89 +/- 3%, respectively. Thus, homogeneous populations of beta 2-adrenoceptors existed in bronchi from both sources. Large populations of beta-adrenoceptors were localized to the bronchial epithelium, submucosal glands, and airway smooth muscle. Asthmatic bronchial tissuemore » featured epithelial damage with exfoliated cells associated with luminal mucus plugs. A thickened basement membrane and airway smooth muscle hyperplasia were also evident. High levels of specific I-CYP binding were also detected over asthmatic bronchial smooth muscle, as assessed by autoradiography and quantitation of specific grain densities. Isoproterenol and fenoterol were 10- and 13-fold less potent, respectively, in bronchi from asthmatic lung than in those from nonasthmatic lung. However, this attenuated responsiveness to beta-adrenoceptor agonists was not caused by reduced beta-adrenoceptor density in asthmatic airways. A defect may exist in the coupling between beta-adrenoceptors and postreceptor mechanisms in severely asthmatic lung.« less

The influence of surface integrin binding patterns on specific biomaterial-cell interactions

NASA Astrophysics Data System (ADS)

Beranek, Maggi Marie

As the future of biomaterials progresses toward bioactivity, the biomaterial surface must control non-specific protein adsorption and encourage selective protein and cell adsorption. Integrins alphavbeta3, alpha 1beta1, alpha5beta1 and alpha Mbeta2 are expressed on cells involved in endothelialization, inflammation, and intimal hyperplasia. These cellular events play a vital role in biomaterial biocompatibility, especially in the vascular environment. The overall hypothesis of these studies is that biomaterial surfaces exhibit selective integrin binding, which then specifies differential cell binding. To test this hypothesis, four specific aims were developed. The first aim was designed to determine whether metal and polymeric biomaterials exhibit selective integrin binding. The tested materials included 316L stainless steel, nitinol, gold, Elgiloy RTM, poly(D, L-lactide-co-glycolide), polycarbonate urethane and expanded polytetrafluoroethylene. Discrete integrin binding patterns were detected microscopically using integrin specific fluorescent antibodies. Stainless steel exhibited high level integrin alpha1beta 1 and low level integrin alphaMbeta2 binding pattern. This suggests that this metal surface should selectively encourage endothelial cell to inflammatory cell binding. In contrast, gold bound ten times the amount of integrin alphaMbeta2 compared to integrin alpha1beta1, which should encourage inflammatory cell adhesion. The 65/35 poly(D, L-lactide-co-glycolide) was the only polymeric biomaterial tested that had integrin binding levels comparable to metal biomaterials. Based on these observations, a combinational biomaterial with a surface pattern of 65/35 poly(D, L-lactide-co-glycolide) dots on a 316L stainless steel background was created. A pattern of high level integrin alpha1beta1 binding and low level integrin alpha Mbeta2 binding on this combinational surface indicates that this surface should selectively favor endothelial cell binding. In the second aim, the response of surface-bound integrins to flow-related shear stress was examined. Based on fluorescent analysis, total alphavbeta 3, alpha1beta1, and alpha5beta 1 appeared to increase on stainless steel after 90-minute low shear stress exposure, whereas only alpha5beta1 appeared to increase when exposed to high shear. 65/35 poly(D, L-lactide-co-glycolide) exhibited increased total binding of alpha5beta1 and alphaMbeta2, when exposed to either shear stress level. Exposure to either shear stress regimen appeared to increase binding of all integrins on the combinational surface. These responses to shear stress suggest differential integrin binding affinity compared to stainless steel. Using antibodies specific to the integrin subunits, the apparent increase in surface-bound integrins was found to be related to a surface disassociation of alpha and beta subunits. The third aim evaluated human aortic endothelial cells and acute monocytic leukemia cells (THP-1) cell binding to the tested biomaterial surfaces under both static and flow conditions. Both stainless steel and the combinational surface had increased endothelial cell binding compared to monocyte attachment. Pre-incubation of the surface with the specific integrins significantly inhibited human aortic endothelial cell binding. Aim four was designed to investigate the influence of surface bound integrins on human aortic endothelial cell migration under shear stress. If biomaterial surface integrin binding patterns are specific, then pre-bound surface integrins should competitively inhibit binding of cellular integrins to the surface. Cell migration distance on to alphavbeta3, alpha 1beta1, and alpha5beta1 pre-incubated stainless steel was decreased ten-fold, and decreased by three-fold on both 65/35 poly(D, L-lactide-coglycolide) and combinational surfaces compared to the respective bare surfaces. In contrast, migration distance on to alphaMbeta2 pre-coated stainless steel and combinational surface was decreased by only sixty percent and only fifty percent on alphaMbeta2 precoated 65/35 poly(D, L -lactide-co-glycolide). These results suggested that surface binding sites are selective and critical in governing endothelial cell migration. In conclusion, these results support the hypothesis that a surface that encourages specific integrin binding would promote differential cell binding. The novel integrin binding model used in this investigation may be a methodology that can be employed to evaluate potential vascular biomaterials.

T lymphocyte recruitment by interleukin-8 (IL-8). IL-8-induced degranulation of neutrophils releases potent chemoattractants for human T lymphocytes both in vitro and in vivo.

PubMed Central

Taub, D D; Anver, M; Oppenheim, J J; Longo, D L; Murphy, W J

1996-01-01

IL-8 has been shown to be a human neutrophil and T cell chemoattractant in vitro. In an effort to assess the in vivo effects of IL-8 on human leukocyte migration, we examined the ability of rhIL-8 to induce human T cell infiltration using a human/mouse model in which SCID mice were administered human peripheral blood lymphocytes intraperitoneally, followed by subcutaneous injections of rhIL-8. rhIL-8 induced predominantly murine neutrophil accumulation by 4 h after administration while recombinant human macrophage inflammatory protein-1beta (rhMIP-1beta) induced both murine monocytes and human T cell infiltration during the same time period as determined by immunohistology. Interestingly, 72 h after chemokine administration, a marked human T cell infiltrate was observed in the IL-8 injection site suggesting that rhIL-8 may be acting indirectly possibly through a murine neutrophil-derived T cell chemoattractant. This hypothesis was confirmed using granulocyte-depleted SCID mice. Moreover, human neutrophils stimulated in vitro with IL-8 were found to release granule-derived factor(s) that induce in vitro T cell and monocyte chemotaxis and chemokinesis. This T cell and monocyte chemotactic activity was detected in extracts of both azurophilic and specific granules. Together, these results demonstrate that neutrophils store and release, upon stimulation with IL-8 or other neutrophil activators, chemoattractants that mediate T cell and monocyte accumulation at sites of inflammation. PMID:8621778

Transient detection of beta-galactosidase activity in hematopoietic cells, following reinjection of retrovirally marked autologous blood progenitors in patients with breast or ovarian cancer receiving high-dose chemotherapy.

PubMed

Bagnis, Claude; Chabannon, Christian; Gravis, Gwenaelle; Imbert, Anne-Marie; Maroc, Christine; Bardin, Florence; Ladaique, Patrick; Viret, Frédéric; Genre, Dominique; Faucher, Catherine; Stoppa, Anne-Marie; Vey, Norbert; Blaise, Didier; Maraninchi, Dominique; Viens, Patrice; Mannoni, Patrice

2002-02-01

The aim of this report is to demonstrate the feasibility and safety of genetically modifying autologous human blood CD34(+) cells in vitro, with a retroviral vector that encodes a marker gene. The fate of genetically modified cells and their progeny was followed in vivo, after reinfusion in patients treated with high-dose chemotherapy for poor-prognosis breast or ovarian carcinomas. Six patients received genetically modified autologous peripheral blood progenitors, together with unmanipulated aphereses, following high-dose chemotherapy. CD34(+) cells were immunoselected from aphereses, and retrovirally transduced by coculture with the retroviral vector producing cell line, to express a nuclear localized version of E. coli beta-galactosidase, encoded by a defective Moloney-murine leukemia virus-derived retroviral vector. Cells were reinfused to the patients after myeloablation, without prior ex vivo selection. Five out of six patients showed the transient presence of low numbers of beta-galactosidase(+) cells, as detected with an immunocytochemical assay, in the peripheral blood, during the first month following infusion. One patient had beta-galactosidase(+) clonogenic progenitors in her marrow at two months after transplantation, including HPP-CFC; intriguingly, this patient had the lowest percentage of X-gal(+) cells in her graft. Patients experienced side effects that are often observed after high-dose chemotherapy. Feasibility and safety of genetic modification of human hematopoietic stem and progenitor cells are demonstrated by this study. Ex vivo or in vivo selection is not mandatory, even in clinical situations where transduced cells have no survival advantage over wild-type cells; however, significant improvements in gene transfer technology are needed to achieve potentially useful levels of expression in such clinical situations.

miR-218 inhibits the invasive ability of glioma cells by direct downregulation of IKK-{beta}

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

Song, Libing, E-mail: lb.song1@gmail.com; Huang, Quan; Chen, Kun

2010-11-05

Research highlights: {yields} miR-218 is markedly downregulated in glioma cell lines and in primary glioma tissues. {yields} Upregulation of miR-218 dramatically reduces the invasive ability of glioma cells. {yields} Ectopic expression of miR-218 inactivates IKK-{beta}/NF-{kappa}B signaling pathway. {yields} miR-218 directly targets the 3'-untranslated region (3'-UTR) of IKK-{beta}. -- Abstract: Aberrant activation of nuclear factor-kappa B (NF-{kappa}B) pathway has been proven to play important roles in the development and progression of cancers. Activation of NF-{kappa}B via the classical pathway is modulated by I{kappa}Bs kinase (IKK-{beta}). However, the mechanism underlying the epigenetic regulation of IKK-{beta}/NF-{kappa}B pathway remains largely unknown. In this study,more » we found that the expression level of miR-218 was markedly downregulated in glioma cell lines and in human primary glioma tissues. Upregulation of miR-218 dramatically reduced the migratory speed and invasive ability of glioma cells. Furthermore, we showed that ectopically expressing miR-218 in glioma cells resulted in downregulation of matrix metalloproteinase-9 (MMP-9) and reduction in NF-{kappa}B transactivity at a transcriptional level, but inhibition of miR-218 enhanced the expression of MMP-9 and transcriptional activity of NF-{kappa}B. Moreover, we showed that miR-218 inactivated the NF-{kappa}B pathway through downregulating IKK-{beta} expression by directly targeting the 3'-untranslated region (3'-UTR) of IKK-{beta}. Taken together, our results suggest that miR-218 plays an important role in preventing the invasiveness of glioma cells, and our results present a novel mechanism of miRNA-mediated direct suppression of IKK-{beta}/NF-{kappa}B pathway in gliomas.« less

Integrin expression and integrin-mediated adhesion in vitro of human multipotent stromal cells (MSCs) to endothelial cells from various blood vessels.

PubMed

Semon, Julie A; Nagy, Lauren H; Llamas, Claire B; Tucker, H Alan; Lee, Ryang Hwa; Prockop, Darwin J

2010-07-01

Multipotent mesenchymal stromal cells (MSCs) home to damaged tissue by processes partly regulated by integrins. Integrin subunits expressed by MSCs were identified by flow cytometry (FC), immunocytochemistry (IC), and a panel of integrin-binding antibodies. In subconfluent cultures, over 80% of MSCs expressed integrin subunits beta1, beta2, and alpha3, 20%-55% expressed alpha1, alpha2, alpha4, alpha5, alpha6, and alphaV, and about 10% expressed beta3 when assayed by FC. None of the cells expressed significant levels of 13 other integrins as assayed by FC, but seven of the 13 integrins were detected by IC: beta5, alpha7, alpha8, alpha9, alpha11, alphaX, and alphaD. Expression of some integrins changed with MSC confluency: integrins beta3, alpha1, alpha3, alpha5, and alphaV increased, and alpha6 decreased. Furthermore, alpha4 was the only integrin to vary among preparations of MSCs from different donors. The results resolved some discrepancies in the literature concerning integrin expression by MSCs. We also investigated the role of specific integrins in MSC adhesion to endothelial cells (ECs) from the pulmonary artery (HPAEC), cardiac-derived microvasculature (HMVEC-C), and umbilical veins (HUVEC). In experiments with blocking antibodies to beta integrins, anti-beta5 reduced MSC adhesion to all types of ECs, anti-beta1 to both HUVEC and HPAEC, anti-beta3 to HUVEC, and anti-beta2 to HMVEC-C. With blocking antibodies to alpha integrins, anti-alphaX reduced adhesion to HPAEC and HMVEC-C, anti-alphaV to HPAEC, and both anti-alpha7 and anti-alphaD to HMVEC-C. Thus, MSCs use diverse integrins to adhere to EC from various blood vessels in vitro.

Effect of peptide aldehydes with IL-1 beta converting enzyme inhibitory properties on IL-1 alpha and IL-1 beta production in vitro.

PubMed

Németh, K; Patthy, M; Fauszt, I; Széll, E; Székely, J I; Bajusz, S

1995-12-01

Tripeptide and pentapeptide aldehydes as substrate-base inhibitors of cysteine proteases were designed in our laboratory for the inhibition of interleukin-1 beta converting enzyme (ICE), a recently described cysteine protease responsible for the processing of IL-1 beta. The biological effectivity of the peptide aldehydes was studied in THP-1 cells and human whole blood. The released and cell-associated IL-1 alpha and IL-1 beta levels were determined by ELISA from the supernatants and cell lysates, respectively. The total IL-1 like bioactivity was assayed by the D10 G4.1 cell proliferation method. The tripeptide aldehyde (Z-Val-His-Asp-H) and pentapeptide aldehyde (Eoc-Ala-Tyr-Val-Ala-Asp-H) significantly reduced IL-1 beta levels in the supernatants in relatively high concentrations (10-100 microM), but the IL-1 alpha release was unaffected by these peptides. However, a considerable decrease in the cell-associated IL-1 beta and IL-1 alpha levels was observed. N-terminal extension of the tripeptide aldehyde yielded even more potent inhibitors. Amino acid substitution at the P2 position did not cause considerable changes in the inhibitory activity. The peptide aldehydes suppressed the IL-1 beta production in a reversible manner, whereas dexamethasone, a glucocorticoid, had a prolonged inhibitory effect. The inhibitory effect of these peptides and that of dexamethasone appeared to be additive. These findings indicate that these peptide aldehydes might be used as IL-beta inhibitory agents in experimental models in which IL-1 beta is a key mediator or ICE is implicated.

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 percentage of cells with a positive reaction to PCNA and Ki 67 antigen. TGF-beta1, isotretinoin and tretinoin added to the culture resulted in the lowest percentage of PCNA positive cells. However, the lowest fraction of Ki 67 positive cells was observed after addition of isotretinoin. The obtained results also confirm the fact that the well-known regulatory proteins Bcl-2 and p53 play an important role in the regulation of apoptosis in the MCF-7 cell line, with lowered Bcl-2 expression accompanying easier apoptotic induction. The majority of the examined compounds act via the p53 pathway although some bypass this important proapoptotic factor.

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.

Rearrangements of genes for the antigen receptor on T cells as markers of lineage and clonality in human lymphoid neoplasms.

PubMed

Waldmann, T A; Davis, M M; Bongiovanni, K F; Korsmeyer, S J

1985-09-26

The T alpha and T beta chains of the heterodimeric T-lymphocyte antigen receptor are encoded by separated DNA segments that recombine during T-cell development. We have used rearrangements of the T beta gene as a widely applicable marker of clonality in the T-cell lineage. We show that the T beta genes are used in both the T8 and T4 subpopulations of normal T cells and that Sézary leukemia, adult T-cell leukemia, and the non-B-lineage acute lymphoblastic leukemias are clonal expansions of T cells. Furthermore, circulating T cells from a patient with the T8-cell-predominantly lymphocytosis associated with granulocytopenia are shown to be monoclonal. Finally, the sensitivity and specificity of this tumor-associated marker have been exploited to monitor the therapy of a patient with adult T-cell leukemia. These unique DNA rearrangements provide insights into the cellular origin, clonality, and natural history of T-cell neoplasia.

Synthetic alleles at position 121 define a functional domain of human interleukin-1 beta.

PubMed

Ambrosetti, D C; Palla, E; Mirtella, A; Galeotti, C; Solito, E; Navarra, P; Parente, L; Melli, M

1996-06-01

The non-conservative substitution of the tyrosine residue at position 121 of human interleukin-1 beta (IL-1 beta) generates protein mutants showing strong reduction of the capacity to induce (a) prostaglandin E2 (PGE2) release from fibroblasts and smooth muscle cells, (b) murine T-cells proliferation and (c) activation of interleukin-6 (IL-6) gene expression. It is generally accepted that these functions are mediated by the type-I interleukin-1 receptor (IL-1RI). However, the mutant proteins maintain the binding affinity to the types-I and II IL-1 receptors, which is the same as the control IL-1 beta, suggesting that this amino acid substitution does not alter the structure of the molecule, except locally. Thus we have identified a new functional site of IL-1 beta different from the known receptor binding region, responsible for fundamental IL-1 beta functions. Moreover, we show that the same mutants maintain at least two hypothalamic functions, that is, the in vitro short-term PGE2 release from rat hypothalamus and the induction of fever in rabbits. This result suggests that there is yet another site of the molecule responsible for the hypothalamic functions, implying that multiple active sites on the IL-1 beta molecule, possibly binding to more than one receptor chain, trigger different signals.

Infection of human T lymphotropic virus-I-specific immune T cell clones by human T lymphotropic virus-I.

PubMed Central

Mitsuya, H; Jarrett, R F; Cossman, J; Cohen, O J; Kao, C S; Guo, H G; Reitz, M S; Broder, S

1986-01-01

Human T lymphotropic virus-I (HTLV-I)-specific T cell lines were established and cloned. K5, an OKT8+ clone bearing multiple proviral integration sites, retained its HTLV-I-specific cytotoxicity and a normal dependence on interleukin 2 (IL-2), indicating that there is a finite number of transforming integration sites. R2, an OKT4+ HTLV-I-infected clone, initially mounted a proliferative response to HTLV-I; but then its IL-2-independent proliferation increased and the antigen specificity was lost. All HTLV-I-infected clones tested including K7, another OKT8+ transformed cytotoxic clone that had lost its reactivity, expressed comparable levels of T cell receptor beta-chain (TCR-beta) messenger (m)RNA. Although clones K5 and K7 had different functional properties, they had the same rearrangement of the TCR-beta gene, suggesting that they had the same clonal origin. These data indicate that HTLV-I-specific T cells retain their immune reactivity for variable periods of time following infection, but then usually lose it; in some cases, however, no alteration in function can be detected. The data also suggest that different consequences can take place in the same clone depending on the pattern of retroviral infection. Images PMID:2877011

Chondrocyte heterogeneity: immunohistologically defined variation of integrin expression at different sites in human fetal knees.

PubMed

Salter, D M; Godolphin, J L; Gourlay, M S

1995-04-01

During development and at maturity different forms of cartilage vary in morphology and macromolecular content. This reflects heterogeneity of chondrocyte activity, in part involving differential interactions with the adjacent extracellular matrix via specialized cell surface receptors such as integrins. We undertook an immunohistological study on a series of human fetal knee joints to assess variation in the expression of integrins by chondrocytes and potential matrix ligands in articular, epiphyseal, growth plate, and meniscal cartilage. The results show that articular chondrocytes (beta 1+, beta 5 alpha V+, alpha 1+, alpha 2+/-, alpha 5+, weakly alpha 6+, alpha V+) differed from epiphyseal (beta 1+, beta 5 alpha V+, alpha 1+/-, alpha 2+/-, alpha 5+, alpha 6+, alpha V+) growth plate (beta 1+, beta 5 alpha V+, alpha 1-, alpha 2-, alpha 5+, alpha 6+, alpha V+), and meniscal cells (beta 1+, beta 5 alpha V+, alpha 1+, strongly alpha 2+, alpha 5+, alpha 6+, alpha V+ in expression of integrin subunits. There was no expression of beta 3, beta 4, beta 6, or alpha 3 by chondrocytes. These results differ from previous reports on the expression of integrins by adult articular cartilage, where alpha 2 and alpha 6 are not seen. Variation in distribution of matrix ligands was also seen. Fibronectin, laminin and Type VI collagen were expressed in all cartilages but there was restricted expression of tenascin, ED-A and ED-B fibronectin isoforms (articular cartilage and meniscus), and vitronectin (absent from growth plate cartilage). Regulated expression of integrins by chondrocytes, associated with changes in the pericellular matrix composition, is of potential importance in control of cartilage differentiation and function in health and disease.

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

Baserga, S.J.; Benz, E.J. Jr.

A number of premature translation termination mutations (nonsense mutations) have been described in the human {alpha}- and {beta}-globin genes. Studies on mRNA isolated from patients with {beta}{sup 0}-thalassemia have shown that for both the {beta}-17 and the {beta}-39 mutations less than normal levels of {beta}-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human {beta}-globin mRNA). In vitro studies using the cloned {beta}-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. Themore » authors have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, they have studied the effect of five nonsense mutations and two missense mutations on the expression of human {beta}-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation.« less

LET and ion-species dependence for cell killing and mutation induction in normal human fibroblasts.

PubMed

Tsuruoka, Chizuru; Suzuki, Masao; Fujitaka, Kazunobu

2003-10-01

We have been studying LET and ion species dependence of RBE values in cell killing and mutation induction. Normal human skin fibroblasts were irradiated with heavy-ion beams such as carbon (290 Mev/u and 135 Mev/u), neon (230 Mev/u and 400 Mev/u), silicon (490 Mev/u) and iron (500 Mev/u) ion beams, generated by Heavy Ion Medical Accelerator in Chiba (HIMAC) at National Institute of Radiological Sciences (NIRS). Cell killing effect was detected as reproductive cell death using a colony formation assay. Mutation induction in hprt locus was detected to measure 6-thioguanine resistant colonies. The RBE-LET curves of cell killing and mutation induction were different each ion beam. So, we plotted RBE for cell killing and mutation induction as function of Z*2/beta2 instead of LET. RBE-Z*2/beta2 curves of cell killing indicated that the discrepancy of RBE-LET curves was reconciled each ion species. But RBE-Z*2/beta2 curves of mutation induction didn't corresponded between carbon- and silicon-ion beams. These results suggested that different biological endpoints may be suitable for different physical parameter, which represent the track structure of energy deposition of ion beams.

Directional and balancing selection in human beta-defensins.

PubMed

Hollox, Edward J; Armour, John A L

2008-04-16

In primates, infection is an important force driving gene evolution, and this is reflected in the importance of infectious disease in human morbidity today. The beta-defensins are key components of the innate immune system, with antimicrobial and cell signalling roles, but also reproductive functions. Here we examine evolution of beta-defensins in catarrhine primates and variation within different human populations. We show that five beta-defensin genes that do not show copy number variation in humans show evidence of positive selection in catarrhine primates, and identify specific codons that have been under selective pressure. Direct haplotyping of DEFB127 in humans suggests long-term balancing selection: there are two highly diverged haplotype clades carrying different variants of a codon that, in primates, is positively selected. For DEFB132, we show that extensive diversity, including a four-state amino acid polymorphism (valine, isoleucine, alanine and threonine at position 93), is present in hunter-gatherer populations, both African and non-African, but not found in samples from agricultural populations. Some, but not all, beta-defensin genes show positive selection in catarrhine primates. There is suggestive evidence of different selective pressures on these genes in humans, but the nature of the selective pressure remains unclear and is likely to differ between populations.

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.

N-acetylcysteine augments adenovirus-mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entry.

PubMed

Jornot, L; Morris, M A; Petersen, H; Moix, I; Rochat, T

2002-01-01

It has previously been shown that oxidants reduce the efficiency of adenoviral transduction in human umbilical vein endothelial cells (HUVECs). In this study, the effect of the antioxidant N-acetylcysteine (NAC) in adenovirus-mediated gene transfer has been investigated. HUVECs were pretreated or not with NAC, and infected with E1E3-deleted adenovirus (Ad) containing the LacZ gene expressed from the RSV-LTR promoter/enhancer in the presence and absence of NAC. Transgene expression was assessed at the protein level (histochemical staining, measurement of beta-Gal activity, and western blot), mRNA level (real-time RT-PCR) and gene level (nuclear run on) 24 h and 48 h after infection. Adenoviral DNA was quantitated by real-time PCR, and cell surface expression of Coxsackie/adenovirus receptors (CAR) was determined by FACS analysis. Pretreatment of cells with NAC prior to Ad infection enhanced beta-Gal activity by two-fold due to an increase in viral DNA, which was related to increased CAR expression. When NAC was present only during the post-infection period, a five-fold increase in beta-Gal activity and LacZ gene transcriptional activity was observed. When NAC was present during both the pretreatment and the post-infection period, beta-Gal activity was further enhanced, by 15-fold. Augmentation of beta-Gal activity was paralleled by an increase in beta-Gal protein and mRNA levels. NAC did not affect the half-life of LacZ mRNA. Pretreatment with NAC prior to Ad infection enhances virus entry, while treatment with NAC post-infection increases transgene transcription. This strategy permits the use of lower adenoviral loads and thus might be helpful for gene therapy of vascular diseases. Copyright 2001 John Wiley & Sons, Ltd.

Pregnancy and labor increase the capacity of human myometrial cells to secrete parathyroid hormone-related protein.

PubMed

Shenberger, J S; Dixon, P S; Choate, J; Helal, K; Shew, R L; Barth, W

2001-02-16

Parathyroid hormone-related protein (PTHrP), a oncofetal gene product possessing smooth muscle relaxant properties, has been found in rat and human uterine smooth muscle cells (USMC) where it is postulated to regulate myometrial tone and/or blood flow. Studies investigating the gestational regulation of PTHrP in human USMC have not been performed. This study was conducted to determine if pregnancy alters the capacity of USMC to secrete or respond to PTHrP. USMC cultures were established from 8 hysterectomy specimens (H) and 7 non-laboring (NP) and 5 laboring term pregnant uterine biopsies (LP). PTHrP secretion was measured at baseline and in response to TGF-beta1 using a immunoradiometric assay. The USMC response to PTHrP was assessed by incubating cultures with human (1-34)PTHrP and measuring cellular cAMP by radioimmunoassay. We found that cultures from the groups did not differ with respect to basal PTHrP secretion. TGF-beta1, on the other hand, produced dose-dependent increases in secreted PTHrP in each group such that LP>NP>H at 12 hrs and LP>NP and H 24 hrs. Maximal responses were found at 24 hrs in cells treated with 10 ng/ml TGF-beta1 (LP: 2034+/-366 vs NP: 1485+/-427; H: 1250+/-202 fmol/mg). Incubation of cultures with PTHrP produced dose-dependent increases in cAMP production, with 10(-7) M increasing levels by 64%. Neither pregnancy nor labor significantly affected the cAMP response. These findings indicate that the human myometrium has the capacity to increase PTHrP secretion during pregnancy and labor through a TGF-beta-dependent pathway. Such findings are consistent with a role of PTHrP in enhancing uterine blood flow.

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 is no longer chemotactic, suggesting that PMNs only use Fn that is constitutively expressed for migration. At higher concentrations of TGF-beta, the Fn released may accumulate basal to the cell, ultimately retarding cellular migration and modulating the chemotactic response.

The EndoC-βH1 cell line is a valid model of human beta cells and applicable for screenings to identify novel drug target candidates.

PubMed

Tsonkova, Violeta Georgieva; Sand, Fredrik Wolfhagen; Wolf, Xenia Asbæk; Grunnet, Lars Groth; Kirstine Ringgaard, Anna; Ingvorsen, Camilla; Winkel, Louise; Kalisz, Mark; Dalgaard, Kevin; Bruun, Christine; Fels, Johannes Josef; Helgstrand, Charlotte; Hastrup, Sven; Öberg, Fredrik Kryh; Vernet, Erik; Sandrini, Michael Paolo Bastner; Shaw, Allan Christian; Jessen, Carsten; Grønborg, Mads; Hald, Jacob; Willenbrock, Hanni; Madsen, Dennis; Wernersson, Rasmus; Hansson, Lena; Jensen, Jan Nygaard; Plesner, Annette; Alanentalo, Tomas; Petersen, Maja Borup Kjær; Grapin-Botton, Anne; Honoré, Christian; Ahnfelt-Rønne, Jonas; Hecksher-Sørensen, Jacob; Ravassard, Philippe; Madsen, Ole D; Rescan, Claude; Frogne, Thomas

2018-02-01

To characterize the EndoC-βH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates. EndoC-βH1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation. Transplantation of EndoC-βH1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-βH1 pseudoislets compared to monolayer cultures for both glucose and incretins. Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate. By screening of various proteins and peptides, we found Bombesin (BB) receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP) to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation. ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST) was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion. Overall, the EndoC-βH1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated insulin secretion capabilities. The cell line has an active cytokine induced caspase 3/7 apoptotic pathway and is responsive to ER stress initiation factors. The cells' ability to proliferate can be further increased by already known compounds as well as by novel peptides and proteins. Based on its robust performance during the functionality assessment assays, the EndoC-βH1 cell line was successfully used as a screening platform for identification of novel anti-diabetic drug candidates. Copyright © 2017 Novo Nordisk A/S. Published by Elsevier GmbH.. All rights reserved.

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

Laouar, A.; Glesne, D.; Huberman, E.

The role of protein kinase C-{beta} (PKC-{beta}) in apoptosis induced by tumor necrosis factor (TNF)-{alpha} and anti-Fas monoclonal antibody (mAb) in the human myeloid HL-60 leukemia cell line was studied by using its variant HL-525, which is deficient in PKC-{beta}. In contrast to the parental HL-60 cells, HL-525 is resistant to TNF-{alpha}-induced apoptosis but sensitive to anti-Fas mAb-induced apoptosis. Both cell types expressed similar levels of the TNF-receptor I, whereas the Fas receptor was detected only in HL-525 cells. Transfecting the HL-525 cells with an expression vector containing PKC-{beta} reestablished their susceptibility to TNF-{alpha}-induced apoptosis. The apoptotic effect of TNF-{alpha}more » in HL-60 and the transfectants was abrogated by fumonisin, an inhibitor of ceramide generation, and by the peptide Ac-YVAD-BoMK, an inhibitor of caspase-1 and -4. Supplementing HL-525 cells with exogenous ceramides bypassed the PKC-{beta} deficiency and induced apoptosis, which was also restrained by the caspase-1 and -4 inhibitor. The apoptotic effect of anti-Fas mAb in HL-525 cells was abrogated by the antioxidants N-acetylcysteine and glutathione and by the peptide z-DEVD-FMK, an inhibitor of caspase-3 and -7. We suggest that TNF-{alpha}-induced apoptosis involves PKC-{beta} and then ceramide and, in turn, caspase-1 and/or -4, whereas anti-Fas mAb-induced apoptosis utilizes reactive oxygen intermediates and, in turn, caspase-3 and/or -7.« less

Nanoparticles for Site Specific Genome Editing

NASA Astrophysics Data System (ADS)

McNeer, Nicole Ali

Triplex-forming peptide nucleic acids (PNAs) can be used to coordinate the recombination of short 50-60 by "donor DNA" fragments into genomic DNA, resulting in site-specific correction of genetic mutations or the introduction of advantageous genetic modifications. Site-specific gene editing in hematopoietic stem and progenitor cells (HSPCs) could result in treatment or cure of inherited disorders of the blood such as beta-thalassemia. Gene editing in HSPCs and differentiated T cells could help combat HIV/AIDs by modifying receptors, such as CCR5, necessary for R5-tropic HIV entry. However, translation of genome modification technologies to clinical practice is limited by challenges in intracellular delivery, especially in difficult-to-transfect hematolymphoid cells. In vivo gene editing could also provide novel treatment for systemic monogenic disorders such as cystic fibrosis, an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane receptor. Here, we have engineered biodegradable nanoparticles to deliver oligonucleotides for site-specific genome editing of disease-relevant genes in human cells, with high efficiency, low toxicity, and editing of clinically relevant cell types. We designed nanoparticles to edit the human beta-globin and CCR5 genes in hematopoietic cells. We show that poly(lactic-co-glycolic acid) (PLGA) nanoparticles can delivery PNA and donor DNA for site-specific gene modification in human hematopoietic cells in vitro and in vivo in NOD-scid IL2rgammanull mice. Nanoparticles delivered by tail vein localized to hematopoietic compartments in the spleen and bone marrow of humanized mice, resulting in modification of the beta-globin and CCR5 genes. Modification frequencies ranged from 0.005 to 20% of cells depending on the organ and cell type, without detectable toxicity. This project developed highly versatile methods for delivery of therapeutics to hematolymphoid cells and hematopoietic stem cells, and will help to translate gene therapies for diseases of the blood and immune system to clinical practice. In addition, we have expanded the use of this technology to an additional nonhematopoietic model system: correction of the human cystic fibrosis transmembrane receptor gene in human bronchial epithelial cells. The work presented here represents (1) the first use of biodegradable nanoparticles for PNA delivery, (2) the first direct in vivo site-specific genome modification in human cells, and (3) the first use of triplex-PNA technology for site-specific genome editing in cystic fibrosis.

