Phosphatidic acid regulates signal output by G protein coupled receptors through direct interaction with phospholipase C-beta(1).

PubMed

Litosch, Irene; Pujari, Rajeshree; Lee, Shawn J

2009-09-01

Phosphatidic acid (PA), generated downstream of monomeric Rho GTPases via phospholipase D (PLD) and additionally by diacylglycerol kinases (DGK), both stimulates phospholipase C-beta(1) (PLC-beta(1)) and potentiates stimulation of PLC-beta(1) activity by Galpha(q) in vitro. PA is a potential candidate for integrating signaling by monomeric and heterotrimeric G proteins to regulate signal output by G protein coupled receptors (GPCR), and we have sought to understand the mechanisms involved. We previously identified the region spanning residues 944-957, lying within the PLC-beta(1) C-terminus alphaA helix and flexible loop of the Galpha(q) binding domain, as required for stimulation of lipase activity by PA in vitro. Regulation by PA does not require residues essential for stimulation by Galpha(q) or GTPase activating activity. The present studies evaluated shorter alanine/glycine replacement mutants and finally point mutations to identify Tyr(952) and Ile(955) as key determinants for regulation by PA, assessed by both in vitro enzymatic and cell-based co-transfection assays. Replacement of Tyr(952) and Ile(955), PLC-beta(1) (Y952G/I955G), results in an 85% loss in stimulation by PA relative to WT-PLC-beta(1) in vitro. COS 7 cells co-transfected with PLC-beta(1) (Y952G/I955G) demonstrate a 10-fold increase in the EC(50) for stimulation and a 60% decrease in maximum stimulation by carbachol via Galpha(q) linked m1 muscarinic receptors, relative to cells co-transfected with WT-PLC-beta(1) but otherwise similar conditions. Residues required for regulation by PA are not essential for stimulation by G protein subunits. WT-PLC-beta(1) and PLC-beta(1) (Y952G/I955G) activity is increased comparably by co-transfection with Galpha(q) and neither is markedly affected by co-transfection with Gbeta(1)gamma(2). Inhibiting PLD-generated PA production by 1-butanol has little effect on maximum stimulation, but shifts the EC(50) for agonist stimulation of WT-PLC-beta(1) by 10-fold, producing a phenotype similar to PLC-beta(1) (Y952G/I955G) with respect to agonist potency. 1-Butanol is without effect on carbachol stimulated PLC activity in cells co-transfected with either PLC-beta(1)(Y952G/I955G) or on endogenous PLC activity, indicating that regulation by PA requires direct interaction with the PLC-beta(1) PA-binding region. These data show that endogenous PA regulates signal output by Galpha(q)-linked GPCRs in transfected cells directly through PLC-beta(1). Galpha(q) and PA may co-ordinate to regulate signaling. Regulation by PA may constitute part of a mechanism that routes receptor signaling to specific PLC isoforms.

Glucocorticoid Signaling Enhances Expression of Glucose-Sensing Molecules in Immature Pancreatic Beta-Like Cells Derived from Murine Embryonic Stem Cells In Vitro.

PubMed

Ghazalli, Nadiah; Wu, Xiaoxing; Walker, Stephanie; Trieu, Nancy; Hsin, Li-Yu; Choe, Justin; Chen, Chialin; Hsu, Jasper; LeBon, Jeanne; Kozlowski, Mark T; Rawson, Jeffrey; Tirrell, David A; Yip, M L Richard; Ku, Hsun Teresa

2018-06-06

Pluripotent stem cells may serve as an alternative source of beta-like cells for replacement therapy of type 1 diabetes; however, the beta-like cells generated in many differentiation protocols are immature. The maturation of endogenous beta cells involves an increase in insulin expression starting in late gestation and a gradual acquisition of the abilities to sense glucose and secrete insulin by week 2 after birth in mice; however, what molecules regulate these maturation processes are incompletely known. In this study, we aim to identify small molecules that affect immature beta cells. A cell-based assay, using pancreatic beta-like cells derived from murine embryonic stem (ES) cells harboring a transgene containing an insulin 1-promoter driven enhanced green fluorescent protein reporter, was used to screen a compound library (NIH Clinical Collection-003). Cortisone, a glucocorticoid, was among five positive hit compounds. Quantitative reverse transcription-polymerase chain reaction analysis revealed that glucocorticoids enhance the gene expression of not only insulin 1 but also glucose transporter-2 (Glut2; Slc2a2) and glucokinase (Gck), two molecules important for glucose sensing. Mifepristone, a pharmacological inhibitor of glucocorticoid receptor (GR) signaling, reduced the effects of glucocorticoids on Glut2 and Gck expression. The effects of glucocorticoids on ES-derived cells were further validated in immature primary islets. Isolated islets from 1-week-old mice had an increased Glut2 and Gck expression in response to a 4-day treatment of exogenous hydrocortisone in vitro. Gene deletion of GR in beta cells using rat insulin 2 promoter-driven Cre crossed with GR flox/flox mice resulted in a reduced gene expression of Glut2, but not Gck, and an abrogation of insulin secretion when islets were incubated in 0.5 mM d-glucose and stimulated by 17 mM d-glucose in vitro. These results demonstrate that glucocorticoids positively regulate glucose sensors in immature murine beta-like cells.

Innovative Approaches to Treating Type 1 Diabetes Addressed in Beta-Cell Replacement Presentations

MedlinePlus

... ability to detect hypoglycemia. It is still considered experimental, however, and, due to a shortage of donors, ... into his or her own body, is another experimental approach for treating degenerative diseases including diabetes. Chad ...

Hb Alesha [beta67(E11)Val-->Met, GTG-->ATG] in an Argentinean girl.

PubMed

Eberle, Silvia Eandi; Noguera, Nélida I; Sciuccati, Gabriela; Bonduel, Mariana; Díaz, Lilian; Staciuk, Raquel; Targovnik, Héctor M; Feliu-Torres, Aurora

2007-01-01

Hb Alesha is caused by a GTG-->ATG mutation at codon 67 of the beta-globin gene, resulting in abnormal beta-globin chains in which the normal beta67(E11) valine is changed to methionine. This hemoglobin (Hb) is also known as Hb Bristol, the first unstable Hb described, since in a fraction of the variant the methionine is modified into an aspartic acid by a posttranslational modification. This replacement disrupts the apolar bonds between the valine and the heme group, producing an unstable Hb and severe hemolysis. We have identified this rare hemoglobinopathy in an Argentinean girl with severe hemolytic anemia, splenomegaly and frequent requirement for red blood cell transfusions.

Structural and biological mimicry of protein surface recognition by [alpha/beta]-peptide foldamers

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

Horne, W. Seth; Johnson, Lisa M.; Ketas, Thomas J.

Unnatural oligomers that can mimic protein surfaces offer a potentially useful strategy for blocking biomedically important protein-protein interactions. Here we evaluate an approach based on combining {alpha}- and {beta}-amino acid residues in the context of a polypeptide sequence from the HIV protein gp41, which represents an excellent testbed because of the wealth of available structural and biological information. We show that {alpha}/{beta}-peptides can mimic structural and functional properties of a critical gp41 subunit. Physical studies in solution, crystallographic data, and results from cell-fusion and virus-infectivity assays collectively indicate that the gp41-mimetic {alpha}/{beta}-peptides effectively block HIV-cell fusion via a mechanism comparablemore » to that of gp41-derived {alpha}-peptides. An optimized {alpha}/{beta}-peptide is far less susceptible to proteolytic degradation than is an analogous {alpha}-peptide. Our findings show how a two-stage design approach, in which sequence-based {alpha} {yields} {beta} replacements are followed by site-specific backbone rigidification, can lead to physical and biological mimicry of a natural biorecognition process.« less

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.

Lack of the central nervous system- and neural crest-expressed forkhead gene Foxs1 affects motor function and body weight.

PubMed

Heglind, Mikael; Cederberg, Anna; Aquino, Jorge; Lucas, Guilherme; Ernfors, Patrik; Enerbäck, Sven

2005-07-01

To gain insight into the expression pattern and functional importance of the forkhead transcription factor Foxs1, we constructed a Foxs1-beta-galactosidase reporter gene "knock-in" (Foxs1beta-gal/beta-gal) mouse, in which the wild-type (wt) Foxs1 allele has been inactivated and replaced by a beta-galactosidase reporter gene. Staining for beta-galactosidase activity reveals an expression pattern encompassing neural crest-derived cells, e.g., cranial and dorsal root ganglia as well as several other cell populations in the central nervous system (CNS), most prominently the internal granule layer of cerebellum. Other sites of expression include the lachrymal gland, outer nuclear layer of retina, enteric ganglion neurons, and a subset of thalamic and hypothalamic nuclei. In the CNS, blood vessel-associated smooth muscle cells and pericytes stain positive for Foxs1. Foxs1beta-gal/beta-gal mice perform significantly better (P < 0.01) on a rotating rod than do wt littermates. We have also noted a lower body weight gain (P < 0.05) in Foxs1beta-gal/lbeta-gal males on a high-fat diet, and we speculate that dorsomedial hypothalamic neurons, expressing Foxs1, could play a role in regulating body weight via regulation of sympathetic outflow. In support of this, we observed increased levels of uncoupling protein 1 mRNA in Foxs1beta-gal/beta-gal mice. This points toward a role for Foxs1 in the integration and processing of neuronal signals of importance for energy turnover and motor function.

Enzyme replacement therapy of Fabry disease.

PubMed

Clarke, Joe T R; Iwanochko, R Mark

2005-08-01

Fabry disease is an X-linked lysosomal storage disease caused by deficiency of the enzyme alpha-galactosidase A and results in pain, progressive renal impairment, cardiomyopathy, and cerebrovascular disease. The results of two major randomized, double-blind, placebo-controlled clinical trials and open-label extensions have shown that replacement of the deficient enzyme with either of two preparations of recombinant human alpha-galactosidase A, agalsidase-alfa, and agalsidase-beta is safe. Biweekly i.v. infusions of 0.2 mg/kg of agalsidase-alfa were associated with a significant decrease in pain and stabilization of renal function. Biweekly infusions of 1 mg/kg of agalsidase-beta were associated with virtually complete clearing of accumulated glycolipid substrate from renal and cutaneous capillary endothelial cells. Several smaller, open-label studies, along with observations made in the course of monitoring large numbers of patients on enzyme replacement therapy, indicated that treatment stabilizes renal function and produces significant improvements in myocardial mass and function. Treatment of Fabry disease by enzyme replacement has a significant impact on at least some serious complications of the disease.

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.

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.

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.

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

Species replacement dominates megabenthos beta diversity in a remote seamount setting.

PubMed

Victorero, Lissette; Robert, Katleen; Robinson, Laura F; Taylor, Michelle L; Huvenne, Veerle A I

2018-03-07

Seamounts are proposed to be hotspots of deep-sea biodiversity, a pattern potentially arising from increased productivity in a heterogeneous landscape leading to either high species co-existence or species turnover (beta diversity). However, studies on individual seamounts remain rare, hindering our understanding of the underlying causes of local changes in beta diversity. Here, we investigated processes behind beta diversity using ROV video, coupled with oceanographic and quantitative terrain parameters, over a depth gradient in Annan Seamount, Equatorial Atlantic. By applying recently developed beta diversity analyses, we identified ecologically unique sites and distinguished between two beta diversity processes: species replacement and changes in species richness. The total beta diversity was high with an index of 0.92 out of 1 and was dominated by species replacement (68%). Species replacement was affected by depth-related variables, including temperature and water mass in addition to the aspect and local elevation of the seabed. In contrast, changes in species richness component were affected only by the water mass. Water mass, along with substrate also affected differences in species abundance. This study identified, for the first time on seamount megabenthos, the different beta diversity components and drivers, which can contribute towards understanding and protecting regional deep-sea biodiversity.

T Cell Determinants Incorporating [beta]-Amino Acid Residues Are Protease Resistant and Remain Immunogenic In Vivo

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

Webb, Andrew I.; Dunstone, Michelle A.; Williamson, Nicholas A.

2010-07-20

A major hurdle in designing successful epitope-based vaccines resides in the delivery, stability, and immunogenicity of the peptide immunogen. The short-lived nature of unmodified peptide-based vaccines in vivo limits their therapeutic application in the immunotherapy of cancers and chronic viral infections as well as their use in generating prophylactic immunity. The incorporation of {beta}-amino acids into peptides decreases proteolysis, yet its potential application in the rational design of T cell mimotopes is poorly understood. To address this, we have replaced each residue of the SIINFEKL epitope individually with the corresponding {beta}-amino acid and examined the resultant efficacy of these mimotopes.more » Some analogs displayed similar MHC binding and superior protease stability compared with the native epitope. Importantly, these analogs were able to generate cross-reactive CTLs in vivo that were capable of lysing tumor cells that expressed the unmodified epitope as a surrogate tumor Ag. Structural analysis of peptides in which anchor residues were substituted with {beta}-amino acids revealed the basis for enhanced MHC binding and retention of immunogenicity observed for these analogs and paves the way for future vaccine design using {beta}-amino acids. We conclude that the rational incorporation of {beta}-amino acids into T cell determinants is a powerful alternative to the traditional homologous substitution of randomly chosen naturally occurring {alpha}-amino acids, and these mimotopes may prove particularly useful for inclusion in epitope-based vaccines.« less

Increasing protein stability by improving beta-turns.

PubMed

Fu, Hailong; Grimsley, Gerald R; Razvi, Abbas; Scholtz, J Martin; Pace, C Nick

2009-11-15

Our goal was to gain a better understanding of how protein stability can be increased by improving beta-turns. We studied 22 beta-turns in nine proteins with 66-370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some beta-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein, Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase alpha-subunit, and Maltose binding protein. Of the 15 single proline mutations, 11 increased stability (Average = 0.8 +/- 0.3; Range = 0.3-1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. On the basis of this and our previous work, we conclude that proteins can generally be stabilized by replacing nonproline residues with proline residues at the i + 1 position of Type I and II beta-turns and at the i position in Type II beta-turns. Other turn positions can sometimes be used if the phi angle is near -60 degrees for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in beta-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in beta-turns that could be replaced by Gly to increase protein stability. Improving beta-turns by substituting Pro residues is a generally useful way of increasing protein stability. 2009 Wiley-Liss, Inc.

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.

Loop III region of platelet-derived growth factor (PDGF) B-chain mediates binding to PDGF receptors and heparin.

PubMed Central

Schilling, D; Reid IV, J D; Hujer, A; Morgan, D; Demoll, E; Bummer, P; Fenstermaker, R A; Kaetzel, D M

1998-01-01

Site-directed mutagenesis of the platelet-derived growth factor (PDGF) B-chain was conducted to determine the importance of cationic amino acid residues (Arg160-Lys161-Lys162; RKK) located within the loop III region in mediating the biological and cell-association properties of the molecule. Binding to both PDGF alpha-and beta-receptors was inhibited by the conversion of all three cationic residues into anionic glutamates (RKK-->EEE), whereas an RKK-->SSS mutant also exhibited a modest loss in affinity for beta-receptors. Replacements with serine at either Arg160 (RKK-->SKK) or at all three positions (RKK-->SSS) had little effect on binding to alpha-receptors. Replacements with either glutamic or serine residues at any of the three positions also resulted in significant inhibition of heparin-binding activity. Furthermore, the RKK-->EEE mutant exhibited decreased association with the cell surface and accumulated in the culture medium as 29-32 kDa forms. Stable transfection of U87 astrocytoma cells with RKK-->EEE mutants of either the A-chain or the B-chain inhibited malignant growth in athymic nude mice. Despite altered receptor-binding activities, each of the loop III mutants retained full mitogenic activity when applied to cultured Swiss 3T3 cells. CD spectrophotometric analysis of the RKK-->EEE mutant revealed a secondary structure indistinguishable from the wild type, with a high degree of beta-sheet structure and random coil content (50% and 43% respectively). These findings indicate an important role of the Arg160-Lys161-Lys162 sequence in mediating the biological and cell-associative activities of the PDGF-BB homodimer, and reveal that the mitogenic activity of PDGF-BB is insufficient to mediate its full oncogenic properties. PMID:9677323

Rapid Immunochromatographic Detection of Serum Anti-α-Galactosidase A Antibodies in Fabry Patients after Enzyme Replacement Therapy.

PubMed

Nakano, Sachie; Tsukimura, Takahiro; Togawa, Tadayasu; Ohashi, Toya; Kobayashi, Masahisa; Takayama, Katsuyoshi; Kobayashi, Yukuharu; Abiko, Hiroshi; Satou, Masatsugu; Nakahata, Tohru; Warnock, David G; Sakuraba, Hitoshi; Shibasaki, Futoshi

2015-01-01

We developed an immunochromatography-based assay for detecting antibodies against recombinant α-galactosidase A proteins in serum. The evaluation of 29 serum samples from Fabry patients, who had received enzyme replacement therapy with agalsidase alpha and/or agalsidase beta, was performed by means of this assay method, and the results clearly revealed that the patients exhibited the same level of antibodies against both agalsidase alpha and agalsidase beta, regardless of the species of recombinant α-galactosidase A used for enzyme replacement therapy. A conventional enzyme-linked immunosorbent assay supported the results. Considering these, enzyme replacement therapy with agalsidase alpha or agalsidase beta would generate antibodies against the common epitopes in both agalsidase alpha and agalsidase beta. Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of α-galactosidase A. As immunochromatography is a handy and simple assay system which can be available at bedside, this assay method would be extremely useful for quick evaluation or first screening of serum antibodies against agalsidase alpha or agalsidase beta in Fabry disease with enzyme replacement therapy.

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.

Rapid Immunochromatographic Detection of Serum Anti-α-Galactosidase A Antibodies in Fabry Patients after Enzyme Replacement Therapy

PubMed Central

Nakano, Sachie; Tsukimura, Takahiro; Togawa, Tadayasu; Ohashi, Toya; Kobayashi, Masahisa; Takayama, Katsuyoshi; Kobayashi, Yukuharu; Abiko, Hiroshi; Satou, Masatsugu; Nakahata, Tohru; Warnock, David G.; Sakuraba, Hitoshi; Shibasaki, Futoshi

2015-01-01

We developed an immunochromatography-based assay for detecting antibodies against recombinant α-galactosidase A proteins in serum. The evaluation of 29 serum samples from Fabry patients, who had received enzyme replacement therapy with agalsidase alpha and/or agalsidase beta, was performed by means of this assay method, and the results clearly revealed that the patients exhibited the same level of antibodies against both agalsidase alpha and agalsidase beta, regardless of the species of recombinant α-galactosidase A used for enzyme replacement therapy. A conventional enzyme-linked immunosorbent assay supported the results. Considering these, enzyme replacement therapy with agalsidase alpha or agalsidase beta would generate antibodies against the common epitopes in both agalsidase alpha and agalsidase beta. Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of α-galactosidase A. As immunochromatography is a handy and simple assay system which can be available at bedside, this assay method would be extremely useful for quick evaluation or first screening of serum antibodies against agalsidase alpha or agalsidase beta in Fabry disease with enzyme replacement therapy. PMID:26083343

The effect of thyroid hormone and a long-acting somatostatin analogue on TtT-97 murine thyrotropic tumors.

PubMed

Woodmansee, W W; Gordon, D F; Dowding, J M; Stolz, B; Lloyd, R V; James, R A; Wood, W M; Ridgway, E C

2000-07-01

Thyroid hormone inhibits thyrotropin (TSH) production and thyrotrope growth. Somatostatin has been implicated as a synergistic factor in the inhibition of thyrotrope function. We have previously shown that pharmacological doses of thyroid hormone (levothyroxine [LT4]) inhibit growth of murine TtT-97 thyrotropic tumors in association with upregulation of somatostatin receptor type 5 (sst5) mRNA and somatostatin receptor binding. In the current study, we examined the effect of physiological thyroid hormone replacement alone or in combination with the long-acting somatostatin analogue, Sandostatin LAR, on thyrotropic tumor growth, thyrotropin growth factor-beta (TSH-beta), and sst5 mRNA expression, as well as somatostatin receptor binding sites. Physiological LT4 replacement therapy resulted in tumor shrinkage in association with increased sst5 mRNA levels, reduced TSH-beta mRNA levels and enhanced somatostatin receptor binding. Sandostatin LAR alone had no effect on any parameter measured. However, Sandostatin LAR combined with LT4 synergistically inhibited TSH-beta mRNA production and reduced final tumor weights to a greater degree. In this paradigm, Sandostatin LAR required a euthyroid status to alter thyrotrope parameters. These data suggest an important interaction between the somatostatinergic system and thyroid hormone in the regulation of thyrotrope cell structure and function.

Enzyme replacement therapy in a patient with Fabry disease and the development of IgE antibodies against agalsidase beta but not agalsidase alpha.

PubMed

Tanaka, Akemi; Takeda, Taisuke; Hoshina, Takao; Fukai, Kazuyoshi; Yamano, Tsunekazu

2010-12-01

Fabry disease is an X-linked inherited lysosomal storage disorder caused by an inborn deficiency of the enzyme α-galactosidase A. Enzyme replacement therapy (ERT) with agalsidase alpha or beta isozymes is an effective treatment. Cross-reactivity of immunoglobulin G (IgG) antibodies with agalsidase alpha and beta has been reported, but no such reaction has been recorded for IgE antibodies. We present the case of a patient with Fabry disease who developed antiagalsidase beta IgE antibodies without cross-reactivity to agalsidase alpha. A 17-year-old boy with Fabry disease had suffered from severe atopic dermatitis since infancy, and he complained for several years of peripheral pain during the summer months and when exercising. Fabry disease was confirmed by family history and a positive enzyme test, and ERT was commenced. Following infusion of agalsidase beta (1.0 mg/kg), the patient complained of a high temperature in his hands and feet, and purulent eczema developed. The infusion dose was reduced to 0.2 mg/kg, but the hyperthermia did not change, although its duration decreased. After three infusions, eosinophilia developed (9.4%; 573 cells/μl blood) and remained unresolved after four infusions with agalsidase beta. Treatment with this enzyme was discontinued, and agalsidase alpha (0.2 mg/kg) started. This produced immediate resolution of the eosinophilia, which has been maintained during follow-up. In conclusion, this patient developed IgE antibodies against agalsidase beta, which demonstrated no cross-reactivity to agalsidase alpha. These findings emphasize the importance of analyzing IgE antibodies against both enzymes when patients exhibit severe infusion-related events.

Estrogen via estrogen receptor beta partially inhibits mandibular condylar cartilage growth.

PubMed

Chen, J; Kamiya, Y; Polur, I; Xu, M; Choi, T; Kalajzic, Z; Drissi, H; Wadhwa, S

2014-11-01

Temporomandibular joint (TMJ) diseases predominantly afflict women, suggesting a role for female hormones in the disease process. However, little is known about the role of estrogen receptor (ER) signaling in regulating mandibular condylar cartilage growth. Therefore, the goal of this study was to examine the effects of altered estrogen levels on the mandibular condylar cartilage in wild type (WT) and ER beta Knockout (KO) mice. 21-day-old female WT (n = 37) and ER beta KO mice (n = 36) were either sham operated or ovariectomized, and treated with either placebo or estradiol. The mandibular condylar cartilage was evaluated by histomorphometry, proliferation was analyzed by double ethynyl-2'-deoxyuridine/bromodeoxyuridine (EdU/BrdU) labeling, and assays on gene and protein expression of chondrocyte maturation markers were performed. In WT mice, ovariectomy caused a significant increase in mandibular condylar cartilage cell numbers, a significant increase in Sox9 expression and a significant increase in proliferation compared with sham operated WT mice. In contrast, ovariectomy did not cause any of these effects in the ER beta KO mice. Estrogen replacement treatment in ovariectomized WT mice caused a significant decrease in ER alpha expression and a significant increase in Sost expression compared with ovariectomized mice treated with placebo. Estrogen replacement treatment in ovariectomized ER beta KO mice caused a significant increase in Col2 expression, no change in ER alpha expression, and a significant increase in Sost expression. Estrogen via ER beta inhibits proliferation and ER alpha expression while estrogen independent of ER beta induces Col2 and Sost expression. Copyright © 2014 China University of Geosciences (Beijing) and Peking University. Published by Elsevier Ltd. All rights reserved.

Determination of the specificity of monoclonal antibodies against Schistosoma mansoni CAA glycoprotein antigen using neoglycoconjugate variants.

PubMed

Carvalho de Souza, Adriana; van Remoortere, Alexandra; Hokke, Cornelis H; Deelder, André M; Vliegenthart, Johannes F G; Kamerling, Johannis P

2005-09-01

The immunogenic O-glycan of circulating anodic antigen (CAA) is a high-molecular-mass polysaccharide with the unique -->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GalpNAc-(1--> repeating unit. To obtain information at the molecular level about the specificity of monoclonal antibodies against CAA, the immunoreactivity of two series of bovine serum albumin-coupled synthetic oligosaccharides related to the CAA O-glycan was monitored using ELISA and surface plasmon resonance spectroscopy. The importance of the axial hydroxyl group of beta-D-GalpNAc for antibody binding was investigated using the following series of analogues: beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->O); beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O); and beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O). In the second series of analogues, beta-D-Glcp6S-(1-->3)-beta-D-GalpNAc-(1-->O), beta-D-GalpNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), and beta-D-Glcp6S-(1-->3)-beta-D-Gal-pNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), the native beta-D-GlcpA moiety was replaced by beta-D-Glcp6S to evaluate the influence of the nature of the charge on antibody recognition. Comparison of the immunoreactivity of these series with that measured for conjugates containing corresponding synthetic CAA fragments showed that the antibody binding levels can be correlated to the antibody specificity to CAA fragments. For the most reactive antibodies, the structural changes chosen (beta-D-GalpNAc replaced by beta-D-GlcpNAc, and beta-D-GlcpA replaced by beta-D-Glcp6S) completely eradicated the binding.

Cloning and characterization of the nagA gene that encodes beta-n-acetylglucosaminidase from Aspergillus nidulans and its expression in Aspergillus oryzae.

PubMed

Kim, Sunhwa; Matsuo, Ichiro; Ajisaka, Katsumi; Nakajima, Harushi; Kitamoto, Katsuhiko

2002-10-01

We isolated a beta-N-acetylglucosaminidase encoding gene and its cDNA from the filamentous fungus Aspergillus nidulans, and designated it nagA. The nagA gene contained no intron and encoded a polypeptide of 603 amino acids with a putative 19-amino acid signal sequence. The deduced amino acid sequence was very similar to the sequence of Candida albicans Hex1 and Trichoderma harzianum Nag1. Yeast cells containing the nagA cDNA under the control of the GAL1 promoter expressed beta-N-acetylglucosaminidase activity. The chromosomal nagA gene of A. nidulans was disrupted by replacement with the argB marker gene. The disruptant strains expressed low levels of beta-N-acetylglucosaminidase activity and showed poor growth on a medium containing chitobiose as a carbon source. Aspergillus oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of beta-N-acetylglucosaminidase in a wheat bran solid culture.

AKT/SGK-sensitive phosphorylation of GSK3 in the regulation of L-selectin and perforin expression as well as activation induced cell death of T-lymphocytes

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

Bhavsar, Shefalee K.; Merches, Katja; Bobbala, Diwakar

2012-08-17

Highlights: Black-Right-Pointing-Pointer Akt/SGK dependent phosphorylation of GSK3{alpha},{beta} regulates T lymphocytes. Black-Right-Pointing-Pointer T cells from mice expressing Akt/SGK insensitive GSK3{alpha},{beta} (gsk3{sup KI}) release less IL-2. Black-Right-Pointing-Pointer CD4{sup +} cells from gsk3{sup KI} mice express less CD62L. Black-Right-Pointing-Pointer CD8{sup +} cells from gsk3{sup KI} mice are relatively resistant to activation induced cell death. Black-Right-Pointing-Pointer Perforin expression is enhanced in gsk3{sup KI} T cells. -- Abstract: Survival and function of T-lymphocytes critically depends on phosphoinositide (PI) 3 kinase. PI3 kinase signaling includes the PKB/Akt and SGK dependent phosphorylation and thus inhibition of glycogen synthase kinase GSK3{alpha},{beta}. Lithium, a known unspecific GSK3 inhibitor protectsmore » against experimental autoimmune encephalomyelitis. The present study explored, whether Akt/SGK-dependent regulation of GSK3 activity is a determinant of T cell survival and function. Experiments were performed in mutant mice in which Akt/SGK-dependent GSK3{alpha},{beta} inhibition was disrupted by replacement of the serine residue in the respective SGK/Akt-phosphorylation consensus sequence by alanine (gsk3{sup KI}). T cells from gsk3{sup KI} mice were compared to T cells from corresponding wild type mice (gsk3{sup WT}). As a result, in gsk3{sup KI} CD4{sup +} cells surface CD62L (L-selectin) was significantly less abundant than in gsk3{sup WT} CD4{sup +} cells. Upon activation in vitro T cells from gsk3{sup KI} mice reacted with enhanced perforin production and reduced activation induced cell death. Cytokine production was rather reduced in gsk3{sup KI} T cells, suggesting that GSK3 induces effector function in CD8{sup +} T cells. In conclusion, PKB/Akt and SGK sensitive phosphorylation of GSK3{alpha},{beta} is a potent regulator of perforin expression and activation induced cell death in T lymphocytes.« less

Neuroimmune response to endogenous and exogenous pyrogens is differently modulated by sex steroids.

PubMed

Mouihate, A; Pittman, Q J

2003-06-01

The objective of this study was to explore whether and how ovarian hormones interact with the febrile response to pyrogens. Estrogen and progesterone treatment of ovariectomized rats was associated with a reduction in lipopolysaccharide (LPS)-induced fever, compared with ovariectomized controls. LPS-fever reduction was accompanied by reduced levels of the inducible cyclooxygenase-2 (COX-2) protein expression in the hypothalamus as well as reduced plasma levels of IL-1beta. The amount of LPS-induced IL-6 in the plasma was not affected by ovarian hormone replacement. In contrast, hypothalamic COX-2 expression in response to intraperitoneal injection of IL-1beta was potentiated by the ovarian hormone replacement. IL-1beta induced a moderate increase in plasma levels of IL-6 that was suppressed by ovarian hormone replacement. These data suggest that ovarian hormone replacement attenuated the proinflammatory response to LPS by suppressing the LPS-induced IL-1beta production and COX-2 expression in the hypothalamus. The markedly different action of ovarian hormones on IL-1beta and LPS effects suggests that this sex hormone modulation of the immune response is a function of the nature of infection and provides further evidence that LPS actions are different from those of IL-1beta.

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.

Phorbol ester and hydrogen peroxide synergistically induce the interaction of diacylglycerol kinase gamma with the Src homology 2 and C1 domains of beta2-chimaerin.

PubMed

Yasuda, Satoshi; Kai, Masahiro; Imai, Shin-ichi; Kanoh, Hideo; Sakane, Fumio

2008-01-01

DGKgamma (diacylglycerol kinase gamma) was reported to interact with beta2-chimaerin, a GAP (GTPase-activating protein) for Rac, in response to epidermal growth factor. Here we found that PMA and H2O2 also induced the interaction of DGKgamma with beta2-chimaerin. It is noteworthy that simultaneous addition of PMA and H2O2 synergistically enhanced the interaction. In this case, PMA was replaceable by DAG (diacylglycerol). The beta2-chimaerin translocation from the cytoplasm to the plasma membrane caused by PMA plus H2O2 was further enhanced by the expression of DGKgamma. Moreover, DGKgamma apparently enhanced the beta2-chimaerin GAP activity upon cell stimulation with PMA. PMA was found to be mainly required for a conversion of beta2-chimaerin into an active form. On the other hand, H2O2 was suggested to induce a release of Zn2+ from the C1 domain of beta2-chimaerin. By stepwise deletion analysis, we demonstrated that the SH2 (Src homology 2) and C1 domains of beta2-chimaerin interacted with the N-terminal half of catalytic region of DGKgamma. Unexpectedly, the SH2 domain of beta2-chimaerin contributes to the interaction independently of phosphotyrosine. Taken together, these results suggest that the functional link between DGKgamma and beta2-chimaerin has a broad significance in response to a wide range of cell stimuli. Our work offers a novel mechanism of protein-protein interaction, that is, the phosphotyrosine-independent interaction of the SH2 domain acting in co-operation with the C1 domain.

The third intracellular loop and carboxyl tail of neurokinin 1 and 3 receptors determine interactions with beta-arrestins.

PubMed

Schmidlin, Fabien; Roosterman, Dirk; Bunnett, Nigel W

2003-10-01

Tachykinins interact with three neurokinin receptors (NKRs) that are often coexpressed by the same cell. Cellular responses to tachykinins depend on the NKR subtype that is activated. We compared the colocalization of NK1R and NK3R with beta-arrestins 1 and 2, which play major roles in receptor desensitization, endocytosis, and signaling. In cells expressing NK1R, the selective agonist Sar-Met-substance P induced rapid translocation of beta-arrestins 1 and 2 from the cytosol to the plasma membrane and then endosomes, indicative of interaction with both isoforms. In contrast, the NK3R interacted transiently with only beta-arrestin 2 at the plasma membrane. Despite these differences, both NK1R and NK3R similarly desensitized, internalized, and activated MAP kinases. Because interactions with beta-arrestins can explain differences in the rate of receptor resensitization, we compared resensitization of agonist-induced Ca2+ mobilization. The NK1R resensitized greater than twofold more slowly than the NK3R. Replacement of intracellular loop 3 and the COOH tail of the NK1R with comparable domains of the NK3R diminished colocalization of the NK1R with beta-arrestin 1 and accelerated resensitization to that of the NK3R. Thus loop 3 and the COOH tail specify colocalization of the NK1R with beta-arrestin 1 and determine the rate of resensitization.

The quest for tissue stem cells in the pancreas and other organs, and their application in beta-cell replacement.

PubMed

Houbracken, Isabelle; Bouwens, Luc

2010-01-01

Adult stem cell research has drawn a lot of attention by many researchers, due to its medical hope of cell replacement or regenerative therapy for diabetes patients. Despite the many research efforts to date, there is no consensus on the existence of stem cells in adult pancreas. Genetic lineage tracing experiments have put into serious doubt whether β-cell neogenesis from stem/progenitor cells takes place postnatally. Different in vitro experiments have suggested centroacinar, ductal, acinar, stellate, or yet unidentified clonigenic cells as candidate β-cell progenitors. As in the rest of the adult stem cell field, sound and promising observations have been made. However, these observations still need to be replicated. As an alternative to committed stem/progenitor cells in the pancreas, transdifferentiation or lineage reprogramming of exocrine acinar and endocrine α-cells may be used to generate new β-cells. At present, it is unclear which approach is most medically promising. This article highlights the progress being made in knowledge about tissue stem cells, their existence and availability for therapy in diabetes. Particular attention is given to the assessment of methods to verify the existence of tissue stem cells.

Insulin therapy at onset of type 2 diabetes mellitus--a new concept.

PubMed

Sahay, B K

2011-04-01

In this study, insulin therapy was initiated at onset of disease in patients whose fasting blood glucose was more than 250 mg/dl. All enrolled subjects were treated with human premixed insulin (30/70) administered subcutaneously twice daily before breakfast and before dinner. A total of 113 subjects entered the study fulfilling the inclusion criteria. Good glycaemic control was achieved in a few days. The dosage requirement of insulin came down gradually after control was achieved as manifest by hypoglycaemia--leading to withdrawal of insulin. Some of them were managed with diet and exercise alone. Others required small doses of oral antidiabetic agents (OAD). There were no cases of secondary failure to OADs. Ten cases are on average duration of follow-up of 10 years. Two cases are under good control with diet and exercise alone, seven on treatment with oral hypoglycemic agents and one of them requiring insulin to maintain HbAlc below 7%. Thus insulin therapy at onset provides an opportunity to correct all the underlying pathogenic mechanisms, i.e., glucotoxicity, lipotoxicity and prevents beta cell apoptosis and suppresses inflammation, leading to beta cell protection. Such timely intervention provides long term benefits, laying the foundation for the concept of beta cell preservation rather that only replacing beta cell function. Hence we propose that all patients with type 2 diabetes should be offered insulin therapy at the onset of their diabetes for a period of 2-4 weeks.

Towards A Possible Therapy for Diabetes Complications

DTIC Science & Technology

2014-12-01

Towards A Possible Therapy for Diabetes Complications PRINCIPAL INVESTIGATOR: Massimo Trucco, M.D...September 2014 4. TITLE AND SUBTITLE Towards A Possible Therapy for Diabetes Complications 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-10-1-1055...of the beta cells. Consistently with this view, the standard of care for diabetic , and especially T1D patients is solely insulin-replacement therapy

Mash1-expressing cells could differentiate to type III cells in adult mouse taste buds.

PubMed

Takagi, Hiroki; Seta, Yuji; Kataoka, Shinji; Nakatomi, Mitsushiro; Toyono, Takashi; Kawamoto, Tatsuo

2018-03-10

The gustatory cells in taste buds have been identified as paraneuronal; they possess characteristics of both neuronal and epithelial cells. Like neurons, they form synapses, store and release transmitters, and are capable of generating an action potential. Like epithelial cells, taste cells have a limited life span and are regularly replaced throughout life. However, little is known about the molecular mechanisms that regulate taste cell genesis and differentiation. In the present study, to begin to understand these mechanisms, we investigated the role of Mash1-positive cells in regulating adult taste bud cell differentiation through the loss of Mash1-positive cells using the Cre-loxP system. We found that the cells expressing type III cell markers-aromatic L-amino acid decarboxylase (AADC), carbonic anhydrase 4 (CA4), glutamate decarboxylase 67 (GAD67), neural cell adhesion molecule (NCAM), and synaptosomal-associated protein 25 (SNAP25)-were significantly reduced in the circumvallate taste buds after the administration of tamoxifen. However, gustducin and phospholipase C beta2 (PLC beta2)-markers of type II taste bud cells-were not significantly changed in the circumvallate taste buds after the administration of tamoxifen. These results suggest that Mash1-positive cells could be differentiated to type III cells, not type II cells in the taste buds.

Safe and Successful Treatment With Agalsidase Beta During Pregnancy in Fabry Disease.

PubMed

Senocak Tasci, Elif; Bicik, Zerrin

2015-09-01

Fabry disease, an X-linked lysosomal storage disorder, is caused by α-galactosidase A deficiency and leads to accumulation of glycospinhgolipids in most tissues, with life-theratening consequences in the kidney, heart, and cerebrovascular system. Enzyme replacement therapy is available as 2 different preparations: agalsidase alfa and agalsidase beta. Enzyme replacement therapy is started as soon as the diagnosis is confirmed, but there is no data available in the literature about its safety during preganacy. Herein, we described 2 patients with Fabry disease who received agalsidase beta during their pregnancy. This report is important as the data about enzyme replacement therapy during pregnancy is restricted with case reports.

Effects of Asp-179 mutations in TEMpUC19 beta-lactamase on susceptibility to beta-lactams.

PubMed Central

Vakulenko, S B; Tóth, M; Taibi, P; Mobashery, S; Lerner, S A

1995-01-01

To examine the effect of disruption of the salt bridge (between Arg-164 and Asp-179 [numbering of Ambler et al. (Biochem J. 267:269-272, 1991)]) that anchors the conserved omega-loop in class A beta-lactamases, we obtained mutant enzymes with each of the 19 other amino acid residues replacing Asp-179 in the TEM beta-lactamase encoded by pUC19 and studied the level of resistance to various beta-lactams conferred by each enzyme. All mutations of Asp-179 compromised the level of resistance to ampicillin, but most of them enhanced resistance to ceftazidime. In contrast, mutations of Asp-179 generally impaired the low levels of resistance to cefepime and aztreonam. One might expect to find clinical isolates with mutant TEM beta-lactamases with replacements of Asp-179 that express an expanded spectrum of resistance to beta-lactams including ceftazidime. PMID:7486939

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.

Safety and efficacy of enzyme replacement therapy in the nephropathy of Fabry disease

PubMed Central

Fervenza, Fernando C; Torra, Roser; Warnock, David G

2008-01-01

Kidney involvement with progressive loss of kidney function (Fabry nephropathy) is an important complication of Fabry disease, an X-linked lysosomal storage disorder arising from deficiency of α-galactosidase activity. Clinical trials have shown that enzyme replacement therapy (ERT) with recombinant human α-galactosidase clears globotriaosylceramide from kidney cells, and can stabilize kidney function in patients with mild to moderate Fabry nephropathy. Recent trials show that patients with more advanced Fabry nephropathy and overt proteinuria do not respond as well to ERT alone, but can benefit from anti-proteinuric therapy given in conjunction with ERT. This review focuses on the use of enzyme replacement therapy with agalsidase-alfa and agalsidase-beta in adults with Fabry nephropathy. The current results are reviewed and evaluated. The issues of dosing of enzyme replacement therapy, the use of adjunctive agents to control urinary protein excretion, and the individual factors that affect disease severity are reviewed. PMID:19707461

Recent progress on normal and malignant pancreatic stem/progenitor cell research: therapeutic implications for the treatment of type 1 or 2 diabetes mellitus and aggressive pancreatic cancer

PubMed Central

Mimeault, M; Batra, S K

2010-01-01

Recent progress on pancreatic stem/progenitor cell research has revealed that the putative multipotent pancreatic stem/progenitor cells and/or more committed beta cell precursors may persist in the pancreatic gland in adult life. The presence of immature pancreatic cells with stem cell-like properties offers the possibility of stimulating their in vivo expansion and differentiation or to use their ex vivo expanded progenies for beta cell replacement-based therapies for type 1 or 2 diabetes mellitus in humans. In addition, the transplantation of either insulin-producing beta cells derived from embryonic, fetal and other tissue-resident adult stem/progenitor cells or genetically modified adult stem/progenitor cells may also constitute alternative promising therapies for treating diabetic patients. The genetic and/or epigenetic alterations in putative pancreatic adult stem/progenitor cells and/or their early progenies may, however, contribute to their acquisition of a dysfunctional behaviour as well as their malignant transformation into pancreatic cancer stem/progenitor cells. More particularly, the activation of distinct tumorigenic signalling cascades, including the hedgehog, epidermal growth factor–epidermal growth factor receptor (EGF–EGFR) system, wingless ligand (Wnt)/β-catenin and/or stromal cell-derived factor-1 (SDF-1)–CXC chemokine receptor 4 (CXCR4) pathways may play a major role in the sustained growth, survival, metastasis and/or drug resistance of pancreatic cancer stem/progenitor cells and their further differentiated progenies. The combination of drugs that target the oncogenic elements in pancreatic cancer stem/progenitor cells and their microenvironment, with the conventional chemotherapeutic regimens, could represent promising therapeutic strategies. These novel targeted therapies should lead to the development of more effective treatments of locally advanced and metastatic pancreatic cancers, which remain incurable with current therapies. PMID:18791122

Excretion profile of boldenone in urine of veal calves fed two different milk replacers.

PubMed

Draisci, R; Merlanti, R; Ferretti, G; Fantozzi, L; Ferranti, C; Capolongo, F; Segato, S; Montesissa, C

2007-03-14

The residue profiles of 17alpha-/17beta-boldenone conjugated (17alpha/beta-Bol) and ADD were investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in urine of male veal calves fed two commercial milk replacers, with different content of cholesterol and phytosterols. The urine samples were collected within 4 h after feeding and further from all the animals. Detectable amounts of 17alpha-Bol conjugated were measured in urine collected from all calves, but the concentrations of 17alpha-Bol were higher in urine from calves receiving the milk replacer with the greater amount of phytosterols. During the whole experiment, 17beta-Bol and ADD were never detected in urine samples collected.

Induction of bphA, encoding biphenyl dioxygenase, in two polychlorinated biphenyl-degrading bacteria, psychrotolerant Pseudomonas strain Cam-1 and mesophilic Burkholderia strain LB400.

PubMed

Master, E R; Mohn, W W

2001-06-01

We investigated induction of biphenyl dioxygenase in the psychrotolerant polychlorinated biphenyl (PCB) degrader Pseudomonas strain Cam-1 and in the mesophilic PCB degrader Burkholderia strain LB400. Using a counterselectable gene replacement vector, we inserted a lacZ-Gm(r) fusion cassette between chromosomal genes encoding the large subunit (bphA) and small subunit (bphE) of biphenyl dioxygenase in Cam-1 and LB400, generating Cam-10 and LB400-1, respectively. Potential inducers of bphA were added to cell suspensions of Cam-10 and LB400-1 incubated at 30 degrees C, and then beta-galactosidase activity was measured. Biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately six times greater than the basal level in cells incubated with pyruvate. In contrast, the beta-galactosidase activities in LB400-1 incubated with biphenyl and in LB400-1 incubated with pyruvate were indistinguishable. At a concentration of 1 mM, most of the 40 potential inducers tested were inhibitory to induction by biphenyl of beta-galactosidase activity in Cam-10. The exceptions were naphthalene, salicylate, 2-chlorobiphenyl, and 4-chlorobiphenyl, which induced beta-galactosidase activity in Cam-10, although at levels that were no more than 30% of the levels induced by biphenyl. After incubation for 24 h at 7 degrees C, biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately four times greater than the basal level in cells incubated with pyruvate. The constitutive level of beta-galactosidase activity in LB400-1 grown at 15 degrees C was approximately five times less than the level in LB400-1 grown at 30 degrees C. Thus, there are substantial differences in the effects of physical and chemical environmental conditions on genetic regulation of PCB degradation in different bacteria.

Modulation of fat-soluble vitamin concentrations and blood mononuclear leukocyte populations in milk replacer-fed calves by dietary vitamin A and beta-carotene.

PubMed

Nonnecke, B J; Horst, R L; Waters, W R; Dubeski, P; Harp, J A

1999-12-01

Dairy calves (n = 18), separated from dams at birth, were fed 1 L of pooled-colostrum. For the remaining 7 wk of the study, they were fed one of three diets consisting of either a custom-formulated milk replacer without vitamin A (controls), or supplemented with retinyl palmitate (equivalent to 32,000 IU of vitamin A/d) or with beta-carotene (equivalent to 20,000 IU of vitamin A/d). Plasma retinol, beta-carotene, and RRR-alpha-tocopherol concentrations were lowest at birth, and increased substantially from birth to 1 wk postpartum in all groups, a probable consequence of ingestion of colostrum. From 1 to 7 wk of age, retinol concentrations were greatest in retinyl palmitate-supplemented calves, intermediate in beta-carotene-supplemented calves and lowest in control calves. At 2, 3, 5, 6, and 7 wk, RRR-alpha-tocopherol concentrations were lower in retinyl palmitate-supplemented calves than in control calves. A negative correlation between plasma retinol and vitamin E concentrations existed from wk 2 to 7, suggesting vitamin A influences the absorption and distribution of RRR-alpha-tocopherol. Supplemental retinyl palmitate, but not beta-carotene, was associated with a reduction in the percentage of blood mononuclear leukocytes expressing CD2, CD4, and CD8-T cell antigens and interleukin-2 receptors. By wk 7, leukocyte populations from retinyl palmitate-supplemented calves were more similar to those from adult cattle than those from control calves, suggesting that supplemental vitamin A, as retinyl palmitate, affects the maturation of the neonatal immune system. Differences in the composition of blood mononuclear leukocyte populations may represent changes in immune competency.

Chimeras of the integrin beta subunit mid-region reveal regions required for heterodimer formation and for activation.

PubMed

Hyland, R H; Douglass, W A; Tan, S M; Law, S K

2001-01-01

A central region of the beta2 integrin subunit, RN (residues D300 to C459), was replaced by the equivalent sequences from beta1 and beta7 to give the chimeras beta2RN1 and beta2RN7. Whilst the former construct failed to form heterodimer at the cell surface with alphaL, the later of these could be expressed together with the alphaL subunit to form a variant LFA-1. Based on recent modelling work, the RN region consists of two parts, one is the C-terminal end of the putative A-domain (RB, residues D300 to A359), and the other the mid-region (BN, residues Y360 to C459). Chimeras exchanging the two component regions were made. Of the four resultant chimeras, only the beta2RB1 chimera failed to support LFA-1 expression. Thus the beta1 specific residues of this region affect the interaction with the alphaL subunit. Whereas the alphaL/beta2RB7 LFA-1 variant is wildtype like with respect to ICAM-1 adhesion, the alphaLbeta2BN1 and alphaLbeta2BN7, as well as the alphaLbeta2RN7, variants are more adhesive than the wildtype. These results suggest that an authentic beta2 mid-region is, in part, required for maintaining the LFA-1 in a resting state.

Agalsidase Benefits Renal Histology in Young Patients with Fabry Disease

PubMed Central

Bostad, Leif; Larsen, Kristin Kampevold; Hirth, Asle; Vikse, Bjørn Egil; Houge, Gunnar; Svarstad, Einar

2012-01-01

The effect of early-onset enzyme replacement therapy on renal morphologic features in Fabry disease is largely unknown. Here, we evaluated the effect of 5 years of treatment with agalsidase alfa or agalsidase beta in 12 consecutive patients age 7–33 years (median age, 16.5 years). We performed renal biopsies at baseline and after 5 years of enzyme replacement therapy; 7 patients had additional biopsies after 1 and 3 years. After a median of 65 months, biopsy findings from all patients showed total clearance of glomerular endothelial and mesangial cell inclusions, and findings from 2 patients showed complete clearance of inclusions from epithelial cells of the distal tubule. The 4 patients who received the highest dose of agalsidase exhibited substantial clearance of podocyte inclusions, and the youngest patient had nearly complete clearance of these inclusions. Linear regression analysis showed a highly significant correlation between podocyte globotriaocylceramide clearance and cumulative agalsidase dose (r=0.804; P=0.002). Microalbuminuria normalized in five patients. In summary, long-term enzyme replacement therapy in young patients can result in complete globotriaocylceramide clearance of mesangial and glomerular endothelial cells across all dosage regimens, and clearance of podocyte inclusions is dose-dependent. PMID:23274955

Photoaffinity labeling and partial purification of the beta cell sulfonylurea receptor using a novel, biologically active glyburide analog

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

Aguilar-Bryan, L.; Nelson, D.A.; Vu, Q.A.

1990-05-15

An iodinated analog of the sulfonylurea, glyburide, has been synthesized which can be labeled to high specific activity and used to photolabel the sulfonylurea receptor. 5-Iodo-2-hydroxy-glyburide, has an iodo group replacing the chlorine at position 5 and a methoxy residue replacing the hydroxy group at position 2 on the benzamido ring. This analog retains biologic activity stimulating insulin secretion from a hamster beta cell line (HIT cells) at the same ED50 (0.4 nM) as glyburide. Scatchard analysis demonstrated high and low affinity binding sites on HIT cell membranes (Kd values of 0.36 nM and 277 nM and Bmax values ofmore » 1.6 and 100 pmol/mg of membrane protein, respectively). Competitive binding assays with unlabeled glyburide or 5-iodo-2-hydroxyglyburide yield Ki values of 0.5 and 1.0 nM, respectively. The analog can be covalently linked by ultraviolet irradiation to a membrane protein of Mr = 140,000. The photolabeling is completely blocked by unlabeled glyburide or the analog. Two other species of Mr = 65,000 and 43,000 are also photolabeled; these may be the low affinity sites. After photolabeling, the receptor has been purified partially by chromatographic procedures and is suitable for obtaining peptide sequence. The 140,000 molecular weight protein is identified as the sulfonylurea receptor since its binding constant, 0.36 nM, is closely correlated with its ability to stimulate insulin secretion (ED50 congruent to 0.4 nM).« less

β1-Adrenergic receptor deficiency in ghrelin-expressing cells causes hypoglycemia in susceptible individuals

PubMed Central

Mani, Bharath K.; Osborne-Lawrence, Sherri; Vijayaraghavan, Prasanna; Hepler, Chelsea; Zigman, Jeffrey M.

2016-01-01

Ghrelin is an orexigenic gastric peptide hormone secreted when caloric intake is limited. Ghrelin also regulates blood glucose, as emphasized by the hypoglycemia that is induced by caloric restriction in mouse models of deficient ghrelin signaling. Here, we hypothesized that activation of β1-adrenergic receptors (β1ARs) localized to ghrelin cells is required for caloric restriction–associated ghrelin release and the ensuing protective glucoregulatory response. In mice lacking the β1AR specifically in ghrelin-expressing cells, ghrelin secretion was markedly blunted, resulting in profound hypoglycemia and prevalent mortality upon severe caloric restriction. Replacement of ghrelin blocked the effects of caloric restriction in β1AR-deficient mice. We also determined that treating calorically restricted juvenile WT mice with beta blockers led to reduced plasma ghrelin and hypoglycemia, the latter of which is similar to the life-threatening, fasting-induced hypoglycemia observed in infants treated with beta blockers. These findings highlight the critical functions of ghrelin in preventing hypoglycemia and promoting survival during severe caloric restriction and the requirement for ghrelin cell–expressed β1ARs in these processes. Moreover, these results indicate a potential role for ghrelin in mediating beta blocker–associated hypoglycemia in susceptible individuals, such as young children. PMID:27548523

Reduction of N-linked xylose and fucose by expression of rat beta1,4-N-acetylglucosaminyltransferase III in tobacco BY-2 cells depends on Golgi enzyme localization domain and genetic elements used for expression.

PubMed

Karg, Saskia R; Frey, Alexander D; Kallio, Pauli T

2010-03-01

Plant-specific N-glycosylation, such as the introduction of core alpha1,3-fucose and beta1,2-xylose residues, is a major obstacle to the utilization of plant cell- or plant-derived recombinant therapeutic proteins. The beta1,4-N-acetylglucosaminyltransferase III (GnTIII) introduces a bisecting GlcNAc residue into N-glycans, which exerts a high level of substrate mediated control over subsequent modifications, for example inhibiting mammalian core fucosylation. Based on similar findings in plants, we used Nicotianatabacum BY-2 cells to study the effects of localization and expression levels of GnTIII in the remodeling of the plant N-glycosylation pathway. The N-glycans produced by the cells expressing GnTIII were partially bisected and practically devoid of the paucimannosidic type which is typical for N-glycans produced by wildtype BY-2 suspension cultured cells. The proportion of human-compatible N-glycans devoid of fucose and xylose could be increased from an average of 4% on secreted protein from wildtype cells to as high as 59% in cells expressing chimeric GnTIII, named GnTIII(A.th.) replacing its native localization domain with the cytoplasmic tail, transmembrane, and stem region of Arabidopsis thaliana mannosidase II. The changes in N-glycosylation observed were dependent on the catalytic activity of GnTIII, as the expression of catalytically inactive GnTIII mutants did not show a significant effect on N-glycosylation. Copyright 2010 Elsevier B.V. All rights reserved.

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.

Molecular characterization of four beta-tubulin genes from dinitroaniline susceptible and resistant biotypes of Eleusine indica.

PubMed

Yamamoto, E; Baird, W V

1999-01-01

Dinitroaniline herbicides are antimicrotubule drugs that bind to tubulins and inhibit polymerization. As a result of repeated application of dinitroaniline herbicides, resistant biotypes of goosegrass (Eleusine indica) developed in previously susceptible wild-type populations. We have previously reported that alpha-tubulin missense mutations correlate with dinitroaniline response phenotypes (Drp) (Plant Cell 10: 297-308, 1998). In order to ascertain associations of other tubulins with dinitroaniline resistance, four beta-tubulin cDNA classes (designated TUB1, TUB2, TUB3, and TUB4) were isolated from dinitroaniline-susceptible and -resistant biotypes. Sequence analysis of the four beta-tubulin cDNA classes identified no missense mutations. Identified nucleotide substitutions did not result in amino acid replacements. These results suggest that the molecular basis of dinitroaniline resistance in goosegrass differs from those of colchicine/dinitroaniline cross-resistant Chlamydomonas reinhardtii and benzimidazole-resistant fungi and yeast. Expression of the four beta-tubulins was highest in inflorescences. This is in contrast to alpha-tubulin TUA1 that is expressed predominantly in roots. Collectively, these results imply that beta-tubulin genes are not associated with dinitroaniline resistance in goosegrass. Phylogenetic analysis of the four beta-tubulins, together with three alpha-tubulins, suggests that the resistant biotype developed independently in multiple locations rather than spreading from one location.

A retrospective analysis of the potential impact of IgG antibodies to agalsidase beta on efficacy during enzyme replacement therapy for Fabry disease.

PubMed

Bénichou, Bernard; Goyal, Sunita; Sung, Crystal; Norfleet, Andrea M; O'Brien, Fanny

2009-01-01

Fabry disease results from a genetic deficiency of alpha-galactosidase A (alpha GAL) and the impaired catabolism of globotriasoylceramide (GL-3) and other glycosphingolipid substrates, which then accumulate pathogenically within most cells. Enzyme replacement therapy (ERT) with agalsidase beta (Fabrazyme), one of two available forms of recombinant human alpha GAL, involves regular intravenous infusions of the therapeutic protein. Immunoglobulin G (IgG) antibodies to recombinant alpha GAL develop in the majority of patients upon repeated infusion. To explore whether anti-alpha GAL IgG interferes with therapeutic efficacy, retrospective analyses were conducted using data obtained from a total of 134 adult male and female patients with Fabry disease who were treated with agalsidase beta at 1mg/kg every 2 weeks for up to 5 years during placebo-controlled trials and the corresponding open-label extension studies. The analyses did not reveal a correlation between anti-alpha GAL IgG titers and the onset of clinical events or the rate of change in estimated GFR during treatment, and no statistically significant association was found between anti-alpha GAL IgG titers and abnormal elevations in plasma GL-3 during treatment. However, a statistically significant association was found between anti-alpha GAL IgG titers and observation of some GL-3 deposition in the dermal capillary endothelial cells of skin during treatment, suggesting that GL-3 clearance may be partially impaired in some patients with high antibody titers. Determination of the long-term impact of circulating anti-alpha GAL IgG antibodies on clinical outcomes will require continued monitoring, and serology testing is recommended as part of the routine care of Fabry disease patients during ERT.

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.

[Nuclear transfer of goat somatic cells transgenic for human lactoferrin].

PubMed

Li, Lan; Shen, Wei; Pan, Qing-Yu; Min, Ling-Jiang; Sun, Yu-Jiang; Fang, Yong-Wei; Deng, Ji-Xian; Pan, Qing-Jie

2006-12-01

Transgenic animal mammary gland bioreactors are being used to produce recombinant proteins with appropriate post-translational modifications, and nuclear transfer of transgenic somatic cells is a more powerful method to produce mammary gland bioreactor. Here we describe efficient gene transfer and nuclear transfer in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene, and the endogenous start condon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer, and some of reconstructed embryos could develop to blastocyst in vitro.

Induction of painless thyroiditis in patients receiving programmed death 1 receptor immunotherapy for metastatic malignancies.

PubMed

Orlov, Steven; Salari, Farnaz; Kashat, Lawrence; Walfish, Paul G

2015-05-01

Immunotherapies against immune checkpoints that inhibit T cell activation [cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed cell death 1 (PD-1)] are emerging and promising treatments for several metastatic malignancies. However, the precise adverse effects of these therapies on thyroid gland function have not been well described. We report on 10 cases of painless thyroiditis syndrome (PTS) from a novel etiology, following immunotherapy with anti-PD-1 monoclonal antibodies (mAb) during treatment for metastatic malignancies. Six patients presented with transient thyrotoxicosis in which thyrotropin binding inhibitory immunoglobulins (TBII) were absent for all, whereas four patients had evidence of positive antithyroid antibodies. All thyrotoxic patients required temporary beta-blocker therapy and had spontaneous resolution of thyrotoxicosis with subsequent hypothyroidism. Four patients presented with hypothyroidism without a detected preceding thyrotoxic phase, occurring 6-8 weeks after initial drug exposure. All of these patients had positive antithyroid antibodies and required thyroid hormone replacement therapy for a minimum of 6 months. Patients receiving anti-PD-1 mAb therapy should be monitored for signs and symptoms of PTS which may require supportive treatment with beta-blockers or thyroid hormone replacement. The anti-PD-1 mAb is a novel exogenous cause of PTS and provides new insight into the possible perturbations of the immune network that may modulate the development of endogenous PTS, including cases of sporadic and postpartum thyroiditis.

Short-term effects of replacing milk with cola beverages on insulin-like growth factor-I and insulin-glucose metabolism: a 10 d interventional study in young men.

PubMed

Hoppe, Camilla; Kristensen, Mette; Boiesen, Marlene; Kudsk, Jane; Fleischer Michaelsen, Kim; Mølgaard, Christian

2009-10-01

In the Western world, a trend towards increased consumption of carbonated soft drinks combined with a decreasing intake of milk is observed. This may affect circulating insulin-like growth factor I (IGF-I) and fasting insulin, as seen in pre-pubertal children. The present study was designed to reflect the trend of replacing milk with carbonated beverages in young men and to study the effects of this replacement on IGF-I, IGF-binding protein 3 (IGFBP-3), IGF-I:IGFBP-3 and glucose-insulin metabolism. A randomised, controlled crossover intervention study, in which eleven men aged 22-29 years were given a low-Ca diet in two 10 d periods with 10 d washout in between. In one period, they drank 2.5 litres of Coca Cola(R) per day and the other period 2.5 litres of semi-skimmed milk. Serum IGF-I, IGFBP-3 (RIA), insulin (fluoro immunoassay) and glucose (Cobas) were determined at baseline and end point of each intervention period. Insulin resistance and beta-cell function were calculated with the homeostasis model assessment. A decrease in serum IGF-I was observed in the cola period compared with the milk period (P < 0.05). No effects of treatment were observed on IGFBP-3, IGF-I:IGFBP-3, insulin, glucose, insulin resistance or beta-cell function. The present study demonstrates that high intake of cola over a 10 d period decreases total IGF-I compared with a high intake of milk, with no effect on glucose-insulin metabolism in adult men. It is unknown whether this is a transient phenomenon or whether it has long-term consequences.

Treatment with agalsidase beta during pregnancy in Fabry disease.

PubMed

Politei, Juan M

2010-04-01

Fabry disease is an X-linked lysosomal storage disease caused by a deficiency of alpha-galactosidase A, which leads to excessive accumulation of glycosphingolipids in most tissues in the body, with life-threatening clinical consequences in the kidney, heart, and cerebrovascular system. Enzyme replacement therapy using exogenously produced alpha-galactosidase has been available for treatment of this multisystem progressive disease since 2001. Two different preparations of enzyme replacement therapy for Fabry disease are available outside of the USA: agalsidase alfa and agalsidase beta. Despite being X-linked, Fabry disease affects many female patients, and this report presents a successful pregnancy of a female patient receiving agalsidase beta.

A bilaminated decellularized scaffold for islet transplantation: Structure, properties and functions in diabetic mice.

PubMed

Wang, Xi; Wang, Kai; Zhang, Wei; Qiang, Ming; Luo, Ying

2017-09-01

Ectopic transplantation of islets provides a beta cell-replacement approach that may allow the recovery of physiological regulation of the blood sugar level in patients with Type I diabetes (T1D). In development of new extrahepatic islet transplantation protocols in support of the islet engraftment, it is pivotal to develop scaffold materials with multifaceted functions to provide beneficial microenvironment, mediate host response in favor of vascularization/islet integration and maintain long-term islet function at the transplantation site. In this study, a new composite bilaminar decellularized scaffold (CDS) was fabricated with differential structural, degradation and mechanical properties by the combination of a fast-degrading porous collagen matrix and a mechanically supportive porcine pericardium. When investigated in the epididymal fat pad in syngeneic mouse models, it was shown that CDS could serve as superior scaffolds to promote islet adhesion and viability, and islet-CDS constructs also allowed rapid reversal of the hyperglycemic condition in the host. The engraftment and effects of islets were achieved at low islet numbers, accompanied by minimal adverse tissue reactions and optimal islet integration with the surrounding fat tissue. The bioactive surface, mechanical/chemical durability and biocompatibility of the CDS may all have played important roles in facilitating the engraftment of islets. Our study provided new insights into scaffold's function in the interplay of cells, materials and host tissue and the extracellular matrix-based scaffolds have potential for clinical translation in the beta cell-replacement therapy to treat T1D. Copyright © 2017 Elsevier Ltd. All rights reserved.

