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  1. Islet Brain 1 Protects Insulin Producing Cells against Lipotoxicity

    PubMed Central

    Brajkovic, Saška; Ferdaoussi, Mourad; Pawlowski, Valérie; Ezanno, Hélène; Plaisance, Valérie; Zmuda, Erik; Hai, Tsonwin; Annicotte, Jean-Sébastien; Waeber, Gérard; Abderrahmani, Amar

    2016-01-01

    Chronic intake of saturated free fatty acids is associated with diabetes and may contribute to the impairment of functional beta cell mass. Mitogen activated protein kinase 8 interacting protein 1 also called islet brain 1 (IB1) is a candidate gene for diabetes that is required for beta cell survival and glucose-induced insulin secretion (GSIS). In this study we investigated whether IB1 expression is required for preserving beta cell survival and function in response to palmitate. Chronic exposure of MIN6 and isolated rat islets cells to palmitate led to reduction of the IB1 mRNA and protein content. Diminution of IB1 mRNA and protein level relied on the inducible cAMP early repressor activity and proteasome-mediated degradation, respectively. Suppression of IB1 level mimicked the harmful effects of palmitate on the beta cell survival and GSIS. Conversely, ectopic expression of IB1 counteracted the deleterious effects of palmitate on the beta cell survival and insulin secretion. These findings highlight the importance in preserving the IB1 content for protecting beta cell against lipotoxicity in diabetes. PMID:26665154

  2. Islet Brain 1 Protects Insulin Producing Cells against Lipotoxicity.

    PubMed

    Brajkovic, Saška; Ferdaoussi, Mourad; Pawlowski, Valérie; Ezanno, Hélène; Plaisance, Valérie; Zmuda, Erik; Hai, Tsonwin; Annicotte, Jean-Sébastien; Waeber, Gérard; Abderrahmani, Amar

    2016-01-01

    Chronic intake of saturated free fatty acids is associated with diabetes and may contribute to the impairment of functional beta cell mass. Mitogen activated protein kinase 8 interacting protein 1 also called islet brain 1 (IB1) is a candidate gene for diabetes that is required for beta cell survival and glucose-induced insulin secretion (GSIS). In this study we investigated whether IB1 expression is required for preserving beta cell survival and function in response to palmitate. Chronic exposure of MIN6 and isolated rat islets cells to palmitate led to reduction of the IB1 mRNA and protein content. Diminution of IB1 mRNA and protein level relied on the inducible cAMP early repressor activity and proteasome-mediated degradation, respectively. Suppression of IB1 level mimicked the harmful effects of palmitate on the beta cell survival and GSIS. Conversely, ectopic expression of IB1 counteracted the deleterious effects of palmitate on the beta cell survival and insulin secretion. These findings highlight the importance in preserving the IB1 content for protecting beta cell against lipotoxicity in diabetes. PMID:26665154

  3. Nanomaterial Solutions for the Protection of Insulin Producing Beta Cells

    NASA Astrophysics Data System (ADS)

    Atchison, Nicole Ann

    Islet transplantation is a promising treatment for type 1 diabetes. However, even with the many successes, islet transplantation has yet to reach its full potential. Limited islet sources, loss of cell viability during isolation and culture, and post-transplant graft loss are a few of the issues preventing extensive use of islet transplantation. The application of biomaterial systems to alleviate some of the stresses affecting islet viability has led to improvements in isolation and transplantation outcomes, but problems persist. In this work we approach two distinct issues affecting islet viability; ischemic conditions and immunological attack post-transplant. Ischemic conditions have been linked to a loss of islet graft function and occur during organ preservation, islet isolation and culture, and after islets are transplanted. We show that liposomal delivery of adenosine triphosphate (ATP) to beta cells can limit cell death and loss of function in ischemic conditions. We demonstrate that by functionalizing liposomes with the fibronectin-mimetic peptide PR_b, delivery of liposomes to porcine islets and rat beta cells is increased compared to nontargeted controls. Additionally, liposomes are shown to protect by providing both ATP and lipids to the ischemic cells. The delivery of ATP was investigated here but application of PR_b functionalized liposomes could be extended to other interesting cargos as well. The second area of investigation involves encapsulation of islets with silica nanoparticles to create a permselective barrier. Silica nanoparticles are an interesting material for encapsulation given their ability to be fine-tuned and further functionalized. We demonstrate that size-tunable, fluorescent silica nanoparticles can be assembled layer-by-layer on the surface of cells and that silica nanoparticle encapsulated islets are able to secrete insulin in response to a glucose challenge.

  4. Improved antioxidative defence protects insulin-producing cells against homocysteine toxicity.

    PubMed

    Scullion, Siobhan M; Hahn, Claudine; Tyka, Karolina; Flatt, Peter R; McClenaghan, Neville H; Lenzen, Sigurd; Gurgul-Convey, Ewa

    2016-08-25

    Homocysteine (HC) is considered to play an important role in the development of metabolic syndrome complications. Insulin-producing cells are prone to HC toxicity and this has been linked to oxidative stress. However, the exact mechanisms remain unknown. Therefore it was the aim of this study to determine the nature of reactive oxygen species responsible for HC toxicity. Chronic exposure of RINm5F and INS1E insulin-producing cells to HC decreased cell viability and glucose-induced insulin secretion in a concentration-dependent manner and led to a significant induction of hydrogen peroxide generation in the cytosolic, but not the mitochondrial compartment of the cell. Cytosolic overexpression of catalase, a hydrogen peroxide detoxifying enzyme, provided a significant protection against viability loss and hydrogen peroxide generation, while mitochondrial overexpression of catalase did not protect against HC toxicity. Overexpression of CuZnSOD, a cytosolic superoxide dismutating enzyme, also protected against HC toxicity. However, the best protection was achieved in the case of a combined overexpression of CuZnSOD and catalase. Incubation of cells in combination with alloxan resulted in a significant increase of HC toxicity and an increase of hydrogen peroxide generation. Overexpression of CuZnSOD or catalase protected against the toxicity of HC plus alloxan, with a superior protection achieved again by combined overexpression. The results indicate that HC induces oxidative stress in insulin-producing cells by stimulation of superoxide radical and hydrogen peroxide generation in the cytoplasm. The low antioxidative defence status makes the insulin-producing cells very vulnerable to HC toxicity. PMID:27317948

  5. Protection against Cytokine Toxicity through Endoplasmic Reticulum and Mitochondrial Stress Prevention by Prostacyclin Synthase Overexpression in Insulin-producing Cells*

    PubMed Central

    Gurgul-Convey, Ewa; Lenzen, Sigurd

    2010-01-01

    Proinflammatory cytokines play a crucial role in the pathogenesis of type 1 diabetes mellitus. One of the cytokine-regulated pathways mediating inflammation in this autoimmune disease is the arachidonic acid metabolism pathway, comprising both the induction of cyclooxygenases and the production of different prostaglandins. Cytokine toxicity is mediated in many cell types, including pancreatic β cells through this pathway. Interestingly, some cell types have been shown to be insensitive to such toxicity, and this correlated with a high expression of prostacyclin synthase (PGIS). Using insulin-producing RINm5F cells as a model for pancreatic β cells, PGIS was overexpressed and exhibited a large protective effect against cytokine toxicity. This protective effect of PGIS against cytokine toxicity correlated with a decreased activation of the transcription factor NFκB and the inducible NO synthase promoter as well as a reduced inducible NO synthase protein expression and nitrite production. A reduction in the cytokine-stimulated endoplasmic reticulum and mitochondrial stress was also found in the PGIS-overexpressing cells. Moreover, cytokine-induced caspase-3 activation and reduction of glucose oxidation and cell proliferation were suppressed. Thus, PGIS overexpression apparently protects insulin-producing cells against cytokine toxicity via suppression of endoplasmic reticulum and mitochondrial stress-mediated cell death pathways. PMID:20159982

  6. Surrogate insulin-producing cells

    PubMed Central

    Wong, Adrianne L.; Hwa, Albert; Hellman, Dov

    2012-01-01

    Diabetes, a large and growing worldwide health concern, affects the functional mass of the pancreatic beta cell, which in turn affects the glucose regulation of the body. Successful transplantation of cadaveric islets and pancreata for patients with uncontrolled type 1 diabetes has provided proof-of-concept for the development of commercial cell therapy approaches to treat diabetes. Three broad issues must be addressed before surrogate insulin-producing cells can become a reality: the development of a surrogate beta-cell source, immunoprotection, and translation. Cell therapy for diabetes is a real possibility, but many questions remain; through the collaborative efforts of multiple stakeholders this may become a reality. PMID:22891077

  7. Transduction of PEP-1-heme oxygenase-1 into insulin-producing INS-1 cells protects them against cytokine-induced cell death

    SciTech Connect

    Lee, Su Jin; Kang, Hyung Kyung; Song, Dong Keun; Eum, Won Sik; Park, Jinseu; Choi, Soo Young; Kwon, Hyeok Yil

    2015-06-05

    Pro-inflammatory cytokines play a crucial role in the destruction of pancreatic β-cells, thereby triggering the development of autoimmune diabetes mellitus. We recently developed a cell-permeable fusion protein, PEP-1-heme oxygenase-1 (PEP-1-HO-1) and investigated the anti-inflammatory effects in macrophage cells. In this study, we transduced PEP-1-HO-1 into INS-1 insulinoma cells and examined its protective effect against cytokine-induced cell death. PEP-1-HO-1 was successfully delivered into INS-1 cells in time- and dose-dependent manner and was maintained within the cells for at least 48 h. Pre-treatment with PEP-1-HO-1 increased the survival of INS-1 cells exposed to cytokine mixture (IL-1β, IFN-γ, and TNF-α) in a dose-dependent manner. PEP-1-HO-1 markedly decreased cytokine-induced production of reactive oxygen species (ROS), nitric oxide (NO), and malondialdehyde (MDA). These protective effects of PEP-1-HO-1 against cytokines were correlated with the changes in the levels of signaling mediators of inflammation (iNOS and COX-2) and cell apoptosis/survival (Bcl-2, Bax, caspase-3, PARP, JNK, and Akt). These results showed that the transduced PEP-1-HO-1 efficiently prevented cytokine-induced cell death of INS-1 cells by alleviating oxidative/nitrosative stresses and inflammation. Further, these results suggested that PEP-1-mediated HO-1 transduction may be a potential therapeutic strategy to prevent β-cell destruction in patients with autoimmune diabetes mellitus. - Highlights: • We showed that PEP-1-HO-1 was efficiently delivered into INS-1 cells. • Transduced PEP-1-HO-1 exerted a protective effect against cytokine-induced cell death. • Transduced PEP-1-HO-1 inhibited cytokine-induced ROS and NO accumulation. • PEP-1-HO-1 suppressed cytokine-induced expression of iNOS, COX-2, and Bax. • PEP-1-HO-1 transduction may be an efficient tool to prevent β-cell destruction.

  8. Insulin-producing cells from embryonic stem cells rescues hyperglycemia via intra-spleen migration.

    PubMed

    Ren, Meng; Shang, Changzhen; Zhong, Xiaomei; Guo, Ruomi; Lao, Guojuan; Wang, Xiaoyi; Cheng, Hua; Min, Jun; Yan, Li; Shen, Jun

    2014-01-01

    Implantation of embryonic stem cells (ESC)-derived insulin-producing cells has been extensively investigated for treatment of diabetes in animal models. However, the in vivo behavior and migration of transplanted cells in diabetic models remains unclear. Here we investigated the location and migration of insulin-producing cells labeled with superparamagnetic iron oxide (SPIO) using a dynamic MRI tracking method. SPIO labeled cells showed hypointense signal under the kidney subcapsules of diabetic mice on MRI, and faded gradually over the visiting time. However, new hypointense signal appeared in the spleen 1 week after transplantation, and became obvious with the time prolongation. Further histological examination proved the immigrated cells were insulin and C-peptide positive cells which were evenly distributed throughout the spleen. These intra-spleen insulin-producing cells maintained their protective effects against hyperglycemia in vivo, and these effects were reversed upon spleen removal. Transplantation of insulin-producing cells through spleen acquired an earlier blood glucose control as compared with that through kidney subcapsules. In summary, our data demonstrate that insulin-producing cells transplanted through kidney subcapsules were not located in situ but migrated into spleen, and rescues hyperglycemia in diabetic models. MRI may provide a novel tracking method for preclinical cell transplantation therapy of diabetes continuously and non-invasively. PMID:25533571

  9. Insulin-producing cells from embryonic stem cells rescues hyperglycemia via intra-spleen migration

    PubMed Central

    Ren, Meng; Shang, Changzhen; Zhong, Xiaomei; Guo, Ruomi; Lao, Guojuan; Wang, Xiaoyi; Cheng, Hua; Min, Jun; Yan, Li; Shen, Jun

    2014-01-01

    Implantation of embryonic stem cells (ESC)-derived insulin-producing cells has been extensively investigated for treatment of diabetes in animal models. However, the in vivo behavior and migration of transplanted cells in diabetic models remains unclear. Here we investigated the location and migration of insulin-producing cells labeled with superparamagnetic iron oxide (SPIO) using a dynamic MRI tracking method. SPIO labeled cells showed hypointense signal under the kidney subcapsules of diabetic mice on MRI, and faded gradually over the visiting time. However, new hypointense signal appeared in the spleen 1 week after transplantation, and became obvious with the time prolongation. Further histological examination proved the immigrated cells were insulin and C-peptide positive cells which were evenly distributed throughout the spleen. These intra-spleen insulin-producing cells maintained their protective effects against hyperglycemia in vivo, and these effects were reversed upon spleen removal. Transplantation of insulin-producing cells through spleen acquired an earlier blood glucose control as compared with that through kidney subcapsules. In summary, our data demonstrate that insulin-producing cells transplanted through kidney subcapsules were not located in situ but migrated into spleen, and rescues hyperglycemia in diabetic models. MRI may provide a novel tracking method for preclinical cell transplantation therapy of diabetes continuously and non-invasively. PMID:25533571