A programmable synthetic lineage-control network that differentiates human IPSCs into glucose-sensitive insulin-secreting beta-like cells

PubMed Central

Saxena, Pratik; Heng, Boon Chin; Bai, Peng; Folcher, Marc; Zulewski, Henryk; Fussenegger, Martin

2016-01-01

Synthetic biology has advanced the design of standardized transcription control devices that programme cellular behaviour. By coupling synthetic signalling cascade- and transcription factor-based gene switches with reverse and differential sensitivity to the licensed food additive vanillic acid, we designed a synthetic lineage-control network combining vanillic acid-triggered mutually exclusive expression switches for the transcription factors Ngn3 (neurogenin 3; OFF-ON-OFF) and Pdx1 (pancreatic and duodenal homeobox 1; ON-OFF-ON) with the concomitant induction of MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A; OFF-ON). This designer network consisting of different network topologies orchestrating the timely control of transgenic and genomic Ngn3, Pdx1 and MafA variants is able to programme human induced pluripotent stem cells (hIPSCs)-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells, whose glucose-stimulated insulin-release dynamics are comparable to human pancreatic islets. Synthetic lineage-control networks may provide the missing link to genetically programme somatic cells into autologous cell phenotypes for regenerative medicine. PMID:27063289

Beta cell chromogranin B is partially segregated in distinct granules and can be released separately from insulin in response to stimulation.

PubMed

Giordano, T; Brigatti, C; Podini, P; Bonifacio, E; Meldolesi, J; Malosio, M L

2008-06-01

We investigated, in three beta cell lines (INS-1E, RIN-5AH, betaTC3) and in human and rodent primary beta cells, the storage and release of chromogranin B, a secretory protein expressed in beta cells and postulated to play an autocrine role. We asked whether chromogranin B is stored together with and discharged in constant ratio to insulin upon various stimuli. The intracellular distribution of insulin and chromogranin B was revealed by immunofluorescence followed by three-dimensional image reconstruction and by immunoelectron microscopy; their stimulated discharge was measured by ELISA and immunoblot analysis of homogenates and incubation media. Insulin and chromogranin B, co-localised in the Golgi complex/trans-Golgi network, appeared largely segregated from each other in the secretory granule compartment. In INS-1E cells, the percentage of granules positive only for insulin or chromogranin B and of those positive for both was 66, 7 and 27%, respectively. In resting cells, both insulin and chromogranin B were concentrated in the granule cores; upon stimulation, chromogranin B (but not insulin) was largely redistributed to the core periphery and the surrounding halo. Strong stimulation with a secretagogue mixture induced parallel release of insulin and chromogranin B, whereas with 3-isobutyl-1-methylxantine and forskolin +/- high glucose release of chromogranin B predominated. Weak, Ca(2+)-dependent stimulation with ionomycin or carbachol induced exclusive release of chromogranin B, suggesting a higher Ca(2+) sensitivity of the specific granules. The unexpected complexity of the beta cell granule population in terms of heterogeneity, molecular plasticity and the differential discharge, could play an important role in physiological control of insulin release and possibly also in beta cell pathology.

Stabilization of mismatch repair gene PMS2 by glycogen synthase kinase 3beta is implicated in the treatment of cervical carcinoma.

PubMed

Zhang, Yuan; Shu, Yi Min; Wang, Shu Fang; Da, Bang Hong; Wang, Ze Hua; Li, Hua Bin

2010-02-23

PMS2 expression loss was reported in a variety of human. However, its importance has not been fully understood in cervical carcinoma. The aim of this study was to determine the expression of PMS2 in cervical carcinoma and evaluate the significance of mismatch repair gene PMS2 regulated by glycogen synthase kinase 3beta (GSK-3beta) in chemosensitivity. We examined PMS2 and phosphorylated GSK-3beta(s9) expression in cervical carcinoma tissues using immunohistochemical staining. Furthermore, we detected PMS2 expression in HeLa cells and evaluate the interaction with GSK-3beta after transfection with GSK-3beta by small interference RNA (siRNA), co-immunoprecipitation and immunoblotting. We also evaluated the effect of PMS2 transfection on HeLa cells' chemosensitivity to cisplatin treatment. We found significant downregulation of PMS2 in cervical carcinoma, which was negatively associated with phosphorylated GSK-3beta (s9). Furthermore, we demonstrated GSK-3beta transfection was able to interact with PMS2 and enhance PMS2 production in HeLa cells, and increased PMS2 production was responsible for enhanced chemosensitivity. Our results provide the evidence that stabilization of PMS2 production by GSK-3beta was important to improve chemosensitization, indicating the significance of GSK-3beta-related PMS2 downregulation in the development of cervical carcinoma and in developing a potential strategy for chemotherapy.

The regulation of progesterone receptor by 17 beta estradiol and tamoxifen in the Zr-75-1 human breast cancer cell line in defined medium.

PubMed

Allegra, J C; Korat, O; Do, H M; Lippman, M

1981-01-01

The regulation of progesterone receptor by 17 beta estradiol and tamoxifen in the ZR-75-1 human breast cancer cell line in defined medium is described. ZR-75-1 cells maintained in serum free hormone supplemented medium minus estradiol lack progesterone receptor activity. Readdition of estradiol to these cells leads to a marked stimulation of progesterone receptor activity (0 to greater than 100 fmols of specifically bound progesterone per million cells). Tamoxifen (10(-6)M-10(-8)M) does not stimulate progesterone receptor activity in this cell line. The presence of progesterone receptor activity is not directly related to growth. Withdrawal of insulin in the continued presence of estradiol has no effect on progesterone receptor concentration although net cell growth ceases. Conversely, withdrawal of estradiol in the continued presence of insulin induces a cessation of net cell growth accompanied by a loss of all progesterone receptor activity within 3-5 days.

Visible red and infrared light alters gene expression in human marrow stromal fibroblast cells.

PubMed

Guo, J; Wang, Q; Wai, D; Zhang, Q Z; Shi, S H; Le, A D; Shi, S T; Yen, S L-K

2015-04-01

This study tested whether or not gene expression in human marrow stromal fibroblast (MSF) cells depends on light wavelength and energy density. Primary cultures of isolated human bone marrow stem cells (hBMSC) were exposed to visible red (VR, 633 nm) and infrared (IR, 830 nm) radiation wavelengths from a light emitting diode (LED) over a range of energy densities (0.5, 1.0, 1.5, and 2.0 Joules/cm2) Cultured cells were assayed for cell proliferation, osteogenic potential, adipogenesis, mRNA and protein content. mRNA was analyzed by microarray and compared among different wavelengths and energy densities. Mesenchymal and epithelial cell responses were compared to determine whether responses were cell type specific. Protein array analysis was used to further analyze key pathways identified by microarrays. Different wavelengths and energy densities produced unique sets of genes identified by microarray analysis. Pathway analysis pointed to TGF-beta 1 in the visible red and Akt 1 in the infrared wavelengths as key pathways to study. TGF-beta protein arrays suggested switching from canonical to non-canonical TGF-beta pathways with increases to longer IR wavelengths. Microarrays suggest RANKL and MMP 10 followed IR energy density dose-response curves. Epithelial and mesenchymal cells respond differently to stimulation by light suggesting cell type-specific response is possible. These studies demonstrate differential gene expression with different wavelengths, energy densities and cell types. These differences in gene expression have the potential to be exploited for therapeutic purposes and can help explain contradictory results in the literature when wavelengths, energy densities and cell types differ. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Revisiting the liver in human yellow fever: virus-induced apoptosis in hepatocytes associated with TGF-beta, TNF-alpha and NK cells activity.

PubMed

Quaresma, Juarez A S; Barros, Vera L R S; Pagliari, Carla; Fernandes, Elaine R; Guedes, Fernanda; Takakura, Cleusa F H; Andrade, Heitor F; Vasconcelos, Pedro F C; Duarte, Maria I S

2006-02-05

Flavivirus infection as dengue and yellow fever persists as a terrible menace to pandemics, due to Aedes prevalence in the Americas. Yellow fever is characterized by hepatocyte damage, with steatosis, apoptosis and necrosis, mainly in the midzonal region of the liver, but the injury mechanism has not been studied at the light of recent knowledge, such as the advances in cell death mechanisms, inflammatory response and cytokine cell expression tools. We studied 53 human liver paraffin embedded blocks from patients who died with yellow fever, all with histological demonstration of higher prevalence of apoptosis over necrosis and mild disproportionate inflammatory response. Viral antigens were found most frequently in hepatocytes from the midzonal area than other lobule areas, as detected by specific immunohistochemistry. Infiltrating cell subpopulations showed mainly CD4+ T lymphocytes, with small numbers of CD8+ cytotoxic lymphocytes, CD20+ B lymphocytes, NKT+ cells and S100+ dendritic cells in the sites of inflammation, as compared to normal and leptospirosis liver blocks. Some cells expressed TNF-alpha and IFN-gamma, but a much more intense proportion of TGF-beta expressing cells were found, suggesting both a Th1 and Th3 patterns of immune response in yellow fever. Most affected hepatocyte presented apoptosis markers that appear at the cell death main pathway in this infection. Viral antigens, which production could interfere in hepatocyte biology, could induce the activation of apoptosis cascade, but TGF-beta was also an apoptosis promoter. Our finding supports the key effect of the yellow fever virus in hepatocyte injury, resulting in prevalence of apoptosis over necrosis, aside from a TGF-beta action induced by the inflammatory response.

Mesenchymal precursor cells maintain the differentiation and proliferation potentials of breast epithelial cells

PubMed Central

2014-01-01

Introduction Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. Studies of the cellular and molecular mechanisms underlying these interactions require ex vivo experimental model systems that recapitulate the complexity of human tissue without compromising the differentiation and proliferation potentials of human primary cells. Methods We isolated and characterized human breast epithelial and mesenchymal precursors from reduction mammoplasty tissue and tagged them with lentiviral vectors. We assembled heterotypic co-cultures and compared mesenchymal and epithelial cells to cells in corresponding monocultures by analyzing growth, differentiation potentials, and gene expression profiles. Results We show that heterotypic culture of non-immortalized human primary breast epithelial and mesenchymal precursors maintains their proliferation and differentiation potentials and constrains their growth. We further describe the gene expression profiles of stromal and epithelial cells in co-cultures and monocultures and show increased expression of the tumor growth factor beta (TGFβ) family member inhibin beta A (INHBA) in mesenchymal cells grown as co-cultures compared with monocultures. Notably, overexpression of INHBA in mesenchymal cells increases colony formation potential of epithelial cells, suggesting that it contributes to the dynamic reciprocity between breast mesenchymal and epithelial cells. Conclusions The described heterotypic co-culture system will prove useful for further characterization of the molecular mechanisms mediating interactions between human normal or neoplastic breast epithelial cells and the stroma, and will provide a framework to test the relevance of the ever-increasing number of oncogenomic alterations identified in human breast cancer. PMID:24916766

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.

Effects of Histone Deacetylase Inhibitor (HDACi); Trichostatin-A (TSA) on the expression of housekeeping genes.

PubMed

Mogal, Ashish; Abdulkadir, Sarki A

2006-04-01

In quantitative RT-PCR (qRT-PCR), analysis of gene expression is dependent on normalization using housekeeping genes such as 18S rRNA, GAPDH and beta actin. However, variability in their expression has been reported to be caused by factors like drug treatment, pathological states and cell-cycle phase. An emerging area of cancer research focuses on identifying the role of epigenetic alterations such as histone modifications and DNA methylation in the initiation and progression of cancer. Histone acetylation is the best studied modification so far and has been probed through the use of histone deacetylase inhibitors (HDACi). Further, modulation of histone acetylation is currently being explored as a therapeutic strategy in the treatment of cancer and HDACis have shown promise in inhibiting tumorigenesis and metastasis. Trichostatin-A (TSA) is the most widely used HDACi. Therefore, we were driven to identify a suitable internal control for RT-PCR following TSA treatment. We performed quantitative RT-PCR analysis using mouse prostate tissue explants, human prostate cancer (LNCaP) cells and human breast cancer (T-47D and ZR-75-1) cells following TSA treatment. Expression of housekeeping genes including 18S rRNA, beta actin, GAPDH and ribosomal highly-basic 23-kDa protein (rb 23-kDa, RPL13A) were compared in vehicle versus TSA treated samples. Our results showed marked variations in 18S rRNA, beta actin mRNA and GAPDH mRNA levels in mouse prostate explants and a human prostate cancer (LNCaP) cell line following TSA treatment. Furthermore, in two human breast cancer cell lines (T-47D and ZR-75-1) 18S rRNA, beta actin mRNA and GAPDH mRNA levels varied significantly. However, RPL13A mRNA levels remained constant in all the conditions tested. Therefore, we recommend use of RPL13A as a standard for normalization during TSA treatment.

Role of Rho/ROCK and p38 MAP kinase pathways in transforming growth factor-beta-mediated Smad-dependent growth inhibition of human breast carcinoma cells in vivo.

PubMed

Kamaraju, Anil K; Roberts, Anita B

2005-01-14

TGF-beta is a multifunctional cytokine known to exert its biological effects through a variety of signaling pathways of which Smad signaling is considered to be the main mediator. At present, the Smad-independent pathways, their interactions with each other, and their roles in TGF-beta-mediated growth inhibitory effects are not well understood. To address these questions, we have utilized a human breast cancer cell line MCF10CA1h and demonstrate that p38 MAP kinase and Rho/ROCK pathways together with Smad2 and Smad3 are necessary for TGF-beta-mediated growth inhibition of this cell line. We show that Smad2/3 are indispensable for TGF-beta-mediated growth inhibition, and that both p38 and Rho/ROCK pathways affect the linker region phosphorylation of Smad2/3. Further, by using Smad3 mutated at the putative phosphorylation sites in the linker region, we demonstrate that phosphorylation at Ser203 and Ser207 residues is required for the full transactivation potential of Smad3, and that these residues are targets of the p38 and Rho/ROCK pathways. We demonstrate that activation of the p38 MAP kinase pathway is necessary for the full transcriptional activation potential of Smad2/Smad3 by TGF-beta, whereas activity of Rho/ROCK is necessary for both down-regulation of c-Myc protein and up-regulation of p21waf1 protein, directly interfering with p21waf1 transcription. Our results not only implicate Rho/ROCK and p38 MAPK pathways as necessary for TGF-beta-mediated growth inhibition, but also demonstrate their individual contributions and the basis for their cooperation with each other.

Placental macrophage contact potentiates the complete replicative cycle of human cytomegalovirus in syncytiotrophoblast cells: role of interleukin-8 and transforming growth factor-beta1.

PubMed

Bácsi, A; Aranyosi, J; Beck, Z; Ebbesen, P; Andirkó, I; Szabó, J; Lampé, L; Kiss, J; Gergely, L; Tóth, F D

1999-10-01

Although syncytiotrophoblast (ST) cells can be infected by human cytomegalovirus (HCMV), in vitro studies have indicated that ST cells do not support the complete viral reproductive cycle, or HCMV replication may occur in less than 3% of ST cells. The present study tested the possibility that placental macrophages might enhance activation of HCMV carried in ST cells and, further, that infected ST cells would be capable of transmitting virus to neighboring macrophages. For this purpose, we studied HCMV replication in ST cells grown alone or cocultured with uninfected placental macrophages. Our results demonstrated that HCMV gene expression in ST cells was markedly upregulated by coculture with macrophages, resulting in release of substantial amounts of infectious virus from HCMV-infected ST cells. After having become permissive for viral replication, ST cells delivered HCMV to the cocultured macrophages, as evidenced by detection of virus-specific antigens in these cells. The stimulatory effect of coculture on HCMV gene expression in ST cells was mediated by marked interleukin-8 (IL-8) and transforming growth factor-beta1 (TGF-beta1) release from macrophages, an effect caused by contact between the different placental cells. Our findings indicate an interactive role for the ST layer and placental macrophages in the dissemination of HCMV among placental tissue. Eventually, these interactions may contribute to the transmission of HCMV from mother to the fetus.

Diabetes-associated dry eye syndrome in a new humanized transgenic model of type 1 diabetes.

PubMed

Imam, Shahnawaz; Elagin, Raya B; Jaume, Juan Carlos

2013-01-01

Patients with Type 1 Diabetes (T1D) are at high risk of developing lacrimal gland dysfunction. We have developed a new model of human T1D using double-transgenic mice carrying HLA-DQ8 diabetes-susceptibility haplotype instead of mouse MHC-class II and expressing the human beta cell autoantigen Glutamic Acid Decarboxylase in pancreatic beta cells. We report here the development of dry eye syndrome (DES) after diabetes induction in our humanized transgenic model. Double-transgenic mice were immunized with DNA encoding human GAD65, either naked or in adenoviral vectors, to induce T1D. Mice monitored for development of diabetes developed lacrimal gland dysfunction. Animals developed lacrimal gland disease (classically associated with diabetes in Non Obese Diabetic [NOD] mice and with T1D in humans) as they developed glucose intolerance and diabetes. Animals manifested obvious clinical signs of dry eye syndrome (DES), from corneal erosions to severe keratitis. Histological studies of peri-bulbar areas revealed lymphocytic infiltration of glandular structures. Indeed, infiltrative lesions were observed in lacrimal/Harderian glands within weeks following development of glucose intolerance. Lesions ranged from focal lymphocytic infiltration to complete acinar destruction. We observed a correlation between the severity of the pancreatic infiltration and the severity of the ocular disease. Our results demonstrate development of DES in association with antigen-specific insulitis and diabetes following immunization with clinically relevant human autoantigen concomitantly expressed in pancreatic beta cells of diabetes-susceptible mice. As in the NOD mouse model and as in human T1D, our animals developed diabetes-associated DES. This specific finding stresses the relevance of our model for studying these human diseases. We believe our model will facilitate studies to prevent/treat diabetes-associated DES as well as human diabetes.

Insertion and deletion mutagenesis of the human cytomegalovirus genome

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

Spaete, R.R.; Mocarski, E.S.

1987-10-01

Studies on human cytomegalovirus (CMV) have been limited by a paucity of molecular genetic techniques available for manipulating the viral genome. The authors have developed methods for site-specific insertion and deletion mutagenesis of CMV utilizing a modified Escherichia coli lacZ gene as a genetic marker. The lacZ gene was placed under the control of the major ..beta.. gene regulatory signals and inserted into the viral genome by homologous recombination, disrupting one of two copies of this ..beta.. gene within the L-component repeats of CMV DNA. They observed high-level expression of ..beta..-galactosidase by the recombinant in a temporally authentic manner, withmore » levels of this enzyme approaching 1% of total protein in infected cells. Thus, CMV is an efficient vector for high-level expression of foreign gene products in human cells. Using back selection of lacZ-deficient virus in the presence of the chromogenic substrate 5-bromo-4-chloro-3-indolyl ..beta..-D-galactoside, they generated random endpoint deletion mutants. Analysis of these mutant revealed that CMV DNA sequences flanking the insert had been removed, thereby establishing this approach as a means of determining whether sequences flanking a lacZ insertion are dispensable for viral growth. In an initial test of the methods, they have shown that 7800 base pairs of one copy of L-component repeat sequences can be deleted without affecting viral growth in human fibroblasts.« less

Crystallization and preliminary X-ray analysis of recombinant human acid beta-glucocerebrosidase, a treatment for Gaucher's disease

NASA Technical Reports Server (NTRS)

Roeber, Dana; Achari, Aniruddha; Manavalan, Partha; Edmunds, Tim; Scott, David L.

2003-01-01

Acid beta-glucocerebrosidase (N-acylsphingosyl-1-O-beta-D-glucoside:glucohydrolase) is a lysosomal glycoprotein that catalyzes the hydrolysis of the glycolipid glucocerebroside to glucose and ceramide. Inadequate levels of this enzyme underly the pathophysiology of Gaucher's disease. Cerezyme (Genzyme Corporation, Cambridge, MA, USA) is a partially deglycosylated form of recombinant human acid beta-glucocerebrosidase that is used in the treatment of Gaucher patients. Although acid beta-glucocerebrosidase belongs to a large family of glycosidases, relatively little is known regarding its structural biology. Here, the crystallization and the initial diffraction analysis of Cerezyme are reported. The crystals are C-centered orthorhombic, with unit-cell parameters a = 285.0, b = 110.2, c = 91.7 A. A 99.9% complete data set has been collected to 2.75 A with an R(sym) of 8.8%.

Crystallization and Preliminary X-ray analysis of Human Recombinant Acid beta-glucocerebrosidase, a treatment for Gaucher's Disease

NASA Technical Reports Server (NTRS)

Roeber, Dana F.; Achari, Aniruddha; Manavalan, Partha; Edmunds, Tim; Scott, David L.; Curreri, Peter A. (Technical Monitor)

2002-01-01

Acid beta-glucocerebrosidase (N-acylsphingosyl - O - beta-D - glucoside:glucohydrolase) is a lysosomal glycoprotein that catalyzes the hydrolysis of the glycolipid glucocerebroside to glucose and ceramide. Inadequate levels of this enzyme underly the pathophysiology of Gaucher's disease. Cerezyme(R) (Genzyme Corporation, Cambridge, MA) is a partially deglycosylated form of recombinant human acid beta-glucocerebrosidase that is commercially available for the treatment of Gaucher patients. Although acid beta-glucocerebrosidase belongs to a large family of glycosidases, relatively little is known regarding its structural biology. We report the crystallization and the initial diffraction analysis of Cerezyme(R). The crystals are C-centered orthorhombic, with unit-cell parameters of a = 285.0 A, b = 110.2 A, and c = 91.7 A. A 99.9 A complete data set has been collected to 2.75 A with an R(sub sym) of 8.8 %.

Magnetic measurements on human erythrocytes: Normal, beta thalassemia major, and sickle

NASA Astrophysics Data System (ADS)

Sakhnini, Lama

2003-05-01

In this article magnetic measurements were made on human erythrocytes at different hemoglobin states (normal and reduced hemoglobin). Different blood samples: normal, beta thalassemia major, and sickle were studied. Beta thalassemia major and sickle samples were taken from patients receiving lifelong blood transfusion treatment. All samples examined exhibited diamagnetic behavior. Beta thalassemia major and sickle samples showed higher diamagnetic susceptibilities than that for the normal, which was attributed to the increase of membrane to hemoglobin volume ratio of the abnormal cells. Magnetic measurements showed that the erythrocytes in the reduced state showed less diamagnetic response in comparison with erythrocytes in the normal state. Analysis of the paramagnetic component of magnetization curves gave an effective magnetic moment of μeff=7.6 μB per reduced hemoglobin molecule. The same procedure was applied to sickle and beta thalassemia major samples and values for μeff were found to be comparable to that of the normal erythrocytes.

Differential regulation of amyloid-. beta. -protein mRNA expression within hippocampal neuronal subpopulations in Alzheimer disease

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

Higgins, G.A.; Lewis, D.A.; Bahmanyar, S.

1988-02-01

The authors have mapped the neuroanatomical distribution of amyloid-..beta..-protein mRNA within neuronal subpopulations of the hippocampal formation in the cynomolgus monkey (Macaca fascicularis), normal aged human, and patients with Alzheimer disease. Amyloid-..beta..-protein mRNA appears to be expressed in all hippocampal neurons, but at different levels of abundance. In the central nervous system of monkey and normal aged human, image analysis shows that neurons of the dentate gyrus and cornu Ammonis fields contain a 2.5-times-greater hybridization signal than is present in neurons of the subiculum and entorhinal cortex. In contrast, in the Alzheimer disease hippocampal formation, the levels of amyloid-..beta..-protein mRNAmore » in the cornu Ammonis field 3 and parasubiculum are equivalent. These findings suggest that within certain neuronal subpopulations cell type-specific regulation of amyloid-..beta..-protein gene expression may be altered in Alzheimer disease.« less

The influence of major dietary fatty acids on insulin secretion and action.

PubMed

López, Sergio; Bermúdez, Beatriz; Abia, Rocío; Muriana, Francisco J G

2010-02-01

To briefly summarize recent advances towards understanding the influence of major dietary fatty acids on beta-cell function and evaluate their implications for insulin resistance. Studies in humans have shown that beta-cell function and insulin sensitivity improve progressively in the postprandial period as the proportion of monounsaturated fatty acids (MUFAs) with respect to saturated fatty acids (SFAs) in dietary fats increases. However, cell-culture experiments have revealed a dichotomy in the ability of fatty acids to moderate hyperactivity of, and induce lipotoxicity in, beta-cells. There are also some novel findings regarding the ability of HDL to protect beta-cells against oxidized LDL-induced apoptosis in vitro and of reconstituted HDL to attenuate insulin resistance in vivo. These findings raise new questions regarding the contribution of dietary fatty acids to insulin secretion and action. These new findings point to a critical role for major dietary fatty acids in the etiology and pathogenesis of diabetes, which appears to be of particular relevance during postprandial periods and mainly depends on the fatty acid type. This underscores the importance of dietary fatty acids in standard diabetes management.

Stabilization of beta-catenin induces pancreas tumor formation.

PubMed

Heiser, Patrick W; Cano, David A; Landsman, Limor; Kim, Grace E; Kench, James G; Klimstra, David S; Taketo, Maketo M; Biankin, Andrew V; Hebrok, Matthias

2008-10-01

beta-Catenin signaling within the canonical Wnt pathway is essential for pancreas development. However, the pathway is normally down-regulated in the adult organ. Increased cytoplasmic and nuclear localization of beta-catenin can be detected in nearly all human solid pseudopapillary neoplasms (SPN), a rare tumor with low malignant potential. Conversely, pancreatic ductal adenocarcinoma (PDA) accounts for the majority of pancreatic tumors and is among the leading causes of cancer death. Whereas activating mutations within beta-catenin and other members of the canonical Wnt pathway are rare, recent reports have implicated Wnt signaling in the development and progression of human PDA. Here, we sought to address the role of beta-catenin signaling in pancreas tumorigenesis. Using Cre/lox technology, we conditionally activated beta-catenin in a subset of murine pancreatic cells in vivo. Activation of beta-catenin results in the formation of large pancreatic tumors at a high frequency in adult mice. These tumors resemble human SPN based on morphologic and immunohistochemical comparisons. Interestingly, stabilization of beta-catenin blocks the formation of pancreatic intraepithelial neoplasia (PanIN) in the presence of an activating mutation in Kras that is known to predispose individuals to PDA. Instead, mice in which beta-catenin and Kras are concurrently activated develop distinct ductal neoplasms that do not resemble PanIN lesions. These results demonstrate that activation of beta-catenin is sufficient to induce pancreas tumorigenesis. Moreover, they indicate that the sequence in which oncogenic mutations are acquired has profound consequences on the phenotype of the resulting tumor.

The Tregs' world according to GARP.

PubMed

Battaglia, Manuela; Roncarolo, Maria Grazia

2009-12-01

Naturally occurring CD4+CD25(high) regulatory T cells (nTreg) are essential for maintaining tolerance. FOXP3 has been established as a molecular marker of nTreg; however, FOXP3 cannot be used as a reliable marker for bona fide human nTreg since effector T cells also up-regulate FOXP3 expression upon activation. Despite the important function of nTreg, the underlying molecular mechanisms of nTreg-mediated suppression are far from defined. Previous studies have demonstrated that the TGF-beta latency-associated peptide (LAP) is expressed on the surface of nTreg, and that immunosuppression can be mediated by membrane TGF-beta; however, it remains unknown how LAP is bound to nTreg and what is the functional significance of its selective expression on activated nTreg. The nTreg's world may now change according to GARP, an orphan toll-like receptor composed of leucine-rich repeats. In this issue of the European Journal of Immunology, a study provides further demonstration that GARP is selectively expressed only in activated human nTreg and nTreg cell clones but not in activated effector T cells, confirming GARP as a bona fide nTreg marker. In addition, GARP binds directly to LAP; yet, GARP over-expression is insufficient to induce modification of latent TGF-beta into active TGF-beta further clarifying its role in nTreg-mediated suppression.

The cathelicidin LL-37 activates human mast cells and is degraded by mast cell tryptase: counter-regulation by CXCL4.

PubMed

Schiemann, Florian; Brandt, Ernst; Gross, Roland; Lindner, Buko; Mittelstädt, Jessica; Sommerhoff, Christian P; Schulmistrat, Jan; Petersen, Frank

2009-08-15

The cathelicidin LL-37 represents a potent antimicrobial and cell-stimulating agent, most abundantly expressed in peripheral organs such as lung and skin during inflammation. Because mast cells (MC) overtake prominent immunomodulatory roles in these organs, we wondered whether interactions exist between MC and LL-37. In this study, we show for the first time to our knowledge that physiological concentrations of LL-37 induce degranulation in purified human lung MC. Intriguingly, as a consequence LL-37 rapidly undergoes limited cleavage by a released protease. The enzyme was identified as beta-tryptase by inhibitor studies and by comparison to the recombinant protease. Examining the resulting LL-37 fragments for their functional activity, we found that none of the typical capacities of intact LL-37, i.e., MC degranulation, bactericidal activity, and neutralization of LPS, were retained. Conversely, we found that another inflammatory protein, the platelet-derived chemokine CXCL4, protects LL-37 from cleavage by beta-tryptase. Interestingly, CXCL4 did not act as a direct enzyme inhibitor, but destabilized active tetrameric beta-tryptase by antagonizing the heparin component required for the integrity of the tetramer. Altogether our results suggest that interaction of LL-37 and MC initiates an effective feedback loop to limit cathelicidin activity during inflammation, whereas CXCL4 may represent a physiological counter-regulator of beta-tryptase activity.

In vivo production of cytokines and beta (C-C) chemokines in human recurrent herpes simplex lesions--do herpes simplex virus-infected keratinocytes contribute to their production?

PubMed

Mikloska, Z; Danis, V A; Adams, S; Lloyd, A R; Adrian, D L; Cunningham, A L

1998-04-01

Recurrent human herpes simplex lesions are infiltrated by macrophages and CD4 and CD8 lymphocytes, which secrete cytokines and chemokines. Vesicle fluid was examined by ELISA for the presence of cytokines and beta (C-C) chemokines. On the first day of the lesion, high concentrations of interleukin (IL)-1beta, and IL-6, moderate concentrations of IL-1alpha and IL-10, and low concentrations of IL-12 and beta chemokines were found; levels of macrophage inflammatory protein (MIP)-1beta were significantly higher than levels of MIP-1alpha and RANTES. At day 3, the concentrations of IL-1beta, IL-6, and MIP-1beta were lower, whereas the levels of IL-10, IL-12, and MIP-1alpha remained similar, and the level of tumor necrosis factor-alpha was now detectable. Herpes simplex virus infection of keratinocytes in vitro stimulated production of beta chemokines followed by IL-12 and then IL-10, IL-1alpha, IL-1beta, and IL-6, indicating a potential role for these events in early recruitment, activation, and interferon-gamma production of CD4 cells in herpetic lesions.

Targeting Alpha5 Beta1 Integrin to Prevent Metastatic Breast Cancer Cell Invasion: PhScN Target Site Definition and Plasma Stability

DTIC Science & Technology

2013-09-01

12192595 12. Yao, H., D. Veine, K. Fay, E. Staszewski, et al., The PHSCN dendrimer as a more potent inhibitor of human breast cancer cell...Z.Z. Zeng, K.S. Fay, et al., Increased potency of the PHSCN dendrimer as an inhibitor of human prostate cancer cell invasion, extravasation, and lung

Anchorage mediated by integrin alpha6beta4 to laminin 5 (epiligrin) regulates tyrosine phosphorylation of a membrane-associated 80-kD protein

PubMed Central

1996-01-01

Detachment of basal keratinocytes from basement membrane signals a differentiation cascade. Two integrin receptors alpha6beta4 and alpha3beta1 mediate adhesion to laminin 5 (epiligrin), a major extracellular matrix protein in the basement membrane of epidermis. By establishing a low temperature adhesion system at 4 degrees C, we were able to examine the exclusive role of alpha6beta4 in adhesion of human foreskin keratinocyte (HFK) and the colon carcinoma cell LS123. We identified a novel 80-kD membrane-associated protein (p80) that is tyrosine phosphorylated in response to dissociation of alpha6beta4 from laminin 5. The specificity of p80 phosphorylation for laminin 5 and alpha6beta4 was illustrated by the lack of regulation of p80 phosphorylation on collagen, fibronectin, or poly-L-lysine surfaces. We showed that blocking of alpha3beta1 function using inhibitory mAbs, low temperature, or cytochalasin D diminished tyrosine phosphorylation of focal adhesion kinase but not p80 phosphorylation. Therefore, under our assay conditions, p80 phosphorylation is regulated by alpha6beta4, while motility via alpha3beta1 causes phosphorylation of focal adhesion kinase. Consistent with a linkage between p80 dephosphorylation and alpha6beta4 anchorage to laminin 5, we found that phosphatase inhibitor sodium vanadate, which blocked the p80 dephosphorylation, prevented the alpha6beta4-dependent cell anchorage to laminin 5 at 4degreesC. In contrast, adhesion at 37 degrees C via alpha3beta1 was unaffected. Furthermore, by in vitro kinase assay, we identified a kinase activity for p80 phosphorylation in suspended HFKs but not in attached cells. The kinase activity, alpha6beta4, and its associated adhesion structure stable anchoring contacts were all cofractionated in the Triton- insoluble cell fraction that lacks alpha3beta1. Thus, regulation of p80 phosphorylation, through the activities of p80 kinase and phosphatase, correlates with alpha6beta4-SAC anchorage to laminin 5 at 4 degrees C in epithelial cells of the skin and intestine. Transmembrane signaling through p80 is an early tyrosine phosphorylation event responsive to and possibly required for anchorage to laminin 5 by HFK and LS123 epithelial cells. PMID:8647901

Characterization of prmt7alpha and beta isozymes from Chinese hamster cells sensitive and resistant to topoisomerase II inhibitors.