Analysis of Transcription Factors Key for Mouse Pancreatic Development Establishes NKX2-2 and MNX1 Mutations as Causes of Neonatal Diabetes in Man

PubMed Central

Flanagan, Sarah E.; De Franco, Elisa; Lango Allen, Hana; Zerah, Michele; Abdul-Rasoul, Majedah M.; Edge, Julie A.; Stewart, Helen; Alamiri, Elham; Hussain, Khalid; Wallis, Sam; de Vries, Liat; Rubio-Cabezas, Oscar; Houghton, Jayne A.L.; Edghill, Emma L.; Patch, Ann-Marie; Ellard, Sian; Hattersley, Andrew T.

2014-01-01

Summary Understanding transcriptional regulation of pancreatic development is required to advance current efforts in developing beta cell replacement therapies for patients with diabetes. Current knowledge of key transcriptional regulators has predominantly come from mouse studies, with rare, naturally occurring mutations establishing their relevance in man. This study used a combination of homozygosity analysis and Sanger sequencing in 37 consanguineous patients with permanent neonatal diabetes to search for homozygous mutations in 29 transcription factor genes important for murine pancreatic development. We identified homozygous mutations in 7 different genes in 11 unrelated patients and show that NKX2-2 and MNX1 are etiological genes for neonatal diabetes, thus confirming their key role in development of the human pancreas. The similar phenotype of the patients with recessive mutations and mice with inactivation of a transcription factor gene support there being common steps critical for pancreatic development and validate the use of rodent models for beta cell development. PMID:24411943

Microporous pure beta-tricalcium phosphate implants for press-fit fixation of anterior cruciate ligament grafts: strength and healing in a sheep model.

PubMed

Mayr, Hermann O; Dietrich, Markwart; Fraedrich, Franz; Hube, Robert; Nerlich, Andreas; von Eisenhart-Rothe, Rüdiger; Hein, Werner; Bernstein, Anke

2009-09-01

A sheep study was conducted to test a press-fit technique using microporous pure beta-tricalcium phosphate (beta-TCP) dowels for fixation of the anterior cruciate ligament (ACL) graft. Microporous (5 mum) cylindrical plugs of beta-TCP (diameter, 7 mm; length, 25 mm) with interconnecting pores were used. The material featured a novel configuration of structure and surface geometry. Implants were tested by use of press-fit fixation of ACL grafts with and without bone blocks in 42 sheep over a period of 24 weeks. Biomechanical, radiologic, histologic, and immunohistochemical evaluations were performed. In load-to-failure tests at 6, 12, and 24 weeks after surgery, the intra-articular graft always failed, not the fixation. Grafts showed bony fixation in the tunnel at 6 weeks and primary healing at the junction of the tunnel and joint after 24 weeks. Tricalcium phosphate was resorbed and simultaneously replaced by bone. Remodeling was still incomplete at 24 weeks. In the sheep model microporous beta-TCP implants used with press-fit fixation of ACL grafts permit early functional rehabilitation. After 6 weeks, the graft is fixed by woven bone or bony integration. Implanted microporous tricalcium phosphate is resorbed and replaced by bone. In a sheep model we showed that primary healing of ACL grafts with resorption and bony replacement of the fixating implant can be achieved by means of press-fit fixation with pure beta-TCP.

A biochemical and physicochemical comparison of two recombinant enzymes used for enzyme replacement therapies of hunter syndrome.

PubMed

Chung, Yo Kyung; Sohn, Young Bae; Sohn, Jong Mun; Lee, Jieun; Chang, Mi Sun; Kwun, Younghee; Kim, Chi Hwa; Lee, Jin Young; Yook, Yeon Joo; Ko, Ah-Ra; Jin, Dong-Kyu

2014-05-01

Mucopolysaccharidosis II (MPS II, Hunter syndrome; OMIM 309900) is an X-linked lysosomal storage disease caused by a deficiency in the enzyme iduronate-2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs). For enzyme replacement therapy (ERT) of Hunter syndrome, two recombinant enzymes, idursulfase (Elaprase(®), Shire Human Genetic Therapies, Lexington, MA) and idursulfase beta (Hunterase(®), Green Cross Corporation, Yongin, Korea), are currently available in Korea. To compare the biochemical and physicochemical differences between idursulfase and idursulfase beta, we examined the formylglycine (FGly) content, specific enzyme activity, mannose-6-phosphate (M6P) content, sialic acid content, and in vitro cell uptake activity of normal human fibroblasts of these two enzymes.The FGly content, which determines the enzyme activity, of idursulfase beta was significantly higher than that of idursulfase (79.4 ± 0.9 vs. 68.1 ± 2.2 %, P < 0.001). In accordance with the FGly content, the specific enzyme activity of idursulfase beta was significantly higher than that of idursulfase (42.6 ± 1.1 vs. 27.8 ± 0.9 nmol/min/μg protein, P < 0.001). The levels of M6P and sialic acid were not significantly different (2.4 ± 0.1 vs 2.4 ± 0.3 mol/mol protein for M6P and 12.3 ± 0.7 vs. 12.4 ± 0.4 mol/mol protein for sialic acid). However, the cellular uptake activity of the normal human fibroblasts in vitro showed a significant difference (Kuptake, 5.09 ± 0.96 vs. 6.50 ± 1.28 nM protein, P = 0.017).In conclusion, idursulfase beta exhibited significantly higher specific enzyme activity than idursulfase, resulting from higher FGly content. These biochemical differences may be partly attributed to clinical efficacy. However, long-term clinical evaluations of Hunter syndrome patients treated with these two enzymes will be needed to demonstrate the clinical implications of significant difference of the enzyme activity and the FGly content.

Lack of tyrosine 320 impairs spontaneous endocytosis and enhances release of HLA-B27 molecules.

PubMed

Santos, Susana G; Antoniou, Antony N; Sampaio, Paula; Powis, Simon J; Arosa, Fernando A

2006-03-01

Several lines of evidence suggest that endocytosis of MHC class I molecules requires conserved motifs within the cytoplasmic domain. In this study, we show, in the C58 rat thymoma cell line transfected with HLA-B27 molecules, that replacement of the highly conserved tyrosine (Tyr320) in the cytoplasmic domain of HLA-B27 does not hamper cell surface expression of beta2-microglobulin H chain heterodimers or formation of misfolded molecules. However, Tyr320 replacement markedly impairs spontaneous endocytosis of HLA-B27. Although wild-type molecules are mostly internalized via endosomal compartments, Tyr320-mutated molecules remain at the plasma membrane in which partial colocalization with endogenous transferrin receptors can be observed, also impairing their endocytosis. Finally, we show that Tyr320 substitution enhances release of cleaved forms of HLA-B27 from the cell surface. These studies show for the first time that Tyr320 is most likely part of a cytoplasmic sorting motif involved in spontaneous endocytosis and shedding of MHC class I molecules.

Pancreatic differentiation of Pdx1-GFP reporter mouse induced pluripotent stem cells.

PubMed

Porciuncula, Angelo; Kumar, Anujith; Rodriguez, Saray; Atari, Maher; Araña, Miriam; Martin, Franz; Soria, Bernat; Prosper, Felipe; Verfaillie, Catherine; Barajas, Miguel

2016-12-01

Efficient induction of defined lineages in pluripotent stem cells constitutes the determinant step for the generation of therapeutically relevant replacement cells to potentially treat a wide range of diseases, including diabetes. Pancreatic differentiation has remained an important challenge in large part because of the need to differentiate uncommitted pluripotent stem cells into highly specialized hormone-secreting cells, which has been shown to require a developmentally informed step-by-step induction procedure. Here, in the framework of using induced pluripotent stem cells (iPSCs) to generate pancreatic cells for pancreatic diseases, we have generated and characterized iPSCs from Pdx1-GFP transgenic mice. The use of a GFP reporter knocked into the endogenous Pdx1 promoter allowed us to monitor pancreatic induction based on the expression of Pdx1, a pancreatic master transcription factor, and to isolate a pure Pdx1-GFP + population for downstream applications. Differentiated cultures timely expressed markers specific to each stage and end-stage progenies acquired a rather immature beta-cell phenotype, characterized by polyhormonal expression even among cells highly expressing the Pdx1-GFP reporter. Our findings highlight the utility of employing a fluorescent protein reporter under the control of a master developmental gene in order to devise novel differentiation protocols for relevant cell types for degenerative diseases such as pancreatic beta cells for diabetes. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Sequence inversion and phenylalanine surrogates at the beta-turn enhance the antibiotic activity of gramicidin S.

PubMed

Solanas, Concepción; de la Torre, Beatriz G; Fernández-Reyes, María; Santiveri, Clara M; Jiménez, M Angeles; Rivas, Luis; Jiménez, Ana I; Andreu, David; Cativiela, Carlos

2010-05-27

A series of gramicidin S (GS) analogues have been synthesized where the Phe (i + 1) and Pro (i + 2) residues of the beta-turn have been swapped while the respective chiralities (D-, L-) at each position are preserved, and Phe is replaced by surrogates with aromatic side chains of diverse size, orientation, and flexibility. Although most analogues preserve the beta-sheet structure, as assessed by NMR, their antibiotic activities turn out to be highly dependent on the bulkiness and spatial arrangement of the aromatic side chain. Significant increases in microbicidal potency against both Gram-positive and Gram-negative pathogens are observed for several analogues, resulting in improved therapeutic profiles. Data indicate that seemingly minor replacements at the GS beta-turn can have significant impact on antibiotic activity, highlighting this region as a hot spot for modulating GS plasticity and activity.

Development and Testing of a 212Pb/212Bi Peptide for Targeting Metastatic Melanoma

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

Fisher, Darrell R.

2012-10-25

The purpose of this project is to develop a new radiolabeled peptide for imaging and treating metastatic melanoma. The immunoconjugate consists of a receptor-specific peptide that targets melanoma cells. The beta-emitter lead-212 (half-life = 10.4 hours) is linked by coordination chemistry to the peptide. After injection, the peptide targets melanoma receptors on the surfaces of melanoma cells. Lead-212 decays to the alpha-emitter bismuth-212 (half-life = 60 minutes). Alpha-particles that hit melanoma cell nuclei are likely to kill the melanoma cell. For cancer cell imaging, the lead-212 is replaced by lead-203 (half-life = 52 hours). Lead-203 emits 279 keV photons (80.1%more » abundance) that can be imaged and measured for biodistribution analysis, cancer imaging, and quantitative dosimetry.« less

Chondrogenesis, osteogenesis and adipogenesis of canine mesenchymal stem cells: a biochemical, morphological and ultrastructural study.

PubMed

Csaki, C; Matis, U; Mobasheri, A; Ye, H; Shakibaei, M

2007-12-01

Musculoskeletal diseases with osteochondrotic articular cartilage defects, such as osteoarthritis, are an increasing problem for humans and companion animals which necessitates the development of novel and improved therapeutic strategies. Canine mesenchymal stem cells (cMSCs) offer significant promise as a multipotent source for cell-based therapies and could form the basis for the differentiation and cultivation of tissue grafts to replace damaged tissue. However, no comprehensive analysis has been undertaken to characterize the ultrastructure of in vitro differentiated cMSCs. The main goal of this paper was to focus on cMSCs and to analyse their differentiation capacity. To achieve this aim, bone marrow cMSCs from three canine patients were isolated, expanded in monolayer culture and characterized with respect to their ability for osteogenic, adipogenic and chondrogenic differentiation capacities. cMSCs showed proliferative potential and were capable of osteogenic, adipogenic and chondrogenic differentiation. cMSCs treated with the osteogenic induction medium differentiated into osteoblasts, produced typical bone matrix components, beta1-integrins and upregulated the osteogenic specific transcription factor Cbfa-1. cMSCs treated with the adipogenic induction medium showed typical adipocyte morphology, produced adiponectin, collagen type I and beta1-integrins, and upregulated the adipogenic specific transcription factor PPAR-gamma. cMSCs treated with the chondrogenic induction medium exhibited a round to oval shape, produced a cartilage-specific extracellular matrix, beta1-integrins and upregulated the chondrogenic specific transcription factor Sox9. These results demonstrate, at the biochemical, morphological and ultrastructural levels, the multipotency of cMSCs and thus highlight their potential therapeutic value for cell-based tissue engineering.

Incorporating beta-turns and a turn mimetic out of context in loop 1 of the WW domain affords cooperatively folded beta-sheets.

PubMed

Kaul, R; Angeles, A R; Jäger, M; Powers, E T; Kelly, J W

2001-06-06

To probe the conformational requirements of loop 1 in the Pin1 WW domain, the residues at the i + 2 and i + 3 positions of a beta-turn within this loop were replaced by dPro-Gly and Asn-Gly, which are known to prefer the conformations required at the i + 1 and i + 2 positions of type II' and type I' beta-turns. Conformational specificity or lack thereof was further examined by incorporating into the i + 2 and i + 3 positions a non-alpha-amino acid-based beta-turn mimetic (4-(2'-aminoethyl)-6-dibenzofuran propionic acid residue, 1), which was designed to replace the i + 1 and i + 2 positions of beta-turns. All these Pin WW variants are monomeric and folded as discerned by analytical ultracentrifugation, NMR, and CD. They exhibit cooperative two-state transitions and display thermodynamic stability within 0.5 kcal/mol of the wild-type WW domain, demonstrating that the acquisition of native structure and stability does not require a specific sequence and, by extension, conformation within loop 1. However, it could be that these loop 1 mutations alter the kinetics of antiparallel beta-sheet folding, which will be addressed by subsequent kinetic studies.

Fabry disease and enzyme replacement therapy in classic patients with same mutation: different formulations--different outcome?

PubMed

Politei, J; Schenone, A B; Cabrera, G; Heguilen, R; Szlago, M

2016-01-01

We describe the results of the multidisciplinary evaluation in patients with Fabry disease and the same genetic mutation and their outcomes using different approved enzyme replacement therapy (ERT). We measured baseline data and serial results of neuropathic pain assessment and renal, cardiac and cerebrovascular functioning. Pain scale showed improvement in all male cases treated with agalsidasa beta. A mild improvement was detected in agalsidasa alfa-treated patients after 1 year with posterior increase. During the agalsidase beta shortage, two male patients were switched to agalsidasa alfa, after 1 year both cases presented an increase in scale values. Renal evolution showed a tendency toward a decrease in proteinuria in patients using agalsidase beta and worsening with agalsidase alfa. We found improvement in two females using agalsidase beta and no changes in the other cases regarding cardiac functioning. Brain magnetic resonance imaging (MRI) showed increase of white matter lesions in four patients. Improvement and stabilization in neuropathic pain, renal and cardiac functioning and brain MRI were found mainly in patients treated with agalsidase beta. Following the reported recommendations on reintroduction of agalsidase beta after the enzyme shortage, we decided to switch all patients to agalsidase beta. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Variable clinical spectrum of the most common inborn error of bile acid metabolism--3beta-hydroxy-Delta 5-C27-steroid dehydrogenase deficiency.

PubMed

Subramaniam, Pushpa; Clayton, Peter T; Portmann, Bernard C; Mieli-Vergani, Giorgina; Hadzić, Nedim

2010-01-01

We studied the clinical features of children with 3beta-hydroxy-Delta 5-C27-steroid dehydrogenase (3beta-HSDH) deficiency presenting to King's College and Great Ormond Street hospitals between 1989 and 2005. The diagnosis was made biochemically by detection of sulphated dihydroxycholenoic acids and trihydroxycholenoic acids in urine by fast atom bombardment mass spectrometry or electrospray ionisation tandem mass spectrophotometry and a plasma bile acid profile showing absent or low cholic and chenodeoxycholic acid levels and high concentrations of 3beta-7 alpha-dihydroxy-5-cholenoic acid and 3beta-7 alpha-12 alpha-trihydroxy-5-cholenoic acid. Eighteen children (12 male) with 3beta-HSDH deficiency were identified and diagnosed at a median age of 1.35 years (range 8 weeks-11 years). The presenting features included neonatal cholestasis (n = 11), rickets (n = 8, 1 of whom also had hypocalcaemic tetany, seizures, and normal liver biochemical markers), hepatomegaly (n = 7), pruritus (n = 3), and steatorrhoea and failure to thrive (n = 3). Ten children had low serum 25-OH vitamin D levels, of whom 8 also had low vitamin E and 6 had low vitamin A serum levels. Liver histology showed giant cell change and hepatocyte disarray in all with added features of cholestasis in 11, bridging fibrosis in 6, micronodular cirrhosis in 1, fatty change in 1, and active lobular and portal inflammation in 1. Five patients were treated with cholic acid and chenodeoxycholic acid (7 mg x kg(-1) x day(-1) of each), 7 with chenodeoxycholic acid only (7-18 mg x kg(-1) x day(-1)), and 1 with cholic acid (8 mg x kg(-1) x day(-1)) only. Repeated liver biopsies performed in 4 patients 6 months after starting replacement therapy showed improved histological changes. Three children died untreated before 5 years of age. After a median follow-up of 5.5 years (range 1-17 years) 12 out of 13 treated children have no clinical signs of liver disease or of fat-soluble vitamin deficiency. 3beta-HSDH deficiency is a rare inborn error of metabolism with diverse clinical features. Early replacement treatment leads to clinical and biochemical control and prevents chronic liver and bone disease, at least in the medium term.

Metabolic Stress and Compromised Identity of Pancreatic Beta Cells

PubMed Central

Swisa, Avital; Glaser, Benjamin; Dor, Yuval

2017-01-01

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

Metabolic Stress and Compromised Identity of Pancreatic Beta Cells.

PubMed

Swisa, Avital; Glaser, Benjamin; Dor, Yuval

2017-01-01

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

Theoretical foundations for quantitative paleogenetics. III - The molecular divergence of nucleic acids and proteins for the case of genetic events of unequal probability

NASA Technical Reports Server (NTRS)

Holmquist, R.; Pearl, D.

1980-01-01

Theoretical equations are derived for molecular divergence with respect to gene and protein structure in the presence of genetic events with unequal probabilities: amino acid and base compositions, the frequencies of nucleotide replacements, the usage of degenerate codons, the distribution of fixed base replacements within codons and the distribution of fixed base replacements among codons. Results are presented in the form of tables relating the probabilities of given numbers of codon base changes with respect to the original codon for the alpha hemoglobin, beta hemoglobin, myoglobin, cytochrome c and parvalbumin group gene families. Application of the calculations to the rabbit alpha and beta hemoglobin mRNAs and proteins indicates that the genes are separated by about 425 fixed based replacements distributed over 114 codon sites, which is a factor of two greater than previous estimates. The theoretical results also suggest that many more base replacements are required to effect a given gene or protein structural change than previously believed.

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

PubMed Central

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

2014-01-01

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

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.

Neural stem cells for disease modeling of Wolman disease and evaluation of therapeutics.

PubMed

Aguisanda, Francis; Yeh, Charles D; Chen, Catherine Z; Li, Rong; Beers, Jeanette; Zou, Jizhong; Thorne, Natasha; Zheng, Wei

2017-06-28

Wolman disease (WD) is a rare lysosomal storage disorder that is caused by mutations in the LIPA gene encoding lysosomal acid lipase (LAL). Deficiency in LAL function causes accumulation of cholesteryl esters and triglycerides in lysosomes. Fatality usually occurs within the first year of life. While an enzyme replacement therapy has recently become available, there is currently no small-molecule drug treatment for WD. We have generated induced pluripotent stem cells (iPSCs) from two WD patient dermal fibroblast lines and subsequently differentiated them into neural stem cells (NSCs). The WD NSCs exhibited the hallmark disease phenotypes of neutral lipid accumulation, severely deficient LAL activity, and increased LysoTracker dye staining. Enzyme replacement treatment dramatically reduced the WD phenotype in these cells. In addition, δ-tocopherol (DT) and hydroxypropyl-beta-cyclodextrin (HPBCD) significantly reduced lysosomal size in WD NSCs, and an enhanced effect was observed in DT/HPBCD combination therapy. The results demonstrate that these WD NSCs are valid cell-based disease models with characteristic disease phenotypes that can be used to evaluate drug efficacy and screen compounds. DT and HPBCD both reduce LysoTracker dye staining in WD cells. The cells may be used to further dissect the pathology of WD, evaluate compound efficacy, and serve as a platform for high-throughput drug screening to identify new compounds for therapeutic development.

Expression of the growth factor pleiotrophin and its receptor protein tyrosine phosphatase beta/zeta in the serum, cartilage and subchondral bone of patients with osteoarthritis.

PubMed

Kaspiris, Angelos; Mikelis, Constantinos; Heroult, Melanie; Khaldi, Lubna; Grivas, Theodoros B; Kouvaras, Ioannis; Dangas, Spyridon; Vasiliadis, Elias; Lioté, Frédéric; Courty, José; Papadimitriou, Evangelia

2013-07-01

Pleiotrophin is a heparin-binding growth factor expressed in embryonic but not mature cartilage, suggesting a role in cartilage development. Elucidation of the molecular changes observed during the remodelling process in osteoarthritis is of paramount importance. This study aimed to investigate serum pleiotrophin levels and expression of pleiotrophin and its receptor protein tyrosine phosphatase beta/zeta in the cartilage and subchondral bone of osteoarthritis patients. Serum samples derived from 16 osteoarthritis patients and 18 healthy donors. Pleiotrophin and receptor protein tyrosine phosphatase beta/zeta in the cartilage and subchondral bone were studied in 29 patients who had undergone total knee or hip replacement for primary osteoarthritis and in 10 control patients without macroscopic osteoarthritis changes. Serum pleiotrophin levels and expression of pleiotrophin in chondrocytes and subchondral bone osteocytes significantly increased in osteoarthritis patients graded Ahlback II to III. Receptor protein tyrosine phosphatase beta/zeta was mainly detected in the subchondral bone osteocytes of patients with moderate osteoarthritis and as disease severity increased, in the osteocytes and bone lining cells of the distant trabeculae. These data render pleiotrophin and receptor protein tyrosine phosphatase beta/zeta promising candidates for further studies towards developing targeted therapeutic schemes for osteoarthritis. Copyright © 2012 Société française de rhumatologie. Published by Elsevier SAS. All rights reserved.

Redox regulation and reaction mechanism of human cystathionine-beta-synthase: a PLP-dependent hemesensor protein.

PubMed

Banerjee, Ruma; Zou, Cheng-Gang

2005-01-01

Cystathionine beta-synthase in mammals lies at a pivotal crossroad in methionine metabolism directing flux toward cysteine synthesis and catabolism. The enzyme exhibits a modular organization and complex regulation. It catalyzes the beta-replacement of the hydroxyl group of serine with the thiolate of homocysteine and is unique in being the only known pyridoxal phosphate-dependent enzyme that also contains heme b as a cofactor. The heme functions as a sensor and modulates enzyme activity in response to redox change and to CO binding. Mutations in this enzyme are the single most common cause of hereditary hyperhomocysteinemia. Elucidation of the crystal structure of a truncated and highly active form of the human enzyme containing the heme- and pyridoxal phosphate binding domains has afforded a structural perspective on mechanistic and mutation analysis studies. The C-terminal regulatory domain containing two CBS motifs exerts intrasteric regulation and binds the allosteric activator, S-adenosylmethionine. Studies with mammalian cells in culture as well as with animal models have unraveled multiple layers of regulation of cystathionine beta-synthase in response to redox perturbations and reveal the important role of this enzyme in glutathione-dependent redox homestasis. This review discusses the recent advances in our understanding of the structure, mechanism, and regulation of cystathionine beta-synthase from the perspective of its physiological function, focusing on the clinically relevant human enzyme.

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

PubMed

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

2010-01-01

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

Three-Dimensional Bioreactor Technologies for the Cocultivation of Human Mesenchymal Stem/Stromal Cells and Beta Cells

PubMed Central

Petry, Florian; Weidner, Tobias; Salzig, Denise

2018-01-01

Diabetes is a prominent health problem caused by the failure of pancreatic beta cells. One therapeutic approach is the transplantation of functional beta cells, but it is difficult to generate sufficient beta cells in vitro and to ensure these cells remain viable at the transplantation site. Beta cells suffer from hypoxia, undergo apoptosis, or are attacked by the host immune system. Human mesenchymal stem/stromal cells (hMSCs) can improve the functionality and survival of beta cells in vivo and in vitro due to direct cell contact or the secretion of trophic factors. Current cocultivation concepts with beta cells are simple and cannot exploit the favorable properties of hMSCs. Beta cells need a three-dimensional (3D) environment to function correctly, and the cocultivation setup is therefore more complex. This review discusses 3D cultivation forms (aggregates, capsules, and carriers) for hMSCs and beta cells and strategies for large-scale cultivation. We have determined process parameters that must be balanced and considered for the cocultivation of hMSCs and beta cells, and we present several bioreactor setups that are suitable for such an innovative cocultivation approach. Bioprocess engineering of the cocultivation processes is necessary to achieve successful beta cell therapy. PMID:29731775

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

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.

Outdoor cultivation of Dunaliella salina KU 11 using brine and saline lake water with raceway ponds in northeastern Thailand.

PubMed

Wu, Zhe; Dejtisakdi, Wipawee; Kermanee, Prasart; Ma, Chunhong; Arirob, Wallop; Sathasivam, Ramaraj; Juntawong, Niran

2017-11-01

To evaluate the potential of algal biotechnology to replace traditional agriculture in northeastern Thailand, an open raceway cultivation system was developed to produce biomass and beta-carotene. Dunaliella salina KU 11 isolated from local saline soil was cultured in open raceway tanks using brine and saline lake water. Grown in modified Johnson's medium (with 2-3.5 M NaCl), the algae reached a maximum cell density on the fourth day (1.8 × 10 6 cells mL -1 ). Increasing KNO 3 and NaHCO 3 from 0.5 and 0.043 g L -1 to 1 and 2.1 g L -1 , respectively, significantly improved the yields of biomass (0.33 g L -1 ) and beta-carotene (19 mg L -1 ). Expected profits for algal production were evaluated, and it was found that this strain was suitable for outdoor cultivation and the developing algal industry in northeastern Thailand could produce high economic benefits (at least $64,120 per year per 0.16 ha). © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Analysis of transcription factors key for mouse pancreatic development establishes NKX2-2 and MNX1 mutations as causes of neonatal diabetes in man.

PubMed

Flanagan, Sarah E; De Franco, Elisa; Lango Allen, Hana; Zerah, Michele; Abdul-Rasoul, Majedah M; Edge, Julie A; Stewart, Helen; Alamiri, Elham; Hussain, Khalid; Wallis, Sam; de Vries, Liat; Rubio-Cabezas, Oscar; Houghton, Jayne A L; Edghill, Emma L; Patch, Ann-Marie; Ellard, Sian; Hattersley, Andrew T

2014-01-07

Understanding transcriptional regulation of pancreatic development is required to advance current efforts in developing beta cell replacement therapies for patients with diabetes. Current knowledge of key transcriptional regulators has predominantly come from mouse studies, with rare, naturally occurring mutations establishing their relevance in man. This study used a combination of homozygosity analysis and Sanger sequencing in 37 consanguineous patients with permanent neonatal diabetes to search for homozygous mutations in 29 transcription factor genes important for murine pancreatic development. We identified homozygous mutations in 7 different genes in 11 unrelated patients and show that NKX2-2 and MNX1 are etiological genes for neonatal diabetes, thus confirming their key role in development of the human pancreas. The similar phenotype of the patients with recessive mutations and mice with inactivation of a transcription factor gene support there being common steps critical for pancreatic development and validate the use of rodent models for beta cell development. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Whole organism high content screening identifies stimulators of pancreatic beta-cell proliferation.

PubMed

Tsuji, Naoki; Ninov, Nikolay; Delawary, Mina; Osman, Sahar; Roh, Alex S; Gut, Philipp; Stainier, Didier Y R

2014-01-01

Inducing beta-cell mass expansion in diabetic patients with the aim to restore glucose homeostasis is a promising therapeutic strategy. Although several in vitro studies have been carried out to identify modulators of beta-cell mass expansion, restoring endogenous beta-cell mass in vivo has yet to be achieved. To identify potential stimulators of beta-cell replication in vivo, we established transgenic zebrafish lines that monitor and allow the quantification of cell proliferation by using the fluorescent ubiquitylation-based cell cycle indicator (FUCCI) technology. Using these new reagents, we performed an unbiased chemical screen, and identified 20 small molecules that markedly increased beta-cell proliferation in vivo. Importantly, these structurally distinct molecules, which include clinically-approved drugs, modulate three specific signaling pathways: serotonin, retinoic acid and glucocorticoids, showing the high sensitivity and robustness of our screen. Notably, two drug classes, retinoic acid and glucocorticoids, also promoted beta-cell regeneration after beta-cell ablation. Thus, this study establishes a proof of principle for a high-throughput small molecule-screen for beta-cell proliferation in vivo, and identified compounds that stimulate beta-cell proliferation and regeneration.

TGF-beta3 is expressed in taste buds and inhibits proliferation of primary cultured taste epithelial cells.

PubMed

Nakamura, Shin-ichi; Kawai, Takayuki; Kamakura, Takashi; Ookura, Tetsuya

2010-01-01

Transforming growth factor-betas (TGF-betas), expressed in various tissues, play important roles in embryonic development and adult tissue homeostasis through their effects on cell proliferation, cell differentiation, cell death, and cell motility. However, expression of TGF-beta signaling components and their biological effect on taste epithelia has not been elucidated. We performed expression analysis of TGF-beta signaling components in taste epithelia and found that the TGF-beta3 mRNA was specifically expressed in taste buds. Type II TGF-betas receptor (TbetaR-II) mRNA was specifically expressed in the tongue epithelia including the taste epithelia. To elucidate the biological function of TGF-beta3 in taste epithelia, we performed proliferation assay with primary cultured taste epithelial cells. In the presence of TGF-beta3, percentage of BrdU-labeled cells decreased significantly, suggesting that the TGF-beta3 inhibited the proliferation of cultured taste epithelial cells through inhibiting cell-cycle entry into S phase. By quantitative reverse transcription-polymerase chain reaction assay, we found that the TGF-beta3 resulted in an increased level of expression of p15Ink4b and p21Cip1, suggesting that the TGF-beta3 inhibited the taste epithelial cell proliferation through inhibiting G1cyclin-Cdk complexes. Taken together, these results suggested that the TGF-beta3 may regulate taste epithelial cell homeostasis through controlling cell proliferation.

Enzyme replacement therapy in Japanese Fabry disease patients: the results of a phase 2 bridging study.

PubMed

Eto, Y; Ohashi, T; Utsunomiya, Y; Fujiwara, M; Mizuno, A; Inui, K; Sakai, N; Kitagawa, T; Suzuki, Y; Mochizuki, S; Kawakami, M; Hosoya, T; Owada, M; Sakuraba, H; Saito, H

2005-01-01

Fabry Disease (alpha-galactosidase A deficiency) is an X-linked hereditary disorder leading to the pathological accumulation of globotriaosylceramide (GL-3) in lysosomes, particularly in the vascular endothelium of the kidney, heart and brain. We report the results of an open-label phase 2 study that was undertaken to evaluate whether ethnic differences exist that would affect agalsidase beta (Fabrazyme) treatment of Fabry patients in the Japanese population, relative to safety and efficacy. The study design mirrored the design of the completed phase 3 clinical trial that led to approval of the product agalsidase beta. The 13 Japanese, male Fabry patients enrolled in the study received the enzyme replacement therapy over a period of 20 weeks as biweekly infusions. All selected efficacy end points showed improvements that were comparable with findings from the phase 3 study. These improvements included reductions of GL-3 accumulation in both kidney and skin capillary endothelial cells to (near) normal levels (92% of patients). Kidney and plasma GL-3 levels decreased by 51.9% and 100%, respectively, by ELISA. Renal function remained normal. Fabry-associated pain, and quality of life, showed improvement over baseline in multiple categories. Related adverse events were mild or moderate in intensity and mostly infusion-associated (fever and rigors). As expected, IgG antibody formation was observed in 85% of the patients, but had no effect on treatment response. These results suggest that treatment with agalsidase beta is safe and effective in Japanese patients with Fabry disease. With regard to safety and efficacy, no differences were observed as compared to the caucasian population.

Bovine dairy complex lipids improve in vitro measures of small intestinal epithelial barrier integrity.

PubMed

Anderson, Rachel C; MacGibbon, Alastair K H; Haggarty, Neill; Armstrong, Kelly M; Roy, Nicole C

2018-01-01

Appropriate intestinal barrier maturation is essential for absorbing nutrients and preventing pathogens and toxins from entering the body. Compared to breast-fed infants, formula-fed infants are more susceptible to barrier dysfunction-associated illnesses. In infant formula dairy lipids are usually replaced with plant lipids. We hypothesised that dairy complex lipids improve in vitro intestinal epithelial barrier integrity. We tested milkfat high in conjugated linoleic acid, beta serum (SureStart™Lipid100), beta serum concentrate (BSC) and a ganglioside-rich fraction (G600). Using Caco-2 cells as a model of the human small intestinal epithelium, we analysed the effects of the ingredients on trans-epithelial electrical resistance (TEER), mannitol flux, and tight junction protein co-localisation. BSC induced a dose-dependent improvement in TEER across unchallenged cell layers, maintained the co-localisation of tight junction proteins in TNFα-challenged cells with increased permeability, and mitigated the TEER-reducing effects of lipopolysaccharide (LPS). G600 also increased TEER across healthy and LPS-challenged cells, but it did not alter the co-location of tight junction proteins in TNFα-challenged cells. SureStart™Lipid100 had similar TEER-increasing effects to BSC when added at twice the concentration (similar lipid concentration). Ultimately, this research aims to contribute to the development of infant formulas supplemented with dairy complex lipids that support infant intestinal barrier maturation.

Bovine dairy complex lipids improve in vitro measures of small intestinal epithelial barrier integrity

PubMed Central

MacGibbon, Alastair K. H.; Haggarty, Neill; Armstrong, Kelly M.; Roy, Nicole C.

2018-01-01

Appropriate intestinal barrier maturation is essential for absorbing nutrients and preventing pathogens and toxins from entering the body. Compared to breast-fed infants, formula-fed infants are more susceptible to barrier dysfunction-associated illnesses. In infant formula dairy lipids are usually replaced with plant lipids. We hypothesised that dairy complex lipids improve in vitro intestinal epithelial barrier integrity. We tested milkfat high in conjugated linoleic acid, beta serum (SureStart™Lipid100), beta serum concentrate (BSC) and a ganglioside-rich fraction (G600). Using Caco-2 cells as a model of the human small intestinal epithelium, we analysed the effects of the ingredients on trans-epithelial electrical resistance (TEER), mannitol flux, and tight junction protein co-localisation. BSC induced a dose-dependent improvement in TEER across unchallenged cell layers, maintained the co-localisation of tight junction proteins in TNFα-challenged cells with increased permeability, and mitigated the TEER-reducing effects of lipopolysaccharide (LPS). G600 also increased TEER across healthy and LPS-challenged cells, but it did not alter the co-location of tight junction proteins in TNFα-challenged cells. SureStart™Lipid100 had similar TEER-increasing effects to BSC when added at twice the concentration (similar lipid concentration). Ultimately, this research aims to contribute to the development of infant formulas supplemented with dairy complex lipids that support infant intestinal barrier maturation. PMID:29304106

A combination of cytokines EGF and CNTF protects the functional beta cell mass in mice with short-term hyperglycaemia.

PubMed

Lemper, Marie; De Groef, Sofie; Stangé, Geert; Baeyens, Luc; Heimberg, Harry

2016-09-01

When the beta cell mass or function declines beyond a critical point, hyperglycaemia arises. Little is known about the potential pathways involved in beta cell rescue. As two cytokines, epidermal growth factor (EGF) and ciliary neurotrophic factor (CNTF), restored a functional beta cell mass in mice with long-term hyperglycaemia by reprogramming acinar cells that transiently expressed neurogenin 3 (NGN3), the current study assesses the effect of these cytokines on the functional beta cell mass after an acute chemical toxic insult. Glycaemia and insulin levels, pro-endocrine gene expression and beta cell origin, as well as the role of signal transducer and activator of transcription 3 (STAT3) signalling, were assessed in EGF+CNTF-treated mice following acute hyperglycaemia. The mice were hyperglycaemic 1 day following i.v. injection of the beta cell toxin alloxan, when the two cytokines were applied. One week later, 68.6 ± 4.6% of the mice had responded to the cytokine treatment and increased their insulin(+) cell number to 30% that of normoglycaemic control mice, resulting in restoration of euglycaemia. Although insulin(-) NGN3(+) cells appeared following acute EGF+CNTF treatment, genetic lineage tracing showed that the majority of the insulin(+) cells originated from pre-existing beta cells. Beta cell rescue by EGF+CNTF depends on glycaemia rather than on STAT3-induced NGN3 expression in acinar cells. In adult mice, EGF+CNTF allows the rescue of beta cells in distress when treatment is given shortly after the diabetogenic insult. The rescued beta cells restore a functional beta cell mass able to control normal blood glucose levels. These findings may provide new insights into compensatory pathways activated early after beta cell loss.

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.

Different susceptibility of rat pancreatic alpha and beta cells to hypoxia.

PubMed

Bloch, Konstantin; Vennäng, Julia; Lazard, Daniel; Vardi, Pnina

2012-06-01

Insulin-producing beta cells are known to be highly susceptible to hypoxia, which is a major factor in their destruction after pancreatic islet transplantation. However, whether the glucagon-producing pancreatic islet alpha cells are sensitive to hypoxia is not known. Our objective was to compare the sensitivity of alpha and beta cells to hypoxia. Isolated rat pancreatic islets were exposed to hypoxia (1% oxygen, 94% N(2), 5% CO(2)) for 3 days. The viability of the alpha and beta cells, as well as the stimulus-specific secretion of glucagon and insulin, was evaluated. A quantitative analysis of the proportion of beta to alpha cells indicated that, under normoxic conditions, islet cells were composed mainly of beta cells (87 ± 3%) with only 13 ± 3% alpha cells. Instead, hypoxia treatment significantly increased the proportion of alpha cells (40 ± 13%) and decreased the proportion of beta cells to 60 ± 13%. Using the fluorescent TUNEL assay we found that only a few percent of beta cells and alpha cells were apoptotic in normoxia. In contrast, hypoxia induced an abundance of apoptotic beta cells (61 ± 22%) and had no effect on the level of apoptosis in alpha cells. In conclusion, this study demonstrates that hypoxia results in severe functional abnormality in both beta and alpha cells while alpha cells display significantly decreased rate of apoptosis compared to intensive apoptotic injury of beta cells. These findings have implications for the understanding of the possible role of hypoxia in the pathophysiology of diabetes.

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

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.

Urocortin 3 Marks Mature Human Primary and Embryonic Stem Cell-Derived Pancreatic Alpha and Beta Cells

PubMed Central

van der Meulen, Talitha; Xie, Ruiyu; Kelly, Olivia G.; Vale, Wylie W.; Sander, Maike; Huising, Mark O.

2012-01-01

The peptide hormone Urocortin 3 (Ucn 3) is abundantly and exclusively expressed in mouse pancreatic beta cells where it regulates insulin secretion. Here we demonstrate that Ucn 3 first appears at embryonic day (E) 17.5 and, from approximately postnatal day (p) 7 and onwards throughout adult life, becomes a unifying and exclusive feature of mouse beta cells. These observations identify Ucn 3 as a potential beta cell maturation marker. To determine whether Ucn 3 is similarly restricted to beta cells in humans, we conducted comprehensive immunohistochemistry and gene expression experiments on macaque and human pancreas and sorted primary human islet cells. This revealed that Ucn 3 is not restricted to the beta cell lineage in primates, but is also expressed in alpha cells. To substantiate these findings, we analyzed human embryonic stem cell (hESC)-derived pancreatic endoderm that differentiates into mature endocrine cells upon engraftment in mice. Ucn 3 expression in hESC-derived grafts increased robustly upon differentiation into mature endocrine cells and localized to both alpha and beta cells. Collectively, these observations confirm that Ucn 3 is expressed in adult beta cells in both mouse and human and appears late in beta cell differentiation. Expression of Pdx1, Nkx6.1 and PC1/3 in hESC-derived Ucn 3+ beta cells supports this. However, the expression of Ucn 3 in primary and hESC-derived alpha cells demonstrates that human Ucn 3 is not exclusive to the beta cell lineage but is a general marker for both the alpha and beta cell lineages. Ucn 3+ hESC-derived alpha cells do not express Nkx6.1, Pdx1 or PC1/3 in agreement with the presence of a separate population of Ucn 3+ alpha cells. Our study highlights important species differences in Ucn 3 expression, which have implications for its utility as a marker to identify mature beta cells in (re)programming strategies. PMID:23251699

Nerve growth factor effect on human primary fibroblastic-keratocytes: possible mechanism during corneal healing.

PubMed

Micera, Alessandra; Lambiase, Alessandro; Puxeddu, Ilaria; Aloe, Luigi; Stampachiacchiere, Barbara; Levi-Schaffer, Francesca; Bonini, Sergio; Bonini, Stefano

2006-10-01

In response to corneal injury, cytokines and growth factors play a crucial role by influencing epithelial-stromal interaction during the healing and reparative processes which may resolve in tissue remodeling and fibrosis. While transforming growth factor-beta1 (TGF-beta1) is considered the main profibrogenic modulator of these process, recently the nerve growth factor (NGF) appears as a pleiotropic modulator of wound-healing and inflammatory responses. Interestingly in the cornea, where NGF, trkA(NGFR) and p75(NTR) are expressed by epithelial cells and keratocytes, the NGF eye-drop induces the healing of neurotrophic or autoimmune corneal ulcers. During corneal healing, quiescent keratocytes are replaced by active fibroblast-like keratocytes/myofibroblasts. While the NGF effect on epithelial cells has been investigated, no data are reported for NGF effects on fibroblastic-keratocytes, during corneal healing. NGF, trkA(NGFR) and p75(NTR) were found expressed by fibroblastic-keratocytes. NGF was able to induce fibroblastic-keratocyte differentiation into myofibroblasts, migration, Metalloproteinase-9 expression/activity and contraction of a 3D collagen gel, without affecting their proliferation and collagen production. These data also show a two-directional control of fibroblastic-keratocytes by NGF and TGF-beta1. To sum up, the findings of this study indicate that NGF can modulate some functional activities of fibroblastic-keratocytes, thus substantiating the healing effects of NGF on corneal wound-healing.

Replacement of the respiratory syncytial virus nonstructural proteins NS1 and NS2 by the V protein of parainfluenza virus 5

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

Tran, Kim C.; He, Biao; Teng, Michael N.

2007-11-10

Paramyxoviruses have been shown to produce proteins that inhibit interferon production and signaling. For human respiratory syncytial virus (RSV), the nonstructural NS1 and NS2 proteins have been shown to have interferon antagonist activity through an unknown mechanism. To understand further the functions of NS1 and NS2, we generated recombinant RSV in which both NS1 and NS2 were replaced by the PIV5 V protein, which has well-characterized IFN antagonist activities ({delta}NS1/2-V). Expression of V was able to partially inhibit IFN responses in {delta}NS1/2-V-infected cells. In addition, the replication kinetics of {delta}NS1/2-V were intermediate between {delta}NS1/2 and wild-type (rA2) in A549 cells.more » However, expression of V did not affect the ability of {delta}NS1/2-V to activate IRF3 nuclear translocation and IFN{beta} transcription. These data indicate that V was able to replace some of the IFN inhibitory functions of the RSV NS1 and NS2 proteins, but also that NS1 and NS2 have functions in viral replication beyond IFN antagonism.« less

Mycophenolate mofetil modulates adhesion receptors of the beta1 integrin family on tumor cells: impact on tumor recurrence and malignancy

PubMed Central

Engl, Tobias; Makarević, Jasmina; Relja, Borna; Natsheh, Iyad; Müller, Iris; Beecken, Wolf-Dietrich; Jonas, Dietger; Blaheta, Roman A

2005-01-01

Background Tumor development remains one of the major obstacles following organ transplantation. Immunosuppressive drugs such as cyclosporine and tacrolimus directly contribute to enhanced malignancy, whereas the influence of the novel compound mycophenolate mofetil (MMF) on tumor cell dissemination has not been explored. We therefore investigated the adhesion capacity of colon, pancreas, prostate and kidney carcinoma cell lines to endothelium, as well as their beta1 integrin expression profile before and after MMF treatment. Methods Tumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1 and 1 μM MMF and compared to unstimulated controls. beta1 integrin analysis included alpha1beta1 (CD49a), alpha2beta1 (CD49b), alpha3beta1 (CD49c), alpha4beta1 (CD49d), alpha5beta1 (CD49e), and alpha6beta1 (CD49f) receptors, and was carried out by reverse transcriptase-polymerase chain reaction, confocal microscopy and flow cytometry. Results Adhesion of the colon carcinoma cell line HT-29 was strongly reduced in the presence of 0.1 μM MMF. This effect was accompanied by down-regulation of alpha3beta1 and alpha6beta1 surface expression and of alpha3beta1 and alpha6beta1 coding mRNA. Adhesion of the prostate tumor cell line DU-145 was blocked dose-dependently by MMF. In contrast to MMF's effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes. Conclusion We conclude that MMF possesses distinct anti-tumoral properties, particularly in colon and prostate carcinoma cells. Adhesion blockage of HT-29 cells was due to the loss of alpha3beta1 and alpha6beta1 surface expression, which might contribute to a reduced invasive behaviour of this tumor entity. The enhancement of integrin beta1 subtypes observed in DU-145 cells possibly causes re-differentiation towards a low-invasive phenotype. PMID:15644133

Inhibin/activin-betaC and -betaE subunits in the Ishikawa human endometrial adenocarcinoma cell line.

PubMed

Kimmich, Tanja; Brüning, Ansgar; Käufl, Stephanie D; Makovitzky, Josef; Kuhn, Christina; Jeschke, Udo; Friese, Klaus; Mylonas, Ioannis

2010-08-01

Inhibins and activins are important regulators of the female reproductive system. Recently, two novel inhibin subunits, named betaC and betaE, have been identified and shown to be expressed in several human tissues. However, only limited data on the expression of these novel inhibin subunits in normal human endometrial tissue and endometrial adenocarcinoma cell lines exist. Samples of proliferative and secretory human endometrium were obtained from five premenopausal, non-pregnant patients undergoing gynecological surgery for benign diseases. Normal endometrial tissue and Ishikawa endometrial adenocarcinoma cell lines were analyzed by immunohistochemistry, immunofluorescence and RT-PCR. Expression of the inhibin betaC and betaE subunits could be demonstrated at the protein level by means of immunohistochemical evaluation and at the transcriptional level by establishing a betaC- and betaE-specific RT-PCR analysis in normal human endometrial tissue and the parental Ishikawa cell line. Interestingly, in a highly de-differentiated subclone of the Ishikawa cell line lacking estrogen receptor expression, the expression of the inhibin-betaC subunit appeared strongly reduced. Here, we show for the first time that the novel inhibin/activin-betaC and -betaE subunits are expressed in normal human endometrium and the estrogen receptor positive human endometrial carcinoma cell line Ishikawa using RT-PCR and immunohistochemical detection methods. Interestingly, the Ishikawa minus cell line (lacking estrogen receptor expression) demonstrated no to minimal expression of the betaC subunit as observed with immunofluorescence and RT-PCR, suggesting a possible hormone- dependency of this subunit in human endometrial cancer cells. Moreover, because the Ishikawa cell line minus is thought to be a more malignant endometrial cell line than its estrogen receptor positive counterpart, inhibin-betaC subunit might be substantially involved in the pathogenesis and malignant transformation in human endometrium.

Influence of fat replacers on chemical composition, proteolysis, texture profiles, meltability and sensory properties of low-fat Kashar cheese.

PubMed

Sahan, Nuray; Yasar, Kurban; Hayaloglu, Ali A; Karaca, Oya B; Kaya, Ahmet

2008-02-01

Changes in chemical composition, proteolysis, lipolysis, texture, melting and sensory properties of low-fat Kashar cheese made with three different fat replacers (Simplesse D-100, Avicel Plus CM 2159 or beta-glucan) were investigated throughout ripening. The low-fat cheeses made with fat replacers were compared with full- and low-fat counterparts as controls. Reduction of fat caused increases in moisture and protein contents and decreases in moisture-in-non fat substance and yield values in low-fat cheeses. The use of fat replacers in the manufacture of low-fat Kashar cheese increased water binding capacity and improved overall quality of the cheeses. Use of fat replacer in low-fat cheese making has enhanced cheese proteolysis. All samples underwent lipolysis during ripening and low-fat cheeses with fat replacers had higher level of total free fatty acid than full- or low-fat control cheeses. Texture attributes and meltability significantly increased with addition of fat replacers. Sensory scores showed that the full-fat cheese was awarded best in all stages of ripening and low-fat variant of Kashar cheeses have inferior quality. However, fat replacers except beta-glucan improved the appearance, texture and flavour attributes of low-fat cheeses. When the fat replacers are compared, the low-fat cheese with Avicel Plus CM 2159 was highly acceptable and had sensory attributes closest to full-fat Kashar cheese.

Can we induce spermatogenesis in the domestic cat using an in vitro tissue culture approach?

PubMed Central

Amaral, Sandra; Tavares, Renata S.; Schlatt, Stefan; Ramalho-Santos, João

2018-01-01

The reduced number of animals in most wild felid populations implies a loss of genetic diversity. The death of juveniles, prior to the production of mature sperm, represents a loss of potential genetic contribution to future populations. Since 2011 mouse testicular organ culture has introduced an alternative mechanism to produce sperm in vitro from immature tissue. However, extension of this technology to other species has remained limited. We have used the domestic cat (Felis catus) as a model for wild felids to investigate spermatogenesis initiation and regulation, with the mouse serving as a control species. Testicular tissue fragments were cultured in control medium or medium supplemented with knockout serum replacement (KSR), AlbuMax, beta-estradiol or AlbuMax plus beta-estradiol. Contrary to expectations, and unlike results obtained in mouse controls, no germ cell differentiation could be detected. The only germ cells observed after six weeks of culture were spermatogonia regardless of the initial stage of tubule development in the donor tissue. Moreover, the number of spermatogonia decreased with time in culture in all media tested, especially in the medium supplemented with KSR, while AlbuMax had a slight protective effect. The combination of AlbuMax and beta-estradiol led to an increase in the area occupied by seminiferous tubules, and thus to an increase in total number of spermatogonial cells. Considering all the media combinations tested the stimulus for felid germ cell differentiation in this type of system seems to be different from the mouse. Studies using other triggers of differentiation and tissue survival factors should be performed to pursue this technology for the genetic diversity preservation in wild felids. PMID:29414992

[Differentiation of human periodontal ligament stem cells into neuron-like cells in vitro].

PubMed

Zhen, Lei; Liu, Hong-Wei

2009-02-01

To isolate and purify the human periodontal ligament stem cells (PDLSC) and investigate the differentiation potentials of PDLSC into neuron-like cells in vitro. PDLSC were isolated and cultivated. PDLSC of passage 2 was plated at a density of 5 x 10(3) per mL. At 80% confluence, the PDLSC were preinduced for 24 hours, and were subsequently replaced with an inducing medium containing certain concentration of 13-mercaptoethanal (beta-ME). After 6 hours of induction, the results were evaluated by morphological observation, immunocytochemical staining for neuron specific enolase (NSE), neurofilament (NF) and glial fibrillary acid protein (GFAP) expression and RT-PCR for NSE, NF, GFAP mRNA. Meanwhile, the uninduced PDLSC were used as a negative control. PDLSC could be differentiate into cells with typical neuronal morphology. Immunohisto-chemistry and RT-PCR confirmed that the induced cells expressed NSE and NF, two marked enzymes of neuron cell. PDLSC can be induced into neuron-like cells in vitro. PDLSC have the capability of multilineage differentiations.

Effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation depend on treatment dose, treatment duration and meal contents

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

Arakawa, Masayuki; Ebato, Chie; Mita, Tomoya

2009-12-18

Beta-cell proliferation is regulated by various metabolic demands including peripheral insulin resistance, obesity, and hyperglycemia. In addition to enhancement of glucose-induced insulin secretion, agonists for glucagon-like peptide-1 receptor (GLP-1R) stimulate proliferation and inhibit apoptosis of beta-cells, thereby probably preserve beta-cell mass. To evaluate the beta-cell preserving actions of GLP-1R agonists, we assessed the acute and chronic effects of exendin-4 on beta-cell proliferation, mass and glucose tolerance in C57BL/6J mice under various conditions. Short-term administration of high-dose exendin-4 transiently stimulated beta-cell proliferation. Comparative transcriptomic analysis showed upregulation of IGF-1 receptor and its downstream effectors in islets. Treatment of mice with exendin-4more » daily for 4 weeks (long-term administration) and feeding high-fat diet resulted in significant inhibition of weight gain and improvement of glucose tolerance with reduced insulin secretion and beta-cell mass. These findings suggest that long-term GLP-1 treatment results in insulin sensitization of peripheral organs, rather than enhancement of beta-cell proliferation and function, particularly when animals are fed high-fat diet. Thus, the effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation largely depend on treatment dose, duration of treatment and meal contents. While GLP-1 enhances proliferation of beta-cells in some diabetic mice models, our results suggest that GLP-1 stimulates beta-cell growth only when expansion of beta-cell mass is required to meet metabolic demands.« less

Characterization and inhibition of beta-adrenergic receptor kinase in intact myocytes.

PubMed

Laugwitz, K L; Kronsbein, K; Schmitt, M; Hoffmann, K; Seyfarth, M; Schömig, A; Ungerer, M

1997-08-01

beta-Adrenergic receptor kinase (beta ARK) phosphorylates and thereby inactivates agonist-occupied beta-adrenergic receptors (beta AR). beta ARK is thought to play an important role in the regulation of cardiac function. Therefore, we studied beta ARK activation and its inhibition in intact smooth muscle cells and in cardiomyoblasts. beta AR agonist-stimulated translocation of beta ARK was monitored by immunofluorescence labelling with specific antibodies and confocal laser scanning microscopy in DDT-MF 2 hamster smooth muscle cells and in H9c2 rat cardiomyoblasts. In unstimulated cells. beta ARK was mainly located in the cytosol. After beta AR agonist stimulation, the beta ARK signal was partially translocated to the membranes. Liposomal gene transfer of the COOH-terminus of beta ARK ('beta ARKmini') as a beta ARK inhibitor led to functional expression of this protein in both cell lines with high efficiency. Western blots with beta ARK antibodies showed a gene concentration-dependent immunoreactivity of the 'beta ARKmini' protein. 'beta ARKmini'-transfected myocytes demonstrated reduced membrane targeting of the beta ARK immuno-fluorescence signal. Additionally, the effect of 'beta ARKmini' on beta AR-induced desensitization of myocytic cAMP accumulation was investigated. In control cells, desensitization with isoproterenol led to a subsequent reduction of beta AR-induced cAMP accumulation. In 'beta ARKmini'-transfected myocytes, this beta AR-induced desensitization was significantly diminished, whereas normal beta AR-induced cAMP accumulation was unaffected. A gene concentration of 2 micrograms 'beta ARKmini' DNA/100,000 cardiomyoblasts, and of 0.7 microgram 'beta ARKmini' DNA/100,000 DDT-MF2 smooth muscle cells led to approximately 5.9- and approximately 5.6-fold overexpressions of 'beta ARKmini' vs. native beta ARK, respectively. These gene doses proved sufficient to attenuate beta-adrenergic desensitization significantly. (1) beta ARK translocation was evidenced in DDT-MF2 smooth muscle cells and in cardiomyoblasts by confocal laser scanning microscopy. (2) Feasibility of 'beta ARKmini' gene transfer to myocytes was demonstrated, and necessary gene doses for beta ARK inhibition were titered. (3) Overexpression of 'beta ARKmini' functionally interacted with endogenous beta-adrenergic signal transduction, leading to sustained cAMP accumulation after prolonged beta-adrenergic stimulation.

Characterization of T cell repertoire changes in acute Kawasaki disease

PubMed Central

1993-01-01

Kawasaki disease (KD) is an acute multisystem vasculitis of unknown etiology that is associated with marked activation of T cells and monocyte/macrophages. Using a quantitative polymerase chain reaction (PCR) technique, we recently found that the acute phase of KD is associated with the expansion of T cells expressing the V beta 2 and V beta 8.1 gene segments. In the present work, we used a newly developed anti-V beta 2 monoclonal antibody (mAb) and studied a new group of KD patients to extend our previous PCR results. Immunofluorescence analysis confirmed that V beta 2-bearing T cells are selectively increased in patients with acute KD. The increase occurred primarily in the CD4 T cell subset. The percentages of V beta 2+ T cells as determined by mAb reactivity and flow cytometry correlated linearly with V beta expression as quantitated by PCR. However, T cells from acute KD patients appeared to express proportionately higher levels of V beta 2 transcripts per cell as compared with healthy controls or convalescent KD patients. Sequence analysis of T cell receptor beta chain genes of V beta 2 and V beta 8.1 expressing T cells from acute KD patients showed extensive junctional region diversity. These data showing polyclonal expansion of V beta 2+ and V beta 8+ T cells in acute KD provide additional insight into the immunopathogenesis of this disease. PMID:8094737

[Exploration of relationship between the expression level of DNA polymerase beta and 60Co gamma-ray radiosensitivity].

PubMed

Cui, Jie; Xu, Xin; Yang, Mo; Chen, Chen; Zhao, Wei; Wu, Mei; Zhang, Zun-zhen

2011-11-01

To explore the relationship between the expression level of DNA polymerase beta (pol beta) and 60Co gamma-ray radiosensitivity and provide a basis on improving the efficiency of radiotherapy theoretically. pol beta wild-type cells (pol beta +/+), pol beta null cells (pol beta -/-) and pol beta overexpressed cells (polp beta oe) were applied as a model system. The radiosensitivity of 60Co gamma-ray on the cell was detected by MTT assay and clone formation assay. The DCFH-DA fluorescent probe was used to examine the cellular ROS after 60Co gamma-rays radiation. MTT assay showed that after radiation by 60Co gamma-rays followed with 72 h incubation, the cell viabilities in the three kinds of cells decreased significantly with a dose-response relationship (r-/+ = -0.976, r-/- = -0.977, r(oe) = -0.982, P<0.05). In addition, the viability of pol beta -/- cell was lower than those of other two kinds of cells at the same dose (P<0.05). Likewise, the colony number and colony formation rate in all tested cells also decreased after exposure to 60Co gamma-rays. The ROS level in the three kinds of cells was enhanced after treatment with 60Co gamma-ray, and the ROS level in pol beta -/- cells was much higher than that in the other two kinds of cells (P<0.05). Cell death caused by 60Co gamma-ray may associated with the DNA oxidative damage mediated by ROS; Overexpression of pol beta could protect against oxidative DNA damage, thus the cell apoptosis/death, thereby leading to reducing the radiosensitivity of 60Co gamma-rays, while null of DNA pol beta could increase radiosensitivity of 60Co gamma-rays by compromising the DNA repair.

Yeast cell-wall products containing beta-glucan plus ascorbic acid affect neonatal Bos taurus calf leukocytes and growth after a transport stressor.

PubMed

Eicher, S D; Wesley, I V; Sharma, V K; Johnson, T R

2010-03-01

The objectives were to ascertain whether a yeast cell-wall derivative that was 1.8% beta-glucan in combination with ascorbyl-2-polyphosphate could improve innate immunity and mediate transportation stress in neonatal calves, and to compare the 1.8% beta-glucan yeast cell-wall derivative with a more purified yeast cell-wall derivative (70% beta-glucan). Treatments were 1) an unsupplemented control (CNT); 2) 113 g of a 1.8% (approximately 2%) beta-glucan derivative of yeast cell walls plus 250 mg of l-ascorbic acid phosphate (BG2); or 3) 150 mg of a purified beta-glucan fraction from yeast cell walls (approximately 70% beta-glucan) plus 250 mg/feeding of l-ascorbic acid phosphate (BG70). Calves (n = 39) were transported for 4 h, placed in outdoor hutches, and randomly assigned to treatments. Treatments (mixed with a milk replacer) were individually fed twice daily for 28 d. Calves were offered calf starter, free choice, throughout the study. Weekly starter intake and BW were measured, and fecal samples were collected for Salmonella Typhimurium and Escherichia coli O157:H7 PCR analysis. Blood was collected immediately before transport (d 0) and on d 3, 7, 10, 14, 21, and 28 after transport. Starter intake and DMI were less (P < 0.05) at d 28 for the BG2 and BG70 treatments compared with the CNT treatment. Hematocrit percentages increased (P = 0.002) throughout the experiment. White blood cell counts (treatment x time interaction, P = 0.066) were less for the calves supplemented with BG70 than for those supplemented with BG2 (P = 0.01) or for CNT calves (P = 0.04) on d 28. Granulocyte counts changed (P = 0.04) throughout the experiment. A trend (P = 0.077) for a treatment x time interaction was detected for peripheral blood mononuclear cell counts (PBMC). Counts of PBMC were greater (P = 0.006) for the BG2 treatment compared with the CNT treatment on d 3. Calves given the BG70 supplement had fewer PBMC than those given the BG2 supplement on d 21 (P = 0.03) and 28 (P = 0.05). Fibrinogen concentrations were affected only by time (P = 0.002). Time effects were detected for phagocytosis (P = 0.005), oxidative burst (P < 0.001), expression of cluster of differentiation 18 (P = 0.001), and increased cluster of differentiation 18 (P = 0.006). Phagocytosis was less (P = 0.05) for calves in the BG70 group than for those in the CNT group. Percentage of calves positive for E. coli O157:H7 was greatest (P

Uneventful pregnancy outcome after enzyme replacement therapy with agalsidase beta in a heterozygous female with Fabry disease: A case report.

PubMed

Germain, Dominique P; Bruneval, Patrick; Tran, Thi-Chien; Balouet, Pierre; Richalet, Bernard; Benistan, Karelle

2010-01-01

No reproductive studies have been performed with enzyme replacement therapy (ERT) for Fabry disease (FD, OMIM 301500), a lysosomal storage disorder. Therefore, use during pregnancy is theoretically contraindicated. We report the first case of agalsidase beta treatment throughout pregnancy. High-range proteinuria remained stable and the patient gave birth to a healthy boy after an uncomplicated pregnancy. The decision to administer ERT during pregnancy should be made on an individual basis, considering the FD status and possible risks. Copyright 2009 Elsevier Masson SAS. All rights reserved.

Degradation of Beta Cloth Covering for a Battery Orbital Replacement Unit in Low Earth Orbit

NASA Technical Reports Server (NTRS)

Gaier, James R.; Waters, Deborah L.; Baldwin, Sammantha; Folz, Angela D.; Loos, Alyssa

2016-01-01

Samples from the beta cloth cover for a battery orbit replaceable unit from the International Space Station (ISS) were characterized using optical and electron microscopy, UV-vis-NIR spectrophotometry, and x-ray energy dispersive spectroscopy. Results showed that in areas where the fabric was exposed to solar radiation the absorptance increased by as much as 20 percent, and the peak difference was in the ultraviolet, indicating that the increased absorptance may have been due to radiation. The emissivity of the material over a temperature range of 300 to 700 K was essentially unchanged.

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.

Evaluation of the transforming growth factor-beta activity in normal and dry eye human tears by CCL-185 cell bioassay.