  10. In vivo regeneration of insulin-producing beta-cells.

    PubMed

    Jun, Hee-Sook

    2010-01-01

    Type 1 and type 2 diabetes mellitus are considered to be caused by defective control of blood glucose resulting from a reduced beta-cell mass. Thus, the restoration of a functional beta-cell mass by replacing the damaged beta-cells or stimulating beta-cell regeneration is a logical approach for the treatment of diabetes. Strategies for increasing the beta-cell mass include stimulating beta-cell replication and differentiation and inhibiting beta-cell death. Treatment with various growth factors such as GLP-1, BTC, HGF, and EGF and forced expression of beta-cell transcription factors such as Pdx-1, NeuroD, and MafA resulted in the regeneration of beta-cells in vivo. Another approach is the administration of stem/progenitor cells, which can differentiate into insulin-producing cells. However, there are no satisfactory methods yet for clinical application. Understanding the mechanisms of the regenerative process of pancreatic beta-cells will pave the way for the development of regenerative medicine for treatment of diabetes. PMID:20217517

  11. Glucose-responsive insulin-producing cells from stem cells.

    PubMed

    Kaczorowski, David J; Patterson, Ethan S; Jastromb, William E; Shamblott, Michael J

    2002-01-01

    Recent success with immunosuppression following islet cell transplantation offers hope that a cell transplantation treatment for type 1 (juvenile) diabetes may be possible if sufficient quantities of safe and effective cells can be produced. For the treatment of type 1 diabetes, the two therapeutically essential functions are the ability to monitor blood glucose levels and the production of corresponding and sufficient levels of mature insulin to maintain glycemic control. Stem cells can replicate themselves and produce cells that take on more specialized functions. If a source of stem cells capable of yielding glucose-responsive insulin-producing (GRIP) cells can be identified, then transplantation-based treatment for type 1 diabetes may become widely available. Currently, stem cells from embryonic and adult sources are being investigated for their ability to proliferate and differentiate into cells with GRIP function. Human embryonic pluripotent stem cells, commonly referred to as embryonic stem (ES) cells and embryonic germ (EG) cells, have received significant attention owing to their broad capacity to differentiate and ability to proliferate well in culture. Their application to diabetes research is of particular promise, as it has been demonstrated that mouse ES cells are capable of producing cells able to normalize glucose levels of diabetic mice, and human ES cells can differentiate into cells capable of insulin production. Cells with GRIP function have also been derived from stem cells residing in adult organisms, here referred to as endogenous stem cell sources. Independent of source, stem cells capable of producing cells with GRIP function may provide a widely available cell transplantation treatment for type 1 diabetes. PMID:12469358

  12. Differentiation of embryonic and adult stem cells into insulin producing cells.

    PubMed

    Zulewski, H

    2008-03-01

    Replacement of insulin producing cells represents an almost ideal treatment for patients with diabetes mellitus type 1. Transplantation of pancreatic islets of Langerhans is successful in experienced centers. The wider application of this therapy, however, is limited by the lack of donor organs. Insulin producing cells generated from stem cells represent an attractive alternative. Stem cells with the potential to differentiate into insulin producing cells include embryonic stem cells (ESC) as well as adult stem cells from various tissues including the pancreas, liver, bone marrow and adipose tissue. The use of human ESC is hampered by ethical concerns but research with human ESC may help us to decipher important steps in the differentiation process in vitro since almost all information available on pancreas development are based on animal studies. The present review summarizes the current knowledge on the development of insulin producing cells from embryonic and adult stem cells with special emphasis on pancreatic, hepatic and human mesenchymal stem cells. PMID:18427390

  13. Endovascular Method for Transplantation of Insulin-Producing Cells to the Pancreas Parenchyma in Swine

    PubMed Central

    Lundberg, J; Stone-Elander, S; Zhang, X-M; Korsgren, O; Jonsson, S; Holmin, S

    2014-01-01

    Insulin-producing cells are transplanted by portal vein injection as an alternative to pancreas transplantation in both clinical and preclinical trials. Two of the main limitations of portal vein transplantation are the prompt activation of the innate immunity and concomitant loss of islets and a small but significant risk of portal vein thrombosis. Furthermore, to mimic physiological release, the insulin-producing cells should instead be located in the pancreas. The trans-vessel wall approach is an endovascular method for penetrating the vessel wall from the inside. In essence, a working channel is established to the parenchyma of organs that are difficult to access by percutaneous technique. In this experiment, we accessed the extra-vascular pancreatic parenchyma in swine by microendovascular technique and injected methylene blue, contrast fluids and insulin-producing cells without acute adverse events. Further, we evaluated the procedure itself by a 1-year angiographical follow-up, without adverse events. This study shows that the novel approach utilizing endovascular minimal invasiveness coupled to accurate trans-vessel wall placement of an injection in the pancreatic parenchyma with insulin-producing cells is possible. In clinical practice, the potential benefits compared to portal vein cell transplantation should significantly improve endocrine function of the graft and potentially reduce adverse events. This study presents one-year follow-up safety data on the microendovascular trans-vessel wall technique and shows that the technique can be used to transplant insulin-producing cells to the swine pancreas parenchyma. PMID:24517268

  14. Adult Stem Cells as a Renewable Source of Insulin-Producing Cells

    PubMed Central

    Jun, Hee-Sook; Park, Eun-Young

    2009-01-01

    Diabetes mellitus is a metabolic disorder resulting from an inadequate mass of insulin-producing pancreatic beta cells. The replacement or restoration of damaged beta cells would be considered the optimal therapeutic options. Islet transplantation seems to be a promising approach for replacement therapy; however, the main obstacle is the shortage of organ donors. As mature beta cells have been shown to be difficult to expand in vitro, regeneration of beta cells from embryonic or adult stem cells or pancreatic progenitor cells is an attractive method to restore the islet cell mass. So far, multiple studies using various strategies have shown direct differentiation of stem and progenitor cells toward insulin-producing cells. The important issue to be solved is how to differentiate these cells into mature functional insulin-producing cells. Further research is required to understand how endogenous beta cells differentiate and to develop methods to regenerate enough functional beta cells for clinically applicable therapies for diabetes. PMID:24855530

  15. Complete Protection of Mice against Lethal Murine Cytomegalovirus Challenge by Immunization with DNA Vaccines Encoding Envelope Glycoprotein Complex III Antigens gH, gL and gO

    PubMed Central

    Wang, Huadong; Huang, Chaoyang; Dong, Jinrong; Yao, Yanfeng; Xie, Zhenyuan; Liu, Xueying; Zhang, Wenjie; Fang, Fang; Chen, Ze

    2015-01-01

    Human cytomegalovirus infects the majority of humanity which may lead to severe morbidity and mortality in newborns and immunocompromised adults. Humoral and cellular immunity are critical for controlling CMV infection. HCMV envelope glycoprotein complexes (gC I, II, III) represent major antigenic targets of antiviral immune responses. The gCIII complex is comprised of three glycoproteins, gH, gL, and gO. In the present study, DNA vaccines expressing the murine cytomegalovirus homologs of the gH, gL, and gO proteins were evaluated for protection against lethal MCMV infection in the mouse model. The results demonstrated that gH, gL, or gO single gene immunization could not yet offer good protection, whereas co-vaccination strategy apparently showed effects superior to separate immunization. Twice immunization with gH/gL/gO pDNAs could provide mice complete protection against lethal salivary gland-derived MCMV (SG-MCMV) challenge, while thrice immunization with pgH/pgL, pgH/pgO or pgL/pgO could not provide full protection. Co-vaccination with gH, gL and gO pDNAs elicited robust neutralizing antibody and cellular immune responses. Moreover, full protection was also achieved by simply passive immunization with anti-gH/gL/gO sera. These data demonstrated that gCIII complex antigens had fine immunogenicity and might be a promising candidate for the development of HCMV vaccines. PMID:25803721

  16. Differentiation of stem cells into insulin-producing cells under the influence of nanostructural polyoxometalates.

    PubMed

    Bâlici, Ştefana; Şuşman, Sergiu; Rusu, Dan; Nicula, Gheorghe Zsolt; Soriţău, Olga; Rusu, Mariana; Biris, Alexandru S; Matei, Horea

    2016-03-01

    Two polyoxometalates (POMs) with W were synthesized by a two-step, self-assembling method. They were used for stimulation of mesenchymal stem cell differentiation into insulin-producing cells. The nanocompounds (tris(vanadyl)-substituted tungsto-antimonate(III) anions [POM1] and tris-butyltin-21-tungsto-9-antimonate(III) anions [POM2]) were characterized by analytical techniques, including ultraviolet-visible, Fourier transform infrared, nuclear magnetic resonance spectroscopy, and transmission electron microscopy. We found that these polyoxotungstates, with 2-4 nm diameters, did not present toxic effects at the tested concentrations. In vitro, POM1 stimulated differentiation of a greater number of dithizone-positive cells (also organized in clusters) than the second nanocompound (POM2). Based on our in vitro studies, we have concluded that both the POMs tested had significant biological activity acting as active stimuli for differentiation of stem cells into insulin-producing cells. PMID:26397720

  17. Differentiation of stem cells into insulin-producing cells: current status and challenges.

    PubMed

    Pokrywczynska, Marta; Krzyzanowska, Sandra; Jundzill, Arkadiusz; Adamowicz, Jan; Drewa, Tomasz

    2013-04-01

    Diabetes mellitus is one of the most serious public health challenges of the twenty-first century. Allogenic islet transplantation is an efficient therapy for type 1 diabetes. However, immune rejection, side effects of immunosuppressive treatment as well as lack of sufficient donor organs limits its potential. In recent years, several promising approaches for generation of new pancreatic β cells have been developed. This review provides an overview of current status of pancreatic and extra-pancreatic stem cells differentiation into insulin-producing cells and the possible application of these cells for diabetes treatment. The PubMed database was searched for English language articles published between 2001 and 2012, using the keyword combinations: diabetes mellitus, differentiation, insulin-producing cells, stem cells. PMID:23283518

  18. A Broadly Cross-protective Vaccine Presenting the Neighboring Epitopes within the VP1 GH Loop and VP2 EF Loop of Enterovirus 71.

    PubMed

    Xu, Longfa; He, Delei; Yang, Lisheng; Li, Zhiqun; Ye, Xiangzhong; Yu, Hai; zhao, Huan; Li, Shuxuan; Yuan, Lunzhi; Qian, Hongliu; Que, Yuqiong; Shih, James Wai Kuo; Zhu, Hua; Li, Yimin; Cheng, Tong; Xia, Ningshao

    2015-01-01

    Human enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the major etiological agents of hand, foot and mouth disease (HFMD) and are often associated with neurological complications. Currently, several vaccine types are being developed for EV71 and CA16. In this study, we constructed a bivalent chimeric virus-like particle (VLP) presenting the VP1 (aa208-222) and VP2 (aa141-155) epitopes of EV71 using hepatitis B virus core protein (HBc) as a carrier, designated HBc-E1/2. Immunization with the chimeric VLPs HBc-E1/2 induced higher IgG titers and neutralization titers against EV71 and CA16 in vitro than immunization with only one epitope incorporated into HBc. Importantly, passive immunization with the recombinant HBc-E2 particles protected neonatal mice against lethal EV71 and CA16 infections. We demonstrate that anti-VP2 (aa141-155) sera bound authentic CA16 viral particles, whereas anti-VP1 (aa208-222) sera could not. Moreover, the anti-VP2 (aa141-155) antibodies inhibited the binding of human serum to virions, which demonstrated that the VP2 epitope is immunodominant between EV71 and CA16. These results illustrated that the chimeric VLP HBc-E1/2 is a promising candidate for a broad-spectrum HFMD vaccine, and also reveals mechanisms of protection by the neighboring linear epitopes of the VP1 GH and VP2 EF loops. PMID:26243660

  19. Noninvasive Tracking of Encapsulated Insulin Producing Cells Labelled with Magnetic Microspheres by Magnetic Resonance Imaging

    PubMed Central

    Yim, Mandy M. W.; Foster, Jayne L.; Oberholzer, Jose

    2016-01-01

    Microencapsulated islets are usually injected free-floating into the peritoneal cavity, so the position of the grafts remains elusive after transplantation. This study aims to assess magnetic resonance imaging (MRI) as a noninvasive means to track microencapsulated insulin producing cells following transplantation. Encapsulated insulin producing cells (MIN6 and human islets) were labelled with magnetic microspheres (MM), assessed for viability and insulin secretion, and imaged in vitro using a clinical grade 3 T MRI and in vivo using both clinical grade 3 T and research grade 11.7 T MRI. Fluorescent imaging demonstrated the uptake of MM by both MIN6 and human islets with no changes in cell morphology and viability. MM labelling did not affect the glucose responsiveness of encapsulated MIN6 and islets in vitro. In vivo encapsulated MM-labelled MIN6 normalized sugar levels when transplanted into diabetic mice. In vitro MRI demonstrated that single microcapsules as well as clusters of encapsulated MM-labelled cells could be visualised clearly in agarose gel phantoms. In vivo encapsulated MM-labelled MIN6 could be visualised more clearly within the peritoneal cavity as discrete hypointensities using the high power 11.7 T but not the clinical grade 3 T MRI. This study demonstrates a method to noninvasively track encapsulated insulin producing cells by MM labelling and MRI.