PubMed

Gros, Laurent; Renodon-Cornière, Axelle; de Saint Vincent, Bruno Robert; Feder, Marcin; Bujnicki, Janusz M; Jacquemin-Sablon, Alain

2006-11-01

By selection of genetic suppressor elements (GSEs) conferring resistance to topoisomerase II inhibitors in Chinese hamster cells (DC-3F), we identified a gene encoding two proteins of 78 and 82 kDa which belong to the protein arginine methyltransferase (PRMT) family. Down-regulation of these enzymes (named PRMT7alpha and beta), either induced by an antisense GSE or as observed in the 9-OH-ellipticine (9-OH-E) resistant mutant DC-3F/9-OH-E, was responsible for cell resistance to various DNA damaging agents. Alternative splicing alterations in the 5'-terminal region and changes of the polyadenylation site of PRMT7 mRNAs were observed in these resistant mutant cells. PRMT7alpha and beta are isoforms of a highly conserved protein containing two copies of a module common to all PRMTs, comprising a Rossmann-fold domain and a beta-barrel domain. The C-terminal repeat appears to be degenerate and catalytically inactive. PRMT7alpha and beta form homo- and hetero-dimers but differ by their sub-cellular localization and in vitro recognize different substrates. PRMT7beta was only observed in Chinese hamster cells while mouse 10T1/2 fibroblasts only contain PRMT7alpha. Surprisingly, in human cells the anti-PRMT7 antibody essentially recognized an approximately 37 kDa peptide, which is not formed during extraction, and a faint band at 78 kDa. Analysis of in vitro and in vivo methylation patterns in cell lines under- or over-expressing PRMT7alpha and beta detected a discrete number of proteins which methylation and/or expression are under the control of these enzymes.

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

An ancient role for nuclear beta-catenin in the evolution of axial polarity and germ layer segregation

NASA Technical Reports Server (NTRS)

Wikramanayake, Athula H.; Hong, Melanie; Lee, Patricia N.; Pang, Kevin; Byrum, Christine A.; Bince, Joanna M.; Xu, Ronghui; Martindale, Mark Q.

2003-01-01

The human oncogene beta-catenin is a bifunctional protein with critical roles in both cell adhesion and transcriptional regulation in the Wnt pathway. Wnt/beta-catenin signalling has been implicated in developmental processes as diverse as elaboration of embryonic polarity, formation of germ layers, neural patterning, spindle orientation and gap junction communication, but the ancestral function of beta-catenin remains unclear. In many animal embryos, activation of beta-catenin signalling occurs in blastomeres that mark the site of gastrulation and endomesoderm formation, raising the possibility that asymmetric activation of beta-catenin signalling specified embryonic polarity and segregated germ layers in the common ancestor of bilaterally symmetrical animals. To test whether nuclear translocation of beta-catenin is involved in axial identity and/or germ layer formation in 'pre-bilaterians', we examined the in vivo distribution, stability and function of beta-catenin protein in embryos of the sea anemone Nematostella vectensis (Cnidaria, Anthozoa). Here we show that N. vectensis beta-catenin is differentially stabilized along the oral-aboral axis, translocated into nuclei in cells at the site of gastrulation and used to specify entoderm, indicating an evolutionarily ancient role for this protein in early pattern formation.

Comparative analysis of Homo sapiens and Mus musculus cyclin-dependent kinase (CDK) inhibitor genes p16 (MTS1) and p15 (MTS2).

PubMed

Jiang, P; Stone, S; Wagner, R; Wang, S; Dayananth, P; Kozak, C A; Wold, B; Kamb, A

1995-12-01

Cyclin-dependent kinase inhibitors are a growing family of molecules that regulate important transitions in the cell cycle. At least one of these molecules, p16, has been implicated in human tumorigenesis while its close homolog, p15, is induced by cell contact and transforming growth factor-beta (TGF-beta). To investigate the evolutionary and functional features of p15 and p16, we have isolated mouse (Mus musculus) homologs of each gene. Comparative analysis of these sequences provides evidence that the genes have similar functions in mouse and human. In addition, the comparison suggests that a gene conversion event is part of the evolution of the human p15 and p16 genes.

Beta-lactams and their potential use as novel anticancer chemotherapeutics drugs.

PubMed

Kuhn, Deborah; Coates, Cristina; Daniel, Kenyon; Chen, Di; Bhuiyan, Mohammad; Kazi, Aslamuzzaman; Turos, Edward; Dou, Q Ping

2004-09-01

The discovery of natural and synthetic antibiotics is one of the most important medical breakthroughs in human history. Many diseases, such as bacterial meningitis, pneumonia, and septicemia, are now curable with the use of antibiotics. Antibiotics are efficacious, generally well tolerated in patients, and have a low toxicity level. It is for these reasons antibiotics remain an attractive target for drug discovery. Traditional beta-lactam antibiotics (e.g. penicillins, penems, cephalosporins) have a bicyclic ring structure that is conformationally rigid and functions to inhibit bacterial cell wall synthesis. In addition to the bactericidal action of antibiotics, it has been discovered that many antibiotics are capable of inhibiting tumor cell growth. There are currently many antitumor antibiotics approved for cancer therapy, which work to inhibit tumor cell growth by DNA intercalation. The use of beta-lactams as prodrugs has also met with success by aiding delivery of the chemotherapeutic directly to tumor sites. Recently, a novel class of N-thiolated monobactams, so termed because they possess a monocyclic ring instead of the bicyclic ring, has been found to induce apoptosis potently and specifically in many tumor cell lines but not in normal, non-transformed cell lines. Other beta-lactams, such as the polyaromatics, have been found to slow or inhibit tumor cell growth, and the 4-alkylidene beta-lactams are capable of inhibiting matrix metalloproteinases and leukocyte elactase activity. These data indicate that synthesis and evaluation of beta-lactams are a promising area for further development in anticancer research.

Physical and functional interaction between integrins and hERG potassium channels.

PubMed

Arcangeli, A; Becchetti, A; Cherubini, A; Crociani, O; Defilippi, P; Guasti, L; Hofmann, G; Pillozzi, S; Olivotto, M; Wanke, E

2004-11-01

Integrins are adhesion receptors capable of transmitting intracellular signals that regulate many different cellular functions. Among integrin-mediated signals, the activation of ion channels can be included. We demonstrated that a long-lasting activation of hERG (human ether-a-go-go-related gene) potassium channels occurs in both human neuroblastoma and leukaemia cells after the activation of the beta1 integrin subunit. This activation is apparently a determining factor inducing neurite extension and osteoclastic differentiation in both the cell types. More recently, we provided evidences that beta1 integrins and hERG channels co-precipitate in both the cell types. Preliminary results suggest that a macromolecular signalling complex indeed occurs between integrins and the hERG1 protein and that hERG channel activity can modulate integrin downstream signalling.

Expanded adipose-derived stem cells suppress mixed lymphocyte reaction by secretion of prostaglandin E2.

PubMed

Cui, Lei; Yin, Shuo; Liu, Wei; Li, Ningli; Zhang, Wenjie; Cao, Yilin

2007-06-01

Multipotent mesenchymal stem cells (MSCs) in adult tissue are known to be less immunogenic and immunosuppressive. Previous study showed that primary cultures of human adipose-derived stem cells (ADSCs) shared their immunomodulatory properties with other MSCs. However, whether passaged human ADSCs can retain their immunomodulatory effect after in vitro expansion remains unknown. In addition, the mechanism of ADSC-mediated immunomodulatory effect remains to be elucidated. This study aimed to investigate these issues by using passaged human ADSCs as an in vitro study model. Flow cytometry showed that passaged ADSCs expressed human leukocyte antigen (HLA) class I but not class II molecules, which could be induced to express to a high level with interferon-gamma (IFN-gamma) treatment. The study found that passaged ADSCs could not elicit lymphocyte proliferation after co-culturing with them, even after IFN-gamma treatment. In addition, either IFN-gamma-treated or non-treated ADSCs could inhibit phytohemagglutinin (PHA)-stimulated lymphocyte proliferation. Moreover, passaged ADSCs could serve as the third-party cells to inhibited two-way mixed lymphocyte reaction (MLR). Further study using a transwell system also showed that this type of immunosuppressive effect was not cell-cell contact dependent. In defining possible soluble factors, we found that passaged ADSCs significantly increased their secretion of prostaglandin E2 (PGE2), but not transforming growth factor-beta (TGF-beta) and hepatocyte growth factor (HGF), when they were co-cultured with MLR. Furthermore, the result demonstrated that only PGE2 production inhibitor indomethacine, but not TGF-beta- and HGF-neutralizing antibodies, could significantly counteract ADSC-mediated suppression on allogeneic lymphocyte proliferation. These results indicated that in vitro expanded ADSCs retain low immunogenicity and immunosuppressive effect, and PGE2 might be the major soluble factor involved in the in vitro inhibition of allogeneic lymphocyte reaction.

A virally inactivated functional growth factor preparation from human platelet concentrates.

PubMed

Su, C-Y; Kuo, Y P; Lin, Y C; Huang, C-T; Tseng, Y H; Burnouf, T

2009-08-01

Human platelet growth factors (HPGF) are essential for tissue regeneration and may replace fetal bovine serum (FBS) in cell therapy. No method for the manufacture of standardized virally inactivated HPGF has been developed yet. Platelet concentrates (PC) were subjected to solvent/detergent (S/D) treatment (1% TnBP/1% Triton X-45), oil extraction, hydrophobic interaction chromatography and sterile filtration. Platelet-derived growth factor (PDGF)-AB, -BB and -AA, transforming growth factor-beta1 (TGF-beta1), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1) and vascular endothelium growth factor (VEGF) were measured by ELISA. Composition in proteins and lipids was determined, protein profiles were obtained by SDS-PAGE, and TnBP and Triton X-45 were assessed by gas chromatography and high-performance liquid chromatography, respectively. Cell growth promoting activity of HPGF was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay using human embryonic kidney (HEK293A) fibroblast and Statens Seruminstitute rabbit corneal (SIRC) epithelial cell lines. The GF preparation contained a mean of 16.66, 2.04, 1.53, 72.19, 0.33, 48.59 and 0.44 ng/ml of PDGF-AB, -BB, -AA, TGF-beta1, EGF, IGF-1 and VEGF, respectively. The protein profile was typical of platelet releasates and had less than 2 p.p.m. of residual S/D agents. MTS assay of HEK293A and SIRC cultures showed that the GF preparation at 10% and 0.1% (v/v), respectively, could successfully replace 10% FBS for cell proliferation. Cell-stimulating activity of HPGF on HEK293A was over twice that of PC releasates. STANDARDIZED and functional virally inactivated HPGF can be prepared from human PC for possible applications in cell therapy and regenerative medicine.

Peginterferon Beta-1a Shows Antitumor Activity as a Single Agent and Enhances Efficacy of Standard of Care Cancer Therapeutics in Human Melanoma, Breast, Renal, and Colon Xenograft Models.

PubMed

Boccia, Antonio; Virata, Cyrus; Lindner, Daniel; English, Nicki; Pathan, Nuzhat; Brickelmaier, Margot; Hu, Xiao; Gardner, Jennifer L; Peng, Liaomin; Wang, Xinzhong; Zhang, Xiamei; Yang, Lu; Perron, Keli; Yco, Grace; Kelly, Rebecca; Gamez, James; Scripps, Thomas; Bennett, Donald; Joseph, Ingrid B; Baker, Darren P

2017-01-01

Because of its tumor-suppressive effect, interferon-based therapy has been used for the treatment of melanoma. However, limited data are available regarding the antitumor effects of pegylated interferons, either alone or in combination with approved anticancer drugs. We report that treatment of human WM-266-4 melanoma cells with peginterferon beta-1a induced apoptotic markers. Additionally, peginterferon beta-1a significantly inhibited the growth of human SK-MEL-1, A-375, and WM-266-4 melanoma xenografts established in immunocompromised mice. Peginterferon beta-1a regressed large, established WM-266-4 xenografts in nude mice. Treatment of SK-MEL-1 tumor-bearing mice with a combination of peginterferon beta-1a and the MEK inhibitor PD325901 ((R)-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide) significantly improved tumor growth inhibition compared with either agent alone. Examination of the antitumor activity of peginterferon beta-1a in combination with approved anticancer drugs in breast and renal carcinomas revealed improved antitumor activity in these preclinical xenograft models, as did the combination of peginterferon beta-1a and bevacizumab in a colon carcinoma xenograft model.

Proliferation of prostate cancer cells and activity of neutral endopeptidase is regulated by bombesin and IL-1beta with IL-1beta acting as a modulator of cellular differentiation.

PubMed

Albrecht, Martin; Doroszewicz, Jolanta; Gillen, Sonja; Gomes, Iara; Wilhelm, Beate; Stief, Thomas; Aumüller, Gerhard

2004-01-01

Neutral endopeptidase (NEP) is a cell-surface bound enzyme that cleaves and inactivates neuropeptides such as bombesin and substance P and is involved in the transition from hormonally regulated androgen-dependent prostate cancer (PC) to androgen-independent PC. Neuropeptides are implicated in growth regulation of different cell types and function as transmitters between the neuroendocrine and the immune system. NEP-expression, enzymatic activity of the membrane bound protein, cell proliferation, procalcitonin (PCT) production, and secretion as well as changes in cell morphology of prostatic cells were evaluated after treatment with the immunomodulatory cytokine interleukin-1beta (IL-1beta), neuropeptides (bombesin, substance P), and neuropeptide-conditioned media derived from a human neuroendocrine cell line. Incubation of LNCaP tumor cells with IL-1beta resulted in a diminished proliferative activity, induction of neurite-like outgrowth which was accompanied by the formation of tubular-type mitochondria typical for neuronal/neuroendocrine cells, and an increased production and secretion of PCT. Conversely, proliferation of prostatic stromal cells was enhanced by the cytokine coming along with an increased number of Golgi-apparatuses and ER-cisternae. Bombesin had an antimitotic effect on LNCaP, but not on stromal cells. Substance P did not influence the growth of any of the cell types investigated, whereas neuropeptide-conditioned media exerted a slightly mitogenic effect on both cell types. The activity of LNCaP cell-surface bound NEP was enhanced by bombesin, but was diminished by substance P and neuropeptide-conditioned media. Proliferation and activity of neuropeptide degrading NEP is regulated differently by immunomodulatory substances in PC cells and cells derived from the prostatic stroma with IL-1beta being a potent modulator of cellular differentiation and a potential target for anticancer drug design in PC cells. Copyright 2003 Wiley-Liss, Inc.

Genus Beta Human Papillomavirus E6 Proteins Vary in Their Effects on the Transactivation of p53 Target Genes

PubMed Central

White, Elizabeth A.; Walther, Johanna; Javanbakht, Hassan

2014-01-01

ABSTRACT The genus beta human papillomaviruses (beta HPVs) cause cutaneous lesions and are thought to be involved in the initiation of some nonmelanoma skin cancers (NMSCs), particularly in patients with the genetic disorder epidermodysplasia verruciformis (EV). We have previously reported that at least two of the genus beta HPV E6 proteins bind to and/or increase the steady-state levels of p53 in squamous epithelial cells. This is in contrast to a well-characterized ability of the E6 proteins of cancer-associated HPVs of genus alpha HPV, which inactivate p53 by targeting its ubiquitin-mediated proteolysis. In this study, we have investigated the ability of genus beta E6 proteins from eight different HPV types to block the transactivation of p53 target genes following DNA damage. We find that the E6 proteins from diverse beta HPV species and types vary in their capacity to block the induction of MDM2, p21, and proapoptotic genes after genotoxic stress. We conclude that some genus beta HPV E6 proteins inhibit at least some p53 target genes, although perhaps not by the same mechanism or to the same degree as the high-risk genus alpha HPV E6 proteins. IMPORTANCE This study addresses the ability of various human papillomavirus E6 proteins to block the activation of p53-responsive cellular genes following DNA damage in human keratinocytes, the normal host cell for HPVs. The E6 proteins encoded by the high-risk, cancer-associated HPV types of genus alpha HPV have a well-established activity to target p53 degradation and thereby inhibit the response to DNA damage. In this study, we have investigated the ability of genus beta HPV E6 proteins from eight different HPV types to block the ability of p53 to transactivate downstream genes following DNA damage. We find that some, but not all, genus beta HPV E6 proteins can block the transactivation of some p53 target genes. This differential response to DNA damage furthers the understanding of cutaneous HPV biology and may help to explain the potential connection between some beta HPVs and cancer. PMID:24850740

TRIM16 inhibits proliferation and migration through regulation of interferon beta 1 in melanoma cells

PubMed Central

Sutton, Selina K.; Koach, Jessica; Tan, Owen; Liu, Bing; Carter, Daniel R.; Wilmott, James S.; Yosufi, Benafsha; Haydu, Lauren E.; Mann, Graham J.; Thompson, John F.; Long, Georgina V.; Liu, Tao; McArthur, Grant; Zhang, Xu Dong; Scolyer, Richard A.; Cheung, Belamy B.; Marshall, Glenn M.

2014-01-01

High basal or induced expression of the tripartite motif protein, TRIM16, leads to reduce cell growth and migration of neuroblastoma and skin squamous cell carcinoma cells. However, the role of TRIM16 in melanoma is currently unknown. TRIM16 protein levels were markedly reduced in human melanoma cell lines, compared with normal human epidermal melanocytes due to both DNA methylation and reduced protein stability. TRIM16 knockdown strongly increased cell migration in normal human epidermal melanocytes, while TRIM16 overexpression reduced cell migration and proliferation of melanoma cells in an interferon beta 1 (IFNβ1)-dependent manner. Chromatin immunoprecipitation assays revealed TRIM16 directly bound the IFNβ1 gene promoter. Low level TRIM16 expression in 91 melanoma patient samples, strongly correlated with lymph node metastasis, and, predicted poor patient prognosis in a separate cohort of 170 melanoma patients with lymph node metastasis. The BRAF inhibitor, vemurafenib, increased TRIM16 protein levels in melanoma cells in vitro, and induced growth arrest in BRAF-mutant melanoma cells in a TRIM16-dependent manner. High levels of TRIM16 in melanoma tissues from patients treated with Vemurafenib correlated with clinical response. Our data, for the first time, demonstrates TRIM16 is a marker of cell migration and metastasis, and a novel treatment target in melanoma. PMID:25333256

Cell Science and Cell Biology Research at MSFC: Summary

NASA Technical Reports Server (NTRS)

2003-01-01

The common theme of these research programs is that they investigate regulation of gene expression in cells, and ultimately gene expression is controlled by the macromolecular interactions between regulatory proteins and DNA. The NASA Critical Path Roadmap identifies Muscle Alterations and Atrophy and Radiation Effects as Very Serious Risks and Severe Risks, respectively, in long term space flights. The specific problem addressed by Dr. Young's research ("Skeletal Muscle Atrophy and Muscle Cell Signaling") is that skeletal muscle loss in space cannot be prevented by vigorous exercise. Aerobic skeletal muscles (i.e., red muscles) undergo the most extensive atrophy during long-term space flight. Of the many different potential avenues for preventing muscle atrophy, Dr. Young has chosen to study the beta-adrenergic receptor (betaAR) pathway. The reason for this choice is that a family of compounds called betaAR agonists will preferentially cause an increase in muscle mass of aerobic muscles (i.e., red muscle) in animals, potentially providing a specific pharmacological solution to muscle loss in microgravity. In addition, muscle atrophy is a widespread medical problem in neuromuscular diseases, spinal cord injury, lack of exercise, aging, and any disease requiring prolonged bedridden status. Skeletal muscle cells in cell culture are utilized as a model system to study this problem. Dr. Richmond's research ("Radiation & Cancer Biology of Mammary Cells in Culture") is directed toward developing a laboratory model for use in risk assessment of cancer caused by space radiation. This research is unique because a human model will be developed utilizing human mammary cells that are highly susceptible to tumor development. This approach is preferential over using animal cells because of problems in comparing radiation-induced cancers between humans and animals.

Noradrenaline inhibits lipopolysaccharide-induced tumor necrosis factor and interleukin 6 production in human whole blood.

PubMed Central

van der Poll, T; Jansen, J; Endert, E; Sauerwein, H P; van Deventer, S J

1994-01-01

Sepsis and lipopolysaccharide (LPS) trigger the systemic release of both cytokines and catecholamines. Cytokines are known to be capable of eliciting a stress hormone response in vivo. The present study sought insight into the effect of noradrenaline on LPS-induced release of tumor necrosis factor alpha (TNF) and interleukin 6 (IL-6) in human whole blood. Whole blood was incubated with LPS for 4 h at 37 degrees C in the presence and absence of noradrenaline and/or specific alpha and beta antagonists and agonists. Noradrenaline caused a dose-dependent inhibition of LPS-induced TNF and IL-6 production. This effect could be completely prevented by addition of the specific beta 1, antagonist metoprolol, while it was not affected by the alpha antagonist phentolamine. Specific beta-adrenergic stimulation by isoprenaline mimicked the inhibiting effect of noradrenaline on LPS-evoked cytokine production, whereas alpha-adrenergic stimulation by phenylephrine had no effect. Fluorescence-activated cell sorter analysis demonstrated that beta-adrenergic stimulation had no effect on LPS binding to and internalization into mononuclear cells or on the expression of CD14, the major receptor for LPS on mononuclear cells. In acute sepsis, enhanced release of noradrenaline may be part of a negative feedback mechanism meant to inhibit ongoing TNF and IL-6 production. PMID:8168970

Cutaneous beta human papillomaviruses and the development of male external genital lesions: A case-control study nested within the HIM Study.

PubMed

Pierce Campbell, Christine M; Gheit, Tarik; Tommasino, Massimo; Lin, Hui-Yi; Torres, B Nelson; Messina, Jane L; Stoler, Mark H; Rollison, Dana E; Sirak, Bradley A; Abrahamsen, Martha; Carvalho da Silva, Roberto J; Sichero, Laura; Villa, Luisa L; Lazcano-Ponce, Eduardo; Giuliano, Anna R

2016-10-01

Cutaneous human papillomaviruses (HPVs) increase the risk of non-melanoma skin cancer in sun-exposed skin. We examined the role of beta-HPV in the development of male external genital lesions (EGLs), a sun-unexposed site. In this nested case-control study (67 men with pathologically-confirmed EGLs and 134 controls), exfoliated cells collected from the surface of lesions and normal genital skin 0, 6, and 12 months preceding EGL development were tested for beta-HPV DNA using a type-specific multiplex genotyping assay. Beta-HPV prevalence was estimated and conditional logistic regression was used to evaluate the association with condyloma, the most common EGL. While beta-HPV prevalence among controls remained stable, the prevalence among cases was lowest on the surface of lesion. Detecting beta-HPV on the normal genital skin was not associated with the presence or development of condyloma. Cutaneous beta-HPV does not appear to be contributing to pathogenesis in male genital skin. Copyright © 2016. Published by Elsevier Inc.

Restoration of normal phenotype in cancer cells

DOEpatents

Bissell, M.J.; Weaver, V.M.

1998-12-08

A method for reversing expression of malignant phenotype in cancer cells is described. The method comprises applying {beta}{sub 1} integrin function-blocking antibody to the cells. The method can be used to assess the progress of cancer therapy. Human breast epithelial cells were shown to be particularly responsive. 14 figs.

Method for restoration of normal phenotype in cancer cells

DOEpatents

Bissell, Mina J.; Weaver, Valerie M.

2000-01-01

A method for reversing expression of malignant phenotype in cancer cells is described. The method comprises applying .beta..sub.1 integrin function-blocking antibody to the cells. The method can be used to assess the progress of cancer therapy. Human breast epithelial cells were shown to be particularly responsive.

Restoration of normal phenotype in cancer cells

DOEpatents

Bissell, Mina J.; Weaver, Valerie M.

1998-01-01

A method for reversing expression of malignant phenotype in cancer cells is described. The method comprises applying .beta..sub.1 integrin function-blocking antibody to the cells. The method can be used to assess the progress of cancer therapy. Human breast epithelial cells were shown to be particularly responsive.

Intramuscular renin-angiotensin system is activated in human muscular dystrophy.

PubMed

Sun, Guilian; Haginoya, Kazuhiro; Dai, Hongmei; Chiba, Yoko; Uematsu, Mitsugu; Hino-Fukuyo, Naomi; Onuma, Akira; Iinuma, Kazuie; Tsuchiya, Shigeru

2009-05-15

To investigate the role of the muscular renin-angiotensin system (RAS) in human muscular dystrophy, we used immunohistochemistry and Western blotting to examine the cellular localization of angiotensin-converting enzyme (ACE), the angiotensin II type 1 receptor (AT1) and the angiotensin II type 2 receptor (AT2) in muscle biopsies from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and congenital muscular dystrophy (CMD). In normal muscle, ACE was expressed in vascular endothelial cells and neuromuscular junctions (NMJs), whereas AT1 was immunolocalized to the smooth muscle cells of blood vessels and intramuscular nerve twigs. AT2 was immunolocalized in the smooth muscle cells of blood vessels. These findings suggest that the RAS has a functional role in peripheral nerves and NMJs. ACE and AT1, but AT2 immunoreactivity were increased markedly in dystrophic muscle as compared to controls. ACE and the AT1 were strongly expressed in the cytoplasm and nuclei of regenerating muscle fibers, fibroblasts, and in macrophages infiltrating necrotic fibers. Double immunolabeling revealed that activated fibroblasts in the endomysium and perimysium of DMD and CMD muscle were positive for ACE and AT1. Triple immunolabeling demonstrated that transforming growth factor-beta1 (TGF-beta1) and ACE were colocalized on the cytoplasm of activated fibroblasts in dystrophic muscle. Furthermore, Western blotting showed increases in the expression of AT1 and TGF-beta1 protein in dystrophic muscle, which coincided with our immunohistochemical results. The overexpression of ACE and AT1 in dystrophic muscle would likely result in the increased production of Ang II, which may act on these cells in an autocrine manner via AT1. The activation of AT1 may induce fibrous tissue formation through overexpression of TGF-beta1, which potently activates fibrogenesis and suppresses regeneration. In conclusion, our results imply that the intramuscular RAS-TGF-beta1 pathway is activated in human muscular dystrophy and plays a role at least partly in the pathophysiology of this disease.

The functional performance of microencapsulated human pancreatic islet-derived precursor cells.

PubMed

Montanucci, Pia; Pennoni, Ilaria; Pescara, Teresa; Blasi, Paolo; Bistoni, Giovanni; Basta, Giuseppe; Calafiore, Riccardo

2011-12-01

We have examined long-term cultured, human islet-derived stem/precursor cells (hIPC). Whole human islets (HI) were obtained by multi-enzymatic digestion of cadaveric donor pancreases, plated on tissue flasks, and allowed to adhere and expand for several in vitro passages, in order to obtain hIPC. We detected specific stem cell markers (Oct-4, Sox-2, Nanog, ABCG2, Klf-4, CD117) in both intact HI and hIPC. Moreover, hIPC while retaining the expression of Glut-2, Pdx-1, CK-19, and ICA-512, started re-expressing Ngn3, thereby indicating acquisition of a specific pancreatic islet beta cell-oriented phenotype identity. The intrinsic plasticity of hIPC was documented by their ability to differentiate into various germ layer-derived cell phenotypes (ie, osteocytic, adipocytic and neural), including endocrine cells associated with insulin secretory capacity. To render hIPC suitable for transplantation we have enveloped them within our highly purified, alginate-based microcapsules. Upon intraperitoneal graft in NOD/SCID mice we have observed that the microcapsules acted as three-dimensional niches favouring post-transplant hIPC differentiation and acquisition of beta cell-like functional competence. Copyright © 2011 Elsevier Ltd. All rights reserved.

Integrins (alpha7beta1) in muscle function and survival. Disrupted expression in merosin-deficient congenital muscular dystrophy.

PubMed Central

Vachon, P H; Xu, H; Liu, L; Loechel, F; Hayashi, Y; Arahata, K; Reed, J C; Wewer, U M; Engvall, E

1997-01-01

Mutations in genes coding for dystrophin, for alpha, beta, gamma, and delta-sarcoglycans, or for the alpha2 chain of the basement membrane component merosin (laminin-2/4) cause various forms of muscular dystrophy. Analyses of integrins showed an abnormal expression and localization of alpha7beta1 isoforms in myofibers of merosin-deficient human patients and mice, but not in dystrophin-deficient or sarcoglycan-deficient humans and animals. It was shown previously that skeletal muscle fibers require merosin for survival and function (Vachon, P.H., F. Loechel, H. Xu, U.M. Wewer, and E. Engvall. 1996. J. Cell Biol. 134:1483-1497). Correction of merosin deficiency in vitro through cell transfection with the merosin alpha2 chain restored the normal localization of alpha7beta1D integrins as well as myotube survival. Overexpression of the apoptosis-suppressing molecule Bcl-2 also promoted the survival of merosin-deficient myotubes, but did not restore a normal expression of alpha7beta1D integrins. Blocking of beta1 integrins in normal myotubes induced apoptosis and severely reduced their survival. These findings (a) identify alpha7beta1D integrins as the de facto receptors for merosin in skeletal muscle; (b) indicate a merosin dependence for the accurate expression and membrane localization of alpha7beta1D integrins in myofibers; (c) provide a molecular basis for the critical role of merosin in myofiber survival; and (d) add new insights to the pathogenesis of neuromuscular disorders. PMID:9312189

Interleukin-1beta may mediate insulin resistance in liver-derived cells in response to adipocyte inflammation.

PubMed

Nov, Ori; Kohl, Ayelet; Lewis, Eli C; Bashan, Nava; Dvir, Irit; Ben-Shlomo, Shani; Fishman, Sigal; Wueest, Stephan; Konrad, Daniel; Rudich, Assaf

2010-09-01

Central obesity is frequently associated with adipose tissue inflammation and hepatic insulin resistance. To identify potential individual mediators in this process, we used in vitro systems and assessed if insulin resistance in liver cells could be induced by secreted products from adipocytes preexposed to an inflammatory stimulus. Conditioned medium from 3T3-L1 adipocytes pretreated without (CM) or with TNFalpha (CM-TNFalpha) was used to treat Fao hepatoma cells. ELISAs were used to assess the concentration of several inflammatory mediators in CM-TNFalpha. CM-TNFalpha-treated Fao cells exhibited about 45% diminution in insulin-stimulated phosphorylation of insulin receptor, insulin receptor substrate proteins, protein kinase B, and glycogen synthase kinase-3 as compared with CM-treated cells, without changes in the total abundance of these protein. Insulin increased glycogenesis by 2-fold in CM-treated Fao cells but not in cells exposed to CM-TNFalpha. Expression of IL-1beta mRNA was elevated 3-fold in TNFalpha-treated adipocytes, and CM-TNFalpha had 10-fold higher concentrations of IL-1beta but not TNFalpha or IL-1alpha. IL-1beta directly induced insulin resistance in Fao, HepG2, and in primary rat hepatocytes. Moreover, when TNFalpha-induced secretion/production of IL-1beta from adipocytes was inhibited by the IL-1 converting enzyme (ICE-1) inhibitor II (Ac-YVAD-CMK), insulin resistance was prevented. Furthermore, liver-derived cells treated with IL-1 receptor antagonist were protected against insulin resistance induced by CM-TNFalpha. Finally, IL-1beta secretion from human omental fat explants correlated with body mass index (R(2) = 0.639, P < 0.01), and the resulting CM induced insulin resistance in HepG2 cells, inhibitable by IL-1 receptor antagonist. Our results suggest that adipocyte-derived IL-1beta may constitute a mediator in the perturbed cross talk between adipocytes and liver cells in response to adipose tissue inflammation.

Differential expression of extracellular matrix molecules and the alpha 6-integrins in the normal and neoplastic prostate.

PubMed Central

Knox, J. D.; Cress, A. E.; Clark, V.; Manriquez, L.; Affinito, K. S.; Dalkin, B. L.; Nagle, R. B.

1994-01-01

The epithelial basal lamina composition and integrin expression profile of normal and neoplastic human prostate was characterized using immunohistochemical analysis of frozen samples. The major components of the basal lamina surrounding normal acini were laminin, type IV collagen, entactin, and type VII collagen with variable amounts of tenascin. The basal lamina of neoplastic acini had a similar composition, except for the loss of type VII collagen, which was observed in all grades of carcinoma. The basal cells of the normal prostate express the alpha 6-, beta 1-, and beta 4-integrin subunits, suggesting that both the alpha 6 beta 1- and alpha 6 beta 4-integrin complexes are formed. In prostate carcinoma there is a complete loss of beta 4 expression and the alpha 6- and beta 1-integrin subunits, which are restricted to the basal and basal lateral surfaces of basal cells, are distributed diffusely throughout the cytoplasmic membrane. The differential expression of type VII collagen and beta 4 are discussed in relationship to their possible role in tumor progression. Images Figure 1 Figure 2 Figure 3 PMID:8030747

Evidence against involvement of the acid lysosomal sphingomyelinase in the tumor-necrosis-factor- and interleukin-1-induced sphingomyelin cycle and cell proliferation in human fibroblasts.