PubMed

Zheng, Xiaofen; De Paiva, Cintia S; Rao, Kavita; Li, De-Quan; Farley, William J; Stern, Michael; Pflugfelder, Stephen C

2010-09-01

To develop a new bioassay method using human lung epithelial cells (CCL-185) to assess activity of transforming growth factor beta (TGF-beta) in human tear fluid from normal subjects and patients with dry eye. Two epithelial cell lines, mink lung cells (CCL-64) and human lung cells (CCL-185), were compared to detect the active form of TGF-beta by BrdU incorporation (quantitation of cell DNA synthesis) and WST assay (metabolic activity of viable cells). The effect of TGF-beta on the growth of CCL-185 cells was observed microscopically. Human tears from normal control subjects and patients with dry eye (DE) with and without Sjögren syndrome were evaluated for TGF-beta concentration by Luminex microbead assay, and TGF-beta activity by the CCL-185 cell growth inhibition bioassay. The metabolic activity of viable CCL-185 cells, measured by WST, was shown to be proportional to the TGF-beta1 concentration (R = 0.919) and confirmed by BrdU assay (R = 0.969). Compared with CCL-185, metabolic activity of viable cells and DNA synthesis, measured by WST and BrdU incorporation assays, were shown to be less proportional to the TGF-beta1 concentration in the CCL-64 line (R = 0.42 and 0.17, respectively). Coincubation with human anti-TGF-beta1 antibody (MAB-240) yielded a dose-dependent inhibition of TGF-beta1 (0.3 ng/mL) activity. CCL-185 cell growth observed microscopically was noted to decrease in response to increasing TGF-beta1 concentrations. Levels of immuodetectable TGF-beta1 and TGF-beta2 were similar in normal and DE tears. TGF-beta bioactivity in DE human tears measured by the CCL-185 cells assay was found to be higher (9777.5 +/- 10481.9 pg/mL) than those in normal controls (4129.3 +/- 1342.9 pg/mL) (P < 0.05). Among patients with DE, TGF-beta bioactivity was highest in those with Sjögren syndrome. Approximately, 79.1% of TGF-beta in DE tears and 37.6% TGF-beta in normal tears were found to be biologically active. The CCL-185 cell assay was found to be a suitable tool for assessing TGF-beta activity in human tears. Tear TGF-beta bioactivity increases in DE, particularly in Sjögren syndrome, where elevated levels of TGF-beta1 transcripts in the conjunctival epithelium have been previously detected.

Structure of the newly found green turtle egg-white ribonuclease.

PubMed

Katekaew, Somporn; Kuaprasert, Buabarn; Torikata, Takao; Kakuta, Yoshimitsu; Kimura, Makoto; Yoneda, Kazunari; Araki, Tomohiro

2010-07-01

Marine green turtle (Chelonia mydas) egg-white ribonuclease (GTRNase) was crystallized from 1.1 M ammonium sulfate pH 5.5 and 30% glycerol using the sitting-drop vapour-diffusion method. The structure of GTRNase has been solved at 1.60 A resolution by the molecular-replacement technique using a model based on the structure of RNase 5 (murine angiogenin) from Mus musculus (46% identity). The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 86.271, b = 34.174, c = 39.738 A, alpha = 90, beta = 102, gamma = 90 degrees . GTRNase consists of three helices and seven beta-strands and displays the alpha+beta folding topology typical of a member of the RNase A superfamily. Superposition of the C(alpha) coordinates of GTRNase and RNase A superfamily members indicates that the overall structure is highly similar to that of angiogenin or RNase 5 from M. musculus (PDB code 2bwl) and RNase A from Bos taurus (PDB code 2blz), with root-mean-square deviations of 3.9 and 2.0 A, respectively. The catalytic residues are conserved with respect to the RNase A superfamily. The three disulfide bridges observed in the reptilian enzymes are conserved in GTRNase, while one further disulfide bond is required for the structural stability of mammalian RNases. GTRNase is expressed in egg white and the fact that its sequence has the highest similarity to that of snapping turtle pancreatic RNase suggests that the GTRNase secreted from oviduct cells to form egg white is probably the product of the same gene as activated in pancreatic cells.

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.

Hb Potomac (101 Glu replaced by Asp): speculations on placental oxygen transport in carriers of high-affinity hemoglobins.

PubMed

Charache, S; Jacobson, R; Brimhall, B; Murphy, E A; Hathaway, P; Winslow, R; Jones, R; Rath, C; Simkovich, J

1978-02-01

Blood from a woman with unexplained erythrocytosis had increased oxygen affinity, but no abnormality could be detected by electrophoresis or chromatography of her hemolysate. Separation of the tryptic peptides of her beta chains disclosed two half-sized peaks in the regions of beta T-11. The faster of these was abnormal, with the structure beta 101 Glu replaced by Asp. The new hemoglobin was called "Potomac." Three of the proband's four surviving siblings and both of her children were carriers. Differences in the ratio of carrier: normal children born to male of female carriers of 23 other high-affinity hemoglobins were not significant. The high proportion of carriers in this kindred was probably due to chance alone, and not because high maternal oxygen affinity interfered with oxygen transport to fetuses with normal hemoglobin.

SIRT1 inhibits proliferation of pancreatic cancer cells expressing pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, by suppression of {beta}-catenin

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

Cho, Il-Rae; Koh, Sang Seok; Department of Functional Genomics, University of Science and Technology, Daejeon 305-333

2012-06-29

Highlights: Black-Right-Pointing-Pointer SIRT1 inhibits protein levels of {beta}-catenin and its transcriptional activity. Black-Right-Pointing-Pointer Nuclear localization of SIRT1 is not required for the decrease of {beta}-catenin expression. Black-Right-Pointing-Pointer SIRT1-mediated degradation of {beta}-catenin is not required for GSK-3{beta} and Siah-1 but for proteosome. Black-Right-Pointing-Pointer SIRT1 activation inhibits proliferation of pancreatic cancer cells expressing PAUF. -- Abstract: Because we found in a recent study that pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, induces a rapid proliferation of pancreatic cells by up-regulation of {beta}-catenin, we postulated that {beta}-catenin might be a target molecule for pancreatic cancer treatment. We thus speculated whether SIRT1, knownmore » to target {beta}-catenin in a colon cancer model, suppresses {beta}-catenin in those pancreatic cancer cells that express PAUF (Panc-PAUF). We further evaluated whether such suppression would lead to inhibition of the proliferation of these cells. The ectopic expression of either SIRT1 or resveratrol (an activator of SIRT1) suppressed levels of {beta}-catenin protein and its transcriptional activity in Panc-PAUF cells. Conversely, suppression of SIRT1 expression by siRNA enhanced {beta}-catenin expression and transcriptional activity. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for reduction of {beta}-catenin. Treatment with MG132, a proteasomal inhibitor, restored {beta}-catenin protein levels, suggesting that SIRT1-mediated degradation of {beta}-catenin requires proteasomal activity. It was reported that inhibition of GSK-3{beta} or Siah-1 stabilizes {beta}-catenin in colon cancer cells, but suppression of GSK-3{beta} or Siah-1 using siRNA in the presence of resveratrol instead diminished {beta}-catenin protein levels in Panc-PAUF cells. This suggests that GSK-3{beta} and Siah-1 are not involved in SIRT1-mediated degradation of {beta}-catenin in the cells. Finally, activation of SIRT1 inhibited the proliferation of Panc-PAUF cells by down-regulation of cyclin-D1, a target molecule of {beta}-catenin. These results suggest that SIRT1 activation may be a therapeutic strategy for treatment of pancreatic cancer cells that express PAUF via the down-regulation of {beta}-catenin.« less

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.

Both conditional ablation and overexpression of E2 SUMO-conjugating enzyme (UBC9) in mouse pancreatic beta cells result in impaired beta cell function.

PubMed

He, Xiaoyu; Lai, Qiaohong; Chen, Cai; Li, Na; Sun, Fei; Huang, Wenting; Zhang, Shu; Yu, Qilin; Yang, Ping; Xiong, Fei; Chen, Zhishui; Gong, Quan; Ren, Boxu; Weng, Jianping; Eizirik, Décio L; Zhou, Zhiguang; Wang, Cong-Yi

2018-04-01

Post-translational attachment of a small ubiquitin-like modifier (SUMO) to the lysine (K) residue(s) of target proteins (SUMOylation) is an evolutionary conserved regulatory mechanism. This modification has previously been demonstrated to be implicated in the control of a remarkably versatile regulatory mechanism of cellular processes. However, the exact regulatory role and biological actions of the E2 SUMO-conjugating enzyme (UBC9)-mediated SUMOylation function in pancreatic beta cells has remained elusive. Inducible beta cell-specific Ubc9 (also known as Ube2i) knockout (KO; Ubc9 Δbeta ) and transgenic (Ubc9 Tg ) mice were employed to address the impact of SUMOylation on beta cell viability and functionality. Ubc9 deficiency or overexpression was induced at 8 weeks of age using tamoxifen. To study the mechanism involved, we closely examined the regulation of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) through SUMOylation in beta cells. Upon induction of Ubc9 deficiency, Ubc9 Δbeta islets exhibited a 3.5-fold higher accumulation of reactive oxygen species (ROS) than Ubc9 f/f control islets. Islets from Ubc9 Δbeta mice also had decreased insulin content and loss of beta cell mass after tamoxifen treatment. Specifically, at day 45 after Ubc9 deletion only 40% of beta cell mass remained in Ubc9 Δbeta mice, while 90% of beta cell mass was lost by day 75. Diabetes onset was noted in some Ubc9 Δbeta mice 8 weeks after induction of Ubc9 deficiency and all mice developed diabetes by 10 weeks following tamoxifen treatment. In contrast, Ubc9 Tg beta cells displayed an increased antioxidant ability but impaired insulin secretion. Unlike Ubc9 Δbeta mice, which spontaneously developed diabetes, Ubc9 Tg mice preserved normal non-fasting blood glucose levels without developing diabetes. It was noted that SUMOylation of NRF2 promoted its nuclear expression along with enhanced transcriptional activity, thereby preventing ROS accumulation in beta cells. SUMOylation function is required to protect against oxidative stress in beta cells; this mechanism is, at least in part, carried out by the regulation of NRF2 activity to enhance ROS detoxification. Homeostatic SUMOylation is also likely to be essential for maintaining beta cell functionality.

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.

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

Elliptical-P cells in the avian perilymphatic interface of the Tegmentum vasculosum

NASA Technical Reports Server (NTRS)

Fermin, C. D.; Lee, D. H.; Martin, D. S.

1995-01-01

Elliptical cells (E-P) are present at the perilymphatic interface lumen (PIL) of the lagena. The E-P cells often separate from the tegmentum vasculosum (TV) and have touching processes that form a monolayer between the K+ rich perilymph and the Na+ rich endolymph, similar to the mammalian Reissner's membrane. We examined the TV of chicks (Gallus domesticus) and quantitated the expression of anti-S100 alphaalphabetabeta and S100 beta. There was a 30% increase of S100 beta saturation in the light cells facing the PIL when compared to other TV light cells. We show that: (1) the dimer anti- S100 alphaalphabetabeta and the monomer anti-S100 beta are expressed preferentially in the light cells and the E-P cells of TV; (2) expression of S100 beta is higher in light cells facing the PIL than in adjacent cells; (3) the expression of the dimer S100 alphaalphabetabeta and monomer S100 beta overlaps in most inner ear cell types, including the cells of the TV, most S100 alphaalphabetabeta positive cells express S 100 beta, but S100 beta positive cells do not always express S100 alphaalphabetabeta; and (4) the S100 beta expression in light cells, the abundant Na+-K+ ATPase on dark cells of the TV, and previously demonstrated co-localization of S100 beta/GABA in sensory cells suggest that S100 beta could have, in the inner ear, a dual neurotrophic-ionic modulating function.

From the rat to the beta cell: a fast and effective technique of separation of Langerhans islets and direct purification of pancreatic beta cells.

PubMed

Tamagno, Gianluca; Vigolo, Simonetta; Olivieri, Massimiliano; Martini, Chiara; De Carlo, Eugenio

2014-01-01

Isolated Langerhans islets represent a useful model for the study of the endocrine pancreas. The possibility to purify pancreatic beta cells from a mixed Langerhans islet cell population may lead towards a dedicated focus on beta cell research. We describe an effective and rapid immunomagnetic technique for the direct purification of beta cells from isolated Langerhans islets of rat. After the sacrifice of the rat, the Langerhans islets were separated by ductal injection of the pancreas with collagenase, altered to a mixed Langerhans islet cell population and incubated with conditioned immunomagnetic beads targeted to the beta cell surface. The beads were previously coated with a specific antibody against the surface of the beta cell, namely K14D10. The suspension of mixed Langerhans islet cells and immunomagnetic K14D10-conditioned beads was pelleted by a magnetic particle concentrator to isolate the bead-bound cells, which were finally suspended in a culture medium. The purified cells were immunoreactive for insulin and no glucagon-positive cells were detected at immunocytochemistry. Real Time PCR confirmed the purification of the pancreatic beta cells. This immunomagnetic technique allows a rapid, effective and consistent purification of beta cells from isolated Langerhans islets in a direct manner by conditioning the immunomagnetic beads only. This technique is easy, fast and reproducible. It promises to be a reliable method for providing purified beta cells for in vitro research.

Expression of S100 beta in sensory and secretory cells of the vertebrate inner ear

NASA Technical Reports Server (NTRS)

Fermin, C. D.; Martin, D. S.

1995-01-01

We evaluated anti-S100 beta expression in the chick (Gallus domesticus) inner ear and determined that: 1) the monomer anti-S100 beta is expressed differentially in the vestibular and auditory perikarya; 2) expression of S100 beta in the afferent nerve terminals is time-related to synapse and myelin formation; 3) the expression of the dimer anti-S100 alpha alpha beta beta and monomer anti-S100 beta overlaps in most inner ear cell types. Most S100 alpha alpha beta beta positive cells express S100 beta, but S100 beta positive cells do not always express S100 alpha alpha beta beta. 4) the expression of S100 beta is diffused over the perikaryal cytoplasm and nuclei of the acoustic ganglia but is concentrated over the nuclei of the vestibular perikarya. 6) S100 beta is expressed in secretory cells, and it is co-localized with GABA in sensory cells. 7) Color thresholding objective quantitation indicates that the amount of S100 beta was higher (mean 22, SD +/- 4) at E19 than at E9 (mean 34, SD +/- 3) in afferent axons. 8) Moreover, S100 beta was unchanged between E11-E19 in the perikaryal cytoplasm, but did change over the nuclei. At E9, 74%, and at E21, 5% of vestibular perikarya were positive. The data suggest that S100 beta may be physically associated with neuronal and ionic controlling cells of the vertebrate inner ear, where it could provide a dual ionic and neurotrophic modulatory function.

Cell surface expression of beta 2-microglobulin (beta 2m) correlates with stages of differentiation in B cell tumours.

PubMed Central

Jones, R A; Scott, C S; Norfolk, D R; Stark, A N; Child, J A

1987-01-01

Cell surface beta 2-microglobulin (beta 2m) densities of malignant B cells were determined by enzyme immunoassay in 97 cases of immunologically defined lymphoproliferative disease. Absolute beta 2m densities were found to depend on disease category with the lowest levels found on cells from chronic lymphocytic leukaemia (mean = 5.6 ng/10(6) cells, n = 27); atypical chronic lymphocytic leukaemia (mean = 5.9 ng/10(6) cells, n = 8); and prolymphocytoid chronic lymphocytic leukaemia variant (mean = 6.0 ng/10(6) cells, n = 16). beta 2m densities for B non-Hodgkin's lymphoma (n = 14) and B prolymphocytic leukaemia (n = 17) cases were 8.1 and 10.0 ng/10(6) cells, respectively, and the highest densities were found on cells from "late-B cell" tumours (mean = 14.3 ng/10(6) cells). Plasma cells from cases of Ig secreting tumours expressed unexpectedly low beta 2m densities (mean = 9.3 ng/10(6) cells; n = 6). PMID:3108331

The chemistry of nicotinamide adenine dinucleotide (NAD) analogues containing C-nucleosides related to nicotinamide riboside.

PubMed

Pankiewicz, Krzysztof W; Watanabe, Kyoichi A; Lesiak-Watanabe, Krystyna; Goldstein, Barry M; Jayaram, Hiremagalur N

2002-04-01

Oncolytic C-nucleosides, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and benzamide riboside (3-beta-D-ribofuranosylbenzamide) are converted in cell into active metabolites thiazole-4-carboxamide- and benzamide adenine dinucleotide, TAD and BAD, respectively. TAD and BAD as NAD analogues were found to bind at the nicotinamide adenine dinucleotide (cofactor NAD) site of inosine monophosphate dehydrogenase (IMPDH), an important target in cancer treatment. The synthesis and evaluation of anticancer activity of a number of C-nucleosides related to tiazofurin and nicotinamide riboside then followed and are reviewed herein. Interestingly, pyridine C-nucleosides (such as C-nicotinamide riboside) are not metabolized into the corresponding NAD analogues in cell. Their conversion by chemical methods is described. As dinucleotides these compounds show inhibition of IMPDH in low micromolar level. Also, the synthesis of BAD in metabolically stable bis(phosphonate) form is discussed indicating the usefulness of such preformed inhibitors in drug development. Among tiazofurin analogues, Franchetti and Grifantini found, that the replacement of the sulfur by oxygen (as in oxazafurin) but not the removal of nitrogen (tiophenfurin) of the thiazole ring resulted in inactive compounds. The anti cancer activity of their synthetic dinucleotide analogues indicate that inactive compounds are not only poorly metabolized in cell but also are weak inhibitors of IMPDH as dinucleotides.

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

PubMed Central

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

1996-01-01

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

Effect of oxygen supply on the size of implantable islet-containing encapsulation devices.

PubMed

Papas, Klearchos K; Avgoustiniatos, Efstathios S; Suszynski, Thomas M

2016-03-01

Beta-cell replacement therapy is a promising approach for the treatment of diabetes but is currently limited by the human islet availability and by the need for systemic immunosuppression. Tissue engineering approaches that will enable the utilization of islets or β-cells from alternative sources (such as porcine islets or human stem cell derived beta cells) and minimize or eliminate the need for immunosuppression have the potential to address these critical limitations. However, tissue engineering approaches are critically hindered by the device size (similar to the size of a large flat screen television) required for efficacy in humans. The primary factor dictating the device size is the oxygen availability to islets to support their viability and function (glucose-stimulated insulin secretion [GSIS]). GSIS is affected (inhibited) at a much higher oxygen partial pressure [pO2] than that of viability (e.g. 10 mmHg as opposed to 0.1 mmHg). Enhanced oxygen supply (higher pO2) than what is available in vivo at transplant sites can have a profound effect on the required device size (potentially reduce it to the size of a postage stamp). This paper summarizes key information on the effect of oxygen on islet viability and function within immunoisolation devices and describes the potential impact of enhanced oxygen supply to devices in vivo on device size reduction.

Demonstration of interleukin-1 beta transcripts in acute myeloblastic leukemic cells by in situ hybridization.

PubMed

Nakamura, M; Kanakura, Y; Furukawa, Y; Ernst, T J; Griffin, J D

1990-07-01

The cells from some patients with acute myeloblastic leukemia will secrete autostimulatory cytokines in tissue culture without the addition of stimulators such as phorbol 12-myristate 13-acetate. Production of interleukin-1 beta (IL-1 beta), for example, has been observed in up to 50% of cases. In order to investigate the nature of the cell secreting IL-1 beta in AML, we used an antisense RNA probe to detect specific IL-1 beta transcripts in individual leukemic cells by in situ hybridization. In fresh, uncultured cells, IL-1 beta transcripts were observed in 1-40% of undifferentiated leukemic blast cells in 17 of 19 cases. In situ hybridization was at least as sensitive as Northern blot analysis in detecting IL-1 beta transcripts. No correlation of IL-1 beta transcript expression with FAB classification was observed. Normal blood and bone marrow mononuclear cells did not contain cells expressing IL-1 beta transcripts. These results support the concept that the regulation of cytokine genes in AML cells is aberrant.

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

Inhibition of AmpC beta-lactamase through a destabilizing interaction in the active site

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

Trehan, I.; Beadle, B.M.; Shoichet, B.K.

2010-03-08

{beta}-Lactamases hydrolyze {beta}-lactam antibiotics, including penicillins and cephalosporins; these enzymes are the most widespread resistance mechanism to these drugs and pose a growing threat to public health. {beta}-Lactams that contain a bulky 6(7){alpha} substituent, such as imipenem and moxalactam, actually inhibit serine {beta}-lactamases and are widely used for this reason. Although mutant serine {beta}-lactamases have arisen that hydrolyze {beta}-lactamase resistant {beta}-lactams (e.g., ceftazidime) or avoid mechanism-based inhibitors (e.g., clavulanate), mutant serine {beta}-lactamases have not yet arisen in the clinic with imipenemase or moxalactamase activity. Structural and thermodynamic studies suggest that the 6(7){alpha} substituents of these inhibitors form destabilizing contacts withinmore » the covalent adduct with the conserved Asn152 in class C {beta}-lactamases (Asn132 in class A {beta}-lactamases). This unfavorable interaction may be crucial to inhibition. To test this destabilization hypothesis, we replaced Asn152 with Ala in the class C {beta}-lactamase AmpC from Escherichia coli and examined the mutant enzyme's thermodynamic stability in complex with imipenem and moxalactam. Consistent with the hypothesis, the Asn152 {yields} Ala substitution relieved 0.44 and 1.10 kcal/mol of strain introduced by imipenem and moxalactam, respectively, relative to the wild-type complexes. However, the kinetic efficiency of AmpC N152A was reduced by 6300-fold relative to that of the wild-type enzyme. To further investigate the inhibitor's interaction with the mutant enzyme, the X-ray crystal structure of moxalactam in complex with N152A was determined to a resolution of 1.83 {angstrom}. Moxalactam in the mutant complex is significantly displaced from its orientation in the wild-type complex; however, moxalactam does not adopt an orientation that would restore competence for hydrolysis. Although Asn152 forces {beta}-lactams with 6(7){alpha} substituents out of a catalytically competent configuration, making them inhibitors, the residue is essential for orienting {beta}-lactam substrates and cannot simply be replaced with a much smaller residue to restore catalytic activity. Designing {beta}-lactam inhibitors that interact unfavorably with this conserved residue when in the covalent adduct merits further investigation.« less

Antidiabetic and Beta Cell-Protection Activities of Purple Corn Anthocyanins

PubMed Central

Hong, Su Hee; Heo, Jee-In; Kim, Jeong-Hyeon; Kwon, Sang-Oh; Yeo, Kyung-Mok; Bakowska-Barczak, Anna M.; Kolodziejczyk, Paul; Ryu, Ok-Hyun; Choi, Moon-Ki; Kang, Young-Hee; Lim, Soon Sung; Suh, Hong-Won; Huh, Sung-Oh; Lee, Jae-Yong

2013-01-01

Antidiabetic and beta cell-protection activities of purple corn anthocyanins (PCA) were examined in pancreatic beta cell culture and db/db mice. Only PCA among several plant anthocyanins and polyphenols showed insulin secretion activity in culture of HIT-T15 cells. PCA had excellent antihyperglycemic activity (in terms of blood glucose level and OGTT) and HbA1c-decreasing activity when compared with glimepiride, a sulfonylurea in db/db mice. In addition, PCA showed efficient protection activity of pancreatic beta cell from cell death in HIT-T15 cell culture and db/db mice. The result showed that PCA had antidiabetic and beta cell-protection activities in pancreatic beta cell culture and db/db mice. PMID:24244813

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

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

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

2010-07-30

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

Trafficking of cell surface beta-amyloid precursor protein: retrograde and transcytotic transport in cultured neurons

PubMed Central

1995-01-01

Amyloid beta-protein (A beta), the principal constituent of senile plaques seen in Alzheimer's disease (AD), is derived by proteolysis from the beta-amyloid precursor protein (beta PP). The mechanism of A beta production in neurons, which are hypothesized to be a rich source of A beta in brain, remains to be defined. In this study, we describe a detailed localization of cell surface beta PP and its subsequent trafficking in primary cultured neurons. Full-length cell surface beta PP was present primarily on perikarya and axons, the latter with a characteristic discontinuous pattern. At growth cones, cell surface beta PP was inconsistently detected. By visualizing the distribution of beta PP monoclonal antibodies added to intact cultures, beta PP was shown to be internalized from distal axons or terminals and retrogradely transported back to perikarya in organelles which colocalized with fluid-phase endocytic markers. Retrograde transport of beta PP was shown in both hippocampal and peripheral sympathetic neurons, the latter using a compartment culture system that isolated cell bodies from distal axons and terminals. In addition, we demonstrated that beta PP from distal axons was transcytotically transported to the surface of perikarya from distal axons in sympathetic neurons. Indirect evidence of this transcytotic pathway was obtained in hippocampal neurons using antisense oligonucleotide to the kinesin heavy chain to inhibit anterograde beta PP transport. Taken together, these results demonstrate novel aspects of beta PP trafficking in neurons, including retrograde axonal transport and transcytosis. Moreover, the axonal predominance of cell surface beta PP is unexpected in view of the recent report of polarized sorting of beta PP to the basolateral domain of MDCK cells. PMID:7721945

Reproductive factors, hormone use, estrogen receptor expression and risk of non small-cell lung cancer in women.

PubMed

Schwartz, Ann G; Wenzlaff, Angela S; Prysak, Geoffrey M; Murphy, Valerie; Cote, Michele L; Brooks, Sam C; Skafar, Debra F; Lonardo, Fulvio

2007-12-20

Estrogen receptor (ER) expression in lung tumors suggests that estrogens may play a role in the development of lung cancer. We evaluated the role of hormone-related factors in determining risk of non-small-cell lung cancer (NSCLC) in women. We also evaluated whether risk factors were differentially associated with cytoplasmic ER-alpha and/or nuclear ER-beta expression-defined NSCLC in postmenopausal women. Population-based participants included women aged 18 to 74 years diagnosed with NSCLC in metropolitan Detroit between November 1, 2001 and October 31, 2005. Population-based controls were identified through random digit dialing, matched to patient cases on race and 5-year age group. Interview data were analyzed for 488 patient cases (241 with tumor ER results) and 498 controls. Increased duration of hormone replacement therapy (HRT) use in quartiles was associated with decreased risk of NSCLC in postmenopausal women (odds ratio = 0.88; 95% CI, 0.78 to 1.00; P = .04), adjusting for age, race, pack-years, education, family history of lung cancer, current body mass index, years exposed to second-hand smoke in the workplace, and obstructive lung disease history. Among postmenopausal women, ever using HRT, increasing HRT duration of use in quartiles, and increasing quartiles of estrogen use were significant predictors of reduced risk of NSCLC characterized as ER-alpha and/or ER-beta positive. None of the hormone-related variables were associated with nuclear ER-alpha- or ER-beta-negative NSCLC. These findings suggest that postmenopausal hormone exposures are associated with reduced risk of ER-alpha- and ER-beta-expressing NSCLC. Understanding tumor characteristics may direct development of targeted treatment for this disease.

Hydrogel-beta-TCP scaffolds and stem cells for tissue engineering bone.

PubMed

Weinand, Christian; Pomerantseva, Irina; Neville, Craig M; Gupta, Rajiv; Weinberg, Eli; Madisch, Ijad; Shapiro, Frederic; Abukawa, Harutsugi; Troulis, Maria J; Vacanti, Joseph P

2006-04-01

Trabecular bone is a material of choice for reconstruction after trauma and tumor resection and for correction of congenital defects. Autologous bone grafts are available in limited shapes and sizes; significant donor site morbidity is another major disadvantage to this approach. To overcome these limitations, we used a tissue engineering approach to create bone replacements in vitro, combining bone-marrow-derived differentiated mesenchymal stem cells (MSCs) suspended in hydrogels and 3-dimensionally printed (3DP) porous scaffolds made of beta-tricalcium-phosphate (beta-TCP). The scaffolds provided support for the formation of bone tissue in collagen I, fibrin, alginate, and pluronic F127 hydrogels during culturing in oscillating and rotating dynamic conditions. Histological evaluation including toluidine blue, alkaline phosphatase, and von Kossa staining was done at 1, 2, 4, and 6 weeks. Radiographic evaluation and high-resolution volumetric CT (VCT) scanning, expression of bone-specific genes and biomechanical compression testing were performed at 6 weeks. Both culture conditions resulted in similar bone tissue formation. Histologically collagen I and fibrin hydrogels specimens had superior bone tissue, although radiopacities were detected only in collagen I samples. VCT scan revealed density values in all but the Pluronic F127 samples, with Houndsfield unit values comparable to native bone in collagen I and fibrin glue samples. Expression of bone-specific genes was significantly higher in the collagen I samples. Pluronic F127 hydrogel did not support formation of bone tissue. All samples cultured in dynamic oscillating conditions had slightly higher mechanical strength than under rotating conditions. Bone tissue can be successfully formed in vitro using constructs comprised of collagen I hydrogel, MSCs, and porous beta-TCP scaffolds.

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

Age-related islet inflammation marks the proliferative decline of pancreatic beta-cells in zebrafish

PubMed Central

Tsakmaki, Anastasia; Mousavy Gharavy, S Neda; Murawala, Priyanka; Konantz, Judith; Birke, Sarah; Hodson, David J; Rutter, Guy A; Bewick, Gavin A

2018-01-01

The pancreatic islet, a cellular community harboring the insulin-producing beta-cells, is known to undergo age-related alterations. However, only a handful of signals associated with aging have been identified. By comparing beta-cells from younger and older zebrafish, here we show that the aging islets exhibit signs of chronic inflammation. These include recruitment of tnfα-expressing macrophages and the activation of NF-kB signaling in beta-cells. Using a transgenic reporter, we show that NF-kB activity is undetectable in juvenile beta-cells, whereas cells from older fish exhibit heterogeneous NF-kB activity. We link this heterogeneity to differences in gene expression and proliferation. Beta-cells with high NF-kB signaling proliferate significantly less compared to their neighbors with low activity. The NF-kB signalinghi cells also exhibit premature upregulation of socs2, an age-related gene that inhibits beta-cell proliferation. Together, our results show that NF-kB activity marks the asynchronous decline in beta-cell proliferation with advancing age. PMID:29624168

Reduced Expression of the Liver/Beta-Cell Glucose Transporter Isoform in Glucose-Insensitive Pancreatic Beta Cells of Diabetic Rats

NASA Astrophysics Data System (ADS)

Thorens, Bernard; Weir, Gordon C.; Leahy, John L.; Lodish, Harvey F.; Bonner-Weir, Susan

1990-09-01

Rats injected with a single dose of streptozocin at 2 days of age develop non-insulin-dependent diabetes 6 weeks later. The pancreatic beta islet cells of these diabetic rats display a loss of glucose-induced insulin secretion while maintaining sensitivity to other secretagogues such as arginine. We analyzed the level of expression of the liver/beta-cell glucose transporter isoform in diabetic islets by immunofluorescence staining of pancreas sections and by Western blotting of islet lysates. Islets from diabetic animals have a reduced expression of this beta-cell-specific glucose transporter isoform and the extent of reduction is correlated with the severity of hyperglycemia. In contrast, expression of this transporter isoform in liver is minimally modified by the diabetes. Thus a decreased expression of the liver/beta-cell glucose transporter isoform in beta cells is associated with the impaired glucose sensing characteristic of diabetic islets; our data suggest that this glucose transporter may be part of the beta-cell glucose sensor.

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

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

Liang Pengfei; Jiang Bimei; Yang Xinghua

2008-10-15

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

Enzyme therapy in Fabry disease: severe adverse events associated with anti-agalsidase cross-reactive IgG antibodies.

PubMed

Tesmoingt, Chloe; Lidove, Olivier; Reberga, Axele; Thetis, Marguerite; Ackaert, Chloe; Nicaise, Pascale; Arnaud, Philippe; Papo, Thomas

2009-11-01

To report a severe adverse event related to enzyme replacement therapy with agalsidase in an hemizygous male patient treated for Fabry disease. Retrospective analysis of clinical, radiological and biochemical data in a patient who suffered adverse events related to both agalsidase alfa and agalsidase beta treatments. A hemizygous male patient was first treated for Fabry disease with agalsidase alfa. After more than 1 year of therapy, infusion-related symptoms necessitated systemic steroids and antihistaminic therapy. Decline in kidney function prompted a switch for agalsidase beta. Anaphylactoid shock occurred after the second infusion. No serum IgE antibodies were disclosed. Skin-test reactivity to agalsidase beta was negative. Following a published rechallenge infusion protocol, agalsidase beta was reintroduced, leading to a second anaphylactoid shock episode. Enzyme replacement therapy was stopped and the patient was treated with symptomatic therapy only. This case was referred to the pharmacovigilance department. The negativity of immunological tests (specific anti-agalsidase IgE antibodies and skin tests) does not rule out the risk of repeated anaphylactoid shock following agalsidase infusion.

Enzyme therapy in Fabry disease: severe adverse events associated with anti-agalsidase cross-reactive IgG antibodies

PubMed Central

Tesmoingt, Chloe; Lidove, Olivier; Reberga, Axele; Thetis, Marguerite; Ackaert, Chloe; Nicaise, Pascale; Arnaud, Philippe; Papo, Thomas

2009-01-01

AIMS To report a severe adverse event related to enzyme replacement therapy with agalsidase in an hemizygous male patient treated for Fabry disease. METHODS Retrospective analysis of clinical, radiological and biochemical data in a patient who suffered adverse events related to both agalsidase alfa and agalsidase beta treatments. RESULTS A hemizygous male patient was first treated for Fabry disease with agalsidase alfa. After more than 1 year of therapy, infusion-related symptoms necessitated systemic steroids and antihistaminic therapy. Decline in kidney function prompted a switch for agalsidase beta. Anaphylactoid shock occurred after the second infusion. No serum IgE antibodies were disclosed. Skin-test reactivity to agalsidase beta was negative. Following a published rechallenge infusion protocol, agalsidase beta was reintroduced, leading to a second anaphylactoid shock episode. Enzyme replacement therapy was stopped and the patient was treated with symptomatic therapy only. This case was referred to the pharmacovigilance department. CONCLUSION The negativity of immunological tests (specific anti-agalsidase IgE antibodies and skin tests) does not rule out the risk of repeated anaphylactoid shock following agalsidase infusion. PMID:19917001

Partitioning diversity into independent alpha and beta components.

PubMed

Jost, Lou

2007-10-01

Existing general definitions of beta diversity often produce a beta with a hidden dependence on alpha. Such a beta cannot be used to compare regions that differ in alpha diversity. To avoid misinterpretation, existing definitions of alpha and beta must be replaced by a definition that partitions diversity into independent alpha and beta components. Such a unique definition is derived here. When these new alpha and beta components are transformed into their numbers equivalents (effective numbers of elements), Whittaker's multiplicative law (alpha x beta = gamma) is necessarily true for all indices. The new beta gives the effective number of distinct communities. The most popular similarity and overlap measures of ecology (Jaccard, Sorensen, Horn, and Morisita-Horn indices) are monotonic transformations of the new beta diversity. Shannon measures follow deductively from this formalism and do not need to be borrowed from information theory; they are shown to be the only standard diversity measures which can be decomposed into meaningful independent alpha and beta components when community weights are unequal.

Relationships of pancreatic beta-cell function with microalbuminuria and glomerular filtration rate in middle-aged and elderly population without type 2 diabetes mellitus: a Chinese community-based analysis.

PubMed

Fu, Shihui; Zhou, Shanjing; Luo, Leiming; Ye, Ping

2017-01-01

Relationships of pancreatic beta-cell function abnormality with microalbuminuria (MA) and glomerular filtration rate (GFR) may differ by age, ethnicity and accompanied diseases. Previous studies were generally conducted in Western adult patients with type 2 diabetes mellitus (T2DM), and it is uncertain whether pancreatic beta-cell function is associated with MA and GFR in Chinese community-dwelling middle-aged and elderly population without T2DM. We therefore examined the relationships of pancreatic beta-cell function with two indices of renal damage, MA and GFR, in Chinese community-dwelling middle-aged and elderly population without T2DM. This analysis focused on 380 Beijing residents older than 45 years who were free of T2DM and completed the evaluation of pancreatic beta-cell function. Median age was 67 (49-80) years. Levels of triglyceride, diastolic blood pressure and homeostasis model assessment-beta (HOMA-beta) index were positively related to urine microalbumin ( P <0.05 for all). Age, low-density lipoprotein cholesterol levels and HOMA-beta index were inversely correlated with GFR, while high-density lipoprotein cholesterol levels were positively correlated with GFR ( P <0.05 for all). In all three adjustment models, there was a significant positive association between HOMA-beta index and MA; subjects with higher beta-cell function had higher odds of MA ( P <0.05 for all). There was no association between HOMA-beta index and GFR <60 mL/min/1.73 m 2 in any model ( P >0.05 for all). Modeling the pancreatic beta-cell function with different adjusted variables provided the same conclusion of association with MA; beta-cell function was positively associated with MA. Additionally, there was a specific difference in the adjusted associations of pancreatic beta-cell function with MA and GFR <60 mL/min/1.73 m 2 ; beta-cell function was not independently associated with GFR <60 mL/min/1.73 m 2 . This result indicated that abnormal pancreatic beta-cell function plays an important role in the development of MA.

TGF{beta} induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

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

Ebi, Masahide; Kataoka, Hiromi, E-mail: hkataoka@med.nagoya-cu.ac.jp; Shimura, Takaya

2010-11-19

Research highlights: {yields} TGF{beta} induces EGFR transactivation through proHB-EGF shedding by activated ADAM members in gastric cancer cells. {yields} TGF{beta} induces nuclear translocation of HB-EGF-CTF cleaved by ADAM members. {yields} TGF{beta} enhances cell growth by EGFR transactivation and HB-EGF-CTF nuclear translocation and ADAM inhibitors block these effects. {yields} Silencing of ADAM17 also blocks EGFR transactivation, HB-EGF-CTF nuclear translocation and cancer cell growth by TGF{beta}. {yields} ADAM17 may play a crucial role in this TGF{beta}-HB-EGF signal transduction. -- Abstract: Background and aims: Transforming growth factor-beta (TGF{beta}) is known to potently inhibit cell growth. Loss of responsiveness to TGF{beta} inhibition on cellmore » growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGF{beta} and HB-EGF signal transduction via ADAM activation. Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGF{beta}. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGF{beta} was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGF{beta} was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown. Result: TGF{beta}-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGF{beta} induced shedding of proHB-EGF allowing HB-EGF-CTF to translocate to the nucleus. ADAM inhibitors blocked this nuclear translocation. TGF{beta} enhanced gastric cancer cell growth and ADAM inhibitors suppressed this effect. EGFR phosphorylation, HB-EGF-CTF nuclear translocation, and cell growth were suppressed in ADAM17 knockdown cells. Conclusion: HB-EGF-CTF nuclear translocation and EGFR transactivation from proHB-EGF shedding mediated by ADAM17 activated by TGF{beta} might be an important pathway of gastric cancer cell proliferation by TGF{beta}.« less

Neurotensin protects pancreatic beta cells from apoptosis.

PubMed

Coppola, Thierry; Béraud-Dufour, Sophie; Antoine, Aurélie; Vincent, Jean-Pierre; Mazella, Jean

2008-01-01

The survival of pancreatic beta cells depends on the balance between external cytotoxic and protective molecular systems. The neuropeptide neurotensin (NT) has been shown to regulate certain functions of the endocrine pancreas including insulin and glucagon release. However, the mechanism of action of NT as well as the identification of receptors involved in the pancreatic functions of the peptide remained to be studied. We demonstrate here that NT is an efficient protective agent of pancreatic beta cells against cytotoxic agents. Both beta-TC3 and INS-1E cell lines and the mouse pancreatic islet cells express the three known NT receptors. The incubation of beta cells with NT protects cells from apoptosis induced either by staurosporine or by IL-1beta. In beta-TC3 cells, NT activates both MAP and PI-3 kinases pathways and strongly reduces the staurosporine or the Il-1beta-induced caspase-3 activity by a mechanism involving Akt activation. The NTSR2 agonist levocabastine displays the same protective effect than NT whereas the NTSR1 antagonist is unable to block the effect of NT suggesting the predominant involvement of the NTSR2 in the action of NT on beta cells. These results clearly indicate for the first time that NT is able to protect endocrine beta cells from external cytotoxic agents, a role well correlated with its release in the circulation after a meal.

Pancreatic islets and insulinoma cells express a novel isoform of group VIA phospholipase A2 (iPLA2 beta) that participates in glucose-stimulated insulin secretion and is not produced by alternate splicing of the iPLA2 beta transcript.

PubMed

Ramanadham, Sasanka; Song, Haowei; Hsu, Fong-Fu; Zhang, Sheng; Crankshaw, Mark; Grant, Gregory A; Newgard, Christopher B; Bao, Shunzhong; Ma, Zhongmin; Turk, John

2003-12-02

Many cells express a group VIA 84 kDa phospholipase A(2) (iPLA(2)beta) that is sensitive to inhibition by a bromoenol lactone (BEL) suicide substrate. Inhibition of iPLA(2)beta in pancreatic islets and insulinoma cells suppresses, and overexpression of iPLA(2)beta in INS-1 insulinoma cells amplifies, glucose-stimulated insulin secretion, suggesting that iPLA(2)beta participates in secretion. Western blotting analyses reveal that glucose-responsive 832/13 INS-1 cells express essentially no 84 kDa iPLA(2)beta-immunoreactive protein but predominantly express a previously unrecognized immunoreactive iPLA(2)beta protein in the 70 kDa region that is not generated by a mechanism of alternate splicing of the iPLA(2)beta transcript. To determine if the 70 kDa-immunoreactive protein is a short isoform of iPLA(2)beta, protein from the 70 kDa region was digested with trypsin and analyzed by mass spectrometry. Such analyses reveal several peptides with masses and amino acid sequences that exactly match iPLA(2)beta tryptic peptides. Peptide sequences identified in the 70 kDa tryptic digest include iPLA(2)beta residues 7-53, suggesting that the N-terminus is preserved. We also report here that the 832/13 INS-1 cells express iPLA(2)beta catalytic activity and that BEL inhibits secretagogue-stimulated insulin secretion from these cells but not the incorporation of arachidonic acid into membrane PC pools of these cells. These observations suggest that the catalytic iPLA(2)beta activity expressed in 832/13 INS-1 cells is attributable to a short isoform of iPLA(2)beta and that this isoform participates in insulin secretory but not in membrane phospholipid remodeling pathways. Further, the finding that pancreatic islets also express predominantly a 70 kDa iPLA(2)beta-immunoreactive protein suggests that a signal transduction role of iPLA(2)beta in the native beta-cell might be attributable to a 70 kDa isoform of iPLA(2)beta.

(beta)-catenin mediates the specification of endoderm cells in ascidian embryos.

PubMed

Imai, K; Takada, N; Satoh, N; Satou, Y

2000-07-01

In the present study, we addressed the role of (beta)-catenin in the specification of embryonic cells of the ascidians Ciona intestinalis and C. savignyi and obtained the following results: (1) During cleavages, (beta)-catenin accumulated in the nuclei of vegetal blastomeres, suggesting that it plays a role in the specification of endoderm. (2) Mis- and/or overexpression of (beta)-catenin induced the development of an endoderm-specific alkaline phosphatase (AP) in presumptive notochord cells and epidermis cells without affecting differentiation of primary lineage muscle cells. (3) Downregulation of (beta)-catenin induced by the overexpression of cadherin resulted in the suppression of endoderm cell differentiation. This suppression was compensated for by the differentiation of extra epidermis cells. (4) Specification of notochord cells did not take place in the absence of endoderm differentiation. Both the overexpression of (beta)-catenin in presumptive notochord cells and the downregulation of (beta)-catenin in presumptive endoderm cells led to the suppression of Brachyury gene expression, resulting in the failure of notochord specification. These results suggest that the accumulation of (beta)-catenin in the nuclei of endoderm progenitor cells is the first step in the process of ascidian endoderm specification.

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.

Immunological characterization of eristostatin and echistatin binding sites on alpha IIb beta 3 and alpha V beta 3 integrins.

PubMed Central

Marcinkiewicz, C; Rosenthal, L A; Mosser, D M; Kunicki, T J; Niewiarowski, S

1996-01-01

Two disintegrins with a high degree of amino acid sequence similarity, echistatin and eristostatin, showed a low level of interaction with Chinese hamster ovary (CHO) cells, but they bound to CHO cells transfected with alpha IIb beta 3 genes (A5 cells) and to CHO cells transfected with alpha v beta 3 genes (VNRC3 cells) in a reversible and saturable manner. Scatchard analysis revealed that eristostatin bound to 816000 sites per A5 cell (Kd 28 nM) and to 200000 sites (Kd 14 nM) per VNRC3 cell respectively. However, VNRC3 cells did not bind to immobilized eristostatin. Echistatin bound to 495000 sites (Kd 53 nM) per A5 cell and to 443000 sites (Kd 20 nM) per VNRC3 cell. As determined by flow cytometry, radiobinding assay and adhesion studies, binding of both disintegrins to A5 cells and resting platelets and binding of echistatin to VNRC3 cells resulted in the expression of ligand-induced binding sites (LIBS) on the beta 3 subunit. Eristostatin inhibited, more strongly than echistatin, the binding of three monoclonal antibodies: OPG2 (RGD motif dependent), A2A9 (alpha IIb beta 3 complex dependent) and 7E3 (alpha IIb beta 3 and alpha v beta 3 complex dependent) to A5 cells, to resting and to activated platelets and to purified alpha IIb beta 3. Experiments in which echistatin and eristostatin were used alone or in combination to inhibit the binding of 7E3 and OPG2 antibodies to resting platelets suggested that these two disintegrins bind to different but overlapping sites on alpha IIb beta 3 integrin. Monoclonal antibody LM 609 and echistatin seemed to bind to different sites on alpha v beta 3 integrin. However, echistatin inhibited binding of 7E3 antibody to VNRC3 cells and to purified alpha v beta 3 suggesting that alpha v beta 3 and alpha IIb beta 3 might share the same epitope to which both echistatin and 7E3 bind. Eristostatin had no effect in these systems, providing further evidence that it binds to a different epitope on alpha v beta 3. PMID:8760368

Evaluation of a low dose, after a standard therapeutic dose, of agalsidase beta during enzyme replacement therapy in patients with Fabry disease.

PubMed

Lubanda, Jean-Claude; Anijalg, Ene; Bzdúch, Vladimír; Thurberg, Beth L; Bénichou, Bernard; Tylki-Szymanska, Anna

2009-04-01

Fabry disease, a genetic deficiency of alpha-galactosidase A, is characterized by pathogenic cellular accumulation of globotriaosylceramide. During clinical trials, recombinant human alpha-galactosidase A (agalsidase beta; Fabrazyme, Genzyme Corporation, Cambridge, MA), infused intravenously at 1.0 mg/kg every 2 weeks for 6 months, cleared or reduced globotriaosylceramide in renal, cardiac, and dermal microvascular endothelia and other cells, with results sustained for up to 5 years in most patients evaluated. This study explored whether a lower dose could maintain globotriaosylceramide clearance achieved with 1.0 mg/kg. Cellular globotriaosylceramide levels were assessed histologically in kidney and skin biopsies from 21 adult Fabry males treated for 6 months at 1.0 mg/kg/2 weeks followed by 18 months at 0.3 mg/kg/2 weeks. In kidney interstitial capillary endothelium, the primary endpoint, globotriaosylceramide clearance was achieved in 100% of patients with 1.0 mg/kg and maintained in 90% with 0.3 mg/kg. In seven other renal cell types and superficial dermal capillary endothelium, globotriaosylceramide reduction or clearance was maintained with 0.3 mg/kg in approximately 70% of patients. A lower dose of agalsidase beta may be sufficient in some, but not all, patients with Fabry disease to maintain the cellular globotriaosylceramide clearance achieved with 1.0 mg/kg/2 weeks. Long-term clinical effects of transitioning to the lower dose have not been evaluated.

TGF-beta1 expression in EL4 lymphoma cells overexpressing growth hormone.

PubMed

Farmer, John T; Weigent, Douglas A

2006-03-01

Our previous studies show that growth hormone overexpression (GHo) upregulates the expression of the IGF-1R and IGF-2R resulting in the protection of the EL4 lymphoma cell line from apoptosis. In this study, we report that GHo also increases TGF-beta1 protein expression measured by luciferase promoter assay, Western analysis, and ELISA. Further, the data show that antibody to TGF-betaR2 decreases TGF-beta1 promoter activity to the level of vector alone control cells. GHo cells treated with (125)I-rh-latent TGF-beta1 showed increased activation of latent TGF-beta1 as measured by an increase in the active 24kDa, TGF-beta1 compared to vector alone control cells. The ability of endogenous GH to increase TGF-beta1 expression is blocked in EL4 cells by antisense but not sense oligodeoxynucleotides or in cells cultured with antibody to growth hormone (GH). The data suggest that endogenous GH may protect from apoptosis through the IGF-1R receptor while limiting cellular growth through increased expression and activation of TGF-beta1.

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

Genetic models rule out a major role of beta cell glycogen in the control of glucose homeostasis.

PubMed

Mir-Coll, Joan; Duran, Jordi; Slebe, Felipe; García-Rocha, Mar; Gomis, Ramon; Gasa, Rosa; Guinovart, Joan J

2016-05-01

Glycogen accumulation occurs in beta cells of diabetic patients and has been proposed to partly mediate glucotoxicity-induced beta cell dysfunction. However, the role of glycogen metabolism in beta cell function and its contribution to diabetes pathophysiology remain poorly understood. We investigated the function of beta cell glycogen by studying glucose homeostasis in mice with (1) defective glycogen synthesis in the pancreas; and (2) excessive glycogen accumulation in beta cells. Conditional deletion of the Gys1 gene and overexpression of protein targeting to glycogen (PTG) was accomplished by Cre-lox recombination using pancreas-specific Cre lines. Glucose homeostasis was assessed by determining fasting glycaemia, insulinaemia and glucose tolerance. Beta cell mass was determined by morphometry. Glycogen was detected histologically by periodic acid-Schiff's reagent staining. Isolated islets were used for the determination of glycogen and insulin content, insulin secretion, immunoblots and gene expression assays. Gys1 knockout (Gys1 (KO)) mice did not exhibit differences in glucose tolerance or basal glycaemia and insulinaemia relative to controls. Insulin secretion and gene expression in isolated islets was also indistinguishable between Gys1 (KO) and controls. Conversely, despite effective glycogen overaccumulation in islets, mice with PTG overexpression (PTG(OE)) presented similar glucose tolerance to controls. However, under fasting conditions they exhibited lower glycaemia and higher insulinaemia. Importantly, neither young nor aged PTG(OE) mice showed differences in beta cell mass relative to age-matched controls. Finally, a high-fat diet did not reveal a beta cell-autonomous phenotype in either model. Glycogen metabolism is not required for the maintenance of beta cell function. Glycogen accumulation in beta cells alone is not sufficient to trigger the dysfunction or loss of these cells, or progression to diabetes.

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

PubMed Central

1987-01-01

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

Mechanistic studies of cancer cell mitochondria- and NQO1-mediated redox activation of beta-lapachone, a potentially novel anticancer agent

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

Li, Jason Z.; Ke, Yuebin; Misra, Hara P.

Beta-lapachone (beta-Lp) derived from the Lapacho tree is a potentially novel anticancer agent currently under clinical trials. Previous studies suggested that redox activation of beta-Lp catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) accounted for its killing of cancer cells. However, the exact mechanisms of this effect remain largely unknown. Using chemiluminescence and electron paramagnetic resonance (EPR) spin-trapping techniques, this study for the first time demonstrated the real-time formation of ROS in the redox activation of beta-lapachone from cancer cells mediated by mitochondria and NQO1 in melanoma B16–F10 and hepatocellular carcinoma HepG2 cancer cells. ES936, a highly selective NQO1 inhibitor, and rotenone,more » a selective inhibitor of mitochondrial electron transport chain (METC) complex I were found to significantly block beta-Lp meditated redox activation in B16–F10 cells. In HepG2 cells ES936 inhibited beta-Lp-mediated oxygen radical formation by ∼ 80% while rotenone exerted no significant effect. These results revealed the differential contribution of METC and NQO1 to beta-lapachone-induced ROS formation and cancer cell killing. In melanoma B16–F10 cells that do not express high NQO1 activity, both NOQ1 and METC play a critical role in beta-Lp redox activation. In contrast, in hepatocellular carcinoma HepG2 cells expressing extremely high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1 (METC plays a minor role). These findings will contribute to our understanding of how cancer cells are selectively killed by beta-lapachone and increase our ability to devise strategies to enhance the anticancer efficacy of this potentially novel drug while minimizing its possible adverse effects on normal cells. - Highlights: • Both isolated mitochondria and purified NQO1 are able to generate ROS by beta-Lp. • The differential roles of mitochondria and NQO1 in mediating redox activation of beta-Lp • In cancer cells with low NQO1 expression, mitochondria play a critical role in beta-Lp redox activation. • In cancer cells with high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1.« less

Chronology of endocrine differentiation and beta-cell neogenesis.

PubMed

Miyatsuka, Takeshi

2016-01-01

Diabetes is a chronic and incurable disease, which results from absolute or relative insulin insufficiency. Therefore, pancreatic beta cells, which are the only type of cell that expresses insulin, is considered to be a potential target for the cure of diabetes. Although the findings regarding beta-cell neogenesis during pancreas development have been exploited to induce insulin-producing cells from non-beta cells, there are still many hurdles towards generating fully functional beta cells that can produce high levels of insulin and respond to physiological signals. To overcome these problems, a solid understanding of pancreas development and beta-cell formation is required, and several mouse models have been developed to reveal the unique features of each endocrine cell type at distinct developmental time points. Here I review our understanding of pancreas development and endocrine differentiation focusing on recent progresses in improving temporal cell labeling in vivo.

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

PubMed

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

2005-06-01

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

Competition between Ski and CREB-binding protein for binding to Smad proteins in transforming growth factor-beta signaling.

PubMed

Chen, Weijun; Lam, Suvana S; Srinath, Hema; Schiffer, Celia A; Royer, William E; Lin, Kai

2007-04-13

The family of Smad proteins mediates transforming growth factor-beta (TGF-beta) signaling in cell growth and differentiation. Smads repress or activate TGF-beta signaling by interacting with corepressors (e.g. Ski) or coactivators (e.g. CREB-binding protein (CBP)), respectively. Specifically, Ski has been shown to interfere with the interaction between Smad3 and CBP. However, it is unclear whether Ski competes with CBP for binding to Smads and whether they can interact with Smad3 at the same binding surface on Smad3. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, we show that Ski-(16-192) interacted directly with a homotrimer of receptor-regulated Smad protein (R-Smad), e.g. Smad2 or Smad3, to form a hexamer; Ski-(16-192) interacted with an R-Smad.Smad4 heterotrimer to form a pentamer. CBP-(1941-1992) was also found to interact directly with an R-Smad homotrimer to form a hexamer and with an R-Smad.Smad4 heterotrimer to form a pentamer. Moreover, these domains of Ski and CBP competed with each other for binding to Smad3. Our mutational studies revealed that domains of Ski and CBP interacted with Smad3 at a portion of the binding surface of the Smad anchor for receptor activation. Our results suggest that Ski negatively regulates TGF-beta signaling by replacing CBP in R-Smad complexes. Our working model suggests that Smad protein activity is delicately balanced by Ski and CBP in the TGF-beta pathway.

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

PubMed

Huang, Yao; Chang, Yongchang

2014-01-01

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

[Modern condition and prospects of improvement of the specialized medical care for acute bone marrow syndrome of radiation etiology].

PubMed

Khalimov, Iu Sh; Grebeniuk, A N; Legeza, V I; Karamullin, M A; Salukhov, V V

2013-01-01

It is shown, that tactics of treatment of acute marrow failure of radiant etiology is based, first of all, on measures of supporting, replaceable and stimulating therapy. The modern means, used for prophylactic and treatment of infectious complications, are resulted. Opportunities and restrictions of transfusion of donor thrombocytes and granulocytes, erythrocytes and chilled plasma are described. Therapeutic efficiency of transplantation of a bone marrow, cells of embryonic liver and stem cells of peripheral or umbilical cord blood is analyzed. It is shown, that the greatest prospects in perfection of the specialized medical aid at acute radiation syndrome are connected to complex application of interleukin-1beta, interleukin-3, granulocyte or granulocyte/macrophage colony stimulated factor, thrombopoietin and others cytokines.

Topologically heterogeneous beta cell adaptation in response to high-fat diet in mice.

PubMed

Ellenbroek, Johanne H; Töns, Hendrica A; de Graaf, Natascha; Loomans, Cindy J; Engelse, Marten A; Vrolijk, Hans; Voshol, Peter J; Rabelink, Ton J; Carlotti, Françoise; de Koning, Eelco J

2013-01-01

Beta cells adapt to an increased insulin demand by enhancing insulin secretion via increased beta cell function and/or increased beta cell number. While morphological and functional heterogeneity between individual islets exists, it is unknown whether regional differences in beta cell adaptation occur. Therefore we investigated beta cell adaptation throughout the pancreas in a model of high-fat diet (HFD)-induced insulin resistance in mice. C57BL/6J mice were fed a HFD to induce insulin resistance, or control diet for 6 weeks. The pancreas was divided in a duodenal (DR), gastric (GR) and splenic (SR) region and taken for either histology or islet isolation. The capacity of untreated islets from the three regions to adapt in an extrapancreatic location was assessed by transplantation under the kidney capsule of streptozotocin-treated mice. SR islets showed 70% increased beta cell proliferation after HFD, whereas no significant increase was found in DR and GR islets. Furthermore, isolated SR islets showed twofold enhanced glucose-induced insulin secretion after HFD, as compared with DR and GR islets. In contrast, transplantation of islets isolated from the three regions to an extrapancreatic location in diabetic mice led to a similar decrease in hyperglycemia and no difference in beta cell proliferation. HFD-induced insulin resistance leads to topologically heterogeneous beta cell adaptation and is most prominent in the splenic region of the pancreas. This topological heterogeneity in beta cell adaptation appears to result from extrinsic factors present in the islet microenvironment.

Revealing Beta-Diversity Patterns of Breeding Bird and Lizard Communities on Inundated Land-Bridge Islands by Separating the Turnover and Nestedness Components

PubMed Central

Si, Xingfeng; Baselga, Andrés; Ding, Ping

2015-01-01

Beta diversity describes changes in species composition among sites in a region and has particular relevance for explaining ecological patterns in fragmented habitats. However, it is difficult to reveal the mechanisms if broad sense beta-diversity indices (i.e. yielding identical values under nestedness and species replacement) are used. Partitioning beta diversity into turnover (caused by species replacement from site to site) and nestedness-resultant components (caused by nested species losses) could provide a unique way to understand the variation of species composition in fragmented habitats. Here, we collected occupancy data of breeding birds and lizards on land-bridge islands in an inundated lake in eastern China. We decomposed beta diversity of breeding bird and lizard communities into spatial turnover and nestedness-resultant components to assess their relative contributions and respective relationships to differences in island area, isolation, and habitat richness. Our results showed that spatial turnover contributed more to beta diversity than the nestedness-resultant component. The degree of isolation had no significant effect on overall beta diversity or its components, neither for breeding birds nor for lizards. In turn, in both groups the nestedness-resultant component increased with larger differences in island area and habitat richness, respectively, while turnover component decreased with them. The major difference among birds and lizards was a higher relevance of nestedness-resultant dissimilarity in lizards, suggesting that they are more prone to local extinctions derived from habitat fragmentation. The dominance of the spatial turnover component of beta diversity suggests that all islands have potential conservation value for breeding bird and lizard communities. PMID:25992559

Cell density and N-cadherin interactions regulate cell proliferation in the sensory epithelia of the inner ear.

PubMed

Warchol, Mark E

2002-04-01

Sensory hair cells in the inner ears of nonmammalian vertebrates can regenerate after injury. In many species, replacement hair cells are produced by the proliferation of epithelial supporting cells. Thus, the ability of supporting cells to undergo renewed proliferation is a key determinant of regenerative ability. The present study used cultures of isolated inner ear sensory epithelia to identify cellular signals that regulate supporting cell proliferation. Small pieces of sensory epithelia from the chicken utricle were cultured in glass microwells. Under those conditions, cell proliferation was inversely related to local cell density. The signaling molecules N-cadherin, beta-catenin, and focal adhesion kinase were immunolocalized in the cultured epithelial cells, and high levels of phosphotyrosine immunoreactivity were present at cell-cell junctions and focal contacts of proliferating cells. Binding of microbeads coated with a function-blocking antibody to N-cadherin inhibited ongoing proliferation. The growth of epithelial cells was also affected by the density of extracellular matrix molecules. The results suggest that cell density, cell-cell contact, and the composition of the extracellular matrix may be critical influences on the regulation of sensory regeneration in the inner ear.

Specific signals involved in the long-term maintenance of radiation-induced fibrogenic differentiation: a role for CCN2 and low concentration of TGF-beta1.

PubMed

Haydont, Valérie; Riser, Bruce L; Aigueperse, Jocelyne; Vozenin-Brotons, Marie-Catherine

2008-06-01

The fibrogenic differentiation of resident mesenchymal cells is a key parameter in the pathogenesis of radiation fibrosis and is triggered by the profibrotic growth factors transforming growth factor (TGF)-beta1 and CCN2. TGF-beta1 is considered the primary inducer of fibrogenic differentiation and is thought to control its long-term maintenance, whereas CCN2 is considered secondary effector of TGF-beta1. Yet, in long-term established fibrosis like that associated with delayed radiation enteropathy, in situ TGF-beta1 deposition is low, whereas CCN2 expression is high. To explore this apparent paradox, cell response to increasing doses of TGF-beta1 was investigated in cells modeling initiation and maintenance of fibrosis, i.e., normal and fibrosis-derived smooth muscle cells, respectively. Activation of cell-specific signaling pathways by low TGF-beta1 doses was demonstrated with a main activation of the Rho/ROCK pathway in fibrosis-derived cells, whereas the Smad pathway was mainly activated in normal cells. This leads to subsequent and cell-specific regulation of the CCN2 gene. These results suggested a specific profibrotic role of CCN2 in fibrosis-initiated cells. Furthermore, the modulation of CCN2 expression by itself and the combination of TGF-beta1 and CCN2 was investigated in fibrosis-derived cells. In fibrosis-initiated cells CCN2 triggered its autoinduction; furthermore, low concentration of TGF-beta1-potentiated CCN2 autoinduction. Our findings showed a differential requirement and action of TGF-beta1 in the fibrogenic response of normal vs. fibrosis-derived cells. This study defines a novel Rho/ROCK but Smad3-independent mode of TGF-beta signaling that may operate during the chronic stages of fibrosis and provides evidence of both specific and combinatorial roles of low TGF-beta1 dose and CCN2.

Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility

PubMed Central

1993-01-01

Coronin is an actin-binding protein in Dictyostelium discoideum that is enriched at the leading edge of the cells and in projections of the cell surface called crowns. The polypeptide sequence of coronin is distinguished by its similarities to the beta-subunits of trimeric G proteins (E. L. de Hostos, B. Bradtke, F. Lottspeich, R. Guggenheim, and G. Gerisch, 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:4097-4104). To elucidate the in vivo function of coronin, null mutants have been generated by gene replacement. The mutant cells lacking coronin grow and migrate more slowly than wild-type cells. When these cor- cells grow in liquid medium they become multinucleate, indicating a role of coronin in cytokinesis. To explore this role, coronin has been localized in mitotic wild-type cells by immunofluorescence labeling. During separation of the daughter cells, coronin is strongly accumulated at their distal portions including the leading edges. This contrasts with the localization of myosin II in the cleavage furrow and suggests that coronin functions independently of the conventional myosin in facilitating cytokinesis. PMID:8380174

[Isolation, purification and primary culture of rat pancreatic beta-cells].