  20. A Modified Method of Insulin Producing Cells' Generation from Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    Czubak, Paweł; Putowski, Lechosław

    2014-01-01

    Type 1 diabetes mellitus is a result of autoimmune destruction of pancreatic insulin producing β-cells and so far it can be cured only by insulin injection, by pancreas transplantation, or by pancreatic islet cells' transplantation. The methods are, however, imperfect and have a lot of disadvantages. Therefore new solutions are needed. The best one would be the use of differentiated mesenchymal stem cells (MSCs). In the present study, we investigated the potential of the bone marrow-derived MSCs line for in vitro differentiation into insulin producing cells (IPSs). We applied an 18-day protocol to differentiate MSCs. Differentiating cells formed cell clusters some of which resembled pancreatic islet-like cells. Using dithizone we confirmed the presence of insulin in the cells. What is more, the expression of proinsulin C-peptide in differentiated IPCs was analyzed by flow cytometry. For the first time, we investigated the influence of growth factors' concentration on IPCs differentiation efficiency. We have found that an increase in the concentration of growth factors up to 60 ng/mL of β-FGF/EGF and 30 ng/mL of activin A/β-cellulin increases the percentage of IPCs. Further increase of growth factors does not show any increase of the percentage of differentiated cells. Our findings suggest that the presented protocol can be adapted for differentiation of insulin producing cells from stem cells. PMID:25405207

  1. A modified method of insulin producing cells' generation from bone marrow-derived mesenchymal stem cells.

    PubMed

    Czubak, Paweł; Bojarska-Junak, Agnieszka; Tabarkiewicz, Jacek; Putowski, Lechosław

    2014-01-01

    Type 1 diabetes mellitus is a result of autoimmune destruction of pancreatic insulin producing β-cells and so far it can be cured only by insulin injection, by pancreas transplantation, or by pancreatic islet cells' transplantation. The methods are, however, imperfect and have a lot of disadvantages. Therefore new solutions are needed. The best one would be the use of differentiated mesenchymal stem cells (MSCs). In the present study, we investigated the potential of the bone marrow-derived MSCs line for in vitro differentiation into insulin producing cells (IPSs). We applied an 18-day protocol to differentiate MSCs. Differentiating cells formed cell clusters some of which resembled pancreatic islet-like cells. Using dithizone we confirmed the presence of insulin in the cells. What is more, the expression of proinsulin C-peptide in differentiated IPCs was analyzed by flow cytometry. For the first time, we investigated the influence of growth factors' concentration on IPCs differentiation efficiency. We have found that an increase in the concentration of growth factors up to 60 ng/mL of β-FGF/EGF and 30 ng/mL of activin A/β-cellulin increases the percentage of IPCs. Further increase of growth factors does not show any increase of the percentage of differentiated cells. Our findings suggest that the presented protocol can be adapted for differentiation of insulin producing cells from stem cells. PMID:25405207

  2. Improving the efficacy of type 1 diabetes therapy by transplantation of immunoisolated insulin-producing cells.

    PubMed

    Ngoc, Phan Kim; Phuc, Pham Van; Nhung, Truong Hai; Thuy, Duong Thanh; Nguyet, Nguyen Thi Minh

    2011-06-01

    Type 1 diabetes occurs when pancreatic islet β-cells are damaged and are thus unable to secrete insulin. Pancreas- or islet-grafting therapy offers highly efficient treatment but is limited by inadequate donor islets or pancreases for transplantation. Stem-cell therapy holds tremendous potential and promises to enhance treatment efficiency by overcoming the limitations of traditional therapies. In this study, we evaluated the efficiency of preclinical diabetic treatment. Diabetes was induced in mice by injections of streptozotocin. Mesenchymal stem cells (MSCs) were derived from mouse bone marrow or human umbilical cord blood and subsequently differentiated into insulin-producing cells. These insulin-producing cells were encapsulated in an alginate membrane to form capsules. Finally, these capsules were grafted into diabetic mice by intraperitoneal injection. Treatment efficiency was evaluated by monitoring body weight and blood glucose levels. Immune reactions after transplantation were monitored by counting total white blood cells. Allografting or xenografting of encapsulated insulin-producing cells (IPCs) reduced blood glucose levels and increased body weight following transplantation. Encapsulation with alginate conferred immune isolation and prevented graft rejection. These results provide further evidence supporting the use of allogeneic or xenogeneic MSCs obtained from bone marrow or umbilical cord blood for treating type 1 diabetes. PMID:21567289

  3. Insulin-Producing Endocrine Cells Differentiated In Vitro From Human Embryonic Stem Cells Function in Macroencapsulation Devices In Vivo

    PubMed Central

    Ambruzs, Dana M.; Moorman, Mark A.; Bhoumik, Anindita; Cesario, Rosemary M.; Payne, Janice K.; Kelly, Jonathan R.; Haakmeester, Carl; Srijemac, Robert; Wilson, Alistair Z.; Kerr, Justin; Frazier, Mauro A.; Kroon, Evert J.; D’Amour, Kevin A.

    2015-01-01

    The PEC-01 cell population, differentiated from human embryonic stem cells (hESCs), contains pancreatic progenitors (PPs) that, when loaded into macroencapsulation devices (to produce the VC-01 candidate product) and transplanted into mice, can mature into glucose-responsive insulin-secreting cells and other pancreatic endocrine cells involved in glucose metabolism. We modified the protocol for making PEC-01 cells such that 73%–80% of the cell population consisted of PDX1-positive (PDX1+) and NKX6.1+ PPs. The PPs were further differentiated to islet-like cells (ICs) that reproducibly contained 73%–89% endocrine cells, of which approximately 40%–50% expressed insulin. A large fraction of these insulin-positive cells were single hormone-positive and expressed the transcription factors PDX1 and NKX6.1. To preclude a significant contribution of progenitors to the in vivo function of ICs, we used a simple enrichment process to remove remaining PPs, yielding aggregates that contained 93%–98% endocrine cells and 1%–3% progenitors. Enriched ICs, when encapsulated and implanted into mice, functioned similarly to the VC-01 candidate product, demonstrating conclusively that in vitro-produced hESC-derived insulin-producing cells can mature and function in vivo in devices. A scaled version of our suspension culture was used, and the endocrine aggregates could be cryopreserved and retain functionality. Although ICs expressed multiple important β cell genes, the cells contained relatively low levels of several maturity-associated markers. Correlating with this, the time to function of ICs was similar to PEC-01 cells, indicating that ICs required cell-autonomous maturation after delivery in vivo, which would occur concurrently with graft integration into the host. Significance Type 1 diabetes (T1D) affects approximately 1.25 million people in the U.S. alone and is deadly if not managed with insulin injections. This paper describes the production of insulin-producing

  4. Potential Factors for the Differentiation of ESCs/iPSCs Into Insulin-Producing Cells.

    PubMed

    Tsugata, Takako; Nikoh, Naruo; Kin, Tatsuya; Saitoh, Issei; Noguchi, Yasufumi; Ueki, Hideo; Watanabe, Masami; James Shapiro, Andrew M; Noguchi, Hirofumi

    2015-02-01

    The low efficiency of in vitro differentiation of human embryonic stem cells (ESCs) or human induced pluripotent stem cells (iPSCs) into insulin-producing cells thus creates a crucial hurdle for the clinical implementation of human pluripotent stem cells (PSCs). In this study, we investigated the key factors for the differentiation of PSCs into insulin-producing cells. We obtained microarray data of HUES8 and HUES6 from two GeneChips (GPL3921: Affymetrix HT Human Genome U133A Array, GPL570: Affymetrix Human Genome U133 Plus 2.0 Array) in a database of GEO (NCBI), since HUES8 can differentiate into pancreatic cells, while HUES6 hardly demonstrates any differentiation at all. The genes with more than fourfold higher expressions in HUES8 compared to HUES6 included RPS4Y1, DDX3Y, EIF1AY, GREM1, GATA6, and NLGN4Y. Since there were four genes, RPS4Y1, DDX3Y, EIF1AY, and NLGN4Y, on the Y chromosome and HUES8 was a male cell line and HUES6 was a female cell line, we excluded these genes in this study. On the other hand, genes with more than fourfold higher expressions in HUES6 compared to HUES8 included NLRP2, EGR1, and SMC3. We next compared iPSCs derived from pancreatic cells (PiPSCs) and iPSCs derived from fibroblasts (FiPSCs). PiPSCs differentiated into insulin-producing cells more easily than FiPSCs because of their epigenetic memory. The gene expressions of GREM1, GATA6, NLRP2, EGR1, and SMC3 in PiPSCs and FiPSCs were also investigated. The expression level of GREM1 and GATA6 in PiPSCs were higher than in FiPSCs. On the other hand, EGR1, which was lower in HUES8 than in HUES6, was predictably lower in PiPSCs than FiPSCs, while NLRP2 and SMC3 were higher in PiPSCs than FiPSCs. These data suggest that the expression of GATA6 and GREM1 and the inhibition of EGR1 may be important factors for the differentiation of PSCs into insulin-producing cells. PMID:26858897

  5. Deoxysphingolipids, novel biomarkers for type 2 diabetes, are cytotoxic for insulin-producing cells.

    PubMed

    Zuellig, Richard A; Hornemann, Thorsten; Othman, Alaa; Hehl, Adrian B; Bode, Heiko; Güntert, Tanja; Ogunshola, Omolara O; Saponara, Enrica; Grabliauskaite, Kamile; Jang, Jae-Hwi; Ungethuem, Udo; Wei, Yu; von Eckardstein, Arnold; Graf, Rolf; Sonda, Sabrina

    2014-04-01

    Irreversible failure of pancreatic β-cells is the main culprit in the pathophysiology of diabetes, a disease that is now a global epidemic. Recently, elevated plasma levels of deoxysphingolipids, including 1-deoxysphinganine, have been identified as a novel biomarker for the disease. In this study, we analyzed whether deoxysphingolipids directly compromise the functionality of insulin-producing Ins-1 cells and primary islets. Treatment with 1-deoxysphinganine induced dose-dependent cytotoxicity with senescent, necrotic, and apoptotic characteristics and compromised glucose-stimulated insulin secretion. In addition, 1-deoxysphinganine altered cytoskeleton dynamics, resulting in intracellular accumulation of filamentous actin and activation of the Rho family GTPase Rac1. Moreover, 1-deoxysphinganine selectively upregulated ceramide synthase 5 expression and was converted to 1-deoxy-dihydroceramides without altering normal ceramide levels. Inhibition of intracellular 1-deoxysphinganine trafficking and ceramide synthesis improved the viability of the cells, indicating that the intracellular metabolites of 1-deoxysphinganine contribute to its cytotoxicity. Analyses of signaling pathways identified Jun N-terminal kinase and p38 mitogen-activated protein kinase as antagonistic effectors of cellular senescence. The results revealed that 1-deoxysphinganine is a cytotoxic lipid for insulin-producing cells, suggesting that the increased levels of this sphingolipid observed in diabetic patients may contribute to the reduced functionality of pancreatic β-cells. Thus, targeting deoxysphingolipid synthesis may complement the currently available therapies for diabetes. PMID:24379346

  6. Selection of insulin-producing rat insulinoma (RINm) cells with improved resistance to oxidative stress.

    PubMed

    Bloch, Konstantin O; Vorobeychik, Marina; Yavrians, Karina; Vardi, Pnina

    2003-06-01

    The defense system against reactive oxygen species is believed to be crucial for the survival of insulin-producing cells after various injuries. The aim of our study was to select a subpopulation of insulin-producing RINm cells with higher resistance to oxidative stress. The cells resistant to hydrogen peroxide (RINmHP) were obtained by repeated exposure of parental RINm cells to 100 and 200 microM hydrogen peroxide (HP). The increased resistance of RINmHP cells to HP was confirmed by three different cytotoxicity assays. In addition, the selected cells also were resistant to the cytotoxic effect of activated rat splenocytes compared to parental cells. The half-life of HP in the RINmHP cell culture medium was about 2.5 times lower than that of the parental cells, corresponding to the increased level of catalase expression and activity in selected cells. The increased defense property of the selected cells was not associated with any significant changes in insulin content and insulin response to a mixture of glucose with isobutyl methyl xanthine or potassium chloride. In conclusion, repeated exposure to HP induces selection of RINm cells with improved resistance to oxidative stress. This improved defense characteristic probably is due to an increased level of catalase expression and activity in the selected cells. PMID:12781331

  7. New method to differentiate human peripheral blood monocytes into insulin producing cells: Human hematosphere culture.

    PubMed

    Hur, Jin; Yang, Ji Min; Choi, Jae-Il; Yun, Ji-Yeon; Jang, Jae Hee; Kim, Joonoh; Kim, Ju-Young; Oh, Il-Young; Yoon, Chang-Hwan; Cho, Hyun-Jai; Park, Young-Bae; Kim, Hyo-Soo

    2012-02-24

    Strategy to differentiate stem cells into insulin producing cells (IPCs) in vitro has been a promising one to get cell source of β-cell replacement therapy for diabetes. It has been suggested that islets and neurons share features and nestin-positive cells could differentiate into IPCs. We have recently developed a three-dimensional culture system using human peripheral blood cells named as blood-born hematosphere (BBHS). Here we showed that most of BBHS were composed of nestin-positive cells. Under the four-stage differentiation protocol for IPCs, we plated nestin-positive BBHS onto fibronectin-coated dish. These cells form islet-like clusters and most of them expressed insulin. Pancreatic specific genes were turned on, such as transcription factors (Pdx-1, Ngn3 and Nkx6.1), genes related to endocrine function (Glut-2 and PC2) or β cell function (Kir6.2, SUR1). Furthermore islet differentiation was confirmed by dithizone (DTZ) staining to detect zinc ion which binds insulin protein within the cells. Finally, IPCs derived from BBHS showed capability to secrete insulin in response to glucose stimulation. Taken together, our novel protocol successfully induced islet-like human insulin producing cells out of BBHS. This strategy of ex vivo expansion of IPCs using BBHS provides an autologous therapeutic cell source for the treatment of diabetes. PMID:22310720

  8. Generation of insulin-producing cells from gnotobiotic porcine skin-derived stem cells

    SciTech Connect

    Yang, Ji Hoon; Lee, Sung Ho; Heo, Young Tae; Uhm, Sang Jun; Lee, Hoon Taek

    2010-07-09

    A major problem in the treatment of type 1 diabetes mellitus is the limited availability of alternative sources of insulin-producing cells for islet transplantation. In this study, we investigated the effect of bone morphogenetic protein 4 (BMP-4) treatments of gnotobiotic porcine skin-derived stem cells (gSDSCs) on their reprogramming and subsequent differentiation into insulin-producing cells (IPCs). We isolated SDSCs from the ear skin of a gnotobiotic pig. During the proliferation period, the cells expressed stem-cell markers Oct-4, Sox-2, and CD90; nestin expression also increased significantly. The cells could differentiate into IPCs after treatments with activin-A, glucagon-like peptide-1 (GLP-1), and nicotinamide. After 15 days in the differentiation medium, controlled gSDSCs began expressing endocrine progenitor genes and proteins (Ngn3, Neuro-D, PDX-1, NKX2.2, NKX6.1, and insulin). The IPCs showed increased insulin synthesis after glucose stimulation. The results indicate that stem cells derived from the skin of gnotobiotic pigs can differentiate into IPCs under the appropriate conditions in vitro. Our three-stage induction protocol could be applied without genetic modification to source IPCs from stem cells in the skin of patients with diabetes for autologous transplantation.