PubMed Central

Andrieu, N; Salvayre, R; Levade, T

1994-01-01

The hydrolysis of sphingomyelin (SPM) has been reported to mediate a number of responses to extracellular agents, including cytokines. The so-called SPM cycle may result from the activation of different types of sphingomyelinases (SPMases). We investigated the hypothetical contribution of acid lysosomal SPMase in the SPM signal-transduction pathway. We examined the ability of human skin fibroblasts with a genetic deficiency of acid lysosomal SPMase activity to respond to tumour necrosis factor alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta). We report that both cytokines promoted SPM hydrolysis in fibroblasts derived from patients with Niemann-Pick disease or I-cell disease, similar to that observed in normal cells. Treatment of normal fibroblasts with cationic amphiphilic drugs resulted in inhibition of acid SPMase activity, but had no effect on cytokine-induced SPM turnover. In addition, TNF-alpha and IL-1 beta stimulated [3H]thymidine incorporation in Niemann-Pick fibroblasts, as in normal cells. Thus our results argue against a role for acid endolysosomal SPMase in mediating the cytokine-induced SPM signalling cascade. Images Figure 2 PMID:7980390

Cytotoxic triterpenoid saponins from the fruits of Aesculus pavia L.

PubMed

Zhang, Zhizhen; Li, Shiyou

2007-08-01

Continued chemical investigation on the fruits of North American Aesculus pavia L. resulted in the isolation and identification of 13 polyhydroxyoleanene pentacyclic triterpenoid saponins, named aesculiosides IIe-IIk (1-7), and IIIa-IIIf (8-13), together with 18 known compounds: aesculiosides Ia-Ie (14-18), IIa-IId (19-22), IVa-IVc (23-25), 3-O-[beta-D-galactopyranosyl(1-->2)]-alpha-L-arabinofuranosyl(1-->3)-beta-D-glucuronopyranosyl-21,22-O-diangeloyl-3beta,15 alpha,16 alpha,21 beta,22 alpha,28-hexahydroxyolean-12-ene (26), 3-O-[beta-D-glucopyranosyl(1-->2)]-alpha-L-arabinofuranosyl(1-->3)-beta-D-glucuronopyranosyl-21,22-O-diangeloyl-3beta,16 alpha,21 beta,22 alpha,24 beta,28-hexahydroxyolean-12-ene (27), 3-O-[beta-D-galactopyranosyl(1-->2)]-alpha-L-arabinofuranosyl(1-->3)-beta-D-glucuronopyranosyl-21,22-O-diangeloyl-3beta,16 alpha,21 beta,22 alpha,28-pentahydroxyolean-12-ene (28), R(1)-barrigenol (29), scopolin (30), and 5-methoxyscopolin (31). The structures of these compounds were elucidated by spectroscopic and chemical analyses. Compounds 14-22 and 26-28 were tested in vitro for their activity against 59 cell lines from nine different human cancers including leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate, and breast. It was found that compounds with two-acyl groups at C-21 and C-22 had cytotoxic activity for all cell lines tested with GI(50) 0.175-8.71 microM, while compounds without acyl groups at C-21 and C-22 had weak or no cytotoxic activity. These results suggest that the acyl groups at C-21 and C-22 are essential for their activity.

Administration of Interleukin-6 Stimulates Multilineage Hematopoiesis and Accelerates Recovery from Radiation-Induced Hematopoietic Depression

DTIC Science & Technology

1991-02-01

Htirano T, Kishimoto T, Nakahata T. Asano S: In vitro hernatopoietic growth factors. J1 Natl Cancer Inst 81: t370. 1989 expansion of the murine...a.i phas S. %’ink A. Billiau A. VanSnick 1: Identification of the nutohlbyrcmiatntrekn.Cllmuolt12. human Zh-kd protein, interferon beta ,, as a B... beta . B-cell %timulatory factor type 2 shares identity T. Takaku F. Akivama Y: In vivo effects of recombinant human with rnsanc~tc-derived

Preferential V beta gene usage and lack of junctional sequence conservation among human T cell receptors specific for a tetanus toxin- derived peptide: evidence for a dominant role of a germline-encoded V region in antigen/major histocompatibility complex recognition

PubMed Central

1992-01-01

To investigate the structural and genetic basis of the T cell response to defined peptide/major histocompatibility (MHC) class II complexes in humans, we established a large panel of T cell clones (61) from donors of different HLA-DR haplotypes and reactive with a tetanus toxin- derived peptide (tt830-844) recognized in association with most DR molecules (universal peptide). By using a bacterial enterotoxin-based proliferation assay and cDNA sequencing, we found preferential use of a particular V beta region gene segment, V beta 2.1, in three of the individuals studied (64%, n = 58), irrespective of whether the peptide was presented by the DR6wcI, DR4w4, or DRw11.1 and DRw11.2 alleles, demonstrating that shared MHC class II antigens are not required for shared V beta gene use by T cell receptors (TCRs) specific for this peptide. V alpha gene use was more heterogeneous, with at least seven different V alpha segments derived from five distinct families encoding alpha chains able to pair with V beta 2.1 chains to form a tt830-844/DR- specific binding site. Several cases were found of clones restricted to different DR alleles that expressed identical V beta and (or very closely related) V alpha gene segments and that differed only in their junctional sequences. Thus, changes in the putative complementary determining region 3 (CDR3) of the TCR may, in certain cases, alter MHC specificity and maintain peptide reactivity. Finally, in contrast to what has been observed in other defined peptide/MHC systems, a striking heterogeneity was found in the junctional regions of both alpha and beta chains, even for TCRs with identical V alpha and/or V beta gene segments and the same restriction. Among 14 anti-tt830-844 clones using the V beta 2.1 gene segment, 14 unique V beta-D-J beta junctions were found, with no evident conservation in length and/or amino acid composition. One interpretation for this apparent lack of coselection of specific junctional sequences in the context of a common V element, V beta 2.1, is that this V region plays a dominant role in the recognition of the tt830-844/DR complex. PMID:1371303

Role of T cell TGF beta signaling in intestinal cytokine responses and helminthic immune modulation

USDA-ARS?s Scientific Manuscript database

Colonization with helminthic parasites down-regulates inflammation in murine colitis and improves activity scores in human inflammatory bowel disease. Helminths induce mucosal regulatory T cells, which are important for intestinal immunologic homeostasis. Regulatory T cell function involves cytoki...

Studies on the interaction of the Sophora japonica lectin and concanavalin A with erythrocytes and lymphocytes.

PubMed Central

Poretz, R D; Barth, R F

1976-01-01

The agglutinating activity of lectins from the seeds of Sophora japonica and Canavalia ensiformis (concanavalin A) with human and murine erythrocytes and lymphocytes have been compared to one another and related to the mitogenic and immunosuppressive properties of these purified proteins. The S. japonica lectin, which demonstrates blood group specificity, is more active than concanavalin A with human erythrocytes, but has a much lower reactivity than concanavalin A with murine red blood cells. Ficin treatment of human erythrocytes results in an increase in agglutinability by both lectins as well as causing the appearance of S. japonica lectin receptors on type O cells. Treatment of murine reythrocytes with ficin alone or followed by beta-galactosidase causes the cells to be more reactive with concanavalin A. Beta-Galactosidase alone has no observable affect on the cells. In contrast, the agglutinability of cells by the S. japonica lectin increases after ficin treatment but is not affected by beta-galaetosidose treatment either after or in the absence of ficinization. Murine lymphocytes react with both lectins in a manner paralleling the agglutination patterns of murine erythrocytes. The S. japonica lectin appears to be devoid of mitogenic and immuno-suppressive activity, in contrast to concanavalin A which suppresses the T helper-dependent antibody response to sheep erythrocytes. These results are discussed in terms of the types of lectin receptors on lymphocytes related to agglutination, induction of blastogenesis and immuno-suppression. PMID:955676

Oxytocin receptors expressed and coupled to Ca2+ signalling in a human vascular smooth muscle cell line.

PubMed

Yazawa, H; Hirasawa, A; Horie, K; Saita, Y; Iida, E; Honda, K; Tsujimoto, G

1996-03-01

1. In a human vascular smooth muscle cell line (HVSMC), binding experiments with [3H]-arginine8-vasopressin (AVP) have shown the existence of a homogeneous population of binding sites with affinity (Kd value) of 0.65 nM and a maximum number of binding sites (Bmax) of 122 fmol mg-1 protein. 2. Nonlabelled compounds compete for [3H]-AVP binding in the HVSMC membrane with an order of potency of oxytocin > lyspressin > or = AVP > Thr4, Gly7-oxytocin > (beta-mercapto-beta-beta-cyclopentamethylenepropionyl-O-Me Tyr2, Arg8) vasopressin > desmopressin > OPC21268 > OPC31260. This order was markedly different from that observed in rat vascular smooth muscle cells (A10), a well-established V1A receptor system. 3. In HVSMC both oxytocin and AVP increased inositol 1,4,5-trisphosphate (IP3) production and [Ca2+]i response, but the efficacy of the responses was greater for oxytocin than AVP. 4. Reverse transcription-polymerase chain reaction (RT-PCR) assay detected only oxytocin receptor but not V1A or V2 receptors in HVSMC, whereas only V1A receptors were found in A10 cells. 5. In conclusion, in HVSMC only oxytocin receptors are expressed among the vasopressin receptor family, and they coupled to phosphatidyl inositol (PI) turnover/Ca2+ signalling. This unexpected observation should provide new insight into the functional role of the oxytocin receptor in a human vascular smooth muscle cell line.

P2X receptor characterization and IL-1/IL-1Ra release from human endothelial cells.

PubMed

Wilson, H L; Varcoe, R W; Stokes, L; Holland, K L; Francis, S E; Dower, S K; Surprenant, A; Crossman, D C

2007-05-01

The pro-inflammatory cytokine, interleukin-1beta (IL-1beta), has been implicated in the pathogenesis of atherosclerosis, potentially via its release from vascular endothelium. Endothelial cells (EC) synthesize IL-1beta in response to inflammatory stimuli, but the demonstration and mechanism of release of IL-1 from ECs remains unclear. In activated monocytes, efficient release of bioactive IL-1beta occurred via activation of ATP-gated P2X(7) receptors (P2X(7)Rs). Activation of P2X(7)R in ECs from human umbilical vein (HUVECs) released IL-1 receptor antagonist (IL-1Ra). The purpose of this study was to provide a quantitative investigation of P2XR expression and function, in parallel with IL-1beta and IL-1Ra synthesis, processing and release, in HUVECs under pro-inflammatory conditions. Quantitative RT-PCR, immunoblotting, ELISA, flow cytometry, and whole-cell patch clamp recordings were used to determine protein expression and receptor function. IL-8-luciferase-reporter was used as an IL-1 sensitive bioassay. HUVECs expressed P2X(4)R and P2X(7)R subtypes and both were significantly up-regulated under inflammatory conditions. P2X(7)R currents were increased 3-fold by inflammatory stimuli, whereas no P2X(4)R-mediated currents were detected. Caspase-1, but not IL-1beta, was present intracellularly under basal conditions; inflammatory stimuli activated the synthesis of intracellular pro-IL-1beta and increased caspase-1 levels. Activation of P2X(7)Rs resulted in low-level release of bioactive IL-1beta and simultaneous release of IL-1Ra. The net biological effect of release was anti-inflammatory. Endothelial P2X(7)Rs induced secretion of both pro- and anti-inflammatory IL-1 receptor ligands, the balance of which may provide a means for altering the inflammatory state of the arterial vessel wall.

Cloning of the cDNA for a hematopoietic cell-specific protein related to CD20 and the {beta} subunit of the high-affinity IgE receptor: Evidence for a family of proteins with four membrane-spanning regions

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

Adra, C.N.; Morrison, P.; Lim, B.

1994-10-11

The authors report the cloning of the cDNA for a human gene whose mRNA is expressed specifically in hematopoietic cells. A long open reading frame in the 1.7-kb mRNA encodes a 214-aa protein of 25 kDa with four hydrophobic regions consistent with a protein that traverses the membrane four times. To reflect the structure and expression of this gene in diverse hematopoietic lineages of lymphoid and myeloid origin, the authors named the gene HTm{sub 4}. The protein is about 20% homologous to two other {open_quotes}four-transmembrane{close_quotes} proteins; the B-cell-specific antigen CD20 and the {beta} subunit of the high-affinity receptor for IgE,more » Fc{sub {epsilon}}RI{beta}. The highest homologies among the three proteins are found in the transmembrane domains, but conserved residues are also recognized in the inter-transmembrane domains and in the N and C termini. Using fluorescence in situ hybridization, they localized HTm{sub 4} to human chromosome 11q12-13.1, where the CD20 and Fc{sub {epsilon}}RI{beta} genes are also located. Both the murine homologue for CD20, Ly-44, and the murine Fc{sub {epsilon}}RI{beta} gene map to the same region in murine chromosome 19. The authors propose that the HTm{sub 4}, CD20, and Fc{sub {epsilon}}RI{beta} genes evolved from the same ancestral gene to form a family of four-transmembrane proteins. It is possible that other related members exist. Similar to CD20 and Fc{sub {epsilon}}RI{beta}, it is likely that Htm{sub 4} has a role in signal transduction and, like Fc{sub {epsilon}}RI{beta}, might be a subunit associated with receptor complexes.« less

Activation of protein kinase C and disruption of endothelial monolayer integrity by sodium arsenite-Potential mechanism in the development of atherosclerosis

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

Pereira, Flavia E.; Coffin, J. Douglas; Beall, Howard D.

2007-04-15

Arsenic exposure has been shown to exacerbate atherosclerosis, beginning with activation of the endothelium that lines the vessel wall. Endothelial barrier integrity is maintained by proteins of the adherens junction (AJ) such as vascular endothelial cadherin (VE-cadherin) and {beta}-catenin and their association with the actin cytoskeleton. In the present study, human aortic endothelial cells (HAECs) were exposed to 1, 5 and 10 {mu}M sodium arsenite [As(III)] for 1, 6, 12 and 24 h, and the effects on endothelial barrier integrity were determined. Immunofluorescence studies revealed formation of actin stress fibers and non-uniform VE-cadherin and {beta}-catenin staining at cell-cell junctions thatmore » were concentration- and time-dependent. Intercellular gaps were observed with a measured increase in endothelial permeability. In addition, concentration-dependent increases in tyrosine phosphorylation (PY) of {beta}-catenin and activation of protein kinase C{alpha} (PKC{alpha}) were observed. Inhibition of PKC{alpha} restored VE-cadherin and {beta}-catenin staining at cell-cell junctions and abolished the As(III)-induced formation of actin stress fibers and intercellular gaps. Endothelial permeability and PY of {beta}-catenin were also reduced to basal levels. These results demonstrate that As(III) induces activation of PKC{alpha}, which leads to increased PY of {beta}-catenin downstream of PKC{alpha} activation. Phosphorylation of {beta}-catenin plausibly severs the association of VE-cadherin and {beta}-catenin, which along with formation of actin stress fibers, results in intercellular gap formation and increased endothelial permeability. To the best of our knowledge, this is the first report demonstrating that As(III) causes a loss of endothelial monolayer integrity, which potentially could contribute to the development of atherosclerosis.« less

Effects of steroid hormones on nuclear membrane and membrane-bound heterochromatin from breast cancer cells evaluated by fractal morphometry.

PubMed

Losa, G A; Graber, R; Baumann, G; Nonnenmacher, T F

1999-10-01

To evaluate the effect of steroid hormones on the ultrastructure of nuclear heterochromatin and perinuclear membranes in human MCF-7 breast cancer cells. MCF-7 cells were cultured briefly (five minutes) in the presence of 10(-9) M estrogen 17 beta-estradiol, a stimulator of cell proliferation and/or 10(-9) M glucocorticoid dexamethasone. Changes in the morphologic complexity of nuclear membrane-bound heterochromatin (NMBHC) and nuclear membranes (ENM) were assessed by means of the fractal capacity dimension, D, a noneuclidean geometric descriptor of complex, irregular bodies. 17 beta-estradiol (10(-9) M) enhanced the ultrastructural irregularity of NMBHC, as documented by the increased value of D, whereas dexamethasone (10(-9) M) reduced it when compared to NMBHC from untreated MCF-7 control cells. In contrast, neither steroid modified ENM ultrastructure. Changes in the nuclear heterochromatin complexity induced by estrogen 17 beta-estradiol occurred concomitantly with functional changes at the cell periphery, such as activation of the phospholipase C, a cell membrane-associated enzyme involved in signal transduction. Dexamethasone reduced the ultrastructural complexity of NMBHC without affecting functional processes. Fractal morphometry proved its usefulness in quantifying early ultrastructural changes in nuclear components induced in MCF-7 cells by steroid hormones, 17 beta-estradiol and dexamethasone.

Syntaxin-4 mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose-stimulated insulin secretion in human pancreatic beta cells.

PubMed

Xie, Li; Zhu, Dan; Dolai, Subhankar; Liang, Tao; Qin, Tairan; Kang, Youhou; Xie, Huanli; Huang, Ya-Chi; Gaisano, Herbert Y

2015-06-01

Of the four exocytotic syntaxins (Syns), much is now known about the role of Syn-1A (pre-docked secretory granules [SGs]) and Syn-3 (newcomer SGs) in insulin exocytosis. Some work was reported on Syn-4's role in biphasic glucose-stimulated insulin secretion (GSIS), but its precise role in insulin SG exocytosis remains unclear. In this paper we examine this role in human beta cells. Endogenous function of Syn-4 in human islets was assessed by knocking down its expression with lentiviral single hairpin RNA (lenti-shRNA)-RFP. Biphasic GSIS was determined by islet perifusion assay. Single-cell analysis of exocytosis of red fluorescent protein (RFP)-positive beta cells (exhibiting near-total depletion of Syn-4) was by patch clamp capacitance measurements (Cm) and total internal reflection fluorescence microscopy (TIRFM), the latter to further assess single SG behaviour. Co-immunoprecipitations were conducted on INS-1 cells to assess exocytotic complexes. Syn-4 knockdown (KD) of 77% in human islets caused a concomitant reduction in cognate Munc18c expression (46%) without affecting expression of other exocytotic proteins; this resulted in reduction of GSIS in the first phase (by 42%) and the second phase (by 40%). Cm of RFP-tagged Syn-4-KD beta cells showed severe inhibition in the readily releasable pool (by 71%) and mobilisation from reserve pools (by 63%). TIRFM showed that Syn-4-KD-induced inhibition of first-phase GSIS was attributed to reduction in exocytosis of both pre-docked and newcomer SGs (which undergo minimal residence or docking time at the plasma membrane before fusion). Second-phase inhibition was attributed to reduction in newcomer SGs. Stx-4 co-immunoprecipitated Munc18c, VAMP2 and VAMP8, suggesting that these exocytotic complexes may be involved in exocytosis of pre-docked and newcomer SGs. Syn-4 is involved in distinct molecular machineries that influence exocytosis of both pre-docked and newcomer SGs in a manner functionally redundant to Syn-1A and Syn-3, respectively; this underlies Syn-4's role in mediating portions of first-phase and second-phase GSIS.

Rapid activation of ERK1/2 and AKT in human breast cancer cells by cadmium

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

Liu Zhiwei; Yu Xinyuan; Shaikh, Zahir A.

2008-05-01

Cadmium (Cd), an endocrine disruptor, can induce a variety of signaling events including the activation of ERK1/2 and AKT. In this study, the involvement of estrogen receptors (ER) in these events was evaluated in three human breast caner cell lines, MCF-7, MDA-MB-231, and SK-BR-3. The Cd-induced signal activation patterns in the three cell lines mimicked those exhibited in response to 17{beta}-estradiol. Specifically, treatment of MCF-7 cells, that express ER{alpha}, ER{beta} and GPR30, to 0.5-10 {mu}M Cd for only 2.5 min resulted in transient phosphorylation of ERK1/2. Cd also triggered a gradual increase and sustained activation of AKT during the 60more » min treatment period. In SK-BR-3 cells, that express only GPR30, Cd also caused a transient activation of ERK1/2, but not of AKT. In contrast, in MDA-MB-231 cells, that express only ER{beta}, Cd was unable to cause rapid activation of either ERK1/2 or AKT. A transient phosphorylation of ER{alpha} was also observed within 2.5 min of Cd exposure in the MCF-7 cells. While the estrogen receptor antagonist, ICI 182,780, did not prevent the effect of Cd on these signals, specific siRNA against hER{alpha} significantly reduced Cd-induced activation of ERK1/2 and completely blocked the activation of AKT. It is concluded that Cd, like estradiol, can cause rapid activation of ERK1/2 and AKT and that these signaling events are mediated by possible interaction with membrane ER{alpha} and GPR30, but not ER{beta}.« less

Dynamin-mediated Nephrin Phosphorylation Regulates Glucose-stimulated Insulin Release in Pancreatic Beta Cells*

PubMed Central

Jeon, Jongmin; Leibiger, Ingo; Moede, Tilo; Walter, Britta; Faul, Christian; Maiguel, Dony; Villarreal, Rodrigo; Guzman, Johanna; Berggren, Per-Olof; Mundel, Peter; Ricordi, Camillo; Merscher-Gomez, Sandra; Fornoni, Alessia

2012-01-01

We have previously demonstrated a role for Nephrin in glucose stimulated insulin release (GSIR). We now hypothesize that Nephrin phosphorylation is required for GSIR and that Dynamin influences Nephrin phosphorylation and function. MIN6-C3 Nephrin-deficient pancreatic beta cells and human islets were transfected with WT-Nephrin or with a mutant Nephrin in which the tyrosine residues responsible for SH2 domain binding were substituted with phenylalanine (3YF-Nephrin). GSIR and live images of Nephrin and vesicle trafficking were studied. Immunoprecipitation experiments and overexpression of WT-Dynamin or dominant negative Dynamin mutant (K44A-Dynamin) in WT-Nephrin, 3YF-Nephrin, or Nephrin siRNA-transfected cells were utilized to study Nephrin-Dynamin interaction. In contrast to WT-Nephrin or to single tyrosine mutants, 3YF-Nephrin did not positively affect GSIR and led to impaired cell-cell contacts and vesicle trafficking. K44A-Dynamin prevented the effect of Nephrin on GSIR in the absence of protein-protein interaction between Nephrin and Dynamin. Nephrin gene silencing abolished the positive effects of WT-Dynamin on GSIR. The effects of protamine sulfate and vanadate on Nephrin phosphorylation and GSIR were studied in MIN6 cells and human islets. WT-Nephrin phosphorylation after glucose occurred at Tyr-1176/1193 and resulted in improved GSIR. On the contrary, protamine sulfate-induced phosphorylation at Tyr-1176/1193/1217 was associated with Nephrin degradation and impaired GSIR. Vanadate, which prevented Nephrin dephosphorylation after glucose stimulation, improved GSIR in human islets and MIN6 cells. In conclusion, Dynamin-dependent Nephrin phosphorylation occurs in response to glucose and is necessary for Nephrin-mediated augmentation of GSIR. Pharmacological modulation of Nephrin phosphorylation may thus facilitate pancreatic beta cell function. PMID:22718751

Proliferation and osteoblastic differentiation of human bone marrow-derived stromal cells on akermanite-bioactive ceramics.

PubMed

Sun, Hongli; Wu, Chengtie; Dai, Kerong; Chang, Jiang; Tang, Tingting

2006-11-01

In the present study, the effects of a calcium magnesium silicate bioactive ceramic (akermanite) on proliferation and osteoblastic differentiation of human bone marrow stromal cells (hBMSC) have been investigated and compared with the classical ceramic (beta-tricalcium phosphate, beta-TCP). Akermanite and beta-TCP disks were seeded with hBMSC and kept in growth medium or osteogenic medium for 10 days. Proliferation and osteoblastic differentiation were evaluated on day 1, 4, 7 and 10. The data from the Alamar Blue assay and lactic acid production assay showed that hBMSC proliferated more significantly on akermanite than on beta-TCP. The analysis of osteoblast-related genes, including alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OC), indicated that akermanite ceramics enhanced the expression of osteoblast-related genes, but type I collagen (COL I) showed no noticeable difference among akermanite and beta-TCP ceramics. Furthermore, this stimulatory effect was observed not only in osteogenic medium, but also in normal growth medium without osteogenic reagents such as l-ascorbic acid, glycerophosphate and dexamethasone. This result suggests that akermanite can promote osteoblastic differentiation of hBMSC in vitro even without osteogenic reagents, and may be used as a bioactive material for bone regeneration and tissue engineering applications.

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.

Tangeretin sensitizes cisplatin-resistant human ovarian cancer cells through downregulation of phosphoinositide 3-kinase/Akt signaling pathway.

PubMed

Arafa, El-Shaimaa A; Zhu, Qianzheng; Barakat, Bassant M; Wani, Gulzar; Zhao, Qun; El-Mahdy, Mohamed A; Wani, Altaf A

2009-12-01

Combination of innocuous dietary components with anticancer drugs is an emerging new strategy for cancer chemotherapy to increase antitumor responses. Tangeretin is a citrus flavonoid known to inhibit cancer cell proliferation. Here, we show an enhanced response of A2780/CP70 and 2008/C13 cisplatin-resistant human ovarian cancer cells to various combination treatments of cisplatin and tangeretin. Pretreatment of cells with tangeretin before cisplatin treatment synergistically inhibited cancer cell proliferation. This combination was effective in activating apoptosis via caspase cascade as well as arresting cell cycle at G(2)-M phase. Moreover, phospho-Akt and its downstream substrates, e.g., NF-kappaB, phospho-GSK-3beta, and phospho-BAD, were downregulated upon tangeretin-cisplatin treatment. The tangeretin-cisplatin-induced apoptosis in A2780/CP70 cells was increased by phosphoinositide-3 kinase (PI3K) inhibition and siRNA-mediated Akt silencing, but reduced by overexpression of constitutively activated Akt and GSK-3beta inhibition. The overall results indicated that tangeretin exposure preconditions cisplatin-resistant human ovarian cancer cells for a conventional response to low-dose cisplatin-induced cell death occurring through downregulation of PI3K/Akt signaling pathway. Thus, effectiveness of tangeretin combinations, as a promising modality in the treatment of resistant cancers, warrants systematic clinical studies.

Effects and underlying mechanisms of curcumin on the proliferation of vascular smooth muscle cells induced by Chol:M{beta}CD

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

Qin Li; Division of Pharmacoproteomics, Institute of Pharmacy and Pharmacology, University of South China, Hengyang 421001; Yang Yunbo

Proliferation of vascular smooth muscle cells (VSMCs) contributes to the development of various cardiovascular diseases. Curcumin, extracted from Curcumae longae, has been shown a variety of beneficial effects on human health, including anti-atherosclerosis by mechanisms poorly understood. In the present study, we attempted to investigate whether curcumin has any effect on VSMCs proliferation and the potential mechanisms involved. Our data showed curcumin concentration-dependently abrogated the proliferation of primary rat VSMCs induced by Chol:M{beta}CD. To explore the underlying cellular and molecular mechanisms, we found that curcumin was capable of restoring caveolin-1 expression which was reduced by Chol:M{beta}CD treatment. Moreover, curcumin abrogatedmore » the increment of phospho-ERK1/2 and nuclear accumulation of ERK1/2 in primary rat VSMCs induced by Chol:M{beta}CD, which led to a suppression of AP-1 promoter activity stimulated by Chol:M{beta}CD. In addition, curcumin was able to reverse cell cycle progression induced by Chol:M{beta}CD, which was further supported by its down-regulation of cyclinD1 and E2F promoter activities in the presence of Chol:M{beta}CD. Taking together, our data suggest curcumin inhibits Chol:M{beta}CD-induced VSMCs proliferation via restoring caveolin-1 expression that leads to the suppression of over-activated ERK signaling and causes cell cycle arrest at G1/S phase. These novel findings support the beneficial potential of curcumin in cardiovascular disease.« less

A role for human MUC4 mucin gene, the ErbB2 ligand, as a target of TGF-beta in pancreatic carcinogenesis.

PubMed

Jonckheere, Nicolas; Perrais, Michaël; Mariette, Christophe; Batra, Surinder K; Aubert, Jean-Pierre; Pigny, Pascal; Van Seuningen, Isabelle

2004-07-29

MUC4: encodes a large transmembrane mucin that is overexpressed in pancreatic adenocarcinomas. The molecular mechanisms responsible for that altered pattern of expression are unknown. TGF-beta, a pleiotropic cytokine, regulates numerous genes involved in pancreatic carcinogenesis via activation of the Smads proteins and MUC4 promoter is rich in Smad-binding elements. Our aim was to study whether the regulation of MUC4 expression by TGF-beta in pancreatic cancer cells was strictly dependent on Smad4 activity. Three pancreatic cancer cell lines, CAPAN-1 (MUC4+/Smad4-), CAPAN-2 (MUC4+/Smad4+) and PANC-1 (MUC4-/Smad4+), were used. By RT-PCR, transfection assays and immunohistochemistry, we show that (i) both MUC4 mRNA and apomucin expression are upregulated by TGF-beta, (ii) Smad2 positively cooperates with Smad4 to activate the promoter, (iii) activation of Smad4 by exogenous TGF-beta induces Smad4 binding to the promoter, (iv) Smad7 and c-ski both inhibit activation by Smad4. When Smad4 is mutated and inactive, TGF-beta activates MUC4 expression via MAPK, PI3K and PKA signaling pathways. Absence of expression in PANC-1 cells is due to histone deacetylation. Altogether, these results indicate that upregulation of MUC4 by TGF-beta is restricted to well-differentiated pancreatic cancer cells, and point out a novel mechanism for TGF-beta as a key molecule in targeting MUC4 overexpression in pancreatic adenocarcinomas.

Galectin-1 suppresses alpha2(I) collagen through Smad3 in renal epithelial cells.

PubMed

Okano, K; Uchida, K; Nitta, K; Hayashida, T

2008-10-01

Transforming growth factor (TGF-beta1) promotes renal fibrogenesis through activation of Smads. Galectin-1 is reported to prevent experimental glomerulonephritis. Here we investigated the fact that transfected galectin-1 significantly suppressed the transcription of alpha2(I) collagen (COL1A2) in TGF-beta1- activated human renal epithelial cells. Conversely, galectin-1 silencing RNA reduced secretion of type I collagen by HKC cells. Galectin-1 significantly decreased activation of a TGF-beta1-responsive reporter construct and of a minimal reporter construct that contains four repeats of the Smad binding element (SBE). Galectin-1 had no effect on phosphorylation of Smad3 at the linker region and C-terminus, whereas it decreased affinity of Smad3 to the SBE. Additionally, the inhibitory effect of galectin-1 disappeared using a mutated reporter construct, 376 m-LUC, in which a potential Smad recognition site within the promoter is mutated. Taken together, the results suggest that galectin-1 decreases Smad3-complex from binding to the SBE, down-regulating transcription of COL1A2 in TGF-beta1-stimulated renal epithelial cells.

EI-2128-1, a novel interleukin-1beta converting enzyme inhibitor produced by Penicillium sp. E-2128.

PubMed

Koizumi, Fumito; Agatsuma, Tsutomu; Ando, Katsuhiko; Kondo, Hidemasa; Saitoh, Yutaka; Matsuda, Yuzuru; Nakanishi, Satoshi

2003-11-01

EI-2128-1, a novel interleukin-1beta converting enzyme (ICE) inhibitor, was isolated from the culture broths of Penicillium sp. E-2128. EI-2128-1 selectively inhibited human recombinant ICE activity with IC50 value of 0.59 microM, without inhibiting elastase and cathepsin B. EI-2128-1 also inhibited mature interleukin-1beta secretion from THP-1 cells induced by LPS with IC50 value of 0.28 microM.

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 disease mechanisms involving chronic ROS production.

Suppression of tumorigenicity by plakoglobin: an augmenting effect of N-cadherin.

PubMed

Simcha, I; Geiger, B; Yehuda-Levenberg, S; Salomon, D; Ben-Ze'ev, A

1996-04-01

Plakoglobin is a major component of the submembranal plaque of adherens junctions and desmosomes in mammalian cells. It is closely related to the Drosophila segment polarity gene armadillo which has a role in the transduction of transmembrane signals that regulate cell fate. Like its close homologue beta-catenin, plakoglobin can associate with the product of the tumor suppressor gene APC that is linked to human colon cancer. We have studied the effect of plakoglobin overexpression, and the cooperation between plakoglobin and N-cadherin, on the morphology and tumorigenic ability of cells either lacking, or expressing cadherin and alpha- and beta-catenin. Overexpression of plakoglobin in SV40-transformed 3T3 (SVT2) cells suppressed the tumorigenicity of the cells in syngeneic mice. Transfection with N-cadherin conferred an epithelial phenotype on the cell culture, but had no significant effect on the tumorigenicity of the cells. Cotransfection of plakoglobin and N-cadherin into SVT2 cells, however, was considerably more effective in tumor suppression than plakoglobin overexpression alone. Finally, transfection of plakoglobin into a human renal carcinoma cell line that expresses neither cadherins nor plakoglobin, or alpha-and beta-catenin, resulted in a dose-dependent suppression of tumor formation by these cells in nude mice. Plakoglobin, in these cells, did not exhibit junctional localization and was diffusely distributed in the cytoplasm, with a significant amount of the protein also localized in the nucleus. The results suggest that plakoglobin can efficiently suppress the tumorigenicity of cells in the presence of, or independently of the cadherin-catenin complex.

Comparison between chondroprotective effects of glucosamine, curcumin, and diacerein in IL-1beta-stimulated C-28/I2 chondrocytes.