PubMed

Liu, Yu-Pu; Lü, Qing-Guo; Tong, Nan-Wei

2009-01-01

To isolate and purify rat pancreatic beta-cells and to explore the best conditions for the primary culture of the pancreatic beta-cells in vitro. The pancreas of Norman Wistar rats were digested by collagenase V. The islets were purified by mesh sieve. The activity of the islets was stimulated by different concentrations of glucose and detected by dithizone dye. The purified islets were put into RPMI-1640 nutritive medium for culture overnight. The cultured islets were digested again with trypsin and DNAase to obtain the suspension containing single pancreatic cells. The beta-cells were separated and purified in a fluorescence-activated cell sorter (FACS) in the medium containing 2.8 mmol/L glucose. The purified beta-cells were identified by immunohistochemistry and glucose stimulating test. Ham's F-10 with different concentrations of glucose and 3-Isobutyl-1-methylxanthine (IBMX) were used as nutritive medium for the primary cell culture for 24 hours. The best conditions for the culture were identified. An average of 550 +/- 90 islets with fine activities were obtained per rat. The purification with FACS obtained about 5688 beta-cells per rat, with a recovery rate of (93.69 +/- 1.26)% and a purity of (85.5 +/- 1.24)%. A concentration of 10.0 mmol/L and 16.0 mmol/L glucose in primary culture for 24 hours produced the highest survival rates of beta-cells, but IBMX did not increase the survival rates of beta-cells. FACS is effective in purifying pancreatic beta-cells from the suspension with a medium containing 2.8 mmol/L glucose. Pancreatic beta-cells maintain relatively high activities in Ham's F-10 medium containing 10.0-16.0 mmol/L glucose in primary culture.

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

Activation of the canonical beta-catenin pathway by histamine.

PubMed

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

2003-12-26

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

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.

The islet beta-cell: fuel responsive and vulnerable.

PubMed

Nolan, Christopher J; Prentki, Marc

2008-10-01

The pancreatic beta-cell senses blood nutrient levels and is modulated by neurohormonal signals so that it secretes insulin according to the need of the organism. Nutrient sensing involves marked metabolic activation, resulting in the production of coupling signals that promote insulin biosynthesis and secretion. The beta-cell's high capacity for nutrient sensing, however, necessitates reduced protection to nutrient toxicity. This potentially explains why in susceptible individuals, chronic fuel surfeit results in beta-cell failure and type 2 diabetes. Here we discuss recent insights into first, the biochemical basis of beta-cell signaling in response to glucose, amino acids and fatty acids, and second, beta-cell nutrient detoxification. We emphasize the emerging role of glycerolipid/fatty acid cycling in these processes.

Apparent inhibition of. beta. -fructosidase secretion by tunicamycin may be explained by breakdown of the unglycosylated protein during secretion. [Daucus carota

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

Faye, L.; Chrispeels, M.J.

1989-03-01

Suspension-cultured carrot (Daucus carota) cells synthesize and secrete {beta}-fructosidase, a glycoprotein with asparagine-linked glycans. Treatment of the cells with tunicamycin completely inhibits the apparent secretion of {beta}-fructosidase as measured by the accumulation of the {sup 35}S-labelled protein in the cell wall or the culture medium. In the past, such a result has been interpreted as an inhibition of secretion by tunicamycin, but we suggest another explanation based on the following results. In the presence of tunicamycin, unglycosylated {beta}-fructosidase is synthesized and is associated with an endoplasmic-reticulum-rich microsomal fraction. Pulse-chase experiments show that the unglycosylated {beta}-fructosidase does not remain in themore » cells and appears to be secreted in the same way as glycosylated {beta}-fructosidase; however, no radioactive, unglycosylated {beta}-fructosidase accumulates extracellularly (cell wall or medium). Protoplasts obtained from carrot cells secrete {beta}-fructosidase protein and activity, and treatment of the protoplasts with tunicamycin results in the synthesis of unglycosylated {beta}-fructosidase. In the presence of tunicamycin, there is no accumulation of {beta}-fructosidase activity or unglycosylated {beta}-fructosidase polypeptide in the protoplast incubation medium. These results are consistent with the interpretation that the glycans of {beta}-fructosidase are necessary for its stability, and that in these suspension-cultured cells, the unglycosylated enzyme is degraded during the last stage(s) of secretion, or immediately after its arrival in the wall.« less

Comparison of osteoblast-like cell responses to calcium silicate and tricalcium phosphate ceramics in vitro.

PubMed

Ni, Siyu; Chang, Jiang; Chou, Lee; Zhai, Wanyin

2007-01-01

Calcium silicate ceramics have been proposed as new bone repair biomaterials, since they have proved to be bioactive, degradable, and biocompatible. Beta-tricalcium phosphate ceramic is a well-known degradable material for bone repair. This study compared the effects of CaSiO3 (alpha-, and beta-CaSiO3) and beta-Ca3(PO4)2 (beta-TCP) ceramics on the early stages of rat osteoblast-like cell attachment, proliferation, and differentiation. Osteoblast-like cells were cultured directly on CaSiO3 (alpha-, and beta-CaSiO3) and beta-TCP ceramics. Attachment of a greater number of cells was observed on CaSiO3 (alpha-, and beta-CaSiO3) ceramics compared with beta-TCP ceramics after incubation for 6 h. SEM observations showed an intimate contact between cells and the substrates, significant cells adhesion, and that the cells spread and grew on the surfaces of all the materials. In addition, the proliferation rate and alkaline phosphatase (ALP) activity of the cells on the CaSiO3 (alpha-, and beta-CaSiO3) ceramics were improved when compared with the beta-TCP ceramics. In the presence of CaSiO3, elevated levels of calcium and silicon in the culture medium were observed throughout the 7-day culture period. In conclusion, the results of the present study revealed that CaSiO3 ceramics showed greater ability to support cell attachment, proliferation, and differentiation than beta-TCP ceramic. 2006 Wiley Periodicals, Inc.

Enhanced sialylation and in vivo efficacy of recombinant human α-galactosidase through in vitro glycosylation

PubMed Central

Sohn, Youngsoo; Lee, Jung Mi; Park, Heung-Rok; Jung, Sung-Chul; Park, Tai Hyun; Oh, Doo-Byoung

2013-01-01

Human α-galactosidase A (GLA) has been used in enzyme replacement therapy for patients with Fabry disease. We expressed recombinant GLA from Chinese hamster ovary cells with very high productivity. When compared to an approved GLA (agalsidase beta), its size and charge were found to be smaller and more neutral. These differences resulted from the lack of terminal sialic acids playing essential roles in the serum half-life and proper tissue targeting. Because a simple sialylation reaction was not enough to increase the sialic acid content, a combined reaction using galactosyltransferase, sialyltransferase, and their sugar substrates at the same time was developed and optimized to reduce the incubation time. The product generated by this reaction had nearly the same size, isoelectric points, and sialic acid content as agalsidase beta. Furthermore, it had better in vivo efficacy to degrade the accumulated globotriaosylceramide in target organs of Fabry mice compared to an unmodified version. [BMB Reports 2013; 46(3): 157-162] PMID:23527859

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.

Pancreatic Beta Cells Synthesize Neuropeptide Y and Can Rapidly Release Peptide Co-Transmitters

PubMed Central

Whim, Matthew D.

2011-01-01

Background In addition to polypeptide hormones, pancreatic endocrine cells synthesize a variety of bioactive molecules including classical transmitters and neuropeptides. While these co-transmitters are thought to play a role in regulating hormone release little is known about how their secretion is regulated. Here I investigate the synthesis and release of neuropeptide Y from pancreatic beta cells. Methodology/Principal Findings NPY appears to be an authentic co-transmitter in neonatal, but not adult, beta cells because (1) early in mouse post-natal development, many beta cells are NPY-immunoreactive whereas no staining is observed in beta cells from NPY knockout mice; (2) GFP-expressing islet cells from an NPY(GFP) transgenic mouse are insulin-ir; (3) single cell RT-PCR experiments confirm that the NPY(GFP) cells contain insulin mRNA, a marker of beta cells. The NPY-immunoreactivity previously reported in alpha and delta cells is therefore likely to be due to the presence of NPY-related peptides. INS-1 cells, a beta cell line, are also NPY-ir and contain NPY mRNA. Using the FMRFamide tagging technique, NPY secretion was monitored from INS-1 beta cells with high temporal resolution. Peptide release was evoked by brief depolarizations and was potentiated by activators of adenylate cyclase and protein kinase A. Following a transient depolarization, NPY-containing dense core granules fused with the cell membrane and discharged their contents within a few milliseconds. Conclusions These results indicate that after birth, NPY expression in pancreatic islets is restricted to neonatal beta cells. The presence of NPY suggests that peptide co-transmitters could mediate rapid paracrine or autocrine signaling within the endocrine pancreas. The FMRFamide tagging technique may be useful in studying the release of other putative islet co-transmitters in real time. PMID:21559341

Calbindin-D(28k) controls [Ca(2+)](i) and insulin release. Evidence obtained from calbindin-d(28k) knockout mice and beta cell lines

NASA Technical Reports Server (NTRS)

Sooy, K.; Schermerhorn, T.; Noda, M.; Surana, M.; Rhoten, W. B.; Meyer, M.; Fleischer, N.; Sharp, G. W.; Christakos, S.

1999-01-01

The role of the calcium-binding protein, calbindin-D(28k) in potassium/depolarization-stimulated increases in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release was investigated in pancreatic islets from calbindin-D(28k) nullmutant mice (knockouts; KO) or wild type mice and beta cell lines stably transfected and overexpressing calbindin. Using single islets from KO mice and stimulation with 45 mM KCl, the peak of [Ca(2+)](i) was 3.5-fold greater in islets from KO mice compared with wild type islets (p < 0.01) and [Ca(2+)](i) remained higher during the plateau phase. In addition to the increase in [Ca(2+)](i) in response to KCl there was also a significant increase in insulin release in islets isolated from KO mice. Evidence for modulation by calbindin of [Ca(2+)](i) and insulin release was also noted using beta cell lines. Rat calbindin was stably expressed in betaTC-3 and betaHC-13 cells. In response to depolarizing concentrations of K(+), insulin release was decreased by 45-47% in calbindin expressing betaTC cells and was decreased by 70-80% in calbindin expressing betaHC cells compared with insulin release from vector transfected betaTC or betaHC cells (p < 0.01). In addition, the K(+)-stimulated intracellular calcium peak was markedly inhibited in calbindin expressing betaHC cells compared with vector transfected cells (225 nM versus 1,100 nM, respectively). Buffering of the depolarization-induced rise in [Ca(2+)](i) was also observed in calbindin expressing betaTC cells. In summary, our findings, using both isolated islets from calbindin-D(28k) KO mice and beta cell lines, establish a role for calbindin in the modulation of depolarization-stimulated insulin release and suggest that calbindin can control the rate of insulin release via regulation of [Ca(2+)](i).

SIRT6-mediated transcriptional suppression of Txnip is critical for pancreatic beta cell function and survival in mice.

PubMed

Qin, Kunhua; Zhang, Ning; Zhang, Zhao; Nipper, Michael; Zhu, Zhenxin; Leighton, Jake; Xu, Kexin; Musi, Nicolas; Wang, Pei

2018-04-01

Better understanding of how genetic and epigenetic components control beta cell differentiation and function is key to the discovery of novel therapeutic approaches to prevent beta cell dysfunction and failure in the progression of type 2 diabetes. Our goal was to elucidate the role of histone deacetylase sirtuin 6 (SIRT6) in beta cell development and homeostasis. Sirt6 endocrine progenitor cell conditional knockout and beta cell-specific knockout mice were generated using the Cre-loxP system. Mice were assayed for islet morphology, glucose tolerance, glucose-stimulated insulin secretion and susceptibility to streptozotocin. Transcriptional regulatory functions of SIRT6 in primary islets were evaluated by RNA-Seq analysis. Reverse transcription-quantitative (RT-q)PCR and immunoblot were used to verify and investigate the gene expression changes. Chromatin occupancies of SIRT6, H3K9Ac, H3K56Ac and active RNA polymerase II were evaluated by chromatin immunoprecipitation. Deletion of Sirt6 in pancreatic endocrine progenitor cells did not affect endocrine morphology, beta cell mass or insulin production but did result in glucose intolerance and defective glucose-stimulated insulin secretion in mice. Conditional deletion of Sirt6 in adult beta cells reproduced the insulin secretion defect. Loss of Sirt6 resulted in aberrant upregulation of thioredoxin-interacting protein (TXNIP) in beta cells. SIRT6 deficiency led to increased acetylation of histone H3 lysine residue at 9 (H3K9Ac), acetylation of histone H3 lysine residue at 56 (H3K56Ac) and active RNA polymerase II at the promoter region of Txnip. SIRT6-deficient beta cells exhibited a time-dependent increase in H3K9Ac, H3K56Ac and TXNIP levels. Finally, beta cell-specific SIRT6-deficient mice showed increased sensitivity to streptozotocin. Our results reveal that SIRT6 suppresses Txnip expression in beta cells via deacetylation of histone H3 and plays a critical role in maintaining beta cell function and viability. Sequence data have been deposited in the National Institutes of Health (NIH) Gene Expression Omnibus (GEO) with the accession code GSE104161.

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.

Differential usage of T-cell receptor V beta gene families by CD4+ and CD8+ T cells in patients with CD8hi common variable immunodeficiency: evidence of a post-thymic effect.

PubMed Central

Duchmann, R; Jaffe, J; Ehrhardt, R; Alling, D W; Strober, W

1996-01-01

In this study, we report that differences between T-cell receptor (TCR) V beta gene family usage in CD4+ and CD8+ T cells are significantly greater in a subgroup of patients with common variable immunodeficiency (CVI) and high levels of activated CD8+ T cells (CD8hi CVI) than in controls (P < 0.001). In CD8hi CVI patients, such differences were also significantly greater for V beta 12 than for other V beta families. As the causes of the differential usage of V beta gene families by CD4+ and CD8+ T cells are under investigation, it was interesting that the combined differences between V beta gene family usage in the CD4+ and CD8+ T-cell subpopulations as a whole were significantly lower than the combined differences between individual V beta gene family usage in either CD4+ or CD8+ T-cell subpopulations (P < 0.001 in both control and CD8hi CVI patients). Further, the pattern of V beta gene family usage in CD4+ T cells was remarkably similar to that in CD8+ T cells in both groups. These data strongly suggest that differences in V beta gene family usage arising from coselection by major histocompatibility complex (MHC) class I versus MHC class II restriction elements do not fundamentally distort 'basic' V beta gene family usage patterns. They also support the concept that differences in CD4+ and CD8+ T-cell V beta gene family usage, which were increased in CD8hi CVI, can arise from high-affinity interactions between disease-associated antigens or superantigens and T cells in the post-thymic T-cell compartment. Images Figure 6 PMID:8666443

A recombinant tail-less integrin beta 4 subunit disrupts hemidesmosomes, but does not suppress alpha 6 beta 4-mediated cell adhesion to laminins

PubMed Central

1995-01-01

To examine the function of the alpha 6 beta 4 integrin we have determined its ligand-binding ability and overexpressed two potentially dominant negative mutant beta 4 subunits, lacking either the cytoplasmic or extracellular domain, in bladder epithelial 804G cells. The results of cell adhesion and radioligand-binding assays showed that alpha 6 beta 4 is a receptor for several laminin isoforms, including laminin 1, 2, 4, and 5. Overexpression of the tail-less or head-less mutant beta 4 subunit did not suppress alpha 6 beta 4-mediated adhesion to laminins, as both types of transfectants adhered to these ligands in the presence of blocking anti-beta 1 antibodies as well as the controls. However, immunofluorescence experiments indicated that the endogenous alpha 6 beta 4 integrin and other hemidesmosomal markers were not concentrated in hemidesmosomes in cells overexpressing tail- less beta 4, while the distribution of these molecules was not altered in cells overexpressing the head-less subunit. Electron microscopic studies confirmed that cells overexpressing tail-less beta 4 had a drastically reduced number of hemidesmosomes, while cells expressing the head-less subunit had a normal number of these structures. Thus, expression of a tail-less, but not a head-less mutant beta 4 subunit leads to a dominant negative effect on hemidesmosome assembly without suppressing initial adhesion to laminins. We conclude that the alpha 6 beta 4 integrin binds to several laminins and plays an essential role in the assembly and/or stability of hemidesmosomes, that alpha 6 beta 4- mediated adhesion and hemidesmosome assembly have distinct requirements, and that it is possible to use a dominant negative approach to selectively interfere with a specific function of an integrin. PMID:7721947

Gene Editing and Human Pluripotent Stem Cells: Tools for Advancing Diabetes Disease Modeling and Beta-Cell Development.

PubMed

Millette, Katelyn; Georgia, Senta

2017-10-05

This review will focus on the multiple approaches to gene editing and address the potential use of genetically modified human pluripotent stem cell-derived beta cells (SC-β) as a tool to study human beta-cell development and model their function in diabetes. We will explore how new variations of CRISPR/Cas9 gene editing may accelerate our understanding of beta-cell developmental biology, elucidate novel mechanisms that establish and regulate beta-cell function, and assist in pioneering new therapeutic modalities for treating diabetes. Improvements in CRISPR/Cas9 target specificity and homology-directed recombination continue to advance its use in engineering stem cells to model and potentially treat disease. We will review how CRISPR/Cas9 gene editing is informing our understanding of beta-cell development and expanding the therapeutic possibilities for treating diabetes and other diseases. Here we focus on the emerging use of gene editing technology, specifically CRISPR/Cas9, as a means of manipulating human gene expression to gain novel insights into the roles of key factors in beta-cell development and function. Taken together, the combined use of SC-β cells and CRISPR/Cas9 gene editing will shed new light on human beta-cell development and function and accelerate our progress towards developing new therapies for patients with diabetes.

Cell-to-cell contact dependence and junctional protein content are correlated with in vivo maturation of pancreatic beta cells.

PubMed

Santos-Silva, Junia Carolina; Carvalho, Carolina Prado de França; de Oliveira, Ricardo Beltrame; Boschero, Antonio Carlos; Collares-Buzato, Carla Beatriz

2012-07-01

In this study, we investigated the cellular distribution of junctional proteins and the dependence on cell-cell contacts of pancreatic beta cells during animal development. Fetus and newborn rat islets, which display a relatively poor insulin secretory response to glucose, present an immature morphology and cytoarchitecture when compared with young and adult islets that are responsive to glucose. At the perinatal stage, beta cells display a low junctional content of neural cell adhesion molecule (N-CAM), α- and β-catenins, ZO-1, and F-actin, while a differential distribution of N-CAM and Pan-cadherin was seen in beta cells and nonbeta cells only from young and adult islets. In the absence of intercellular contacts, the glucose-stimulated insulin secretion was completely blocked in adult beta cells, but after reaggregation they partially reestablished the secretory response to glucose. By contrast, neonatal beta cells were poorly responsive to sugar, regardless of whether they were arranged as intact islets or as isolated cells. Interestingly, after 10 days of culturing, neonatal beta cells, known to display increased junctional protein content in vitro, became responsive to glucose and concomitantly dependent on cell-cell contacts. Therefore, our data suggest that the developmental acquisition of an adult-like insulin secretory pattern is paralleled by a dependence on direct cell-cell interactions.

Islet immunity and beta cell reserve of indigenous Black South Africans with ketoacidosis at initial diagnosis of diabetes.

PubMed

Ekpebegh, Chukwuma; Longo-Mbenza, Benjamin; Blanco-Blanco, Ernesto

2013-01-01

Islet immunity and beta cell reserve status were utilized to classify persons with ketoacidosis as the initial manifestation of diabetes. The clinical features of the various diabetes classes were also characterized. Prospective cross sectional study. Nelson Mandela Academic Hospital, Mthatha, Eastern Cape Province, South Africa. Indigenous Black South Africans with ketoacidosis as the initial manifestation of diabetes. Islet immunity and beta cell reserve were respectively assessed using serum anti-glutamic acid decarboxylase 65 (GAD) antibody and serum C-peptide after 1 mg of intravenous glucagon. Serum anti-GAD 65 antibody > or = 5 units/L and < 5 units/L, respectively defined anti-GAD 65 positive (A+) and negative (A-). Replete (beta+) and deplete (beta-) beta cell reserve were serum C-peptide after glucagon injection of > or = 0.5 ng/mL and < 0.5 ng/mL, respectively. The proportions of patients with A+beta-, A+beta+, A-beta- and A-beta+ and their clinical characteristics were determined. Of the 38 males and 33 females who participated in the study, patients were categorized in various classes: A-beta+, 46.5% (n=33/ 71); A-beta-, 26.8% (n=19/71); A+beta-, 22.5% (n=16/71); and A+beta+, 4.2% (n=3/71). The ages of the various classes were: 41.8 +/- 13.8 years for A-beta+ (n=33); 36.5 +/- 14.6 years for A-beta- (n=19); and 20.6 +/- 7.1 years for the combination of A+beta- with A+beta+ (n=19) (P<.0001, P<.0001 for the combination of A+beta- and A+beta+ vs A-beta+, P=.001 for the combination of A+beta- and A+beta+ vs A-beta-and P=.2 for A-beta- vs A-beta+. The clinical features of type 2 diabetes were most prevalent in A-beta+ class while the A+beta- and A+beta+ groups had the clinical profile of type 1A diabetes. Most of the indigenous Black South African patients with ketoacidosis as the initial manifestation of diabetes had islet immunity, beta cell reserve status and clinical profiles of type 2 diabetes.

Intermittent fasting preserves beta-cell mass in obesity-induced diabetes via the autophagy-lysosome pathway.

PubMed

Liu, Haiyan; Javaheri, Ali; Godar, Rebecca J; Murphy, John; Ma, Xiucui; Rohatgi, Nidhi; Mahadevan, Jana; Hyrc, Krzysztof; Saftig, Paul; Marshall, Connie; McDaniel, Michael L; Remedi, Maria S; Razani, Babak; Urano, Fumihiko; Diwan, Abhinav

2017-01-01

Obesity-induced diabetes is characterized by hyperglycemia, insulin resistance, and progressive beta cell failure. In islets of mice with obesity-induced diabetes, we observe increased beta cell death and impaired autophagic flux. We hypothesized that intermittent fasting, a clinically sustainable therapeutic strategy, stimulates autophagic flux to ameliorate obesity-induced diabetes. Our data show that despite continued high-fat intake, intermittent fasting restores autophagic flux in islets and improves glucose tolerance by enhancing glucose-stimulated insulin secretion, beta cell survival, and nuclear expression of NEUROG3, a marker of pancreatic regeneration. In contrast, intermittent fasting does not rescue beta-cell death or induce NEUROG3 expression in obese mice with lysosomal dysfunction secondary to deficiency of the lysosomal membrane protein, LAMP2 or haplo-insufficiency of BECN1/Beclin 1, a protein critical for autophagosome formation. Moreover, intermittent fasting is sufficient to provoke beta cell death in nonobese lamp2 null mice, attesting to a critical role for lysosome function in beta cell homeostasis under fasting conditions. Beta cells in intermittently-fasted LAMP2- or BECN1-deficient mice exhibit markers of autophagic failure with accumulation of damaged mitochondria and upregulation of oxidative stress. Thus, intermittent fasting preserves organelle quality via the autophagy-lysosome pathway to enhance beta cell survival and stimulates markers of regeneration in obesity-induced diabetes.

Hypersomnolence with beta-adrenergic blockers.

PubMed

Thachil, J; Zeller, J R; Kochar, M S

1987-11-01

An elderly, mildly demented, hypertensive male patient developed hypersomnolence on administration of propranolol for treatment of hypertension; no other cause for hypersomnolence was detected. Upon replacement of propranolol with atenolol, he felt better but continued to be quite somnolent. When atenolol was discontinued, he reported to have lack of sleep. On readministration of subtherapeutic doses of the same beta-adrenergic blocking agents, he once again experienced excessive sleepiness. By discontinuing beta-blocking agents and introducing captopril, he felt much better, became pleasant and talkative, and blood pressure was well controlled. Beta antagonists are important drugs in the management of many cardiovascular problems. Propranolol, a lipophilic beta-blocking agent, and atenolol, a hydrophilic beta-blocking agent, are two of the major agents currently used clinically in the United States. Numerous neuropsychiatric side-effects of the beta-adrenergic blocking drugs have been reported, but hypersomnolence is not readily recognized as one of them.

Molecular role of TGF-beta, secreted from a new type of CD4+ suppressor T cell, NY4.2, in the prevention of autoimmune IDDM in NOD mice.

PubMed

Han, H S; Jun, H S; Utsugi, T; Yoon, J W

1997-06-01

A new type of CD4+ T cell clone (NY4.2) isolated from pancreatic islet-infiltrated lymphocytes of acutely diabetic non-obese diabetic (NOD) mice prevents the development of insulin-dependent diabetes mellitus (IDDM) in NOD mice, as well as the recurrence of autoimmune diabetes in syngeneic islet-transplanted NOD mice. It has been demonstrated that the cytokine TGF-beta, secreted from the cells of this clone, is the substance which prevents autoimmune IDDM. This investigation was initiated to determine the molecular role TGF-beta plays in the prevention of autoimmune IDDM by determining its effect on IL-2-induced signal transduction in Con A-activated NOD mouse splenocytes and HT-2 cells. First, we determined whether TGF-beta, secreted from NY4.2 T cells, inhibits IL-2-dependent T cell proliferation in HT-2 cells (IL-2-dependent T cell line) and NOD splenocytes. We found that TGF-beta suppresses IL-2-dependent T cell proliferation. Second, we determined whether TGF-beta inhibits the activation of Janus kinases (JAKs), as well as signal transducers and activators of transcription (STAT) proteins, involved in an IL-2-induced signalling pathway that normally leads to the proliferation of T cells. We found that TGF-beta inhibited tyrosine phosphorylation of JAK1, JAK3, STAT3 and STAT5 in Con A blasts from NOD splenocytes and HT-2 cells. Third, we examined whether TGF-beta inhibits the cooperation between STAT proteins and mitogen-activated protein kinase (MAPK), especially extracellular signal-regulated kinase 2 (ERK2). We found that TGF-beta inhibited the association of STAT3 and STAT5 with ERK2 in Con A blasts from NOD splenocytes and HT-2 cells. On the basis of these observations, we conclude that TGF-beta may interfere with signal transduction via inhibition of the IL-2-induced JAK/STAT pathway and inhibition of the association of STAT proteins with ERK2 in T cells from NOD splenocytes, resulting in the inhibition of IL-2-dependent T cell proliferation. TGF-beta-mediated suppression of T cell activation may be responsible for the prevention of effector T cell-mediated autoimmune IDDM in NOD mice by TGF-beta-producing CD4+ suppressor T cells.

Hypothyroidism in utero stimulates pancreatic beta cell proliferation and hyperinsulinaemia in the ovine fetus during late gestation.

PubMed

Harris, Shelley E; De Blasio, Miles J; Davis, Melissa A; Kelly, Amy C; Davenport, Hailey M; Wooding, F B Peter; Blache, Dominique; Meredith, David; Anderson, Miranda; Fowden, Abigail L; Limesand, Sean W; Forhead, Alison J

2017-06-01

Thyroid hormones are important regulators of growth and maturation before birth, although the extent to which their actions are mediated by insulin and the development of pancreatic beta cell mass is unknown. Hypothyroidism in fetal sheep induced by removal of the thyroid gland caused asymmetric organ growth, increased pancreatic beta cell mass and proliferation, and was associated with increased circulating concentrations of insulin and leptin. In isolated fetal sheep islets studied in vitro, thyroid hormones inhibited beta cell proliferation in a dose-dependent manner, while high concentrations of insulin and leptin stimulated proliferation. The developing pancreatic beta cell is therefore sensitive to thyroid hormone, insulin and leptin before birth, with possible consequences for pancreatic function in fetal and later life. The findings of this study highlight the importance of thyroid hormones during pregnancy for normal development of the fetal pancreas. Development of pancreatic beta cell mass before birth is essential for normal growth of the fetus and for long-term control of carbohydrate metabolism in postnatal life. Thyroid hormones are also important regulators of fetal growth, and the present study tested the hypotheses that thyroid hormones promote beta cell proliferation in the fetal ovine pancreatic islets, and that growth retardation in hypothyroid fetal sheep is associated with reductions in pancreatic beta cell mass and circulating insulin concentration in utero. Organ growth and pancreatic islet cell proliferation and mass were examined in sheep fetuses following removal of the thyroid gland in utero. The effects of triiodothyronine (T 3 ), insulin and leptin on beta cell proliferation rates were determined in isolated fetal ovine pancreatic islets in vitro. Hypothyroidism in the sheep fetus resulted in an asymmetric pattern of organ growth, pancreatic beta cell hyperplasia, and elevated plasma insulin and leptin concentrations. In pancreatic islets isolated from intact fetal sheep, beta cell proliferation in vitro was reduced by T 3 in a dose-dependent manner and increased by insulin at high concentrations only. Leptin induced a bimodal response whereby beta cell proliferation was suppressed at the lowest, and increased at the highest, concentrations. Therefore, proliferation of beta cells isolated from the ovine fetal pancreas is sensitive to physiological concentrations of T 3 , insulin and leptin. Alterations in these hormones may be responsible for the increased beta cell proliferation and mass observed in the hypothyroid sheep fetus and may have consequences for pancreatic function in later life. © 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

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

PubMed

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

2011-01-01

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

Interleukin-1{beta} regulates cell proliferation and activity of extracellular matrix remodelling enzymes in cultured primary pig heart cells

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

Zitta, Karina; Brandt, Berenice; Wuensch, Annegret

Research highlights: {yields} Levels of IL-1{beta} are increased in the pig myocardium after infarction. {yields} Cultured pig heart cells possess IL-1 receptors. {yields} IL-1{beta} increases cell proliferation of pig heart cells in-vitro. {yields} IL-1{beta} increases MMP-2 and MMP-9 activity in pig heart cells in-vitro. {yields} IL-1{beta} may be important for tissue remodelling events after myocardial infarction. -- Abstract: After myocardial infarction, elevated levels of interleukins (ILs) are found within the myocardial tissue and IL-1{beta} is considered to play a major role in tissue remodelling events throughout the body. In the study presented, we have established a cell culture model ofmore » primary pig heart cells to evaluate the effects of different concentrations of IL-1{beta} on cell proliferation as well as expression and activity of enzymes typically involved in tissue remodelling. Primary pig heart cell cultures were derived from three different animals and stimulated with recombinant pig IL-1{beta}. RNA expression was detected by RT-PCR, protein levels were evaluated by Western blotting, activity of matrix metalloproteinases (MMPs) was quantified by gelatine zymography and cell proliferation was measured using colorimetric MTS assays. Pig heart cells express receptors for IL-1 and application of IL-1{beta} resulted in a dose-dependent increase of cell proliferation (P < 0.05 vs. control; 100 ng/ml; 24 h). Gene expression of caspase-3 was increased by IL-1{beta} (P < 0.05 vs. control; 100 ng/ml; 3 h), and pro-caspase-3 but not active caspase was detected in lysates of pig heart cells by Western blotting. MMP-2 gene expression as well as enzymatic activities of MMP-2 and MMP-9 were increased by IL-1{beta} (P < 0.05 vs. control; 100 ng/ml; 3 h for gene expression, 48 and 72 h for enzymatic activities of MMP-2 and MMP-9, respectively). Our in vitro data suggest that IL-1{beta} plays a major role in the events of tissue remodelling in the heart. Combined with our recently published in vivo data (Meybohm et al., PLoS One, 2009), the results presented here strongly suggest IL-1{beta} as a key molecule guiding tissue remodelling events after myocardial infarction.« less

Immunotherapy for Alzheimer's disease: DNA- and protein-based epitope vaccines.

PubMed

Davtyan, Hayk; Petrushina, Irina; Ghochikyan, Anahit

2014-01-01

Active immunotherapy for Alzheimer's disease (AD) is aimed to induce antibodies specific to amyloid-beta (Aβ) that are capable to reduce the level of Aβ in the CNS of Alzheimer's disease patients. First clinical trial AN-1792 that was based on vaccination with full-length Aβ42 showed that safe and effective AD vaccine should induce high titers of anti-Aβ antibodies without activation of harmful autoreactive T cells. Replacement of self-T cell epitope with foreign epitope, keeping self-B cell epitope intact, may allow to induce high titers of anti-Aβ antibodies while avoiding the activation of T cells specific to Aβ. Here we describe the protocols for evaluation of AD DNA- or multiple antigenic peptide (MAP)-based epitope vaccines composed of Aβ(1-11) B cell epitope fused to synthetic T cell epitope PADRE (Aβ(1-11)-PADRE). All protocols could be used for testing any epitope vaccine constructed in your lab and composed of other T cell epitopes using the appropriate peptides in tests for evaluation of humoral and cellular immune responses.

Identification of beta-2 as a key cell adhesion molecule in PCa cell neurotropic behavior: a novel ex vivo and biophysical approach.

PubMed

Jansson, Keith H; Castillo, Deborah G; Morris, Joseph W; Boggs, Mary E; Czymmek, Kirk J; Adams, Elizabeth L; Schramm, Lawrence P; Sikes, Robert A

2014-01-01

Prostate cancer (PCa) is believed to metastasize through the blood/lymphatics systems; however, PCa may utilize the extensive innervation of the prostate for glandular egress. The interaction of PCa and its nerve fibers is observed in 80% of PCa and is termed perineural invasion (PNI). PCa cells have been observed traveling through the endoneurium of nerves, although the underlying mechanisms have not been elucidated. Voltage sensitive sodium channels (VSSC) are multimeric transmembrane protein complexes comprised of a pore-forming α subunit and one or two auxiliary beta (β) subunits with inherent cell adhesion molecule (CAM) functions. The beta-2 isoform (gene SCN2B) interacts with several neural CAMs, while interacting putatively with other prominent neural CAMs. Furthermore, beta-2 exhibits elevated mRNA and protein levels in highly metastatic and castrate-resistant PCa. When overexpressed in weakly aggressive LNCaP cells (2BECFP), beta-2 alters LNCaP cell morphology and enhances LNCaP cell metastasis associated behavior in vitro. We hypothesize that PCa cells use beta-2 as a CAM during PNI and subsequent PCa metastasis. The objective of this study was to determine the effect of beta-2 expression on PCa cell neurotropic metastasis associated behavior. We overexpressed beta-2 as a fusion protein with enhanced cyan fluorescence protein (ECFP) in weakly aggressive LNCaP cells and observed neurotropic effects utilizing our novel ex vivo organotypic spinal cord co-culture model, and performed functional assays with neural matrices and atomic force microscopy. With increased beta-2 expression, PCa cells display a trend of enhanced association with nerve axons. On laminin, a neural CAM, overexpression of beta-2 enhances PCa cell migration, invasion, and growth. 2BECFP cells exhibit marked binding affinity to laminin relative to LNECFP controls, and recombinant beta-2 ectodomain elicits more binding events to laminin than BSA control. Functional overexpression of VSSC beta subunits in PCa may mediate PCa metastatic behavior through association with neural matrices.

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

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

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

2005-02-01

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

Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells.

PubMed

Rostovskaya, Maria; Bredenkamp, Nicholas; Smith, Austin

2015-10-19

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification. © 2015 The Authors.

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.

Orphan nuclear receptor TLX activates Wnt/beta-catenin signalling to stimulate neural stem cell proliferation and self-renewal.

PubMed

Qu, Qiuhao; Sun, Guoqiang; Li, Wenwu; Yang, Su; Ye, Peng; Zhao, Chunnian; Yu, Ruth T; Gage, Fred H; Evans, Ronald M; Shi, Yanhong

2010-01-01

The nuclear receptor TLX (also known as NR2E1) is essential for adult neural stem cell self-renewal; however, the molecular mechanisms involved remain elusive. Here we show that TLX activates the canonical Wnt/beta-catenin pathway in adult mouse neural stem cells. Furthermore, we demonstrate that Wnt/beta-catenin signalling is important in the proliferation and self-renewal of adult neural stem cells in the presence of epidermal growth factor and fibroblast growth factor. Wnt7a and active beta-catenin promote neural stem cell self-renewal, whereas the deletion of Wnt7a or the lentiviral transduction of axin, a beta-catenin inhibitor, led to decreased cell proliferation in adult neurogenic areas. Lentiviral transduction of active beta-catenin led to increased numbers of type B neural stem cells in the subventricular zone of adult brains, whereas deletion of Wnt7a or TLX resulted in decreased numbers of neural stem cells retaining bromodeoxyuridine label in the adult brain. Both Wnt7a and active beta-catenin significantly rescued a TLX (also known as Nr2e1) short interfering RNA-induced deficiency in neural stem cell proliferation. Lentiviral transduction of an active beta-catenin increased cell proliferation in neurogenic areas of TLX-null adult brains markedly. These results strongly support the hypothesis that TLX acts through the Wnt/beta-catenin pathway to regulate neural stem cell proliferation and self-renewal. Moreover, this study suggests that neural stem cells can promote their own self-renewal by secreting signalling molecules that act in an autocrine/paracrine mode.

Neural cell adhesion molecule-deficient beta-cell tumorigenesis results in diminished extracellular matrix molecule expression and tumour cell-matrix adhesion.

PubMed

Håkansson, Joakim; Xian, Xiaojie; He, Liqun; Ståhlberg, Anders; Nelander, Sven; Samuelsson, Tore; Kubista, Mikael; Semb, Henrik

2005-01-01

To understand by which mechanism neural cell adhesion molecule (N-CAM) limits beta tumour cell disaggregation and dissemination, we searched for potential downstream genes of N-CAM during beta tumour cell progression by gene expression profiling. Here, we show that N-CAM-deficient beta-cell tumorigenesis is associated with changes in the expression of genes involved in cell-matrix adhesion and cytoskeletal dynamics, biological processes known to affect the invasive and metastatic behaviour of tumour cells. The extracellular matrix (ECM) molecules emerged as the primary target, i.e. N-CAM deficiency resulted in down-regulated mRNA expression of a broad range of ECM molecules. Consistent with this result, deficient deposition of major ECM stromal components, such as fibronectin, laminin 1 and collagen IV, was observed. Moreover, N-CAM-deficient tumour cells displayed defective matrix adhesion. These results offer a potential mechanism for tumour cell disaggregation during N-CAM-deficient beta tumour cell progression. Prospective consequences of these findings for the role of N-CAM in beta tumour cell dissemination are discussed.

The involvement of AMPK/GSK3-beta signals in the control of metastasis and proliferation in hepato-carcinoma cells treated with anthocyanins extracted from Korea wild berry Meoru

PubMed Central

2014-01-01

Background Activation of the Wnt pathway is known to promote tumorigenesis and tumor metastasis, and targeting Wnt pathway inhibition has emerged as an attractive approach for controlling tumor invasion and metastasis. The major pathway for inhibiting Wnt is through the degradation of β-catenin by the GSK3-beta/CK1/Axin/APC complex. It was found that Hep3B hepato-carcinoma cells respond to anthocyanins through GSK3-beta-induced suppression of beta-catenin; however, they cannot dephosphorylate GSK3-beta without AMPK activation. Methods We tested the effects of anthocyanins on proliferation and apoptosis by MTT and Annexin V-PI staining in vitro. Mouse xenograft models of hepato-carcinomas were established by inoculation with Hep3B cells, and mice were injected with 50 mg/kg/ml of anthocyanins. In addition, protein levels of p-GSK3-beta, beta-catenin, p-AMPK, MMP-9, VEGF, and Ang-1 were also analyzed using western blot. Results Anthocyanins decrease phospho-GSK3-beta and beta-catenin expression in an in vivo tumor xenograft model, increase AMPK activity in this model, and inhibit cell migration and invasion, possibly by inhibiting MMP-2 (in vitro) and the panendothelial marker, CD31 (in vivo). To elucidate the role of the GSK3-beta/beta-catenin pathway in cancer control, we conditionally inactivated this pathway, using activated AMPK for inhibition. Further, we showed that AMPK siRNA treatment abrogated the ability of anthocyanins to control cell proliferation and metastatic potential, and Compound C, an AMPK inhibitor, could not restore GSK3-beta regulation, as exhibited by anthocyanins in Hep3B cells. Conclusion These observations imply that the AMPK-mediated GSK3-beta/beta-catenin circuit plays crucial roles in inhibiting cancer cell proliferation and metastasis in anthocyanin-treated hepato-carcinoma cells of Meoru origin. PMID:24666969

The J beta segment of the T cell receptor contributes to the V beta-specific T cell expansion caused by staphylococcal enterotoxin B and Urtica dioica superantigens.

PubMed

Musette, P; Galelli, A; Truffa-Bachi, P; Peumans, W; Kourilsky, P; Gachelin, G

1996-03-01

We have used a new polymerase chain reaction-based technique to analyze at the clonal level the CDR3 diversity and the J beta usage associated with the V beta-dependent T cell receptor (TCR) recognition of two superantigens: the staphylococcal enterotoxin B and the Urtica dioica agglutinin. Our results show that subset of J beta elements is preferentially expanded in a given V beta family, independently of the nature of the superantigen. By contrast, the CDR3 loop does not contribute significantly to the T cell expansion induced by the superantigens. We conclude that the J beta segment of the TCR beta chain, but not the CDR3 region, participates in superantigen binding, presumably by influencing the quaternary structure of the TCR beta chain.

Changes on the Pancreas in Experimental Diabetes and the Effect of Lycopene on These Changes: Pdx-1, Ngn-3, and Nestin Expressions.

PubMed

Sandikci, Mustafa; Karagenc, Levent; Yildiz, Mustafa

2017-12-01

The aim of the present study was to investigate changes occurring in the number of beta cells, as well as the expressions of Ngn-3, nestin and Pdx-1 of pancreatic progenitor cells in the pancreas of experimentally-induced adult diabetic rats and to determine the effect of orally-administered lycopene on these changes. Following the administration of 50 mg/kg streptozotocin to rats, four groups of animals were established: control + corn oil, control + lycopene, diabetic + corn oil and diabetic + lycopene. The animals in the control + lycopene and diabetic + lycopene groups received 4 mg/kg lycopene for a period of four weeks. The expressions of insulin, Ngn-3, nestin, and Pdx-1 were determined through immunohistochemistry in sections taken from pancreas tissue samples at the end of the experiment. The number of insulin-positive cells was found to be significantly low in the diabetic groups compared to the control groups. In addition, the presence of Ngn-3 and nestin-positive cells within the exocrine pancreas surrounding the islands was noted in the diabetic groups. Lycopene, in general did not have any effect in any of the parameters analyzed in the present study. It is suggested that these cells would function as stem cells to replace the lost beta-cell population. It is also suggested that it is possible to demonstrate the antioxidant effects of lycopene in the pancreas of diabetic rats by increasing the dose and duration of lycopene administration. Anat Rec, 300:2200-2207, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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.

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

PubMed

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

2009-01-01

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

AMP-activated protein kinase (AMPK) cross-talks with canonical Wnt signaling via phosphorylation of {beta}-catenin at Ser 552

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

Zhao, Junxing; Yue, Wanfu; Zhu, Mei J.

AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism; its activity is regulated by a plethora of physiological conditions, exercises and many anti-diabetic drugs. Recent studies show that AMPK involves in cell differentiation but the underlying mechanism remains undefined. Wingless Int-1 (Wnt)/{beta}-catenin signaling pathway regulates the differentiation of mesenchymal stem cells through enhancing {beta}-catenin/T-cell transcription factor 1 (TCF) mediated transcription. The objective of this study was to determine whether AMPK cross-talks with Wnt/{beta}-catenin signaling through phosphorylation of {beta}-catenin. C3H10T1/2 mesenchymal cells were used. Chemical inhibition of AMPK and the expression of a dominant negative AMPK decreased phosphorylation ofmore » {beta}-catenin at Ser 552. The {beta}-catenin/TCF mediated transcription was correlated with AMPK activity. In vitro, pure AMPK phosphorylated {beta}-catenin at Ser 552 and the mutation of Ser 552 to Ala prevented such phosphorylation, which was further confirmed using [{gamma}-{sup 32}P]ATP autoradiography. In conclusion, AMPK phosphorylates {beta}-catenin at Ser 552, which stabilizes {beta}-catenin, enhances {beta}-catenin/TCF mediated transcription, expanding AMPK from regulation of energy metabolism to cell differentiation and development via cross-talking with the Wnt/{beta}-catenin signaling pathway.« less

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

PubMed

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

2010-07-01

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

Inhibitory effect of dimeric beta peptide on the recurrence and metastasis of hepatocellular carcinoma in vitro and in mice.

PubMed

Wang, Song-Mei; Zhu, Jun; Pan, Luan-Feng; Liu, Yin-Kun

2008-05-21

To block the adhesion of tumor cells to the extracellular matrix, and prevent tumor metastasis and recurrence, the dimer of the beta peptide (DLYYLMDLSYSMKGGDLYYLMDLSYSMK, beta2) was designed and synthesized and its anti-adhesion and anti-invasion effects on hepatocellular carcinoma cells were assessed. Additionally, its influence on the metastasis and recurrence of mouse hepatocellular carcinoma was measured. The anti-adhesion effect of beta2 on the highly metastatic hepatocellular carcinoma cell line HCCLM6 cells and fibronectin (FN) was assayed by the MTT assay. The inhibition of invasion of HCCLM6 cells by beta2 was observed using a Transwell (modified Boyden chamber) and matrigel. Using the hepatocellular carcinoma metastasis model and LCI-D20 nude mice, the influence of beta2 on the metastasis and recurrence of hepatocellular carcinoma after early resection was investigated. HCCLM6 cells co-incubated with 100 mumol/L, 50 micromol/L, 20 micromol/L or 10 micromol/L beta2 for 3 h showed an obvious decrease in adhesion to FN. The adhesion inhibition ratios were 11.8%, 21.7%, 29.6% and 48.7%, respectively. Additionally, HCCLM6 cells cultured with 100 mumol/L beta2 had a dramatic decrease in cell invasion. beta2 was also observed to inhibit the incisal edge recurrence and the distant metastasis of nude mice hepatocellular carcinoma after early resection (P < 0.05). The beta2 peptide can specifically block the adhesion and invasion of HCCLM6 cells, and can inhibit HCC recurrence and metastasis of LCI-D20 model posthepatectomy in vivo. Thus, beta2 should be further studied as a new anti-tumor drug.

Inhibition of beta-adrenergic receptor trafficking in adult cardiocytes by MAP4 decoration of microtubules.

PubMed

Cheng, Guangmao; Qiao, Fei; Gallien, Thomas N; Kuppuswamy, Dhandapani; Cooper, George

2005-03-01

Decreased beta-adrenergic receptor (beta-AR) number occurs both in animal models of cardiac hypertrophy and failure and in patients. beta-AR recycling is an important mechanism for the beta-AR resensitization that maintains a normal complement of cell surface beta-ARs. We have shown that 1) in severe pressure overload cardiac hypertrophy, there is extensive microtubule-associated protein 4 (MAP4) decoration of a dense microtubule network; and 2) MAP4 microtubule decoration inhibits muscarinic acetylcholine receptor recycling in neuroblastoma cells. We asked here whether MAP4 microtubule decoration inhibits beta-AR recycling in adult cardiocytes. [(3)H]CGP-12177 was used as a beta-AR ligand, and feline cardiocytes were isolated and infected with adenovirus containing MAP4 (AdMAP4) or beta-galactosidase (Adbeta-gal) cDNA. MAP4 decorated the microtubules extensively only in AdMAP4 cardiocytes. beta-AR agonist exposure reduced cell surface beta-AR number comparably in AdMAP4 and Adbeta-gal cardiocytes; however, after agonist withdrawal, the cell surface beta-AR number recovered to 78.4 +/- 2.9% of the pretreatment value in Adbeta-gal cardiocytes but only to 56.8 +/- 1.4% in AdMAP4 cardiocytes (P < 0.01). This result was confirmed in cardiocytes isolated from transgenic mice having cardiac-restricted MAP4 overexpression. In functional terms of cAMP generation, beta-AR agonist responsiveness of AdMAP4 cells was 47% less than that of Adbeta-gal cells. We conclude that MAP4 microtubule decoration interferes with beta-AR recycling and that this may be one mechanism for beta-AR downregulation in heart failure.

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

Reduction of high-affinity beta2-adrenergic receptor binding by hyperforin and hyperoside on rat C6 glioblastoma cells measured by fluorescence correlation spectroscopy.

PubMed

Prenner, Lars; Sieben, Anne; Zeller, Karin; Weiser, Dieter; Häberlein, Hanns

2007-05-01

Beta-adrenergic receptors (beta-AR) are potential targets for antidepressants. Desensitization and downregulation of beta-AR are discussed as possible modes of action for antidepressants. We have investigated the effects of hyperforin and hyperoside, compounds with potentially antidepressant activity from St. John's Wort, on the binding behavior and dynamics of beta2-AR in living rat C6 glioblastoma cells, compared to desipramine (desmethylimipramine; DMI) by means of fluorescence correlation spectroscopy (FCS) and fluorescence microscopy. FCS-binding studies with the fluorescently labeled ligand Alexa532-noradrenaline (Alexa532-NA) binding to beta2-AR of C6 cells showed a significant reduction in total beta2-AR binding after preincubation with hyperforin and hyperoside for 3 days, respectively, which was also found for DMI. This was mainly observed in high-affinity receptor-ligand complexes with hindered lateral mobility (D2 = 1.1 (+/-0.4) microm2/s) in the biomembrane. However, internalization of beta2-AR was found neither in z-scans of these C6 cells nor in HEK 293 cells stably transfected with GFP-tagged beta2-adrenergic receptors (beta2AR-GFP) after incubation up to 6 days with either DMI, hyperforin, or hyperoside. Thus, under these conditions reduction of beta2-AR binding was not mediated by receptor internalization. Additionally, preincubation of C6 cells with DMI, hyperforin, and hyperoside led to a loss of second messenger cAMP after beta2-adrenergic stimulating conditions with terbutaline. Our current results indicate that hyperforin and hyperoside from St. John's Wort, as well as DMI, reduce beta2-adrenergic sensitivity in C6 cells, emphasizing the potential usefulness of St. John's Wort dry extracts in clinical treatment of depressive symptoms.

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

PubMed

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

2008-03-15

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

Differential expression of E-cadherin at the surface of rat beta-cells as a marker of functional heterogeneity.

PubMed

Bosco, Domenico; Rouiller, Dominique G; Halban, Philippe A

2007-07-01

The aim of this study was to assess whether the expression of E-cadherin at the surface of rat beta-cells is regulated by insulin secretagogues and correlates with insulin secretion. When cultured under standard conditions, virtually all beta-cells expressed E-cadherin observed by immunofluorescence, but heterogeneous staining was observed. Using fluorescence-activated cell sorting (FACS), two beta-cell sub-populations were sorted: one that was poorly labeled ('ECad-low') and another that was highly labeled ('ECad-high'). After 1-h stimulation with 16.7 mM glucose, insulin secretion (reverse hemolytic plaque assay) from individual ECad-high beta-cells was higher than that from ECad-low beta-cells. Ca2+-dependent beta-cell aggregation was increased at 16.7 mM glucose when compared with 2.8 mM glucose. E-cadherin at the surface of beta-cells was increased after 18 h at 11.1 and 22.2 mM glucose when compared with 2.8 mM glucose, with the greatest increase at 22.2 mM glucose + 0.5 mM isobutylmethylxanthine (IBMX). While no labeling was detected on freshly trypsinized cells, the proportion of stained cells increased in a time-dependent manner during culture for 1, 3, and 24 h. This recovery was faster when cells were incubated at 16.7 vs 2.8 mM glucose. Cycloheximide inhibited expression of E-cadherin at 2.8 mM glucose, but not at 16.7 mM, while depolymerization of actin by either cytochalasin B or latrunculin B increased surface E-cadherin at low glucose. In conclusion, these results show that expression of E-cadherin at the surface of islet beta-cells is controlled by secretagogues including glucose, correlates with insulin secretion, and can serve as a surface marker of beta-cell function.

Analysis of polyethylene wear debris using micro-Raman spectroscopy: a report on the presence of beta-carotene.

PubMed

Hahn, D W; Wolfarth, D L; Parks, N L

1997-04-01

This paper describes micro-Raman spectroscopy of ultra-high molecular weight polyethylene wear debris isolated from revised knee replacements. The novel application of micro-Raman spectroscopy to the analysis of in vivo-generated wear debris was used to evaluate the chemical nature of individual, retrieved polyethylene particles. The analysis revealed the presence of beta-carotene on particles from both synovial fluid and tissue samples. Raman analysis of retrieved polyethylene tibial inserts also revealed localized beta-carotene signals within the primary wear region. In this paper, a mechanism is suggested that may account for the coupling of beta-carotene and polyethylene wear debris. We also discuss the origin of beta-carotene within the implanted joint and the implications that beta-carotene, an anti-oxidant, has for the overall host response to polyethylene orthopedic components.

Influence and timing of arrival of murine neural crest on pancreatic beta cell development and maturation.

PubMed

Plank, Jennifer L; Mundell, Nathan A; Frist, Audrey Y; LeGrone, Alison W; Kim, Thomas; Musser, Melissa A; Walter, Teagan J; Labosky, Patricia A

2011-01-15

Interactions between cells from the ectoderm and mesoderm influence development of the endodermally-derived pancreas. While much is known about how mesoderm regulates pancreatic development, relatively little is understood about how and when the ectodermally-derived neural crest regulates pancreatic development and specifically, beta cell maturation. A previous study demonstrated that signals from the neural crest regulate beta cell proliferation and ultimately, beta cell mass. Here, we expand on that work to describe timing of neural crest arrival at the developing pancreatic bud and extend our knowledge of the non-cell autonomous role for neural crest derivatives in the process of beta cell maturation. We demonstrated that murine neural crest entered the pancreatic mesenchyme between the 26 and 27 somite stages (approximately 10.0 dpc) and became intermingled with pancreatic progenitors as the epithelium branched into the surrounding mesenchyme. Using a neural crest-specific deletion of the Forkhead transcription factor Foxd3, we ablated neural crest cells that migrate to the pancreatic primordium. Consistent with previous data, in the absence of Foxd3, and therefore the absence of neural crest cells, proliferation of insulin-expressing cells and insulin-positive area are increased. Analysis of endocrine cell gene expression in the absence of neural crest demonstrated that, although the number of insulin-expressing cells was increased, beta cell maturation was significantly impaired. Decreased MafA and Pdx1 expression illustrated the defect in beta cell maturation; we discovered that without neural crest, there was a reduction in the percentage of insulin-positive cells that co-expressed Glut2 and Pdx1 compared to controls. In addition, transmission electron microscopy analyses revealed decreased numbers of characteristic insulin granules and the presence of abnormal granules in insulin-expressing cells from mutant embryos. Together, these data demonstrate that the neural crest is a critical regulator of beta cell development on two levels: by negatively regulating beta cell proliferation and by promoting beta cell maturation. Copyright © 2010 Elsevier Inc. All rights reserved.

Comparison of six bone-graft substitutes regarding to cell seeding efficiency, metabolism and growth behaviour of human mesenchymal stem cells (MSC) in vitro.

PubMed

Seebach, Caroline; Schultheiss, Judith; Wilhelm, Kerstin; Frank, Johannes; Henrich, Dirk

2010-07-01

Various synthetic bone-graft substitutes are used commercially as osteoconductive scaffolds in the treatment of bone defects and fractures. The role of bone-graft substitutes is changing from osteoconductive conduits for growth to an delivery system for biologic fracture treatments. Achieving optimal bone regeneration requires biologics (e.g. MSC) and using the correct scaffold incorporated into a local environment for bone regeneration. The need for an unlimited supply with high quality bone-graft substitutes continue to find alternatives for bone replacement surgery. This in vitro study investigates cell seeding efficiency, metabolism, gene expression and growth behaviour of MSC sown on six commercially clinical available bone-graft substitutes in order to define their biological properties: synthetic silicate-substituted porous hydroxyapatite (Actifuse ABX), synthetic alpha-TCP (Biobase), synthetic beta-TCP (Vitoss), synthetic beta-TCP (Chronos), processed human cancellous allograft (Tutoplast) and processed bovines hydroxyapatite ceramic (Cerabone). 250,000 MSC derived from human bone marrow (n=4) were seeded onto the scaffolds, respectively. On days 2, 6 and 10 the adherence of MSC (fluorescence microscopy) and cellular activity (MTT assay) were analysed. Osteogenic gene expression (cbfa-1) was analysed by RT-PCR and scanning electron microscopy was performed. The highest number of adhering cells was found on Tutoplast (e.g. day 6: 110.0+/-24.0 cells/microscopic field; p<0.05) followed by Chronos (47.5+/-19.5, p<0.05), Actifuse ABX (19.1+/-4.4), Biobase (15.7+/-9.9), Vitoss (8.8+/-8.7) and Cerabone (8.1+/-2.2). MSC seeded onto Tutoplast showed highest metabolic activity and gene expression of cbfa-1. These data are confirmed by scanning electron microscopy. The cell shapes varied from round-shaped cells to wide spread cells and cell clusters, depending on the bone-graft substitutes. Processed human cancellous allograft is a well-structured and biocompatible scaffold for ingrowing MSC in vitro. Of all other synthetical scaffolds, beta-tricalcium phosphate (Chronos) have shown the best growth behaviour for MSC. Our results indicate that various bone-graft substitutes influence cell seeding efficiency, metabolic activity and growth behaviour of MSC in different manners. We detected a high variety of cellular integration of MSC in vitro, which may be important for bony integration in the clinical setting. 2010 Elsevier Ltd. All rights reserved.

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.

Tumor-produced, active Interleukin-1 {beta} regulates gene expression in carcinoma-associated fibroblasts

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

Dudas, Jozsef, E-mail: Jozsef.Dudas@i-med.ac.at; Fullar, Alexandra, E-mail: fullarsz@gmail.com; 1st Institute of Pathology and Experimental Cancer Research, Semmelweis University, Ulloei ut 26, H-1085 Budapest

2011-09-10

Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial-mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1{beta} (IL1-{beta}) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-{beta} expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-{beta} processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated withmore » IL1-{beta}. IL1-{beta} signaling was investigated by western blot and immunocytochemistry. IL1-{beta}-regulated genes were analyzed by real-time qPCR. SCC-25 cells produced 16 kD active IL1-{beta}, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NF{kappa}B{alpha}. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-{beta} reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-{beta}-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-{beta} in the tumor cells leads to IL1-{beta}-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression. -- Graphical abstract: SCC-25 cells produce active, processed IL1-{beta}. PDL fibroblasts possess receptor for IL1-{beta}, and its expression is increased 4.56-times in the presence of SCC-25 tumor cells. IL1-{beta} receptor expression in fibroblasts, especially in CAFs represents a major option in coordination of fibroblast and tumor behavior. A key event in IL1-{beta} signaling, the phosphorylation of IRAK1, occurred in co-cultured fibroblasts, which has lead to nuclear translocation of NF{kappa}B{alpha}, and finally to induction of several genes, including BDNF, IRF1, IL-6 and COX-2. The most enhanced induction was found for IL-6 and COX-2.« less

Beta-lactamase induction and cell wall metabolism in Gram-negative bacteria

PubMed Central

Zeng, Ximin; Lin, Jun

2013-01-01

Production of beta-lactamases, the enzymes that degrade beta-lactam antibiotics, is the most widespread and threatening mechanism of antibiotic resistance. In the past, extensive research has focused on the structure, function, and ecology of beta-lactamases while limited efforts were placed on the regulatory mechanisms of beta-lactamases. Recently, increasing evidence demonstrate a direct link between beta-lactamase induction and cell wall metabolism in Gram-negative bacteria. Specifically, expression of beta-lactamase could be induced by the liberated murein fragments, such as muropeptides. This article summarizes current knowledge on cell wall metabolism, beta-lactam antibiotics, and beta-lactamases. In particular, we comprehensively reviewed recent studies on the beta-lactamase induction by muropeptides via two major molecular mechanisms (the AmpG–AmpR–AmpC pathway and BlrAB-like two-component regulatory system) in Gram-negative bacteria. The signaling pathways for beta-lactamase induction offer a broad array of promising targets for the discovery of new antibacterial drugs used for combination therapies. Therefore, to develop effective mitigation strategies against the widespread beta-lactam resistance, examination of the molecular basis of beta-lactamase induction by cell wall fragment is highly warranted. PMID:23734147

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

Amino acid substitutions enhancing thermostability of Bacillus polymyxa beta-glucosidase A.

PubMed Central

Lopez-Camacho, C; Salgado, J; Lequerica, J L; Madarro, A; Ballestar, E; Franco, L; Polaina, J

1996-01-01

Mutations enhancing the thermostability of beta-glucosidase A of Bacillus polymyxa, a family 1 glycosyl hydrolase, have been obtained after hydroxylamine mutagenesis of a plasmid containing the bglA gene, transformation of Escherichia coli with the mutagenized plasmid, and identification of transformant colonies that showed beta-glucosidase activity after a thermal treatment that inactivated the wild-type enzyme. Two additive mutations have been characterized that cause replacement of glutamate at position 96 by lysine and of methionine at position 416 by isoleucine respectively. The thermoresistant mutant enzymes showed increased resistance to other denaturing agents, such as pH and urea, while their kinetic parameters did not change. CD spectra indicated that the E96K replacement caused an increase in alpha-helix content. The observed effect of the M416I mutation is consistent with the lower content of cysteine and methionine found in family 1 enzymes of thermophilic species compared with similar ones from mesophilic organisms. PMID:8615777

β-Arrestin2 plays a key role in the modulation of the pancreatic beta cell mass in mice.

PubMed

Ravier, Magalie A; Leduc, Michele; Richard, Joy; Linck, Nathalie; Varrault, Annie; Pirot, Nelly; Roussel, Morgane M; Bockaert, Joël; Dalle, Stéphane; Bertrand, Gyslaine

2014-03-01

Beta cell failure due to progressive secretory dysfunction and limited expansion of beta cell mass is a key feature of type 2 diabetes. Beta cell function and mass are controlled by glucose and hormones/neurotransmitters that activate G protein-coupled receptors or receptor tyrosine kinases. We have investigated the role of β-arrestin (ARRB)2, a scaffold protein known to modulate such receptor signalling, in the modulation of beta cell function and mass, with a specific interest in glucagon-like peptide-1 (GLP-1), muscarinic and insulin receptors. β-arrestin2-knockout mice and their wild-type littermates were fed a normal or a high-fat diet (HFD). Glucose tolerance, insulin sensitivity and insulin secretion were assessed in vivo. Beta cell mass was evaluated in pancreatic sections. Free cytosolic [Ca(2+)] and insulin secretion were determined using perifused islets. The insulin signalling pathway was evaluated by western blotting. Arrb2-knockout mice exhibited impaired glucose tolerance and insulin secretion in vivo, but normal insulin sensitivity compared with wild type. Surprisingly, the absence of ARRB2 did not affect glucose-stimulated insulin secretion or GLP-1- and acetylcholine-mediated amplifications from perifused islets, but it decreased the islet insulin content and beta cell mass. Additionally, there was no compensatory beta cell mass expansion through proliferation in response to the HFD. Furthermore, Arrb2 deletion altered the islet insulin signalling pathway. ARRB2 is unlikely to be involved in the regulation of insulin secretion, but it is required for beta cell mass plasticity. Additionally, we provide new insights into the mechanisms involved in insulin signalling in beta cells.