  9. Cold-sensing regulates Drosophila growth through insulin-producing cells

    PubMed Central

    Li, Qiaoran; Gong, Zhefeng

    2015-01-01

    Across phyla, body size is linked to climate. For example, rearing fruit flies at lower temperatures results in bigger body sizes than those observed at higher temperatures. The underlying molecular basis of this effect is poorly understood. Here we provide evidence that the temperature-dependent regulation of Drosophila body size depends on a group of cold-sensing neurons and insulin-producing cells (IPCs). Electrically silencing IPCs completely abolishes the body size increase induced by cold temperature. IPCs are directly innervated by cold-sensing neurons. Stimulation of these cold-sensing neurons activates IPCs, promotes synthesis and secretion of Drosophila insulin-like peptides and induces a larger body size, mimicking the effects of rearing the flies in cold temperature. Taken together, these findings reveal a neuronal circuit that mediates the effects of low temperature on fly growth. PMID:26648410

  10. Cold-sensing regulates Drosophila growth through insulin-producing cells.

    PubMed

    Li, Qiaoran; Gong, Zhefeng

    2015-01-01

    Across phyla, body size is linked to climate. For example, rearing fruit flies at lower temperatures results in bigger body sizes than those observed at higher temperatures. The underlying molecular basis of this effect is poorly understood. Here we provide evidence that the temperature-dependent regulation of Drosophila body size depends on a group of cold-sensing neurons and insulin-producing cells (IPCs). Electrically silencing IPCs completely abolishes the body size increase induced by cold temperature. IPCs are directly innervated by cold-sensing neurons. Stimulation of these cold-sensing neurons activates IPCs, promotes synthesis and secretion of Drosophila insulin-like peptides and induces a larger body size, mimicking the effects of rearing the flies in cold temperature. Taken together, these findings reveal a neuronal circuit that mediates the effects of low temperature on fly growth. PMID:26648410

  11. Extrinsic Factors Involved in the Differentiation of Stem Cells into Insulin-Producing Cells: An Overview

    PubMed Central

    Wong, Rebecca S. Y.

    2011-01-01

    Diabetes mellitus is a chronic disease with many debilitating complications. Treatment of diabetes mellitus mainly revolves around conventional oral hypoglycaemic agents and insulin replacement therapy. Recently, scientists have turned their attention to the generation of insulin-producing cells (IPCs) from stem cells of various sources. To date, many types of stem cells of human and animal origins have been successfully turned into IPCs in vitro and have been shown to exert glucose-lowering effect in vivo. However, scientists are still faced with the challenge of producing a sufficient number of IPCs that can in turn produce sufficient insulin for clinical use. A careful choice of stem cells, methods, and extrinsic factors for induction may all be contributing factors to successful production of functional beta-islet like IPCs. It is also important that the mechanism of differentiation and mechanism by which IPCs correct hyperglycaemia are carefully studied before they are used in human subjects. PMID:21747828

  12. Insight into Insulin Secretion from Transcriptome and Genetic Analysis of Insulin-Producing Cells of Drosophila

    PubMed Central

    Cao, Jian; Ni, Julie; Ma, Wenxiu; Shiu, Vanessa; Milla, Luis A.; Park, Sangbin; Spletter, Maria L.; Tang, Sheng; Zhang, Jun; Wei, Xing; Kim, Seung K.; Scott, Matthew P.

    2014-01-01

    Insulin-producing cells (IPCs) in the Drosophila brain produce and release insulin-like peptides (ILPs) to the hemolymph. ILPs are crucial for growth and regulation of metabolic activity in flies, functions analogous to those of mammalian insulin and insulin-like growth factors (IGFs). To identify components functioning in IPCs to control ILP production, we employed genomic and candidate gene approaches. We used laser microdissection and messenger RNA sequencing to characterize the transcriptome of larval IPCs. IPCs highly express many genes homologous to genes active in insulin-producing β-cells of the mammalian pancreas. The genes in common encode ILPs and proteins that control insulin metabolism, storage, secretion, β-cell proliferation, and some not previously linked to insulin production or β-cell function. Among these novelties is unc-104, a kinesin 3 family gene, which is more highly expressed in IPCs compared to most other neurons. Knockdown of unc-104 in IPCs impaired ILP secretion and reduced peripheral insulin signaling. Unc-104 appears to transport ILPs along axons. As a complementary approach, we tested dominant-negative Rab genes to find Rab proteins required in IPCs for ILP production or secretion. Rab1 was identified as crucial for ILP trafficking in IPCs. Inhibition of Rab1 in IPCs increased circulating sugar levels, delayed development, and lowered weight and body size. Immunofluorescence labeling of Rab1 showed its tight association with ILP2 in the Golgi of IPCs. Unc-104 and Rab1 join other proteins required for ILP transport in IPCs. PMID:24558258

  13. Insulin-producing cells derived from stem cells: recent progress and future directions.

    PubMed

    Santana, A; Enseñat-Waser, R; Arribas, María Isabel; Reig, J A; Roche, E

    2006-01-01

    Type 1 diabetes is characterized by the selective destruction of pancreatic beta-cells caused by an autoimmune attack. Type 2 diabetes is a more complex pathology which, in addition to beta-cell loss caused by apoptotic programs, includes beta-cell dedifferentiation and peripheric insulin resistance. beta-Cells are responsible for insulin production, storage and secretion in accordance to the demanding concentrations of glucose and fatty acids. The absence of insulin results in death and therefore diabetic patients require daily injections of the hormone for survival. However, they cannot avoid the appearance of secondary complications affecting the peripheral nerves as well as the eyes, kidneys and cardiovascular system. These afflictions are caused by the fact that external insulin injection does not mimic the tight control that pancreatic-derived insulin secretion exerts on the body's glycemia. Restoration of damaged beta-cells by transplantation from exogenous sources or by endocrine pancreas regeneration would be ideal therapeutic options. In this context, stem cells of both embryonic and adult origin (including beta-cell/islet progenitors) offer some interesting alternatives, taking into account the recent data indicating that these cells could be the building blocks from which insulin secreting cells could be generated in vitro under appropriate culture conditions. Although in many cases insulin-producing cells derived from stem cells have been shown to reverse experimentally induced diabetes in animal models, several concerns need to be solved before finding a definite medical application. These refer mainly to the obtainment of a cell population as similar as possible to pancreatic beta-cells, and to the problems related with the immune compatibility and tumor formation. This review will summarize the different approaches that have been used to obtain insulin-producing cells from embryonic and adult stem cells, and the main problems that hamper the

  14. Insulin - producing cells derived from stem cells: recent progress and future directions

    PubMed Central

    Santana, A; Enseñat - Waser, R; Arribas, Maria Isabel; Reig, J A; Roche, E

    2006-01-01

    Type 1 diabetes is characterized by the selective destruction of pancreatic β-cells caused by an autoimmune attack. Type 2 diabetes is a more complex pathology which, in addition to β-cell loss caused by apoptotic programs, includes β-cell dedifferentiation and peripheric insulin resistance. β-Cells are responsible for insulin production, storage and secretion in accordance to the demanding concentrations of glucose and fatty acids. The absence of insulin results in death and therefore diabetic patients require daily injections of the hormone for survival. However, they cannot avoid the appearance of secondary complications affecting the peripheral nerves as well as the eyes, kidneys and cardiovascular system. These afflictions are caused by the fact that external insulin injection does not mimic the tight control that pancreaticderived insulin secretion exerts on the body’s glycemia. Restoration of damaged β-cells by transplantation from exogenous sources or by endocrine pancreas regeneration would be ideal therapeutic options. In this context, stem cells of both embryonic and adult origin (including β-cell/islet progenitors) offer some interesting alternatives, taking into account the recent data indicating that these cells could be the building blocks from which insulin secreting cells could be generated in vitro under appropriate culture conditions. Although in many cases insulin-producing cells derived from stem cells have been shown to reverse experimentally induced diabetes in animal models, several concerns need to be solved before finding a definite medical application. These refer mainly to the obtainment of a cell population as similar as possible to pancreatic β-cells, and to the problems related with the immune compatibility and tumor formation. This review will summarize the different approaches that have been used to obtain insulin-producing cells from embryonic and adult stem cells, and the main problems that hamper the clinical

  15. GH deficiency status combined with GH receptor polymorphism affects response to GH in children.

    PubMed

    Valsesia, Armand; Chatelain, Pierre; Stevens, Adam; Peterkova, Valentina A; Belgorosky, Alicia; Maghnie, Mohamad; Antoniazzi, Franco; Koledova, Ekaterina; Wojcik, Jerome; Farmer, Pierre; Destenaves, Benoit; Clayton, Peter

    2015-12-01

    Meta-analysis has shown a modest improvement in first-year growth response to recombinant human GH (r-hGH) for carriers of the exon 3-deleted GH receptor (GHRd3) polymorphism but with significant interstudy variability. The associations between GHRd3 and growth response to r-hGH over 3 years in relation to severity of GH deficiency (GHD) were investigated in patients from 14 countries. Treatment-naïve pre-pubertal children with GHD were enrolled from the PREDICT studies (NCT00256126 and NCT00699855), categorized by peak GH level (peak GH) during provocation test: ≤4 μg/l (severe GHD; n=45) and >4 to <10 μg/l mild GHD; n=49) and genotyped for the GHRd3 polymorphism (full length (fl/fl, fl/d3, d3/d3). Gene expression (GE) profiles were characterized at baseline. Changes in growth (height (cm) and SDS) over 3 years were measured. There was a dichotomous influence of GHRd3 polymorphism on response to r-hGH, dependent on peak GH level. GH peak level (higher vs lower) and GHRd3 (fl/fl vs d3 carriers) combined status was associated with height change over 3 years (P<0.05). GHRd3 carriers with lower peak GH had lower growth than subjects with fl/fl (median difference after 3 years -3.3 cm; -0.3 SDS). Conversely, GHRd3 carriers with higher peak GH had better growth (+2.7 cm; +0.2 SDS). Similar patterns were observed for GH-dependent biomarkers. GE profiles were significantly different between the groups, indicating that the interaction between GH status and GHRd3 carriage can be identified at a transcriptomic level. This study demonstrates that responses to r-hGH depend on the interaction between GHD severity and GHRd3 carriage. PMID:26340968

  16. GH deficiency status combined with GH receptor polymorphism affects response to GH in children

    PubMed Central

    Valsesia, Armand; Chatelain, Pierre; Stevens, Adam; Peterkova, Valentina A; Belgorosky, Alicia; Maghnie, Mohamad; Antoniazzi, Franco; Koledova, Ekaterina; Wojcik, Jerome; Farmer, Pierre; Destenaves, Benoit; Clayton, Peter

    2015-01-01

    Meta-analysis has shown a modest improvement in first-year growth response to recombinant human GH (r-hGH) for carriers of the exon 3-deleted GH receptor (GHRd3) polymorphism but with significant interstudy variability. The associations between GHRd3 and growth response to r-hGH over 3 years in relation to severity of GH deficiency (GHD) were investigated in patients from 14 countries. Treatment-naïve pre-pubertal children with GHD were enrolled from the PREDICT studies (NCT00256126 and NCT00699855), categorized by peak GH level (peak GH) during provocation test: ≤4 μg/l (severe GHD; n=45) and >4 to <10 μg/l mild GHD; n=49) and genotyped for the GHRd3 polymorphism (full length (fl/fl, fl/d3, d3/d3). Gene expression (GE) profiles were characterized at baseline. Changes in growth (height (cm) and SDS) over 3 years were measured. There was a dichotomous influence of GHRd3 polymorphism on response to r-hGH, dependent on peak GH level. GH peak level (higher vs lower) and GHRd3 (fl/fl vs d3 carriers) combined status was associated with height change over 3 years (P<0.05). GHRd3 carriers with lower peak GH had lower growth than subjects with fl/fl (median difference after 3 years −3.3 cm; −0.3 SDS). Conversely, GHRd3 carriers with higher peak GH had better growth (+2.7 cm; +0.2 SDS). Similar patterns were observed for GH-dependent biomarkers. GE profiles were significantly different between the groups, indicating that the interaction between GH status and GHRd3 carriage can be identified at a transcriptomic level. This study demonstrates that responses to r-hGH depend on the interaction between GHD severity and GHRd3 carriage. PMID:26340968

  17. Three-dimensional differentiation of adipose-derived mesenchymal stem cells into insulin-producing cells.

    PubMed

    Khorsandi, Layasadat; Khodadadi, Ali; Nejad-Dehbashi, Fereshteh; Saremy, Sadegh

    2015-09-01

    The aim of this study is to evaluate the collagen/hyaluronic acid (Col/HA) scaffold effect on the differentiation of insulin-producing cells (IPCs) from adipose-derived mesenchymal stem cells (ASCs). In this experimental study, ASCs were cultured and seeded in a Col/HA scaffold (3D culture) and then treated with induction media. After induction, the presence of IPCs was evaluated using gene expression (PDX-1, GLUT-2 and insulin) analysis and immunocytochemistry, while functional maturity was determined by measuring insulin release in response to low- and high-glucose media. The induced IPCs were morphologically similar to pancreatic islet-like cells. Expression of the islet-associated genes PDX-1, GLUT-2 and insulin genes in 3D-cultured cells was markedly higher than the 2D-cultured cells exposure differentiation media. Compared to the 2D culture of ASCs-derived IPCs, the insulin release from 3D ASCs-derived IPCs showed a nearly 4-fold (p < 0.05) increase when exposed to a high glucose (25 mmol) medium. The percentage of insulin-positive cells in the 3D experimental group showed an approximately 4-fold increase compared to the 2D experimental culture cells. The results of this study demonstrated that the COL/HA scaffold can enhance the differentiation of IPCs from rat ASCs. PMID:25795142

  18. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells.

    PubMed

    Rezania, Alireza; Bruin, Jennifer E; Arora, Payal; Rubin, Allison; Batushansky, Irina; Asadi, Ali; O'Dwyer, Shannon; Quiskamp, Nina; Mojibian, Majid; Albrecht, Tobias; Yang, Yu Hsuan Carol; Johnson, James D; Kieffer, Timothy J

    2014-11-01

    Transplantation of pancreatic progenitors or insulin-secreting cells derived from human embryonic stem cells (hESCs) has been proposed as a therapy for diabetes. We describe a seven-stage protocol that efficiently converts hESCs into insulin-producing cells. Stage (S) 7 cells expressed key markers of mature pancreatic beta cells, including MAFA, and displayed glucose-stimulated insulin secretion similar to that of human islets during static incubations in vitro. Additional characterization using single-cell imaging and dynamic glucose stimulation assays revealed similarities but also notable differences between S7 insulin-secreting cells and primary human beta cells. Nevertheless, S7 cells rapidly reversed diabetes in mice within 40 days, roughly four times faster than pancreatic progenitors. Therefore, although S7 cells are not fully equivalent to mature beta cells, their capacity for glucose-responsive insulin secretion and rapid reversal of diabetes in vivo makes them a promising alternative to pancreatic progenitor cells or cadaveric islets for the treatment of diabetes. PMID:25211370

  19. The GH/IGF-1 axis in ageing and longevity

    PubMed Central

    List, Edward O.; Berryman, Darlene E.; Murrey, John W.