PubMed

Toegel, S; Wu, S Q; Piana, C; Unger, F M; Wirth, M; Goldring, M B; Gabor, F; Viernstein, H

2008-10-01

To compare the effects of glucosamine (GlcN), curcumin, and diacerein in immortalized human C-28/I2 chondrocytes at the cellular and the gene expression level. This study aimed to provide insights into the proposed beneficial effects of these agents and to assess the applicability of the C-28/I2 cell line as a model for the evaluation of chondroprotective action. Interleukin-1beta (IL-1beta)-stimulated C-28/I2 cells were cultured in the presence of GlcN, curcumin, and diacerein prior to the evaluation of parameters such as viability, morphology and proliferation. The impact of GlcN, curcumin, and diacerein on gene expression was determined using quantitative real-time RT-PCR (qPCR). At the transcriptional level, 5 mM GlcN and 50 microM diacerein increased the expression of cartilage-specific genes such as aggrecan (AGC) and collagen type II (COL2), while reducing collagen type I (COL1) mRNA levels. Moreover, the IL-1beta-mediated shift in gene expression pattern was antagonized by GlcN and diacerein. These effects were associated with a significant reduction in cellular proliferation and the development of chondrocyte-specific cell morphology. In contrast, curcumin was not effective at lower concentrations but even damaged the cells at higher amounts. Both GlcN and diacerein promoted a differentiated chondrocytic phenotype of immortalized human C-28/I2 chondrocytes by altering proliferation, morphology, and COL2/COL1 mRNA ratios. Moreover, both agents antagonized inhibitory effects of IL-1beta by enhancing AGC and COL2 as well as by reducing COL1 mRNA levels.

Functional expression of IL-12 receptor by human eosinophils: IL-12 promotes eosinophil apoptosis.

PubMed

Nutku, E; Zhuang, Q; Soussi-Gounni, A; Aris, F; Mazer, B D; Hamid, Q

2001-07-15

In murine models of allergic inflammation, IL-12 has been shown to decrease tissue eosinophilia, but the underlying mechanisms are not known. We evaluated the expression of IL-12R and the effect of IL-12 on eosinophil survival. In situ hybridization demonstrated the presence of mRNA and immunoreactivity for IL-12Rbeta1 and -beta2 subunits in human peripheral blood eosinophils. Surface expression of IL-12Rbeta1 and -beta2 subunits on freshly isolated human eosinophils was optimally expressed after incubation with PMA. To determine the functional significance of IL-12R studies, we studied cell viability and apoptosis. Morphological analysis and propidium iodide staining for cell cycle demonstrated that recombinant human IL-12 increased in vitro human eosinophil apoptosis in a dose-dependent manner. Addition of IL-5 together with IL-12 abrogated eosinophil apoptosis, suggesting that IL-12 and IL-5 have antagonistic effects. Our findings provide evidence for a novel role for IL-12 in regulating eosinophil function by increasing eosinophil apoptosis.

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 in HOSE and CSOC, and equivalent SnoN mRNA induction after TGF-beta treatment. Surprising, TGF-beta-induced Ski degradation was not observed in HOSE or CSOC, suggesting that Ski may not function as a TGF-beta/Smad corepressor in ovarian epithelial cells. These data implied that the TGF-beta/Smad pathway remains functional in CSOC, although CSOC cells are resistant to antimitogenic TGF-beta effects. CSOC resistance to TGF-beta coincided with the loss of c-myc down-regulation. These data suggest that TGF-beta/Smad signaling is blocked downstream of Smad complex formation or that an alternate signaling pathway other than TGF-beta/Smad may transmit TGF-beta-induced cell cycle arrest in the ovarian epithelium.

TGF-beta1 release from biodegradable polymer microparticles: its effects on marrow stromal osteoblast function

NASA Technical Reports Server (NTRS)

Lu, L.; Yaszemski, M. J.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

2001-01-01

BACKGROUND: Controlled release of transforming growth factor-beta1 (TGF-beta1) to a bone defect may be beneficial for the induction of a bone regeneration cascade. The objectives of this work were to assess the feasibility of using biodegradable polymer microparticles as carriers for controlled TGF-beta1 delivery and the effects of released TGF-beta1 on the proliferation and differentiation of marrow stromal cells in vitro. METHODS: Recombinant human TGF-beta1 was incorporated into microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG). Fluorescein isothiocynate-labeled bovine serum albumin (FITC-BSA) was co-encapsulated as a porogen. The effects of PEG content (0, 1, or 5% by weight [wt%]) and buffer pH (3, 5, or 7.4) on the protein release kinetics and the degradation of PLGA were determined in vitro for as long as 28 days. Rat marrow stromal cells were seeded on a biodegradable poly(propylene fumarate) (PPF) substrate. The dose response and biological activity of released TGF-beta1 was determined after 3 days in culture. The effects of TGF-beta1 released from PLGA/PEG microparticles on marrow stromal cell proliferation and osteoblastic differentiation were assessed during a 21-day period. RESULTS: TGF-beta1 was encapsulated along with FITC-BSA into PLGA/PEG blend microparticles and released in a multiphasic fashion including an initial burst for as long as 28 days in vitro. Increasing the initial PEG content resulted in a decreased cumulative mass of released proteins. Aggregation of FITC-BSA occurred at lower buffer pH, which led to decreased release rates of both proteins. The degradation of PLGA was increased at higher PEG content and significantly accelerated at acidic pH conditions. Rat marrow stromal cells cultured on PPF substrates showed a dose response to TGF-beta1 released from the microparticles similar to that of added TGF-beta1, indicating that the activity of TGF-beta1 was retained during microparticle fabrication and after growth factor release. At an optimal TGF-beta1 dosage of 1.0 ng/ml after 3 days, the released TGF-beta1 enhanced the proliferation and osteoblastic differentiation of marrow stromal cells over 21 days of culture, with increased total cell number, alkaline phosphatase activity, and osteocalcin production. CONCLUSIONS: PLGA/PEG blend microparticles can serve as delivery vehicles for controlled release of TGF-beta1, and the released growth factor enhances marrow stromal cell proliferation and osteoblastic differentiation in vitro. CLINICAL RELEVANCE: Controlled release of TGF-beta1 from PLGA/PEG microparticles is representative of emerging tissue engineering technologies that may modulate cellular responses to encourage bone regeneration at a skeletal defect site.

Transforming growth factor beta-1 expression in macrophages of human chronic periapical diseases.

PubMed

Liang, Z-Z; Li, J; Huang, S-G

2017-03-30

The objective of this study was to observe the distribution of macrophages (MPs) expressing transforming growth factor beta-1 (TGF-β1) in tissue samples from patients with different human chronic periapical diseases. In this study, samples were collected from 75 volunteers, who were divided into three groups according to classified standards, namely, healthy control (N = 25), periapical granuloma (N = 25), and periapical cyst (N = 25). The samples were fixed in 10% buffered formalin for more than 48 h, dehydrated, embedded, and stained with hematoxylin and eosin for histopathology. Double immunofluorescence was conducted to analyze the expression of TGF-β-CD14 double-positive MPs in periapical tissues. The number of double-positive cells (cells/mm 2 ) were significantly higher in the chronic periapical disease tissues (P < 0.01) compared to that in the control tissue; in addition, the density of TGF-β1-CD14 double positive cells was significantly higher in the periapical cyst group than in the periapical granuloma group (P < 0.01). The number of TGF-β1 expressing macrophages varied with human chronic periapical diseases. The TGF-β1-CD14 double-positive cells might play an important role in the pathology of human chronic periapical diseases.

Sex steroid hormone metabolism takes place in human ocular cells.

PubMed

Coca-Prados, Miguel; Ghosh, Sikha; Wang, Yugang; Escribano, Julio; Herrala, Annakaisa; Vihko, Pirkko

2003-08-01

Steroids are potentially important mediators in the pathophysiology of ocular diseases. In this study, we report on the gene expression in the human eye of a group of enzymes, the 17beta-hydroxysteroid dehydrogenases (17HSDs), involved in the biosynthesis and inactivation of sex steroid hormones. In the eye, the ciliary epithelium, a neuroendocrine secretory epithelium, co-expresses the highest levels of 17HSD2 and 5 mRNAs, and in lesser level 17HSD7 mRNA. The regulation of gene expression of these enzymes was investigated in vitro in cell lines, ODM-C4 and chronic open glaucoma (GCE), used as cell models of the human ciliary epithelium. The estrogen, 17beta-estradiol (10(-7) M) and androgen agonist, R1881 (10(-8) M) elicited in ODM-C4 and GCE cells over a 24 h time course a robust up-regulation of 17HSD7 mRNA expression. 17HSD2 was up-regulated by estradiol in ODM-C4 cells, but not in GCE cells. Under steady-state conditions, ODM-C4 cells exhibited a predominant 17HSD2 oxidative enzymatic activity. In contrast, 17HSD2 activity was low or absent in GCE cells. Our collective data suggest that cultured human ciliary epithelial cells are able to metabolize estrogen, androgen and progesterone, and that 17HSD2 and 7 in these cells are sex steroid hormone-responsive genes and 17HSD7 is responsible to keep on intra/paracrine estrogenic milieu.

The effects of dan-shen root on cardiomyogenic differentiation of human placenta-derived mesenchymal stem cells

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

Li, Kun; Li, Shi-zheng, E-mail: ychozon@yahoo.com.cn; Zhang, Yun-li

2011-11-11

Highlights: Black-Right-Pointing-Pointer Conditional medium and dan-shen root were used for cardiomyogenic differentiation. Black-Right-Pointing-Pointer They all could induce hPDMSCs to differentiate into cardiomyocytes. Black-Right-Pointing-Pointer The induction effect of the latter was slightly higher compared to that of the former. Black-Right-Pointing-Pointer Dan-shen root could be a good inducer for cardiomyogenic differentiation. -- Abstract: The aim of this study was to search for a good inducer agent using for cardiomyogenic differentiation of stem cells. Human placenta-derived mesenchymal stem cells (hPDMSCs) were isolated and incubated in enriched medium. Fourth passaged cells were treated with 10 mg/L dan-shen root for 20 days. Morphologic characteristics weremore » analyzed by confocal and electron microscopy. Expression of {alpha}-sarcomeric actin was analyzed by immunohistochemistry. Expression of cardiac troponin-I (TnI) was analyzed by immunohistofluorescence. Atrial natriuretic factor (ANF) and beta-myocin heavy chain ({beta}-MHC) were detected by reverse transcriptase polymerase chain reaction (RT-PCR). hPDMSCs treated with dan-shen root gradually formed a stick-like morphology and connected with adjoining cells. On the 20th day, most of the induced cells stained positive with {alpha}-sarcomeric actin and TnI antibody. ANF and {beta}-MHC were also detected in the induced cells. Approximately 80% of the cells were successfully transdifferentiated into cardiomyocytes. In conclusion, dan-shen root is a good inducer agent used for cardiomyogenic differentiation of hPDMSCs.« less

Apigenin inhibits HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and {beta}4 integrin function in MDA-MB-231 breast cancer cells

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

Lee, W.-J.; Chen, W.-K.; Wang, C.-J.

2008-01-15

Hepatocyte growth factor (HGF) and its receptor, Met, known to control invasive growth program have recently been shown to play crucial roles in the survival of breast cancer patients. The diet-derived flavonoids have been reported to possess anti-invasion properties; however, knowledge on the pharmacological and molecular mechanisms in suppressing HGF/Met-mediated tumor invasion and metastasis is poorly understood. In our preliminary study, we use HGF as an invasive inducer to investigate the effect of flavonoids including apigenin, naringenin, genistein and kaempferol on HGF-dependent invasive growth of MDA-MB-231 human breast cancer cells. Results show that apigenin presents the most potent anti-migration andmore » anti-invasion properties by Boyden chamber assay. Furthermore, apigenin represses the HGF-induced cell motility and scattering and inhibits the HGF-promoted cell migration and invasion in a dose-dependent manner. The effect of apigenin on HGF-induced signaling activation involving invasive growth was evaluated by immunoblotting analysis, it shows that apigenin blocks the HGF-induced Akt phosphorylation but not Met, ERK, and JNK phosphorylation. In addition to MDA-MB-231 cells, apigenin exhibits inhibitory effect on HGF-induced Akt phosphorylation in hepatoma SK-Hep1 cells and lung carcinoma A549 cells. By indirect immunofluorescence microscopy assay, apigenin inhibits the HGF-induced clustering of {beta}4 integrin at actin-rich adhesive site and lamellipodia through PI3K-dependent manner. Treatment of apigenin inhibited HGF-stimulated integrin {beta}4 function including cell-matrix adhesion and cell-endothelial cells adhesion in MDA-MB-231 cells. By Akt-siRNA transfection analysis, it confirmed that apigenin inhibited HGF-promoted invasive growth involving blocking PI3K/Akt pathway. Finally, we evaluated the effect of apigenin on HGF-promoted metastasis by lung colonization of tumor cells in nude mice and organ metastasis of tumor cells in chick embryo. By histological and gross examination of mouse lung and real-time PCR analysis of human alu in host tissues, it showed that apigenin, wortmannin, as well as anti-{beta}4 antibody all inhibit HGF-promoted metastasis. These data support the inhibitory effect of apigenin on HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and integrin {beta}4 function.« less

Modulation of lens cell adhesion molecules by particle beams

NASA Technical Reports Server (NTRS)

McNamara, M. P.; Bjornstad, K. A.; Chang, P. Y.; Chou, W.; Lockett, S. J.; Blakely, E. A.

2001-01-01

Cell adhesion molecules (CAMs) are proteins which anchor cells to each other and to the extracellular matrix (ECM), but whose functions also include signal transduction, differentiation, and apoptosis. We are testing a hypothesis that particle radiations modulate CAM expression and this contributes to radiation-induced lens opacification. We observed dose-dependent changes in the expression of beta 1-integrin and ICAM-1 in exponentially-growing and confluent cells of a differentiating human lens epithelial cell model after exposure to particle beams. Human lens epithelial (HLE) cells, less than 10 passages after their initial culture from fetal tissue, were grown on bovine corneal endothelial cell-derived ECM in medium containing 15% fetal bovine serum and supplemented with 5 ng/ml basic fibroblast growth factor (FGF-2). Multiple cell populations at three different stages of differentiation were prepared for experiment: cells in exponential growth, and cells at 5 and 10 days post-confluence. The differentiation status of cells was characterized morphologically by digital image analysis, and biochemically by Western blotting using lens epithelial and fiber cell-specific markers. Cultures were irradiated with single doses (4, 8 or 12 Gy) of 55 MeV protons and, along with unirradiated control samples, were fixed using -20 degrees C methanol at 6 hours after exposure. Replicate experiments and similar experiments with helium ions are in progress. The intracellular localization of beta 1-integrin and ICAM-1 was detected by immunofluorescence using monoclonal antibodies specific for each CAM. Cells known to express each CAM were also processed as positive controls. Both exponentially-growing and confluent, differentiating cells demonstrated a dramatic proton-dose-dependent modulation (upregulation for exponential cells, downregulation for confluent cells) and a change in the intracellular distribution of the beta 1-integrin, compared to unirradiated controls. In contrast, there was a dose-dependent increase in ICAM-1 immunofluorescence in confluent, but not exponentially-growing cells. These results suggest that proton irradiation downregulates beta 1-integrin and upregulates ICAM-1, potentially contributing to cell death or to aberrant differentiation via modulation of anchorage and/or signal transduction functions. Quantification of the expression levels of the CAMs by Western analysis is in progress.

Expansion and redifferentiation of chondrocytes from osteoarthritic cartilage: cells for human cartilage tissue engineering.

PubMed

Hsieh-Bonassera, Nancy D; Wu, Iwen; Lin, Jonathan K; Schumacher, Barbara L; Chen, Albert C; Masuda, Koichi; Bugbee, William D; Sah, Robert L

2009-11-01

To determine if selected culture conditions enhance the expansion and redifferentiation of chondrocytes isolated from human osteoarthritic cartilage with yields appropriate for creation of constructs for treatment of joint-scale cartilage defects, damage, or osteoarthritis. Chondrocytes isolated from osteoarthritic cartilage were analyzed to determine the effects of medium supplement on cell expansion in monolayer and then cell redifferentiation in alginate beads. Expansion was assessed as cell number estimated from DNA, growth rate, and day of maximal growth. Redifferentiation was evaluated quantitatively from proteoglycan and collagen type II content, and qualitatively by histology and immunohistochemistry. Using either serum or a growth factor cocktail (TFP: transforming growth factor beta1, fibroblast growth factor 2, and platelet-derived growth factor type bb), cell growth rate in monolayer was increased to 5.5x that of corresponding conditions without TFP, and cell number increased 100-fold within 17 days. In subsequent alginate bead culture with human serum or transforming growth factor beta1 and insulin-transferrin-selenium-linoleic acid-bovine serum albumin, redifferentiation was enhanced with increased proteoglycan and collagen type II production. Effects of human serum were dose dependent, and 5% or higher induced formation of chondron-like structures with abundant proteoglycan-rich matrix. Chondrocytes from osteoarthritic cartilage can be stimulated to undergo 100-fold expansion and then redifferentiation, suggesting that they may be useful as a cell source for joint-scale cartilage tissue engineering.

Apolipoprotein CIII-induced THP-1 cell adhesion to endothelial cells involves pertussis toxin-sensitive G protein- and protein kinase C alpha-mediated nuclear factor-kappaB activation.

PubMed

Kawakami, Akio; Aikawa, Masanori; Nitta, Noriko; Yoshida, Masayuki; Libby, Peter; Sacks, Frank M

2007-01-01

Plasma apolipoprotein CIII (apoCIII) independently predicts risk for coronary heart disease (CHD). We recently reported that apoCIII directly enhances adhesion of human monocytes to endothelial cells (ECs), and identified the activation of PKC alpha as a necessary upstream event of enhanced monocyte adhesion. This study tested the hypothesis that apoCIII activates PKC alpha in human monocytic THP-1 cells, leading to NF-kappaB activation. Among inhibitors specific to PKC activators, phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609 limited apoCIII-induced PKC alpha activation and THP-1 cell adhesion. ApoCIII increased PC-PLC activity in THP-1 cells, resulting in PKC alpha activation. Pertussis toxin (PTX) inhibited apoCIII-induced PC-PLC activation and subsequent PKC alpha activation, implicating PTX-sensitive G protein pathway. ApoCIII further activated nuclear factor-kappaB (NF-kappaB) through PKC alpha in THP-1 cells and augmented beta1-integrin expression. The NF-kappaB inhibitor peptide SN50 partially inhibited apoCIII-induced beta1-integrin expression and THP-1 cell adhesion. ApoCIII-rich VLDL had similar effects to apoCIII alone. PTX-sensitive G protein pathway participates critically in PKC alpha stimulation in THP-1 cells exposed to apoCIII, activating NF-kappaB, and increasing beta1-integrin. This action causes monocytic cells to adhere to endothelial cells. Furthermore, because leukocyte NF-kappaB activation contributes to inflammatory aspects of atherogenesis, apoCIII may stimulate diverse inflammatory responses through monocyte activation.

Effect of beta2-adrenoceptor agonists and other cAMP-elevating agents on inflammatory gene expression in human ASM cells: a role for protein kinase A.

PubMed

Kaur, Manminder; Holden, Neil S; Wilson, Sylvia M; Sukkar, Maria B; Chung, Kian Fan; Barnes, Peter J; Newton, Robert; Giembycz, Mark A

2008-09-01

In diseases such as asthma, airway smooth muscle (ASM) cells play a synthetic role by secreting inflammatory mediators such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, or IL-8 and by expressing surface adhesion molecules, including ICAM-1. In the present study, PGE(2), forskolin, and short-acting (salbutamol) and long-acting (salmeterol and formoterol) beta(2)-adrenoceptor agonists reduced the expression of ICAM-1 and the release of GM-CSF evoked by IL-1beta in ASM cells. IL-1beta-induced IL-8 release was also repressed by PGE(2) and forskolin, whereas the beta(2)-adrenoceptor agonists were ineffective. In each case, repression of these inflammatory indexes was prevented by adenoviral overexpression of PKIalpha, a highly selective PKA inhibitor. These data indicate a PKA-dependent mechanism of repression and suggest that agents that elevate intracellular cAMP, and thereby activate PKA, may have a widespread anti-inflammatory effect in ASM cells. Since ICAM-1 and GM-CSF are highly NF-kappaB-dependent genes, we used an adenoviral-delivered NF-kappaB-dependent luciferase reporter to examine the effects of forskolin and the beta(2)-adrenoceptor agonists on NF-kappaB activation. There was no effect on luciferase activity measured in the presence of forskolin or beta(2)-adrenoceptor agonists. This finding is consistent with the observation that IL-1beta-induced expression of IL-6, a known NF-kappaB-dependent gene in ASM, was also unaffected by beta(2)-adrenoceptor agonists, forskolin, PGE(2), 8-bromo-cAMP, or rolipram. Collectively, these results indicate that repression of IL-1beta-induced ICAM-1 expression and GM-CSF release by cAMP-elevating agents, including beta(2)-adrenoceptor agonists, may not occur through a generic effect on NF-kappaB.

Live Candida albicans suppresses production of reactive oxygen species in phagocytes.

PubMed

Wellington, Melanie; Dolan, Kristy; Krysan, Damian J

2009-01-01

Production of reactive oxygen species (ROS) is an important aspect of phagocyte-mediated host responses. Since phagocytes play a crucial role in the host response to Candida albicans, we examined the ability of Candida to modulate phagocyte ROS production. ROS production was measured in the murine macrophage cell line J774 and in primary phagocytes using luminol-enhanced chemiluminescence. J774 cells, murine polymorphonuclear leukocytes (PMN), human monocytes, and human PMN treated with live C. albicans produced significantly less ROS than phagocytes treated with heat-killed C. albicans. Live C. albicans also suppressed ROS production in murine bone marrow-derived macrophages from C57BL/6 mice, but not from BALB/c mice. Live C. albicans also suppressed ROS in response to external stimuli. C. albicans and Candida glabrata suppressed ROS production by phagocytes, whereas Saccharomyces cerevisiae stimulated ROS production. The cell wall is the initial point of contact between Candida and phagocytes, but isolated cell walls from both heat-killed and live C. albicans stimulated ROS production. Heat-killed C. albicans has increased surface exposure of 1,3-beta-glucan, a cell wall component that can stimulate phagocytes. To determine whether surface 1,3-beta-glucan exposure accounted for the difference in ROS production, live C. albicans cells were treated with a sublethal dose of caspofungin to increase surface 1,3-beta-glucan exposure. Caspofungin-treated C. albicans was fully able to suppress ROS production, indicating that suppression of ROS overrides stimulatory signals from 1,3-beta-glucan. These studies indicate that live C. albicans actively suppresses ROS production in phagocytes in vitro, which may represent an important immune evasion mechanism.

Comparison of the effects of agalsidase alfa and agalsidase beta on cultured human Fabry fibroblasts and Fabry mice.

PubMed

Sakuraba, Hitoshi; Murata-Ohsawa, Mai; Kawashima, Ikuo; Tajima, Youichi; Kotani, Masaharu; Ohshima, Toshio; Chiba, Yasunori; Takashiba, Minako; Jigami, Yoshifumi; Fukushige, Tomoko; Kanzaki, Tamotsu; Itoh, Kohji

2006-01-01

We compared two recombinant alpha-galactosidases developed for enzyme replacement therapy for Fabry disease, agalsidase alfa and agalsidase beta, as to specific alpha-galactosidase activity, stability in plasma, mannose 6-phosphate (M6P) residue content, and effects on cultured human Fabry fibroblasts and Fabry mice. The specific enzyme activities of agalsidase alfa and agalsidase beta were 1.70 and 3.24 mmol h(-1) mg protein(-1), respectively, and there was no difference in stability in plasma between them. The M6P content of agalsidase beta (3.6 mol/mol protein) was higher than that of agalsidase alfa (1.3 mol/mol protein). The administration of both enzymes resulted in marked increases in alpha-galactosidase activity in cultured human Fabry fibroblasts, and Fabry mouse kidneys, heart, spleen and liver. However, the increase in enzyme activity in cultured fibroblasts, kidneys, heart and spleen was higher when agalsidase beta was used. An immunocytochemical analysis revealed that the incorporated recombinant enzyme degraded the globotriaosyl ceramide accumulated in cultured Fabry fibroblasts in a dose-dependent manner, with the effect being maintained for at least 7 days. Repeated administration of agalsidase beta apparently decreased the number of accumulated lamellar inclusion bodies in renal tubular cells of Fabry mice.

Structure of the human transcobalamin beta domain in four distinct states

PubMed Central

Bloch, Joël S.; Ruetz, Markus; Kräutler, Bernhard

2017-01-01

Vitamin B12 (cyanocobalamin, CNCbl) is an essential cofactor-precursor for two biochemical reactions in humans. When ingested, cobalamins (Cbl) are transported via a multistep transport system into the bloodstream, where the soluble protein transcobalamin (TC) binds Cbl and the complex is taken up into the cells via receptor mediated endocytosis. Crystal structures of TC in complex with CNCbl have been solved previously. However, the initial steps of holo-TC assembly have remained elusive. Here, we present four crystal structures of the beta domain of human TC (TC-beta) in different substrate-bound states. These include the apo and CNCbl-bound states, providing insight into the early steps of holo-TC assembly. We found that in vitro assembly of TC-alpha and TC-beta to a complex was Cbl-dependent. We also determined the structure of TC-beta in complex with cobinamide (Cbi), an alternative substrate, shedding light on the specificity of TC. We finally determined the structure of TC-beta in complex with an inhibitory antivitamin B12 (anti-B12). We used this structure to model the binding of anti-B12 into full-length holo-TC and could rule out that the inhibitory function of anti-B12 was based on an inability to form a functional complex with TC. PMID:28910388

Antiviral activity of 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodocytosine against human cytomegalovirus in human skin fibroblasts.

PubMed Central

Colacino, J M; Lopez, C

1985-01-01

2'-Deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodocytosine (FIAC) was shown to be a selective anti-human cytomegalovirus agent in vitro with a 50% antiviral effective dose of 0.6 microM (J. M. Colacino and C. Lopez, Antimicrob. Agents Chemother. 26:505-508, 1983) and a 50% cell growth inhibitory dose of 8 microM. Antiviral activity was more readily reversed with 10-fold excess thymidine, whereby the 50% effective dose was increased to 11.3 microM. FIAC-induced cytotoxicity was more readily reversed with 10-fold excess of deoxycytidine, whereby the 50% inhibitory dose was increased to greater than 100 microM. Thymidine was unable to reverse completely the antiviral activity of FIAC. Although, the extent of phosphorylation of thymidine, deoxycytidine, and deoxyuridine was 6-, 4-, and 4-fold greater, respectively, in human cytomegalovirus-infected cell lysates than in uninfected cell lysates, the extent of phosphorylation of FIAC was only 1.3-fold greater in human cytomegalovirus-infected cell lysates than in uninfected cell lysates. By comparison, the extent of FIAC phosphorylation was 500 times greater in herpes simplex virus type 1-infected cells than in uninfected cell lysates. Methotrexate was 400 times more effective against human cytomegalovirus replication than it was against herpes simplex virus type 1 replication, indicating that thymidylate synthetase may be important for human cytomegalovirus replication. However, 10 microM FIAC did not inhibit thymidylate synthetase activity in uninfected or virus-infected cells as determined by their metabolism of [6-3H]deoxyuridine in the presence or absence of drug. FIAC at 1 microM suppresses and FIAC at 10 microM completely inhibits human cytomegalovirus DNA replication as indicated by Southern blot analysis. This inhibition was reversible. FIAC incorporation into the DNA of human cytomegalovirus strain AD169-infected cells was stimulated relative to that in nondividing, uninfected cells. Images PMID:3010842

Beta 2-Microglobulin: A Novel Therapeutic Target for the Treatment of Human Prostate Cancer Bone Metastasis

DTIC Science & Technology

2009-03-14

H, Sodek J, Zhau HE, Chung LW. Human osteocalcin and bone sialoprotein mediating osteomimicry of prostate cancer cells: role of cAMP-dependent...with mesenchymal phenotype b2-m b2-Microglobulin BSP Bone sialoprotein C4-2 Lineage derivative cells from LNCaP C4-2B C4-2 cells metastasized to bone...OPN) and bone sialoprotein (BSP), and RANKL, collectively allow- ing cancer cells to survive and thrive in the bone microenvironment [7–9]. Previous

PGE(2) inhibition of TGF-beta1-induced myofibroblast differentiation is Smad-independent but involves cell shape and adhesion-dependent signaling.

PubMed

Thomas, Peedikayil E; Peters-Golden, Marc; White, Eric S; Thannickal, Victor J; Moore, Bethany B

2007-08-01

Myofibroblasts are pathogenic in pulmonary fibrotic disease due to their exuberant production of matrix rich in collagen that interferes with gas exchange and the ability of these cells to contract and distort the alveolar space. Transforming growth factor-beta1 (TGF-beta1) is a well-known inducer of myofibroblast differentiation. TGF-beta1-induced transformation of fibroblasts to apoptosis-resistant myofibroblasts is adhesion-dependent and focal adhesion kinase (FAK)-mediated. Prostaglandin E(2) (PGE(2)) inhibits this differentiation via E prostanoid receptor 2 (EP2) signaling and cAMP elevation, but whether PGE(2) does so by interfering with TGF-beta1 signaling is unknown. Thus we examined the effects of PGE(2) in the presence and absence of TGF-beta1 stimulation on candidate signaling pathways in human lung fibroblasts. We now demonstrate that PGE(2) does not interfere with TGF-beta1-induced Smad phosphorylation or its translocation to the nucleus. Rather, PGE(2) has dramatic effects on cell shape and cytoskeletal architecture and disrupts the formation of appropriate focal adhesions. PGE(2) treatment diminishes TGF-beta1-induced phosphorylation of paxillin, STAT-3, and FAK and, in turn, limits activation of the protein kinase B (PKB/Akt) pathway. These alterations do not, however, result in increased apoptosis within the first 24 h of treatment. Interestingly, the effects of PGE(2) stimulation alone do not always mirror the effects of PGE(2) in the presence of TGF-beta1, indicating that the context for EP2 signaling is different in the presence of TGF-beta1. Taken together, our results demonstrate that PGE(2) has the potential to limit TGF-beta1-induced myofibroblast differentiation via adhesion-dependent, but Smad-independent, pathways.

IL1{beta}-mediated Stromal COX-2 signaling mediates proliferation and invasiveness of colonic epithelial cancer cells

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

Zhu, Yingting, E-mail: yitizhu@yahoo.com; Tissue Tech Inc, Miami, FL 33173; Zhu, Min

2012-11-15

COX-2 is a major inflammatory mediator implicated in colorectal inflammation and cancer. However, the exact origin and role of COX-2 on colorectal inflammation and carcinogenesis are still not well defined. Recently, we reported that COX-2 and iNOS signalings interact in colonic CCD18Co fibroblasts. In this article, we investigated whether activation of COX-2 signaling by IL1{beta} in primary colonic fibroblasts obtained from normal and cancer patients play a critical role in regulation of proliferation and invasiveness of human colonic epithelial cancer cells. Our results demonstrated that COX-2 level was significantly higher in cancer associated fibroblasts than that in normal fibroblasts withmore » or without stimulation of IL-1{beta}, a powerful stimulator of COX-2. Using in vitro assays for estimating proliferative and invasive potential, we discovered that the proliferation and invasiveness of the epithelial cancer cells were much greater when the cells were co-cultured with cancer associated fibroblasts than with normal fibroblasts, with or without stimulation of IL1{beta}. Further analysis indicated that the major COX-2 product, prostaglandin E{sub 2}, directly enhanced proliferation and invasiveness of the epithelial cancer cells in the absence of fibroblasts. Moreover, a selective COX-2 inhibitor, NS-398, blocked the proliferative and invasive effect of both normal and cancer associate fibroblasts on the epithelial cancer cells, with or without stimulation of IL-1{beta}. Those results indicate that activation of COX-2 signaling in the fibroblasts plays a major role in promoting proliferation and invasiveness of the epithelial cancer cells. In this process, PKC is involved in the activation of COX-2 signaling induced by IL-1{beta} in the fibroblasts.« less

The TGF-beta-induced gene product, betaig-h3: its biological implications in peritoneal dialysis.

PubMed

Park, Sun-Hee; Choi, Soon-Youn; Kim, Mi-Hyung; Oh, Eun-Joo; Ryu, Hye Myung; Kim, Chan-Duck; Kim, In-San; Kim, Yong-Lim

2008-01-01

TGF-beta is involved in peritoneal changes during long-term peritoneal dialysis (PD). TGF-beta induces betaig-h3 in several cell lines, and betaig-h3 may be a marker for biologically active TGF-beta. However, no study has reported induction of betaig-h3 in human peritoneal mesothelial cells (HPMCs) or its involvement in PD-related peritoneal membrane changes. We used cultured HPMCs to investigate the biological roles of betaig-h3 during mesothelial cell injury and repair, employing the adhesion, spreading, scratching and cell migration assays. Changes in betaig-h3 expression after high glucose exposure in vivo were also evaluated using an animal chronic PD model. In vitro, TGF-beta1 induced betaig-h3 in cultured HPMCs, and betaig-h3-mediated mesothelial cell adhesion occurred via alphavbeta3 integrin. betaig-h3 enhanced mesothelial cell adhesion and migration and, in part, wound healing during mesothelial cell injury. The animal study demonstrated that compared to the control group, betaig-h3 concentrations in the dialysate effluent increased in the dialysis group with alterations in peritoneal structure and function during PD, and betaig-h3 positively correlated with peritoneal solute transport. Immunohistochemical and immunoblotting results showed that betaig-h3 localizes in the mesothelium and submesothelial matrix of the parietal peritoneum, and in the vascular endothelium of omentum. betaig-h3 protein expression was higher in the dialysis group. In vitro, betaig-h3 induced by TGF-beta1 in HPMCs improved adhesion and migration of HPMCs during wound healing. In the chronic infusion model of PD, betaig-h3 played a role in the functional deterioration of the peritoneal membrane, which is associated with fibrosis.