Mutant HNF-1{alpha} and mutant HNF-1{beta} identified in MODY3 and MODY5 downregulate DPP-IV gene expression in Caco-2 cells

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

Gu Ning; Laboratory of Neurochemistry, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto; Adachi, Tetsuya

2006-08-04

Dipeptidylpeptidase IV (DPP-IV) is a well-documented drug target for the treatment of type 2 diabetes. Hepatocyte nuclear factors (HNF)-1{alpha} and HNF-1{beta}, known as the causal genes of MODY3 and MODY5, respectively, have been reported to be involved in regulation of DPP-IV gene expression. But, it is not completely clear (i) that they play roles in regulation of DPP-IV gene expression, and (ii) whether DPP-IV gene activity is changed by mutant HNF-1{alpha} and mutant HNF-1{beta} in MODY3 and MODY5. To explore these questions, we investigated transactivation effects of wild HNF-1{alpha} and 13 mutant HNF-1{alpha}, as well as wild HNF-1{beta} and 2more » mutant HNF-1{beta}, on DPP-IV promoter luciferase gene in Caco-2 cells by means of a transient experiment. Both wild HNF-1{alpha} and wild HNF-1{beta} significantly transactivated DPP-IV promoter, but mutant HNF-1{alpha} and mutant HNF-1{beta} exhibited low transactivation activity. Moreover, to study whether mutant HNF-1{alpha} and mutant HNF-1{beta} change endogenous DPP-IV enzyme activity, we produced four stable cell lines from Caco-2 cells, in which wild HNF-1{alpha} or wild HNF-1{beta}, or else respective dominant-negative mutant HNF-1{alpha}T539fsdelC or dominant-negative mutant HNF-1{beta}R177X, was stably expressed. We found that DPP-IV gene expression and enzyme activity were significantly increased in wild HNF-1{alpha} cells and wild HNF-1{beta} cells, whereas they decreased in HNF-1{alpha}T539fsdelC cells and HNF-1{beta}R177X cells, compared with DPP-IV gene expression and enzyme activity in Caco-2 cells. These results suggest that both wild HNF-1{alpha} and wild HNF-1{beta} have a stimulatory effect on DPP-IV gene expression, but that mutant HNF-1{alpha} and mutant HNF-1{beta} attenuate the stimulatory effect.« less

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

Effect of interlukin-1beta on proliferation of gastric epithelial cells in culture.

PubMed

Beales, Ian L P

2002-04-05

Helicobacter pylori is the main risk factor for the development of non-cardia gastric cancer. Increased proliferation of the gastric mucosa is a feature of H. pylori infection. Mucosal interkeukin-1beta production is increased in H. pylori infection and IL-1beta genotypes associated with increased pro-inflammatory activity are risk factors for the development of gastric cancer. The effect of IL-1beta on gastric epithelial cell proliferation has been examined in this study. AGS cells were cultured with IL-1beta. DNA synthesis was assed by [3H]thymidine incorporation and total viable cell numbers by MTT assay. IL-1beta dose dependently increased DNA synthesis and cell numbers. The enhanced proliferation was blocked by interleukin-1 receptor antagonist. Addition of neutralising antibody to GM-CSF reduced IL-1beta-stimulated proliferation by 31 +/- 4 %. GM-CSF alone significantly stimulated proliferation. Addition or neutralisation of IL-8 had no effect on basal or IL-1beta-stimulated proliferation. The tyrosine kinase inhibitor genistein completely blocked IL-1beta-stimulated proliferation and inhibition of the extracellular signal related kinase pathway with PD 98059 inhibited IL-1beta stimulated proliferation by 58 +/- 5 %. IL-1beta stimulates proliferation in gastric epithelial cells. Autocrine stimulation by GM-CSF contributes to this proliferative response. Signalling via tyrosine kinase activity is essential to the mitogenic response to IL-1beta. The extracellular signal related kinase pathway is involved in, but not essential to downstream signalling. IL-1beta may contribute to the hyperproliferation seen in H. pylori- infected gastric mucosa, and be involved in the carcinogenic process.

Interpreting beta-diversity components over time to conserve metacommunities in highly dynamic ecosystems.

PubMed

Ruhí, Albert; Datry, Thibault; Sabo, John L

2017-12-01

The concept of metacommunity (i.e., a set of local communities linked by dispersal) has gained great popularity among community ecologists. However, metacommunity research mostly addresses questions on spatial patterns of biodiversity at the regional scale, whereas conservation planning requires quantifying temporal variation in those metacommunities and the contributions that individual (local) sites make to regional dynamics. We propose that recent advances in diversity-partitioning methods may allow for a better understanding of metacommunity dynamics and the identification of keystone sites. We used time series of the 2 components of beta diversity (richness and replacement) and the contributions of local sites to these components to examine which sites controlled source-sink dynamics in a highly dynamic model system (an intermittent river). The relative importance of the richness and replacement components of beta diversity fluctuated over time, and sample aggregation led to underestimation of beta diversity by up to 35%. Our literature review revealed that research on intermittent rivers would benefit greatly from examination of beta-diversity components over time. Adequately appraising spatiotemporal variability in community composition and identifying sites that are pivotal for maintaining biodiversity at the landscape scale are key needs for conservation prioritization and planning. Thus, our framework may be used to guide conservation actions in highly dynamic ecosystems when time-series data describing biodiversity across sites connected by dispersal are available. © 2017 Society for Conservation Biology.

Islets of Langerhans in the parakeet, Psittacula krameri.

PubMed

Gupta, Y K; Kumar, S

1980-01-01

The pancreatic gland of Psittacula krameri is divisible into 4 lobes i.e. dorsal, ventral, third and splenic. The endocrine part is composed of alpha 1-, alpha 2- and beta-cells. The islets are of 4 kinds viz., alpha islets (having alpha 1- and alpha 2-cells), beta islets (having beta- and alpha 1-cells), pure beta islets (consisting of beta-cells exclusively) and mixed islets (with beta-, alpha 1- and alpha 2-cells). The distribution of alpha islets is mostly restricted to the splenic and third lobes whereas the beta islets are found in all 4 lobes. Though the alpha islets are only few in the dorsal lobe, their size is best developed in the third and dorsal lobes. Sometimes beta and alpha islets are present in very close proximity but their cells never mingle. An interesting feature was the complete absence of alpha islets from the ventral lobe.A relative abundance of alpha 2- cells in this bird seems to be associated with its comparatively higher blood glucose level and frugivorous habit. Tinctorial reactions suggest that the insulin content of the endocrine pancreas is low. There were no seasonal changes in the islet tissue of P. krameri.

Deficiency of bone marrow beta3-integrin enhances non-functional neovascularization.

PubMed

Watson, Alan R; Pitchford, Simon C; Reynolds, Louise E; Direkze, Natalie; Brittan, Mairi; Alison, Malcolm R; Rankin, Sara; Wright, Nicholas A; Hodivala-Dilke, Kairbaan M

2010-03-01

beta3-Integrin is a cell surface adhesion and signalling molecule important in the regulation of tumour angiogenesis. Mice with a global deficiency in beta3-integrin show increased pathological angiogenesis, most likely due to increased vascular endothelial growth factor receptor 2 expression on beta3-null endothelial cells. Here we transplanted beta3-null bone marrow (BM) into wild-type (WT) mice to dissect the role of BM beta3-integrin deficiency in pathological angiogenesis. Mice transplanted with beta3-null bone marrow show significantly enhanced angiogenesis in subcutaneous B16F0 melanoma and Lewis lung carcinoma (LLC) cell models and in B16F0 melanoma lung metastasis when compared with tumours grown in mice transplanted with WT bone marrow. The effect of bone marrow beta3-integrin deficiency was also assessed in the RIPTAg mouse model of pancreatic tumour growth. Again, angiogenesis in mice lacking BM beta3-integrin was enhanced. However, tumour weight between the groups was not significantly altered, suggesting that the enhanced blood vessel density in the mice transplanted with beta3-null bone marrow was not functional. Indeed, we demonstrate that in mice transplanted with beta3-null bone marrow a significant proportion of tumour blood vessels are non-functional when compared with tumour blood vessels in WT-transplanted controls. Furthermore, beta3-null-transplanted mice showed an increased angiogenic response to VEGF in vivo when compared with WT-transplanted animals. BM beta3-integrin deficiency affects the mobilization of progenitor cells to the peripheral circulation. We show that VEGF-induced mobilization of endothelial progenitor cells is enhanced in mice transplanted with beta3-null bone marrow when compared with WT-transplanted controls, suggesting a possible mechanism underlying the increased blood vessel density seen in beta3-null-transplanted mice. In conclusion, although BM beta3-integrin is not required for pathological angiogenesis, our studies demonstrate a role for BM beta3-integrin in VEGF-induced mobilization of bone marrow-derived cells to the peripheral circulation and for the functionality of those vessels in which BM-derived cells become incorporated.

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.

Boldenone, boldione, and milk replacers in the diet of veal calves: the effects of phytosterol content on the urinary excretion of boldenone metabolites.

PubMed

Gallina, G; Ferretti, G; Merlanti, R; Civitareale, C; Capolongo, F; Draisci, R; Montesissa, C

2007-10-03

Twenty-six veal calves were split into two groups and fed two milk replacers with a different content of phytosterols for 26 days; then, 14 calves (7 animals from each diet) were kept as controls and 12 calves (6 per diet) received daily, per os, a combination of 17beta-boldenone (17beta-Bol) and androsta-1,4-dien-3,17-dione (ADD) for 38 days. The urinary elimination of 17 alpha-/17beta-boldenone conjugates (17 alpha/beta-Bol) and androsta-1,4-dien-3,17-dione (ADD) was followed by liquid chromatography-tandem mass spectrometry from all of the animals until slaughtering. In urine from treated animals, 17 alpha-Bol concentrations, despite a great variability, were greater than 17beta-Bol, both detected always as conjugates. At days 1, 2, and 3, the mean urine concentration of 17 alpha-Bol was higher than 12 ng/mL. A remarkable decrease was observed during the following days, but the 17 alpha-Bol concentration was still higher than the attention level of 2 ng/mL in 58% of the samples; the concentration of 17beta-Bol was around the action level of 1 ng/mL; two days after treatment withdrawal, no 17beta-Bol was detected in the urine. In urine from control animals, the 17 alpha-Bol concentration was strictly related to the phytosterol content of the diet, while, in urine from treated animals, the much higher 17 alpha-Bol levels were not modified by the production from diet precursors. The results confirmed that a 17 alpha-Bol level higher than 2 ng/mL should be considered as evidence of suspected illegal treatment and that the urinary excretion of 17beta-Bol is due to exogenous administration of 17beta-Bol. The discontinuous rate of elimination of both 17 alpha- and 17beta-Bol, despite the daily administration of 17beta-Bol plus ADD, indicates the necessity for further research to detect other urinary boldenone metabolites to strength surveillance strategy.

Impaired compensatory beta-cell function and growth in response to high-fat diet in LDL receptor knockout mice

PubMed Central

Oliveira, Ricardo B d; Carvalho, Carolina P d F; Polo, Carla C; Dorighello, Gabriel d G; Boschero, Antônio C; Oliveira, Helena C F d; Collares-Buzato, Carla B

2014-01-01

In this study, we investigated the effect of low density lipoprotein receptor (LDLr) deficiency on gap junctional connexin 36 (Cx36) islet content and on the functional and growth response of pancreatic beta-cells in C57BL/6 mice fed a high-fat (HF) diet. After 60 days on regular or HF diet, the metabolic state and morphometric islet parameters of wild-type (WT) and LDLr−/− mice were assessed. HF diet-fed WT animals became obese and hypercholesterolaemic as well as hyperglycaemic, hyperinsulinaemic, glucose intolerant and insulin resistant, characterizing them as prediabetic. Also they showed a significant decrease in beta-cell secretory response to glucose. Overall, LDLr−/− mice displayed greater susceptibility to HF diet as judged by their marked cholesterolaemia, intolerance to glucose and pronounced decrease in glucose-stimulated insulin secretion. HF diet induced similarly in WT and LDLr−/− mice, a significant decrease in Cx36 beta-cell content as revealed by immunoblotting. Prediabetic WT mice displayed marked increase in beta-cell mass mainly due to beta-cell hypertrophy/replication. Nevertheless, HF diet-fed LDLr−/− mice showed no significant changes in beta-cell mass, but lower islet–duct association (neogenesis) and higher beta-cell apoptosis index were seen as compared to controls. The higher metabolic susceptibility to HF diet of LDLr−/− mice may be explained by a deficiency in insulin secretory response to glucose associated with lack of compensatory beta-cell expansion. PMID:24853046

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.

Expression of the leukemia-associated CBF{beta}/SMMHC chimeric gene causes transformation of 3T3 cells

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

Hajra, A.; Liu, P.; Collins, E.S.

1994-09-01

A pericentric inversion of chromosome 16 (inv(16)(p13;q22)) is consistently seen in acute myeloid leukemia of the M4Eo subtype. This inversion fuses almost the entire coding region of the gene encoding of the {beta} subunit of the heterodimeric transcription factor CBF/PEBP2 to the region of the MYH11 gene encoding the rod domain for the smooth muscle myosin heavy chain (SMMHC). To investigate the biological properties of the CBF{beta}/SMMHC fusion protein, we have generated 3T3 cell lines that stably express the CBF{beta}/SMMHC chimeric cDNA or the normal, nonchimeric CBF{beta} and SMMHC cDNAs. 3T3 cells expressing CBF{beta}/SMMHC acquire a transformed phenotype, as indicatedmore » by altered cell morphology, formation of foci, and growth in soft agar. Cells constitutively overexpressing the normal CBF{beta} cDNA or the rod region of SMMHC remain nontransformed. Western blot analysis using antibodies to CBF{beta} and the SMMHC rod demonstrates that stably transfected cells express the appropriate chimeric or normal protein. Electrophoretic mobility shift assays reveal that cells transformed by the chimeric cDNA do not have a CBF-DNA complex of the expected mobility, but instead contain a large complex with CBF DNA-binding activity that fails to migrate out of the gel wells. In order to define the regions of CBF{beta}/SMMHC necessary for 3T3 transformation, we have stably transfected cells with mutant CBF{beta}/SMMHC cDNAs containing various deletions of the coding region. Analysis of these cell lines indicates that the transformation property of CBF{beta}/SMMHC requires regions of CBF{beta} known to be necessary for association with the DNA-binding CBF{alpha} subunit, and also requires an intact SMMHC carboxyl terminus, which is necessary for formation of the coiled coil domain of the myosin rod.« less

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

PubMed

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

2008-01-01

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

A transmission infrared cell design for temperature-controlled adsorption and reactivity studies on heterogeneous catalysts

NASA Astrophysics Data System (ADS)

Cybulskis, Viktor J.; Harris, James W.; Zvinevich, Yury; Ribeiro, Fabio H.; Gounder, Rajamani

2016-10-01

A design is presented for a versatile transmission infrared cell that can interface with an external vacuum manifold to undergo in situ gas treatments and receive controlled doses of various adsorbates and probe molecules, allowing characterization of heterogeneous catalyst surfaces in order to identify and quantify active sites and adsorbed surface species. Critical design characteristics include customized temperature control for operation between cryogenic and elevated temperatures (100-1000 K) and modified Cajon fittings for operation over a wide pressure range (10-2-103 Torr) that eliminates the complications introduced when using sealants or flanges to secure cell windows. The customized, hand-tightened Cajon fittings simplify operation of the cell compared to previously reported designs, because they allow for rapid cell assembly and disassembly and, in turn, replacement of catalyst samples. In order to validate the performance of the cell, transmission infrared spectroscopic experiments are reported to characterize the Brønsted and Lewis acid sites present in H-beta and H-mordenite zeolites using cryogenic adsorption of CO (<150 K).

A transmission infrared cell design for temperature-controlled adsorption and reactivity studies on heterogeneous catalysts.

PubMed

Cybulskis, Viktor J; Harris, James W; Zvinevich, Yury; Ribeiro, Fabio H; Gounder, Rajamani

2016-10-01

A design is presented for a versatile transmission infrared cell that can interface with an external vacuum manifold to undergo in situ gas treatments and receive controlled doses of various adsorbates and probe molecules, allowing characterization of heterogeneous catalyst surfaces in order to identify and quantify active sites and adsorbed surface species. Critical design characteristics include customized temperature control for operation between cryogenic and elevated temperatures (100-1000 K) and modified Cajon fittings for operation over a wide pressure range (10 -2 -10 3 Torr) that eliminates the complications introduced when using sealants or flanges to secure cell windows. The customized, hand-tightened Cajon fittings simplify operation of the cell compared to previously reported designs, because they allow for rapid cell assembly and disassembly and, in turn, replacement of catalyst samples. In order to validate the performance of the cell, transmission infrared spectroscopic experiments are reported to characterize the Brønsted and Lewis acid sites present in H-beta and H-mordenite zeolites using cryogenic adsorption of CO (<150 K).

{beta}-Catenin regulates airway smooth muscle contraction.

PubMed

Jansen, Sepp R; Van Ziel, Anna M; Baarsma, Hoeke A; Gosens, Reinoud

2010-08-01

beta-Catenin is an 88-kDa member of the armadillo family of proteins that is associated with the cadherin-catenin complex in the plasma membrane. This complex interacts dynamically with the actin cytoskeleton to stabilize adherens junctions, which play a central role in force transmission by smooth muscle cells. Therefore, in the present study, we hypothesized a role for beta-catenin in the regulation of smooth muscle force production. beta-Catenin colocalized with smooth muscle alpha-actin (sm-alpha-actin) and N-cadherin in plasma membrane fractions and coimmunoprecipitated with sm-alpha-actin and N-cadherin in lysates of bovine tracheal smooth muscle (BTSM) strips. Moreover, immunocytochemistry of cultured BTSM cells revealed clear and specific colocalization of sm-alpha-actin and beta-catenin at the sites of cell-cell contact. Treatment of BTSM strips with the pharmacological beta-catenin/T cell factor-4 (TCF4) inhibitor PKF115-584 (100 nM) reduced beta-catenin expression in BTSM whole tissue lysates and in plasma membrane fractions and reduced maximal KCl- and methacholine-induced force production. These changes in force production were not accompanied by changes in the expression of sm-alpha-actin or sm-myosin heavy chain (MHC). Likewise, small interfering RNA (siRNA) knockdown of beta-catenin in BTSM strips reduced beta-catenin expression and attenuated maximal KCl- and methacholine-induced contractions without affecting sm-alpha-actin or sm-MHC expression. Conversely, pharmacological (SB-216763, LiCl) or insulin-induced inhibition of glycogen synthase kinase-3 (GSK-3) enhanced the expression of beta-catenin and augmented maximal KCl- and methacholine-induced contractions. We conclude that beta-catenin is a plasma membrane-associated protein in airway smooth muscle that regulates active tension development, presumably by stabilizing cell-cell contacts and thereby supporting force transmission between neighboring cells.

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.

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.

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.

Insulin-producing cells could not mimic the physiological regulation of insulin secretion performed by pancreatic beta cells

PubMed Central

2013-01-01

Objective The aim of this study was to compare the difference between insulin-producing cells (IPCs) and normal human pancreatic beta cells both in physiological function and morphological features in cellular level. Methods The levels of insulin secretion were measured by enzyme-linked immunosorbent assay. The insulin gene expression was determined by real-time quantitative polymerase chain reaction. The morphological features were detected by atomic force microscopy (AFM) and laser confocal scanning microscopy. Results IPCs and normal human pancreatic beta cells were similar to each other under the observation in AFM with the porous structure features in the cytoplasm. Both number of membrane particle size and average roughness of normal human beta cells were higher than those of IPCs. Conclusions Our results firstly revealed that the cellular ultrastructure of IPCs was closer to that of normal human pancreatic beta cells, but they still could not mimic the physiological regulation of insulin secretion performed by pancreatic beta cells. PMID:23421382

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.

Gender-specific effects of endogenous testosterone: female alpha-estrogen receptor-deficient C57Bl/6J mice develop glomerulosclerosis.

PubMed

Elliot, S J; Berho, M; Korach, K; Doublier, S; Lupia, E; Striker, G E; Karl, M

2007-08-01

Young female mice on a C57Bl/6J (B6) background are considered glomerulosclerosis (GS)-resistant but aging B6 mice develop mild GS. Estrogen deficiency accelerates while estrogen replacement retards GS in young sclerosis-prone oligosyndactyly mutant mice on an ROP background. To explore the effects of sex hormones on glomerular structure and function in the context of gender and genetic background, we studied mice in which the estrogen-receptor (ER) genes alpha- or -beta were deleted (alpha- or betaER knockout (KO)) and crossed into the B6 background. We also studied ovariectomized (Ovx) B6 mice given testosterone. Male and female betaERKO and male alphaERKO mice had no glomerular dysfunction at 9 months of age; however, alphaERKO female mice displayed albuminuria and GS. Ovx prevented glomerular dysfunction in alphaERKO female mice by eliminating endogenous testosterone production while exogenous testosterone induced GS in Ovx B6 mice. Androgen receptor (AR) expression and function was found in microdissected glomeruli and cultured mesangial cells. Testosterone compared to placebo increased both AR expression and TGF-beta1 mRNA levels in glomeruli isolated from female B6 mice. Estrogen deficiency had no deleterious effects on the glomeruli in B6 mice. Our study shows that genetic traits strongly influence the GS-promoting effects of estrogen deficiency while testosterone induces GS in a gender-specific manner.

The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells.

PubMed

Collombat, Patrick; Xu, Xiaobo; Ravassard, Philippe; Sosa-Pineda, Beatriz; Dussaud, Sébastien; Billestrup, Nils; Madsen, Ole D; Serup, Palle; Heimberg, Harry; Mansouri, Ahmed

2009-08-07

We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell mass and curing diabetes in animals that have been chemically depleted of beta cells.

Genome-wide analysis of DNA methylation variations caused by chronic glucolipotoxicity in beta-cells.

PubMed

Hu, Y; Xu, X-H; He, K; Zhang, L-L; Wang, S-K; Pan, Y-Q; He, B-S; Feng, T-T; Mao, X-M

2014-02-01

There is a growing body of literature suggesting the role of interactions between genes and the environment in development of type 2 diabetes mellitus (T2DM). However, the interplay between environment and genetic in developing and progressing T2MD is not fully understood. To determine the effects of high-glucose-lipid on the status of DNA methylation in beta cells, and clarify the mechanism of glucolipotoxicity on beta-cell deterioration, the DNA methylation profile was detected in beta-cells cultured with high-glucose-lipid medium.We utilized a high throughput NimbleGen RN34 CpG Island & Promoter Microarray to investigate the DNA methylation profile in beta-cells cultured with high-glucose-lipid medium. To validate the results of microarray, the immunoprecipitation (MeDIP) PCR was used to test the methylation status of some selected genes. The mRNA and protein expression of insulin and Tcf7l2 in these cells were quantified by RT-PCR and western blot, respectively.We have identified a lot of loci which experienced aberrant DNA methylation in beta-cells cultured with high-glucose-lipid medium. The results of MeDIP PCR were consistency to the microarray. An opposite regulation in transcription and translation of Tcf7l2 gene was found. Furthermore, the insulin mRNA and protein expression in beta-cells also decreased after cultured with high-glucose-lipid medium compared with the control cells.We conclude that chronic glucolipotoxicity could induce aberrant DNA methylation of some genes and may affect these genes expression in beta-cells, which might contribute to beta-cell function failure in T2DM and be helpful to explain, at least partially, the mechanism of glucolipotoxicity on beta-cells deterioration. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York.

Pathological features of olfactory neuroblastoma in an axolotl (Ambystoma mexicanum).

PubMed

Shioda, Chieko; Uchida, Kazuyuki; Nakayama, Hiroyuki

2011-08-01

A one-year-old, female Mexican axolotl (Ambystoma mexicanum) had a rough-surfaced, polypoid, pink tumor mass of approximately 10 mm in diameter in the oral cavity. Histologically, the tumor extended from the ethmoturbinate region and into the oral cavity and had replaced some of the maxillary bone tissue. The tumor mass was composed of a lobular architecture of small round-shaped tumor cells with occasional Flexner-Wintersteiner-like rosette formation. There were no metastatic lesions in the other organs. Immunohistochemically, the tumor cells were partly positive for several neural markers (class III beta-tubulin, S-100 protein, and doublecortin) and intensely positive for an epithelial marker (cytokeratin AE1/AE3). These results suggest that the present tumor originated from neuroectodermal tissue. Considering the location and histological and immunohistochemical features of the tumor, a diagnosis of olfactory neuroblastoma was made.

Therapeutic evaluation of GM2 gangliosidoses by ELISA using anti-GM2 ganglioside antibodies.

PubMed

Tsuji, Daisuke; Higashine, Yukari; Matsuoka, Kazuhiko; Sakuraba, Hitoshi; Itoh, Kohji

2007-03-01

GM2 gangliosidoses, including Tay-Sachs disease, Sandhoff disease and the AB variant, comprise deficiencies of beta-hexosaminidase isozymes and GM2 ganglioside activator protein associated with accumulation of GM2 ganglioside (GM2) in lysosomes and neurosomatic clinical manifestations. A simple assay system for intracellular quantification of GM2 is required to evaluate the therapeutic effects on GM2-gangliosidoses. We newly established a cell-ELISA system involving anti-GM2 monoclonal antibodies for measuring GM2 storage in fibroblasts from Tay-Sachs and Sandhoff disease patients. We succeeded in detecting the corrective effect of enzyme replacement on elimination of GM2 in the cells with this ELISA system. This simple and sensitive system should be useful as additional diagnosis tool as well as therapeutic evaluation of GM2 gangliosidoses.

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.

Genetics Home Reference: sickle cell disease

MedlinePlus

... of beta-globin; this abnormality is called beta thalassemia . In people with sickle cell disease , at least ... globin. If mutations that produce hemoglobin S and beta thalassemia occur together, individuals have hemoglobin S- beta thalassemia (HbSBetaThal) ...

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

Wang, Jiying; Ohno-Matsui, Kyoko, E-mail: k.ohno.oph@tmd.ac.jp; Morita, Ikuo

Highlights: Black-Right-Pointing-Pointer Cholesterol-treated RPE produces more A{beta} than non-treated RPE. Black-Right-Pointing-Pointer Neprilysin expression and activity decreased in cholesterol-treated RPE. Black-Right-Pointing-Pointer {alpha}-Secretase expression and activity decreased in cholesterol-treated RPE. Black-Right-Pointing-Pointer Cholesterol-enriched diet induced subRPE deposits in aged mice. Black-Right-Pointing-Pointer A{beta} were present in cholesterol-enriched-diet-induced subRPE deposits in aged mice. -- Abstract: Subretinally-deposited amyloid {beta} (A{beta}) is a main contributor of developing age-related macular degeneration (AMD). However, the mechanism causing A{beta} deposition in AMD eyes is unknown. Hypercholesterolemia is a significant risk for developing AMD. Thus, we investigated the effects of cholesterol on A{beta} production in retinal pigment epithelial (RPE) cells inmore » vitro and in the mouse retina in vivo. RPE cells isolated from senescent (12-month-old) C57BL/6 mice were treated with 10 {mu}g/ml cholesterol for 48 h. A{beta} amounts in culture supernatants were measured by ELISA. Activity and expression of enzymes and proteins that regulate A{beta} production were examined by activity assay and real time PCR. The retina of mice fed cholesterol-enriched diet was examined by transmission electron microscopy. Cholesterol significantly increased A{beta} production in cultured RPE cells. Activities of A{beta} degradation enzyme; neprilysin (NEP) and anti-amyloidogenic secretase; {alpha}-secretase were significantly decreased in cell lysates of cholesterol-treated RPE cells compared to non-treated cells, but there was no change in the activities of {beta}- or {gamma}-secretase. mRNA levels of NEP and {alpha}-secretase (ADAM10 and ADAM17) were significantly lower in cholesterol-treated RPE cells than non-treated cells. Senescent (12-month-old) mice fed cholesterol-enriched chow developed subRPE deposits containing A{beta}, whereas age-matched mice fed standard rodent chow diet did not. Activities and mRNA levels of NEP and {alpha}-secretase were significantly lower in native RPE cells freshly isolated from cholesterol-enriched chow fed mice compared to standard rodent chow fed mice. These findings suggest that cholesterol enhances subretinal A{beta} accumulation by modulating the activities of enzymes degrading and processing A{beta} in RPE cells in senescent subjects.« less

Beta2 toxin is not involved in in vitro cell cytotoxicity caused by human and porcine cpb2-harbouring Clostridium perfringens.

PubMed

Allaart, Janneke G; van Asten, Alphons J A M; Vernooij, Johannes C M; Gröne, Andrea

2014-06-25

Clostridium perfringens is a common cause of intestinal disease in animals and humans. Its pathogenicity is attributed to the toxins it can produce, including the beta2 toxin. The presence of cpb2, the gene encoding the beta2 toxin, has been associated with diarrhoea in neonatal piglets and humans. However, the exact role of the beta2 toxin in the development of diarrhoea is still unknown. In this study we investigated the level of cytotoxicity to porcine IPI-21 and human Caco-2 cell-lines caused by porcine and human cpb2-harbouring C. perfringens and the significance of the beta2 toxin for the induction of cell cytotoxicity. Supernatants of porcine cpb2-harbouring C. perfringens strains were cytotoxic to both cell lines. Cell cytotoxicity caused by supernatant of human cpb2-harbouring C. perfringens strains was variable among strains. However, removal of the beta2 toxin by anti-beta2 toxin antibodies or degradation of the beta2 toxin by trypsin did not reduce the cytotoxic effect of any of the supernatants. These data suggest that beta2 toxin does not play a role in the development of cell cytotoxicity in in vitro experiments. In vivo studies are necessary to definitely define the role of beta2 toxin in the development of cell cytotoxicity and subsequent diarrhoea. Copyright © 2014 Elsevier B.V. All rights reserved.

Epidermal differentiation during ontogeny and after hatching in the snake Liasis fuscus (Pythonidae, Serpentes, Reptilia), with emphasis on the formation of the shedding complex.

PubMed

Alibardi, L; Thompson, M B

2003-04-01

Differentiation and localization of keratin in the epidermis during embryonic development and up to 3 months posthatching in the Australian water python, Liasis fuscus, was studied by ultrastructural and immunocytochemical methods. Scales arise from dome-like folds in the skin that produce tightly imbricating scales. The dermis of these scales is completely differentiated before any epidermal differentiation begins, with a loose dermis made of mesenchymal cells beneath the differentiating outer scale surface. At this stage (33) the embryo is still unpigmented and two layers of suprabasal cells contain abundant glycogen. At Stage 34 (beginning of pigmentation) the first layers of cells beneath the bilayered periderm (presumptive clear and oberhautchen layers) have not yet formed a shedding complex, within which prehatching shedding takes place. At Stage 35 the shedding complex, consisting of the clear and oberhautchen layers, is discernible. The clear layer contains a fine fibrous network that faces the underlying oberhautchen, where the spinulae initially contain a core of fibrous material and small beta-keratin packets. Differentiation continues at Stage 36 when the beta-layer forms and beta-keratin packets are deposited both on the fibrous core of the oberhautchen and within beta-cells. Mesos cells are produced from the germinal layer but remain undifferentiated. At Stage 37, before hatching, the beta-layer is compact, the mesos layer contains mesos granules, and cells of the alpha-layer are present but are not yet keratinized. They are still only partially differentiated a few hours after hatching, when a new shedding complex is forming underneath. Using antibodies against chick scale beta-keratin resolved at high magnification with immunofluorescent or immunogold conjugates, we offer the first molecular confirmation that in snakes only the oberhautchen component of the shedding complex and the underlying beta cells contain beta-keratin. Initially, there is little immunoreactivity in the small beta-packets of the oberhautchen, but it increases after fusion with the underlying cells to produce the syncytial beta layer. The beta-keratin packets coalesce with the tonofilaments, including those attached to desmosomes, which rapidly disappear in both oberhautchen and beta-cells as differentiation progresses. The labeling is low to absent in forming mesos-cells beneath the beta-layer. This study further supports the hypothesis that the shedding complex in lepidosaurian reptiles evolved after there was a segregation between alpha-keratogenic cells from beta-keratogenic cells during epidermal renewal. Copyright 2003 Wiley-Liss, Inc.

Cell cycle-related metabolism and mitochondrial dynamics in a replication-competent pancreatic beta-cell line.

PubMed

Montemurro, Chiara; Vadrevu, Suryakiran; Gurlo, Tatyana; Butler, Alexandra E; Vongbunyong, Kenny E; Petcherski, Anton; Shirihai, Orian S; Satin, Leslie S; Braas, Daniel; Butler, Peter C; Tudzarova, Slavica

2017-01-01

Cell replication is a fundamental attribute of growth and repair in multicellular organisms. Pancreatic beta-cells in adults rarely enter cell cycle, hindering the capacity for regeneration in diabetes. Efforts to drive beta-cells into cell cycle have so far largely focused on regulatory molecules such as cyclins and cyclin-dependent kinases (CDKs). Investigations in cancer biology have uncovered that adaptive changes in metabolism, the mitochondrial network, and cellular Ca 2+ are critical for permitting cells to progress through the cell cycle. Here, we investigated these parameters in the replication-competent beta-cell line INS 832/13. Cell cycle synchronization of this line permitted evaluation of cell metabolism, mitochondrial network, and cellular Ca 2+ compartmentalization at key cell cycle stages. The mitochondrial network is interconnected and filamentous at G1/S but fragments during the S and G2/M phases, presumably to permit sorting to daughter cells. Pyruvate anaplerosis peaks at G1/S, consistent with generation of biomass for daughter cells, whereas mitochondrial Ca 2+ and respiration increase during S and G2/M, consistent with increased energy requirements for DNA and lipid synthesis. This synchronization approach may be of value to investigators performing live cell imaging of Ca 2+ or mitochondrial dynamics commonly undertaken in INS cell lines because without synchrony widely disparate data from cell to cell would be expected depending on position within cell cycle. Our findings also offer insight into why replicating beta-cells are relatively nonfunctional secreting insulin in response to glucose. They also provide guidance on metabolic requirements of beta-cells for the transition through the cell cycle that may complement the efforts currently restricted to manipulating cell cycle to drive beta-cells through cell cycle.

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.

[Enzyme replacement therapy in patients with Fabry disease: state of the art and review of the literature].

PubMed

Riccio, Eleonora; Capuano, Ivana; Visciano, Bianca; Marchetiello, Cristina; Petrillo, Fortunato; Pisani, Antonio

2013-01-01

Anderson-Fabry disease is a hereditary X-linked lysosomal storage disorder caused by a deficiency of the lysosomal enzyme alpha galactosidase A. It results in the accumulation of the glycosphingolypid globotrioasoyl ceramide (Gb3 in different cells and organs, resulting in a multi-system pathology including end organ failure. Patients with Fabry disease present clinically with cardiac, renal and neurological involvement; both life expectancy and quality of life are severely compromised. The current causal treatment for Fabry disease is enzyme replacement therapy (ERT), available since 2001. The two recombinant preparations available for ERT are agalsidase alfa (Replagal) and agalsidase beta (Fabrazyme). They have both been showed to have positive effect on kidney and heart, on the symptoms of pain and quality of life. Few data to date are available on comparison of the two preparations of ERT. This article reviews evidence of the literature and shows our personal experience about the safety and efficacy of ERT.

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.

Beta-catenin interacts with low-molecular-weight protein tyrosine phosphatase leading to cadherin-mediated cell-cell adhesion increase.

PubMed

Taddei, Maria Letizia; Chiarugi, Paola; Cirri, Paolo; Buricchi, Francesca; Fiaschi, Tania; Giannoni, Elisa; Talini, Doriana; Cozzi, Giacomo; Formigli, Lucia; Raugei, Giovanni; Ramponi, Giampietro

2002-11-15

Beta-catenin plays a dual role as a major constituent of cadherin-based adherens junctions and also as a transcriptional coactivator. In normal ephitelial cells, at adherens junction level, beta-catenin links cadherins to the actin cytoskeleton. The structure of adherens junctions is dynamically regulated by tyrosine phosphorylation. In particular, cell-cell adhesion can be negatively regulated through the tyrosine phosphorylation of beta-catenin. Furthermore, the loss of beta-catenin-cadherin association has been correlated with the transition from a benign tumor to an invasive, metastatic cancer. Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) is a ubiquitous PTP implicated in the regulation of mitosis and cytoskeleton rearrangement. Here we demonstrate that the amount of free cytoplasmic beta-catenin is decreased in NIH3T3, which overexpresses active LMW-PTP, and this results in a stronger association between cadherin complexes and the actin-based cytoskeleton with respect to control cells. Confocal microscopy analysis shows that beta-catenin colocalizes with LMW-PTP at the plasma membrane. Furthermore, we provide evidence that beta-catenin is able to associate with LMW-PTP both in vitro and in vivo. Moreover, overexpression of active LMW-PTP strongly potentiates cadherin-mediated cell-cell adhesion, whereas a dominant-negative form of LMW-PTP induces the opposite phenotype, both in NIH3T3 and in MCF-7 carcinoma cells. On the basis of these results, we propose that the stability of cell-cell contacts at the adherens junction level is positively influenced by LMW-PTP expression, mainly because of the beta-catenin and LMW-PTP interaction at the plasma membrane level with consequent dephosphorylation.

[The effect of 18beta-glycyrrhetinic acid on gap junction among cerebral arteriolar smooth muscle cells in Wistar rat and spontaneously hypertensive rat].

PubMed

Chen, Xin-Yan; Si, Jun-Qiang; Li, Li; Zhao, Lei; Wei, Li-Li; Jiang, Xue-Wei; Ma, Ke-Tao

2013-05-01

This study compared Wistar rat with spontaneously hypertensive rat (SHR) on the electrophysiology and coupling force of the smooth muscle cells in the cerebral arteriolar segments and observe the influence of 18beta-glycyrrhetinic acid(18beta-GA) on the gap junctions between the arterial smooth muscle cells. The outer layer's connective tissue of the cerebral arteriolar segments was removed. Whole-cell patch clamp recordings were used to observe the 18beta-GA's impaction on the arteriolar segment membrane's input capacitance (C(input)), input conductance (G(input)) and input resistance (R(input)) of the smooth muscle cells. (1) The C(input) and G(input) of the SHR arteriolar segment smooth muscle cells was much higher than the Wistar rats, there was significant difference (P < 0.05). (2) 18beta-GA concentration-dependently reduced C(input) and G(input) (or increase R(input)) on smooth muscle cells in arteriolar segment. IC50 of 18beta-GA suppression's G(input) of the Wistar rat and SHR were 1.7 and 2.0 micromol/L respectively, there was not significant difference (P > 0.05). After application of 18beta-GA concentration > or = 100 micrmol/L, the C(input), G(input) and R(input) of the single smooth muscle cells was very close. Gap junctional coupling is enhanced in the SHR cerebral arterial smooth muscle cells. 18beta-GA concentration-dependent inhibits Wistar rat's and SHR cerebral arteriolar gap junctions between arterial smooth muscle cells. The inhibitory potency is similar between the two different rats. When 18beta-GA concentration is > or = 100 micromol/L, it can completely block gap junctions between arteriolar smooth muscle cells.

Gene silencing of beta-catenin in melanoma cells retards their growth but promotes the formation of pulmonary metastasis in mice.

PubMed

Takahashi, Yuki; Nishikawa, Makiya; Suehara, Tetsuya; Takiguchi, Naomi; Takakura, Yoshinobu

2008-11-15

Altered expression of beta-catenin, a key component of the Wnt signaling pathway, is involved in a variety of cancers because increased levels of beta-catenin protein are frequently associated with enhanced cellular proliferation. Although our previous study demonstrated that gene silencing of beta-catenin in melanoma B16-BL6 cells by plasmid DNA (pDNA) expressing short-hairpin RNA targeting the gene (pshbeta-catenin) markedly suppressed their growth in vivo, gene silencing of beta-catenin could promote tumor metastasis by the rearranging cell adhesion complex. In this study, we investigated how silencing of beta-catenin affects metastatic aspects of melanoma cells. Transfection of B16-BL6 cells with pshbeta-catenin significantly reduced the amount of cadherin protein, a cell adhesion molecule binding to beta-catenin, with little change in its mRNA level. Cadherin-derived fragments were detected in culture media of B16-BL6 cells transfected with pshbeta-catenin, suggesting that cadherin is shed from the cell surface when the expression of beta-catenin is reduced. The mobility of B16-BL6 cells transfected with pshbeta-catenin was greater than that of cells transfected with any of the control pDNAs. B16-BL6 cells stably transfected with pshbeta-catenin (B16/pshbeta-catenin) formed less or an equal number of tumor nodules in the lung than cells stably transfected with other plasmids when injected into mice via the tail vein. However, when subcutaneously inoculated, B16/pshbeta-catenin cells formed more nodules in the lung than the other stably transfected cells. These results raise concerns about the gene silencing of beta-catenin for inhibiting tumor growth, because it promotes tumor metastasis by reducing the amount of cadherin in tumor cells. (c) 2008 Wiley-Liss, Inc.

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

Co-culture of clonal beta cells with GLP-1 and glucagon-secreting cell line impacts on beta cell insulin secretion, proliferation and susceptibility to cytotoxins.

PubMed

Green, Alastair D; Vasu, Srividya; Moffett, R Charlotte; Flatt, Peter R

2016-06-01

We investigated the direct effects on insulin releasing MIN6 cells of chronic exposure to GLP-1, glucagon or a combination of both peptides secreted from GLUTag L-cell and αTC1.9 alpha-cell lines in co-culture. MIN6, GLUTag and αTC1.9 cell lines exhibited high cellular hormone content and release of insulin, GLP-1 and glucagon, respectively. Co-culture of MIN6 cells with GLUTag cells significantly increased cellular insulin content, beta-cell proliferation, insulin secretory responses to a range of established secretogogues and afforded protection against exposure cytotoxic concentrations of glucose, lipid, streptozotocin or cytokines. Benefits of co-culture of MIN6 cells with αTC1.9 alphacells were limited to enhanced beta-cell proliferation with marginal positive actions on both insulin secretion and cellular protection. In contrast, co-culture of MIN6 with GLUTag cells plus αTC1.9 cells, markedly enhanced both insulin secretory responses and protection against beta-cell toxins compared with co-culture with GLUTag cells alone. These data indicate important long-term effects of conjoint GLP-1 and glucagon exposure on beta-cell function. This illustrates the possible functional significance of alpha-cell GLP-1 production as well as direct beneficial effects of dual agonism at beta-cell GLP-1 and glucagon receptors. Copyright © 2016 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

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.

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.

Akt interacts directly with Smad3 to regulate the sensitivity to TGF-beta induced apoptosis.

PubMed

Conery, Andrew R; Cao, Yanna; Thompson, E Aubrey; Townsend, Courtney M; Ko, Tien C; Luo, Kunxin

2004-04-01

Transforming growth factor beta (TGF-beta) induces both apoptosis and cell-cycle arrest in some cell lines, but only growth arrest in others. It is not clear how this differential response to TGF-beta is specified. Smad proteins are critical mediators of TGF-beta signalling. After stimulation by TGF-beta, Smad2 and Smad3 become phosphorylated by the activated TGF-beta receptor kinases, oligomerize with Smad4, translocate to the nucleus and regulate the expression of TGF-beta target genes. Here we report that the sensitivity to TGF-beta induced apoptosis is regulated by crosstalk between the Akt/PKB serine/threonine kinase and Smad3 through a mechanism that is independent of Akt kinase activity. Akt interacts directly with unphosphorylated Smad3 to sequester it outside the nucleus, preventing its phosphorylation and nuclear translocation. This results in inhibition of Smad3-mediated transcription and apoptosis. Furthermore, the ratio of Smad3 to Akt correlates with the sensitivity of cells to TGF-beta induced apoptosis. Alteration of this ratio changes the apoptotic, but not the growth-inhibitory, responses of cells to TGF-beta. These findings identify an important determinant of sensitivity to TGF-beta-induced apoptosis that involves crosstalk between the TGF-beta and phosphatidylinositol-3-OH kinase (PI(3)K) pathways.

Regulation of GM-CSF-induced dendritic cell development by TGF-beta1 and co-developing macrophages.

PubMed

Yamaguchi, Y

1998-01-01

Using a culture system of bone marrow progenitor cells with GM-CSF and TGF-beta1, a study was performed to analyze the effect of TGF-beta1 on the development of dendritic cells (DC) and to elucidate the regulatory role of macrophages co-developing with dendritic cells. The results demonstrate that DC generated in the presence of TGF-beta1 were immature with respect to the expression of CD86, nonspecific esterase activity and cell shape. Such inhibitory effects of TGF-beta1 were dependent on FcR+ macrophages, which were depleted by panning. TGF-beta1 did not appear to inhibit the commitment of progenitor cells to the DC lineage. In addition, TGF-beta1 also acted directly on the intermediate stage of DC to prevent their over-maturation, which results in a preferential decrease in MHC class II, but not in CD86, in the presence of TNF-alpha. FcR+ suppressive macrophages were also shown to facilitate DC maturation when stimulated via FcR-mediated signals even in the presence of TGF-beta1. These results indicate that TGF-beta1 indirectly and directly regulate the development of DC and that co-developing macrophages have a regulatory role in DC maturation.

Successful management of enzyme replacement therapy in related fabry disease patients with severe adverse events by switching from agalsidase Beta (fabrazyme(®)) to agalsidase alfa (replagal (®)).

PubMed

Tsuboi, Kazuya; Yamamoto, Hiroshi; Somura, Fuji; Goto, Hiromi

2015-01-01

Enzyme replacement therapy (ERT) is the only approved therapy for Fabry disease. In June 2009, there was a worldwide shortage of agalsidase beta, necessitating dose reductions or switching to agalsidase alfa in some patients. We present two cases of Fabry disease (a parent and a child) who received agalsidase beta for 27 months at the licensed dose and 10 months at a reduced dose, followed by a switch to agalsidase alfa for 28 months. Case 1, a 26-year-old male had severe coughing and fatigue during ERT with agalsidase beta requiring antitussive and asthmatic drug therapy. After switching to agalsidase alfa, the coughing gradually resolved completely. Case 2, a 62-year-old female had advanced cardiac manifestations at the time of diagnosis. Despite receiving ERT with the approved dose of agalsidase beta, she experienced aggravation of congestive heart failure and was hospitalized. After switching to agalsidase alfa with standard care in heart disease, BNP level, echocardiographic parameters, eGFR rate and lyso-Gb3 levels were improved or stabilized. We report on two Fabry disease patients who experienced severe adverse events while on approved and/or reduced doses of agalsidase beta. Switching to agalsidase alfa associated with standard care in heart disease led to resolution or improvement in the cardiorespiratory status. And reduction in dose associated with standard care in respiratory disease was useful for decrease in cough and fatigue. Plasma BNP level was useful for monitoring heart failure and the effects of ERT.

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.

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.

Basics and applications of stem cells in the pancreas.

PubMed

Sekine, Keisuke; Taniguchi, Hideki

2012-11-01

Enormous efforts have been made to establish pancreatic stem/progenitor cells as a source for regenerative medicine for the treatment of diabetes mellitus. In recent years, it has been recognized that the self-renewal of beta cells is the dominant process involved in postnatal beta-cell regeneration and expansion. Nevertheless, several in-vitro studies have suggested that ductal or as yet unidentified cells are candidates for pancreatic stem/progenitor cells that can differentiate into multilineage cells, including insulin(+) cells. The question remains as to whether beta cells are generated postnatally from stem/progenitor cells other than pre-existing beta cells. Furthermore, mutated pancreatic stem cells are considered to be prospective candidates for cancer stem cells or tumor-initiating cells. This review highlights recent progress in pancreatic stem/progenitor cell research.

HCG-. beta. -subunit radioimmunoassay: potential error in HCG measurement related to choice of labeled antigen. [/sup 125/I

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

Tyrey, L.; Hammond, C.B.

1976-05-15

Antiserum generated against the hormone-specific ..beta..-subunit of hCG was used with different labeled antigens to measure circulating hCG in patients having trophoblastic disease. When /sup 125/I-hCG..beta.. served as the labeled antigen, a small number of patient sera failed to show parallelism with the second IS-hCG reference and erroneous estimates of hormone concentrations were obtained. Replacement of the /sup 125/I-hCG..beta.. with labeled hCG corrected the nonparallelism exhibited by these samples. Inhibition curves obtained with purified hCG and hCG..beta.. suggested that both the nonparallelism and its correction with the change in labeled antigen would be consistent with the possibility that this assaymore » aberration may result from the presence of free hCG..beta.. in these sera. (auth)« less

Hb Molfetta [beta126(H4)Val-->Leu, GTG-->CTG]: a new, silent, neutral beta chain variant found in an Italian woman.

PubMed

Qualtieri, Antonio; Le, Pera Maria; Pedace, Vera; Magariello, Angela; Brancati, Carlo

2002-02-01

We have identified a new neutral hemoglobin variant in a pregnant Italian woman, that resulted from a GTG-->CTG replacement at codon 126 of the beta chain, corresponding to a Val-->Leu amino acid change at position beta126(H4). Thermal and isopropanol stability tests were normal and there were no abnormal clinical features. Routine electrophoretic and ion exchange chromatographic methods for hemoglobin separation failed to show this variant, but reversed phase high performance liquid chromatography revealed an abnormal peak eluting near the normal beta chain. No abnormal tryptic peptide was revealed on the high performance liquid chromatographic elution pattern of the total globin digest. The mutation was determined at the DNA level by amplification of the three beta exons by polymerase chain reaction and direct sequencing of one exon that showed an abnormal migration on single strand conformational polymorphism analysis.

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.

Osteocalcin protects pancreatic beta cell function and survival under high glucose conditions

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

Kover, Karen, E-mail: kkover@cmh.edu; University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108; Yan, Yun

Diabetes is characterized by progressive beta cell dysfunction and loss due in part to oxidative stress that occurs from gluco/lipotoxicity. Treatments that directly protect beta cell function and survival in the diabetic milieu are of particular interest. A growing body of evidence suggests that osteocalcin, an abundant non-collagenous protein of bone, supports beta cell function and proliferation. Based on previous gene expression data by microarray, we hypothesized that osteocalcin protects beta cells from glucose-induced oxidative stress. To test our hypothesis we cultured isolated rat islets and INS-1E cells in the presence of normal, high, or high glucose ± osteocalcin for up tomore » 72 h. Oxidative stress and viability/mitochondrial function were measured by H{sub 2}O{sub 2} assay and Alamar Blue assay, respectively. Caspase 3/7 activity was also measured as a marker of apoptosis. A functional test, glucose stimulated insulin release, was conducted and expression of genes/protein was measured by qRT-PCR/western blot/ELISA. Osteocalcin treatment significantly reduced high glucose-induced H{sub 2}O{sub 2} levels while maintaining viability/mitochondrial function. Osteocalcin also significantly improved glucose stimulated insulin secretion and insulin content in rat islets after 48 h of high glucose exposure compared to untreated islets. As expected sustained high glucose down-regulated gene/protein expression of INS1 and BCL2 while increasing TXNIP expression. Interestingly, osteocalcin treatment reversed the effects of high glucose on gene/protein expression. We conclude that osteocalcin can protect beta cells from the negative effects of glucose-induced oxidative stress, in part, by reducing TXNIP expression, thereby preserving beta cell function and survival. - Highlights: • Osteocalcin reduces glucose-induced oxidative stress in beta cells. • Osteocalcin preserves beta cell function and survival under stress conditions. • Osteocalcin reduces glucose-induced TXNIP expression in beta cells.« less

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.

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

PubMed

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

2009-10-09

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

Release of IL-1beta via IL-1beta-converting enzyme in a skin dendritic cell line exposed to 2,4-dinitrofluorobenzene.

PubMed

Matos, Teresa J; Jaleco, Sara P; Gonçalo, Margarida; Duarte, Carlos B; Lopes, M Celeste

2005-08-14

We used a mouse fetal skin dendritic cell line (FSDC) to study the effect of the strong allergen 2,4-dinitrofluorobenzene (DNFB) on interleukin (IL)-1beta release and IL-1beta receptor immunoreactivity. Stimulation with DNFB (30 minutes) increased IL-1 release without changing the mRNA levels of the protein. Furthermore, DNFB increased transiently the interleukin-1beta-converting enzyme (ICE) activity, as measured with its fluorogenic substrate Z-Tyr-Val-Ala-Asp-AFC. The ICE inhibitor Z-YVAD-FMK prevented the release of IL-1beta evoked by DNFB. Incubation of the cells with DNFB (30 minutes) strongly increased IL-1beta receptor immunoreactivity. The rapid effect of DNFB on the release of mature IL-1beta, without inducing an increase of IL-1beta mRNA in FSDC, suggests a posttranslational modification of pro-IL-1beta by ICE activity.

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

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

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.

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.

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.

Mono-(2-ethylhexyl) phthalate (MEHP) regulates glucocorticoid metabolism through 11{beta}-hydroxysteroid dehydrogenase 2 in murine gonadotrope cells

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

Hong, Dun; Department of Orthopedics, Taizhou Hospital, Wenzhou Medical College, Lin Hai, ZJ 317000; Li, Xing-Wang

2009-11-13

Di-(2-ethylhexyl) phthalate (DEHP) and its metabolite mono-(2-ethylhexyl) phthalate (MEHP) have been classified as toxicants to the reproductive system at the testis level and DEHP may also impair reproductive axis function at the pituitary levels. However, MEHP is 10-fold more potent than DEHP in toxicity and little is known about the toxicological effect of MEHP on pituitary. In this study, we demonstrated that 11{beta}-hydroxysteroid dehydrogenase type 2 (11{beta}-HSD2), not 11{beta}-HSD1, is strongly expressed in murine gonadotrope L{beta}T2 cells. Interestingly, MEHP inhibited Hsd11b2 mRNA level and 11{beta}-HSD2 enzyme activity in L{beta}T2 cells at as low as 10{sup -7} M. Corticosterone (CORT) atmore » a concentration of 10{sup -6} M significantly inhibited L{beta}T2 cell proliferation after 2-day culture, and 10{sup -6} M RU486, an antagonist of glucocorticoid receptor (GR), reversed this inhibition. However, in the presence of 10{sup -5} or 10{sup -4} M MEHP, the minimal concentration of CORT to inhibit the proliferation of L{beta}T2 cells was lowered to 10{sup -7} M, and 10{sup -6} M RU486 was not able to completely reverse the CORT effect. In conclusion, along with the regulation of GR, 11{beta}-HSD2 may have a key role in glucocorticoid metabolism in L{beta}T2 cells. MEHP may participate in the glucocorticoid metabolism in L{beta}T2 cells through inhibition of 11{beta}-HSD2 enzyme activity. Such perturbation may be of pathological significance as MEHP may interfere with the reproductive system at pituitary level through regulation of glucocorticoid metabolism, especially in neonates with higher risk of phthalates exposure.« less

The Golgi localization of phosphatidylinositol transfer protein beta requires the protein kinase C-dependent phosphorylation of serine 262 and is essential for maintaining plasma membrane sphingomyelin levels.

PubMed

van Tiel, Claudia M; Westerman, Jan; Paasman, Marten A; Hoebens, Martha M; Wirtz, Karel W A; Snoek, Gerry T

2002-06-21

Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165) as a minor phosphorylation site. The phospholipid transfer activities of wild-type PI-TP beta and PI-TP beta(S262A) were identical, whereas PI-TP beta(S165A) was completely inactive. PKC-dependent phosphorylation of Ser(262) also had no effect on the transfer activity of PI-TP beta. To investigate the role of Ser(262) in the functioning of PI-TP beta, wtPI-TP beta and PI-TP beta(S262A) were overexpressed in NIH3T3 fibroblast cells. Two-dimensional PAGE analysis of cell lysates was used to separate PI-TP beta from its phosphorylated form. After Western blotting, wtPI-TP beta was found to be 85% phosphorylated, whereas PI-TP beta(S262A) was not phosphorylated. In the presence of the PKC inhibitor GF 109203X, the phosphorylated form of wtPI-TP beta was strongly reduced. Immunolocalization showed that wtPI-TP beta was predominantly associated with the Golgi membranes. In the presence of the PKC inhibitor, wtPI-TP beta was distributed throughout the cell similar to what was observed for PI-TP beta(S262A). In contrast to wtPI-TP beta overexpressors, cells overexpressing PI-TP beta(S262A) were unable to rapidly replenish sphingomyelin in the plasma membrane upon degradation by sphingomyelinase. This implies that PKC-dependent association with the Golgi complex is a prerequisite for PI-TP beta to express its effect on sphingomyelin metabolism.

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

Involvement of DNA polymerase beta in repairing oxidative damages induced by antitumor drug adriamycin

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

Liu Shukun; Wu Mei; Zhang Zunzhen, E-mail: zhangzunzhen@163.co

2010-08-01

Adriamycin (ADM) is a widely used antineoplastic drug. However, the increasing cellular resistance has become a serious limitation to ADM clinical application. The most important mechanism related to ADM-induced cell death is oxidative DNA damage mediated by reactive oxygen species (ROS). Base excision repair (BER) is a major pathway in the repair of DNA single strand break (SSB) and oxidized base. In this study, we firstly applied the murine embryo fibroblasts wild-type (pol {beta} +/+) and homozygous pol {beta} null cell (pol {beta} -/-) as a model to investigate ADM DNA-damaging effects and the molecular basis underlying these effects. Here,more » cellular sensitivity to ADM was examined using colorimetric assay and colony forming assay. ADM-induced cellular ROS level and the alteration of superoxide dismutase (SOD) activity were measured by commercial kits. Further, DNA strand break, chromosomal damage and gene mutation were assessed by comet assay, micronucleus test and hprt gene mutation assay, respectively. The results showed that pol {beta} -/- cells were more sensitive to ADM compared with pol {beta} +/+ cells and more severe SSB and chromosomal damage as well as higher hprt gene mutation frequency were observed in pol {beta} -/- cells. ROS level in pol {beta} -/- cells increased along with decreased activity of SOD. These results demonstrated that pol {beta} deficiency could enable ROS accumulation with SOD activity decrease, further elevate oxidative DNA damage, and subsequently result in SSB, chromosome cleavage as well as gene mutation, which may be partly responsible for the cytotoxicity of ADM and the hypersensitivity of pol {beta} -/- cells to ADM. These findings suggested that pol {beta} is vital for repairing oxidative damage induced by ADM.« less

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

PubMed Central

Rodriguez-Artigas, Sandra M.; Ballester, Rodrigo

2016-01-01

Beta-diversity, defined as spatial replacement in species composition, is crucial to the understanding of how local communities assemble. These changes can be driven by environmental or geographic factors (such as geographic distance), or a combination of the two. Spiders have been shown to be good indicators of environmental quality. Accordingly, spiders are used in this work as model taxa to establish whether there is a decrease in community similarity that corresponds to geographic distance in the grasslands of the Campos & Malezales ecoregion (Corrientes). Furthermore, the influence of climactic factors and local vegetation heterogeneity (environmental factors) on assemblage composition was evaluated. Finally, this study evaluated whether the differential dispersal capacity of spider families is a factor that influences their community structure at a regional scale. Spiders were collected with a G-Vac from vegetation in six grassland sites in the Campos & Malezales ecoregion that were separated by a minimum of 13 km. With this data, the impact of alpha-diversity and different environmental variables on the beta-diversity of spider communities was analysed. Likewise, the importance of species replacement and nesting on beta-diversity and their contribution to the regional diversity of spider families with different dispersion capacities was evaluated. The regional and site-specific inventories obtained were complete. The similarity between spider communities declined as the geographic distance between sites increased. Environmental variables also influenced community composition; stochastic events and abiotic forces were the principal intervening factors in assembly structure. The differential dispersal capacity of spider groups also influenced community structure at a regional scale. The regional beta-diversity, as well as species replacement, was greater in high and intermediate vagility spiders; while nesting was greater in spiders with low dispersion capacity. Geographic distance, among other factors (climate, and active and passive dispersion capacity), explains assembly structure and the decrease spider community similarity between geographically distant sites. Spiders with the highest dispersal capacity showed greater species replacement. This may be due to the discontinuity (both natural and anthropic) of the grasslands in this ecoregion, which limits the dispersal capacity of these spiders, and their close dependence on microhabitats. The dispersal capacity of the least vagile spiders is limited by geographic distance and biotic factors, such as competition, which could explain the nesting observed between their communities. PMID:27123380

Transforming growth factor beta induced FoxP3+ regulatory T cells suppress Th1 mediated experimental colitis.

PubMed

Fantini, M C; Becker, C; Tubbe, I; Nikolaev, A; Lehr, H A; Galle, P; Neurath, M F

2006-05-01

The imbalance between effector and regulatory T cells plays a central role in the pathogenesis of inflammatory bowel diseases. In addition to the thymus, CD4+CD25+ regulatory T cells can be induced in the periphery from a population of CD25- T cells by treatment with transforming growth factor beta (TGF-beta). Here, we analysed the in vivo function of TGF-beta induced regulatory T (Ti-Treg) cells in experimental colitis. Ti-Treg cells were generated in cell culture in the presence or absence of TGF-beta and tested for their regulatory potential in experimental colitis using the CD4+CD62L+ T cell transfer model. Ti-Treg cells significantly suppressed Th1 mediated colitis on CD4+CD62L+ T cell transfer in vivo, as shown by high resolution endoscopy, histology, immunohistochemistry, and cytokine analysis. Further analysis of in vivo and in vitro expanded Ti-Treg cells showed that exogenous interleukin 2 (IL-2) was crucial for survival and expansion of these cells. Our data suggest that regulatory Ti-Treg cells expand by TGF-beta and exogenous IL-2 derived from effector T cells at the site of inflammation. In addition to Tr1 and thymic CD4+CD25+ T cells, peripheral Ti-Treg cells emerge as a class of regulatory T cells with therapeutic potential in T cell mediated chronic intestinal inflammation.

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

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

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

2010-09-03

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

Requirement for Pdx1 in specification of latent endocrine progenitors in zebrafish

PubMed Central

2011-01-01

Background Insulin-producing beta cells emerge during pancreas development in two sequential waves. Recently described later-forming beta cells in zebrafish show high similarity to second wave mammalian beta cells in developmental capacity. Loss-of-function studies in mouse and zebrafish demonstrated that the homeobox transcription factors Pdx1 and Hb9 are both critical for pancreas and beta cell development and discrete stage-specific requirements for these genes have been uncovered. Previously, exocrine and endocrine cell recovery was shown to follow loss of pdx1 in zebrafish, but the progenitor cells and molecular mechanisms responsible have not been clearly defined. In addition, interactions of pdx1 and hb9 in beta cell formation have not been addressed. Results To learn more about endocrine progenitor specification, we examined beta cell formation following morpholino-mediated depletion of pdx1 and hb9. We find that after early beta cell reduction, recovery occurs following loss of either pdx1 or hb9 function. Unexpectedly, simultaneous knockdown of both hb9 and pdx1 leads to virtually complete and persistent beta cell deficiency. We used a NeuroD:EGFP transgenic line to examine endocrine cell behavior in vivo and developed a novel live-imaging technique to document emergence and migration of late-forming endocrine precursors in real time. Our data show that Notch-responsive progenitors for late-arising endocrine cells are predominantly post mitotic and depend on pdx1. By contrast, early-arising endocrine cells are specified and differentiate independent of pdx1. Conclusions The nearly complete beta cell deficiency after combined loss of hb9 and pdx1 suggests functional cooperation, which we clarify as distinct roles in early and late endocrine cell formation. A novel imaging approach permitted visualization of the emergence of late endocrine cells within developing embryos for the first time. We demonstrate a pdx1-dependent progenitor population essential for the formation of duct-associated, second wave endocrine cells. We further reveal an unexpectedly low mitotic activity in these progenitor cells, indicating that they are set aside early in development. PMID:22034951

Human beta-cell precursors mature into functional insulin-producing cells in an immunoisolation device: implications for diabetes cell therapies.