    2014-01-01

    Secretion of growth hormone (GH), and consequently that of insulin-like growth factor 1 (IGF-1), declines over time until only low levels can be detected in individuals aged ≥60 years. This phenomenon, which is known as the ‘somatopause’, has led to recombinant human GH being widely promoted and abused as an antiageing drug, despite lack of evidence of efficacy. By contrast, several mutations that decrease the tone of the GH/IGF-1 axis are associated with extended longevity in mice. In humans, corresponding or similar mutations have been identified, but whether these mutations alter longevity has yet to be established. The powerful effect of reduced GH activity on lifespan extension in mice has generated the hypothesis that pharmaceutically inhibiting, rather than increasing, GH action might delay ageing. Moreover, mice as well as humans with reduced activity of the GH/IGF-1 axis are protected from cancer and diabetes mellitus, two major ageing-related morbidities. Here, we review data on mouse strains with alterations in the GH/IGF-1 axis and their effects on lifespan. The outcome of corresponding or similar mutations in humans is described, as well as the potential mechanisms underlying increased longevity and the therapeutic benefits and risks of medical disruption of the GH/IGF-1 axis in humans. PMID:23591370

  20. Ligand-bound Thyroid Hormone Receptor Contributes to Reprogramming of Pancreatic Acinar Cells into Insulin-producing Cells*

    PubMed Central

    Furuya, Fumihiko; Shimura, Hiroki; Asami, Keiichi; Ichijo, Sayaka; Takahashi, Kazuya; Kaneshige, Masahiro; Oikawa, Yoichi; Aida, Kaoru; Endo, Toyoshi; Kobayashi, Tetsuro

    2013-01-01

    One goal of diabetic regenerative medicine is to instructively convert mature pancreatic exocrine cells into insulin-producing cells. We recently reported that ligand-bound thyroid hormone receptor α (TRα) plays a critical role in expansion of the β-cell mass during postnatal development. Here, we used an adenovirus vector that expresses TRα driven by the amylase 2 promoter (AdAmy2TRα) to induce the reprogramming of pancreatic acinar cells into insulin-producing cells. Treatment with l-3,5,3-triiodothyronine increases the association of TRα with the p85α subunit of phosphatidylinositol 3-kinase (PI3K), leading to the phosphorylation and activation of Akt and the expression of Pdx1, Ngn3, and MafA in purified acinar cells. Analyses performed with the lectin-associated cell lineage tracing system and the Cre/loxP-based direct cell lineage tracing system indicate that newly synthesized insulin-producing cells originate from elastase-expressing pancreatic acinar cells. Insulin-containing secretory granules were identified in these cells by electron microscopy. The inhibition of p85α expression by siRNA or the inhibition of PI3K by LY294002 prevents the expression of Pdx1, Ngn3, and MafA and the reprogramming to insulin-producing cells. In immunodeficient mice with streptozotocin-induced hyperglycemia, treatment with AdAmy2TRα leads to the reprogramming of pancreatic acinar cells to insulin-producing cells in vivo. Our findings suggest that ligand-bound TRα plays a critical role in β-cell regeneration during postnatal development via activation of PI3K signaling. PMID:23595988

  1. Ligand-bound thyroid hormone receptor contributes to reprogramming of pancreatic acinar cells into insulin-producing cells.

    PubMed

    Furuya, Fumihiko; Shimura, Hiroki; Asami, Keiichi; Ichijo, Sayaka; Takahashi, Kazuya; Kaneshige, Masahiro; Oikawa, Yoichi; Aida, Kaoru; Endo, Toyoshi; Kobayashi, Tetsuro

    2013-05-31

    One goal of diabetic regenerative medicine is to instructively convert mature pancreatic exocrine cells into insulin-producing cells. We recently reported that ligand-bound thyroid hormone receptor α (TRα) plays a critical role in expansion of the β-cell mass during postnatal development. Here, we used an adenovirus vector that expresses TRα driven by the amylase 2 promoter (AdAmy2TRα) to induce the reprogramming of pancreatic acinar cells into insulin-producing cells. Treatment with l-3,5,3-triiodothyronine increases the association of TRα with the p85α subunit of phosphatidylinositol 3-kinase (PI3K), leading to the phosphorylation and activation of Akt and the expression of Pdx1, Ngn3, and MafA in purified acinar cells. Analyses performed with the lectin-associated cell lineage tracing system and the Cre/loxP-based direct cell lineage tracing system indicate that newly synthesized insulin-producing cells originate from elastase-expressing pancreatic acinar cells. Insulin-containing secretory granules were identified in these cells by electron microscopy. The inhibition of p85α expression by siRNA or the inhibition of PI3K by LY294002 prevents the expression of Pdx1, Ngn3, and MafA and the reprogramming to insulin-producing cells. In immunodeficient mice with streptozotocin-induced hyperglycemia, treatment with AdAmy2TRα leads to the reprogramming of pancreatic acinar cells to insulin-producing cells in vivo. Our findings suggest that ligand-bound TRα plays a critical role in β-cell regeneration during postnatal development via activation of PI3K signaling. PMID:23595988

  2. Examination of a Viral Infection Mimetic Model in Human iPS Cell-Derived Insulin-Producing Cells and the Anti-Apoptotic Effect of GLP-1 Analogue

    PubMed Central

    Baden, Megu Yamaguchi; Fukui, Kenji; Hosokawa, Yoshiya; Iwahashi, Hiromi; Imagawa, Akihisa; Shimomura, Iichiro

    2015-01-01

    Aims Viral infection is associated with pancreatic beta cell destruction in fulminant type 1 diabetes mellitus. The aim of this study was to investigate the acceleration and protective mechanisms of beta cell destruction by establishing a model of viral infection in pancreatic beta cells. Methods Polyinosinic:polycytidylic acid was transfected into MIN6 cells and insulin-producing cells differentiated from human induced pluripotent stem cells via small molecule applications. Gene expression was analyzed by real-time PCR, and apoptosis was evaluated by caspase-3 activity and TUNEL staining. The anti-apoptotic effect of Exendin-4 was also evaluated. Results Polyinosinic:polycytidylic acid transfection led to elevated expression of the genes encoding IFNα, IFNβ, CXCL10, Fas, viral receptors, and IFN-inducible antiviral effectors in MIN6 cells. Exendin-4 treatment suppressed the elevated gene expression levels and reduced polyinosinic:polycytidylic acid-induced apoptosis both in MIN6 cells and in insulin-producing cells from human induced pluripotent stem cells. Glucagon-like peptide-1 receptor, protein kinase A, and phosphatidylinositol-3 kinase inhibitors counteracted the anti-apoptotic effect of Exendin-4. Conclusions Polyinosinic:polycytidylic acid transfection can mimic viral infection, and Exendin-4 exerted an anti-apoptotic effect both in MIN6 and insulin-producing cells from human induced pluripotent stem cells. PMID:26659307

  3. Transplantation of insulin-producing cells to treat diabetic rats after 90% pancreatectomy

    PubMed Central

    Yu, Ya-Bin; Bian, Jian-Min; Gu, Dian-Hua

    2015-01-01

    AIM: To investigate the effects of transplantation of insulin-producing cells (IPCs) in the treatment of diabetic rats after 90% pancreatectomy. METHODS: Human umbilical cord mesenchymal stem cells (UCMSCs) were isolated and induced into IPCs using differentiation medium. Differentiated cells were examined by dithizone (DTZ) staining, reverse transcription-polymerase chain reaction (RT-PCR), and real-time RT-PCR. C-peptide release, both spontaneously and after glucose challenge, was measured by ELISA. IPCs were then transplanted into Sprague-Dawley rats after 90% pancreatectomy and blood glucose levels and body weight were measured. RESULTS: The differentiated cells were positive for DTZ staining and expressed pancreatic β-cell related genes. C-peptide release by the differentiated cells increased after glucose challenge (380.6 ± 15.32 pmol/L vs 272.4 ± 15.32 pmol/L, P < 0.05). Further, in the cell transplantation group, blood sugar levels were significantly lower than in the sham group 2 wk after transplantation (18.7 ± 2.5 mmol/L vs 25.8 ± 1.25 mmol/L, P < 0.05). Glucose tolerance tests showed that 45 min after intraperitoneal glucose injection, blood glucose levels were significantly lower on day 56 after transplantation of IPCs (12.5 ± 4.7 mmol/L vs 42.2 ± 9.3 mmol/L, P < 0.05). CONCLUSION: Our results show that UCMSCs can differentiate into islet-like cells in vitro under certain conditions, which can function as IPCs both in vivo and in vitro. PMID:26074696

  4. The Drosophila TNF Eiger Is an Adipokine that Acts on Insulin-Producing Cells to Mediate Nutrient Response.

    PubMed

    Agrawal, Neha; Delanoue, Renald; Mauri, Alessandra; Basco, Davide; Pasco, Matthieu; Thorens, Bernard; Léopold, Pierre

    2016-04-12

    Adaptation of organisms to ever-changing nutritional environments relies on sensor tissues and systemic signals. Identification of these signals would help understand the physiological crosstalk between organs contributing to growth and metabolic homeostasis. Here we show that Eiger, the Drosophila TNF-α, is a metabolic hormone that mediates nutrient response by remotely acting on insulin-producing cells (IPCs). In the condition of nutrient shortage, a metalloprotease of the TNF-α converting enzyme (TACE) family is active in fat body (adipose-like) cells, allowing the cleavage and release of adipose Eiger in the hemolymph. In the brain IPCs, Eiger activates its receptor Grindelwald, leading to JNK-dependent inhibition of insulin production. Therefore, we have identified a humoral connexion between the fat body and the brain insulin-producing cells relying on TNF-α that mediates adaptive response to nutrient deprivation. PMID:27076079

  5. From the Cover: Cell-replacement therapy for diabetes: Generating functional insulin-producing tissue from adult human liver cells

    NASA Astrophysics Data System (ADS)

    Sapir, Tamar; Shternhall, Keren; Meivar-Levy, Irit; Blumenfeld, Tamar; Cohen, Hamutal; Skutelsky, Ehud; Eventov-Friedman, Smadar; Barshack, Iris; Goldberg, Iris; Pri-Chen, Sarah; Ben-Dor, Lya; Polak-Charcon, Sylvie; Karasik, Avraham; Shimon, Ilan; Mor, Eytan; Ferber, Sarah

    2005-05-01

    Shortage in tissue availability from cadaver donors and the need for life-long immunosuppression severely restrict the large-scale application of cell-replacement therapy for diabetic patients. This study suggests the potential use of adult human liver as alternate tissue for autologous beta-cell-replacement therapy. By using pancreatic and duodenal homeobox gene 1 (PDX-1) and soluble factors, we induced a comprehensive developmental shift of adult human liver cells into functional insulin-producing cells. PDX-1-treated human liver cells express insulin, store it in defined granules, and secrete the hormone in a glucose-regulated manner. When transplanted under the renal capsule of diabetic, immunodeficient mice, the cells ameliorated hyperglycemia for prolonged periods of time. Inducing developmental redirection of adult liver offers the potential of a cell-replacement therapy for diabetics by allowing the patient to be the donor of his own insulin-producing tissue. pancreas | transdifferentiation

  6. Peribiliary Glands as a Niche of Extrapancreatic Precursors Yielding Insulin-Producing Cells in Experimental and Human Diabetes.

    PubMed

    Carpino, Guido; Puca, Rosa; Cardinale, Vincenzo; Renzi, Anastasia; Scafetta, Gaia; Nevi, Lorenzo; Rossi, Massimo; Berloco, Pasquale B; Ginanni Corradini, Stefano; Reid, Lola M; Maroder, Marella; Gaudio, Eugenio; Alvaro, Domenico

    2016-05-01

    Peribiliary glands (PBGs) are niches in the biliary tree and containing heterogeneous endodermal stem/progenitors cells that can differentiate, in vitro and in vivo, toward pancreatic islets. The aim of this study was to evaluate, in experimental and human diabetes, proliferation of cells in PBGs and differentiation of the biliary tree stem/progenitor cells (BTSCs) toward insulin-producing cells. Diabetes was generated in mice by intraperitoneal injection of a single dose of 200 mg/kg (N = 12) or 120 mg/kg (N = 12) of streptozotocin. Liver, pancreas, and extrahepatic biliary trees were en bloc dissected and examined. Cells in PBGs proliferated in experimental diabetes, and their proliferation was greatest in the PBGs of the hepatopancreatic ampulla, and inversely correlated with the pancreatic islet area. In rodents, the cell proliferation in PBGs was characterized by the expansion of Sox9-positive stem/progenitor cells that gave rise to insulin-producing cells. Insulin-producing cells were located mostly in PBGs in the portion of the biliary tree closest to the duodenum, and their appearance was associated with upregulation of MafA and Gli1 gene expression. In patients with type 2 diabetes, PBGs at the level of the hepatopancreatic ampulla contained cells showing signs of proliferation and pancreatic fate commitment. In vitro, high glucose concentrations induced the differentiation of human BTSCs cultures toward pancreatic beta cell fates. The cells in PBGs respond to diabetes with proliferation and differentiation towards insulin-producing cells indicating that PBG niches may rescue pancreatic islet impairment in diabetes. These findings offer important implications for the pathophysiology and complications of this disease. Stem Cells 2016;34:1332-1342. PMID:26850087

  7. Mitochondrial Respiration in Insulin-Producing β-Cells: General Characteristics and Adaptive Effects of Hypoxia

    PubMed Central

    Ma, Zuheng; Scholz, Hanne; Björklund, Anneli; Grill, Valdemar

    2015-01-01

    Objective To provide novel insights on mitochondrial respiration in β-cells and the adaptive effects of hypoxia. Methods and Design Insulin-producing INS-1 832/13 cells were exposed to 18 hours of hypoxia followed by 20–22 hours re-oxygenation. Mitochondrial respiration was measured by high-resolution respirometry in both intact and permeabilized cells, in the latter after establishing three functional substrate-uncoupler-inhibitor titration (SUIT) protocols. Concomitant measurements included proteins of mitochondrial complexes (Western blotting), ATP and insulin secretion. Results Intact cells exhibited a high degree of intrinsic uncoupling, comprising about 50% of oxygen consumption in the basal respiratory state. Hypoxia followed by re-oxygenation increased maximal overall respiration. Exploratory experiments in peremabilized cells could not show induction of respiration by malate or pyruvate as reducing substrates, thus glutamate and succinate were used as mitochondrial substrates in SUIT protocols. Permeabilized cells displayed a high capacity for oxidative phosphorylation for both complex I- and II-linked substrates in relation to maximum capacity of electron transfer. Previous hypoxia decreased phosphorylation control of complex I-linked respiration, but not in complex II-linked respiration. Coupling control ratios showed increased coupling efficiency for both complex I- and II-linked substrates in hypoxia-exposed cells. Respiratory rates overall were increased. Also previous hypoxia increased proteins of mitochondrial complexes I and II (Western blotting) in INS-1 cells as well as in rat and human islets. Mitochondrial effects were accompanied by unchanged levels of ATP, increased basal and preserved glucose-induced insulin secretion. Conclusions Exposure of INS-1 832/13 cells to hypoxia, followed by a re-oxygenation period increases substrate-stimulated respiratory capacity and coupling efficiency. Such effects are accompanied by up-regulation of