Withangulatin I, a new cytotoxic withanolide from Physalis angulata.

PubMed

Lee, Shwu-Woan; Pan, Min-Hsiung; Chen, Chiu-Ming; Chen, Zong-Tsi

2008-02-01

A new withanolide, withangulatin I (2), was isolated from the whole plant of Physalis angulata. Its structure was established as (20S,22R)-15alpha-acetoxy-5beta,6beta-epoxy-14alpha-hydroxy-1,4-dioxo-witha-2,16,24-trienolide on the basis of chemical and spectroscopic methods including 2D-NMR and circular dichroism (CD) experiments. Withangulatin A (1) and withangulatin I (2) were tested for their cytotoxic activities against two human cancer cell lines, colorectal carcinoma COLO 205 and gastric carcinoma AGS, in vitro. Compounds 1 and 2 exhibited inhibitory activities against these two human cancer cells with IC(50) values of 16.6 and 1.8 and 53.6 and 65.4 muM, respectively.

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

Rouyer-Fessard, P.; Garel, M.C.; Domenget, C.

The soluble pool of alpha hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, beta A hemoglobin chain labeled with ({sup 3}H)N-ethylmaleimide. This pool of soluble alpha chains was 0.067 {plus minus} 0.017% of hemoglobin in blood of normal adult, 0.11 {plus minus} 0.03% in heterozygous beta thalassemia and ranged from 0.26 to 1.30% in homozygous beta thalassemia intermedia. This elevated pool of soluble alpha chains observed in human beta thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% inmore » the most dense red blood cells. The amount of insoluble alpha chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In beta thalassemia intermedia the amount of insoluble alpha chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using ({sup 3}H)N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups.« less

Activation of PKC{beta}{sub II} and PKC{theta} is essential for LDL-induced cell proliferation of human aortic smooth muscle cells via Gi-mediated Erk1/2 activation and Egr-1 upregulation

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

Heo, Kyung-Sun; Department of Pharmacy, Chungnam National University, Yuseong, Daejeon; Kim, Dong-Uk

Native LDL may be a mitogenic stimulus of VSMC proliferation in lesions where endothelial disruption occurs. Recent studies have demonstrated that the mitogenic effects of LDL are accompanied by Erk1/2 activation via an unknown G-protein-coupled receptor (GPCR). In this article, we report that LDL translocated PKC{beta}{sub II} and PKC{theta} from cytosol to plasma membrane, and inhibition of PKC{beta}{sub II} and PKC{theta} decreased LDL effects via the deactivation of Erk1/2. Moreover, pertussis toxin, but not cholera toxin or heparin, inhibited LDL-induced translocation of PKC{beta}{sub II} and PKC{theta}, suggesting that Gi protein plays a role in LDL effects. Of LPA, S1P, andmore » LDL, whose signaling is conveyed via Gi/o proteins, only LDL induced translocation of PKC{beta}{sub II} and PKC{theta}. Inhibition of PKC{beta}{sub II} or PKC{theta}, as well as of Erk1/2 and GPCR, decreases LDL-induced upregulation of Egr-1, which is critical for cell proliferation. This is the first report, to our knowledge, that the participation of PKC{theta} in VSMC proliferation is unique.« less

Thermogelling chitosan and collagen composite hydrogels initiated with beta-glycerophosphate for bone tissue engineering.

PubMed

Wang, Limin; Stegemann, Jan P

2010-05-01

Chitosan and collagen type I are naturally derived materials used as cell carriers because of their ability to mimic the extracellular environment and direct cell function. In this study beta-glycerophosphate (beta-GP), an osteogenic medium supplement and a weak base, was used to simultaneously initiate gelation of pure chitosan, pure collagen, and chitosan-collagen composite materials at physiological pH and temperature. Adult human bone marrow-derived stem cells (hBMSC) encapsulated in such hydrogels at chitosan/collagen ratios of 100/0, 65/35, 25/75, and 0/100 wt% exhibited high viability at day 1 after encapsulation, but DNA content dropped by about half over 12 days in pure chitosan materials while it increased twofold in materials containing collagen. Collagen-containing materials compacted more strongly and were significantly stiffer than pure chitosan gels. In monolayer culture, exposure of hBMSC to beta-GP resulted in decreased cell metabolic activity that varied with concentration and exposure time, but washing effectively removed excess beta-GP from hydrogels. The presence of chitosan in materials resulted in higher expression of osterix and bone sialoprotein genes in medium with and without osteogenic supplements. Chitosan also increased alkaline phosphatase activity and calcium deposition in osteogenic medium. Chitosan-collagen composite materials have potential as matrices for cell encapsulation and delivery, or as in situ gel-forming materials for tissue repair. Copyright 2010 Elsevier Ltd. All rights reserved.

Sonic hedgehog protein promotes proliferation and chondrogenic differentiation of bone marrow-derived mesenchymal stem cells in vitro.

PubMed

Warzecha, Jörg; Göttig, Stephan; Brüning, Christian; Lindhorst, Elmar; Arabmothlagh, Mohammad; Kurth, Andreas

2006-10-01

Sonic hedgehog (Shh) protein is known to be an important signaling protein in early embryonic development. Also, Shh is involved in the induction of early cartilaginous differentiation of mesenchymal cells in the limb and in the spine. The impact of Shh on adult stem cells, human bone marrow-derived mesenchymal stem cells (MSCs), was tested. The MSCs were treated either with recombinant Sonic hedgehog protein (r-Shh) or with transforming growth factor-beta 1 (TGF-beta(1)) as a positive control in vitro for 3 weeks. The effects on cartilaginous differentiation and proliferation were assayed. MSCs when treated with either Shh or TGF-beta(1) showed expression of cartilage markers aggrecan, Sox9, CEP-68, and collagen type II and X within 3 weeks. Only r-Shh-treated cells showed a very strong cell proliferation and much higher BrdU incorporation in cell assay systems. These are the first data that indicate an important role of Shh for the induction of cartilage production by MSCs in vitro.

Stimulation of the follicular bulge LGR5+ and LGR6+ stem cells with the gut-derived human alpha defensin 5 results in decreased bacterial presence, enhanced wound healing, and hair growth from tissues devoid of adnexal structures.

PubMed

Lough, Denver; Dai, Hui; Yang, Mei; Reichensperger, Joel; Cox, Lisa; Harrison, Carrie; Neumeister, Michael W

2013-11-01

Discovery of leucine-rich repeat-containing G-protein-coupled receptors 5 and 6 (LGR5 and LGR6) as markers of adult epithelial stem cells of the skin and intestine permits researchers to draw on the intrinsic cellular fundamentals of wound healing and proliferation dynamics of epithelial surfaces. In this study, the authors use the intestine-derived human alpha defensin 5 to stimulate epithelial proliferation, bacterial reduction, and hair production in burn wound beds to provide the field with initial insight on augmenting wound healing in tissues devoid of adnexal stem cells. Murine third-degree burn wound beds were treated with (1) intestine-derived human alpha defensin 5, (2) skin-derived human beta defensin 1, and (3) sulfadiazine to determine their roles in wound healing, bacterial reduction, and hair growth. The human alpha defensin 5 peptide significantly enhanced wound healing and reduced basal bacterial load compared with human beta defensin 1 and sulfadiazine. Human alpha defensin 5 was the only therapy to induce LGR stem cell migration into the wound bed. In addition, gene heat mapping showed significant mRNA up-regulation of key wound healing and Wnt pathway transcripts such as Wnt1 and Wisp1. Ex vivo studies showed enhanced cell migration in human alpha defensin 5-treated wounds compared with controls. Application of human alpha defensin 5 increases LGR stem cell migration into wound beds, leading to enhanced healing, bacterial reduction, and hair production through the augmentation of key Wnt and wound healing transcripts. These findings can be used to derive gut protein-based therapeutics in wound healing.

Mechanical unloading of the failing human heart fails to activate the protein kinase B/Akt/glycogen synthase kinase-3beta survival pathway.

PubMed

Razeghi, Peter; Bruckner, Brian A; Sharma, Saumya; Youker, Keith A; Frazier, O H; Taegtmeyer, Heinrich

2003-01-01

Left ventricular assist device (LVAD) support of the failing human heart improves myocyte function and increases cell survival. One potential mechanism underlying this phenomenon is activation of the protein kinase B (PKB)/Akt/glycogen synthase kinase-3beta (GSK-3beta) survival pathway. Left ventricular tissue was obtained both at the time of implantation and explantation of the LVAD (n = 11). Six patients were diagnosed with idiopathic dilated cardiomyopathy, 4 patients with ischemic cardiomyopathy and 1 patient with peripartum cardiomyopathy. The mean duration of LVAD support was 205 +/- 35 days. Myocyte diameter and phosphorylation of ERK were used as indices for reverse remodeling. Transcript levels of genes required for the activation of PKB/Akt (insulin-like growth factor-1, insulin receptor substrate-1) were measured by quantitative RT-PCR. In addition, we measured the relative activity of PKB/Akt and GSK-3beta, and assayed for molecular and histological indices of PKB/Akt activation (cyclooxygenase mRNA levels and glycogen levels). Myocyte diameter and phosphorylation of ERK decreased with LVAD support. In contrast, none of the components of the PKB/Akt/GSK-3beta pathway changed significantly with mechanical unloading. The PKB/Akt/GSK-3beta pathway is not activated during LVAD support. Other signaling pathways must be responsible for the improvement of cellular function and cell survival during LVAD support. Copyright 2003 S. Karger AG, Basel

A cytotoxic and apoptosis-inducing sesquiterpenoid isolated from the aerial parts of Artemisia princeps PAMPANINI (Sajabalssuk).

PubMed

Bang, Myun-Ho; Han, Min-Woo; Song, Myoung-Chong; Cho, Jin-Gyeong; Chung, Hae-Gon; Jeong, Tae-Sook; Lee, Kyung-Tae; Choi, Myung-Sook; Kim, Se-Young; Baek, Nam-In

2008-08-01

Repeated silica gel and octadecyl silica gel (ODS) column chromatography of the aerial parts of Artemisia princeps PAMPANINI (Sajabalssuk) led to the isolation of a new sesquiterpenoid, 3-((S)-2-methylbutyryloxy)-costu-1(10),4(5)-dien-12,6 alpha-olide (2), along with two previously reported sesquiterpenoids: 8 alpha-angeloyloxy-3beta,4 beta-epoxy-6 beta H,7 alpha H,8 beta H-guaia-1(10),11(13)-dien-12,6 alpha-olide (1, carlaolide B) and 3beta,4 beta-epoxy-8 alpha-isobutyryloxy-6 beta H,7 alpha H,8 beta H-guaia-1(10),11(13)-dien-12,6 alpha-olide (3, carlaolide A). The structure of compound 2 was elucidated by spectroscopic data analysis, including one dimensional (1D) and two dimensional (2D) nuclear magnetic resonance (NMR) experiments. Of the isolates, compound 2 exhibited potent cytotoxicity against human cervix adenocarcinoma cells and induced apoptosis.

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

Regulation of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase expression and activity in the hypophysectomized rat ovary: Interactions between the stimulatory effect of human chorionic gonadotropin and the luteolytic effect of prolactin

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

Martel, C.; Labrie, C.; Dupont, E.

1990-12-01

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) catalyzes an obligatory step in the conversion of pregnenolone and other 5-ene-3 beta-hydroxysteroids into progesterone as well as precursors of all androgens and estrogens in the ovary. Since 3 beta-HSD is likely to be an important target for regulation by pituitary hormones, we have studied the effect of chronic treatment with LH (hCG), FSH, and PRL on ovarian 3 beta-HSD expression and activity in hypophysectomized adult female rats. Human CG (hCG) (10 IU, twice a day (bid)), ovine FSH (0.5 microgram, bid), and ovine PRL (1 mg, bid) were administered,more » singly or in combination, for a period of 10 days starting 15 days after hypophysectomy. In hypophysectomized rats, PRL exerted a potent inhibitory effect on all the parameters studied. In fact, PRL caused a 81% decrease in ovarian 3 beta-HSD mRNA content accompanied by a similar decrease in 3 beta-HSD activity and protein levels. In addition, ovarian weight decreased by 40% whereas serum progesterone fell dramatically from 1.92 nmol/liter to undetectable levels after treatment with PRL. Whereas hCG alone had only slight stimulatory effects on 3 beta-HSD mRNA, protein content and activity levels, treatment with the gonadotropin partially or completely reversed the potent inhibitory effects of oPRL on all the parameters measured. FSH, on the other hand, had no significant effect on 3 beta-HSD expression and activity. In situ hybridization experiments using the 35S-labeled rat ovary 3 beta-HSD cDNA probe show that the inhibitory effect of PRL is exerted primarily on luteal cell 3 beta-HSD expression and activity. On the other hand, it can be seen that hCG stimulates 3 beta-HSD mRNA accumulation in interstitial cells.« less

Regional localization of the human integrin {beta}{sub 6} gene (ITGB6) to chromosome 2q24-q31

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

Fernandez-Ruiz, E.; Sanchez-Madrid, F.

The heterodimer {alpha}{sub v}{beta}{sub 6} acts as a fibronectin receptor for human carcinoma cells. The authors report here the regional localization of the {beta}{sub 6} gene to 2q24-q31 by fluorescence in situ hybridization coupled with GTG-banding. This gene is located close to the region to which genes coding for the {alpha} subunits of the integrins VLA-4 and vitronectin receptor (ITGA4 and ITGAV, respectively) have been previously mapped (2q31-q32). These data suggest a proximal position of the integrin {beta}{sub 6} locus (ITGB6) on this integrin gene cluster. Futhermore, double-labeling in situ hybridization experiments performed with {alpha}{sub 4} and {alpha}{sub v} probesmore » indicated a telomeric position of ITGAV with respect to ITGA4. 22 refs., 2 figs.« less

Calcium Co-regulates Oxidative Metabolism and ATP Synthase-dependent Respiration in Pancreatic Beta Cells

PubMed Central

De Marchi, Umberto; Thevenet, Jonathan; Hermant, Aurelie; Dioum, Elhadji; Wiederkehr, Andreas

2014-01-01

Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)+ ratio. PMID:24554722

Granulocyte-macrophage and macrophage colony-stimulating factors differentially regulate alpha v integrin expression on cultured human macrophages.

PubMed

De Nichilo, M O; Burns, G F

1993-03-15

The colony-stimulating factors (CSFs) greatly influence mature macrophage function in vitro: macrophage (M)-CSF induces maturation of monocytes and enhances differentiated cell function; granulocyte-macrophage (GM)-CSF stimulates a variety of antimicrobial functions. In vivo M-CSF is thought to promote differentiation, and GM-CSF is thought to potentiate the inflammatory response. One mechanism by which these differential effects may be achieved is through the receptor-mediated interaction of macrophages with their extracellular matrix. Here we show that M-CSF induces specifically the expression of the alpha v beta 5 integrin receptor, whereas GM-CSF rapidly induces mRNA and surface expression of the alpha v beta 3 integrin. The M-CSF-treated cells acquire a flattened epitheloid phenotype, and on vitronectin the alpha v beta 5 is located in adhesion plaques. These cells do not bind collagen or laminin. In contrast, cells treated with GM-CSF adopt an elongated phenotype on a number of substrates, including collagen and laminin, and express alpha v beta 3 at the leading edge of cells on vitronectin. These results suggest that a primary means by which the CSFs exert their individual effects on mature cells may be through regulating integrin expression.

Repression of TGF-beta signaling by the oncogenic protein SKI in human melanomas: consequences for proliferation, survival, and metastasis.

PubMed

Medrano, Estela E

2003-05-19

Transforming growth factor-beta (TGF-beta ) has dual and paradoxical functions as a tumor suppressor and promoter of tumor progression and metastasis. TGF-Ji-mediated growth inhibition is gradually lost during melanoma tumor progression, but there are no measurable defects at the receptor level. Furthermore, melanoma cells release high levels of TGF-beta to the microenvironment, which upon activation induces matrix deposition, angiogenesis, survival, and transition to more aggressive phenotypes. The SKI and SnoN protein family associate with and repress the activity of Smad2, Smad3, and Smad4, three members of the TGF-fl signaling pathway. SKI also facilitates cell-cycle progression by targeting the RB pathway by at least two ways: it directly associates with RB and represses its activity when expressed at high levels, and indirectly, it represses Smad-mediated induction of p21(Waf-1) This results in increased CDK2 activity, RB phosphorylation,and inactivation. Therefore, high levels of SKI result in lesions to the RB pathway in a manner similar to p16 (INK4a) loss. SKI mRNA and protein levels dramatically increase during human melanoma tumor progression. In addition,the SKI protein shifts from nuclear localization in intraepidermal melanoma cells to nuclear and cytoplasmic in invasive and metastatic melanomas. Here, I discuss the basis for repression of intracellular TGF-beta signaling by SKI, some additional activities of this protein, and propose that by disrupting multiple tumor suppressor pathways, SKI functions as a melanoma oncogene.

Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells.

PubMed Central

Marui, N; Offermann, M K; Swerlick, R; Kunsch, C; Rosen, C A; Ahmad, M; Alexander, R W; Medford, R M

1993-01-01

Oxidative stress and expression of the vascular cell adhesion molecule-1 (VCAM-1) on vascular endothelial cells are early features in the pathogenesis of atherosclerosis and other inflammatory diseases. Regulation of VCAM-1 gene expression may be coupled to oxidative stress through specific reduction-oxidation (redox) sensitive transcriptional or posttranscriptional regulatory factors. In cultured human umbilical vein endothelial (HUVE) cells, the cytokine interleukin 1 beta (IL-1 beta) activated VCAM-1 gene expression through a mechanism that was repressed approximately 90% by the antioxidants pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC). Furthermore, PDTC selectively inhibited the induction of VCAM-1, but not intercellular adhesion molecule-1 (ICAM-1), mRNA and protein accumulation by the cytokine tumor necrosis factor-alpha (TNF alpha) as well as the noncytokines bacterial endotoxin lipopolysaccharide (LPS) and double-stranded RNA, poly(I:C) (PIC). PDTC also markedly attenuated TNF alpha induction of VCAM-1-mediated cellular adhesion. In a distinct pattern, PDTC partially inhibited E-selectin gene expression in response to TNF alpha but not to LPS, IL-1 beta, or PIC. TNF alpha and LPS-mediated transcriptional activation of the human VCAM-1 promoter through NF-kappa B-like DNA enhancer elements and associated NF-kappa B-like DNA binding proteins was inhibited by PDTC. These studies suggest a molecular linkage between an antioxidant sensitive transcriptional regulatory mechanism and VCAM-1 gene expression that expands on the notion of oxidative stress as an important regulatory signal in the pathogenesis of atherosclerosis. Images PMID:7691889

Benefit evaluation of space processing of biological materials

NASA Technical Reports Server (NTRS)

1977-01-01

A rational analytical basis for the evaluation of potential benefits of space processing of biological materials is described. A preliminary evaluation of three candidate space processed biological materials was accomplished. Materials investigated were human lymphocytes, urokinase, and Beta cells. Separation of lymphocyte groups was considered in order to improve the matching of donors and recipients for kidney transplantation, while urokinase was examined in regard to treatment of thromboembolic diseases. Separation of Beta cells was studied since it could provide a highly effective means for the treatment of juvenile-onset diabetes.

Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells

NASA Technical Reports Server (NTRS)

Hatton, Jason P.; Gaubert, Francois; Cazenave, Jean-Pierre; Schmitt, Didier; Hashemi, B. B. (Principal Investigator); Hughes-Fulford, M. (Principal Investigator)

2002-01-01

Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.

Expression of L1-CAM and ADAM10 in human colon cancer cells induces metastasis.

PubMed

Gavert, Nancy; Sheffer, Michal; Raveh, Shani; Spaderna, Simone; Shtutman, Michael; Brabletz, Thomas; Barany, Francis; Paty, Phillip; Notterman, Daniel; Domany, Eytan; Ben-Ze'ev, Avri

2007-08-15

L1-CAM, a neuronal cell adhesion receptor, is also expressed in a variety of cancer cells. Recent studies identified L1-CAM as a target gene of beta-catenin-T-cell factor (TCF) signaling expressed at the invasive front of human colon cancer tissue. We found that L1-CAM expression in colon cancer cells lacking L1-CAM confers metastatic capacity, and mice injected in their spleen with such cells form liver metastases. We identified ADAM10, a metalloproteinase that cleaves the L1-CAM extracellular domain, as a novel target gene of beta-catenin-TCF signaling. ADAM10 overexpression in colon cancer cells displaying endogenous L1-CAM enhanced L1-CAM cleavage and induced liver metastasis, and ADAM10 also enhanced metastasis in colon cancer cells stably transfected with L1-CAM. DNA microarray analysis of genes induced by L1-CAM in colon cancer cells identified a cluster of genes also elevated in a large set of human colon carcinoma tissue samples. Expression of these genes in normal colon epithelium was low. These results indicate that there is a gene program induced by L1-CAM in colon cancer cells that is also present in colorectal cancer tissue and suggest that L1-CAM can serve as target for colon cancer therapy.

6-Shogaol, an active constituent of dietary ginger, induces autophagy by inhibiting the AKT/mTOR pathway in human non-small cell lung cancer A549 cells.

PubMed

Hung, Jen-Yu; Hsu, Ya-Ling; Li, Chien-Te; Ko, Ying-Chin; Ni, Wen-Chiu; Huang, Ming-Shyan; Kuo, Po-Lin

2009-10-28

This study is the first study to investigate the anticancer effect of 6-shogaol in human non-small cell lung cancer A549 cells. 6-Shogaol inhibited cell proliferation by inducing autophagic cell death, but not, predominantly, apoptosis. Pretreatment of cells with 3-methyladenine (3-MA), an autophagy inhibitor, suppressed 6-shogaol mediated antiproliferation activity, suggesting that induction of autophagy by 6-shogaol is conducive to cell death. We also found that 6-shogaol inhibited survival signaling through the AKT/mTOR signaling pathway by blocking the activation of AKT and downstream targets, including the mammalian target of rapamycin (mTOR), forkhead transcription factors (FKHR) and glycogen synthase kinase-3beta (GSK-3beta). Phosphorylation of both of mTOR's downstream targets, p70 ribosomal protein S6 kinase (p70S6 kinase) and 4E-BP1, was also diminished. Overexpression of AKT by AKT cDNA transfection decreased 6-shogaol mediated autophagic cell death, supporting inhibition of AKT beneficial to autophagy. Moreover, reduction of AKT expression by siRNA potentiated 6-shogaol's effect, also supporting inhibition of AKT beneficial to autophagy. Taken together, these findings suggest that 6-shogaol may be a promising chemopreventive agent against human non-small cell lung cancer.

New discoveries on the biology and detection of human chorionic gonadotropin

PubMed Central

Cole, Laurence A

2009-01-01

Human chorionic gonadotropin (hCG) is a glycoprotein hormone comprising 2 subunits, alpha and beta joined non covalently. While similar in structure to luteinizing hormone (LH), hCG exists in multiple hormonal and non-endocrine agents, rather than as a single molecule like LH and the other glycoprotein hormones. These are regular hCG, hyperglycosylated hCG and the free beta-subunit of hyperglycosylated hCG. For 88 years regular hCG has been known as a promoter of corpus luteal progesterone production, even though this function only explains 3 weeks of a full gestations production of regular hCG. Research in recent years has explained the full gestational production by demonstration of critical functions in trophoblast differentiation and in fetal nutrition through myometrial spiral artery angiogenesis. While regular hCG is made by fused villous syncytiotrophoblast cells, extravillous invasive cytotrophoblast cells make the variant hyperglycosylated hCG. This variant is an autocrine factor, acting on extravillous invasive cytotrophoblast cells to initiate and control invasion as occurs at implantation of pregnancy and the establishment of hemochorial placentation, and malignancy as occurs in invasive hydatidiform mole and choriocarcinoma. Hyperglycosylated hCG inhibits apoptosis in extravillous invasive cytotrophoblast cells promoting cell invasion, growth and malignancy. Other non-trophoblastic malignancies retro-differentiate and produce a hyperglycosylated free beta-subunit of hCG (hCG free beta). This has been shown to be an autocrine factor antagonizing apoptosis furthering cancer cell growth and malignancy. New applications have been demonstrated for total hCG measurements and detection of the 3 hCG variants in pregnancy detection, monitoring pregnancy outcome, determining risk for Down syndrome fetus, predicting preeclampsia, detecting pituitary hCG, detecting and managing gestational trophoblastic diseases, diagnosing quiescent gestational trophoblastic disease, diagnosing placental site trophoblastic tumor, managing testicular germ cell malignancies, and monitoring other human malignancies. There are very few molecules with such wide and varying functions as regular hCG and its variants, and very few tests with such a wide spectrum of clinical applications as total hCG. PMID:19171054

In vitro cell injury by oxidized low density lipoprotein involves lipid hydroperoxide-induced formation of alkoxyl, lipid, and peroxyl radicals.

PubMed Central

Coffey, M D; Cole, R A; Colles, S M; Chisolm, G M

1995-01-01

Mounting evidence supports current theories linking lipoprotein oxidation to atherosclerosis. We sought the cellular biochemical mechanism by which oxidized LDL inflicts cell injury. Inhibitors of candidate pathways of cell death were used to treat human fibroblast target cells exposed to oxidized LDL.. Ebselen, which degrades lipid hydroperoxides, inhibited oxidized LDL toxicity, consistent with our recent report that 7 beta-hydroperoxycholesterol (7 beta-OOH chol) is the major cytotoxin of oxidized LDL. Intracellular chelation of metal ions inhibited, while preloading cells with iron enhanced, toxicity, Inhibition of oxidized LDL and 7 beta-OOH chol toxicity by 2-keto-4-thiolmethyl butyric acid, a putative alkoxyl radical scavenger and by vitamin E, probucol and diphenylphenylenediamine, putative scavengers of peroxyl radicals was consistent with the involvement of these radicals in the lethal sequence. Cell death was thus postulated to occur due to lipid peroxidation via a sequence involving lipid hydroperoxide-induced, iron-mediated formation of alkoxyl, lipid, and peroxyl radicals. Pathways involving other reactive oxygen species, new protein synthesis, or altered cholesterol metabolism were considered less likely, since putative inhibitors failed to lessen toxicity. Understanding the mechanism of cell injury by oxidized LDL and its toxic moiety, 7 beta-OOH chol, may indicate specific interventions in the cell injury believed to accompany vascular lesion development. PMID:7560078

Cell cycle and adhesion defects in mice carrying a targeted deletion of the integrin beta4 cytoplasmic domain.

PubMed Central

Murgia, C; Blaikie, P; Kim, N; Dans, M; Petrie, H T; Giancotti, F G

1998-01-01

The cytoplasmic domain of the integrin beta4 subunit mediates both association with the hemidesmosomal cytoskeleton and recruitment of the signaling adaptor protein Shc. To examine the significance of these interactions during development, we have generated mice carrying a targeted deletion of the beta4 cytoplasmic domain. Analysis of homozygous mutant mice indicates that the tail-less alpha6beta4 binds efficiently to laminin 5, but is unable to integrate with the cytoskeleton. Accordingly, these mice display extensive epidermal detachment at birth and die immmediately thereafter from a syndrome resembling the human disease junctional epidermolysis bullosa with pyloric atresia (PA-JEB). In addition, we find a significant proliferative defect. Specifically, the number of precursor cells in the intestinal epithelium, which remains adherent to the basement membrane, and in intact areas of the skin is reduced, and post-mitotic enterocytes display increased levels of the cyclin-dependent kinase inhibitor p27(Kip). These findings indicate that the interactions mediated by the beta4 tail are crucial for stable adhesion of stratified epithelia to the basement membrane and for proper cell-cycle control in the proliferative compartments of both stratified and simple epithelia. PMID:9670011

RACK1 binds to Smad3 to modulate transforming growth factor-beta1-stimulated alpha2(I) collagen transcription in renal tubular epithelial cells.

PubMed

Okano, Kazuhiro; Schnaper, H William; Bomsztyk, Karol; Hayashida, Tomoko

2006-09-08

Although it is clear that transforming growth factor-beta1 (TGF-beta1) is critical for renal fibrogenesis, the complexity of the involved mechanisms is increasingly apparent. TGF-beta1 stimulates phosphorylation of Smad2/3 and activates other signaling molecules as well. The molecular link between these other kinases and Smads is not known. We sought new binding partners for Smad3 in renal cells and identified receptor for activated protein kinase C 1 (RACK1) as a novel binding partner of Smad3. The linker region of Smad3 and the tryptophan-aspartic acid repeat 6 and 7 of RACK1 are sufficient for the association. RACK1 also interacts with Smad3 in the human kidney epithelial cell line, HKC. Silencing RACK1 increases transcriptional activity of TGF-beta1-responsive promoter sequences of the Smad binding element (SBE), p3TP-Lux, and alpha2(I) collagen. Conversely, overexpressed RACK1 negatively modulates alpha2(I) collagen transcriptional activity in TGF-beta1-stimulated cells. RACK1 did not affect phosphorylation of Smad3 at the C terminus or in the linker region. However, RACK1 reduced direct binding of Smad3 to the SBE motif. Mutating a RACK1 tyrosine at residue 246, but not at 228, decreased the inhibitory effect of RACK1 on both alpha2(I) collagen promoter activity and Smad binding to SBE induced by TGF-beta1. These results suggest that RACK1 modulates transcription of alpha2(I) collagen by TGF-beta1 through interference with Smad3 binding to the gene promoter.

Beta 1D integrin displaces the beta 1A isoform in striated muscles: localization at junctional structures and signaling potential in nonmuscle cells.

PubMed

Belkin, A M; Zhidkova, N I; Balzac, F; Altruda, F; Tomatis, D; Maier, A; Tarone, G; Koteliansky, V E; Burridge, K

1996-01-01

The cytoplasmic domains of integrins provide attachment of these extracellular matrix receptors to the cytoskeleton and play a critical role in integrin-mediated signal transduction. In this report we describe the identification, expression, localization, and initial functional characterization of a novel form of beta 1 integrin, termed beta 1D. This isoform contains a unique alternatively spliced cytoplasmic domain of 50 amino acids, with the last 24 amino acids encoded by an additional exon. Of these 24 amino acids, 11 are conserved when compared to the beta 1A isoform, but 13 are unique (Zhidkova, N. I., A. M. Belkin, and R. Mayne. 1995. Biochem. Biophys. Res. Commun. 214:279-285; van der Flier, A., I. Kuikman, C. Baudoin, R, van der Neuf, and A. Sonnenberg. 1995. FEBS Lett. 369:340-344). Using an anti-peptide antibody against the beta 1D integrin subunit, we demonstrated that the beta 1D isoform is synthesized only in skeletal and cardiac muscles, while very low amounts of beta 1A were detected by immunoblot in striated muscles. Whereas beta 1A could not be detected in adult skeletal muscle fibers and cardiomyocytes by immunofluorescence, beta 1D was localized to the sarcolemma of both cell types. In skeletal muscle, beta 1D was concentrated in costameres, myotendinous, and neuromuscular junctions. In cardiac muscle this beta 1 isoform was found in costamers and intercalated discs. beta 1D was associated with alpha 7A and alpha 7B in adult skeletal muscle. In cardiomyocytes of adult heart, alpha 7B was the major partner for the beta 1D isoform. beta 1D could not be detected in proliferating C2C12 myoblasts, but it appeared immediately after myoblast fusion and its amount continued to rise during myotube growth and maturation. In contrast, expression of the beta 1A isoform was downregulated during myodifferentiation in culture and it was completely displaced by beta 1D in mature differentiated myotubes. We also analyzed some functional properties of the beta 1D integrin subunit. Expression of human beta 1D in CHO cells led to its localization at focal adhesions. Clustering of this integrin isoform on the cell surface stimulated tyrosine phosphorylation of pp125FAK (focal adhesion kinase) and caused transient activation of mitogen-activated protein (MAP) kinases. These data indicate that beta 1D and beta 1A integrin isoforms are functionally similar with regard to integrin-mediated signaling.

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 protein.

Expression of 11beta-hydroxysteroid-dehydrogenase type 2 in human thymus.

PubMed

Almanzar, Giovanni; Mayerl, Christina; Seitz, Jan-Christoph; Höfner, Kerstin; Brunner, Andrea; Wild, Vanessa; Jahn, Daniel; Geier, Andreas; Fassnacht, Martin; Prelog, Martina

2016-06-01

11beta-hydroxysteroid-dehydrogenase type 2 (11β-HSD2) is a high affinity dehydrogenase which rapidly inactivates physiologically-active glucocorticoids to protect key tissues. 11β-HSD2 expression has been described in peripheral cells of the innate and the adaptive immune system as well as in murine thymus. In absence of knowledge of 11β-HSD2 expression in human thymus, the study aimed to localize 11β-HSD2 in human thymic tissue. Thymic tissue was taken of six healthy, non-immunologically impaired male infants below 12months of age with congenital heart defects who had to undergo correction surgery. 11β-HSD2 protein expression was analyzed by immunohistochemistry and Western blot. Kidney tissue, peripheral blood mononuclear cells (PBMCs) and human umbilical vein endothelial cells (HUVEC) were taken as positive controls. Significant expression of 11β-HSD2 protein was found at single cell level in thymus parenchyma, at perivascular sites of capillaries and small vessels penetrating the thymus lobuli and within Hassall's bodies. The present study demonstrates that 11β-HSD2 is expressed in human thymus with predominant perivascular expression and also within Hassall's bodies. To our knowledge, this is the first report confirming 11β-HSD2 expression at the protein level in human thymic tissue underlining a potential role of this enzyme in regulating glucocorticoid function at the thymic level. Copyright © 2016 Elsevier Inc. All rights reserved.