PubMed

Lee, Seung-Hee; Hao, Ergeng; Savinov, Alexei Y; Geron, Ifat; Strongin, Alex Y; Itkin-Ansari, Pamela

2009-04-15

Islet transplantation is limited by the need for chronic immunosuppression and the paucity of donor tissue. As new sources of human beta-cells are developed (e.g., stem cell-derived tissue), transplanting them in a durable device could obviate the need for immunosuppression, while also protecting the patient from any risk of tumorigenicity. Here, we studied (1) the survival and function of encapsulated human beta-cells and their progenitors and (2) the engraftment of encapsulated murine beta-cells in allo- and autoimmune settings. Human islets and human fetal pancreatic islet-like cell clusters were encapsulated in polytetrafluorethylene devices (TheraCyte) and transplanted into immunodeficient mice. Graft survival and function was measured by immunohistochemistry, circulating human C-peptide levels, and blood glucose levels. Bioluminescent imaging was used to monitor encapsulated neonatal murine islets. Encapsulated human islet-like cell clusters survived, replicated, and acquired a level of glucose responsive insulin secretion sufficient to ameliorate hyperglycemia in diabetic mice. Bioluminescent imaging of encapsulated murine neonatal islets revealed a dynamic process of cell death followed by regrowth, resulting in robust long-term allograft survival. Further, in the non-obese diabetic (NOD) mouse model of type I diabetes, encapsulated primary beta-cells ameliorated diabetes without stimulating a detectable T-cell response. We demonstrate for the first time that human beta-cells function is compatible with encapsulation in a durable, immunoprotective device. Moreover, our study suggests that encapsulation of beta-cells before terminal differentiation will be a successful approach for new cell-based therapies for diabetes, such as those derived from stem cells.

C/EBPβ Promotes STAT3 Expression and Affects Cell Apoptosis and Proliferation in Porcine Ovarian Granulosa Cells.

PubMed

Yuan, Xiaolong; Zhou, Xiaofeng; He, Yingting; Zhong, Yuyi; Zhang, Ailing; Zhang, Zhe; Zhang, Hao; Li, Jiaqi

2018-06-13

Previous studies suggest that signal transducer and activator of transcription 3 (STAT3) and CCAAT/enhancer binding protein beta (C/EBPβ) play an essential role in ovarian granulosa cells (GCs) for mammalian follicular development. Several C/EBPβ putative binding sites were previously predicted on the STAT3 promoter in mammals. However, the molecular regulation of C/EBPβ on STAT3 and their effects on cell proliferation and apoptosis remain virtually unexplored in GCs. Using porcine GCs as a model, the 5′-deletion, luciferase report assay, mutation, chromatin immunoprecipitation, Annexin-V/PI staining and EdU assays were applied to investigate the molecular mechanism for C/EBPβ regulating the expression of STAT3 and their effects on the cell proliferation and apoptosis ability. We found that over and interfering with the expression of C/EBPβ significantly increased and decreased the messenger RNA (mRNA) and protein levels of STAT3 , respectively. The dual luciferase reporter assay showed that C/EBPβ directly bound at −1397/−1387 of STAT3 to positively regulate the mRNA and protein expressions of STAT3 . Both C/EBPβ and STAT3 were observed to inhibit cell apoptosis and promote cell proliferation. Furthermore, C/EBPβ might enhance the antiapoptotic and pro-proliferative effects of STAT3 . These results would be of great insight in further exploring the molecular mechanism of C/EBPβ and STAT3 on the function of GCs and the development of ovarian follicles in mammals.

Blood vessel crosstalk during organogenesis – Focus on Pancreas

PubMed Central

Azizoglu, D. Berfin; Cleaver, Ondine

2016-01-01

Blood vessels form a highly branched, interconnected and largely stereotyped network of tubes that sustains every organ and tissue in vertebrates. How vessels come to take on their particular architecture, or how they are ‘patterned’, and in turn, how they influence surrounding tissues are fundamental questions of organogenesis. Decades of work have begun to elucidate how endothelial progenitors arise and home to precise locations within tissues, integrating attractive and repulsive cues to build vessels where they are needed. Conversely, more recent findings have revealed an exciting facet of blood vessel interaction with tissues, where vascular cells provide signals to developing organs and progenitors therein. Here, we discuss the exchange of reciprocal signals between endothelial cells (ECs) and neighboring tissues during embryogenesis, with a special focus on the developing pancreas. Understanding the mechanisms driving both sides of these interactions will be crucial to the development of therapies, from improving organ regeneration to efficient production of cell based therapies. Specifically, elucidating the interface of the vasculature with pancreatic lineages, including endocrine cells, will instruct approaches such as generation of replacement beta cells for Type I diabetes. PMID:27328421

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

PubMed

Mathis, Ute; Schaeffel, Frank

2010-06-01

In the chicken model of myopia, it has first been shown that imposing defocus to the retina results in active remodelling of the sclera which, in turn, results in axial length changes of the eye. Transforming growth factor-beta (TGF-beta) is one of the scleral growth modulators but its cellular localization in the fundal layers, colocalization and function are not well known. The aim of the current study was to investigate the cellular distribution of the three isoforms TGF-beta1, 2 and 3 by immunohistochemical labelling. Furthermore, the effects of visual experience that induces refractive errors on TGF-beta2 labelling were examined. Transversal cryostat sections of the fundal layers were analyzed by indirect immunofluorescent labelling and cell counts. Visual experience was changed by having the chicks wear either diffusers, or positive or negative lenses of 7D power in front of the right eyes for various periods of time. Left eyes served as uncovered controls. All TGF-beta isoforms were localized in both scleral layers. In choroid, diffuse labelling of all isoforms was found. In retina, TGF-beta1 and 3 were detected in bipolar, amacrine and ganglion cells and TGF-beta2 in amacrine and ganglion cells. To further characterize these cells, double-labelling with known amacrine and bipolar cell markers was performed (calbindin, cellular retinoic acid binding protein (CRABP), Islet1, Lim3 and protein kinase C (PKC)). TGF-beta1, 2 and 3 could be colocalized with calbindin and CRABP in single amacrine cells. TGF-beta1-positive bipolar cells were immunoreactive to Lim3. TGF-beta1 and 3 were never colocalized with PKC in bipolar cells. Also, colocalization with peptides known to be involved in myopia development in chicks, such as glucagon, or vasointestinal polypeptide and the key enzyme for dopamine synthesis, tyrosine hydroxylase, was not observed. Lenses or diffusers, worn by the chicks for various periods of time, had no effect on TGF-beta2 immunoreactivity in choroid or sclera, or on the number of TGF-beta2 (active and latent form) expressing amacrine cells. This result did not change when the two identified populations of TGF-beta2 expressing amacrine cells (one calbindin-positive and the other CRABP-positive) were separately considered. Also no modulation was seen in choroid, although an earlier study had found changes in TGF-beta2 mRNA after lens treatment. The lack of any visually-induced changes in retina or choroid suggests that TGF-beta may not represent a key molecule in the retino-choroidal signalling cascade although it has previously been shown to have a primary role in scleral remodelling. Copyright 2010 Elsevier Ltd. All rights reserved.

Enzyme replacement therapy for Fabry disease: lessons from two alpha-galactosidase A orphan products and one FDA approval.

PubMed

Desnick, Robert J

2004-07-01

Two virtually identical products have been developed for enzyme replacement therapy for the treatment of Fabry disease, which is a rare and debilitating genetic disease caused by decreased activity of the lysosomal enzyme alpha-galactosidase A. Lack of this enzyme results in progressive tissue accumulation of globotriaosylceramide (GL-3), resulting in life-threatening renal, cardiac and cerebrovascular complications. Both enzyme replacement products, agalsidase alfa (Replagal; Transkaryotic Therapies, Cambridge, MA, USA) and agalsidase beta (Fabrazyme; Genzyme Corporation, Cambridge, MA, USA), were approved by the European Agency for the Evaluation of Medicinal Products in 2001; agalsidase alfa at a recommended dose of 0.2 mg/kg and agalsidase beta at a recommended dose of 1 mg/kg. In the US, however, orphan drug laws dictated that only one of these products could be approved. In April 2003, after a rigorous evaluation of both products by the US FDA, this approval was granted to agalsidase beta. This decision reflected clinical trial design, how dosages were determined, antibody effects and the ability of each product to demonstrate either clinical efficacy or reduction of tissue storage of GL-3 in major organs of pathology when administered at the recommended dosage. The process also highlighted important issues in the evaluation of drugs to treat life-threatening genetic diseases for which the pathological basis is well-defined.

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.

Functional characterization of the beta-adrenergic receptor subtypes expressed by CA1 pyramidal cells in the rat hippocampus.

PubMed

Hillman, Kristin L; Doze, Van A; Porter, James E

2005-08-01

Recent studies have demonstrated that activation of the beta-adrenergic receptor (AR) using the selective beta-AR agonist isoproterenol (ISO) facilitates pyramidal cell long-term potentiation in the cornu ammonis 1 (CA1) region of the rat hippocampus. We have previously analyzed beta-AR genomic expression patterns of 17 CA1 pyramidal cells using single cell reverse transcription-polymerase chain reaction, demonstrating that all samples expressed the beta2-AR transcript, with four of the 17 cells additionally expressing mRNA for the beta1-AR subtype. However, it has not been determined which beta-AR subtypes are functionally expressed in CA1 for these same pyramidal neurons. Using cell-attached recordings, we tested the ability of ISO to increase pyramidal cell action potential (AP) frequency in the presence of subtype-selective beta-AR antagonists. ICI-118,551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol] and butoxamine [alpha-[1-(t-butylamino)ethyl]-2,5-dimethoxybenzyl alcohol) hydrochloride], agents that selectively block the beta2-AR, produced significant parallel rightward shifts in the concentration-response curves for ISO. From these curves, apparent equilibrium dissociation constant (K(b)) values of 0.3 nM for ICI-118,551 and 355 nM for butoxamine were calculated using Schild regression analysis. Conversely, effective concentrations of the selective beta1-AR antagonists CGP 20712A [(+/-)-2-hydroxy-5-[2-([2-hydroxy-3-(4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy)propyl]amino)ethoxy]-benzamide methanesulfonate] and atenolol [4-[2'-hydroxy-3'-(isopropyl-amino)propoxy]phenylacetamide] did not significantly affect the pyramidal cell response to ISO. However, at higher concentrations, atenolol significantly decreased the potency for ISO-mediated AP frequencies. From these curves, an apparent atenolol K(b) value of 3162 nM was calculated. This pharmacological profile for subtype-selective beta-AR antagonists indicates that beta2-AR activation is mediating the increased AP frequency. Knowledge of functional AR expression in CA1 pyramidal neurons will aid future long-term potentiation studies by allowing selective manipulation of specific beta-AR subtypes.

Radiation leukemia virus-induced thymic lymphomas express a restricted repertoire of T-cell receptor V beta gene products.

PubMed Central

Sen-Majumdar, A; Weissman, I L; Hansteen, G; Marian, J; Waller, E K; Lieberman, M

1994-01-01

We have investigated the phenotypic changes that take place during the process of neoplastic transformation in the thymocytes of C57BL/Ka mice infected by the radiation leukemia virus (RadLV). By the combined use of antibodies against the envelope glycoprotein gp70 of RadLV, the transformation-associated cell surface marker 1C11, and the CD3-T-cell receptor (TCR) complex, we found that in the RadLV-infected thymus, the earliest expression of viral gp70 is in 1C11hi cells; a small but significant percentage of these cells also express CD3. A first wave of viral replication, manifested by the expression of high levels of gp70 in thymocytes (over 70% positive), reaches a peak at 2 weeks; during this period, no significant changes are observed in the expression of 1C11 or CD3. The population of gp70+ cells is drastically reduced at 3 to 4 weeks after infection. However, a second cohort of gp70+ cells appears after 4 weeks, and these cells express high levels of 1C11 and TCR determinants as well. RadLV-induced lymphomas differ from normal thymocytes in their CD4 CD8 phenotype, with domination by one or more subsets. Characterization of TCR gene rearrangements in RadLV-induced lymphomas shows that most of these tumors are clonal or oligoclonal with respect to the J beta 2 TCR gene, while the J beta 1 TCR gene is rearranged in a minority (4 of 11) of lymphomas. TCR V beta repertoire analysis of 12 tumors reveals that 6 (50%) express exclusively the V beta 6 gene product, 2 (17%) are V beta 5+, and 1 (8%) each are V beta 8+ and V beta 9+. In normal C57BL/Ka mice, V beta 6 is expressed on 12%, V beta 5 is expressed on 9%, V beta 8 is expressed on 22%, and V beta 9 is expressed on 4% of TCRhi thymocytes. Thus, it appears that RadLV-induced thymic lymphomas are not randomly selected with respect to expressed TCR V beta type. Images PMID:8289345

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.

Functional Connectivity in Islets of Langerhans from Mouse Pancreas Tissue Slices

PubMed Central

Stožer, Andraž; Gosak, Marko; Dolenšek, Jurij; Perc, Matjaž; Marhl, Marko; Rupnik, Marjan Slak; Korošak, Dean

2013-01-01

We propose a network representation of electrically coupled beta cells in islets of Langerhans. Beta cells are functionally connected on the basis of correlations between calcium dynamics of individual cells, obtained by means of confocal laser-scanning calcium imaging in islets from acute mouse pancreas tissue slices. Obtained functional networks are analyzed in the light of known structural and physiological properties of islets. Focusing on the temporal evolution of the network under stimulation with glucose, we show that the dynamics are more correlated under stimulation than under non-stimulated conditions and that the highest overall correlation, largely independent of Euclidean distances between cells, is observed in the activation and deactivation phases when cells are driven by the external stimulus. Moreover, we find that the range of interactions in networks during activity shows a clear dependence on the Euclidean distance, lending support to previous observations that beta cells are synchronized via calcium waves spreading throughout islets. Most interestingly, the functional connectivity patterns between beta cells exhibit small-world properties, suggesting that beta cells do not form a homogeneous geometric network but are connected in a functionally more efficient way. Presented results provide support for the existing knowledge of beta cell physiology from a network perspective and shed important new light on the functional organization of beta cell syncitia whose structural topology is probably not as trivial as believed so far. PMID:23468610

GLP-1 mediates antiapoptotic effect by phosphorylating Bad through a beta-arrestin 1-mediated ERK1/2 activation in pancreatic beta-cells.

PubMed

Quoyer, Julie; Longuet, Christine; Broca, Christophe; Linck, Nathalie; Costes, Safia; Varin, Elodie; Bockaert, Joël; Bertrand, Gyslaine; Dalle, Stéphane

2010-01-15

Strategies based on activating GLP-1 receptor (GLP-1R) are intensively developed for the treatment of type 2 diabetes. The exhaustive knowledge of the signaling pathways linked to activated GLP-1R within the beta-cells is of major importance. In beta-cells, GLP-1 activates the ERK1/2 cascade by diverse pathways dependent on either Galpha(s)/cAMP/cAMP-dependent protein kinase (PKA) or beta-arrestin 1, a scaffold protein. Using pharmacological inhibitors, beta-arrestin 1 small interfering RNA, and islets isolated from beta-arrestin 1 knock-out mice, we demonstrate that GLP-1 stimulates ERK1/2 by two temporally distinct pathways. The PKA-dependent pathway mediates rapid and transient ERK1/2 phosphorylation that leads to nuclear translocation of the activated kinases. In contrast, the beta-arrestin 1-dependent pathway produces a late ERK1/2 activity that is restricted to the beta-cell cytoplasm. We further observe that GLP-1 phosphorylates the cytoplasmic proapoptotic protein Bad at Ser-112 but not at Ser-155. We find that the beta-arrestin 1-dependent ERK1/2 activation engaged by GLP-1 mediates the Ser-112 phosphorylation of Bad, through p90RSK activation, allowing the association of Bad with the scaffold protein 14-3-3, leading to its inactivation. beta-Arrestin 1 is further found to mediate the antiapoptotic effect of GLP-1 in beta-cells through the ERK1/2-p90RSK-phosphorylation of Bad. This new regulatory mechanism engaged by activated GLP-1R involving a beta-arrestin 1-dependent spatiotemporal regulation of the ERK1/2-p90RSK activity is now suspected to participate in the protection of beta-cells against apoptosis. Such signaling mechanism may serve as a prototype to generate new therapeutic GLP-1R ligands.

Vertebrate beta-thymosins: conserved synteny reveals the relationship between those of bony fish and of land vertebrates.

PubMed

Edwards, John

2010-03-05

Using conservation of synteny I show how the four thymosins expressed by teleost fish are related to the three of tetrapods, which is not evident from their protein sequences. This clarification was aided by identification of a novel thymosin of reptilians that replaces the beta10 thymosin of mammals. Recent reconstruction of the ancestral vertebrate genome suggests that divergence of beta-thymosins began with duplication preceding the two rounds of whole genome duplication. Copyright (c) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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

Antiproteinuric therapy and fabry nephropathy: sustained reduction of proteinuria in patients receiving enzyme replacement therapy with agalsidase-beta.

PubMed

Tahir, Hindia; Jackson, Leslie L; Warnock, David G

2007-09-01

This report describes an open-label, nonrandomized, prospective evaluation of the effects of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker therapy on patients who have Fabry disease and also received enzyme replacement therapy with agalsidase-beta, given at 1 mg/kg body wt every 2 wk. Previous placebo-controlled phase III and phase IV trials with agalsidase-beta demonstrated clearing of globotriaosylceramide from vascular endothelia but little effect on proteinuria or progressive loss of kidney function in patients with Fabry disease and severe chronic kidney disease marked by overt proteinuria and/or estimated GFR <60 ml/min per 1.73 m2. Angiotensin-converting enzyme inhibitor and/or angiotensin receptor blocker therapy is the standard of care for patients with proteinuric kidney diseases, but their use is challenging in patients with Fabry disease and low or low-normal baseline systemic BP. A group of patients with Fabry disease were treated with antiproteinuric therapy, in conjunction with agalsidase-beta; sustained reductions in proteinuria with stabilization of kidney function were achieved in a group of six patients who had severe Fabry nephropathy; the progression rate was -0.23 +/- 1.12 ml/min per 1.73 m2 per yr with 30 mo of follow-up.

Fabry disease in children: agalsidase-beta enzyme replacement therapy.

PubMed

Borgwardt, L; Feldt-Rasmussen, U; Rasmussen, A K; Ballegaard, M; Meldgaard Lund, A

2013-05-01

Fabry disease is a rare, multiorgan disease. The most serious complications involve the kidney, brain and heart. This study aims to assess the effect of enzyme replacement therapy (ERT) using agalsidase-beta in children with Fabry disease. We carried out a nationwide, descriptive and observational retrospective cohort study of 10 children (9-16 years at baseline), who underwent regular systematic investigations for 1-8 years after initiation of ERT with agalsidase-beta (Fabryzyme®, Genzyme). Ophthalmological, echocardiographic abnormalities and hypohidrosis were found at baseline and during the follow-up period. Serious kidney, heart or brain involvement had not developed at the last follow-up examination. For the majority of the patients improvements were found concerning headache, acroparaesthesias and gastrointestinal pain during the follow-up period. The level of energy and physical activity also increased. Treatment with agalsidase-beta was associated with a reduction of neuropathic and abdominal pain and headache. Although all aspects of the Fabry pain phenotype cannot be treated with ERT, the observed effects were clinically significant in the lives of the majority of Fabry children and together with the absence of serious Fabry manifestations at last follow-up, we argue that early initiation of ERT may be considered. © 2012 John Wiley & Sons A/S.

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.

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

Liang, Wenqing, E-mail: liangwenqing_1234@126.com; Yang, Chengwei; Qian, Yu

Highlights: Black-Right-Pointing-Pointer {beta}-Catenin expression were markedly down-regulated by CTNNB1 shRNA. Black-Right-Pointing-Pointer CTNNB1 shRNA could inhibit the proliferation of RPMI8226 cells. Black-Right-Pointing-Pointer Significantly profound apoptotic cell death in CTNNB1 shRNA cells. Black-Right-Pointing-Pointer In vivo, CTNNB1 silence led to a growth inhibition of myeloma growth. Black-Right-Pointing-Pointer c-myc and {beta}-catenin in the expression cells of cleaved caspase-3 were increased. -- Abstract: Multiple myeloma (MM) is thrombogenic as a consequence of multiple hemostatic effects. Overexpression of {beta}-catenin has been observed in several types of malignant tumors, including MM. However, the relationship between {beta}-catenin expression and MM remains unclear. In the present study, RNA interferencemore » was used to inhibit {beta}-catenin expression in RPMI8226 cells. RT-PCR and Western blotting analyses showed that {beta}-catenin mRNA and protein expression were markedly down-regulated by CTNNB1 shRNA. Western blotting showed that the protein levels of cyclin D1 and glutamine synthetase were downregulated and supported the transcriptional regulatory function of {beta}-catenin. The MTT assay showed that CTNNB1 shRNA could have significant inhibitory effects on the proliferation of RPMI8226 cells. The TOPflash reporter assay demonstrated significant downregulation after CTNNB1 shRNA transfection in RPMI8226 cells. Flow cytometric analyses also showed significantly profound apoptosis in CTNNB1 shRNA cells. We found CTNNB1 silence led to growth inhibition of MM growth in vivo. Immunohistochemical analyses showed that c-myc and {beta}-catenin were reduced in CTNNB1 shRNA tumor tissues, but that expression of cleaved caspase-3 was increased. These results show that {beta}-catenin could be a new therapeutic agent that targets the biology of MM cells.« less

Impaired expression of importin/karyopherin {beta}1 leads to post-implantation lethality

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

Miura, Katsutaka; Yoshinobu, Kumiko; Imaizumi, Takashi

2006-03-03

Importin {beta}1 (Imp{beta})/karyopherin {beta}1 (Kpnb1) mediates the nuclear import of a large variety of substrates. This study aimed to investigate the requirement for the Kpnb1 gene in mouse development, using a gene trap line, B6-CB-Ayu8108 {sup GtgeoIMEG} (Ayu8108 {sup geo}), in which the trap vector was inserted into the promoter region of the Kpnb1 gene, but in reverse orientation of the Kpnb1 gene. Ayu8108 {sup geo/geo} homozygous embryos could develop to the blastocyst stage, but died before embryonic day 5.5, and expression of the Kpnb1 gene in homozygous blastocysts was undetectable. We also replaced the {beta}geo gene with Imp{beta} cDNAmore » through Cre-mediated recombination to rescue Imp{beta} expression. Homozygous mice for the rescued allele Ayu8108 {sup Imp{beta}}{sup /Imp{beta}} were born and developed normally. These results demonstrated that the cause of post-implantation lethality of Ayu8108 {sup geo/geo} homozygous embryos was impaired expression of the Kpnb1 gene, indicating indispensable roles of Imp{beta}1 in early development of mice.« less

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.

Overexpression of {alpha}-catenin increases osteoblastic differentiation in mouse mesenchymal C3H10T1/2 cells

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

Kim, Dohee; Yang, Jae-Yeon; Shin, Chan Soo, E-mail: csshin@snu.ac.kr

2009-05-15

{alpha}- and {beta}-Catenin link cadherins to the actin-based cytoskeleton at adherens junctions and regulate cell-cell adhesion. Although roles of cadherins and canonical Wnt-/{beta}-catenin-signaling in osteoblastic differentiation have been extensively studied, the role of {alpha}-catenin is not known. Murine embryonic mesenchymal stem cells, C3H10T1/2 cells, were transduced with retrovirus encoding {alpha}-catenin (MSCV-{alpha}-catenin-HA-GFP). In the presence of Wnt-3A conditioned medium or osteogenic medium ({beta}-glycerol phosphate and ascorbic acid), cells overexpressing {alpha}-catenin showed enhanced osteoblastic differentiation as measured by alkaline phosphatase (ALP) staining and ALP activity assay compared to cells transduced with empty virus (MSCV-GFP). In addition, mRNA expression of osteocalcin and Runx2more » was significantly increased compared to control. Cell aggregation assay revealed that {alpha}-catenin overexpression has significantly increased cell-cell aggregation. However, cellular {beta}-catenin levels (total, cytoplasmic-nuclear ratio) and {beta}-catenin-TCF/LEF transcriptional activity did not change by overexpression of {alpha}-catenin. Knock-down of {alpha}-catenin using siRNA decreased osteoblastic differentiation as measured by ALP assay. These results suggest that {alpha}-catenin overexpression increases osteoblastic differentiation by increasing cell-cell adhesion rather than Wnt-/{beta}-catenin-signaling.« less

GARP (LRRC32) is essential for the surface expression of latent TGF-beta on platelets and activated FOXP3+ regulatory T cells.

PubMed

Tran, Dat Q; Andersson, John; Wang, Rui; Ramsey, Heather; Unutmaz, Derya; Shevach, Ethan M

2009-08-11

TGF-beta family members are highly pleiotropic cytokines with diverse regulatory functions. TGF-beta is normally found in the latent form associated with latency-associated peptide (LAP). This latent complex can associate with latent TGFbeta-binding protein (LTBP) to produce a large latent form. Latent TGF-beta is also found on the surface of activated FOXP3(+) regulatory T cells (Tregs), but it is unclear how it is anchored to the cell membrane. We show that GARP or LRRC32, a leucine-rich repeat molecule of unknown function, is critical for tethering TGF-beta to the cell surface. We demonstrate that platelets and activated Tregs co-express latent TGF-beta and GARP on their membranes. The knockdown of GARP mRNA with siRNA prevented surface latent TGF-beta expression on activated Tregs and recombinant latent TGF-beta1 is able to bind directly with GARP. Confocal microscopy and immunoprecipitation strongly support their interactions. The role of TGF-beta on Tregs appears to have dual functions, both for Treg-mediated suppression and infectious tolerance mechanism.

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

PubMed Central

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

2012-01-01

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

Adenomatous polyposis coli protein (APC)-independent regulation of beta-catenin/Tcf-4 mediated transcription in intestinal cells.

PubMed Central

Baulida, J; Batlle, E; García De Herreros, A

1999-01-01

Alterations in the transcriptional activity of the beta-catenin-Tcf complex have been associated with the earlier stages of colonic transformation. We show here that the activation of protein kinase C by the phorbol ester PMA in several intestinal cell lines increases the levels of beta-catenin detected in the nucleus and augments the transcriptional activity mediated by beta-catenin. The response to PMA was not related to modifications in the cytosolic levels of beta-catenin and was observed not only in cells with wild-type adenomatous polyposis coli protein (APC) but also in APC-deficient cells. Binding assays in vitro revealed that PMA facilitates the interaction of the beta-catenin with the nuclear structure. Our results therefore show that beta-catenin-mediated transcription can be regulated independently of the presence of APC. PMID:10567241

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

Clinical observations on enzyme replacement therapy in patients with Fabry disease and the switch from agalsidase beta to agalsidase alfa.

PubMed

Lin, Hsiang-Yu; Huang, Yu-Hsiu; Liao, Hsuan-Chieh; Liu, Hao-Chuan; Hsu, Ting-Rong; Shen, Chia-I; Li, Shao-Tzu; Li, Cheng-Fang; Lee, Li-Hong; Lee, Pi-Chang; Huang, Chun-Kai; Chiang, Chuan-Chi; Lin, Shuan-Pei; Niu, Dau-Ming

2014-04-01

Fabry disease is an X-linked inherited lysosomal storage disease that can be treated with the enzymes of agalsidase beta (Fabrazyme) and agalsidase alfa (Replagal). Since June 2009, viral contamination of Genzyme's production facility has resulted in a worldwide shortage of agalsidase beta, leading to the switch to agalsidase alfa for patients with Fabry disease in Taiwan. The medical records were retrospectively reviewed for nine male patients with Fabry disease from the start of agalsidase beta treatment until the switch to agalsidase alfa for at least 1 year. After 12-112 months of enzyme replacement therapy (ERT), decreased plasma globotriaosylsphingosine (lyso-Gb3) was found in five out of seven patients, indicating improvement in disease severity. Among the six patients with available echocardiographic data at baseline and after ERT, all six experienced reductions of left ventricular mass index. Renal function, including microalbuminuria and estimated glomerular filtration rate, showed stability after ERT. Mainz Severity Score Index scores revealed that all nine patients remained stable at 12 months after switching to agalsidase alfa. ERT improved or stabilized cardiac status and stabilized renal function, while reducing plasma lyso-Gb3. ERT was well tolerated, even among the three patients who had hypersensitivity reactions. The switch of ERT from agalsidase beta to agalsidase alfa appears to be safe after 1 year of follow-up for Taiwanese patients with Fabry disease. Copyright © 2013. Published by Elsevier B.V.

Analysis and separation of residues important for the chemoattractant and antimicrobial activities of beta-defensin 3.

PubMed

Taylor, Karen; Clarke, David J; McCullough, Bryan; Chin, Wutharath; Seo, Emily; Yang, De; Oppenheim, Joost; Uhrin, Dusan; Govan, John R W; Campopiano, Dominic J; MacMillan, Derek; Barran, Perdita; Dorin, Julia R

2008-03-14

beta-Defensins are important in mammalian immunity displaying both antimicrobial and chemoattractant activities. Three canonical disulfide intramolecular bonds are believed to be dispensable for antimicrobial activity but essential for chemoattractant ability. However, here we show that HBD3 (human beta-defensin 3) alkylated with iodoactemide and devoid of any disulfide bonds is still a potent chemoattractant. Furthermore, when the canonical six cysteine residues are replaced with alanine, the peptide is no longer active as a chemoattractant. These findings are replicated by the murine ortholog Defb14. We restore the chemoattractant activity of Defb14 and HBD3 by introduction of a single cysteine in the fifth position (Cys V) of the beta-defensin six cysteine motif. In contrast, a peptide with a single cysteine at the first position (Cys I) is inactive. Moreover, a range of overlapping linear fragments of Defb14 do not act as chemoattractants, suggesting that the chemotactic activity of this peptide is not dependent solely on an epitope surrounding Cys V. Full-length peptides either with alkylated cysteine residues or with cysteine residues replaced with alanine are still strongly antimicrobial. Defb14 peptide fragments were also tested for antimicrobial activity, and peptides derived from the N-terminal region display potent antimicrobial activity. Thus, the chemoattractant and antimicrobial activities of beta-defensins can be separated, and both of these functions are independent of intramolecular disulfide bonds. These findings are important for further understanding of the mechanism of action of defensins and for therapeutic design.

Digestion of Yeasts and Beta-1,3-Glucanases in Mosquito Larvae: Physiological and Biochemical Considerations.

PubMed

Souza, Raquel Santos; Diaz-Albiter, Hector Manuel; Dillon, Vivian Maureen; Dillon, Rod J; Genta, Fernando Ariel

2016-01-01

Aedes aegypti larvae ingest several kinds of microorganisms. In spite of studies regarding mosquito digestion, little is known about the nutritional utilization of ingested cells by larvae. We investigated the effects of using yeasts as the sole nutrient source for A. aegypti larvae. We also assessed the role of beta-1,3-glucanases in digestion of live yeast cells. Beta-1,3-glucanases are enzymes which hydrolyze the cell wall beta-1,3-glucan polyssacharide. Larvae were fed with cat food (controls), live or autoclaved Saccharomyces cerevisiae cells and larval weight, time for pupation and adult emergence, larval and pupal mortality were measured. The presence of S. cerevisiae cells inside the larval gut was demonstrated by light microscopy. Beta-1,3-glucanase was measured in dissected larval samples. Viability assays were performed with live yeast cells and larval gut homogenates, with or without addition of competing beta-1,3-glucan. A. aegypti larvae fed with yeast cells were heavier at the 4th instar and showed complete development with normal mortality rates. Yeast cells were efficiently ingested by larvae and quickly killed (10% death in 2 h, 100% in 48 h). Larvae showed beta-1,3-glucanase in head, gut and rest of body. Gut beta-1,3-glucanase was not derived from ingested yeast cells. Gut and rest of body activity was not affected by the yeast diet, but head homogenates showed a lower activity in animals fed with autoclaved S. cerevisiae cells. The enzymatic lysis of live S. cerevisiae cells was demonstrated using gut homogenates, and this activity was abolished when excess beta-1,3-glucan was added to assays. These results show that live yeast cells are efficiently ingested and hydrolyzed by A. aegypti larvae, which are able to fully-develop on a diet based exclusively on these organisms. Beta-1,3-glucanase seems to be essential for yeast lytic activity of A. aegypti larvae, which possess significant amounts of these enzyme in all parts investigated.

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

NASA Technical Reports Server (NTRS)

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

1999-01-01

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

A critical role for beta cell M3 muscarinic acetylcholine receptors in regulating insulin release and blood glucose homeostasis in vivo.

PubMed

Gautam, Dinesh; Han, Sung-Jun; Hamdan, Fadi F; Jeon, Jongrye; Li, Bo; Li, Jian Hua; Cui, Yinghong; Mears, David; Lu, Huiyan; Deng, Chuxia; Heard, Thomas; Wess, Jürgen

2006-06-01

One of the hallmarks of type 2 diabetes is that pancreatic beta cells fail to release sufficient amounts of insulin in the presence of elevated blood glucose levels. Insulin secretion is modulated by many hormones and neurotransmitters including acetylcholine, the major neurotransmitter of the peripheral parasympathetic nervous system. The physiological role of muscarinic acetylcholine receptors expressed by pancreatic beta cells remains unclear at present. Here, we demonstrate that mutant mice selectively lacking the M3 muscarinic acetylcholine receptor subtype in pancreatic beta cells display impaired glucose tolerance and greatly reduced insulin release. In contrast, transgenic mice selectively overexpressing M3 receptors in pancreatic beta cells show a profound increase in glucose tolerance and insulin release. Moreover, these mutant mice are resistant to diet-induced glucose intolerance and hyperglycemia. These findings indicate that beta cell M3 muscarinic receptors play a key role in maintaining proper insulin release and glucose homeostasis.

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.

Mechanism of ion transport by avian salt gland primary cell cultures.

PubMed

Lowy, R J; Dawson, D C; Ernst, S A

1989-06-01

Confluent sheets formed from primary culture of avian salt gland secretory cells exhibit a short-circuit current (Isc) in response to cholinergic and beta-adrenergic stimulation [Lowy, R. J., D. C. Dawson, and S. A. Ernst. Am J. Physiol. 249 (Cell Physiol. 18): C41-C47, 1985]. To establish the ionic basis for the Isc, transmural fluxes of 22Na and 36Cl were measured. Under short-circuit conditions there was little net flux of either ion in the absence of agonists. Addition of carbachol elevated net serosal-to-mucosal Cl flux to 1.71 mu eq.h-1.cm-2, whereas a smaller increase to 0.85 mu eq.h-1.cm-2 occurred with isoproterenol. Neither agonist altered net Na flux. The stimulated Isc accounted for 70% of the net Cl flux induced by carbachol and nearly 100% of that induced by isoproterenol. Replacement of Cl by gluconate or Na by choline abolished (carbachol) or greatly reduced (isoproterenol) the Isc, which could be restored in a dose-dependent fashion by ion restitution. Active ion transport was preferentially inhibited by basal (vs. apical) addition of ouabain, furosemide, or barium. The results provide evidence that cholinergic and beta-adrenergic agonists elicit active transmural Cl secretion. They further suggest that transport is dependent on the Na+-K+-adenosine-triphosphatase, a Na-Cl cotransport process, and a basal K conductance, all features of a secondary active Cl secretory mechanism.

Activation of c-Raf-1 kinase signal transduction pathway in alpha(7) integrin-deficient mice.

PubMed

Saher, G; Hildt, E

1999-09-24

Integrin alpha(7)-deficient mice develop a novel form of muscular dystrophy. Here we report that deficiency of alpha(7) integrin causes an activation of the c-Raf-1/mitogen-activated protein (MAP) 2 kinase signal transduction pathway in muscle cells. The observed activation of c-Raf-1/MAP2 kinases is a specific effect, because the alpha(7) integrin deficiency does not cause unspecific stress as determined by measurement of the Hsp72/73 level and activity of the JNK2 kinase. Because an increased level of activated FAK was found in muscle of alpha(7) integrin-deficient mice, the activation of c-Raf-1 kinase is triggered most likely by an integrin-dependent pathway. In accordance with this, in the integrin alpha(7)-deficient mice, part of the integrin beta(1D) variant in muscle is replaced by the beta(1A) variant, which permits the FAK activation. A recent report describes that integrin activity can be down-modulated by the c-Raf-1/MAP2 kinase pathway. Specific activation of the c-Raf-1/MAP2 kinases by cell-permeable peptides in skeletal muscle of rabbits causes degeneration of muscle fibers. Therefore, we conclude that in alpha(7) integrin-deficient mice, the continuous activation of c-Raf-1 kinase causes a permanent reduction of integrin activity diminishing integrin-dependent cell-matrix interactions and thereby contributing to the development of the dystrophic phenotype.

Role of 17 beta-estradiol on type IV collagen fibers volumetric density in the basement membrane of bladder wall.

PubMed

de Fraga, Rogerio; Dambros, Miriam; Miyaoka, Ricardo; Riccetto, Cássio Luís Zanettini; Palma, Paulo César Rodrigues

2007-10-01

The authors quantified the type IV collagen fibers volumetric density in the basement membrane of bladder wall of ovariectomized rats with and without estradiol replacement. This study was conducted on 40 Wistar rats (3 months old) randomly divided in 4 groups: group 1, remained intact (control); group 2, submitted to bilateral oophorectomy and daily replacement 4 weeks later of 17 beta-estradiol for 12 weeks; group 3, sham operated and daily replacement 4 weeks later of sesame oil for 12 weeks; and group 4, submitted to bilateral oophorectomy and killed after 12 weeks. It was used in immunohistochemistry evaluation using type IV collagen polyclonal antibody to stain the fibers on paraffin rat bladder sections. The M-42 stereological grid system was used to analyze the fibers. Ovariectomy had an increase effect on the volumetric density of the type IV collagen fibers in the basement membrane of rat bladder wall. Estradiol replacement in castrated animals demonstrated a significative difference in the stereological parameters when compared to the castrated group without hormonal replacement. Surgical castration performed on rats induced an increasing volumetric density of type IV collagen fibers in the basement membrane of rats bladder wall and the estradiol treatment had a significant effect in keeping a low volumetric density of type IV collagen fibers in the basement membrane of rats bladder wall.

Calcium Channels in Postnatal Development of Rat Pancreatic Beta Cells and Their Role in Insulin Secretion

PubMed Central

García-Delgado, Neivys; Velasco, Myrian; Sánchez-Soto, Carmen; Díaz-García, Carlos Manlio; Hiriart, Marcia

2018-01-01

Pancreatic beta cells during the first month of development acquire functional maturity, allowing them to respond to variations in extracellular glucose concentration by secreting insulin. Changes in ionic channel activity are important for this maturation. Within the voltage-gated calcium channels (VGCC), the most studied channels are high-voltage-activated (HVA), principally L-type; while low-voltage-activated (LVA) channels have been poorly studied in native beta cells. We analyzed the changes in the expression and activity of VGCC during the postnatal development in rat beta cells. We observed that the percentage of detection of T-type current increased with the stage of development. T-type calcium current density in adult cells was higher than in neonatal and P20 beta cells. Mean HVA current density also increased with age. Calcium current behavior in P20 beta cells was heterogeneous; almost half of the cells had HVA current densities higher than the adult cells, and this was independent of the presence of T-type current. We detected the presence of α1G, α1H, and α1I subunits of LVA channels at all ages. The Cav 3.1 subunit (α1G) was the most expressed. T-type channel blockers mibefradil and TTA-A2 significantly inhibited insulin secretion at 5.6 mM glucose, which suggests a physiological role for T-type channels at basal glucose conditions. Both, nifedipine and TTA-A2, drastically decreased the beta-cell subpopulation that secretes more insulin, in both basal and stimulating glucose conditions. We conclude that changes in expression and activity of VGCC during the development play an important role in physiological maturation of beta cells. PMID:29556214

Regulation of calcium-permeable TRPV2 channel by insulin in pancreatic beta-cells.

PubMed

Hisanaga, Etsuko; Nagasawa, Masahiro; Ueki, Kohjiro; Kulkarni, Rohit N; Mori, Masatomo; Kojima, Itaru

2009-01-01

Calcium-permeable cation channel TRPV2 is expressed in pancreatic beta-cells. We investigated regulation and function of TRPV2 in beta-cells. Translocation of TRPV2 was assessed in MIN6 cells and cultured mouse beta-cells by transfecting TRPV2 fused to green fluorescent protein or TRPV2 containing c-Myc tag in the extracellular domain. Calcium entry was assessed by monitoring fura-2 fluorescence. In MIN6 cells, TRPV2 was observed mainly in cytoplasm in an unstimulated condition. Addition of exogenous insulin induced translocation and insertion of TRPV2 to the plasma membrane. Consistent with these observations, insulin increased calcium entry, which was inhibited by tranilast, an inhibitor of TRPV2, or by knockdown of TRPV2 using shRNA. A high concentration of glucose also induced translocation of TRPV2, which was blocked by nefedipine, diazoxide, and somatostatin, agents blocking glucose-induced insulin secretion. Knockdown of the insulin receptor attenuated insulin-induced translocation of TRPV2. Similarly, the effect of insulin on TRPV2 translocation was not observed in a beta-cell line derived from islets obtained from a beta-cell-specific insulin receptor knockout mouse. Knockdown of TRPV2 or addition of tranilast significantly inhibited insulin secretion induced by a high concentration of glucose. Likewise, cell growth induced by serum and glucose was inhibited by tranilast or by knockdown of TRPV2. Finally, insulin-induced translocation of TRPV2 was observed in cultured mouse beta-cells, and knockdown of TRPV2 reduced insulin secretion induced by glucose. TRPV2 is regulated by insulin and is involved in the autocrine action of this hormone on beta-cells.

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.

Interleukin-1 beta induced synthesis of protein kinase C-delta and protein kinase C-epsilon in EL4 thymoma cells: possible involvement of phosphatidylinositol 3-kinase.

PubMed

Varley, C L; Royds, J A; Brown, B L; Dobson, P R

2001-01-01

We present evidence here that the proinflammatory cytokine, interleukin-1 beta (IL-1 beta) stimulates a significant increase in protein kinase C (PKC)-epsilon and PKC-delta protein levels and increases PKC-epsilon, but not PKC-delta, transcripts in EL4 thymoma cells. Incubation of EL4 cells with IL-1 beta induced protein synthesis of PKC-epsilon (6-fold increase) by 7 h and had a biphasic effect on PKC-delta levels with peaks at 4 h (2-fold increase) and 24 h (4-fold increase). At the level of mRNA, PKC-epsilon, but not PKC-delta levels, were induced after incubation of EL4 cells with IL-1 beta. The signalling mechanisms utilized by IL-1 beta to induce the synthesis of these PKC isoforms were investigated. Two phosphatidylinositol (PI) 3-kinase-specific inhibitors, wortmannin and LY294002, inhibited IL-1 beta-induced synthesis of PKC-epsilon. However, the PI 3-kinase inhibitors had little effect on the IL-1 beta-induced synthesis of PKC-delta in these cells. Our results indicate that IL-1 beta induced both PKC-delta and PKC-epsilon expression over different time periods. Furthermore, our evidence suggests that IL-1 beta induction of PKC-epsilon, but not PKC-delta, may occur via the PI 3-kinase pathway. Copyright 2001 S. Karger AG, Basel

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

Alpha cells secrete acetylcholine as a non-neuronal paracrine signal priming human beta cell function

PubMed Central

Rodriguez-Diaz, Rayner; Dando, Robin; Jacques-Silva, M. Caroline; Fachado, Alberto; Molina, Judith; Abdulreda, Midhat; Ricordi, Camillo; Roper, Stephen D.; Berggren, Per-Olof; Caicedo, Alejandro

2011-01-01

Acetylcholine is a neurotransmitter that plays a major role in the function of the insulin secreting pancreatic beta cell1,2. Parasympathetic innervation of the endocrine pancreas, the islets of Langerhans, has been shown to provide cholinergic input to the beta cell in several species1,3,4, but the role of autonomic innervation in human beta cell function is at present unclear. Here we show that, in contrast to mouse islets, cholinergic innervation of human islets is sparse. Instead, we find that the alpha cells of the human islet provide paracrine cholinergic input to surrounding endocrine cells. Human alpha cells express the vesicular acetylcholine transporter and release acetylcholine when stimulated with kainate or a lowering in glucose concentration. Acetylcholine secretion by alpha cells in turn sensitizes the beta cell response to increases in glucose concentration. Our results demonstrate that in human islets acetylcholine is a paracrine signal that primes the beta cell to respond optimally to subsequent increases in glucose concentration. We anticipate these results to revise models about neural input and cholinergic signaling in the endocrine pancreas. Cholinergic signaling within the islet represents a potential therapeutic target in diabetes5, highlighting the relevance of this advance to future drug development. PMID:21685896

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.

Beta-catenin-mediated transactivation and cell-cell adhesion pathways are important in curcumin (diferuylmethane)-induced growth arrest and apoptosis in colon cancer cells.

PubMed

Jaiswal, Aruna S; Marlow, Benjamin P; Gupta, Nirupama; Narayan, Satya

2002-12-05

The development of nontoxic natural agents with chemopreventive activity against colon cancer is the focus of investigation in many laboratories. Curcumin (feruylmethane), a natural plant product, possesses such chemopreventive activity, but the mechanisms by which it prevents cancer growth are not well understood. In the present study, we examined the mechanisms by which curcumin treatment affects the growth of colon cancer cells in vitro. Results showed that curcumin treatment causes p53- and p21-independent G(2)/M phase arrest and apoptosis in HCT-116(p53(+/+)), HCT-116(p53(-/-)) and HCT-116(p21(-/-)) cell lines. We further investigated the association of the beta-catenin-mediated c-Myc expression and the cell-cell adhesion pathways in curcumin-induced G(2)/M arrest and apoptosis in HCT-116 cells. Results described a caspase-3-mediated cleavage of beta-catenin, decreased transactivation of beta-catenin/Tcf-Lef, decreased promoter DNA binding activity of the beta-catenin/Tcf-Lef complex, and decreased levels of c-Myc protein. These activities were linked with decreased Cdc2/cyclin B1 kinase activity, a function of the G(2)/M phase arrest. The decreased transactivation of beta-catenin in curcumin-treated HCT-116 cells was unpreventable by caspase-3 inhibitor Z-DEVD-fmk, even though the curcumin-induced cleavage of beta-catenin was blocked in Z-DEVD-fmk pretreated cells. The curcumin treatment also induced caspase-3-mediated degradation of cell-cell adhesion proteins beta-catenin, E-cadherin and APC, which were linked with apoptosis, and this degradation was prevented with the caspase-3 inhibitor. Our results suggest that curcumin treatment impairs both Wnt signaling and cell-cell adhesion pathways, resulting in G(2)/M phase arrest and apoptosis in HCT-116 cells.

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.

[The mechanism of vasculogenesis: the critical role of transforming growth factor-beta 1 in the formation of vessel-like structures during the differentiation in vitro of murine embryonic stem cells].

PubMed

Tsung, H C; Yao, Z

1996-09-01

When ES-5 cells were transfected with an exogenous porcine TGF-beta 1 gene, one can obtain clones of genetically modified ES cells with over-expression of the transfected gene. We called the genetically modified ES-5 cells as ES-T cells. When ES-T cells were used to study their differentiation in vitro by all trans-retinoic acid (RA), it was soon noticed that embryoid bodies of ES-T cells can exclusively differentiate into endothelial cells and vessel-like structures, but not in their parent ES-5 cells. The above result is the first indication that the differentiation of tubular structures in embryoid bodies of ES-T cells may somehow be related to TGF-beta 1. To demonstrate further the role of TGF-beta 1 in the formation of vessel-like structures, the cultured ES-5 cells in the presence of added rhTGF-beta 1 were closely followed in the course of their differentiation. We have, thus, demonstrated the promoting effects of exogenous rhTGF-beta 1 in the formation of vessel-like structures, morphologically similar to those structures derived from ES-T6 cells, during the differentiation of ES-5 cells, both in monolayer culture, in three dimensional collagen gel and in embryoid bodies cultured on gelatin-coated tissue culture wells. Addition of suitable amount of anti-TGF-beta 1 monoclonal antibody IgG (TB21) to the culture medium of embryoid bodies of ES-T6 cells could effectively abolish the formation of vessel-like structures induced by retinoic acid. The percentage of the inhibition was very high, giving a figure comparable to that of atypical vessel-like structures formed in the control embryoid bodies from their parent ES-5 cells. The flat epithelial-like cells and round cells differentiated from embryoid bodies of ES-T6 cells were stained rather strongly for laminin and type IV collagen by immunofluorescent procedure. The above results indicate clearly that TGF-beta 1 is a crucial factor in organizing the differentiated derivatives (endothelial-like cells and their immediate progenitor cells) from ES-T6 cells to form vessel-like structures, and that the role of TGF-beta 1 in vasculogenesis might be performed, in part, through the modulation of the composition and organization of the extracellular matrix. In addition, the enhanced expression of bFGF mRNA in derivatives differentiated from both ES-5 cells treated with rhTGF-beta 1 and ES-T6 cells were detected by Northern blot analysis. Thus, aside from its effects on extracellular matrix, TGF-beta 1 might also modulate the bioactivity of bFGF in relation to the growth of vascular endothelial cells in the present system.

Ultrasound-mediated interferon {beta} gene transfection inhibits growth of malignant melanoma

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

Yamaguchi, Kazuki; Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180; Feril, Loreto B., E-mail: ferilism@yahoo.com

2011-07-22

Highlights: {yields} Successful ultrasound-mediated transfection of melanoma (C32) cells with IFN-{beta} genes both in vitro and in vivo. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited proliferation of melanoma cells in vitro. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited melanoma tumor growth in vivo. -- Abstract: We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon {beta} (IFN-{beta}) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-{beta} in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-{beta} genes mixed with microbubbles. Successful sonotransfection with IFN-{beta} gene in vitro was confirmed by ELISA,more » which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-{beta} gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.« less

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

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.

Vesicle-associated membrane protein 2 mediates trafficking of {alpha}5{beta}1 integrin to the plasma membrane

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

Hasan, Nazarul; Hu, Chuan, E-mail: chuan.hu@louisville.edu

2010-01-01

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of {alpha}5{beta}1 integrin. VAMP2 was present on vesicles containing endocytosed {beta}1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cellmore » surface {alpha}5{beta}1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of {alpha}5{beta}1, without altering cell surface expression of {alpha}2{beta}1 integrin or {alpha}3{beta}1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of {alpha}5{beta}1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.« less

Unique Aspects of Cryptochrome in Chronobiology and Metabolism, Pancreatic β-Cell Dysfunction, and Regeneration: Research into Cysteine414-Alanine Mutant CRY1.

PubMed

Okano, Satoshi

2016-01-01

Cryptochrome proteins (CRYs), which can bind noncovalently to cofactor (chromophore) flavin adenine dinucleotide (FAD), occur widely among organisms. CRYs play indispensable roles in the generation of circadian rhythm in mammals. Transgenic mice (Tg mice), ubiquitously expressing mouse CRY1 having a mutation in which cysteine414 (the zinc-binding site of CRY1) being replaced with alanine, display unique phenotypes in their circadian rhythms. Moreover, male Tg mice exhibit symptoms of diabetes characterized by beta-cell dysfunction, resembling human maturity onset diabetes of the young (MODY). The lowered proliferation of β -cells is a primary cause of age-dependent β -cell loss. Furthermore, unusually enlarged duct-like structures developed prominently in the Tg mice pancreases. The duct-like structures contained insulin-positive cells, suggesting neogenesis of β -cells in the Tg mice. This review, based mainly on the author's investigation of the unique features of Tg mice, presents reported results and recent findings related to molecular processes associated with mammalian cryptochromes, especially their involvement in the regulation of metabolism. New information is described with emphasis on the aspects of islet architecture, pancreatic β -cell dysfunction, and regeneration.

Nanoscale organization of {beta}{sub 2}-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells

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

Vobornik, Dusan; Rouleau, Yanouchka; Haley, Jennifer

2009-04-24

Adrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize {beta}{sub 2}-adrenergic receptors ({beta}{sub 2}AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the {beta}{sub 2}AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline,more » the size of domains containing the {beta}{sub 2}AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of {beta}{sub 2}AR are observed in the membrane of the HEK293 cells that stably overexpress {beta}{sub 2}AR-GFP and {beta}{sub 2}AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use {beta}{sub 2}AR-Venus fusion proteins as models for {beta}{sub 2}AR function. These observations are critical for designing future cell models and assays based on {beta}{sub 2}AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.« less

Tumor evasion of the immune system by converting CD4+CD25- T cells into CD4+CD25+ T regulatory cells: role of tumor-derived TGF-beta.

PubMed

Liu, Victoria C; Wong, Larry Y; Jang, Thomas; Shah, Ali H; Park, Irwin; Yang, Ximing; Zhang, Qiang; Lonning, Scott; Teicher, Beverly A; Lee, Chung

2007-03-01

CD4+CD25+ T regulatory (T(reg)) cells were initially described for their ability to suppress autoimmune diseases in animal models. An emerging interest is the potential role of T(reg) cells in cancer development and progression because they have been shown to suppress antitumor immunity. In this study, CD4+CD25- T cells cultured in conditioned medium (CM) derived from tumor cells, RENCA or TRAMP-C2, possess similar characteristics as those of naturally occurring T(reg) cells, including expression of Foxp3, a crucial transcription factor of T(reg) cells, production of low levels of IL-2, high levels of IL-10 and TGF-beta, and the ability to suppress CD4+CD25- T cell proliferation. Further investigation revealed a critical role of tumor-derived TGF-beta in converting CD4+CD25- T cells into T(reg) cells because a neutralizing Ab against TGF-beta, 1D11, completely abrogated the induction of T(reg) cells. CM from a nontumorigenic cell line, NRP-152, or irradiated tumor cells did not convert CD4+CD25- T cells to T(reg) cells because they produce low levels of TGF-beta in CM. Finally, we observed a reduced tumor burden in animals receiving 1D11. The reduction in tumor burden correlated with a decrease in tumor-derived TGF-beta. Treatment of 1D11 also reduced the conversion of CD4+ T cells into T(reg) cells and subsequent T(reg) cell-mediated suppression of antitumor immunity. In summary, we have demonstrated that tumor cells directly convert CD4+CD25- T cells to T(reg) cells through production of high levels of TGF-beta, suggesting a possible mechanism through which tumor cells evade the immune system.

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.

17betaE2 promotes cell proliferation in endometriosis by decreasing PTEN via NFkappaB-dependent pathway.

PubMed

Zhang, Hui; Zhao, Xingbo; Liu, Shu; Li, Jijun; Wen, Zeqing; Li, Mingjiang

2010-04-12

The objective of this study was to explore the mechanism of phosphatase and tensin homolog (PTEN) loss in endometriosis. We found that aberrant PTEN expression and mitogen-activated protein kinases (MAPK)/ERK, phosphoinositide 3-kinase (PI3K)/AKt, and nuclear factor-kappaB (NFkappaB) signaling overactivities coexisted in endometriosis. In vitro, 17beta-estradiol rapidly activated the 3 pathways in endometriotic cells and specific inhibitions on the 3 pathways respectively blocked 17beta-estradiol-induced cell proliferation. 17beta-estradiol suppressed PTEN transcription and expression in endometriotic cells which was abolished by specific NFkappaB inhibition. Total/nuclear PTEN-loss and MAPK/ERK, PI3K/AKt, and NFkappaB signal overactivities coexist in endometriosis. In vitro, 17beta-estradiol can promotes cell proliferation in endometriosis by activating PI3K/AKt pathway via an NFkappaB/PTEN-dependent pathway. For the first time we propose the possibility of the presence of a positive feedback-loop: 17beta-estradiol-->high NFkappaB-->low PTEN-->high PI3K-->high NFkappaB, in endometriosis, which may finally promote the proliferation of ectopic endometrial epithelial cells and in turn contributes to the progression of the disease.

Beta-Adrenergic Receptor Population is Up-Regulated in Chicken Skeletal Muscle Cells Treated with Forskolin

NASA Technical Reports Server (NTRS)

Bridge, K. Y.; Young, R. B.; Vaughn, J. R.

1998-01-01

Skeletal muscle hypertrophy is promoted by in vivo administration of beta-adrenergic receptor (betaAR) agonists. These compounds presumably exert their physiological action through the betaAR, and alterations in the population of betaAR could potentially change the ability of the cell to respond to the betaAR agonists. Since the intracellular chemical signal generated by the betaAR is cyclic AMP (cAMP), experiments were initiated in primary chicken muscle cell cultures to determine if artificial elevation of intracellular cAMP by treatment with forskolin would alter the population of functional betaAR expressed on the surface of muscle cells. Chicken skeletal muscle cells after 7 days in culture were employed for the experiments because muscle cells have attained a steady state with respect to muscle protein metabolism at this stage. Cells were treated with 0-10 microM forskolin for a total of three days. At the end of the 1, 2, and 3 day treatment intervals, the concentration of cAMP and the betaAR population were measured. Receptor population was measured in intact muscle cell cultures as the difference between total binding of [H-3]CGP-12177 and non-specific binding of [H-3]CGP-12177 in the presence of 1 microM propranolol. Intracellular cAMP concentration was measured by radioimmunoassay. The concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. Increasing concentrations of forskolin also led to an increase in betaAR population, with a maximum increase of approximately 50% at 10 microM. This increase in PAR population was apparent after only 1 day of treatment, and the pattern of increase was maintained for all 3 days of the treatment period. Thus, increasing the intracellular concentration of cAMP leads to up-regulation of betaAR population. The effect of forskolin on the quantity and apparent synthesis rate of the heavy chain of myosin (mhc) were also investigated. A maximum increase of 50% in the quantity of mhc was observed at 0.2 microM forskolin, but higher concentrations of forskolin reduced the quantity of mhc back to control levels.

A human beta cell line with drug inducible excision of immortalizing transgenes

PubMed Central

Benazra, Marion; Lecomte, Marie-José; Colace, Claire; Müller, Andreas; Machado, Cécile; Pechberty, Severine; Bricout-Neveu, Emilie; Grenier-Godard, Maud; Solimena, Michele; Scharfmann, Raphaël; Czernichow, Paul; Ravassard, Philippe

2015-01-01

Objectives Access to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-βH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen. Methods We used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC βH2 line. The resulting EndoC-βH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion. Results We showed that EndoC-βH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 μg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable function for 5 weeks in culture. Conclusions EndoC βH3 cell line represents a powerful tool that allows, using a simple and efficient procedure, the massive production of functional non-proliferative human beta cells. Such cells are close to genuine human beta cells and maintain a stable phenotype for 5 weeks in culture. PMID:26909308

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

PubMed

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

2006-06-15

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

Effect of electrical stimulation on beta-adrenergic receptor population and cyclic amp production in chicken and rat skeletal muscle cell cultures

NASA Technical Reports Server (NTRS)

Young, R. B.; Bridge, K. Y.; Strietzel, C. J.

2000-01-01

Expression of the beta-adrenergic receptor (betaAR) and its coupling to cyclic AMP (cAMP) synthesis are important components of the signaling system that controls muscle atrophy and hypertrophy, and the goal of this study was to determine if electrical stimulation in a pattern simulating slow muscle contraction would alter the betaAR response in primary cultures of avian and mammalian skeletal muscle cells. Specifically, chicken skeletal muscle cells and rat skeletal muscle cells that had been grown for 7 d in culture were subjected to electrical stimulation for an additional 2 d at a pulse frequency of 0.5 pulses/sec and a pulse duration of 200 msec. In chicken skeletal muscle cells, the betaAR population was not significantly affected by electrical stimulation; however, the ability of these cells to synthesize cyclic AMP was reduced by approximately one-half. In contrast, the betaAR population in rat muscle cells was increased slightly but not significantly by electrical stimulation, and the ability of these cells to synthesize cyclic AMP was increased by almost twofold. The basal levels of intracellular cyclic AMP in neither rat muscle cells nor chicken muscle cells were affected by electrical stimulation.

SU-E-J-03: A Comprehensive Comparison Between Alpha and Beta Emitters for Cancer Radioimmunotherapy

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

Huang, C.Y.; Guatelli, S; Oborn, B

2014-06-01

Purpose: The purpose of this study is to perform a comprehensive comparison of the therapeutic efficacy and cytotoxicity of alpha and beta emitters for Radioimmunotherapy (RIT). For each stage of cancer development, specific models were built for the separate objectives of RIT to be addressed:a) kill isolated cancer cells in transit in the lymphatic and vascular circulation,b) regress avascular cell clusters,c) regress tumor vasculature and tumors. Methods: Because of the nature of short range, high LET alpha and long energy beta radiation and heterogeneous antigen expression among cancer cells, the microdosimetric approach is essential for the RIT assessment. Geant4 basedmore » microdosimetric models are developed for the three different stages of cancer progression: cancer cells, cell clusters and tumors. The energy deposition, specific energy resulted from different source distribution in the three models was calculated separately for 4 alpha emitting radioisotopes ({sup 211}At, {sup 213}Bi, {sup 223}Ra and {sup 225}Ac) and 6 beta emitters ({sup 32}P, {sup 33}P, {sup 67}Cu, {sup 90}Y, {sup 131}I and {sup 177}Lu). The cell survival, therapeutic efficacy and cytotoxicity are determined and compared between alpha and beta emitters. Results: We show that internal targeted alpha radiation has advantages over beta radiation for killing isolated cancer cells, regressing small cell clusters and also solid tumors. Alpha particles have much higher dose specificity and potency than beta particles. They can deposit 3 logs more dose than beta emitters to single cells and solid tumor. Tumor control probability relies on deep penetration of radioisotopes to cancer cell clusters and solid tumors. Conclusion: The results of this study provide a quantitative understanding of the efficacy and cytotoxicity of RIT for each stage of cancer development.« less

GLP-1-oestrogen attenuates hyperphagia and protects from beta cell failure in diabetes-prone New Zealand obese (NZO) mice.

PubMed

Schwenk, Robert W; Baumeier, Christian; Finan, Brian; Kluth, Oliver; Brauer, Christine; Joost, Hans-Georg; DiMarchi, Richard D; Tschöp, Matthias H; Schürmann, Annette

2015-03-01

Oestrogens have previously been shown to exert beta cell protective, glucose-lowering effects in mouse models. Therefore, the recent development of a glucagon-like peptide-1 (GLP-1)-oestrogen conjugate, which targets oestrogen into cells expressing GLP-1 receptors, offers an opportunity for a cell-specific and enhanced beta cell protection by oestrogen. The purpose of this study was to compare the effects of GLP-1 and GLP-1-oestrogen during beta cell failure under glucolipotoxic conditions. Male New Zealand obese (NZO) mice were treated with daily s.c. injections of GLP-1 and GLP-1-oestrogen, respectively. Subsequently, the effects on energy homeostasis and beta cell integrity were measured. In order to clarify the targeting of GLP-1-oestrogen, transcription analyses of oestrogen-responsive genes in distinct tissues as well as microarray analyses in pancreatic islets were performed. In contrast to GLP-1, GLP-1-oestrogen significantly decreased food intake resulting in a substantial weight reduction, preserved normoglycaemia, increased glucose tolerance and enhanced beta cell protection. Analysis of hypothalamic mRNA profiles revealed elevated expression of Pomc and Leprb. In livers from GLP-1-oestrogen-treated mice, expression of lipogenic genes was attenuated and hepatic triacylglycerol levels were decreased. In pancreatic islets, GLP-1-oestrogen altered the mRNA expression to a pattern that was similar to that of diabetes-resistant NZO females. However, conventional oestrogen-responsive genes were not different, indicating rather indirect protection of pancreatic beta cells. GLP-1-oestrogen efficiently protects NZO mice against carbohydrate-induced beta cell failure by attenuation of hyperphagia. In this regard, targeted delivery of oestrogen to the hypothalamus by far exceeds the anorexigenic capacity of GLP-1 alone.

Membrane protein GARP is a receptor for latent TGF-beta on the surface of activated human Treg.