  8. Identification of miRNAs Involved in Reprogramming Acinar Cells into Insulin Producing Cells

    PubMed Central

    Teichenne, Joan; Morró, Meritxell; Casellas, Alba; Jimenez, Veronica; Tellez, Noelia; Leger, Adrien; Bosch, Fatima; Ayuso, Eduard

    2015-01-01

    Reprogramming acinar cells into insulin producing cells using adenoviral (Ad)-mediated delivery of Pdx1, Ngn3 and MafA (PNM) is an innovative approach for the treatment of diabetes. Here, we aimed to investigate the molecular mechanisms involved in this process and in particular, the role of microRNAs. To this end, we performed a comparative study of acinar-to-β cell reprogramming efficiency in the rat acinar cell line AR42J and its subclone B13 after transduction with Ad-PNM. B13 cells were more efficiently reprogrammed than AR42J cells, which was demonstrated by a strong activation of β cell markers (Ins1, Ins2, IAPP, NeuroD1 and Pax4). miRNome panels were used to analyze differentially expressed miRNAs in acinar cells under four experimental conditions (i) non-transduced AR42J cells, (ii) non-transduced B13 cells, (iii) B13 cells transduced with Ad-GFP vectors and (iv) B13 cells transduced with Ad-PNM vectors. A total of 59 miRNAs were found to be differentially expressed between non-transduced AR42J and B13 cells. Specifically, the miR-200 family was completely repressed in B13 cells, suggesting that these cells exist in a less differentiated state than AR42J cells and as a consequence they present a greater plasticity. Adenoviral transduction per se induced dedifferentiation of acinar cells and 11 miRNAs were putatively involved in this process, whereas 8 miRNAs were found to be associated with PNM expression. Of note, Ad-PNM reprogrammed B13 cells presented the same levels of miR-137-3p, miR-135a-5p, miR-204-5p and miR-210-3p of those detected in islets, highlighting their role in the process. In conclusion, this study led to the identification of miRNAs that might be of compelling importance to improve acinar-to-β cell conversion for the future treatment of diabetes. PMID:26690959

  9. PDX1-engineered embryonic stem cell-derived insulin producing cells regulate hyperglycemia in diabetic mice

    PubMed Central

    2012-01-01

    Background Type 1 diabetes can be treated by the transplantation of cadaveric whole pancreata or isolated pancreatic islets. However, this form of treatment is hampered by the chronic shortage of cadaveric donors. Embryonic stem (ES) cell-derived insulin producing cells (IPCs) offer a potentially novel source of unlimited cells for transplantation to treat type 1 and possibly type 2 diabetes. However, thus far, the lack of a reliable protocol for efficient differentiation of ES cells into IPCs has hindered the clinical exploitation of these cells. Methods To efficiently generate IPCs using ES cells, we have developed a double transgenic ES cell line R1Pdx1AcGFP/RIP-Luc that constitutively expresses pancreatic β-cell-specific transcription factor pancreatic and duodenal homeobox gene 1 (Pdx1) as well as rat insulin promoter (RIP) driven luciferase reporter. We have established several protocols for the reproducible differentiation of ES cells into IPCs. The differentiation of ES cells into IPCs was monitored by immunostaining as well as real-time quantitative RT-PCR for pancreatic β-cell-specific markers. Pancreatic β-cell specific RIP became transcriptionally active following the differentiation of ES cells into IPCs and induced the expression of the luciferase reporter. Glucose stimulated insulin secretion by the ES cell-derived IPCs was measured by ELISA. Further, we have investigated the therapeutic efficacy of ES cell-derived IPCs to correct hyperglycemia in syngeneic streptozotocin (STZ)-treated diabetic mice. The long term fate of the transplanted IPCs co-expressing luciferase in syngeneic STZ-induced diabetic mice was monitored by real time noninvasive in vivo bioluminescence imaging (BLI). Results We have recently demonstrated that spontaneous in vivo differentiation of R1Pdx1AcGFP/RIP-Luc ES cell-derived pancreatic endoderm-like cells (PELCs) into IPCs corrects hyperglycemia in diabetic mice. Here, we investigated whether R1Pdx1AcGFP/RIP-Luc ES cells

  10. Insulin producing cells established using non-integrated lentiviral vector harboring PDX1 gene

    PubMed Central

    Boroujeni, Zahra Niki; Aleyasin, Ahmad

    2013-01-01

    AIM: To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene. METHODS: In this study, human adipose tissue derived stem cells (hADSCs) were obtained from abdominal adipose tissues by liposuction, selected by plastic adhesion, and characterized by flow cytometric analysis. Human ADSCs were differentiated into adipocytes and osteocytes using differentiating medium to confirm their multipotency. Non-integrated lentiviruses harboring PDX1 (Non-integrated LV-PDX1) were constructed using specific plasmids (pLV-HELP, pMD2G, LV-105-PDX1-1). Then, hADSCs were transduced with non-integrated LV-PDX1. After transduction, ADSCsPDX1+ were cultured in high glucose DMEM medium supplement by B27, nicotinamide and βFGF for 21 d. Expressions of PDX1 and insulin were detected at protein level by immunofluorescence analysis. Expressions of PDX1, neurogenin3 (Ngn3), glucagon, glucose transporter2 (Glut2) and somatostatin as specific marker genes were investigated at mRNA level by quantitative RT-PCR. Insulin secretion of hADSCsPDX1+ in the high-glucose medium was detected by electrochemiluminescence test. Human ADSCsPDX1+ were implanted into hyperglycemic rats. RESULTS: Human ADSCs exhibited their fibroblast-like morphology and made colonies after 7-10 d of culture. Determination of hADSCs identified by FACS analysis showed that hADSCs were positive for mesenchymal cell markers and negative for hematopoietic cell markers that guaranteed the lack of hematopoietic contamination. In vitro differentiation of hADSCs into osteocytes and adipocytes were detected by Alizarin red and Oil red O staining and confirmed their multilineage differentiation ability. Transduced hADSCs+PDX1 became round and clusters in the differentiation medium. The appropriate expression of PDX1 and insulin proteins was confirmed using immunocytochemistry analysis. Significant expressions of PDX1, Ngn3, glucagon, Glut2 and

  11. LysGH15 kills Staphylococcus aureus without being affected by the humoral immune response or inducing inflammation.

    PubMed

    Zhang, Lei; Li, Dong; Li, Xinwei; Hu, Liyuan; Cheng, Mengjun; Xia, Feifei; Gong, Pengjuan; Wang, Bin; Ge, Jinli; Zhang, Hao; Cai, Ruopeng; Wang, Yanmei; Sun, Changjiang; Feng, Xin; Lei, Liancheng; Han, Wenyu; Gu, Jingmin

    2016-01-01

    The lysin LysGH15, derived from the staphylococcal phage GH15, exhibits a wide lytic spectrum and highly efficient lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we found that LysGH15 did not induce resistance in MRSA or methicillin-sensitive S. aureus (MSSA) strains after repeated treatment. Although LysGH15 triggered the generation of LysGH15-specific antibodies in mice, these antibodies did not block lytic activity in vitro (nor the binding capacity of LysGH15). More importantly, when the antibody titre was highest in mice immunized with LysGH15, a single intravenous injection of LysGH15 was sufficient to protect mice against lethal infection with MRSA. These results indicated that LysGH15-specific antibodies did not affect the killing efficiency of LysGH15 against MRSA in vitro or in vivo. LysGH15 also reduced pro-inflammatory cytokines in mice with lethal infections. Furthermore, a high-dose LysGH15 injection did not cause significant adverse effects or pathological changes in the main organs of treated animals. These results provide further evidence for the administration of LysGH15 as an alternative strategy for the treatment of infections caused by MRSA. PMID:27385518

  12. LysGH15 kills Staphylococcus aureus without being affected by the humoral immune response or inducing inflammation

    PubMed Central

    Zhang, Lei; Li, Dong; Li, Xinwei; Hu, Liyuan; Cheng, Mengjun; Xia, Feifei; Gong, Pengjuan; Wang, Bin; Ge, Jinli; Zhang, Hao; Cai, Ruopeng; Wang, Yanmei; Sun, Changjiang; Feng, Xin; Lei, Liancheng; Han, Wenyu; Gu, Jingmin

    2016-01-01

    The lysin LysGH15, derived from the staphylococcal phage GH15, exhibits a wide lytic spectrum and highly efficient lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we found that LysGH15 did not induce resistance in MRSA or methicillin-sensitive S. aureus (MSSA) strains after repeated treatment. Although LysGH15 triggered the generation of LysGH15-specific antibodies in mice, these antibodies did not block lytic activity in vitro (nor the binding capacity of LysGH15). More importantly, when the antibody titre was highest in mice immunized with LysGH15, a single intravenous injection of LysGH15 was sufficient to protect mice against lethal infection with MRSA. These results indicated that LysGH15-specific antibodies did not affect the killing efficiency of LysGH15 against MRSA in vitro or in vivo. LysGH15 also reduced pro-inflammatory cytokines in mice with lethal infections. Furthermore, a high-dose LysGH15 injection did not cause significant adverse effects or pathological changes in the main organs of treated animals. These results provide further evidence for the administration of LysGH15 as an alternative strategy for the treatment of infections caused by MRSA. PMID:27385518

  13. Factors expressed by murine embryonic pancreatic mesenchyme enhance generation of insulin-producing cells from hESCs.

    PubMed

    Guo, Tingxia; Landsman, Limor; Li, Na; Hebrok, Matthias

    2013-05-01

    Islet transplantation has proven to be a successful strategy to restore normoglycemia in patients with type 1 diabetes (T1D). However, the dearth of cadaveric islets available for transplantation hampers the widespread application of this treatment option. Although human embryonic stem cells and induced pluripotent stem cells are capable of generating insulin-producing cells in vitro when provided with the appropriate inductive cues, the insulin-expressing cells that develop behave more like immature β-cells with minimal sensitivity to glucose stimulation. Here, we identify a set of signaling factors expressed in mouse embryonic mesenchyme during the time when foregut and pancreatic progenitors are specified and test their activities during in vitro differentiation of human embryonic stem cells. Several of the identified factors work in concert to expand the pancreatic progenitor pool. Interestingly, transforming growth factor (TGF)-β ligands, most potent in inducing pancreatic progenitors, display strong inhibitory effects on subsequent endocrine cell differentiation. Treatment with TGF-β ligands, followed by the addition of a TGF-β receptor antagonist, dramatically increased the number of insulin-producing cells in vitro, demonstrating the need for dynamic temporal regulation of TGF-β signaling during in vitro differentiation. These studies illustrate the need to precisely mimic the in vivo conditions to fully recapitulate pancreatic lineage specification in vitro. PMID:23305648

  14. Elevated GH/IGF-I promotes mammary tumors in high-fat, but not low-fat, fed mice.

    PubMed

    Gahete, Manuel D; Córdoba-Chacón, José; Lantvit, Daniel D; Ortega-Salas, Rosa; Sanchez-Sanchez, Rafael; Pérez-Jiménez, Francisco; López-Miranda, José; Swanson, Steven M; Castaño, Justo P; Luque, Raúl M; Kineman, Rhonda D

    2014-11-01

    Growth hormone (GH) and/or insulin-like growth factor I (IGF-I) are thought to promote breast cancer based on reports showing circulating IGF-I levels correlate, in epidemiological studies, with breast cancer risk. Also, mouse models with developmental GH/IGF-I deficiency/resistance are less susceptible to genetic- or chemical-induced mammary tumorigenesis. However, given the metabolic properties of GH, medical strategies have been considered to raise GH to improve body composition and metabolic function in elderly and obese patients. Since hyperlipidemia, inflammation, insulin resistance and obesity increase breast cancer risk, elevating GH may serve to exacerbate cancer progression. To better understand the role GH/IGF-I plays in tumor formation, this study used unique mouse models to determine if reducing GH/IGF-I in adults protects against 7,12-dimethylbenz[α]anthracene (DMBA)-induced mammary tumor development, and if moderate elevations in endogenous GH/IGF-I alter DMBA-induced tumorigenesis in mice fed a standard-chow diet or in mice with altered metabolic function due to high-fat feeding. We observed that adult-onset isolated GH-deficient mice, which also have reduced IGF-I levels, were less susceptible to DMBA-treatment. Specifically, fewer adult-onset isolated GH-deficient mice developed mammary tumors compared with GH-replete controls. In contrast, chow-fed mice with elevated endogenous GH/IGF-I (HiGH mice) were not more susceptible to DMBA-treatment. However, high-fat-fed, HiGH mice showed reduced tumor latency and increased tumor incidence compared with diet-matched controls. These results further support a role of GH/IGF-I in regulating mammary tumorigenesis but suggest the ultimate consequences of GH/IGF-I on breast tumor development are dependent on the diet and/or metabolic status. PMID:25085903

  15. Usefulness of urinary growth hormone (GH) measurement for evaluating endogenous GH secretion in acromegaly.

    PubMed

    Mauri, M; Picó, A M; Alfayate, R; Dominguez, J R; Cámara, R; Miralles, C

    1993-01-01

    We investigated the relationship between urinary growth hormone (u-GH) and spontaneous 24-hour plasma GH secretion in 15 acromegalic patients. To measure u-GH, we have developed a method based on concentrating the sample by centrifugal ultrafiltration and then performing an immunoradiometric assay using commercially available reagents. u-GH correlated well with the integrated concentration of plasma GH (r = 0.66, p < 0.02). Additionally, u-GH excretion in acromegalic patients was significantly higher than in the control group (190 +/- 100 vs. 3.89 +/- 0.56 pg/min, mean +/- SEM, p < 0.001). Immunoreactive u-GH showed the same elution pattern in Sephadex G-75 as standard or labeled hGH, proving that the substance measured in urine is authentic GH. In conclusion, u-GH appears to be a simple, noninvasive and inexpensive test for evaluating GH secretion in active acromegaly. PMID:8406334

  16. Three-dimensional printed polymeric system to encapsulate human mesenchymal stem cells differentiated into islet-like insulin-producing aggregates for diabetes treatment

    PubMed Central

    Sabek, Omaima M; Farina, Marco; Fraga, Daniel W; Afshar, Solmaz; Ballerini, Andrea; Filgueira, Carly S; Thekkedath, Usha R; Grattoni, Alessandro; Gaber, A Osama

    2016-01-01

    Diabetes is one of the most prevalent, costly, and debilitating diseases in the world. Pancreas and islet transplants have shown success in re-establishing glucose control and reversing diabetic complications. However, both are limited by donor availability, need for continuous immunosuppression, loss of transplanted tissue due to dispersion, and lack of vascularization. To overcome the limitations of poor islet availability, here, we investigate the potential of bone marrow–derived mesenchymal stem cells differentiated into islet-like insulin-producing aggregates. Islet-like insulin-producing aggregates, characterized by gene expression, are shown to be similar to pancreatic islets and display positive immunostaining for insulin and glucagon. To address the limits of current encapsulation systems, we developed a novel three-dimensional printed, scalable, and potentially refillable polymeric construct (nanogland) to support islet-like insulin-producing aggregates’ survival and function in the host body. In vitro studies showed that encapsulated islet-like insulin-producing aggregates maintained viability and function, producing steady levels of insulin for at least 4 weeks. Nanogland—islet-like insulin-producing aggregate technology here investigated as a proof of concept holds potential as an effective and innovative approach for diabetes cell therapy. PMID:27152147

  17. 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

  18. Differentiation of PDX1 gene-modified human umbilical cord mesenchymal stem cells into insulin-producing cells in vitro.