Evaluating the potential of poly(beta-amino ester) nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells.

PubMed

Bhise, Nupura S; Wahlin, Karl J; Zack, Donald J; Green, Jordan J

2013-01-01

Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts. A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling. 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents, including Lipofectamine® 2000, FuGENE® HD, and 25 kDa branched polyethylenimine, for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa, and enabled coexpression of exogenously delivered genes, as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation, but not by poly(beta-amino ester) reprogramming, could be differentiated toward the neuronal lineage, specifically pseudostratified optic cups. This study shows that certain nonviral reprogramming methods may not necessarily be safer than viral approaches and that maximizing exogenous gene expression of reprogramming factors is not sufficient to ensure successful reprogramming.

Effect of beta-agonists on LAM progression and treatment.

PubMed

Le, Kang; Steagall, Wendy K; Stylianou, Mario; Pacheco-Rodriguez, Gustavo; Darling, Thomas N; Vaughan, Martha; Moss, Joel

2018-01-30

Lymphangioleiomyomatosis (LAM), a rare disease of women, is associated with cystic lung destruction resulting from the proliferation of abnormal smooth muscle-like LAM cells with mutations in the tuberous sclerosis complex (TSC) genes TSC1 and/or TSC2 The mutant genes and encoded proteins are responsible for activation of the mechanistic target of rapamycin (mTOR), which is inhibited by sirolimus (rapamycin), a drug used to treat LAM. Patients who have LAM may also be treated with bronchodilators for asthma-like symptoms due to LAM. We observed stabilization of forced expiratory volume in 1 s over time in patients receiving sirolimus and long-acting beta-agonists with short-acting rescue inhalers compared with patients receiving only sirolimus. Because beta-agonists increase cAMP and PKA activity, we investigated effects of PKA activation on the mTOR pathway. Human skin TSC2 +/- fibroblasts or LAM lung cells incubated short-term with isoproterenol (beta-agonist) showed a sirolimus-independent increase in phosphorylation of S6, a downstream effector of the mTOR pathway, and increased cell growth. Cells incubated long-term with isoproterenol, which may lead to beta-adrenergic receptor desensitization, did not show increased S6 phosphorylation. Inhibition of PKA blocked the isoproterenol effect on S6 phosphorylation. Thus, activation of PKA by beta-agonists increased phospho-S6 independent of mTOR, an effect abrogated by beta-agonist-driven receptor desensitization. In agreement, retrospective clinical data from patients with LAM suggested that a combination of bronchodilators in conjunction with sirolimus may be preferable to sirolimus alone for stabilization of pulmonary function.

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

Shen, Longxiang; Glowacki, Julie; Zhou, Shuanhu, E-mail: szhou@rics.bwh.harvard.edu

The WNT signaling pathway plays important roles in the self-renewal and differentiation of mesenchymal stem cells (MSCs). Little is known about WNT signaling in adipocyte differentiation of human MSCs. In this study, we tested the hypothesis that canonical and non-canonical WNTs differentially regulate in vitro adipocytogenesis in human MSCs. The expression of adipocyte gene PPAR{gamma}2, lipoprotein lipase, and adipsin increased during adipocytogenesis of hMSCs. Simultaneously, the expression of canonical WNT2, 10B, 13, and 14 decreased, whereas non-canonical WNT4 and 11 increased, and WNT5A was unchanged. A small molecule WNT mimetic, SB-216763, increased accumulation of {beta}-catenin protein, inhibited induction of WNT4more » and 11 and inhibited adipocytogenesis. In contrast, knockdown of {beta}-catenin with siRNA resulted in spontaneous adipocytogenesis. These findings support the view that canonical WNT signaling inhibits and non-canonical WNT signaling promotes adipocytogenesis in adult human marrow-derived mesenchymal stem cells.« less

Loss of tumorigenic potential by human lung tumor cells in the presence of antisense RNA specific to the ectopically synthesized alpha subunit of human chorionic gonadotropin.

PubMed

Rivera, R T; Pasion, S G; Wong, D T; Fei, Y B; Biswas, D K

1989-06-01

A clonal strain of human lung tumor cells in culture (ChaGo), derived from a bronchogenic carcinoma, synthesizes and secretes large amounts of alpha (alpha) and a comparatively lower level of beta (beta) subunit of the glycoprotein hormone, human chorionic gonadotropin (HCG). ChaGo cells lost their characteristic anchorage-independent growth phenotype in the presence of anti-alpha-HCG antibody. The effect of the antibody was partially reversed by addition of alpha-HCG to the culture medium. ChaGo cells were transfected with an expression vector (pRSV-anti-alpha-HCG), that directs synthesis of RNA complementary to alpha-HCG mRNA. The transfectants produced alpha-HCG antisense RNA which was associated with the reduced level of alpha-HCG. Transfectants also displayed several altered phenotypic properties, including altered morphology, less mitosis, reduced growth rate, loss of anchorage-independent growth, and loss of tumorigenicity in nude mice. Treatment of transfectants with 8,bromo-cAMP resulted in increased accumulation of alpha-HCG mRNA, no change in the level of alpha-HCG antisense RNA, release of the inhibition of [3H]thymidine incorporation, and restoration of anchorage-independent growth phenotype. The overexpression of c-myc, observed in ChaGo cells, was unaffected by the reduced level of alpha-HCG. These results suggest that ectopic synthesis of the alpha subunit of HCG plays a functional role in the transformation of these human lung cells.

Recombinant follicle-stimulating hormone: new biotechnology for infertility.

PubMed

Prevost, R R

1998-01-01

The frequency of infertility in developed countries is approximately 8-10%. New drugs are available for assisted reproduction techniques. Two recombinant follicle-stimulating hormone (FSH) products, follitropin-beta (Follistim in the United States, Puregon in Europe) and follitropin-alpha (Gonal-F), join compounds derived through transfecting nonhuman cell lines with genetic material capable of replicating identical amino acid sequences to human compounds. The cell line used for recombinant (r)-FSH production is the Chinese hamster ovary (CHO). Previously, the only agents that showed benefit in controlled ovulatory stimulation were derived from the urine of menopausal women. Those compounds contain additional substances, such as urinary proteins and various amounts of luteininzing hormone. The amino acid sequence of r-FSH is identical to that of human FSH, but the two recombinant products exist in many different isoforms and differ from each other and from human FSH due to varied carbohydrate side chains. Due to variation in the carbohydrate side chains, follitropin-beta in solution has a higher pH than urine-derived FSH, which enhances receptor affinity and therefore is a greater inducer of folliculogenesis. Follitropin-beta does not cause endogenous production of anti-CHO or anti-FSH antibodies, and is well tolerated.

Cell-type specific effects of endocytosis inhibitors on 5-hydroxytryptamine(2A) receptor desensitization and resensitization reveal an arrestin-, GRK2-, and GRK5-independent mode of regulation in human embryonic kidney 293 cells.

PubMed

Gray, J A; Sheffler, D J; Bhatnagar, A; Woods, J A; Hufeisen, S J; Benovic, J L; Roth, B L

2001-11-01

The effect of endocytosis inhibitors on 5-hydroxytryptamine(2A) (5-HT(2A)) receptor desensitization and resensitization was examined in transiently transfected human embryonic kidney (HEK) 293 cells and in C6 glioma cells that endogenously express 5-HT(2A) receptors. In HEK-293 cells, 5-HT(2A) receptor desensitization was unaffected by cotransfection with a dominant-negative mutant of dynamin (DynK44A), a truncation mutant of arrestin-2 [Arr2(319-418)], or by two well-characterized chemical inhibitors of endocytosis: concanavalin A (conA) and phenylarsine oxide (PAO). In contrast, beta 2-adrenergic receptor desensitization was significantly potentiated by each of these treatments in HEK-293 cells. In C6 glioma cells, however, DynK44A, Arr2(319-418), conA, and PAO each resulted in the potentiation of 5-HT(2A) and beta-adrenergic receptor desensitization. The cell-type-specific effect of Arr2(319-418) on 5-HT(2A) receptor desensitization was not related to the level of GRK2 or GRK5 expression. Interestingly, although beta 2-adrenergic receptor resensitization was potently blocked by cotransfection with DynK44A, 5-HT(2A) receptor resensitization was enhanced, suggesting the existence of a novel cell-surface mechanism for 5-HT(2A) receptor resensitization in HEK-293 cells. In addition, Arr2(319-418) had no effect on 5-HT(2A) receptor resensitization in HEK-293 cells, although it attenuated the resensitization of the beta 2-adrenergic receptor. However, in C6 glioma cells, both DynK44A and Arr2(319-418) significantly reduced 5-HT(2A) receptor resensitization. Taken together, these results provide the first convincing evidence of cell-type-specific roles for endocytosis inhibitors in regulating GPCR activity. Additionally, these results imply that novel GRK and arrestin-independent mechanisms of 5-HT(2A) receptor desensitization and resensitization exist in HEK-293 cells.

Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation.

PubMed

Russ, Holger A; Landsman, Limor; Moss, Christopher L; Higdon, Roger; Greer, Renee L; Kaihara, Kelly; Salamon, Randy; Kolker, Eugene; Hebrok, Matthias

2016-01-01

Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation.

Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation

PubMed Central

Russ, Holger A.; Landsman, Limor; Moss, Christopher L.; Higdon, Roger; Greer, Renee L.; Kaihara, Kelly; Salamon, Randy; Kolker, Eugene; Hebrok, Matthias

2016-01-01

Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation. PMID:26681951

Targeting of MPEG-protected polyamino acid carrier to human E-selectin in vitro.

PubMed

Kang, H W; Weissleder, R; Bogdanov, A

2002-01-01

Targeted diagnostic agents are expected to have a significant impact in molecular imaging of cell-surface associated markers of proliferation, inflammation and angiogenesis. In this communication, we describe a new class of targeted polyamino acid-based protected graft copolymers (PGC) of poly-(L-lysine) and methyl poly-(ethylene glycol) (PGC) covalently conjugated with a monoclonal antibody fragment, F(ab')(2). We utilized targeted PGC conjugates as carriers of near-infrared indocyanine fluorophores (Cy5.5) for optical imaging of endothelial cell populations expressing IL-1 beta inducible proinflammatory marker E-selectin. We compared two conjugation chemistries, involving either introduction of sulfhydryl group to F(ab')(2), or via direct attachment of the antibody fragment directly to the chemically activated PGC. Both PGC-based targeted agents demonstrated high binding specificity (20-30 fold over non-specific uptake) and were utilized for imaging E-selectin expression on human endothelial cells activated with IL-1 beta.

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

Choi, Yoon Pyo; Kim, Baek Gil; Department of Pathology, Yonsei University College of Medicine, Seoul

Highlights: Black-Right-Pointing-Pointer The potential of targeting ILK and integrins for highly aggressive ovarian cancer. Black-Right-Pointing-Pointer Unanticipated synergistic effect for the combination of ILK/{beta}4 integrin. Black-Right-Pointing-Pointer Combination of ILK/{beta}4 integrin effectively inhibited the PI3K/Akt/Rac1 cascade. Black-Right-Pointing-Pointer Targeting of {beta}4 integrin/ILK had potent inhibitory effects in ovarian cancer. -- Abstract: Integrins and integrin-linked kinase (ILK) are essential to cancerous invasion because they mediate physical interactions with the extracellular matrix, and regulate oncogenic signaling pathways. The purpose of our study is to determine whether deletion of {beta}1 and {beta}4 integrin and ILK, alone or in combination, has antitumoral effects in ovarian cancer. Expressionmore » of {beta}1 and {beta}4 integrin and ILK was analyzed by immunohistochemistry in 196 ovarian cancer tissue samples. We assessed the effects of depleting these molecules with shRNAs in ovarian cancer cells by Western blot, conventional RT-PCR, cell proliferation, migration, invasion, and in vitro Rac1 activity assays, and in vivo xenograft formation assays. Overexpression of {beta}4 integrin and ILK in human ovarian cancer specimens was found to correlate with tumor aggressiveness. Depletion of these targets efficiently suppresses ovarian cancer cell proliferation, migration, and invasion in vitro and xenograft tumor formation in vivo. We also demonstrated that single depletion of ILK or combination depletion of {beta}4 integrin/ILK inhibits phosphorylation of downstream signaling targets, p-Ser 473 Akt and p-Thr202/Tyr204 Erk1/2, and activation of Rac1, as well as reduce expression of MMP-2 and MMP-9 and increase expression of caspase-3 in vitro. In conclusion, targeting {beta}4 integrin combined with ILK can instigate the latent tumorigenic potential and abrogate the invasive potential in ovarian cancer.« less

Insulin sensitivity and beta-cell function in healthy cats: assessment with the use of the hyperglycemic glucose clamp.

PubMed

Slingerland, L I; Robben, J H; van Haeften, T W; Kooistra, H S; Rijnberk, A

2007-05-01

A hyperglycemic clamp (HGC) was developed for use in conscious cats. In 21 healthy, normal glucose tolerant cats glucose disposal rate (M), insulin sensitivity (ISI (HGC)), and beta-cell response (I) at arterial plasma glucose of 9 mmol.l (-1) were measured. The HGC was tolerated well and steady state glucose infusion was achieved. Compared to values reported for humans, M values for the cats were low, which appeared to relate to both a low ISI (HGC) and a low I. HGC measures correlated with fasting plasma glucose and insulin concentrations as well as with their HOMA (homeostasis model assessment) and QUICKI (quantitative insulin sensitivity check index) counterparts. Also, I and ISI (HGC) correlated with their counterparts derived from intravenous glucose tolerance tests. In conclusion, this is the first report of hyperglycemic glucose clamping in cats. The procedure (HGC) allows for measurements of glucose disposal, beta-cell response and insulin sensitivity. Compared to human data, both insulin sensitivity and insulin secretion appeared to be low in cats. This is compatible with the carnivorous nature of this species, for which insulin resistance would be advantageous during periods of restricted food availability.

Ovarian carcinoma ascites spheroids adhere to extracellular matrix components and mesothelial cell monolayers.

PubMed

Burleson, Kathryn M; Casey, Rachael C; Skubitz, Keith M; Pambuccian, Stephan E; Oegema, Theodore R; Skubitz, Amy P N

2004-04-01

Ovarian carcinoma cells form multicellular aggregates, or spheroids, in the peritoneal cavity of patients with advanced disease. The current paradigm that ascites spheroids are non-adhesive leaves their contribution to ovarian carcinoma dissemination undefined. Here, spheroids obtained from ovarian carcinoma patients' ascites were characterized for their ability to adhere to molecules encountered in the peritoneal cavity, with the goal of establishing their potential to contribute to ovarian cancer spread. Spheroids were recovered from the ascites fluid of 11 patients with stage III or stage IV ovarian carcinoma. Adhesion assays to extracellular matrix (ECM) proteins and human mesothelial cell monolayers were performed for each of the ascites spheroid samples. Subsequently, inhibition assays were performed to identify the cell receptors involved. Most ascites samples adhered moderately to fibronectin and type I collagen, with reduced adhesion to type IV collagen and laminin. Monoclonal antibodies against the beta1 integrin subunit partially inhibited this adhesion. Ascites spheroids also adhered to hyaluronan. Additionally, spheroids adhered to live, but not fixed, human mesothelial cell monolayers, and this adhesion was partially mediated by beta1 integrins. The cellular content of the ascites fluid has often been considered non-adhesive, but our findings are the first to suggest that patient-derived ascites spheroids can adhere to mesothelial extracellular matrix via beta1 integrins, indicating that spheroids should not be ignored in the dissemination of ovarian cancer.

The intracoelomic route: a new approach for in utero human cord blood stem cell transplantation.

PubMed

Noia, Giuseppe; Pierelli, Luca; Bonanno, Giuseppina; Monego, Giovanni; Perillo, Alessandro; Rutella, Sergio; Cavaliere, Anna Franca; Straface, Gianluca; Fortunato, Giuseppe; Cesari, Elena; Scambia, Giovanni; Terzano, Marinella; Iannace, Enrico; Zelano, Giovanni; Michetti, Fabrizio; Leone, Giuseppe; Mancuso, Salvatore

2004-01-01

The intracoelomic route for in utero hematopoietic stem cell transplantation has been evaluated in pre-immune fetal sheep and the engraftment characteristics defined. Twelve ovine fetuses (gestational ages: 40-45 days) received intracoelomic transplants of human CD3-depleted (50 x 10(6) per lamb) or CD34-selected (1-2 x 10(5) per lamb) cord blood hematopoietic stem cells. Engraftment was evaluated from cell suspension of the liver, spleen, bone marrow and thymus by flow cytometry, cloning assays and polymerase chain reaction (PCR) analysis for human beta(2)-microglobulin gene. The engraftment of liver samples was also evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescent in situ hybridization (FISH) and immunohistochemistry. Four fetuses (33%) aborted shortly after intracoelomic transplantation and were not evaluable for engraftment. Engraftment was detected in 4 fetuses obtained from cesarean delivery on day 70 after transplantation of CD3-depleted cord blood cells. The degree of engraftment in these 4 fetuses ranged from 6 to 22% in the different organs (as revealed by antigenic analysis of human CD45 with flow cytometry). Three fetuses obtained after cesarean section at 102 (No. 435184) and 105 (Nos 915293, 037568) days and 1 fetus delivered at term, which received CD34-selected cord blood cells, had human engraftment with 10, 32, 20 and 10% CD45+ cells in bone marrow, respectively. A further check of human chimerism was done at 1 year after birth of the fetus delivered at term and 7.6% of bone marrow chimerism was detected. In 6 out of 8 fetuses evaluable for human engraftment, chimerism was confirmed by PCR analysis for human beta(2)-microglobulin which also identified human cells in brain, spinal cord, heart, lung and skeletal muscle. On liver samples, FISH and RT-PCR confirmed the xenograft of human cells and the immunohistochemical analysis detected human markers of hematopoietic and hepatic lineage of differentiation. This preliminary study indicates that intracoelomic transplantation of human hematopoietic stem cells in fetal lambs is feasible and effective in terms of hematopoietic engraftment. Copyright 2004 S. Karger AG, Basel

Effects of transforming growth factor-beta1 on cell motility, collagen gel contraction, myofibroblastic differentiation, and extracellular matrix expression of human adipose-derived stem cell.

PubMed

Kakudo, Natsuko; Kushida, Satoshi; Suzuki, Kenji; Ogura, Tsunetaka; Notodihardjo, Priscilla Valentin; Hara, Tomoya; Kusumoto, Kenji

2012-12-01

Human adipose-derived stem cells (ASCs) are adult pluripotent stem cells, and their usefulness in plastic surgery has garnered attention in recent years. Although, there have been expectations that ASCs might function in wound repair and regeneration, no studies to date have examined the role of ASCs in the mechanism that promotes wound-healing. Transforming growth factor-beta1 (TGF-β1) is a strong candidate cytokine for the triggering of mesenchymal stem cell migration, construction of extracellular matrices, and differentiation of ASCs into myofibroblasts. Cell proliferation, motility, and differentiation, as well as extracellular matrix production, play an important role in wound-healing. We have evaluated the capacity of ASCs to proliferate and their potential to differentiate into phenotypic myofibroblasts, as well as their cell motility and collagen gel contraction ability, when cultured with TGF-β1. Cell motility was analyzed using a wound-healing assay. ASCs that differentiated into myofibroblasts expressed the gene for alpha-smooth muscle actin, and its protein expression was detected immunohistochemically. The extracellular matrix expression in ASCs was evaluated using real-time RT-PCR. Based on the results, we conclude that human ASCs have the potential for cell motility, extracellular matrix gene expression, gel contraction, and differentiation into myofibroblasts and, therefore, may play an important role in the wound-healing process.

Dose responses for adaption to low doses of (60)Co gamma rays and (3)H beta particles in normal human fibroblasts.

PubMed

Broome, E J; Brown, D L; Mitchel, R E J

2002-08-01

The dose response for adaption to radiation at low doses was compared in normal human fibroblasts (AG1522) exposed to either (60)Co gamma rays or (3)H beta particles. Cells were grown in culture to confluence and exposed at either 37 degrees C or 0 degrees C to (3)H beta-particle or (60)Co gamma-ray adapting doses ranging from 0.1 mGy to 500 mGy. These cells, and unexposed control cells, were allowed to adapt during a fixed 3-h, 37 degrees C incubation prior to a 4-Gy challenge dose of (60)Co gamma rays. Adaption was assessed by measuring micronucleus frequency in cytokinesis-blocked, binucleate cells. No adaption was detected in cells exposed to (60)Co gamma radiation at 37 degrees C after a dose of 0.1 mGy given at a low dose rate or to 500 mGy given at a high dose rate. However, low-dose-rate exposure (1-3 mGy/min) to any dose between 1 and 500 mGy from either radiation, delivered at either temperature, caused cells to adapt and reduced the micronucleus frequency that resulted from the subsequent 4-Gy exposure. Within this dose range, the magnitude of the reduction was the same, regardless of the dose or radiation type. These results demonstrate that doses as low as (on average) about one track per cell (1 mGy) produce the same maximum adaptive response as do doses that deposit many tracks per cell, and that the two radiations were not different in this regard. Exposure at a temperature where metabolic processes, including DNA repair, were inactive (0 degrees C) did not alter the result, indicating that the adaptive response is not sensitive to changes in the accumulation of DNA damage within this range. The results also show that the RBE for low doses of tritium beta-particle radiation is 1, using adaption as the end point.

Acceleration of Smad2 and Smad3 phosphorylation via c-Jun NH(2)-terminal kinase during human colorectal carcinogenesis.

PubMed

Yamagata, Hideo; Matsuzaki, Koichi; Mori, Shigeo; Yoshida, Katsunori; Tahashi, Yoshiya; Furukawa, Fukiko; Sekimoto, Go; Watanabe, Toshihiko; Uemura, Yoshiko; Sakaida, Noriko; Yoshioka, Kazuhiko; Kamiyama, Yasuo; Seki, Toshihito; Okazaki, Kazuichi

2005-01-01

Conversion of normal epithelial cells to tumors is associated with a shift in transforming growth factor-beta (TGF-beta) function: reduction of tumor suppressor activity and increase of oncogenic activity. However, specific mechanisms of this functional alteration during human colorectal carcinogenesis remain to be elucidated. TGF-beta signaling involves Smad2/3 phosphorylated at linker regions (pSmad2/3L) and COOH-terminal regions (pSmad2/3C). Using antibodies specific to each phosphorylation site, we herein showed that Smad2 and Smad3 were phosphorylated at COOH-terminal regions but not at linker regions in normal colorectal epithelial cells and that pSmad2/3C were located predominantly in their nuclei. However, the linker regions of Smad2 and Smad3 were phosphorylated in 31 sporadic colorectal adenocarcinomas. In particular, late-stage invasive and metastatic cancers typically showed a high degree of phosphorylation of Smad2/3L. Their extent of phosphorylation in 11 adenomas was intermediate between those in normal epithelial cells and adenocarcinomas. Whereas pSmad2L remained in the cytoplasm, pSmad3L was located exclusively in the nuclei of Ki-67-immunoreactive adenocarcinomas. In contrast, pSmad3C gradually decreased as the tumor stage progressed. Activated c-Jun NH(2)-terminal kinase in cancers could directly phosphorylate Smad2/3L. Although Mad homology 2 region sequencing in the Smad4 gene revealed a G/A substitution at codon 361 in one adenocarcinoma, the mutation did not correlate with phosphorylation. No mutations in the type II TGF-beta receptor and Smad2 genes were observed in the tumors. In conclusion, pSmad3C, which favors tumor suppressor activity of TGF-beta, was found to decrease, whereas c-Jun NH(2)-terminal kinase tended to induce the phosphorylation of Smad2/3L in human colorectal adenoma-carcinoma sequence.

Paradigm Shift in Thyroid Hormone Mechanism of Action | Center for Cancer Research

Cancer.gov

Thyroid hormone (TH) is one of the primary endocrine regulators of human metabolism and homeostasis. Acting through three forms of the thyroid hormone receptor (THR; alpha-1, beta-1, and beta-2), TH regulates target gene expression in nearly every cell in the body, modulating fundamental processes, such as basal metabolic rate, long bone growth, and neural maturation. TH is

Cigarette smoke toxicants as substrates and inhibitors for human cytosolic SULTs

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

Yasuda, Shin; Idell, Steven; Fu Jian

2007-05-15

The current study was designed to examine the role of sulfation in the metabolism of cigarette smoke toxicants and clarify whether these toxicants, by serving as substrates for the cytosolic sulfotransferases (SULTs), may interfere with the sulfation of key endogenous compounds. By metabolic labeling, [{sup 35}S]sulfated species were found to be generated and released into the media of HepG2 human hepatoma cells and primary human lung endothelial cells labeled with [{sup 35}S]sulfate in the presence of cigarette smoke extract (CSE). Concomitantly, several [{sup 35}S]sulfated metabolites observed in the medium in the absence of CSE either decreased or disappeared. Eleven previouslymore » prepared human cytosolic SULTs were tested for sulfating activity with CSE and known cigarette smoke toxicants as substrates. Activity data revealed SULT1A1, SULT1A2, SULT1A3, and SULT1C2 as major enzymes responsible for their sulfation. To examine their inhibitory effects on the sulfation of 17{beta}-estradiol by SULT1A1, enzymatic assays were performed in the presence of three representative toxicant compounds, namely N-hydroxy-4-aminobiphenyl (N-OH-4-ABP), 4-aminobiphenyl (4-ABP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). IC{sub 50} values determined for the sulfation of 17{beta}-estradiol by SULT1A1 were 11.8 {mu}M, 28.2 {mu}M, and 500 {mu}M, respectively, for N-OH-4-ABP, 4-ABP and PhIP. Kinetic analyses indicated that the mechanism underlying the inhibition of 17{beta}-estradiol sulfation by these cigarette smoke toxicants is of a mixed competitive-noncompetitive type. Metabolic labeling experiments clearly showed inhibition of the production of [{sup 35}S]sulfated 17{beta}-estradiol by N-OH-4-ABP in a concentration-dependent manner in HepG2 cells. Taken together, these results suggest that sulfation plays a significant role in the metabolism of cigarette smoke compounds. By serving as substrates for SULTs, cigarette smoke toxicants may interfere with the metabolism of 17{beta}-estradiol and other endogenous compounds.« less

Purification and ATPase activity of human ABCA1.

PubMed

Takahashi, Kei; Kimura, Yasuhisa; Kioka, Noriyuki; Matsuo, Michinori; Ueda, Kazumitsu

2006-04-21

ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein metabolism. Apolipoprotein A-I binds to ABCA1 and cellular cholesterol and phospholipids, mainly phosphatidylcholine, are loaded onto apoA-I to form pre-beta high density lipoprotein (HDL). It is proposed that ABCA1 translocates phospholipids and cholesterol directly or indirectly to form pre-beta HDL. To explore the mechanism of ABCA1-mediated pre-beta HDL formation, we expressed human ABCA1 in insect Sf9 cells and purified it. Trypsin limited-digestion of purified ABCA1 in the detergent-soluble form suggested that it retained conformation similar to ABCA1 expressed in the membranes of human fibroblast WI-38 cells. Purified ABCA1 showed robust ATPase activity when reconstituted in liposomes made of synthetic phosphatidylcholine. ABCA1 showed lower ATPase activity when reconstituted in liposomes containing phosphatidylserine, phosphatidylethanolamine, or phosphatidylglycerol and also showed weak specificity in acyl chain species. ATPase activity was reduced by the addition of cholesterol and decreased by 25% in the presence of 20% cholesterol. Beta-sitosterol and campesterol showed similar inhibitory effects but stigmasterol did not, suggesting structure-specific interaction between ABCA1 and sterols. Glibenclamide suppressed ABCA1 ATPase, suggesting that it inhibits apoA-I-dependent cellular cholesterol efflux by suppressing ABCA1 ATPase activity. These results suggest that the ATPase activity of ABCA1 is stimulated preferentially by phospholipids with choline head groups, phosphatidylcholine and sphingomyelin. This study with purified human ABCA1 provides the first biochemical basis of the mechanism for HDL formation mediated by ABCA1.

BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling.

PubMed

He, Xi C; Zhang, Jiwang; Tong, Wei-Gang; Tawfik, Ossama; Ross, Jason; Scoville, David H; Tian, Qiang; Zeng, Xin; He, Xi; Wiedemann, Leanne M; Mishina, Yuji; Li, Linheng

2004-10-01

In humans, mutations in BMPR1A, SMAD4 and PTEN are responsible for juvenile polyposis syndrome, juvenile intestinal polyposis and Cowden disease, respectively. The development of polyposis is a common feature of these diseases, suggesting that there is an association between BMP and PTEN pathways. The mechanistic link between BMP and PTEN pathways and the related etiology of juvenile polyposis is unresolved. Here we show that conditional inactivation of Bmpr1a in mice disturbs homeostasis of intestinal epithelial regeneration with an expansion of the stem and progenitor cell populations, eventually leading to intestinal polyposis resembling human juvenile polyposis syndrome. We show that BMP signaling suppresses Wnt signaling to ensure a balanced control of stem cell self-renewal. Mechanistically, PTEN, through phosphatidylinosital-3 kinase-Akt, mediates the convergence of the BMP and Wnt pathways on control of beta-catenin. Thus, BMP signaling may control the duplication of intestinal stem cells, thereby preventing crypt fission and the subsequent increase in crypt number.

Aprotinin stimulates angiogenesis and human endothelial cell migration through the growth factor pleiotrophin and its receptor protein tyrosine phosphatase beta/zeta.

PubMed

Koutsioumpa, Marina; Hatziapostolou, Maria; Mikelis, Constantinos; Koolwijk, Pieter; Papadimitriou, Evangelia

2009-01-14

Pleiotrophin is an 18 kDa secreted polypeptide growth factor with direct pro-angiogenic and tumorigenic properties. Pleiotrophin is a substrate for proteolytic enzymes, such as plasmin, leading to proteolytic fragments with distinct activities on endothelial cell activation in vitro or angiogenesis in vivo. Aprotinin is a naturally occurring broad spectrum protease inhibitor, used widely in cardiac surgery due to its ability to inhibit plasmin and reduce perioperative bleeding. Since we have seen that aprotinin inhibits proteolysis of pleiotrophin by plasmin, the aim of the present study was to evaluate the possible role of pleiotrophin in the effects of aprotinin on angiogenesis and human endothelial cell migration. Our data demonstrate that aprotinin, in a concentration-dependent manner, is angiogenic in the chicken embryo chorioallantoic membrane assay in vivo and induces human endothelial cell migration in vitro. Aprotinin inhibits pleiotrophin proteolysis and induces expression and secretion of pleiotrophin through an AP-1-dependent transcriptional activation of the pleiotrophin gene, and pleiotrophin seems to mediate the stimulatory effects of aprotinin on cell migration through its receptor protein tyrosine phosphatase beta/zeta. The stimulatory effect of aprotinin on pleiotrophin expression and cell migration may explain, at least partly, the problems observed with the clinical use of aprotinin.