PubMed

Stockis, Julie; Colau, Didier; Coulie, Pierre G; Lucas, Sophie

2009-12-01

Human Treg and Th clones secrete the latent form of TGF-beta, in which the mature TGF-beta protein is bound to the latency-associated peptide (LAP), and is thereby prevented from binding to the TGF-beta receptor. We previously showed that upon TCR stimulation, human Treg clones but not Th clones produce active TGF-beta and bear LAP on their surface. Here, we show that latent TGF-beta, i.e. both LAP and mature TGF-beta, binds to glycoprotein A repetitions predominant (GARP), a transmembrane protein containing leucine rich repeats, which is present on the surface of stimulated Treg clones but not on Th clones. Membrane localization of latent TGF-beta mediated by binding to GARP may be necessary for the ability of Treg to activate TGF-beta upon TCR stimulation. However, it is not sufficient as lentiviral-mediated expression of GARP in human Th cells induces binding of latent TGF-beta to the cell surface, but does not result in the production of active TGF-beta upon stimulation of these Th cells.

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

PubMed

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

2004-12-01

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

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.

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

PubMed

Danner, S; Lohse, M J

1997-07-16

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

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

PubMed

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

2010-03-01

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

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)

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

PubMed Central

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

1992-01-01

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

Electrical Stimulation Decreases Coupling Efficiency Between Beta-Adrenergic Receptors and Cyclic AMP Production in Cultured Muscle Cells

NASA Technical Reports Server (NTRS)

Young, R. B.; Bridge, K. Y.

1999-01-01

Electrical stimulation of skeletal muscle cells in culture is an effective way to simulate the effects of muscle contraction and its effects on gene expression in muscle cells. Expression of the beta-adrenergic receptor and its coupling to cyclic AMP synthesis are important components of the signaling system that controls muscle atrophy and hypertrophy, and the goal of this project was to determine if electrical stimulation altered the beta-adrenergic response in muscle cells. Chicken skeletal muscle cells that had been grown for seven days in culture were subjected to electrical stimulation for an additional two days at a pulse frequency of 0.5 pulses/sec and a pulse duration of 200 msec. At the end of this two-day stimulation period, beta-adrenergic receptor population was measured by the binding of tritium-labeled CGP-12177 to muscle cells, and coupling to cAMP synthesis was measured by Radioimmunoassay (RIA) after treating the cells for 10 min with the potent (beta)AR agonist, isoproterenol. The number of beta adrenergic receptors and the basal levels of intracellular cyclic AMP were not affected by electrical stimulation. However, the ability of these cells to synthesize cyclic AMP was reduced by approximately 50%. Thus, an enhanced level of contraction reduces the coupling efficiency of beta-adrenergic receptors for cyclic AMP production.

Impact of Vitamin D Replacement on Markers of Glucose Metabolism and Cardio-Metabolic Risk in Women with Former Gestational Diabetes--A Double-Blind, Randomized Controlled Trial.

PubMed

Yeow, Toh Peng; Lim, Shueh Lin; Hor, Chee Peng; Khir, Amir S; Wan Mohamud, Wan Nazaimoon; Pacini, Giovanni

2015-01-01

Gestational Diabetes Mellitus (GDM) and vitamin D deficiency are related to insulin resistance and impaired beta cell function, with heightened risk for future development of diabetes. We evaluated the impact of vitamin D supplementation on markers of glucose metabolism and cardio metabolic risk in Asian women with former GDM and hypovitaminosis D. In this double blind, randomized controlled trial, 26 participants were randomized to receive either daily 4000 IU vitamin D3 or placebo capsules. 75 g Oral Glucose Tolerance Test (OGTT) and biochemistry profiles were performed at baseline and 6 month visits. Mathematical models, using serial glucose, insulin and C peptide measurements from OGTT, were employed to calculate insulin sensitivity and beta cell function. Thirty three (76%) women with former GDM screened had vitamin D level of <50 nmol/L at baseline. Supplementation, when compared with placebo, resulted in increased vitamin D level (+51.1 nmol/L vs 0.2 nmol/L, p<0.001) and increased fasting insulin (+20% vs 18%, p = 0.034). The vitamin D group also demonstrated a 30% improvement in disposition index and an absolute 0.2% (2 mmol/mol) reduction in HbA1c. There was no clear change in insulin sensitivity or markers of cardio metabolic risk. This study highlighted high prevalence of vitamin D deficiency among Asian women with former GDM. Six months supplementation with 4000 IU of vitamin D3 safely restored the vitamin D level, improved basal pancreatic beta-cell function and ameliorated the metabolic state. There was no effect on markers of cardio metabolic risk. Further mechanistic studies exploring the role of vitamin D supplementation on glucose homeostasis among different ethnicities may be needed to better inform future recommendations for these women with former GDM at high risk of both hypovitaminosis D and future diabetes.

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.

Isolation of mouse pancreatic alpha, beta, duct and acinar populations with cell surface markers.

PubMed

Dorrell, Craig; Grompe, Maria T; Pan, Fong Cheng; Zhong, Yongping; Canaday, Pamela S; Shultz, Leonard D; Greiner, Dale L; Wright, Chris V; Streeter, Philip R; Grompe, Markus

2011-06-06

Tools permitting the isolation of live pancreatic cell subsets for culture and/or molecular analysis are limited. To address this, we developed a collection of monoclonal antibodies with selective surface labeling of endocrine and exocrine pancreatic cell types. Cell type labeling specificity and cell surface reactivity were validated on mouse pancreatic sections and by gene expression analysis of cells isolated using FACS. Five antibodies which marked populations of particular interest were used to isolate and study viable populations of purified pancreatic ducts, acinar cells, and subsets of acinar cells from whole pancreatic tissue or of alpha or beta cells from isolated mouse islets. Gene expression analysis showed the presence of known endocrine markers in alpha and beta cell populations and revealed that TTR and DPPIV are primarily expressed in alpha cells whereas DGKB and GPM6A have a beta cell specific expression profile. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

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

Campbell, K.S.

Norepinephrine has previously been demonstrated by this laboratory to potentiate the in vitro T-dependent antibody response through the stimulation of {beta}-adrenergic receptors. The role of {beta}-adrenergic receptor subtypes in norepinephrine-induced potentiation of the antibody responses was examined with selective {beta}-adrenergic antagonists. The antagonists were metoprolol ({beta}{sub 1}-selective), ICI 118-551 ({beta}{sub 2}-selective), and propranolol ({beta}-non-selective). Both propranolol and ICI 118-551 blocked norepinephrine-induced potentiation of the antibody response, but metoprolol was ineffective. Receptor binding competition of antagonists with the radioligant, ({sup 3}H)CGP-12177 was examined and results were analyzed with the computer program, LIGAND. Competition by ICI 118-551 identified 75% {beta}{sub 2}- andmore » 25% {beta}{sub 1}-adrenergic receptors on splenic mononuclear cells. Enriched T lymphocytes exhibited 75% {beta}{sub 2}-adrenergic receptors, while enriched B lymphocytes contained 90% {beta}{sub 2}-adrenergic receptors as identified by ICI 118-551. Greater than twice as many total receptors were identified on B lymphocytes than T lymphocytes. A T cell lymphoma contained about 60% {beta}{sub 2}-receptors, while 100% were {beta}{sub 2} receptors on a B cell lymphoma, as assessed by ICI 118-551. Results support a heterogeneous {beta}-adrenergic receptor population on T lymphocytes and a more homogeneous {beta}{sub 2}-population on B lymphocytes.« less

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.

Isoreserpine promotes {beta}-catenin degradation via Siah-1 up-regulation in HCT116 colon cancer cells

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

Gwak, Jungsug; Song, Taeyun; Song, Jie-Young

2009-09-25

Aberrant accumulation of intracellular {beta}-catenin in intestinal epithelial cells is a frequent early event during the development of colon cancer. To identify small molecules that decrease the level of intracellular {beta}-catenin, we performed cell-based chemical screening using genetically engineered HEK293 reporter cells to detect compounds that inhibit TOPFlash reporter activity, which was stimulated by Wnt3a-conditioned medium. We found that isoreserpine promoted the degradation of intracellular {beta}-catenin by up-regulation of Siah-1 in HEK293 and HCT116 colon cancer cells. Moreover, isoreserpine repressed the expression of {beta}-catenin/T-cell factor (TCF)-dependent genes, such as cyclin D1 and c-myc, resulting in the suppression of HCT116 cellmore » proliferation. Our findings suggest that isoreserpine can potentially be used as a chemotherapeutic agent against colon cancer.« less

Application of microalgal fucoxanthin for the reduction of colon cancer risk: inhibitory activity of fucoxanthin against beta-glucuronidase and DLD-1 cancer cells.

PubMed

Kawee-Ai, Arthitaya; Kim, Sang Moo

2014-07-01

Intestinal bacterial beta-glucuronidases are capable of retoxifying compounds that have been detoxified by liver glucuronidation and are also known to accelerate colon cancer invasion and metastasis. In this study, fucoxanthin extracted from the microalga Phaeodactylum tricornutum was investigated for its inhibitory activity against Escherichia coli beta-glucuronidase and DLD-1 cancer cells. Fucoxanthin inhibited beta-glucuronidase in a concentration-dependent manner with an IC50 value of 2.32 mM and a mixed inhibition type. Fucoxanthin had more potent inhibitory activity on beta-glucuronidase at 37 degrees C and in alkaline conditions. Fucoxanthin also inhibited the beta-glucuronidase activity of DLD-1 cancer cells at a concentration of 20-50 microM. The presence of beta-glucuronidase and substrate in the medium decreased the inhibitory activity of fucoxanthin against DLD-1 cancer cells. Therefore, microalgal fucoxanthin might prevent colon cancer because of its strong beta-glucuronidase inhibitory activity and could be utilized as a novel functional ingredient of food and pharmaceutical supplements.

Design of retrovirus vectors for transfer and expression of the human. beta. -globin gene

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

Miller, A.D.; Bender, M.A.; Harris, E.A.S.

1988-11-01

Regulated expression of the human ..beta..-globin gene has been demonstrated in cultured murine erythroleukemia cells and in mice after retrovirus-mediated gene transfer. However, the low titer of recombinant viruses described to date results in relatively inefficient gene transfer, which limits their usefulness for animal studies and for potential gene therapy in humans for diseases involving defective ..beta..-globin genes. The authors found regions that interfered with virus production within intron 2 of the ..beta..-globin gene and on both sides of the gene. The flanking regions could be removed, but intron 2 was required for ..beta..-globin expression. Inclusion of ..beta..-globin introns necessitatesmore » an antisense orientation of the gene within the retrovirus vector. However, they found no effect of the antisense ..beta..-globin transcription on virus production. A region downstream of the ..beta..-globin gene that stimulates expression of the gene in transgenic mice was included in the viruses without detrimental effects on virus titer. Virus titers of over 10/sup 6/ CFU/ml were obtained with the final vector design, which retained the ability to direct regulated expression of human ..beta..-globin in murine erythroleukemia cells. The vector also allowed transfer and expression of the human ..beta..-globin gene in hematopoietic cells (CFU-S cells) in mice.« less

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

Burgess, Antony W., E-mail: burgess@ludwig.edu.au; Faux, Maree C.; Layton, Meredith J.

In this brief overview we discuss the association between Wnt signaling and colon cell biology and tumorigenesis. Our current understanding of the role of Apc in the {beta}-catenin destruction complex is compared with potential roles for Apc in cell adhesion and migration. The requirement for phosphorylation in the proteasomal-mediated degradation of {beta}-catenin is contrasted with roles for phospho-{beta}-catenin in the activation of transcription, cell adhesion and migration. The synergy between Myb and {beta}-catenin regulation of transcription in crypt stem cells during Wnt signaling is discussed. Finally, potential effects of growth factor regulatory systems, Apc or truncated-Apc on crypt morphogenesis, stemmore » cell localization and crypt fission are considered.« less

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.

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.

The Role of Oxidative Stress and Hypoxia in Pancreatic Beta-Cell Dysfunction in Diabetes Mellitus.

PubMed

Gerber, Philipp A; Rutter, Guy A

2017-04-01

Metabolic syndrome is a frequent precursor of type 2 diabetes mellitus (T2D), a disease that currently affects ∼8% of the adult population worldwide. Pancreatic beta-cell dysfunction and loss are central to the disease process, although understanding of the underlying molecular mechanisms is still fragmentary. Recent Advances: Oversupply of nutrients, including glucose and fatty acids, and the subsequent overstimulation of beta cells, are believed to be an important contributor to insulin secretory failure in T2D. Hypoxia has also recently been implicated in beta-cell damage. Accumulating evidence points to a role for oxidative stress in both processes. Although the production of reactive oxygen species (ROS) results from enhanced mitochondrial respiration during stimulation with glucose and other fuels, the expression of antioxidant defense genes is unusually low (or disallowed) in beta cells. Not all subjects with metabolic syndrome and hyperglycemia go on to develop full-blown diabetes, implying an important role in disease risk for gene-environment interactions. Possession of common risk alleles at the SLC30A8 locus, encoding the beta-cell granule zinc transporter ZnT8, may affect cytosolic Zn 2+ concentrations and thus susceptibility to hypoxia and oxidative stress. Loss of normal beta-cell function, rather than total mass, is increasingly considered to be the major driver for impaired insulin secretion in diabetes. Better understanding of the role of oxidative changes, its modulation by genes involved in disease risk, and effects on beta-cell identity may facilitate the development of new therapeutic strategies to this disease. Antioxid. Redox Signal. 26, 501-518.

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.

Heat shock protein 90{beta}: A novel mediator of vitamin D action

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

Angelo, Giana; Mineral Bioavailability Laboratory, 711 Washington Street, Boston, MA 02111; Lamon-Fava, Stefania

2008-03-14

We investigated the role of Heat shock protein 90 (Hsp90) in vitamin D action in Caco-2 cells using geldanamycin (GA) to block Hsp90 function and RNA interference to reduce Hsp90{beta} expression. When cells were exposed to GA, vitamin D-mediated gene expression and transcriptional activity were inhibited by 69% and 54%, respectively. Gel shift analysis indicated that GA reduced vitamin D-mediated DNA binding activity of the vitamin D receptor (VDR). We tested the specific role of Hsp90{beta} by knocking down its expression with stably expressed short hairpin RNA. Vitamin D-induced gene expression and transcriptional activity were reduced by 90% and 80%,more » respectively, in Hsp90{beta}-deficient cells. Nuclear protein for VDR and RXR{alpha}, its heterodimer partner, were not reduced in Hsp90{beta}-deficient cells. These findings indicate that Hsp90{beta} is needed for optimal vitamin D responsiveness in the enterocyte and demonstrate a specific role for Hsp90{beta} in VDR signaling.« less

Mechanisms of integrin-vascular endothelial growth factor receptor cross-activation in angiogenesis.

PubMed

Mahabeleshwar, Ganapati H; Feng, Weiyi; Reddy, Kumar; Plow, Edward F; Byzova, Tatiana V

2007-09-14

The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between beta3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and beta3 integrin appears to be synergistic, because VEGFR-2 activation induces beta3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesion- and growth factor-induced beta3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent beta3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and beta3 integrin. Moreover, c-Src mediates growth factor-induced beta3 integrin activation, ligand binding, beta3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro- and antiangiogenic strategies.

Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

PubMed

Koyama, T; Hughes, R C

1992-12-25

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.

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

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.

Inflammatory Response in Islet Transplantation

PubMed Central

Kanak, Mazhar A.; Kunnathodi, Faisal; Lawrence, Michael C.; Levy, Marlon F.

2014-01-01

Islet cell transplantation is a promising beta cell replacement therapy for patients with brittle type 1 diabetes as well as refractory chronic pancreatitis. Despite the vast advancements made in this field, challenges still remain in achieving high frequency and long-term successful transplant outcomes. Here we review recent advances in understanding the role of inflammation in islet transplantation and development of strategies to prevent damage to islets from inflammation. The inflammatory response associated with islets has been recognized as the primary cause of early damage to islets and graft loss after transplantation. Details on cell signaling pathways in islets triggered by cytokines and harmful inflammatory events during pancreas procurement, pancreas preservation, islet isolation, and islet infusion are presented. Robust control of pre- and peritransplant islet inflammation could improve posttransplant islet survival and in turn enhance the benefits of islet cell transplantation for patients who are insulin dependent. We discuss several potent anti-inflammatory strategies that show promise for improving islet engraftment. Further understanding of molecular mechanisms involved in the inflammatory response will provide the basis for developing potent therapeutic strategies for enhancing the quality and success of islet transplantation. PMID:24883060

Tissue strands as "bioink" for scale-up organ printing.

PubMed

Yu, Yin; Ozbolat, Ibrahim T

2014-01-01

Organ printing, takes tissue spheroids as building blocks together with additive manufacturing technique to engineer tissue or organ replacement parts. Although a wide array of cell aggregation techniques has been investigated, and gained noticeable success, the application of tissue spheroids for scale-up tissue fabrication is still worth investigation. In this paper, we introduce a new micro-fabrication technique to create tissue strands at the scale of 500-700μm as a "bioink" for future robotic tissue printing. Printable alginate micro-conduits are used as semi-permeable capsules for tissue strand fabrication. Mouse insulinoma beta TC3 cell tissue strands were formed upon 4 days post fabrication with reasonable mechanical strength, high cell viability close to 90%, and tissue specific markers expression. Fusion was readily observed between strands when placing them together as early as 24h. Also, tissue strands were deposited with human umbilical vein smooth muscle cells (HUVSMCs) vascular conduits together to fabricated miniature pancreatic tissue analog. Our study provided a novel technique using tissue strands as "bioink" for scale-up bioprinting of tissues or organs.

Grape seed procyanidin extract modulates proliferation and apoptosis of pancreatic beta-cells.

PubMed

Cedó, Lídia; Castell-Auví, Anna; Pallarès, Victor; Blay, Mayte; Ardévol, Anna; Arola, Lluís; Pinent, Montserrat

2013-05-01

Grape seed procyanidin extract (GSPE) modulates glucose homeostasis and insulinemia in several animal models. Under pathological conditions, insulin levels are dependent on pancreatic beta-cell functionality, as well as on the beta-cell mass expansion or apoptosis in the pancreas. In this study, we analysed the effects of GSPE on modulating apoptosis and proliferation in beta-cells. We tested the effects of GSPE in the INS-1E pancreatic beta-cell line, either under basal or altered conditions with high glucose, insulin or palmitate levels. GSPE enhanced the pro-apoptotic effect of high glucose and showed clear antiproliferative effects under high glucose, insulin and palmitate conditions. These antiproliferative effects are likely due to high molecular weight compounds contained in the extract. GSPE also modulated pro- and anti-apoptotic markers in the pancreas of rats fed a cafeteria diet, with the effect depending on the dose of GSPE and duration of treatment. Thus, GSPE is able to modulate apoptosis and proliferation of beta-cells under altered, but not basal, conditions. Copyright © 2012 Elsevier Ltd. All rights reserved.

What Is the Role of Nutritional Supplements in Support of Total Hip Replacement and Total Knee Replacement Surgeries? A Systematic Review.

PubMed

Burgess, Louise C; Phillips, Stuart M; Wainwright, Thomas W

2018-06-25

Nutritional supplements can influence outcomes for individuals undergoing major surgery, particularly in older persons whose functional reserve is limited. Accelerating recovery from total hip replacement (THR) and total knee replacement (TKR) may offer significant benefits. Therefore, we explored the role of nutritional supplements in improving recovery following THR and TKR. A systematic review was conducted to source randomized clinical trials that tested nutritional supplements in cohorts of THR or TKR patients. Our search yielded nine relevant trials. Intake of a carbohydrate-containing fluid is reported to improve insulin-like growth factor levels, reduce hunger, nausea, and length of stay, and attenuate the decrease in whole-body insulin sensitivity and endogenous glucose release. Amino acid supplementation is reported to reduce muscle atrophy and accelerate return of functional mobility. One paper reported a suppressive effect of beta-hydroxy beta-methylbutyrate, L-arginine, and L-glutamine supplementation on muscle strength loss following TKR. There is limited evidence for nutritional supplementation in THR and TKR pathways; however, the low risk profile and potential benefits to adjunctive treatment methods, such as exercise programs, suggest nutritional supplements may have a role. Optimizing nutritional status pre-operatively may help manage the surgical stress response, with a particular benefit for undernourished, frail, or elderly individuals.

Langerhans cells beta 2-adrenoceptors: role in migration, cytokine production, Th priming and contact hypersensitivity.

PubMed

Maestroni, Georges J M; Mazzola, Paola

2003-11-01

We showed that norepinephrine (NE) hampers IL-12 and stimulates IL-10 production via adrenoceptors (ARs) in bone marrow-derived dendritic cells (BMDC) influencing their Th priming ability. Others have shown that Langerhans cells (LC) express mRNA for beta1-, beta2- and alpha1(A)-(ARs) and that catecholamines may inhibit the antigen-presenting capability via beta2-ARs. Here, we show that also BMDC express mRNA for beta1-, beta2-, alpha2(A)- and alpha2(C)-ARs. Inhibition of IL-12 is mediated by both beta2- and alpha2(A)-ARs, while stimulation of IL-10 by beta2-ARs only. In addition, LC migration, the contact hypersensitivity response (CHS) and production of IFN-gamma and IL-2 in draining lymph node cells is increased in mice treated topically with the beta2-AR antagonist ICI 118,551 during FITC sensitization. Activation of beta2-ARs in BMDC before adoptive transfer could reduce both migration and CHS response to FITC. Finally, preincubation of BMDC with LPS in presence of the specific beta2-AR agonist salbutamol impaired their chemotactic response to CCL19 and CCL21 and this effect was neutralized by anti-IL-10 mAb. We suggest that the physiological activation of beta2-ARs in DC (LC) results in stimulation of IL-10 which in turn restrains DC (LC) migration influencing antigen presentation and the consequent CHS response.

Wnt/beta-Catenin, Foxa2, and CXCR4 Axis Controls Prostate Cancer Progression

DTIC Science & Technology

2014-07-01

NT1 cells that over-expressing Foxa2. The reason we used NT1 cells for the Foxa2 over-expressing experiments is that NT1 is an AR-expressing... cells . We have also established NT1 cells over-expressing a dominant active beta-catenin. We have characterized these cells . Our research found: 1...expression profiles of control NT1 , NT1 /Foxa2, and NT1 /beta-catenin cells Figure 1. We did RT-PCR to examine the expression of key

The effect of TRAIL molecule on cell viability in in vitro beta cell culture.

PubMed

Tekmen, I; Ozyurt, D; Pekçetin, C; Buldan, Z

2007-06-01

Insulin-dependent diabetes mellitus (IDDM) is an organ-specific autoimmune disorder triggered by autoreactive T cells directed to pancreas beta-cell antigens. In this disorder, more than 90% of beta cells are destroyed. Cell death may be mediated via soluble or membrane-bound cell death ligands. One of these ligands may be tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF-alpha superfamily. In the present study, we examined whether TRAIL had cytotoxic effects on adult rat pancreas beta cell cultures and INS1-E rat insulinoma cell line cultures or not. In this study, cell destruction models were built with TRAIL concentrations of 10, 100 and 1000 ng. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used for evaluating cell viability. It was detected that cell cultures with TRAIL added showed no differences statistically when compared with control cultures containing no toxic additions. These results showed that TRAIL did not have significant cytotoxic effects on pancreas beta cell culture and INS-1E rat insulinoma cell line cultures. Detection of the expression of TRAIL receptors and natural apoptosis inhibitor proteins will be favourable to investigate the resistance mechanisms to TRAIL-induced cell death in this cell culture system.

Phytomedicines and Nutraceuticals: Alternative Therapeutics for Sickle Cell Anemia

PubMed Central

Imaga, Ngozi Awa

2013-01-01

Sickle cell anemia is a genetically inherited disease in which the “SS” individual possesses an abnormal beta globin gene. A single base substitution in the gene encoding the human β-globin subunit results in replacement of β6 glutamic acid by valine, leading to the devastating clinical manifestations of sickle cell disease. This substitution causes drastic reduction in the solubility of sickle cell hemoglobin (HbS) when deoxygenated. Under these conditions, the HbS molecules polymerize to form long crystalline intracellular mass of fibers which are responsible for the deformation of the biconcave disc shaped erythrocyte into a sickle shape. First-line clinical management of sickle cell anemia include, use of hydroxyurea, folic acid, amino acids supplementation, penicillinprophylaxis, and antimalarial prophylaxis to manage the condition and blood transfusions to stabilize the patient's hemoglobin level. These are quite expensive and have attendant risk factors. However, a bright ray of hope involving research into antisickling properties of medicinal plants has been rewarding. This alternative therapy using phytomedicines has proven to not only reduce crisis but also reverse sickling (in vitro). The immense benefits of phytomedicines and nutraceuticals used in the management of sickle cell anemia are discussed in this paper. PMID:23476125

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

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

PubMed

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

1998-12-01

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

Beta-1,4-glucanase-like protein from the cyanobacterium Synechocystis PCC6803 is a beta-1,3-1,4-glucanase and functions in salt stress tolerance.

PubMed

Tamoi, Masahiro; Kurotaki, Hideki; Fukamizo, Tamo

2007-07-01

In the present study, we characterized the gene (Cyanobase accession number slr0897) designated Ssglc encoding a beta-1,4-glucanase-like protein (SsGlc) from Synechocystis PCC6803. The deduced amino acid sequence for Ssglc showed a high degree of similarity to sequences of GH (glycoside hydrolase) family 9 beta-1,4-glucanases (cellulases) from various sources. Surprisingly, the recombinant protein obtained from the Escherichia coli expression system was able to hydrolyse barley beta-glucan and lichenan (beta-1,3-1,4-glucan), but not cellulose (beta-1,4-glucan), curdlan (beta-1,3-glucan), or laminarin (beta-1,3-1,6-glucan). A 1H-NMR analysis of the enzymatic products revealed that the enzyme hydrolyses the beta-1,4-glycosidic linkage of barley beta-glucan through an inverting mechanism. The data indicated that SsGlc was a novel type of GH9 glucanase which could specifically hydrolyse the beta-1,3-1,4-linkage of glucan. The growth of mutant Synechocystis cells in which the Ssglc gene was disrupted by a kanamycin-resistance cartridge gene was almost the same as that of the wild-type cells under continuous light (40 micromol of photons/m2 per s), a 12 h light (40 micromol of photons/m2 per s)/12 h dark cycle, cold stress (4 degrees C), and high light stress (200 micromol of photons/m2 per s). However, under salt stress (300-450 mM NaCl), growth of the Ssglc-disrupted mutant cells was significantly inhibited as compared with that of the wild-type cells. The Ssglc-disrupted mutant cells showed a decreased rate of O2 consumption and NaHCO3-dependent O2 evolution as compared with the wild-type cells under salt stress. Under osmotic stress (100-400 mM sorbitol), there was no difference in growth between the wild-type and the Ssglc-disrupted mutant cells. These results suggest that SsGlc functions in salt stress tolerance in Synechocystis PCC6803.

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.

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

PubMed Central

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

2009-01-01

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

Neuroprotective effect of cannabidiol, a non-psychoactive component from Cannabis sativa, on beta-amyloid-induced toxicity in PC12 cells.

PubMed

Iuvone, Teresa; Esposito, Giuseppe; Esposito, Ramona; Santamaria, Rita; Di Rosa, Massimo; Izzo, Angelo A

2004-04-01

Abstract Alzheimer's disease is widely held to be associated with oxidative stress due, in part, to the membrane action of beta-amyloid peptide aggregates. Here, we studied the effect of cannabidiol, a major non-psychoactive component of the marijuana plant (Cannabis sativa) on beta-amyloid peptide-induced toxicity in cultured rat pheocromocytoma PC12 cells. Following exposure of cells to beta-amyloid peptide (1 micro g/mL), a marked reduction in cell survival was observed. This effect was associated with increased reactive oxygen species (ROS) production and lipid peroxidation, as well as caspase 3 (a key enzyme in the apoptosis cell-signalling cascade) appearance, DNA fragmentation and increased intracellular calcium. Treatment of the cells with cannabidiol (10(-7)-10(-4)m) prior to beta-amyloid peptide exposure significantly elevated cell survival while it decreased ROS production, lipid peroxidation, caspase 3 levels, DNA fragmentation and intracellular calcium. Our results indicate that cannabidiol exerts a combination of neuroprotective, anti-oxidative and anti-apoptotic effects against beta-amyloid peptide toxicity, and that inhibition of caspase 3 appearance from its inactive precursor, pro-caspase 3, by cannabidiol is involved in the signalling pathway for this neuroprotection.

Transient receptor potential channel M5 and phospholipaseC-beta2 colocalizing in zebrafish taste receptor cells.

PubMed

Yoshida, Yuki; Saitoh, Kana; Aihara, Yoshiko; Okada, Shinji; Misaka, Takumi; Abe, Keiko

2007-10-08

In mammals, transient receptor potential (TRP) channel M5 (TRPM5) is coexpressed with phospholipaseC-beta2 (PLC-beta2) in the taste receptor cells, and both PLC-beta2 and TRPM5 are essential elements in the signal transduction of sweet, bitter and umami stimuli. In this study, we identified the zebrafish homologue of TRPM5 (zfTRPM5) and examined its expression in the gustatory system by in-situ hybridization. Using a transgenic zebrafish line that expressed green fluorescent protein under the control of the PLC-beta2 promoter, we showed that zfTRPM5 is expressed in green fluorescent protein-labeled cells of the taste buds. These results demonstrate that zfTRPM5 and PLC-beta2 colocalize in zebrafish taste receptor cells, suggesting their crucial roles in taste signaling via the fish taste receptors.

Impact of small-molecule glucokinase activator on glucose metabolism and beta-cell mass.

PubMed

Nakamura, Akinobu; Terauchi, Yasuo; Ohyama, Sumika; Kubota, Junko; Shimazaki, Hiroko; Nambu, Tadahiro; Takamoto, Iseki; Kubota, Naoto; Eiki, Junichi; Yoshioka, Narihito; Kadowaki, Takashi; Koike, Takao

2009-03-01

We investigated the effect of glucokinase activator (GKA) on glucose metabolism and beta-cell mass. We analyzed four mouse groups: wild-type mice and beta-cell-specific haploinsufficiency of glucokinase gene (Gck(+/-)) mice on a high-fat (HF) diet. Each genotype was also treated with GKA mixed in the HF diet. Rodent insulinoma cells and isolated islets were used to evaluate beta-cell proliferation by GKA. After 20 wk on the above diets, there were no differences in body weight, lipid profiles, and liver triglyceride content among the four groups. Glucose tolerance was improved shortly after the GKA treatment in both genotypes of mice. beta-Cell mass increased in wild-type mice compared with Gck(+/-) mice, but a further increase was not observed after the administration of GKA in both genotypes. Interestingly, GKA was able to up-regulate insulin receptor substrate-2 (Irs-2) expression in insulinoma cells and isolated islets. The administration of GKA increased 5-bromo-2-deoxyuridine (BrdU) incorporation in insulinoma cells, and 3 d administration of GKA markedly increased BrdU incorporation in mice treated with GKA in both genotypes, compared with those without GKA. In conclusion, GKA was able to chronically improve glucose metabolism for mice on the HF diet. Although chronic GKA administration failed to cause a further increase in beta-cell mass in vivo, GKA was able to increase beta cell proliferation in vitro and with a 3-d administration in vivo. This apparent discrepancy can be explained by a chronic reduction in ambient blood glucose levels by GKA treatment.

Amino acid sequence requirements at residues 69 and 238 for the SME-1 beta-lactamase to confer resistance to beta-lactam antibiotics.

PubMed

Majiduddin, Fahd K; Palzkill, Timothy

2003-03-01

Carbapenem antibiotics have been used to counteract resistant strains of bacteria harboring beta-lactamases and extended-spectrum beta-lactamases. Four enzymes from the class A group of beta-lactamases, NMC-A, IMI-1, SME-1, and KPC-1, efficiently hydrolyze carbapenem antibiotics. Sequence comparisons and structural information indicate that cysteines at amino acid residues 69 and 238, which are conserved in all four of these enzymes, form a disulfide bond that is unique to these beta-lactamases. To test whether this disulfide bond is required for catalytic activity, the codons for residues Cys69 and Cys238 were randomized individually and simultaneously by PCR-based mutagenesis to create random replacement libraries for these positions. Mutants that were able to confer resistance to ampicillin, imipenem, or cefotaxime were selected from these libraries. The results indicate that positions Cys69 and Cys238 are critical for hydrolysis of all of the antibiotics tested, suggesting that the disulfide bond is generally required for this enzyme to catalyze the hydrolysis of beta-lactam antibiotics.

Presumptive identification of streptococci with a new test system.

PubMed Central

Facklam, R R; Thacker, L G; Fox, B; Eriquez, L

1982-01-01

A test is described that could replace bacitracin susceptibility for presumptive identification of group A streptococci as well as 6.5% NaCl agar tolerance for presumptive identification of enterococcal streptococci. The L-pyrrolidonyl-beta-naphthylamide test, based on hydrolysis of pyrrolidonyl-beta-naphthylamide, was used in conjunction with the CAMP and bile-esculin tests to presumptively identify the streptococci. Among the beta-hemolytic streptococci; 98% of 50 group A, 98% of 46 group B, and 100% of 70 strains that were not group A, B, or D were correctly identified by the new presumptive test scheme. Among the non-beta-hemolytic streptococci; 96% of 74 group D enterococcal, 100% of 30 group D nonenterococcal, and 82% of 112 viridans strains were correctly identified by the new presumptive test scheme. PMID:7050157

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.

Enzyme replacement therapy for Anderson-Fabry disease.

PubMed

El Dib, Regina; Gomaa, Huda; Carvalho, Raíssa Pierri; Camargo, Samira E; Bazan, Rodrigo; Barretti, Pasqual; Barreto, Fellype C

2016-07-25

Anderson-Fabry disease is an X-linked defect of glycosphingolipid metabolism. Progressive renal insufficiency is a major source of morbidity, additional complications result from cardio- and cerebro-vascular involvement. Survival is reduced among affected males and symptomatic female carriers.This is an update of a Cochrane review first published in 2010, and previously updated in 2013. To evaluate the effectiveness and safety of enzyme replacement therapy compared to other interventions, placebo or no interventions, for treating Anderson-Fabry disease. We searched the Cystic Fibrosis and Genetic Disorders Group's Inborn Errors of Metabolism Trials Register (date of the most recent search: 08 July 2016). We also searched 'Clinical Trials' on The Cochrane Library, MEDLINE, Embase and LILACS (date of the most recent search: 24 September 2015). Randomized controlled trials of agalsidase alfa or beta in participants diagnosed with Anderson-Fabry disease. Two authors selected relevant trials, assessed methodological quality and extracted data. Nine trials comparing either agalsidase alfa or beta in 351 participants fulfilled the selection criteria.Both trials comparing agalsidase alfa to placebo reported on globotriaosylceramide concentration in plasma and tissue; aggregate results were non-significant. One trial reported pain scores measured by the Brief Pain Inventory severity, there was a statistically significant improvement for participants receiving treatment at up to three months, mean difference -2.10 (95% confidence interval -3.79 to -0.41; at up to five months, mean difference -1.90 (95% confidence interval -3.65 to -0.15); and at up to six months, mean difference -2.00 (95% confidence interval -3.66 to -0.34). There was a significant difference in the Brief Pain Inventory pain-related quality of life at over five months and up to six months, mean difference -2.10 (95% confidence interval -3.92 to -0.28) but not at other time points. Death was not an outcome in either of the trials.One of the three trials comparing agalsidase beta to placebo reported on globotriaosylceramide concentration in plasma and tissue and showed significant improvement: kidney, mean difference -1.70 (95% confidence interval -2.09 to -1.31); heart, mean difference -0.90 (95% confidence interval -1.18 to -0.62); and composite results (renal, cardiac, and cerebrovascular complications and death), mean difference -4.80 (95% confidence interval -5.45 to -4.15). There was no significant difference between groups for death; no trials reported on pain.Only two trials compared agalsidase alfa to agalsidase beta. One of them showed no significant difference between the groups regarding adverse events, risk ratio 0.36 (95% confidence interval 0.08 to 1.59), or any serious adverse events; risk ratio 0.30; (95% confidence interval 0.03 to 2.57).Two trials compared different dosing schedules of agalsidase alfa. One of them involved three different doses (0.2 mg/kg every two weeks; 0.1 mg/kg weekly and; 0.2 mg/kg weekly), the other trial evaluated two further doses to the dosage schedules: 0.4 mg/kg every week and every other week. Both trials failed to show significant differences with various dosing schedules on globotriaosylceramide levels. No significant differences were found among the schedules for the primary efficacy outcome of self-assessed health state, or for pain scores.One trial comparing agalsidase alfa to agalsidase beta showed no significant difference for any adverse events such as dyspnoea and hypertension.The methodological quality of the included trials was generally unclear for the random sequence generation and allocation concealment. Trials comparing enzyme replacement therapy to placebo show significant improvement with enzyme replacement therapy in regard to microvascular endothelial deposits of globotriaosylceramide and in pain-related quality of life. There is, however, no evidence identifying if the alfa or beta form is superior or the optimal dose or frequency of enzyme replacement therapy. With regards to safety, adverse events (i.e., rigors, fever) were more significant in the agalsidase beta as compared to placebo. The long-term influence of enzyme replacement therapy on risk of morbidity and mortality related to Anderson-Fabry disease remains to be established. This review highlights the need for continued research into the use of enzyme replacement therapy for Anderson-Fabry disease.

beta. -adrenergic relaxation of smooth muscle: differences between cells and tissues

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

Scheid, C.R.

1987-09-01

The present studies were carried out in an attempt to resolve the controversy about the Na/sup +/ dependence of ..beta..-adrenergic relaxation in smooth muscle. Previous studies on isolated smooth muscle cells from the toad stomach had suggested that at least some of the actions of ..beta..-adrenergic agents, including a stimulatory effect on /sup 45/Ca efflux, were dependent on the presence of a normal transmembrane Na/sup +/ gradient. Studies by other investigators using tissues derived from mammalian sources had suggested that the relaxing effect of ..beta..-adrenergic agents was Na/sup +/ independent. Uncertainty remained as to whether these discrepancies reflected differences betweenmore » cells and tissues or differences between species. Thus, in the present studies, the authors utilized both tissues and cells from the same source, the stomach muscle of the toad Bufo marinus, and assessed the Na/sup +/ dependence of ..beta..-adrenergic relaxation. They found that elimination of a normal Na/sup +/ gradient abolished ..beta..-adrenergic relaxation of isolated cells. In tissues, however, similar manipulations had no effect on relaxation. The reasons for this discrepancy are unclear but do not appear to be attributable to changes in smooth muscle function following enzymatic dispersion. Thus the controversy concerning the mechanisms of ..beta..-adrenergic relaxation may reflect inherent differences between tissues and cells.« less

Dietary toxins, endoplasmic reticulum (ER) stress and diabetes.

PubMed

Hettiarachchi, Kalindi D; Zimmet, Paul Z; Myers, Mark A

2008-05-01

The incidence of Type 1 diabetes has been increasing at a rate too rapid to be due to changes in genetic risk. Instead changes in environmental factors are the likely culprit. The endoplasmic reticulum (ER) plays an important role in the production of newly synthesized proteins and interference with these processes leads to ER stress. The insulin-producing beta cells are particularly prone to ER stress as a result of their heavy engagement in insulin production. Increasing evidence suggests ER stress is central to initiation and progression of Type 1 diabetes. An early environmental exposure, such as toxins and viral infections, can impart a significant physiological load on beta cells to initiate abnormal processing of proinsulin, ER stress and insulin secretory defects. Release of altered proinsulin from the beta cells early in life may trigger autoimmunity in those with genetic susceptibility leading to cytokine-induced nitric oxide production and so exacerbating ER stress in beta cells, ultimately leading to apoptosis of beta cells and diabetes. Here we suggest that ER stress is an inherent cause of beta cell dysfunction and environmental factors, in particular dietary toxins derived from Streptomyces in infected root vegetables, can impart additional stress that aggravates beta cell death and progression to diabetes. Furthermore, we propose that the increasing incidence of Type 1 diabetes may be accounted for by increased dietary exposure to ER-stress-inducing Streptomyces toxins.

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.

The physiology of rodent beta-cells in pancreas slices.

PubMed

Rupnik, M

2009-01-01

Beta-cells in pancreatic islets form complex syncytia. Sufficient cell-to-cell electrical coupling seems to ensure coordinated depolarization pattern and insulin release that can be further modulated by rich innervation. The complex structure and coordinated action develop after birth during fast proliferation of the endocrine tissue. These emergent properties can be lost due to various reasons later in life and can lead to glucose intolerance and diabetes mellitus. Pancreas slice is a novel method of choice to study the physiology of beta-cells still embedded in their normal cellulo-social context. I present major advantages, list drawbacks and provide an overview on recent advances in our understanding of the physiology of beta-cells using the pancreas slice approach.

Endoglin inhibits ERK-induced c-Myc and cyclin D1 expression to impede endothelial cell proliferation

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

Pan, Christopher C.; Bloodworth, Jeffrey C.; Mythreye, Karthikeyan

2012-08-03

Highlights: Black-Right-Pointing-Pointer Endoglin inhibits ERK activation in endothelial cells. Black-Right-Pointing-Pointer Endoglin is a regulator of c-Myc and cyclin D1 expression. Black-Right-Pointing-Pointer {beta}-arrestin2 interaction with endoglin is required for ERK/c-Myc repression. Black-Right-Pointing-Pointer Endoglin impedes cellular proliferation by targeting ERK-induced mitogenic signaling. -- Abstract: Endoglin is an endothelial-specific transforming growth factor beta (TGF-{beta}) co-receptor essential for angiogenesis and vascular remodeling. Endoglin regulates a wide range of cellular processes, including cell adhesion, migration, and proliferation, through TGF-{beta} signaling to canonical Smad and Smad-independent pathways. Despite its overall pro-angiogenic role in the vasculature, the underlying mechanism of endoglin action is poorly characterized. We previouslymore » identified {beta}-arrestin2 as a binding partner that causes endoglin internalization from the plasma membrane and inhibits ERK signaling towards endothelial migration. In the present study, we examined the mechanistic role of endoglin and {beta}-arrestin2 in endothelial cell proliferation. We show that endoglin impedes cell growth through sustained inhibition of ERK-induced c-Myc and cyclin D1 expression in a TGF-{beta}-independent manner. The down-regulation of c-Myc and cyclin D1, along with growth-inhibition, are reversed when the endoglin/{beta}-arrestin2 interaction is disrupted. Given that TGF-{beta}-induced Smad signaling potently represses c-Myc in most cell types, our findings here show a novel mechanism by which endoglin augments growth-inhibition by targeting ERK and key downstream mitogenic substrates.« less

Switch from agalsidase beta to agalsidase alfa in the enzyme replacement therapy of patients with Fabry disease in Latin America.

PubMed

Ripeau, Diego; Amartino, Hernán; Cedrolla, Martín; Urtiaga, Luis; Urdaneta, Bella; Cano, Marilis; Valdez, Rita; Antongiovanni, Norberto; Masllorens, Francisca

2017-01-01

There are currently two available enzyme replacement therapies for Fabry disease and little information regarding efficacy and safety of switching therapies. Between 2009 and 2012 there was a worldwide shortage of agalsidase beta and patients on that enzyme were switched to agalsidase alfa. This retrospective observational study assessed a 2-year period of efficacy and safety in a population of Fabry patients, in Argentina (30 patients) and Venezuela (3 patients), who switched therapies from algasidase beta to agalsidase alfa. Thirty-three patients completed 24-months follow-up after the switch (age 32.4 ± 2.0, range 10.0-55.9 years; male: female 23:10). Measures of renal function such as estimated glomerular filtration rate remained almost unchanged in 31 patients without end stage renal disease over the 2 years after switching and urine protein excretion continued stable. Cardiac functional parameters: left ventricular mass index, interventricular septum, left ventricular posterior wall showed no significant change from baseline in the 33 patients. Quality of life, pain and disease severity scores were mostly unchanged after 24-months and agalsidase alfa was generally well tolerated. Our findings showed there is no significant change in the efficacy measured through the renal or cardiac function, quality of life, pain, disease severity scoring and safety for at least 2 years after switching from agalsidase beta to agalsidase alfa.

Beta-catenin regulates vitamin C biosynthesis and cell survival in murine liver.

PubMed

Nejak-Bowen, Kari N; Zeng, Gang; Tan, Xinping; Cieply, Benjamin; Monga, Satdarshan P

2009-10-09

Because the Wnt/beta-catenin pathway plays multiple roles in liver pathobiology, it is critical to identify gene targets that mediate such diverse effects. Here we report a novel role of beta-catenin in controlling ascorbic acid biosynthesis in murine liver through regulation of expression of regucalcin or senescence marker protein 30 and L-gulonolactone oxidase. Reverse transcription-PCR, Western blotting, and immunohistochemistry demonstrate decreased regucalcin expression in beta-catenin-null livers and greater expression in beta-catenin overexpressing transgenic livers, HepG2 hepatoma cells (contain constitutively active beta-catenin), regenerating livers, and in hepatocellular cancer tissues that exhibit beta-catenin activation. Interestingly, coprecipitation and immunofluorescence studies also demonstrate an association of beta-catenin and regucalcin. Luciferase reporter and chromatin immunoprecipitation assays verified a functional TCF-4-binding site located between -163 and -157 (CTTTGCA) on the regucalcin promoter to be critical for regulation by beta-catenin. Significantly lower serum ascorbate levels were observed in beta-catenin knock-out mice secondary to decreased expression of regucalcin and also of L-gulonolactone oxidase, the penultimate and last (also rate-limiting) steps in the synthesis of ascorbic acid, respectively. These mice also show enhanced basal hepatocyte apoptosis. To test if ascorbate deficiency secondary to beta-catenin loss and regucalcin decrease was contributing to apoptosis, beta-catenin-null hepatocytes or regucalcin small interfering RNA-transfected HepG2 cells were cultured, which exhibited significant apoptosis that was alleviated by the addition of ascorbic acid. Thus, through regucalcin and L-gulonolactone oxidase expression, beta-catenin regulates vitamin C biosynthesis in murine liver, which in turn may be one of the mechanisms contributing to the role of beta-catenin in cell survival.

Regulation of interleukin-1beta and interleukin-8 production by agonists of mu and delta opiate receptors in vitro.

PubMed

Gein, S V; Gorshkova, K G; Tendryakova, S P

2009-07-01

The studies reported here showed that beta-endorphin at concentrations of 10(-7)-10(-11) M increased interleukin-1beta (IL-1beta) production in unfractionated leukocyte suspensions both in the presence of 0.1 microg/ml lipopolysaccharide (LPS) and in cultures not stimulated with LPS. Interleukin-8 (IL-8) production by leukocytes was inhibited by beta-endorphin at concentrations of 10(-7) and 10(-11) M in the presence of LPS. The stimulatory effect of beta-endorphin on IL-1beta production was not blocked by naloxone or naltrindole. Suppression of IL-8 production was blocked by naloxone and naltrindole. In the mononuclear cell and neutrophil fractions, beta-endorphin and the delta agonist DADLE increased IL-1beta synthesis in both the spontaneous and stimulated versions of the test, while beta-endorphin and the delta agonist DADLE inhibited IL-8 production in the mononuclear cell and neutrophil fractions only in LPS-stimulated cultures. The mu agonist DAGO had no effect on IL-1beta production by mononuclear cells or neutrophils, though it suppressed LPS-induced secretion of IL-8 by neutrophils.

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.

Liver X receptor activation inhibits PC-3 prostate cancer cells via the beta-catenin pathway.

PubMed

Youlin, Kuang; Li, Zhang; Weiyang, He; Jian, Kang; Siming, Liang; Xin, Gou

2017-03-01

Liver X receptors (LXRs) are nuclear receptors family of ligand-dependent transcription factors that play a crucial role in regulating cholesterol metabolism and inflammation. Recent studies show that LXR agonists exhibit anti-cancer activities in a variety of cancer cell lines including prostate. To further identify the potential mechanisms of LXRα activation on prostate cancer, we investigated the effect of LXR agonist T0901317 on PC3 prostate cancer cell and in which activity of beta-catenin pathway involved. Prostate cancer PC3 cells were transfected with LXR-a siRNA and treated with LXR activator T0901317. qRT-PCR and western blot were used to detect the LXR-a expression. beta-catenin, cyclin D1 and c-MYC were analyzed by western blot. Cell apoptosis was examined by flow cytometry and Cell proliferation was assessed by Cell Counting Kit-8 assay. Cell migration was detected by Transwell chambers. Data showed that T0901317 significantly inhibited PC3 cell proliferation as well as invasion and increased apoptosis in vitro. Furthermore, we found that LXRα activation induced the reduction of beta-catenin expression in PC3 cells, and this inhibitory effect could be totally abolished when cells were treated with LXRα. Meanwhile, the expression of beta-catenin target gene cyclin D1 and c-MYC were also decreased. This study provided additional evidence that LXR activation inhibited PC-3 prostate cancer cells via suppressing beta-catenin pathway. Copyright © 2016 Elsevier GmbH. All rights reserved.

Pancreatic stellate cells are activated by proinflammatory cytokines: implications for pancreatic fibrogenesis.

PubMed

Apte, M V; Haber, P S; Darby, S J; Rodgers, S C; McCaughan, G W; Korsten, M A; Pirola, R C; Wilson, J S

1999-04-01

The pathogenesis of pancreatic fibrosis is unknown. In the liver, stellate cells play a major role in fibrogenesis by synthesising increased amounts of collagen and other extracellular matrix (ECM) proteins when activated by profibrogenic mediators such as cytokines and oxidant stress. To determine whether cultured rat pancreatic stellate cells produce collagen and other ECM proteins, and exhibit signs of activation when exposed to the cytokines platelet derived growth factor (PDGF) or transforming growth factor beta (TGF-beta). Cultured pancreatic stellate cells were immunostained for the ECM proteins procollagen III, collagen I, laminin, and fibronectin using specific polyclonal antibodies. For cytokine studies, triplicate wells of cells were incubated with increasing concentrations of PDGF or TGF-beta. Cultured pancreatic stellate cells stained strongly positive for all ECM proteins tested. Incubation of cells with 1, 5, and 10 ng/ml PDGF led to a significant dose related increase in cell counts as well as in the incorporation of 3H-thymidine into DNA. Stellate cells exposed to 0.25, 0.5, and 1 ng/ml TGF-beta showed a dose dependent increase in alpha smooth muscle actin expression and increased collagen synthesis. In addition, TGF-beta increased the expression of PDGF receptors on stellate cells. Pancreatic stellate cells produce collagen and other extracellular matrix proteins, and respond to the cytokines PDGF and TGF-beta by increased proliferation and increased collagen synthesis. These results suggest an important role for stellate cells in pancreatic fibrogenesis.

Effects of 18beta-glycyrrhetinic acid on the junctional complex and steroidogenesis in rat adrenocortical cells.

PubMed

Huang, Shih-Horng; Wu, Jiahn-Chun; Hwang, Ra-Der; Yeo, Hui-Lin; Wang, Seu-Mei

2003-09-01

Cellular junctions play important roles in cell differentiation, signal transduction, and cell function. This study investigated their function in steroid secretion by adrenal cells. Immunofluorescence staining revealed the presence of gap junctions and adherens junctions between adrenal cells. The major gap junction protein, connexin43, was seen as a linear dotted pattern of the typical gap junction plaques, in contrast to alpha-, beta-, and gamma-catenin, which were seen as continuous, linear staining of cell-cell adherens junction. Treatment with 18beta-glycyrrhetinic acid, a gap junction inhibitor, reduced the immunoreactivity of these proteins in a time- and dose-dependent manner, and caused the gap junction and adherens junction to separate longitudinally from the cell-cell contact sites, indicating the structural interdependency of these two junctions. Interestingly, 18beta-glycyrrhetinic acid stimulated a two- to three-fold increase in steroid production in these adrenal cells lacking intact cell junctions. These data raise the question of the necessity for cell communication for the endocrine function of adrenal cells. Pharmacological analyses indicated that the steroidogenic effect of 18beta-glycyrrhetinic acid was partially mediated by extracellular signal-related kinase and calcium/calmodulin-dependent kinase, a pathway distinct from the protein kinase A signaling pathway already known to mediate steroidogenesis in adrenal cells. Copyright 2003 Wiley-Liss, Inc.

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

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

Activated type I TGFbeta receptor (Alk5) kinase confers enhancedsurvival to mammary epithelial cells and accelerates mammary tumorprogression

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

Muraoka-Cook, Rebecca S.; Shin, Incheol; Yi, Jae Youn

2005-01-02

The transforming growth factor-betas (TGF{beta}s) are members of a large superfamily of pleiotropic cytokines that also includes the activins and the bone morphogenetic proteins (BMPs). Members of the TGF{beta} family regulate complex physiological processes such cell proliferation, differentiation, adhesion, cell-cell and cell-matrix interactions, motility, and cell death, among others (Massague, 1998). Dysregulation of TGF{beta} signaling contributes to several pathological processes including cancer, fibrosis, and auto-immune disorders (Massague et al., 2000). The TGF{beta}s elicit their biological effects by binding to type II and type I transmembrane receptor serine-threonine kinases (T{beta}RII and T{beta}RI) which, in turn, phosphorylated Smad 2 and Smad 3.more » Phosphorylated Smad 2/3 associate with Smad 4 and, as a heteromeric complex, translocate to the nucleus where they regulate gene transcription. The inhibitory Smad7 down regulates TGF{beta} signaling by binding to activated T{beta}RI and interfering with its ability to phosphorylate Smad 2/3 (Derynck and Zhang, 2003; Shi and Massague, 2003). Signaling is also regulated by Smad proteolysis. TGF{beta} receptor-mediated activation results in multi-ubiquitination of Smad 2 in the nucleus and subsequent degradation of Smad 2 by the proteasome (Lo and Massague, 1999). Activation of TGF{beta} receptors also induces mobilization of a Smad 7-Smurf complex from the nucleus to the cytoplasm; this complex recognizes the activated receptors and mediates their ubiquitination and internalization via caveolin-rich vesicles, leading to termination of TGF{beta} signaling (Di Guglielmo et al., 2003). Other signal transducers/pathways have been implicated in TGF{beta} actions. These include the extracellular signal-regulated kinase (Erk), c-Jun N-terminal kinase (Jnk), p38 mitogen-activated protein kinase (MAPK), protein phosphatase PP2A, phosphatidylinositol-3 kinase (PI3K), and the family of Rho GTPases [reviewed in (Derynck and Zhang, 2003)]. Although signaling by Smads has been shown to be causally associated with the anti-proliferative effect of TGF{beta} (Datto et al., 1999; Liu et al., 1997), the role of non-Smad effectors on mediating the cellular effects of TGF{beta} is less well characterized.« less

Species-specific vesicular monoamine transporter 2 (VMAT2) expression in mammalian pancreatic beta cells: implications for optimising radioligand-based human beta cell mass (BCM) imaging in animal models

PubMed Central

Hartwig, N. R.; Kalmbach, N.; Klietz, M.; Anlauf, M.; Eiden, L. E.; Weihe, E.

2014-01-01

Aims/hypothesis Imaging of beta cell mass (BCM) is a major challenge in diabetes research. The vesicular monoamine transporter 2 (VMAT2) is abundantly expressed in human beta cells. Radiolabelled analogues of tetrabenazine (TBZ; a low-molecular-weight, cell-permeant VMAT2-selective ligand) have been employed for pancreatic islet imaging in humans. Since reports on TBZ-based VMAT2 imaging in rodent pancreas have been fraught with confusion, we compared VMAT2 gene expression patterns in the mouse, rat, pig and human pancreas, to identify appropriate animal models with which to further validate and optimise TBZ imaging in humans. Methods We used a panel of highly sensitive VMAT2 antibodies developed against equivalently antigenic regions of the transporter from each species in combination with immunostaining for insulin and species-specific in situ hybridisation probes. Individual pancreatic islets were obtained by laser-capture microdissection and subjected to analysis of mRNA expression of VMAT2. Results The VMAT2 protein was not expressed in beta cells in the adult pancreas of common mouse or rat laboratory strains, in contrast to its expression in beta cells (but not other pancreatic endocrine cell types) in the pancreas of pigs and humans. VMAT2- and tyrosine hydroxylase co-positive (catecholaminergic) innervation was less abundant in humans than in rodents. VMAT2-positive mast cells were identified in the pancreas of all species. Conclusions/interpretation Primates and pigs are suitable models for TBZ imaging of beta cells. Rodents, because of a complete lack of VMAT2 expression in the endocrine pancreas, are a ‘null’ model for assessing interference with BCM measurements by VMAT2-positive mast cells and sympathetic innervation in the pancreas. PMID:23404442

Integrin alphaIIb-subunit cytoplasmic domain mutations demonstrate a requirement for tyrosine phosphorylation of beta3-subunits in actin cytoskeletal organization.

PubMed

Yamodo, Innocent H; Blystone, Scott D

2004-01-01

Using truncated or mutated alphaIIb integrin cytoplasmic domains fused to the alphaV extracellular domain and expressed with the beta3 integrin subunit, we demonstrate that the double mutation of proline residues 998 and 999 to alanine (PP998/999AA), previously shown to disturb the C-terminal conformation of the alphaIIb integrin cytoplasmic domain, prevents tyrosine phosphorylation of beta3 integrin induced by Arg-Gly-Asp peptide ligation. This mutation also inhibits integrin mediated actin assembly and cell adhesion to vitronectin. In contrast, progressive truncation of the alphaIIb-subunit cytoplasmic domain did not reproduce these effects. Interestingly, the PP998/999AA mutations of alphaIIb did not affect beta3 tyrosine phosphorylation, cell adhesion, or actin polymerization induced by manganese. Exogenous addition of manganese was sufficient to rescue beta3 phosphorylation, cell adhesion, and actin assembly in cells expressing the PP998/999AA mutation when presented with a vitronectin substrate. Further, induction of the high affinity conformation of this mutant beta3 integrin by incubation with either Arg-Gly-Asp peptide or exogenous manganese was equivalent. These results suggest that the extracellular structure of beta3 integrins in the high affinity conformation is not directly related to the structure of the cytoplasmic face of the integrin. Moreover, the requirement for beta3 phosphorylation is demonstrated without mutation of the beta3 subunit. In support of our previous hypothesis of a role for beta3 phosphorylation in adhesion, these studies demonstrate a strong correlation between beta3 tyrosine phosphorylation and assembly of a cytoskeleton competent to support firm cell adhesion.

Distinct single-cell morphological dynamics under beta-lactam antibiotics

PubMed Central

Yao, Zhizhong; Kahne, Daniel; Kishony, Roy

2012-01-01

Summary The bacterial cell wall is conserved in prokaryotes, stabilizing cells against osmotic stress. Beta-lactams inhibit cell wall synthesis and induce lysis through a bulge-mediated mechanism; however, little is known about the formation dynamics and stability of these bulges. To capture processes of different timescales, we developed an imaging platform combining automated image analysis with live cell microscopy at high time resolution. Beta-lactam killing of Escherichia coli cells proceeded through four stages: elongation, bulge formation, bulge stagnation and lysis. Both the cell wall and outer membrane (OM) affect the observed dynamics; damaging the cell wall with different beta-lactams and compromising OM integrity cause different modes and rates of lysis. Our results show that the bulge formation dynamics is determined by how the cell wall is perturbed. The OM plays an independent role in stabilizing the bulge once it is formed. The stabilized bulge delays lysis, and allows recovery upon drug removal. PMID:23103254

Beta-Actin Is Required for Proper Mouse Neural Crest Ontogeny

PubMed Central

Tondeleir, Davina; Noelanders, Rivka; Bakkali, Karima; Ampe, Christophe

2014-01-01

The mouse genome consists of six functional actin genes of which the expression patterns are temporally and spatially regulated during development and in the adult organism. Deletion of beta-actin in mouse is lethal during embryonic development, although there is compensatory expression of other actin isoforms. This suggests different isoform specific functions and, more in particular, an important function for beta-actin during early mammalian development. We here report a role for beta-actin during neural crest ontogeny. Although beta-actin null neural crest cells show expression of neural crest markers, less cells delaminate and their migration arrests shortly after. These phenotypes were associated with elevated apoptosis levels in neural crest cells, whereas proliferation levels were unchanged. Specifically the pre-migratory neural crest cells displayed higher levels of apoptosis, suggesting increased apoptosis in the neural tube accounts for the decreased amount of migrating neural crest cells seen in the beta-actin null embryos. These cells additionally displayed a lack of membrane bound N-cadherin and dramatic decrease in cadherin-11 expression which was more pronounced in the pre-migratory neural crest population, potentially indicating linkage between the cadherin-11 expression and apoptosis. By inhibiting ROCK ex vivo, the knockout neural crest cells regained migratory capacity and cadherin-11 expression was upregulated. We conclude that the presence of beta-actin is vital for survival, specifically of pre-migratory neural crest cells, their proper emigration from the neural tube and their subsequent migration. Furthermore, the absence of beta-actin affects cadherin-11 and N-cadherin function, which could partly be alleviated by ROCK inhibition, situating the Rho-ROCK signaling in a feedback loop with cadherin-11. PMID:24409333

The Role of Oxidative Stress and Hypoxia in Pancreatic Beta-Cell Dysfunction in Diabetes Mellitus

PubMed Central

Gerber, Philipp A.

2017-01-01

Abstract Significance: Metabolic syndrome is a frequent precursor of type 2 diabetes mellitus (T2D), a disease that currently affects ∼8% of the adult population worldwide. Pancreatic beta-cell dysfunction and loss are central to the disease process, although understanding of the underlying molecular mechanisms is still fragmentary. Recent Advances: Oversupply of nutrients, including glucose and fatty acids, and the subsequent overstimulation of beta cells, are believed to be an important contributor to insulin secretory failure in T2D. Hypoxia has also recently been implicated in beta-cell damage. Accumulating evidence points to a role for oxidative stress in both processes. Although the production of reactive oxygen species (ROS) results from enhanced mitochondrial respiration during stimulation with glucose and other fuels, the expression of antioxidant defense genes is unusually low (or disallowed) in beta cells. Critical Issues: Not all subjects with metabolic syndrome and hyperglycemia go on to develop full-blown diabetes, implying an important role in disease risk for gene–environment interactions. Possession of common risk alleles at the SLC30A8 locus, encoding the beta-cell granule zinc transporter ZnT8, may affect cytosolic Zn2+ concentrations and thus susceptibility to hypoxia and oxidative stress. Future Directions: Loss of normal beta-cell function, rather than total mass, is increasingly considered to be the major driver for impaired insulin secretion in diabetes. Better understanding of the role of oxidative changes, its modulation by genes involved in disease risk, and effects on beta-cell identity may facilitate the development of new therapeutic strategies to this disease. Antioxid. Redox Signal. 26, 501–518. PMID:27225690

cGMP may have trophic effects on beta cell function comparable to those of cAMP, implying a role for high-dose biotin in prevention/treatment of diabetes.

PubMed

McCarty, Mark F

2006-01-01

Incretin hormones have trophic effects on beta cell function that can aid prevention and treatment of diabetes. cAMP is the primary mediator of these effects, and has been shown to potentiate glucose-stimulated insulin secretion, promote proper beta cells differentiation by increasing expression of the crucial transcription factor PDX-1, and prevent beta cell apoptosis. cGMP's role in beta cell function has received far less scrutiny, but there is emerging evidence that it may have a trophic impact on beta cell function analogous to that of cAMP. An increase in plasma glucose boosts beta cell production of cGMP, which acts as a feed-forward mediator to enhance glucose-stimulated insulin secretion. cGMP also has an anti-apoptotic effect in beta cells, and there is now indirect evidence that it promotes expression of PDX-1. Supraphysiological concentrations of biotin can directly activate guanylate cyclase, and there is limited evidence that high intakes of this vitamin can be therapeutically beneficial in diabetics and in rodent models of diabetes. Beneficial effects of cGMP on muscle insulin sensitivity and on control of hepatic glucose output may contribute to biotin's utility in diabetes. The fact that nitric oxide/cGMP exert a range of favorable effects on vascular health should further encourage exploration of biotin's preventive and therapeutic potential. If an appropriate high-dose biotin regimen could achieve a modest systemic increase in guanylate cyclase activity, without entailing unacceptable side effects or risks, such a regimen might have considerable potential for promoting vascular health and preventing or managing diabetes.