    PubMed

    He, Dongmei; Wang, Juan; Gao, Yangjun; Zhang, Yuan

    2011-12-01

    Mesenchymal stem cells (MSCs) have significant advantages over other stem cell types, and greater potential for immediate clinical application. MSCs would be an interesting cellular source for treatment of type 1 diabetes. In this study, MSCs from human umbilical cord were differentiated into functional insulin-producing cells in vitro by introduction of the pancreatic and duodenal homeobox factor 1 (PDX1) and in the presence of induction factors. The expressions of cell surface antigens were detected by flow cytometry. After induction in an adipogenic medium or an osteogenic medium, the cells were observed by Oil Red O staining and alkaline phosphatase staining. Recombinant adenovirus carrying the PDX1 gene was constructed and MSCs were infected by the recombinant adenovirus, then treated with several inducing factors for differentiation into islet β-like cells. The expression of the genes and protein related to islet β-cells was detected by immunocytochemistry, RT-PCR and Western blot analysis. Insulin and C-peptide secretion were assayed. Our results show that the morphology and immunophenotype of MSCs from human umbilical cord were similar to those present in human bone marrow. The MSCs could be induced to differentiate into osteocytes and adipocytes. After induction by recombined adenovirus vector with induction factors, MSCs were aggregated and presented islet-like bodies. Dithizone staining of these cells was positive. The genes' expression related to islet β-cells was found. After induction, insulin and C-peptide secretion in the supernatant were significantly increased. In conclusion, our results demonstrated that PDX1 gene-modified human umbilical cord mesenchymal stem cells could be differentiated into insulin-producing cells in vitro. PMID:21837359

  19. Insulin-Producing Cells Differentiated from Human Bone Marrow Mesenchymal Stem Cells In Vitro Ameliorate Streptozotocin-Induced Diabetic Hyperglycemia

    PubMed Central

    Xin, Ying; Jiang, Xin; Wang, Yishu; Su, Xuejin; Sun, Meiyu; Zhang, Lihong; Tan, Yi; Wintergerst, Kupper A.; Li, Yan; Li, Yulin

    2016-01-01

    Background The two major obstacles in the successful transplantation of islets for diabetes treatment are inadequate supply of insulin-producing tissue and immune rejection. Induction of the differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) into insulin-producing cells (IPCs) for autologous transplantation may alleviate those limitations. Methods hMSCs were isolated and induced to differentiate into IPCs through a three-stage differentiation protocol in a defined media with high glucose, nicotinamide, and exendin-4. The physiological characteristics and functions of IPCs were then evaluated. Next, about 3 × 106 differentiated cells were transplanted into the renal sub-capsular space of streptozotocin (STZ)-induced diabetic nude mice. Graft survival and function were assessed by immunohistochemistry, TUNEL staining and measurements of blood glucose levels in the mice. Results The differentiated IPCs were characterized by Dithizone (DTZ) positive staining, expression of pancreatic β-cell markers, and human insulin secretion in response to glucose stimulation. Moreover, 43% of the IPCs showed L-type Ca2+ channel activity and similar changes in intracellular Ca2+ in response to glucose stimulation as that seen in pancreatic β-cells in the process of glucose-stimulated insulin secretion. Transplantation of functional IPCs into the renal subcapsular space of STZ-induced diabetic nude mice ameliorated the hyperglycemia. Immunofluorescence staining revealed that transplanted IPCs sustainably expressed insulin, c-peptide, and PDX-1 without apparent apoptosis in vivo. Conclusions IPCs derived from hMSCs in vitro can ameliorate STZ-induced diabetic hyperglycemia, which indicates that these hMSCs may be a promising approach to overcome the limitations of islet transplantation. PMID:26756576

  20. GH13 amylosucrases and GH70 branching sucrases, atypical enzymes in their respective families.

    PubMed

    Moulis, Claire; André, Isabelle; Remaud-Simeon, Magali

    2016-07-01

    Amylosucrases and branching sucrases are α-retaining transglucosylases found in the glycoside-hydrolase families 13 and 70, respectively, of the clan GH-H. These enzymes display unique activities in their respective families. Using sucrose as substrate and without mediation of nucleotide-activated sugars, amylosucrase catalyzes the formation of an α-(1 → 4) linked glucan that resembles amylose. In contrast, the recently discovered branching sucrases are unable to catalyze polymerization of glucosyl units as they are rather specific for dextran branching through α-(1 → 2) or α-(1 → 3) branching linkages depending on the enzyme regiospecificity. In addition, GH13 amylosucrases and GH70 branching sucrases are naturally promiscuous and can glucosylate different types of acceptor molecules including sugars, polyols, or flavonoids. Amylosucrases have been the most investigated glucansucrases, in particular to control product profiles or to successfully develop tailored α-transglucosylases able to glucosylate various molecules of interest, for example, chemically protected carbohydrates that are planned to enter in chemoenzymatic pathways. The structural traits of these atypical enzymes will be described and compared, and an overview of the potential of natural or engineered enzymes for glycodiversification and chemoenzymatic synthesis will be highlighted. PMID:27141938

  1. A Variant of GJD2, Encoding for Connexin 36, Alters the Function of Insulin Producing β-Cells

    PubMed Central

    Cigliola, Valentina; Populaire, Celine; Pierri, Ciro L.; Deutsch, Samuel; Haefliger, Jacques-Antoine; Fadista, João; Lyssenko, Valeriya; Groop, Leif; Rueedi, Rico; Thorel, Fabrizio; Herrera, Pedro Luis; Meda, Paolo

    2016-01-01

    Signalling through gap junctions contributes to control insulin secretion and, thus, blood glucose levels. Gap junctions of the insulin-producing β-cells are made of connexin 36 (Cx36), which is encoded by the GJD2 gene. Cx36-null mice feature alterations mimicking those observed in type 2 diabetes (T2D). GJD2 is also expressed in neurons, which share a number of common features with pancreatic β-cells. Given that a synonymous exonic single nucleotide polymorphism of human Cx36 (SNP rs3743123) associates with altered function of central neurons in a subset of epileptic patients, we investigated whether this SNP also caused alterations of β-cell function. Transfection of rs3743123 cDNA in connexin-lacking HeLa cells resulted in altered formation of gap junction plaques and cell coupling, as compared to those induced by wild type (WT) GJD2 cDNA. Transgenic mice expressing the very same cDNAs under an insulin promoter revealed that SNP rs3743123 expression consistently lead to a post-natal reduction of islet Cx36 levels and β-cell survival, resulting in hyperglycemia in selected lines. These changes were not observed in sex- and age-matched controls expressing WT hCx36. The variant GJD2 only marginally associated to heterogeneous populations of diabetic patients. The data document that a silent polymorphism of GJD2 is associated with altered β-cell function, presumably contributing to T2D pathogenesis. PMID:26959991

  2. Human iPS Cell-Derived Insulin Producing Cells Form Vascularized Organoids under the Kidney Capsules of Diabetic Mice

    PubMed Central

    Raikwar, Sudhanshu P.; Kim, Eun-Mi; Sivitz, William I.; Allamargot, Chantal; Thedens, Daniel R.; Zavazava, Nicholas

    2015-01-01

    Type 1 diabetes (T1D) is caused by autoimmune disease that leads to the destruction of pancreatic β-cells. Transplantation of cadaveric pancreatic organs or pancreatic islets can restore normal physiology. However, there is a chronic shortage of cadaveric organs, limiting the treatment of the majority of patients on the pancreas transplantation waiting list. Here, we hypothesized that human iPS cells can be directly differentiated into insulin producing cells (IPCs) capable of secreting insulin. Using a series of pancreatic growth factors, we successfully generated iPS cells derived IPCs. Furthermore, to investigate the capability of these cells to secrete insulin in vivo, the differentiated cells were transplanted under the kidney capsules of diabetic immunodeficient mice. Serum glucose levels gradually declined to either normal or near normal levels over 150 days, suggesting that the IPCs were secreting insulin. In addition, using MRI, a 3D organoid appeared as a white patch on the transplanted kidneys but not on the control kidneys. These organoids showed neo-vascularization and stained positive for insulin and glucagon. All together, these data show that a pancreatic organ can be created in vivo providing evidence that iPS cells might be a novel option for the treatment of T1D. PMID:25629318

  3. A Systematic Comparison of Purification and Normalization Protocols for Quantitative MicroRNA Expressional Profiling in Insulin-Producing Cells

    PubMed Central

    Vestergaard, Anna Lindeløv; Blankestijn, Maaike; Stahl, Jonathan Lucien; Pallesen, Emil Marek Heymans; Bang-Berthelsen, Claus Heiner; Pociot, Flemming; Novotny, Guy Wayne; Lundh, Morten; Mandrup-Poulsen, Thomas

    2016-01-01

    As microRNAs (miRs) are gaining increasing attention as key regulators of cellular processes, expressional quantification is widely applied. However, in the processing of relatively quantified data, the importance of testing the stability of several reference mRNAs and/or miRs and choosing among these for normalization is often overlooked, potentially leading to biased results. Here, we have optimized the purification of miR-enriched total RNA from pancreatic insulin-producing INS-1 cells. Additionally, we optimized and analyzed miR expression by a qPCR-based microarray and by specific qPCR and tested the stability of candidate reference mRNAs and miRs. Hence, this study gives a widely applicable example on how to easily and systematically test and decide how to normalize miR quantification. We suggest that caution in the interpretation of miR quantification studies that do not comprise stability analysis should be exerted. PMID:27338345

  4. Induced ICER I{gamma} down-regulates cyclin A expression and cell proliferation in insulin-producing {beta} cells

    SciTech Connect

    Inada, Akari; Weir, Gordon C.; Bonner-Weir, Susan . E-mail: susan.bonner-weir@joslin.harvard.edu

    2005-04-15

    We have previously found that cyclin A expression is markedly reduced in pancreatic {beta}-cells by cell-specific overexpression of repressor inducible cyclic AMP early repressor (ICER I{gamma}) in transgenic mice. Here we further examined regulatory effects of ICER I{gamma} on cyclin A gene expression using Min6 cells, an insulin-producing cell line. The cyclin A promoter luciferase assay showed that ICER I{gamma} directly repressed cyclin A gene transcription. In addition, upon ICER I{gamma} overexpression, cyclin A mRNA levels markedly decreased, thereby confirming an inhibitory effect of ICER I{gamma} on cyclin A expression. Suppression of cyclin A results in inhibition of BrdU incorporation. Under normal culture conditions endogenous cyclin A is abundant in these cells, whereas ICER is hardly detectable. However, serum starvation of Min6 cells induces ICER I{gamma} expression with a concomitant very low expression level of cyclin A. Cyclin A protein is not expressed unless the cells are in active DNA replication. These results indicate a potentially important anti-proliferative effect of ICER I{gamma} in pancreatic {beta} cells. Since ICER I{gamma} is greatly increased in diabetes as well as in FFA- or high glucose-treated islets, this effect may in part exacerbate diabetes by limiting {beta}-cell proliferation.

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

    PubMed

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

    2003-06-10

    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. PMID:12756298

  6. 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.

  7. Differentiation of Human Mesenchymal Stem Cells into Insulin Producing Cells by Using A Lentiviral Vector Carrying PDX1

    PubMed Central

    Allahverdi, Amir; Abroun, Saied; Jafarian, Arefeh; Soleimani, Masoud; Taghikhani, Mohammad; Eskandari, Fatemeh

    2015-01-01

    Objective Type I diabetes is an immunologically-mediated devastation of insulin producing cells (IPCs) in the pancreatic islet. Stem cells that produce β-cells are a new promising tool. Adult stem cells such as mesenchymal stem cells (MSCs) are self renewing multi potent cells showing capabilities to differentiate into ectodermal, mesodermal and endodermal tissues. Pancreatic and duodenal homeobox factor 1 (PDX1) is a master regulator gene required for embryonic development of the pancreas and is crucial for normal pancreatic islets activities in adults. Materials and Methods We induced the over-expression of the PDX1 gene in human bone marrow MSCs (BM-MSCs) by Lenti-PDX1 in order to generate IPCs. Next, we examine the ability of the cells by measuring insulin/c-peptide production and INSULIN and PDX1 gene expressions. Results After transduction, MSCs changed their morphology at day 5 and gradually differentiated into IPCs. INSULIN and PDX1 expressions were confirmed by real time polymerase chain reaction (RT-PCR) and immunostaining. IPC secreted insulin and C-peptide in the media that contained different glucose concentrations. Conclusion MSCs differentiated into IPCs by genetic manipulation. Our result showed that lentiviral vectors could deliver PDX1 gene to MSCs and induce pancreatic differentiation. PMID:26199902

  8. Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors.