Targeting innate immunity to downmodulate adaptive immunity and reverse type 1 diabetes

PubMed Central

Itoh, Arata; Ridgway, William M

2017-01-01

Type 1 diabetes (T1D) is characterized by specific destruction of pancreatic insulin-producing beta cells accompanied by evidence of beta-cell-directed autoimmunity such as autoreactive T cells and islet autoantibodies (IAAs). Currently, T1D cannot be prevented or reversed in humans. T1D is easy to prevent in the nonobese diabetic (NOD) spontaneous mouse model but reversing new-onset T1D in mice is more difficult. Since the discovery of the T-cell receptor in the 1980s and the subsequent identification of autoreactive T cells directed toward beta-cell antigens (eg, insulin, glutamic acid decarboxylase), the dream of antigen-specific immunotherapy has dominated the field with its promise of specificity and limited side effects. While such approaches have worked in the NOD mouse, however, dozens of human trials have failed. Broader immunosuppressive approaches (originally cyclosporine, subsequently anti-CD3 antibody) have shown partial successes (e.g., prolonged C peptide preservation) but no major therapeutic efficacy or disease reversal. Human prevention trials have failed, despite the ease of such approaches in the NOD mouse. In the past 50 years, the incidence of T1D has increased dramatically, and one explanation is the “hygiene hypothesis”, which suggests that decreased exposure of the innate immune system to environmental immune stimulants (e.g., bacterial products such as Toll-like receptor (TLR) 4-stimulating lipopolysaccharide [LPS]) dramatically affects the adaptive immune system and increases subsequent autoimmunity. We have tested the role of innate immunity in autoimmune T1D by treating acute-onset T1D in NOD mice with anti-TLR4/MD-2 agonistic antibodies and have shown a high rate of disease reversal. The TLR4 antibodies do not directly stimulate T cells but induce tolerogenic antigen-presenting cells (APCs) that mediate decreased adaptive T-cell responses. Here, we review our current knowledge and suggest future prospects for targeting innate immunity in T1D immunotherapy. PMID:28580341

Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor

PubMed Central

1994-01-01

Interleukin 15 (IL-15) is a novel cytokine that has recently been cloned and expressed. Whereas it has no sequence homology with IL-2, IL- 15 interacts with components of the IL-2 receptor (IL-2R). In the present study we performed a functional analysis of recombinant IL-15 on phenotypically and functionally distinct populations of highly purified human natural killer (NK) cells. The CD56bright subset of human NK cells constitutively expresses the high affinity IL-2R and exhibits a brisk proliferative response after the binding of picomolar amounts of IL-2. Using a proliferation assay, IL-15 demonstrated a very steep dose-response curve that was distinct from the dose-response curve for IL-2. The proliferative effects of IL-15 could be abrogated by anti-IL-2R beta (p75), but not by anti-IL-2R alpha (p55). The proliferative effects of IL-2 on CD56bright NK cells could be inhibited by both antibodies. CD56dim NK cells express the intermediate affinity IL-2R in the absence of the high affinity IL-2R. Activation of CD56dim NK cells by IL-15 was similar to that of IL-2 as measured by enhanced NK cytotoxic activity, antibody-dependent cellular cytotoxicity, and NK cell production of interferon gamma, tumor necrosis factor alpha, and granulocyte/macrophage colony-stimulating factor. The IL-15-enhanced NK cytotoxic activity could be completely blocked by anti-IL-2R beta monoclonal antibody. The binding of radiolabeled IL-2 and IL-15 to CD56dim NK cells was inhibited in the presence of anti-IL-2R beta. Scatchard analysis of radiolabeled IL-15 and IL-2 binding to NK- enriched human lymphocytes revealed the presence of high and intermediate affinity receptors for both ligands. IL-15 is a ligand that activates human NK cells through components of the IL-2R in a pattern that is similar but not identical to that of IL-2. Unlike IL-2, IL-15 is produced by activated monocytes/macrophages. The discovery of IL-15 may increase our understanding of how monocytes/macrophages participate in the regulation of NK cell function. PMID:7523571

Gastroesophageal reflux disease-associated esophagitis induces endogenous cytokine production leading to motor abnormalities.

PubMed

Rieder, Florian; Cheng, Ling; Harnett, Karen M; Chak, Amitabh; Cooper, Gregory S; Isenberg, Gerard; Ray, Monica; Katz, Jeffry A; Catanzaro, Andrew; O'Shea, Robert; Post, Anthony B; Wong, Richard; Sivak, Michael V; McCormick, Thomas; Phillips, Manijeh; West, Gail A; Willis, Joseph E; Biancani, Piero; Fiocchi, Claudio

2007-01-01

Gastroesophageal reflux disease is a condition frequently associated with esophagitis and motor abnormalities. Recent evidence suggests that proinflammatory cytokines, such as interleukin (IL)-1beta and IL-6, may be implicated because they reduce esophageal muscle contractility, but these results derive from in vitro or animal models of esophagitis. This study used human esophageal cells and tissues to identify the cellular source of cytokines in human esophagitis investigate whether cytokines can be induced by gastric refluxate, and examine whether esophageal tissue- or cell-derived mediators affect muscle contractility. Endoscopic mucosal biopsy specimens were obtained from patients with and without esophagitis, organ-cultured, and undernatants were assessed for cytokine content. The cytokine profile of esophageal epithelial, fibroblast, and muscle cells was analyzed, and esophageal mucosa and cell products were tested in an esophageal circular muscle contraction assay. The mucosa of esophagitis patients produced significantly greater amounts of IL-1beta and IL-6 compared with those of control patients. Cultured esophageal epithelial cells produced IL-6, as did fibroblasts and muscle cells. Epithelial cells exposed to buffered, but not denatured, gastric juice produced IL-6. Undernatants of mucosal biopsy cultures from esophagitis patients reduced esophageal muscle contraction, as did supernatants from esophageal epithelial cell cultures. The human esophagus produces cytokines capable of reducing contractility of esophageal muscle cells. Exposure to gastric juice is sufficient to stimulate esophageal epithelial cells to produce IL-6, a cytokine able to alter esophageal contractility. These results indicate that classic cytokines are important mediators of the motor disturbances associated with human esophageal inflammation.

H2O2 accelerates cellular senescence by accumulation of acetylated p53 via decrease in the function of SIRT1 by NAD+ depletion.

PubMed

Furukawa, Ayako; Tada-Oikawa, Saeko; Kawanishi, Shosuke; Oikawa, Shinji

2007-01-01

It has been reported that p53 acetylation, which promotes cellular senescence, can be regulated by the NAD(+)-dependent deacetylase SIRT1, the human homolog of yeast Sir2, a protein that modulates lifespan. To clarify the role of SIRT1 in cellular senescence induced by oxidative stress, we treated normal human diploid fibroblast TIG-3 cells with H(2)O(2) and examined DNA cleavage, depletion of intracellular NAD(+), expression of p21, SIRT1, and acetylated p53, cell cycle arrest, and senescence-associated beta-galactosidase (SA-beta-gal) activity. DNA cleavage was observed immediately in TIG-3 cells treated with H(2)O(2), though no cell death was observed. NAD(+) levels in TIG-3 cells treated with H(2)O(2) were also decreased significantly. Pre-incubation with the poly (ADP-ribose) polymerase (PARP) inhibitor resulted in preservation of intracellular NAD(+) levels. The amount of acetylated p53 was increased in TIG-3 cells at 4h after H(2)O(2) treatment, while there was little to no decrease in SIRT1 protein expression. The expression level of p21 was increased at 12h and continued to increase for up to 24h. Additionally, exposure of TIG-3 cells to H(2)O(2) induced cell cycle arrest at 24h and increased SA-beta-gal activity at 48h. This pathway likely plays an important role in the acceleration of cellular senescence by oxidative stress.

p53-dependent control of cell death by nicastrin: lack of requirement for presenilin-dependent gamma-secretase complex.

PubMed

Pardossi-Piquard, Raphaëlle; Dunys, Julie; Giaime, Emilie; Guillot-Sestier, Marie-Victoire; St George-Hyslop, Peter; Checler, Frédéric; Alves da Costa, Cristine

2009-04-01

Nicastrin (NCT) is a component of the presenilin (PS)-dependent gamma-secretase complexes that liberate amyloid beta-peptides from the beta-Amyloid Precursor Protein. Several lines of evidence indicate that the members of these complexes could also contribute to the control of cell death. Here we show that over-expression of NCT increases the viability of human embryonic kidney (HEK293) cells and decreases staurosporine (STS)- and thapsigargin (TPS)-induced caspase-3 activation in various cell lines from human and neuronal origins by Akt-dependent pathway. NCT lowers p53 expression, transcriptional activity and promoter transactivation and reduces p53 phosphorylation. NCT-associated protection against STS-stimulated cell death was completely abolished by p53 deficiency. Conversely, the depletion of NCT drastically enhances STS-induced caspase-3 activation and p53 pathway and favored p53 nuclear translocation. We examined whether NCT protective function depends on PS-dependent gamma-secretase activity. First, a 29-amino acid deletion known to reduce NCT-dependent amyloid beta-peptide production did not affect NCT-associated protective phenotype. Second, NCT still reduces STS-induced caspase-3 activation in fibroblasts lacking PS1 and PS2. Third, the gamma-secretase inhibitor DFK167 did not affect NCT-mediated reduction of p53 activity. Altogether, our study indicates that NCT controls cell death via phosphoinositide 3-kinase/Akt and p53-dependent pathways and that this function remains independent of the activity and molecular integrity of the gamma-secretase complexes.

Role of adapter function in oncoprotein-mediated activation of NF-kappaB. Human T-cell leukemia virus type I Tax interacts directly with IkappaB kinase gamma.

PubMed

Jin, D Y; Giordano, V; Kibler, K V; Nakano, H; Jeang, K T

1999-06-18

Mechanisms by which the human T-cell leukemia virus type I Tax oncoprotein activates NF-kappaB remain incompletely understood. Although others have described an interaction between Tax and a holo-IkappaB kinase (IKK) complex, the exact details of protein-protein contact are not fully defined. Here we show that Tax binds to neither IKK-alpha nor IKK-beta but instead complexes directly with IKK-gamma, a newly characterized component of the IKK complex. This direct interaction with IKK-gamma correlates with Tax-induced IkappaB-alpha phosphorylation and NF-kappaB activation. Thus, our findings establish IKK-gamma as a key molecule for adapting an oncoprotein-specific signaling to IKK-alpha and IKK-beta.

Effects of extracts of Coriolus versicolor (I'm-Yunity) on cell-cycle progression and expression of interleukins-1 beta,-6, and -8 in promyelocytic HL-60 leukemic cells and mitogenically stimulated and nonstimulated human lymphocytes.

PubMed

Hsieh, Tze-Chen; Kunicki, Jan; Darzynkiewicz, Zbigniew; Wu, Joseph M

2002-10-01

The goal of this in vitro study was to test the cytostatic and cytotoxic activities of extracts derived from the polysaccharopeptide (PSP), I'm-Yunity (Integrated Chinese Medicine Holdings Ltd., Kowloon, Hong Kong) prepared from strain Cov-1 of the mushroom Coriolus versicolor. Different volumes of 70% ethanol and water extracts of I'm-Yunity were incubated with cultures of human promyelocytic leukemic HL-60 cells, and compared to nontreated control cells. At various times after treatment, cells were harvested and analyzed with respect to: (1). proliferation and cell cycle phase distribution, (2). induction of apoptosis, and (3). changes in expression of the immunomodulating cytokines interleukin (IL)-1 beta, IL-6, and IL-8. To test whether extracts also affected normal cells, similar experiments were also performed using isolated peripheral blood lymphocytes from healthy volunteers, with and without stimulation by the mitogen phytohemagglutinin (PHA). The ability of extracts to affect the secretion of IL-1 beta, IL-6, and IL-8 were assessed by enzyme-linked immunosorbent assay. HL-60 cells incubated with various amounts (1, 3, 5, 7.5, and 10 micro l/mL) of the extracts for 1-3 days showed dose-dependent, time-dependent growth suppression and decrease in cell viability. Flow cytometric analysis revealed partial cell arrest in the G(1) phase at less than 5 micro L/mL and induction of apoptosis at 10 micro L/mL or more of ethanol and water extracts, with the latter exhibiting more pronounced inhibition than the former. Experiments performed with lymphocytes demonstrated that extracts of I'm-Yunity alone were without effect; moreover, they also did not affect the lymphocyte response to PHA. Water extract of I'm-Yunity also significantly increased IL-1 beta and IL-6 while substantially lowering IL-8. I'm-Yunity acts selectively in HL-60 leukemic cells, resulting in cell cycle restriction through the G(1)/S checkpoint and the induction of apoptosis.

Sterols isolated from Nuruk (Rhizopus oryzae KSD-815) inhibit the migration of cancer cells.

PubMed

Lee, Dae-Young; Lee, Sang-Jin; Kwak, Ho-Young; Jung, Lakoon; Heo, Jieun; Hong, Sungyoul; Kim, Gye-Won; Baek, Nam-In

2009-11-01

An activity-guided fractionation method was used to isolate anticancer components from Nuruk (Rhizopus oryzae KSD-815:KSD-815). Dried powder of KSD-815 was extracted with 80% methanol and partitioned successively using nhexane, ethyl acetate, n-butanol, and water. The n-hexane and n-butanol fractions showed a strong antimigratory effect on human cancer cells. Both of these fractions were subjected to separation and purification procedures using silica gel, octadecyl silica gel, and Sephadex LH-20 column chromatographies to afford four purified compounds. These were identified as ergosterol peroxide (1), stigmast- 5-en-3beta,7beta-diol (2), ergosta-7,22-dien-3beta,5alpha,6beta,9alpha-tetraol (3), and daucosterol (4), respectively, by spectroscopic methods such as nuclear magnetic resonance spectrometry, mass spectrometry, and infrared spectroscopy, and comparison with those in the literature. Compounds 1-4 were isolated from KSD-815 for the first time. Compounds 1 and 4 inhibited the migration of MDA-MB-231 cells at concentrations lower than 20 micronM.

Novel Humanized mice to test Therapeutics for Human Type 1 Diabetes

DTIC Science & Technology

2014-01-06

performed in non- obese diabetic (NOD) mice, the closest animal model for human T1D, have identified different immune cells involved in pancreatic β-cell...periphery. Each MHC class II chain contributes to the formation of a groove where the peptide is embedded (78). 17 The polygenetic factor underlying...diabetes mellitus in non- obese diabetic mice by transgenes encoding modified I-A beta-chain or normal I-E alpha-chain. Nature 345:727-9 63. Marek

Regulation of aromatase activity in bone-derived cells: possible role of mitogen-activated protein kinase.

PubMed

Shozu, M; Sumitani, H; Murakami, K; Segawa, T; Yang, H J; Inoue, M

2001-12-01

Fetal human osteoblast-like cells and the THP-1 cell line that differentiates into macrophage/osteoblast-like cells in the presence of Vitamin D3 and which possesses high aromatase activity, constitute a useful model with which to study the regulation of aromatase in bone. We showed that dexamethasone (DEX)-induced aromatase activity in the THP-1 cell line is completely suppressed by forskolin and by dibutyryl cAMP. We therefore investigated the contribution of mitogen-activated protein kinase (MAPK) to the regulation of aromatase, because cAMP inhibits MAPK in many cells. We examined the role of MAPK on aromatase activity using PD98059, a selective inhibitor of MEK-1. PD98059 (100 microM) reduced DEX+interleukin (IL)-1beta-induced aromatase activity in human osteoblast-like cells by more than 90%, whereas 50% of the aromatase mRNA concentration was retained compared with the control incubated with DEX+IL-1beta. PD98059 (50 microM) reduced the activity of aromatase in THP-1 cells by 80% without significantly affecting the mRNA level. These results indicated that MAPK plays an important role in aromatase activation at the post-transcriptional level.

Negative allosteric modulators that target human alpha4beta2 neuronal nicotinic receptors.

PubMed

Henderson, Brandon J; Pavlovicz, Ryan E; Allen, Jerad D; González-Cestari, Tatiana F; Orac, Crina M; Bonnell, Andrew B; Zhu, Michael X; Boyd, R Thomas; Li, Chenglong; Bergmeier, Stephen C; McKay, Dennis B

2010-09-01

Allosteric modulation of neuronal nicotinic acetylcholine receptors (nAChRs) is considered to be one of the most promising approaches for therapeutics. We have previously reported on the pharmacological activity of several compounds that act as negative allosteric modulators (NAMs) of nAChRs. In the following studies, the effects of 30 NAMs from our small chemical library on both human alpha4beta2 (Halpha4beta2) and human alpha3beta4 (Halpha3beta4) nAChRs expressed in human embryonic kidney ts201 cells were investigated. During calcium accumulation assays, these NAMs inhibited nAChR activation with IC(50) values ranging from 2.4 microM to more than 100 microM. Several NAMs showed relative selectivity for Halpha4beta2 nAChRs with IC(50) values in the low micromolar range. A lead molecule, KAB-18, was identified that shows relative selectivity for Halpha4beta2 nAChRs. This molecule contains three phenyl rings, one piperidine ring, and one ester bond linkage. Structure-activity relationship (SAR) analyses of our data revealed three regions of KAB-18 that contribute to its relative selectivity. Predictive three-dimensional quantitative SAR (comparative molecular field analysis and comparative molecular similarity indices analysis) models were generated from these data, and a pharmacophore model was constructed to determine the chemical features that are important for biological activity. Using docking approaches and molecular dynamics on a Halpha4beta2 nAChR homology model, a binding mode for KAB-18 at the alpha/beta subunit interface that corresponds to the predicted pharmacophore is described. This binding mode was supported by mutagenesis studies. In summary, these studies highlight the importance of SAR, computational, and molecular biology approaches for the design and synthesis of potent and selective antagonists targeting specific nAChR subtypes.

Estradiol coupling to human monocyte nitric oxide release is dependent on intracellular calcium transients: evidence for an estrogen surface receptor.

PubMed

Stefano, G B; Prevot, V; Beauvillain, J C; Fimiani, C; Welters, I; Cadet, P; Breton, C; Pestel, J; Salzet, M; Bilfinger, T V

1999-10-01

We tested the hypothesis that estrogen acutely stimulates constitutive NO synthase (cNOS) activity in human peripheral monocytes by acting on an estrogen surface receptor. NO release was measured in real time with an amperometric probe. 17beta-estradiol exposure to monocytes stimulated NO release within seconds in a concentration-dependent manner, whereas 17alpha-estradiol had no effect. 17beta-estradiol conjugated to BSA (E2-BSA) also stimulated NO release, suggesting mediation by a membrane surface receptor. Tamoxifen, an estrogen receptor inhibitor, antagonized the action of both 17beta-estradiol and E2-BSA, whereas ICI 182,780, a selective inhibitor of the nuclear estrogen receptor, had no effect. We further showed, using a dual emission microfluorometry in a calcium-free medium, that the 17beta-estradiol-stimulated release of monocyte NO was dependent on the initial stimulation of intracellular calcium transients in a tamoxifen-sensitive process. Leeching out the intracellular calcium stores abolished the effect of 17beta-estradiol on NO release. RT-PCR analysis of RNA obtained from the cells revealed a strong estrogen receptor-alpha amplification signal and a weak beta signal. Taken together, a physiological dose of estrogen acutely stimulates NO release from human monocytes via the activation of an estrogen surface receptor that is coupled to increases in intracellular calcium.

Sitagliptin inhibit human lymphocytes proliferation and Th1/Th17 differentiation in vitro.

PubMed

Pinheiro, Marcelo Maia; Stoppa, Caroline Lais; Valduga, Claudete Justina; Okuyama, Cristina Eunice; Gorjão, Renata; Pereira, Regina Mara Silva; Diniz, Susana Nogueira

2017-03-30

Dipeptidyl peptidase-4 (DPP-4) inhibitors are a new class of anti-diabetic agents that are widely used in clinical practice to improve glycemic control in patients with type 2 diabetes. DPP-4 is also known as lymphocyte cell surface protein, CD26, and plays an important role in T-cell immunity. Recent studies suggest that DPP-4 inhibitors improve beta-cell function and attenuate autoimmunity in type 1 diabetic mouse models. To investigate the direct effect of DPP4 in immune response, human peripheral blood mononuclear cells (PBMC) from healthy volunteers were obtained by Ficoll gradient and cultivated in the absence (control) or presence of phytohemagglutinin (PHA), or stimulated with PHA and treated with sitagliptin. The immune modulation mechanisms analyzed were: cell proliferation, by MTT assay; cytokine quantification by ELISA or cytometric bead array (CBA), Th1/Th2/Th17 phenotyping by flow cytometric analysis and CD26 gene expression by real time PCR. The results showed that sitagliptin treatment inhibited the proliferation of PBMC-PHA stimulated cells in a dose dependent manner and decreased CD26 expression by these cells, suggesting that sitagliptin may interfere in CD26 expression, dimerization and cell signaling. Sitagliptin treatment not only inhibited IL-10 (p<0.05) and IFN-gamma (p=0.07) cytokines, but also completely abolish IL-6 expression by PBMCs (p<0.001). On the other hand, IL-4 were secreted in culture supernatants from sitagliptin treated cells. A statistically significant increase (p<0.05) in the ratio of TGF-beta/proliferation index after sitagliptin treatment (2627.97±1351.65), when comparing to untreated cells (646.28±376.94), was also demonstrated, indicating higher TGF-beta1 production by viable cells in cultures. Sitagliptin treatment induced a significantly (p<0.05) decrease in IL-17 and IFN-gamma intracellular expression compared with PHA alone. Also, the percentage of T CD4 + IL-17 + , T CD4 + IFNgamma + and T CD4 + IL-4 + cells were significantly reduced (p<0.05) by sitagliptin. Our data demonstrated an immunosuppressive effect of sitagliptin on Th1, Th17 and Th2 lymphocytes differentiation that leads to the generation of regulatory TGF-beta1 secreting cells with low CD26 gene expression that may influence the state of pancreatic beta-cells and controlling DM1 patients. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Adhesive interactions of human multiple myeloma cell lines with different extracellular matrix molecules.

PubMed

Kibler, C; Schermutzki, F; Waller, H D; Timpl, R; Müller, C A; Klein, G

1998-06-01

Multiple myeloma represents a human B cell malignancy which is characterized by a predominant localization of the malignant cell clone within the bone marrow. With the exception of the terminal stage of the disease the myeloma tumor cells do not circulate in the peripheral blood. The bone marrow microenvironment is believed to play an important role in homing, proliferation and terminal differentiation of myeloma cells. Here we have studied the expression of several extracellular matrix (ECM) molecules in the bone marrow of multiple myeloma patients and analyzed their adhesive capacities with four different human myeloma-derived cell lines. All ECM molecules analyzed (tenascin, laminin, fibronectin, collagen types I, III, V and VI) could be detected in bone marrow cryostat sections of multiple myeloma patients. Adhesion assays showed that only laminin, the microfibrillar collagen type VI and fibronectin were strong adhesive components for the myeloma cell lines U266, IM-9, OPM-2 and NCI-H929. Tenascin and collagen type I were only weak adhesive substrates for these myeloma cells. Adhesion to laminin and fibronectin was beta 1-integrin-mediated since addition of anti-beta 1-integrin antibodies could inhibit the binding of the four different cell types to both matrix molecules. In contrast, integrins do not seem to be involved in binding of the myeloma cells to collagen type VI. Instead, inhibition of binding by heparin suggested that membrane-bound heparan sulfate proteoglycans are responsible ligands for binding to collagen type VI. Adhesion assays with several B-cell lines resembling earlier differentiation stages revealed only weak interactions with tenascin and no interactions with collagen type VI, laminin or fibronectin. In summary, the interactions of human myeloma cells with the extracellular matrix may explain the specific retention of the plasma cells within the bone marrow.

Three mouse models of human thalassemia.

PubMed Central

Martinell, J; Whitney, J B; Popp, R A; Russell, L B; Anderson, W F

1981-01-01

Three types of mice with globin gene mutations, called 352HB, 27HB, and Hbath-J, appear to be true animal models of human thalassemia. Expression of the alpha-globin genes in three stocks of mice, each one heterozygous for one of the alpha-globin mutations, was examined at the polypeptide, RNA, and DNA levels. alpha-Globin polypeptide chains, relative to beta-globin chains in heterozygous thalassemic mice, are present at approximately 80% of normal. The ratios of alpha-globin to beta-globin RNA sequences are also 75-80% of normal, exactly reflecting the alpha-globin to beta-globin chain ratios. In the case of mutant 352HB, at least one alpha-globin gene is deleted. Thalassemic mouse erythroid cells appear to compensate partially for the loss of half of their alpha-globin genes. Images PMID:6946454

[Survey of recent clinical trials of the prevention and immunointervention of type 1 diabetes mellitus].

PubMed

Boerschmann, H; Walter, M; Achenbach, P; Ziegler, A-G

2010-02-01

Immunomodulatory strategies in the management of type 1 diabetes mellitus (T1DM) have as their primary target the prevention of initiating islet autoimmunity (primary-), the secondary one is the progression to diabetes (secondary-) in non-diabetic persons at risk, and the decline of beta-cell function in new-onset patients (tertiary-prevention). This article reviews four recent immunointervention trials in patients with T1DM. (1) The Pre-POINT study is a primary prevention trial that will test whether vaccination with oral or nasal insulin can prevent the progression of islet autoimmunity and of T1DM in autoantibody-negative children who are genetically at high diabetes risk. (2) The Cord Blood study is a tertiary immunointervention trial that will test whether administration of autologous umbilical cord blood to children with T1DM can lead to regeneration of pancreatic islet insulin-producing beta-cells and improved blood glucose control. (3) The GAD Vaccination study will test whether vaccination with alum-formulated rhGAD65 (recombinant human glutamic acid decarboxylate) can preserve beta-cell function in 320 children with newly diagnosed T1DM, as has been suggested in a recent phase II study. (4) The AIDA study will test the beta-cell protective effect of interleukin-1-receptor antagonist Anakinra in 80 patients with T1DM, which has recently been shown to improve beta-cell function in patients with type 2 diabetes. Copyright Georg Thieme Verlag KG Stuttgart . New York.

Beta Cell Breakthroughs

MedlinePlus

... First successful pancreas transplant in a human. 1994: Experiments with islet transplantation begin. 1999: "Edmonton protocol" results ... initial results in 2000. Though complex, the Edmonton experiments managed to succeed where so many others had ...

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.

A Novel Anti-Beta2-Microglobulin Antibody Inhibition of Androgen Receptor Expression, Survival, and Progression in Prostate Cancer Cells

DTIC Science & Technology

2009-05-31

Konaka H, Sodek J, Zhau HE, Chung LW. Human Osteocalcin and Bone Sialoprotein Mediating Osteomimicry of Prostate Cancer Cells: Role of cAMP... sialoprotein mediating osteomimicry of prostate cancer cells: role of cAMP-dependent protein kinase A signaling path- way. Cancer Res 2005;65:2303^13

Contractile properties of early human embryonic stem cell-derived cardiomyocytes: beta-adrenergic stimulation induces positive chronotropy and lusitropy but not inotropy.

PubMed

Pillekamp, Frank; Haustein, Moritz; Khalil, Markus; Emmelheinz, Markus; Nazzal, Rewa; Adelmann, Roland; Nguemo, Filomain; Rubenchyk, Olga; Pfannkuche, Kurt; Matzkies, Matthias; Reppel, Michael; Bloch, Wilhelm; Brockmeier, Konrad; Hescheler, Juergen

2012-08-10

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide the unique opportunity to study the very early development of the human heart. The aim of this study was to investigate the effect of calcium and beta-adrenergic stimulation on the contractile properties of early hESC-CMs. Beating clusters containing hESC-CMs were co-cultured in vitro with noncontractile slices of neonatal murine ventricles. After 5-7 days, when beating clusters had integrated morphologically into the damaged tissue, isometric force measurements were performed during spontaneous beating as well as during electrical field stimulation. Spontaneous beating stopped when extracellular calcium ([Ca²⁺](ec)) was removed or after administration of the Ca²⁺ channel blocker nifedipine. During field stimulation at a constant rate, the developed force increased with incremental concentrations of [Ca²⁺](ec). During spontaneous beating, rising [Ca²⁺](ec) increased beating rate and developed force up to a [Ca²⁺](ec) of 2.5 mM. When [Ca²⁺](ec) was increased further, spontaneous beating rate decreased, whereas the developed force continued to increase. The beta-adrenergic agonist isoproterenol induced a dose-dependent increase of the frequency of spontaneous beating; however, it did not significantly change the developed force during spontaneous contractions or during electrical stimulation at a constant rate. Force developed by early hESC-CMs depends on [Ca²⁺](ec) and on the L-type Ca²⁺ channel. The lack of an inotropic reaction despite a pronounced chronotropic response after beta-adrenergic stimulation most likely indicates immaturity of the sarcoplasmic reticulum. For cell-replacement strategies, further maturation of cardiac cells has to be achieved either in vitro before or in vivo after transplantation.

Activation of the Wnt/β-catenin pathway in pancreatic beta cells during the compensatory islet hyperplasia in prediabetic mice

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

Maschio, D. A.; Oliveira, R. B.; Santos, M. R.

The Wnt/β-catenin signaling pathway, also known as the canonical Wnt pathway, plays a role in cell proliferation and differentiation in several tissues/organs. It has been recently described in humans a relationship between type 2 diabetes (T2DM) and mutation in the gene encoding the transcription factor TCF7L2 associated to the Wnt/β-catenin pathway. In the present study, we demonstrated that hyperplastic pancreatic islets from prediabetic mice fed a high-fat diet (HFD) for 60 d displayed nuclear translocation of active β-catenin associated with significant increases in protein content and gene expression of β-catenin as well as of cyclins D1, D2 and c-Myc (target genesmore » of the Wnt pathway) but not of Tcf7l2 (the transcription factor). Meanwhile, these alterations were not observed in pancreatic islets from 30 d HFD-fed mice, that do not display significant beta cell hyperplasia. These data suggest that the Wnt/β-catenin pathway is activated in pancreatic islets during prediabetes and may play a role in the induction of the compensatory beta cell hyperplasia observed at early phase of T2DM. - Highlights: • Exposure to high-fat diet for 60 days induced prediabetes and beta cell mass expansion. • Hyperplastic pancreatic islets displayed nuclear translocation of active β-catenin. • Hyperplastic islets showed increased expression of target genes of the Wnt/β-catenin pathway. • Wnt/β-catenin pathway is activated during compensatory beta cell hyperplasia in mice.« less

Regenerative medicine for diabetes: differentiation of human pluripotent stem cells into functional β-cells in vitro and their proposed journey to clinical translation.

PubMed

Bose, Bipasha; Katikireddy, Kishore Reddy; Shenoy, P Sudheer

2014-01-01

Diabetes is a group of metabolic diseases, rising globally at an alarming rate. Type 1 (juvenile diabetes) is the autoimmune version of diabetes where the pancreas is unable to produce insulin, whereas type 2 (adult onset diabetes) is caused due to insulin resistance of the cells. In either of the cases, elevated blood glucose levels are observed which leads to progressive comorbidity like renal failure, cardiovascular disease, retinopathy, etc. Metformin, sulphonyl urea group of drugs, as well as insulin injections are the available therapies. In advanced cases of diabetes, the drug alone or drug in combination with insulin injections are not able to maintain a steady level of blood glucose. Moreover, frequent insulin injections are rather cumbersome for the patient. So, regenerative medicine could be a permanent solution for fighting diabetes. Islet transplantation has been tried with a limited amount of success on a large population of diabetics because of the shortage of cadaveric pancreas. Therefore, the best proposed alternative is regenerative medicine involving human pluripotent stem cell (hPSC)-derived beta islet transplantation which can be obtained in large quantities. Efficient protocols for in vitro differentiation of hPSC into a large number of sustained insulin-producing beta cells for transplantation will be considered to be a giant leap to address global rise in diabetic cases. Although most of the protocols mimic in vivo pancreatic development in humans, considerable amount of lacuna persists for near-perfect differentiation strategies. Moreover, beta islets differentiated from hPSC have not yet been successfully translated under clinical scenario. © 2014 Elsevier Inc. All rights reserved.

Defective pigment granule biogenesis and aberrant behavior caused by mutations in the Drosophila AP-3beta adaptin gene ruby.

PubMed Central

Kretzschmar, D; Poeck, B; Roth, H; Ernst, R; Keller, A; Porsch, M; Strauss, R; Pflugfelder, G O

2000-01-01

Lysosomal protein trafficking is a fundamental process conserved from yeast to humans. This conservation extends to lysosome-like organelles such as mammalian melanosomes and insect eye pigment granules. Recently, eye and coat color mutations in mouse (mocha and pearl) and Drosophila (garnet and carmine) were shown to affect subunits of the heterotetrameric adaptor protein complex AP-3 involved in vesicle trafficking. Here we demonstrate that the Drosophila eye color mutant ruby is defective in the AP-3beta subunit gene. ruby expression was found in retinal pigment and photoreceptor cells and in the developing central nervous system. ruby mutations lead to a decreased number and altered size of pigment granules in various cell types in and adjacent to the retina. Humans with lesions in the related AP-3betaA gene suffer from Hermansky-Pudlak syndrome, which is caused by defects in a number of lysosome-related organelles. Hermansky-Pudlak patients have a reduced skin pigmentation and suffer from internal bleeding, pulmonary fibrosis, and visual system malfunction. The Drosophila AP-3beta adaptin also appears to be involved in processes other than eye pigment granule biogenesis because all ruby allele combinations tested exhibited defective behavior in a visual fixation paradigm. PMID:10790396

Leupaxin acts as a mediator in prostate carcinoma progression through deregulation of p120catenin expression.

PubMed

Kaulfuss, S; von Hardenberg, S; Schweyer, S; Herr, A M; Laccone, F; Wolf, S; Burfeind, P

2009-11-12

Recently, we could show that the focal adhesion protein leupaxin (LPXN) is expressed in human prostate carcinomas (PCa) and induces invasiveness of androgen-independent PCa cells. In this study we show that LPXN enhanced the progression of existing PCa in vivo by breeding transgenic mice with prostate-specific LPXN expression and TRAMP mice (transgenic adenocarcinoma of mouse prostate). Double transgenic LPXN/TRAMP mice showed a significant increase in poorly differentiated PCa and distant metastases as compared with control TRAMP mice. Additional studies on primary PCa cells generated from both transgenic backgrounds confirmed the connection regarding LPXN overexpression and increased motility and invasiveness of PCa cells. One mediator of LPXN-induced invasion was found to be the cell-cell adhesion protein p120catenin (p120CTN). Both in vitro and in vivo experiments revealed that p120CTN expression negatively correlates with LPXN expression, followed by a redistribution of beta-catenin. Downregulation of LPXN using small interfering RNAs (siRNAs) resulted in a membranous localization of beta-catenin, whereas strong nuclear accumulation of beta-catenin was observed in p120CTN knockdown cells leading to enhanced transcription of the beta-catenin target gene matrix metalloprotease-7. In conclusion, the present results indicate that LPXN enhances the progression of PCa through downregulation of p120CTN expression and that LPXN could function as a marker for aggressive PCa in the future.