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.

β-MSCs: successful fusion of MSCs with β-cells results in a β-cell like phenotype.

PubMed

Azizi, Zahra; Lange, Claudia; Paroni, Federico; Ardestani, Amin; Meyer, Anke; Wu, Yonghua; Zander, Axel R; Westenfelder, Christof; Maedler, Kathrin

2016-08-02

Bone marrow mesenchymal stromal cells (MSC) have anti-inflammatory, anti-apoptotic and immunosuppressive properties and are a potent source for cell therapy. Cell fusion has been proposed for rapid generation of functional new reprogrammed cells. In this study, we aimed to establish a fusion protocol of bone marrow-derived human MSCs with the rat beta-cell line (INS-1E) as well as human isolated pancreatic islets in order to generate insulin producing beta-MSCs as a cell-based treatment for diabetes.Human eGFP+ puromycin+ MSCs were co-cultured with either stably mCherry-expressing rat INS-1E cells or human dispersed islet cells and treated with phytohemagglutinin (PHA-P) and polyethylene glycol (PEG) to induce fusion. MSCs and fused cells were selected by puromycin treatment.With an improved fusion protocol, 29.8 ± 2.9% of all MSCs were β-MSC heterokaryons based on double positivity for mCherry and eGFP.After fusion and puromycin selection, human NKX6.1 and insulin as well as rat Neurod1, Nkx2.2, MafA, Pdx1 and Ins1 mRNA were highly elevated in fused human MSC/INS-1E cells, compared to the mixed control population. Such induction of beta-cell markers was confirmed in fused human MSC/human dispersed islet cells, which showed elevated NEUROD1, NKX2.2, MAFA, PDX1 and insulin mRNA compared to the mixed control. Fused cells had higher insulin content and improved insulin secretion compared to the mixed control and insulin positive beta-MSCs also expressed nuclear PDX1. We established a protocol for fusion of human MSCs and beta cells, which resulted in a beta cell like phenotype. This could be a novel tool for cell-based therapies of diabetes.

Soybean beta-conglycinin peptone suppresses food intake and gastric emptying by increasing plasma cholecystokinin levels in rats.

PubMed

Nishi, Takashi; Hara, Hiroshi; Tomita, Fusao

2003-02-01

Cholecystokinin (CCK) is an important physiologic mediator that regulates satiety and gastric emptying. We demonstrated previously that soybean peptone acts directly on rat small intestinal mucosal cells to stimulate CCK release. In the present study, we examined the effects of beta-conglycinin, a major component of soy protein, and its peptone on food intake and gastric emptying after an intraduodenal infusion of beta-conglycinin peptone in relation to CCK release and interaction with the mucosal cell membrane. Intraduodenal infusion of beta-conglycinin peptone inhibited food intake in a dose-dependent manner, but that of whole soy peptone or camostat did not. The suppression of food intake by beta-conglycinin peptone was abolished by an intravenous injection of devazepide, a selective peripheral CCK receptor antagonist. The beta-conglycinin peptone infusion strongly suppressed gastric emptying with marked increases in portal CCK levels. We also observed that the beta-conglycinin peptone dose dependently and more potently stimulated CCK release from isolated dispersed mucosal cells of the rat jejunum than did beta-conglycinin itself. This stimulation corresponded to the binding activity of the peptide or protein to solubilized components of the rat jejunum membrane as evaluated by surface plasmon biosensor. These results indicate that beta-conglycinin peptone suppresses food intake, and this effect may be due to beta-conglycinin peptone in the lumen stimulating endogenous CCK release with direct acceptance to the intestinal 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.

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

TGF-beta1 modulates focal adhesion kinase expression in rat intestinal epithelial IEC-6 cells via stimulatory and inhibitory Smad binding elements.

PubMed

Walsh, Mary F; Ampasala, Dinakar R; Rishi, Arun K; Basson, Marc D

2009-02-01

TGF-beta and FAK modulate cell migration, differentiation, proliferation and apoptosis, and TGF-beta promotes FAK transcription in intestinal epithelial cells via Smad-dependent and independent pathways. We utilized a 1320 bp FAK promoter-luciferase construct to characterize basal and TGF-beta-mediated FAK gene transcription in IEC-6 cells. Inhibiting JNK or Akt negated TGF-beta-stimulated promoter activity; ERK inhibition did not block the TGF-beta effect but increased basal activity. Co-transfection with Co-Smad4 enhanced the TGF-beta response while the inhibitory Smad7 abolished it. Serial deletions sequentially removing the four Smad binding elements (SBE) in the 5' untranslated region of the promoter revealed that the two most distal SBE's are positive regulators while SBE3 exerts a negative influence. Mutational deletion of two upstream p53 sites enhanced basal but did not affect TGF-beta-stimulated increases in promoter activity. TGF-beta increased DNA binding of Smad4, phospho-Smad2/3 and Runx1/AML1a to the most distal 435 bp containing 3 SBE and 2 AML1a sites by ChIP assay. However, although point mutation of SBE1 ablated the TGF-beta-mediated rise in SV40-promoter activity, mutation of AML1a sites did not. TGF-beta regulation of FAK transcription reflects a complex interplay between positive and negative non-Smad signals and SBE's, the last independent of p53 or AML1a.

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

Segregation of two spectrin isoforms: polarized membrane-binding sites direct polarized membrane skeleton assembly.

PubMed

Dubreuil, R R; Maddux, P B; Grushko, T A; MacVicar, G R

1997-10-01

Spectrin isoforms are often segregated within specialized plasma membrane subdomains where they are thought to contribute to the development of cell surface polarity. It was previously shown that ankyrin and beta spectrin are recruited to sites of cell-cell contact in Drosophila S2 cells expressing the homophilic adhesion molecule neuroglian. Here, we show that neuroglian has no apparent effect on a second spectrin isoform (alpha beta H), which is constitutively associated with the plasma membrane in S2 cells. Another membrane marker, the Na,K-ATPase, codistributes with ankyrin and alpha beta spectrin at sites of neuroglian-mediated contact. The distributions of these markers in epithelial cells in vivo are consistent with the order of events observed in S2 cells. Neuroglian, ankyrin, alpha beta spectrin, and the Na,K-ATPase colocalize at the lateral domain of salivary gland cells. In contrast, alpha beta H spectrin is sorted to the apical domain of salivary gland and somatic follicle cells. Thus, the two spectrin isoforms respond independently to positional cues at the cell surface: in one case an apically sorted receptor and in the other case a locally activated cell-cell adhesion molecule. The results support a model in which the membrane skeleton behaves as a transducer of positional information within cells.

Rev-erb beta regulates the Srebp-1c promoter and mRNA expression in skeletal muscle cells

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

Ramakrishnan, Sathiya N.; Lau, Patrick; Crowther, Lisa M.

2009-10-30

The nuclear hormone receptor, Rev-erb beta operates as a transcriptional silencer. We previously demonstrated that exogenous expression of Rev-erb{beta}{Delta}E in skeletal muscle cells increased Srebp-1c mRNA expression. We validated these in vitro observations by injection of an expression vector driving Rev-erb{beta}{Delta}E expression into mouse tibialis muscle that resulted in increased Srebp-1c mRNA expression. Paradoxically, Rev-erb{beta} siRNA expression in skeletal muscle cells repressed Srebp-1c expression, and indicated that Rev-erb{beta} expression was necessary for Srebp-1c expression. ChIP analysis demonstrated that Rev-erb{beta} was recruited to the Srebp-1c promoter. Moreover, Rev-erb{beta} trans-activated the Srebp-1c promoter, in contrast, Rev-erb{beta} efficiently repressed the Rev-erb{alpha} promoter, amore » previously characterized target gene. Finally, treatment with the Rev-erb agonist (hemin) (i) increased the trans-activation of the Srebp-1c promoter by Rev-erb{beta}; and (ii) increased Rev-erb{beta} and Srebp-1c mRNA expression. These data suggest that Rev-erb{beta} has the potential to activate gene expression, and is a positive regulator of Srebp-1c, a regulator of lipogenesis.« less

Tyrosine residues 654 and 670 in {beta}-cat enin are crucial in regulation of Met-{beta}-catenin interactions

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

Zeng, Gang; Apte, Udayan; Micsenyi, Amanda

2006-11-01

{beta}-catenin, a key component of the canonical Wnt pathway, is also regulated by tyrosine phosphorylation that regulates its association to E-cadherin. Previously, we reported its association with the hepatocyte growth factor (HGF) receptor Met at the membrane. HGF induced Met-{beta}-catenin dissociation and nuclear translocation of {beta}-catenin, which was tyrosine-phosphorylation-dependent. Here, we further investigate the Met-{beta}-catenin interaction by selectively mutating several tyrosine residues, alone or in combination, in {beta}-catenin. The mutants were subcloned into FLAG-CMV vector and stably transfected into rat hepatoma cells, which were treated with HGF. All single or double-mutant-transfected cells continued to show HGF-induced nuclear translocation of FLAG-{beta}-cateninmore » except the mutations affecting 654 and 670 simultaneously (Y654/670F), which coincided with the lack of formation of {beta}-catenin-TCF complex and DNA synthesis, in response to the HGF treatment. In addition, the Y654/670F-transfected cells also showed no phosphorylation of {beta}-catenin or dissociation from Met in response to HGF. Thus, intact 654 and 670 tyrosine residues in {beta}-catenin are crucial in HGF-mediated {beta}-catenin translocation, activation and mitogenesis.« less

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

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.

Agglutination of like-charged red blood cells induced by binding of beta2-glycoprotein I to outer cell surface.

PubMed

Lokar, Marusa; Urbanija, Jasna; Frank, Mojca; Hägerstrand, Henry; Rozman, Blaz; Bobrowska-Hägerstrand, Malgorzata; Iglic, Ales; Kralj-Iglic, Veronika

2008-08-01

Plasma protein-mediated attractive interaction between membranes of red blood cells (RBCs) and phospholipid vesicles was studied. It is shown that beta(2)-glycoprotein I (beta(2)-GPI) may induce RBC discocyte-echinocyte-spherocyte shape transformation and subsequent agglutination of RBCs. Based on the observed beta(2)-GPI-induced RBC cell shape transformation it is proposed that the hydrophobic portion of beta(2)-GPI molecule protrudes into the outer lipid layer of the RBC membrane and increases the area of this layer. It is also suggested that the observed agglutination of RBCs is at least partially driven by an attractive force which is of electrostatic origin and depends on the specific molecular shape and internal charge distribution of membrane-bound beta(2)-GPI molecules. The suggested beta(2)-GPI-induced attractive electrostatic interaction between like-charged RBC membrane surfaces is qualitatively explained by using a simple mathematical model within the functional density theory of the electric double layer, where the electrostatic attraction between the positively charged part of the first domains of bound beta(2)-GPI molecules and negatively charged glycocalyx of the adjacent RBC membrane is taken into account.

Conversion of immortal liver progenitor cells into pancreatic endocrine progenitor cells by persistent expression of Pdx-1.

PubMed

Jin, Cai-Xia; Li, Wen-Lin; Xu, Fang; Geng, Zhen H; He, Zhi-Ying; Su, Juan; Tao, Xin-Rong; Ding, Xiao-Yan; Wang, Xin; Hu, Yi-Ping

2008-05-01

The conversion of expandable liver progenitor cells into pancreatic beta cells would provide a renewable cell source for diabetes cell therapy. Previously, we reported the establishment of liver epithelial progenitor cells (LEPCs). In this work, LEPCs were modified into EGFP/Pdx-1 LEPCs, cells with stable expression of both Pdx-1 and EGFP. Unlike previous work, with persistent expression of Pdx-1, EGFP/Pdx-1 LEPCs acquired the phenotype of pancreatic endocrine progenitor cells rather than giving rise to insulin-producing cells directly. EGFP/Pdx-1 LEPCs proliferated vigorously and expressed the crucial transcription factors involved in beta cell development, including Ngn3, NeuroD, Nkx2.2, Nkx6.1, Pax4, Pax6, Isl1, MafA and endogenous Pdx-1, but did not secrete insulin. When cultured in high glucose/low serum medium supplemented with cytokines, EGFP/Pdx-1 LEPCs stopped proliferating and gave rise to functional beta cells without any evidence of exocrine or other islet cell lineage differentiation. When transplanted into diabetic SCID mice, EGFP/Pdx-1 LEPCs ameliorated hyperglycemia by secreting insulin in a glucose regulated manner. Considering the limited availability of beta cells, we propose that our experiments will provide a framework for utilizing the immortal liver progenitor cells as a renewable cell source for the generation of functional pancreatic beta cells.

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.

Enzyme replacement therapy in patients with Fabry disease: state of the art and review of the literature.

PubMed

Pisani, Antonio; Visciano, Bianca; Roux, Graciana Diez; Sabbatini, Massimo; Porto, Caterina; Parenti, Giancarlo; Imbriaco, Massimo

2012-11-01

Anderson-Fabry disease is an X-linked lysosomal storage disorder resulting from the deficiency of the hydrolytic enzyme alpha galactosidase A, with consequent accumulation of globotrioasoyl ceramide in cells and tissues of the body, resulting in a multi-system pathology including end organ failure. In the classical phenotype, cardiac failure, renal failure and stroke result in a reduced median life expectancy. The current causal treatment for Fabry disease is the enzyme replacement therapy (ERT): two different products, Replagal (agalsidase alfa) and Fabrazyme (agalsidase beta), have been commercially available in Europe for almost 10 years and they are both indicated for long-term treatment. In fact, clinical trials, observational studies and registry data have provided many evidences for safety and efficacy of ERT in improving symptoms of pain, gastrointestinal disturbances, hypohidrosis, left ventricular mass index, glomerular filtration rate and quality of life. Few data are available on comparison of the two treatments and on the clinical course of the disease. This article reviews the published evidence for clinical efficacy of the two available enzyme preparations. Copyright © 2012 Elsevier Inc. All rights reserved.

Progressive renal failure despite long-term biweekly enzyme replacement therapy in a patient with Fabry disease secondary to a new α-galactosidase mutation of Leu311Arg (L311R).

PubMed

Suzuki, Keisuke; Miura, Naoto; Kitagawa, Wataru; Suzuki, Shinkichi; Komatsuda, Atsushi; Nishikawa, Kazuhiro; Watanabe, Daisuke; Imai, Hirokazu

2011-12-01

A 37-year-old Japanese man affected by Fabry disease secondary to a novel mutation of Leu311Arg (L311R) in α-galactosidase demonstrated progressive renal failure despite biweekly enzyme replacement therapy (ERT) for approximately 10 years. Kidney biopsy revealed foamy glomerular epithelial cells, compatible with the typical pathologic features of Fabry disease. The patient entered a phase III study of Replagal (agalsidase alfa) in 2001, allowing him to continue ERT with biweekly dosing for almost 10 years. During 2 years of that period, he was continued on Fabrazyme (agalsidase beta) biweekly dosing. His estimated GFR was calculated to decrease by 9.9 mL/min/1.73 m(2) per year. Patients with Fabry disease have been reported to have a mean decrease in GFR of 12.2 ± 8.1 mL/min/1.73 m(2) per year. This result suggests that biweekly ERT is only mildly effective at preventing loss of kidney function.

Generation of stable cell line by using chitosan as gene delivery system.

PubMed

Şalva, Emine; Turan, Suna Özbaş; Ekentok, Ceyda; Akbuğa, Jülide

2016-08-01

Establishing stable cell lines are useful tools to study the function of various genes and silence or induce the expression of a gene of interest. Nonviral gene transfer is generally preferred to generate stable cell lines in the manufacturing of recombinant proteins. In this study, we aimed to establish stable recombinant HEK-293 cell lines by transfection of chitosan complexes preparing with pDNA which contain LacZ and GFP genes. Chitosan which is a cationic polymer was used as gene delivery system. Stable HEK-293 cell lines were established by transfection of cells with complexes which were prepared with chitosan and pVitro-2 plasmid vector that contains neomycin drug resistance gene, beta gal and GFP genes. The transfection efficiency was shown with GFP expression in the cells using fluorescence microscopy. Beta gal protein expression in stable cells was examined by beta-galactosidase assay as enzymatically and X-gal staining method as histochemically. Full complexation was shown in the above of 1/1 ratio in the chitosan/pDNA complexes. The highest beta-galactosidase activity was obtained with transfection of chitosan complexes. Beta gal gene expression was 15.17 ng/ml in the stable cells generated by chitosan complexes. In addition, intensive blue color was observed depending on beta gal protein expression in the stable cell line with X-gal staining. We established a stable HEK-293 cell line that can be used for recombinant protein production or gene expression studies by transfecting the gene of interest.

Fenoterol, a beta(2)-adrenoceptor agonist, inhibits LPS-induced membrane-bound CD14, TLR4/CD14 complex, and inflammatory cytokines production through beta-arrestin-2 in THP-1 cell line.

PubMed

Wang, Wei; Xu, Ming; Zhang, You-yi; He, Bei

2009-11-01

To investigate the molecular mechanism and signaling pathway by which fenoterol, a beta(2)-adrenergic receptor (beta(2)-AR) agonist, produces anti-inflammatory effects. THP-1, a monocytic cell line, was used to explore the mechanism of beta(2)-AR stimulation in LPS-induced secretion of inflammatory cytokines and changes of toll-like receptors (TLRs). We labeled TLR4 and CD14 using monoclonal anti-TLR4 PE-conjugated and anti-CD14 FITC-conjugated antibodies in THP-1 cells stimulated by beta(2)-AR in the presence or absence of lipopolysaccharide (LPS) and small, interfering RNA (siRNA)-mediated knockdown of beta-arrestin-2, and then analyzed their changes in distribution by flow cytometry, Western blotting and confocal analysis. LPS-induced membrane-bound CD14, TLR4/CD14 complex levels and elevation of inflammatory cytokines were all significantly reduced by pre-incubation of fenoterol (P<0.05). However, the total level of CD14 and TLR4 was not significantly changed. Interestingly, confocal microscopy revealed redistribution of CD14 and TLR4/CD14 complex under beta(2)-AR stimulation. Furthermore, siRNA-mediated knockdown of beta-arrestin-2 eliminated the anti-inflammatory effects and redistribution of CD14 and TLR4/CD14 complex stimulated by beta(2)-AR. beta(2)-AR agonist exerts its anti-inflammatory effects by down-regulating TLR signaling in THP-1 cells, potentially resulting from beta-arrestin-2 mediated redistribution of CD14 and TLR14/CD14 complex.

A novel chimeric peptide with antimicrobial activity.

PubMed

Alaybeyoglu, Begum; Akbulut, Berna Sariyar; Ozkirimli, Elif

2015-04-01

Beta-lactamase-mediated bacterial drug resistance exacerbates the prognosis of infectious diseases, which are sometimes treated with co-administration of beta-lactam type antibiotics and beta-lactamase inhibitors. Antimicrobial peptides are promising broad-spectrum alternatives to conventional antibiotics in this era of evolving bacterial resistance. Peptides based on the Ala46-Tyr51 beta-hairpin loop of beta-lactamase inhibitory protein (BLIP) have been previously shown to inhibit beta-lactamase. Here, our goal was to modify this peptide for improved beta-lactamase inhibition and cellular uptake. Motivated by the cell-penetrating pVEC sequence, which includes a hydrophobic stretch at its N-terminus, our approach involved the addition of LLIIL residues to the inhibitory peptide N-terminus to facilitate uptake. Activity measurements of the peptide based on the 45-53 loop of BLIP for enhanced inhibition verified that the peptide was a competitive beta-lactamase inhibitor with a K(i) value of 58 μM. Incubation of beta-lactam-resistant cells with peptide decreased the number of viable cells, while it had no effect on beta-lactamase-free cells, indicating that this peptide had antimicrobial activity via beta-lactamase inhibition. To elucidate the molecular mechanism by which this peptide moves across the membrane, steered molecular dynamics simulations were carried out. We propose that addition of hydrophobic residues to the N-terminus of the peptide affords a promising strategy in the design of novel antimicrobial peptides not only against beta-lactamase but also for other intracellular targets. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

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.

Oligodendrocytes in brain and optic nerve express the beta3 subunit isoform of Na,K-ATPase.

PubMed

Martín-Vasallo, P; Wetzel, R K; García-Segura, L M; Molina-Holgado, E; Arystarkhova, E; Sweadner, K J

2000-09-01

The Na,K-ATPase, which catalyzes the active transport of Na(+) and K(+), has two principal subunits (alpha and beta) that have several genetically distinct isoforms. Most of these isoforms are expressed in the nervous system, but certain ones are preferentially expressed in glia and others in neurons. Of the beta isoforms, beta1 predominates in neurons and beta2 in astrocytes, although there are some exceptions. Here we demonstrate that beta3 is expressed in rat and mouse white matter oligodendrocytes. Immunofluorescence microscopy identified beta3 in oligodendrocytes of rat brain white matter in typical linear arrays of cell bodies between fascicles of axons. The intensity of stain peaked at 20 postnatal days. beta3 was identified in cortical oligodendrocytes grown in culture, where it was expressed in processes and colocalized with antibody to galactocerebroside. In the mouse and rat optic nerve, beta3 stain was seen in oligodendrocytes, where it colocalized with carbonic anhydrase II. For comparison, optic nerve was stained for the beta1 and beta2 subunits, showing distinct patterns of labelling of axons (beta1) and astrocytes (beta2). The C6 glioma cell line was also found to express the beta3 isoform preferentially. Since beta3 was not found at detectable levels in astrocytes, this suggests that C6 is closer to oligodendrocytes than astrocytes in the glial cell lineage. Copyright 2000 Wiley-Liss, Inc.

The transplantation of neural stem cells and predictive factors in hematopoietic recovery in irradiated mice.

PubMed

Filip, S; Mokrý, J; Karbanová, J; Vávrová, J; Vokurková, J; Bláha, M; English, D

2005-04-01

A number of surprising observations have shown that stem cells, in suitable conditions, have the ability to produce a whole spectrum of cell types, regardless, whether these tissues are derived from the same germ layer or not. This phenomenon is called stem cell plasticity, which means that tissue-specific stem cells are mutually interchangeable. In our experiments, as a model, we used neural stem cells (NSCs) harvested from fetal (E14-15) neocortex and beta-galactosidase positive. In the first experiment we found that on days 12 and 30 after sub-lethal irradiation (LD 8.5 Gy) and (beta-galactosidase(+)) NSCs transplantation all mice survived, just as the group with bone marrow transplantation. Moreover, the bone marrow of mice transplanted NSCs contained the number of CFU-GM colonies with beta-galactosidase(+) cells which was as much as 50% higher. These differences were statistically significant, p<0.001. In the second experiment, we studied kinetics of (beta-galactosidase(+)) NSCs after their transplantation to sub-lethally irradiated mice. Histochemistry of tissues was performed on days 12 and 30 post-transplantation, and beta-galactosidase(+) cells were detected with the help of histochemical examination of removed tissues (lung, liver, spleen, thymus, and skeletal muscle). In tissues removed on day 12 post-transplantation, we found a significantly higher number of beta-galactosidase(+) cells in the spleen and thymus on day 30. While we presumed the presence beta-galactosidase(+) cells in the spleen, as spleen and reticuloendothelial system represent an important retaining system for different cell types, the presence of beta-galactosidase(+) cells in the thymus was rather surprising but very interesting. This indicates a certain mutual and close interconnection of transplanted stem cells and immune system in an adult organism. In the third experiment, we verified the mutual interchange of Sca-1 surface antigen in the bone marrow cells and NSCs before transplantation. Analysis of this antigen showed 24.8% Sca-1 positive cells among the bone marrow cells, while NSCs were Sca-1 negative. Our experiments show that NSCs share hemopoietic identity and may significantly influence the recovery of damaged hematopoiesis but do not have typical superficial markers as HSCs. This result is important for the determination of predictive factors for hemopoiesis recovery, for stem cell plasticity and for their use in the cell therapy.

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

PubMed

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

1992-05-15

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.

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.

Mediation of glucolipotoxicity in INS-1 rat insulinoma cells by small heterodimer partner interacting leucine zipper protein (SMILE).

PubMed

Lee, Kyeong-Min; Seo, Ye Jin; Kim, Mi-Kyung; Seo, Hyun-Ae; Jeong, Ji-Yun; Choi, Hueng-Sik; Lee, In-Kyu; Park, Keun-Gyu

2012-03-23

Sustained elevations of glucose and free fatty acid concentration have deleterious effects on pancreatic beta cell function. One of the hallmarks of such glucolipotoxicity is a reduction in insulin gene expression, resulting from decreased insulin promoter activity. Sterol regulatory element binding protein-1c (SREBP-1c), a lipogenic transcription factor, is related to the development of beta cell dysfunction caused by elevated concentrations of glucose and free fatty acid. Small heterodimer partner (SHP) interacting leucine zipper protein (SMILE), also known as Zhangfei, is a novel protein which interacts with SHP that mediates glucotoxicity in INS-1 rat insulinoma cells. Treatment of INS-1 cells with high concentrations of glucose and palmitate increased SREBP-1c and SMILE expression, and decreased insulin gene expression. Adenovirus-mediated overexpression of SREBP-1c in INS-1 cells induced SMILE expression. Moreover, adenovirus-mediated overexpression of SMILE (Ad-SMILE) in INS-1 cells impaired glucose-stimulated insulin secretion as well as insulin gene expression. Ad-SMILE overexpression also inhibited the expression of beta-cell enriched transcription factors including pancreatic duodenal homeobox factor-1, beta cell E box transactivator 2 and RIPE3b1/MafA, in INS-1 cells. Finally, in COS-1 cells, expression of SMILE inhibited the insulin promoter activity induced by these same beta-cell enriched transcription factors. These results collectively suggest that SMILE plays an important role in the development of beta cell dysfunction induced by glucolipotoxicity. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

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.

Beta-cell function, incretin effect, and incretin hormones in obese youth along the span of glucose tolerance from normal to prediabetes to Type 2 diabetes

USDA-ARS?s Scientific Manuscript database

Using the hyperglycemic and euglycemic clamp, we demonstrated impaired Beta-cell function in obese youth with increasing dysglycemia. Herein we describe oral glucose tolerance test (OGTT)-modeled Beta-cell function and incretin effect in obese adolescents spanning the range of glucose tolerance. Bet...

In vitro differentiation of rat bone marrow mesenchymal stem cells into hepatocytes.

PubMed

Feng, Zhihui; Li, Changying; Jiao, Shuxian; Hu, Bin; Zhao, Lin

2011-01-01

To investigate the mechanism and regulation of differentiation from bone marrow mesenchymal stem cells (BMSCs) into hepatocytes and to find a new source for therapies of hepatic diseases. We isolated BMSCs for subsequent differentiation in the presence of hepatocyte growth factor (HGF) or beta-nerve growth factor (beta-NGF). Cell morphology was observed and cell surface phenotypings were detected by flow cytometry. a1-antitrypsin (AAT) expression of the hepatocytes was confirmed by immunocytochemistry and albumin expression was validated by real time PCR and western blotting. The expression of high-affinity nerve growth factor receptor (TrkA) and the activation of Erk pathway were detected by western blotting. Hepatocyte functional activity was confirmed by uptake of indocyanine green (ICG) assay. Small round cells appeared in the presence of HGF on day 10 or beta-NGF on day 12. Differentiated cells expressed albumin and had functional characteristics of hepatocytes, such as uptake of ICG. BMSCs were positive for TrkA. HGF and beta-NGF significantly upregulated the protein levels of phospho-Erk. BMSCs could differentiate into hepatocytes in the differentiation media including HGF or beta-NGF. Combination of HGF and beta-NGF significantly increased the efficiency of hepatic differentiation.

[Expression of EP-CAM, beta-catenin in the carcinogenesis of squamous cell carcinoma of uterine cervix].

PubMed

Yang, Jian-zhu; Zhang, Xiang-hong; Wu, Wen-xin; Yan, Xia; Liu, Yan-li; Wang, Jun-ling; Wang, Feng-rong

2003-07-01

To study the expression of EP-CAM, beta-catenin in the carcinogenesis of squamous cell carcinoma of uterine cervix. The expressions of EP-CAM and beta-catenin were detected with immunohistochemical stain in 14 cases of normal cervical squamous epithelium, 32 cases of cervical intraepithelial neoplasia (CIN) and 38 cases of cervical invasive squamous cell carcinoma. The over-expression rates of EP-CAM were 0, 7.1%, 20.0%, 62.5% and 55.3% for normal cervical epithelium, CINI, CINII, CINIII and carcinoma groups. The EP-CAM over-expression rates in CINIII and cervical carcinoma groups were significantly higher than those in normal epithelium and CINI groups (P < 0.001). No aberrant expression of beta-catenin was shown in normal cervical epithelium, while the aberrant expression rates of beta-catenin in CINI, CINII, CINIII and cervical carcinoma group were 28.6%, 40.0%, 62.5% and 84.2%. The aberrant expression rate of beta-catenin increased with the increase in degree of CIN and development of cervical carcinoma. The over-expression rate of EP-CAM was reversely related to the differentiation of cervical squamous cell carcinoma (P < 0.001). EP-CAM and beta-catenin may be involved in the carcinogenesis of squamous cell carcinoma of uterine cervix. The over-expression of EP-CAM and aberrant expression of beta-catenin may serve as markers of squamous carcinogenesis of uterine cervix.

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.

[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.

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.

Reduced expression of TGF beta is associated with advanced disease in transitional cell carcinoma.

PubMed Central

Coombs, L. M.; Pigott, D. A.; Eydmann, M. E.; Proctor, A. J.; Knowles, M. A.

1993-01-01

The gene structure and expression of the related peptide regulatory factors TGF beta 1 and TGF beta 2 were studied in a panel of seven urothelial carcinoma cell lines and 40 transitional cell carcinomas. The latter comprised 15 grade 1, 18 grade 2 and 5 grade 3 tumours and two cases of carcinoma in situ. Control tissues included ten matched 'field' biopsies and 17 other biopsies including 11 biopsies of macroscopically normal urothelium, two of which were from patients with no history of bladder cancer. No amplification of rearrangements of either TGF beta 1 or TGF beta 2 were detected in any sample. A complex pattern of expression or the two genes was found in the urothelial cell lines. High, but variable levels of the 2.5 kb TGF beta 1 transcript were detected and lower and more variable levels of the three (4.1 kb, 5.1 kb and 6.5 kb) transcripts of TGF beta 2 were detected. Although those cell lines expressing most TGF beta 1 tended to express less TGF beta 2 transcript there was no clear-cut relationship. In comparison, no TGF beta 2 transcript was identified in any primary transitional cell carcinoma or control tissue. Markedly reduced or undetectable levels of TGF beta 1 transcript were detected in 4/15 (26%) grade 1, 5/18 (28%) grade 2 and 3/5 (60%) grade 3 tumours. There was no clear relationship to tumour stage, lymphocytic infiltration or stromal content of the tumours. Clinical review one year after the 2 year period of tumour collection showed that 6/9 (66%) of patients with tumours with reduced levels of transcript had died or had disease which was not controllable by local resection and 3/9 (33%) had developed tumour re-occurrences. In comparison, in the group with normal levels of expression of TGF beta 1, 3/18 (17%) had disease which was not controllable by local means, 9/18 (50%) had tumour re-occurrence and 6/18 (33%) had no evidence of disease. The association of reduced expression of TGF beta 1 and advanced disease was statistically significant P < 0.02 (Fisher's test). Although the sample size is small, we suggest that the loss of expression of TGF beta 1 may be a potential marker of progressive disease or prognosis in transitional cell carcinoma and warrants further study. Images Figure 1 Figure 2 Figure 3 PMID:8439507

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

PubMed

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

2010-03-01

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

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

PubMed

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

2003-05-01

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

Immune Interventions to Preserve Beta Cell Function in Type 1 Diabetes

PubMed Central

Ehlers, Mario R.

2015-01-01

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

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

PubMed

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

2014-09-01

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

Aggregation and lack of secretion of most newly synthesized proinsulin in non-beta-cell lines.

PubMed

Zhu, Yong Lian; Abdo, Alexander; Gesmonde, Joan F; Zawalich, Kathleen C; Zawalich, Walter; Dannies, Priscilla S

2004-08-01

Myoblasts transfected with HB10D insulin secrete more hormone than those transfected with wild-type insulin, as published previously, indicating that production of wild-type insulin is not efficient in these cells. The ability of non-beta-cells to produce insulin was examined in several cell lines. In clones of neuroendocrine GH(4)C(1) cells stably transfected with proinsulin, two thirds of (35)S-proinsulin was degraded within 3 h of synthesis, whereas (35)S-prolactin was stable. In transiently transfected neuroendocrine AtT20 cells, half of (35)S-proinsulin was degraded within 3 h after synthesis, whereas (35)S-GH was stable. In transiently transfected fibroblast COS cells, (35)S-proinsulin was stable for longer, but less than 10% was secreted 8 h after synthesis. Proinsulin formed a concentrated patch detected by immunofluorescence in transfected cells that did not colocalize with calreticulin or BiP, markers for the endoplasmic reticulum, but did colocalize with membrin, a marker for the cis-medial Golgi complex. Proinsulin formed a Lubrol-insoluble aggregate within 30 min after synthesis in non-beta-cells but not in INS-1E cells, a beta-cell line that normally produces insulin. More than 45% of (35)S-HB10D proinsulin was secreted from COS cells 3 h after synthesis, and this mutant formed less Lubrol-insoluble aggregate in the cells than did wild-type hormone. These results indicate that proinsulin production from these non-beta-cells is not efficient and that proinsulin aggregates in their secretory pathways. Factors in the environment of the secretory pathway of beta-cells may prevent aggregation of proinsulin to allow efficient production.

Dominant expression of interleukin-10 and transforming growth factor-beta genes in activated T-cells of chronic active Epstein-Barr virus infection.

PubMed

Ohga, Shouichi; Nomura, Akihiko; Takada, Hidetoshi; Tanaka, Tamami; Furuno, Kenji; Takahata, Yasushi; Kinukawa, Naoko; Fukushima, Noriyasu; Imai, Shosuke; Hara, Toshiro

2004-11-01

Chronic active Epstein-Barr virus (EBV) infection is a chronic mononucleosis syndrome associated with clonal proliferation of EBV-carrying T-/natural killer (NK)-cells. High levels of circulating EBV and activated T-cells are sustained during the prolonged disease course, whereas it is not clear how ectopic EBV infection in T-/NK-cells has been established and maintained. To assess the biological role of activated T-cells in chronic active EBV infection (CAEBV), EBV DNA and cellular gene expressions in peripheral T-cells were quantified in CAEBV and infectious mononucleosis (IM) patients. In CAEBV, HLA-DR(+) T-cells had higher viral load and larger amounts of IFN gamma, IL-10, transforming growth factor-beta (TGF beta), and cytotoxic T lymphocyte antigen-4 (CTLA4) mRNA than HLA-DR(-)T-cells. HLA-DR(+) T cells of IM patients transcribed more IFN gamma and IL-10 than their HLA-DR(-)T cells. Expression levels of IFN gamma and forkhead box p3 (Foxp3) in CAEBV HLA-DR(+) T-cells were higher than in IM HLA-DR(+) T-cells. The effective variables to discriminate the positivity of HLA-DR were IL-10, IFN gamma, CTLA4, TGF beta, and IL-2 in the order of statistical weight. EBV load in CAEBV T-cells correlated with the expression levels of only IL-10 and TGF beta. These results suggest that CAEBV T-cells are activated to transcribe IFN gamma, IL-10, and TGF beta excessively, and the latter two genes are expressed preferentially in the EBV-infected subsets. The dominant expression of regulatory cytokines in T-cells may imply a viral evasion mechanism in the disease.

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

PubMed Central

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

1994-01-01

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

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.

Enhanced IL-1{beta}-induced IL-8 production in cystic fibrosis lung epithelial cells is dependent of both mitogen-activated protein kinases and NF-{kappa}B signaling

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

Muselet-Charlier, Celine; Universite Pierre et Marie Curie-Paris 6, Paris, UMR-S719, F-75012; Roque, Telma

2007-06-01

Transcription nuclear factor-{kappa}B (NF-{kappa}B) is hyperactivated in cystic fibrosis (CF) lung epithelial cells, and participates in exaggerated IL-8 production in the CF lung. We recently found that rapid activation of NF-{kappa}B occurred in a CF lung epithelial IB3-1 cell line (CF cells) upon IL-1{beta} stimulation, which was not observed in its CFTR-corrected lung epithelial S9 cell line (corrected cells). To test whether other signaling pathways such as that of mitogen-activated protein kinases (MAPKs) could be involved in IL-1{beta}-induced IL-8 production of CF cells, we investigated ERK1/2, JNK, and p38MAP signaling compared to NF-{kappa}B. Within 30 min, exposure to IL-1{beta} causedmore » high activation of NF-{kappa}B, ERK1/2, p38MAP but not JNK in CF cells compared to corrected cells. Treatment of IL-1{beta}-stimulated CF cells with a series of chemical inhibitors of NF-{kappa}B, ERK1/2, and p38MAP, when used separately, reduced slightly IL-8 production. However, when used together, these inhibitors caused a blockade in IL-1{beta}-induced IL-8 production in CF cells. Understanding of the cross-talk between NF-{kappa}B and MAPKs signaling in CF lung epithelial cells may help in developing new therapeutics to reduce lung inflammation in patients with CF.« less

Methods of Fabricating Scintillators with Radioisotopes for Beta Battery Applications

NASA Technical Reports Server (NTRS)

Rensing, Noa M.; Squillante, Michael R.; Tieman, Timothy C.; Higgins, William; Shiriwadkar, Urmila

2013-01-01

Technology has been developed for a class of self-contained, long-duration power sources called beta batteries, which harvest the energy contained in the radioactive emissions from beta decay isotopes. The new battery is a significant improvement over the conventional phosphor/solar cell concept for converting this energy in three ways. First, the thin phosphor is replaced with a thick scintillator that is transparent to its own emissions. By using a scintillator sufficiently thick to completely stop all the beta particles, efficiency is greatly improved. Second, since the energy of the beta particles is absorbed in the scintillator, the semiconductor photodetector is shielded from radiation damage that presently limits the performance and lifetime of traditional phosphor converters. Finally, instead of a thin film of beta-emitting material, the isotopes are incorporated into the entire volume of the thick scintillator crystal allowing more activity to be included in the converter without self-absorption. There is no chemical difference between radioactive and stable strontium beta emitters such as Sr-90, so the beta emitter can be uniformly distributed throughout a strontium based scintillator crystal. When beta emitter material is applied as a foil or thin film to the surface of a solar cell or even to the surface of a scintillator, much of the radiation escapes due to the geometry, and some is absorbed within the layer itself, leading to inefficient harvesting of the energy. In contrast, if the emitting atoms are incorporated within the scintillator, the geometry allows for the capture and efficient conversion of the energy of particles emitted in any direction. Any gamma rays associated with secondary decays or Bremsstrahlung photons may also be absorbed within the scintillator, and converted to lower energy photons, which will in turn be captured by the photocell or photodiode. Some energy will be lost in this two-stage conversion process (high-energy particle to low-energy photons to electric current). The geometric advantage partially offsets this as well, since the absorption depth of high-energy beta radiation is much larger than the depth of a p-n junction. Thus, in a p-n junction device, much of the radiation is absorbed far away from the junction, and the electron- hole pairs are not all effectively collected. In contrast, with a transparent scintillator the radiation can be converted to light in a larger volume, and all of the light can be collected in the active region of the photodiode. Finally, the new device is more practical because it can be used at much higher power levels without unduly shortening its lifetime. While the crystal structure of scintillators is also subject to radiation damage, their performance is far more tolerant of defects than that of semiconductor junctions. This allows the scintillator- based approach to use both higher energy isotopes and larger quantities of the isotopes. It is projected that this technology has the potential to produce a radioisotope battery with up to twice the efficiency of presently used systems.

Advancement Of Tritium Powered Betavoltaic Battery Systems

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

Staack, G.; Gaillard, J.; Hitchcock, D.

2015-10-14

Due to their decades-long service life and reliable power output under extreme conditions, betavoltaic batteries offer distinct advantages over traditional chemical batteries, especially in applications where frequent battery replacement is hazardous, or cost prohibitive. Although many beta emitting isotopes exist, tritium is considered ideal in betavoltaic applications for several reasons: 1) it is a “pure” beta emitter, 2) the beta is not energetic enough to damage the semiconductor, 3) it has a moderately long half-life, and 4) it is readily available. Unfortunately, the widespread application of tritium powered betavoltaics is limited, in part, by their low power output. This researchmore » targets improving the power output of betavoltaics by increasing the flux of beta particles to the energy conversion device (the p-n junction) through the use of low Z nanostructured tritium trapping materials.« less

The synergistic effect of beta-boswellic acid and Nurr1 overexpression on dopaminergic programming of antioxidant glutathione peroxidase-1-expressing murine embryonic stem cells.

PubMed

Abasi, M; Massumi, M; Riazi, G; Amini, H

2012-10-11

Parkinson's disease (PD) is a neurodegenerative disorder in which the nigro-striatal dopaminergic (DAergic) neurons have been selectively lost. Due to side effects of levodopa, a dopamine precursor drug, recently cell replacement therapy for PD has been considered. Lack of sufficient amounts of, embryos and ethical problems regarding the use of dopamine-rich embryonic neural cells have limited the application of these cells for PD cell therapy. Therefore, many investigators have focused on using the pluripotent stem cells to generate DAergic neurons. This study is aimed first to establish a mouse embryonic stem (mES) cell line that can stably co-express Nurr1 (Nuclear receptor subfamily 4, group A, member 2) transcription factor in order to efficiently generate DAergic neurons, and glutathione peroxidase-1 (GPX-1) to protect the differentiated DAergic-like cells against oxidative stress. In addition to genetic engineering of ES cells, the effect of Beta-boswellic acid (BBA) on DAergic differentiation course of mES cells was sought in the present study. To that end, the feeder-independent CGR8 mouse embryonic stem cells were transduced by Nurr1- and GPX-1-harboring Lentiviruses and the generated Nurr1/GPX-1-expresssing ES clones were characterized and verified. Gene expression analyses demonstrated that BBA treatment and overexpression of Nurr1 has a synergistic effect on derivation of DAergic neurons from Nurr1/GPX-1-expressing ES cells. The differentiated cells could exclusively synthesize and secrete dopamine in response to stimuli. Overexpression of GPX-1 in genetically engineered Nurr1/GPX-1-ES cells increased the viability of these cells during their differentiation into CNS stem cells. In conclusion, the results demonstrated that Nurr1-overexpressing feeder-independent ES cells like the feeder-dependent ES cells, can be efficiently programmed into functional DAergic neurons and additional treatment of cells by BBA can even augment this efficiency. GPX-1 overexpression in Nurr1/GPX-1-ES cells increases the viability of differentiated CNS stem-like cells. The result of this study may have impact on future stem cell therapy of PD. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

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.

Rat leucine-rich protein binds and activates the promoter of the beta isoform of Ca2+/calmodulin-dependent protein kinase II gene.

PubMed

Ochiai, Nagahiro; Masumoto, Shuji; Sakagami, Hiroyuki; Yoshimura, Yoshiyuki; Yamauchi, Takashi

2007-05-01

We previously found the neuronal cell-type specific promoter and binding partner of the beta isoform of Ca(2+)/calmodulin-dependent protein kinase II (beta CaM kinase II) in rat brain [Donai, H., Morinaga, H., Yamauchi, T., 2001. Genomic organization and neuronal cell type specific promoter activity of beta isoform of Ca(2+)/calmodulin-dependent protein kinase II of rat brain. Mol. Brain Res. 94, 35-47]. In the present study, we purified a protein that binds specifically a promoter region of beta CaM kinase II gene from a nuclear extract of the rat cerebellum using DEAE-cellulose column chromatography, ammonium sulfate fractionation, gel filtration and polyacrylamide gel electrophoresis. The purified protein was identified as rat leucine-rich protein 157 (rLRP157) using tandem mass spectrometry. Then, we prepared its cDNA by reverse transcriptase-polymerase chain reaction (RT-PCR) from poly(A)(+)RNA of rat cerebellum. The rLRP157 cDNA was introduced into mouse neuroblastomaxrat glioma hybrid NG108-15 cells, and cells stably expressing rLRP157 (NG/LRP cells) were isolated. Binding of rLRP157 with the promoter sequence was confirmed by electrophoretic mobility shift assay using nuclear extract of NG/LRP cells. A luciferase reporter gene containing a promoter of beta CaM kinase II was transiently expressed in NG/LRP cells. Under the conditions, the promoter activity was enhanced about 2.6-fold in NG/LRP cells as compared with wild-type cells. The expression of rLRP157 mRNA was paralleled with that of beta CaM kinase II in the adult and embryo rat brain detected by in situ hybridization. Nuclear localization of rLRP157 was confirmed using GFP-rLRP157 fusion protein investigated under a confocal microscope. These results indicate that rLRP157 is one of the proteins binding to, and regulating the activity of, the promoter of beta CaM kinase II.

Localization of beta-D-glucosidase activity and glucovanillin in vanilla bean (Vanilla planifolia Andrews).

PubMed

Odoux, E; Escoute, J; Verdeil, J-L; Brillouet, J-M

2003-09-01

The morphology, anatomy and histology of mature green vanilla beans were examined by light and transmission electron microscopy. Beans have a triangular cross-section with a central cavity containing seeds. Each angle is lined with tubular cells, or papillae, while the cavity sides consist of placental laminae. The epicarp and endocarp are formed by one or two layers of very small cells, while the mesocarp contains large, highly vacuolarized cells, the cytoplasm being restricted to a thin layer along the cell walls. The radial distributions of glucovanillin and beta-glucosidase activity, measured on p-nitrophenyl-beta-glucopyranoside and glucovanillin, are superimposable and show how beta-glucosidase activity increases from the epicarp towards the placental zone, whereas glucovanillin is exclusively located in the placentae and papillae. Subcellular localization of beta-glucosidase activity was achieved by incubating sections of vanilla beans in a buffer containing 5-bromo-4-chloro-3-indolyl-beta-d-glucopyranoside as a substrate. Activity was observed in the cytoplasm (and/or the periplasm) of mesocarp and endocarp cells, with a more diffuse pattern observed in the papillae. A possible mechanism for the hydrolysis of glucovanillin and release of the aromatic aglycon vanillin involves the decompartmentation of cytoplasmic (and/or periplasmic) beta-glucosidase and vacuolar glucovanillin.

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

PubMed

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

2008-05-23

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

Expression of the alpha and beta subunits of Ca2+/calmodulin kinase II in the cerebellum of jaundiced Gunn rats during development: a quantitative light microscopic analysis.

PubMed

Conlee, J W; Shapiro, S M; Churn, S B

2000-04-01

The homozygous (jj) jaundiced Gunn rat model for hyperbilirubinemia displays pronounced cerebellar hypoplasia. To examine the cellular mechanisms involved in bilirubin toxicity, this study focused on the effect of hyperbilirubinemia on calcium/calmodulin-dependent kinase II (CaM kinase II). CaM kinase II is a neuronally enriched enzyme which performs several important functions. Immunohistochemical analysis of alternating serial sections were performed using monoclonal antibodies for the alpha and beta subunits of CaM kinase II. Measurements were made of the total numbers of stained cells in each of the deep cerebellar nuclei and of Purkinje and granule cell densities in cerebellar lobules II, VI, and IX. The beta subunit was present in Purkinje cells and deep cerebellar nuclei of both groups at all ages, but only granule cells which had migrated through the Purkinje cell layer showed staining for beta subunit; external granule cells were completely negative. Many Purkinje cells had degenerated in the older animals, and the percent of granule cells stained for beta subunit was significantly reduced. The alpha subunit was found exclusively in Purkinje cells, although its appearance was delayed in the jaundiced animals. Sulfadimethoxine was administered to some jj rats 24 h or 15 days prior to sacrifice to increase brain bilirubin concentration. Results showed that bilirubin exposure modulated both alpha and beta CaM kinase II subunit expression in selective neuronal populations, but sulfadimethoxine had no acute effect on enzyme immunoreactivity. Thus, developmental expression of the alpha and beta subunits of CaM kinase II was affected by chronic bilirubin exposure during early postnatal development of jaundiced Gunn rats.

Practice advisory: The utility of EEG theta/beta power ratio in ADHD diagnosis

PubMed Central

Gloss, David; Varma, Jay K.; Pringsheim, Tamara; Nuwer, Marc R.

2016-01-01

Objective: To evaluate the evidence for EEG theta/beta power ratio for diagnosing, or helping to diagnose, attention-deficit/hyperactivity disorder (ADHD). Methods: We identified relevant studies and classified them using American Academy of Neurology criteria. Results: Two Class I studies assessing the ability of EEG theta/beta power ratio and EEG frontal beta power to identify patients with ADHD correctly identified 166 of 185 participants. Both studies evaluated theta/beta power ratio and frontal beta power in suspected ADHD or in syndromes typically included in an ADHD differential diagnosis. A bivariate model combining the diagnostic studies shows that the combination of EEG frontal beta power and theta/beta power ratio has relatively high sensitivity and specificity but is insufficiently accurate. Conclusions: It is unknown whether a combination of standard clinical examination and EEG theta/beta power ratio increases diagnostic certainty of ADHD compared with clinical examination alone. Recommendations: Level B: Clinicians should inform patients with suspected ADHD and their families that the combination of EEG theta/beta power ratio and frontal beta power should not replace a standard clinical evaluation. There is a risk for significant harm to patients from ADHD misdiagnosis because of the unacceptably high false-positive diagnostic rate of EEG theta/beta power ratio and frontal beta power. Level R: Clinicians should inform patients with suspected ADHD and their families that the EEG theta/beta power ratio should not be used to confirm an ADHD diagnosis or to support further testing after a clinical evaluation, unless such diagnostic assessments occur in a research setting. PMID:27760867

Linking TGF-beta-mediated Cdc25A inhibition and cytoskeletal regulation through RhoA/p160(ROCK) signaling.

PubMed

Brown, Kimberly; Bhowmick, Neil A

2004-04-01

Transforming growth factor-beta (TGF-beta) can mediate G(1)/S cell-cycle inhibition and changes in the cytoskeletal organization through multiple parallel downstream signaling pathways. Recent findings regarding TGF-beta-mediated cell-cycle checkpoint control and epithelial to mesenchymal transition have converged to the RhoA/p160(ROCK) signaling pathway. The activation of TGF-beta-mediated p160(ROCK)rapidly inhibits the Cdc25A phosphatase as a component of the G(1)/S checkpoint control at the time cytoskeletal re-organization occurs. This can be likened to the ability to preserve genomic integrity in circumstances of genotoxic stress. The inactivation of the RhoA/p160(ROCK) pathway may be a mechanism by which cancer cells bypass growth inhibition even in the presence of TGF-beta.

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

Castillo, Gaelle del; Murillo, Miguel M.; IDIBELL-Institut de Recerca Oncologica, Gran Via s/n, Km 2.7, 08907 L'Hospitalet, Barcelona

Transforming growth factor-beta (TGF-{beta}) induces apoptosis in fetal rat hepatocytes. However, a subpopulation of these cells survives, concomitant with changes in phenotype, reminiscent of an epithelial-mesenchymal transition (EMT). We have previously suggested that EMT might confer cell resistance to apoptosis (Valdes et al., Mol. Cancer Res., 1: 68-78, 2002). However, the molecular mechanisms responsible for this resistance are not explored yet. In this work, we have isolated and subcultured the population of hepatocytes that suffered the EMT process and are resistant to apoptosis (TGF-{beta}-treated fetal hepatocytes: T{beta}T-FH). We prove that they secrete mitogenic and survival factors, as analyzed by themore » proliferative and survival capacity of conditioned medium. Inhibition of the epidermal growth factor receptor (EGFR) sensitizes T{beta}T-FH to die after serum withdrawal. T{beta}T-FH expresses high levels of transforming growth factor-alpha (TGF-{alpha}) and heparin-binding EGF-like growth factor (HB-EGF) and shows constitutive activation of the EGFR pathway. A blocking anti-TGF-{alpha} antibody restores the capacity of cells to die. TGF-{beta}, which is expressed by T{beta}T-FH, mediates up-regulation of TGF-{alpha} and HB-EGF expression in those cells. In summary, results suggest that an autocrine loop of TGF-{beta} confers resistance to apoptosis after an EMT process in hepatocytes, through the increase in the expression of EGFR ligands.« less

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

Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.

PubMed Central

Larsson, S; Hotchkiss, G; Andäng, M; Nyholm, T; Inzunza, J; Jansson, I; Ahrlund-Richter, L

1994-01-01

We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels. Images PMID:8036151

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

PubMed

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

2006-11-01

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

Antenatal glucocorticoid treatment alters Na+ uptake in renal proximal tubule cells from adult offspring in a sex-specific manner.

PubMed

Su, Yixin; Bi, Jianli; Pulgar, Victor M; Figueroa, Jorge; Chappell, Mark; Rose, James C

2015-06-01

We have shown a sex-specific effect of fetal programming on Na(+) excretion in adult sheep. The site of this effect in the kidney is unknown. Therefore, we tested the hypothesis that renal proximal tubule cells (RPTCs) from adult male sheep exposed to betamethasone (Beta) before birth have greater Na(+) uptake than do RPTCs from vehicle-exposed male sheep and that RPTCs from female sheep similarly exposed are not influenced by antenatal Beta. In isolated RPTCs from 1- to 1.5-yr-old male and female sheep, we measured Na(+) uptake under basal conditions and after stimulation with ANG II. To gain insight into the mechanisms involved, we also measured nitric oxide (NO) levels, ANG II receptor mRNA levels, and expression of Na(+)/H(+) exchanger 3. Basal Na(+) uptake increased more in cells from Beta-exposed male sheep than in cells from vehicle-exposed male sheep (400% vs. 300%, P < 0.00001). ANG II-stimulated Na(+) uptake was also greater in cells from Beta-exposed males. Beta exposure did not increase Na(+) uptake by RPTCs from female sheep. NO production was suppressed more by ANG II in RPTCs from Beta-exposed males than in RPTCs from either vehicle-exposed male or female sheep. Our data suggest that one site of the sex-specific effect of Beta-induced fetal programming in the kidney is the RPTC and that the enhanced Na(+) uptake induced by antenatal Beta in male RPTCs may be related to the suppression of NO in these cells. Copyright © 2015 the American Physiological Society.

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.

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.

IL-1 beta and IL-6 in mouse parotid acinar cells: characterization of synthesis, storage, and release.

PubMed

Tanda, N; Ohyama, H; Yamakawa, M; Ericsson, M; Tsuji, T; McBride, J; Elovic, A; Wong, D T; Login, G R

1998-01-01

Synthesis, storage, and secretion of the proinflammatory cytokine interleukin-1 beta (IL-1 beta) and the anti-inflammatory cytokine IL-6 have not been established in normal exocrine gland secretory cells. Parotid glands and isolated acinar cells prepared from BALB/c mice were homogenized for RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR). IL-1 beta and IL-6 enzyme-linked immunosorbent assays (ELISAs) were done on supernatants prepared from mouse parotid acinar cell (MPAC) preparations unstimulated or stimulated between 0 and 10 min with 10(-5) M norepinephrine at 37 degrees C. MPACs were fixed in paraformaldehyde, frozen sectioned for light and electron microscopy, and labeled with antibodies to IL-1 beta and IL-6. Mouse specific riboprobes to IL-1 and IL-6 were used for in situ hybridization. RT-PCR yielded the expected IL-1 (336-bp) and IL-6 (614-bp) mRNA products. By ELISA, stimulated MPACs showed a significant increase in IL-1 beta (P < 0.03) and IL-6 (P < 0.01) release into supernatants by 10 min that paralleled the time course of amylase release. In situ hybridization showed the presence of transcripts for IL-1 and IL-6 in glandular epithelial cells. Gold-labeled IL-1 beta and IL-6 were significantly higher (P < 0.01) in granules than in the nucleus and cytoplasm. This study shows that MPACs synthesize IL-1 beta and IL-6 and release these cytokines from their granules after alpha- and beta-adrenergic stimulation.

Acorus tatarinowii Schott extract protects PC12 cells from amyloid-beta induced neurotoxicity.

PubMed

An, Hong-Mei; Li, Guo-Wen; Lin, Chen; Gu, Chao; Jin, Miao; Sun, Wen-Xian; Qiu, Ming-Feng; Hu, Bing

2014-05-01

Amyloid-beta induced neurotoxicity has been identified as a major cause of Alzheimer's disease. Acorus tatarinowii Schott is one of the most frequently used Chinese herbs for Alzheimer's disease treatment. However, the effects of Acorus tatarinowii Schott on amyloid-beta mediated nerve cell damage remains unknown. In the present study, neuronal differentiated PC12 cells were used as a model to evaluate the effects of A. tatarinowii Schott extract (ATSE) against Abeta25-35 induced neurotoxicity. The results showed pretreatment with ATSE significantly protected PC12 cells from Abeta25-35 induced cell death, lactate dehydrogenase release, DNA damage, mitochondrial dysfunction and cytochrome c release from mitochondria. In addition, pretreatment with ATSE also significantly inhibited Abeta25-35 induced caspase-3 activation and reactive oxygen species generation in PC12 cells. These observations suggested that ATSE protects PC12 cells from amyloid-beta induced neurotoxicity.

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.

Improvement of Fabry Disease-Related Gastrointestinal Symptoms in a Significant Proportion of Female Patients Treated with Agalsidase Beta: Data from the Fabry Registry.

PubMed

Wilcox, William R; Feldt-Rasmussen, Ulla; Martins, Ana Maria; Ortiz, Alberto; Lemay, Roberta M; Jovanovic, Ana; Germain, Dominique P; Varas, Carmen; Nicholls, Katherine; Weidemann, Frank; Hopkin, Robert J

2018-01-01

Fabry disease, an X-linked inherited lysosomal storage disorder, is caused by mutations in the gene encoding α-galactosidase, GLA. In patients with Fabry disease, glycosphingolipids accumulate in various cell types, triggering a range of cellular and tissue responses that result in a wide spectrum of organ involvement. Although variable, gastrointestinal symptoms are among the most common and significant early clinical manifestations; they tend to persist into adulthood if left untreated. To further understand the effects of sustained enzyme replacement therapy (ERT) with agalsidase beta on gastrointestinal symptoms in heterozygotes, a data analysis of female patients enrolled in the Fabry Registry was conducted. To be included, females of any age must have received agalsidase beta (average dose 1.0 mg/kg every 2 weeks) for at least 2.5 years. Measured outcomes were self-reported gastrointestinal symptoms (abdominal pain, diarrhea). Outcomes at baseline and last follow-up, and their change from baseline to last follow-up, were assessed. Relevant data were available for 168 female patients. Mean age at the start of ERT was 43 years and mean treatment duration 5.7 years. Baseline pre-treatment abdominal pain was reported by 45% of females and diarrhea by 39%. At last follow-up, 31% reported abdominal pain (p < 0.01) and 27% diarrhea (p < 0.01). The results of this Fabry Registry analysis suggest that while on sustained treatment with agalsidase beta (1.0 mg/kg every 2 weeks), both abdominal pain and diarrhea improved in many female patients with Fabry disease.

Peptide fragments of a beta-defensin derivative with potent bactericidal activity.

PubMed

Reynolds, Natalie L; De Cecco, Martin; Taylor, Karen; Stanton, Chloe; Kilanowski, Fiona; Kalapothakis, Jason; Seo, Emily; Uhrin, Dusan; Campopiano, Dominic; Govan, John; Macmillan, Derek; Barran, Perdita; Dorin, Julia R

2010-05-01

Beta-defensins are known to be both antimicrobial and able to chemoattract various immune cells. Although the sequences of paralogous genes are not highly conserved, the core defensin structure is retained. Defb14-1C(V) has bactericidal activity similar to that of its parent peptide (murine beta-defensin Defb14) despite all but one of the canonical six cysteines being replaced with alanines. The 23-amino-acid N-terminal half of Defb14-1C(V) is a potent antimicrobial while the C-terminal half is not. Here, we use a library of peptide derivatives to demonstrate that the antimicrobial activity can be localized to a particular region. Overlapping fragments of the N-terminal region were tested for their ability to kill Gram-positive and Gram-negative bacteria. We demonstrate that the most N-terminal fragments (amino acids 1 to 10 and 6 to 17) are potent antimicrobials against Gram-negative bacteria whereas fragments based on sequence more C terminal than amino acid 13 have very poor activity against both Gram-positive and -negative types. We further test a series of N-terminal deletion peptides in both their monomeric and dimeric forms. We find that bactericidal activity is lost against both Gram types as the deletion region increases, with the point at which this occurs varying between bacterial strains. The dimeric form of the peptides is more resistant to the peptide deletions, but this is not due just to increased charge. Our results indicate that the primary sequence, together with structure, is essential in the bactericidal action of this beta-defensin derivative peptide and importantly identifies a short fragment from the peptide that is a potent bactericide.

Crystal structure of a dimeric mannose-specific agglutinin from garlic: quaternary association and carbohydrate specificity.

PubMed

Chandra, N R; Ramachandraiah, G; Bachhawat, K; Dam, T K; Surolia, A; Vijayan, M

1999-01-22

A mannose-specific agglutinin, isolated from garlic bulbs, has been crystallized in the presence of a large excess of alpha-d-mannose, in space group C2 and cell dimensions, a=203.24, b=43.78, c=79.27 A, beta=112.4 degrees, with two dimers in the asymmetric unit. X-ray diffraction data were collected up to a nominal resolution of 2.4 A and the structure was solved by molecular replacement. The structure, refined to an R-factor of 22.6 % and an Rfree of 27.8 % reveals a beta-prism II fold, similar to that in the snowdrop lectin, comprising three antiparallel four-stranded beta-sheets arranged as a 12-stranded beta-barrel, with an approximate internal 3-fold symmetry. This agglutinin is, however, a dimer unlike snowdrop lectin which exists as a tetramer, despite a high degree of sequence similarity between them. A comparison of the two structures reveals a few substitutions in the garlic lectin which stabilise it into a dimer and prevent tetramer formation. Three mannose molecules have been identified on each subunit. In addition, electron density is observed for another possible mannose molecule per dimer resulting in a total of seven mannose molecules in each dimer. Although the mannose binding sites and the overall structure are similar in the subunits of snowdrop and garlic lectin, their specificities to glycoproteins such as GP120 vary considerably. These differences appear, in part, to be a direct consequence of the differences in oligomerisation, implying that variation in quaternary association may be a mode of achieving oligosaccharide specificity in bulb lectins. Copyright 1998 Academic Press.

[Recent advances in the treatment of type 1 diabetes mellitus].

PubMed

Schloot, N C; Roden, M; Bornstein, S R; Brendel, M D

2011-02-01

INTERVENTIONAL APPROACHES TO BETA CELL PRESERVATION: In a pilot study, initial attempts at primary prevention by preserving islet beta cells have been successful with highly hydrolyzed milk formula in children who are at high genetic risk of diabetes. Attempts at secondary prevention by intranasal application in children with a high-risk HLA genotype and positive islet autoantibodies have been disappointing. But in tertiary prevention anti-inflammatory, antigen-directed and T-cell targeted treatment has been partially successful in slowing down the destruction of beta cells. BIOLOGICAL BETA CELL SUBSTITUTION: Transplantation of a vascularised pancreas or islet cells results in disease regression and the prevention of secondary/tertiary complications of diabetes. A principal aim is the avoidance of frequent, severe hypoglycaemic episodes resulting from markedly reduced awareness of hypoglycaemia or its counter-regulation. © Georg Thieme Verlag KG Stuttgart · New York.

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.