    PubMed

    Koblas, Tomas; Leontovyc, Ivan; Loukotova, Sarka; Kosinova, Lucie; Saudek, Frantisek

    2016-01-01

    Direct reprogramming of pancreatic nonendocrine cells into insulin-producing β-cells represents a promising approach for the treatment of insulin-dependent diabetes. However, its clinical application is limited by the potential for insertional mutagenesis associated with the viral vectors currently used for cell reprogramming. With the aim of developing a nonintegrative reprogramming strategy for derivation of insulin-producing cells, here, we evaluated a new approach utilizing synthetic messenger RNAs encoding reprogramming transcription factors. Administration of synthetic mRNAs encoding three key transcription regulators of β-cell differentiation-Pdx1, Neurogenin3, and MafA-efficiently reprogrammed the pancreatic exocrine cells into insulin-producing cells. In addition to the insulin genes expression, the synthetic mRNAs also induced the expressions of genes important for proper pancreatic β-cell function, including Sur1, Kir6.2, Pcsk1, and Pcsk2. Pretreating cells with the chromatin-modifying agent 5-Aza-2'-deoxycytidine further enhanced reprogramming efficiency, increasing the proportion of insulin-producing cells from 3.5 ± 0.9 to 14.3 ± 1.9% (n = 4). Moreover, 5-Aza-2'-deoxycytidine pretreatment enabled the reprogrammed cells to respond to glucose challenge with increased insulin secretion. In conclusion, our results support that the reprogramming of pancreatic exocrine cells into insulin-producing cells, induced by synthetic mRNAs encoding pancreatic transcription factors, represents a promising approach for cell-based diabetes therapy. PMID:27187823

  9. Functional characterization of GH-like homolog in amphioxus reveals an ancient origin of GH/GH receptor system.

    PubMed

    Li, Mengyang; Gao, Zhan; Ji, Dongrui; Zhang, Shicui

    2014-12-01

    Amphioxus belongs to the subphylum cephalochordata, an extant representative of the most basal chordates. Despite many studies on the endocrine system of amphioxus, no evidence showed the presence of pituitary hormones. In this study, we clearly demonstrated the existence of a functional GH-like hormone in amphioxus, which is able to bind purified GH receptors, stimulate IGF-I expression, promote growth rate of fish, and rescue embryonic defects caused by a shortage of GH. We also showed the presence of a GH/prolactin-like-binding protein containing the entire hormone binding domain of GH/prolactin receptors in amphioxus, which is widely expressed among tissues, and interacts with the GH-like hormone. It is clear from these results that the GH/GH receptor-like system is present in amphioxus and, hence, in all classes of chordates. Notably, the GH-like hormone appears to be the only member of the vertebrate pituitary hormones family in amphioxus, suggesting that the hormone is the ancestral peptide that originated first in the molecular evolution of the pituitary hormones family in chordates. These data collectively suggest that a vertebrate-like neuroendocrine axis setting has already emerged in amphioxus, which lays a foundation for subsequent formation of hypothalamic-pituitary system in vertebrates. PMID:25333966

  10. Mechanisms of hepatocyte growth factor-mediated signaling in differentiation of pancreatic ductal epithelial cells into insulin-producing cells

    SciTech Connect

    Li, Xin-Yu; Zhan, Xiao-Rong; Lu, Chong; Liu, Xiao-Min; Wang, Xiao-Chen

    2010-07-30

    Research highlights: {yields} A hypothesis that the differentiation of PDEC is through MAPKs or PI3K/AKT pathways. {yields} Determine if kinases (ERK1/2, p38, JNK, and AKT) are activated in these pathways. {yields} Determine signal pathway(s) that may effect on HGF-induced differentiation of PDEC. {yields} PI3K-AKT pathway is involved in the differentiation of PDECs induced by HGF. {yields} MEK-ERK pathway effect on the proliferation of PDECs but not the differentiation. -- Abstract: Pancreatic ductal epithelial cells (PDECs) were induced to differentiate into insulin-producing cells by hepatocyte growth factor (HGF) in our previous study, but the mechanism through which this induction occurs is still unknown. HGF is a ligand that activates a tyrosine kinase encoded by the c-Met proto-oncogene. This activation is followed by indirect activation of multiple downstream signal transduction pathways (including MAPKs and the PI3K/AKT signaling pathways) that initiate various biological effects. Therefore, we speculated that the differentiation of PDECs is through either the MAPK signaling pathway or the PI3K/AKT signaling pathway. To test this hypothesis, isolated PDECs from adult rats were stimulated by adding HGF to their medium for 28 days. Then, the expression levels of several protein kinases, including MAPKs (ERK1/2, p38, and JNK) and AKT, were determined by Western blotting to determine if specific protein kinases are activated in these pathways. Subsequently, re-isolated from adult rats and cultured PDECs were pre-treated with specific inhibitors of proteins shown to be activated in these signaling pathways; these cells were then induced to differentiate by the addition of HGF. The expression levels of protein kinases were determined by Western blotting, and the differentiation rate of insulin-positive cells was determined by flow cytometry. The change of PDEC differentiation rates were compared between the groups in which cells with or without inhibitors

  11. Aromatic malononitriles stimulate the resistance of insulin-producing beta-cells to oxidants and inflammatory cytokines.

    PubMed

    Turpaev, Kyril; Welsh, Nils

    2016-08-01

    We presently report that treatment with tyrphostin AG-126 (2-(3-hydroxy-4-nitrobenzylidene)malononitrile) and ten other aromatic malononitrile compounds (AMN) improves the resistance of insulin-producing βTC6, RIN-5AH, and MIN6 cells to oxidative stress and pro-inflammatory cytokines. On the molecular level AMN compounds promote nuclear accumulation of the Nrf2 transcription factor and expression of the cytoprotective genes heme ogygenase 1 (HO-1) and NAD(P)H/quinone oxidoreductase 1 (NQO1), inhibit cytokine-dependent inducible nitric oxide synthase (iNOS) induction, suppress intracellular production of reactive oxygen species in βTC6 and counteract to impairments of glucose-stimulated insulin secretion induced by pro-inflammatory cytokines in MIN6 cells. Nrf2 up-regulation and HO-1 induction by AG-126 are attenuated at the presence of siRNA against Nrf2 and brusatol, an inhibitor of the Nrf2 signaling pathway. Our present results indicate that in respect of inhibition of IL-1β-dependent iNOS induction, βTC6 cells are more sensitive to EMK 1071 (2-((5-methylthiophen-2-yl)methylene)malononitrile) and EMK 31 (2-(4-hydroxy-3-methoxybenzylidene)malononitrile) as compared to other analyzed AMN compounds. We suggest that the ability of AMN compounds to inhibit iNOS induction and other cytokine-induced transcriptional events might be a tool to achieve improved β-cell survival and functionality. PMID:27178899

  12. Differentiation of P19 embryonal carcinoma stem cells into insulin-producing cells promoted by pancreas-conditioned medium.

    PubMed

    Mansouri, Akram; Esmaeili, Fariba; Nejatpour, Azadeh; Houshmand, Fariba; Shabani, Leila; Ebrahimie, Esmaeil

    2016-07-01

    The ability of embryonal carcinoma )EC (stem cells to generate insulin-producing cells (IPCs) is still unknown. We examined the trophic effects of pancreas-conditioned medium (PCM) on in vitro production of IPCs. Initially, P19 EC cells were characterized by the expression of stem cell markers, Oct3/4, Sox-2 and Nanog. To direct differentiation, P19-derived embryoid bodies (EBs) were induced by selection of nestin-positive cells and treatment with different concentrations of PCM. Morphological studies documented the presence of islet-like cell IPCs clusters. The differentiated cells were immunoreactive for β cell-specific proteins, including insulin, proinsulin, C-peptide and insulin receptor-β. The expression of genes related to pancreatic β cell development and function (PDX-1, INS1, INS2, EP300 and CREB1) was confirmed by qPCR. During differentiation, the expression of EP300 and CREB1 increased by 2.5 and 3.1 times, respectively. In contrast, a sharp decrease in the expression of Oct3/4, Sox-2 and Nanog by 4, 1.5 and 1.5 times, respectively, was observed. The differentiated cells were functionally active, synthesizing and secreting insulin in a glucose-regulated manner. Network prediction highlighted crosstalk between PDX-1 transcription factor and INS2 ligand in IPC generation and revealed positive regulatory effects of EP300, CREB1, PPARA, EGR, KIT, GLP1R, and PKT2 on activation of PDX-1 and INS2. This is the first report of the induction of IPC differentiation from EC cells by using neonate mouse PCM. Since P19 EC cells are widely available, easily cultured without feeders and do not require special growth conditions, they would provide a valuable tool for studying pancreatic β cell differentiation and development. Copyright © 2016 John Wiley & Sons, Ltd. PMID:25044225

  13. In vitro differentiation of human umbilical cord Wharton’s jelly mesenchymal stromal cells to insulin producing clusters

    PubMed Central

    Nekoei, Seideh Masoomeh; Azarpira, Negar; Sadeghi, Ladan; Kamalifar, Sulmaz

    2015-01-01

    AIM: To investigate the differentiation of human Wharton’s jelly derived mesenchymal stromal cells (WJ-MSCs) to insulin producing clusters (IPC) this study was conducted. METHODS: The umbilical cords samples were collected from full term caesarian section mothers and the WJ-MSCS were cultured from tissue explants in High glucose-Dulbecco’s Modified Eagle Medium (H-DMEM); H-DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics. The expression of CD90, CD44, CD105, CD34 and CD133 as well as osteogenic and adipogenic differentiation of cells in appropriate medium were also evaluated. The cells were differentiated toward IPC with changing the culture medium and adding the small molecules such as nicotinic acid, epidermal growth factor, and exendin-4 during 3 wk period. The gene expression of PDX1, NGN3, Glut2, insulin was monitored by reveres transcription polymerase chain reaction method. The differentiated clusters were stained with Dithizone (DTZ) which confirms the presence of insulin granules. The insulin challenge test (low and high glucose concentration in Krebs-Ringer HEPES buffer) was also used to evaluate the functional properties of differentiated clusters. RESULTS: WJ-MSCS were positive for mesenchymal surface markers (CD90, CD44, CD105), and negative for CD34 and CD133. The accumulation of lipid vacuoles and deposition of calcium mineral in cells were considered as adipogenic and osteogenic potential of WJ-MSCS. The cells also expressed the transcriptional factors such as Nanog and OCT4. During this three step differentiation, the WJ-MSCS morphology was gradually changed from spindle shaped cells in to epithelioid cells and eventually to three dimensional clusters. The clusters expressed PDX1, NGN3, Glut2, and insulin. The cells became bright red color when stained with DTZ and the insulin secretion was also confirmed. In glucose challenge test a significant increase in insulin secretion from 0.91 ± 0.04 μIu/mL (2.8 mmol/L glucose) to

  14. Laminin 411 acts as a potent inducer of umbilical cord mesenchymal stem cell differentiation into insulin-producing cells

    PubMed Central

    2014-01-01

    Background Diabetes mellitus (DM) is an incurable metabolic disease constituting a major threat to human health. Insulin-producing cells (IPCs) differentiated from mesenchymal stem cells (MSCs) hold great promise in the treatment of DM. The development of an efficient IPC induction system is a crucial step for the clinical application of IPCs for DM. Laminin 411 is a key component of the basement membrane and is involved in the regulation of cell differentiation; however, little is known about a role of laminin 411 in the regulation of IPC differentiation from human MSCs. Methods MSCs were isolated from human umbilical cord (UC-MSCs) and expanded in an in vitro culture system. UC-MSCs were then cultured in the IPC induction and differentiation medium in the presence of laminin 411. Flow cytometry, Quantitative realtime PCR, immunofluorescence staining, ELISA, Western blotting and other techniques were applied to determine IPC generation, insulin expression and related mechanisms. To evaluate potential therapeutic efficacy of IPCs induced from UC-MSCs, a type-1 diabetes (T1DM) rat model was generated using streptozotocin. Blood glucose, insulin levels, and survival of rats were monitored periodically following intravenous injection of the tested cells. Results Laminin 411 markedly induced the expression of the genes Foxa2 and Sox17, markers for pancreatic precursor cells, efficiently induced IPC differentiation from MSCs, and up-regulated insulin expression at both mRNA and protein levels. Furthermore, the expression of the genes known to govern insulin expression including Pdx1 and Ngn3 was markedly induced by laminin 411, which suggests that Pdx1 and Ngn3 signaling pathways are involved in laminin 411 induced-insulin expression machinery. More importantly, administration of laminin 411-induced IPCs rapidly and significantly down-regulated fasting blood glucose levels, significantly reduced the HbA1c concentration and markedly improved the symptoms and survival of

  15. Growth hormone (GH) binding and effects of GH analogs in transgenic mice

    SciTech Connect

    Bartke, A.; Steger, R.W.; Turyn, D.

    1994-12-31

    Overexpression of human (h) or bovine (b) growth hormone (GH) in transgenic mice is associated with marked (2- to 12-fold) and significant increase in hepatic binding of GH and prolactin (PRL). This is due to an increase in the number of GH and PRL receptors (GHR, PRLR) per mg of microsomal protein without changes in binding affinity. Comparison of results obtained in transgenic animals expressing bGH with a mouse metallothionein (MT) or a rat phosphoenolpyruvate carboxykinase (PEPCK) promoter suggests that effects of bGH on hepatic GHR and PRLR do not require GH overexpression during fetal life and, within the dose range tested, the effects on PRLR are not dose dependent. The increase in hepatic GHR was accompanied by significant increases in plasma GH-binding protein (GHBP) and in mean residence time of injected GH. Thus life-long elevation of peripheral GH levels alters the availability of both free GH and GHR. Site-directed in vitro mutagenesis was used to produce hGH and bGH analogs mutated within one of the sites involved in binding to GHR and PRLR. Mutating hGH to produce amino acid identity with bGH at Position 11, 18 (within Helix 1), 57, or 60 (within the loop between Helix 1 and 2) did not affect binding to GHR in vitro, or somatotropic activity in transgenic mice in vivo but reduced lactogenic activity in Nb{sub 2} cells by 22%-45%. Mutations of bGH designed to produce amino acid identity with hGH at one to four of the corresponding positions in the bGH molecule did not interfere with binding to GHR or somatotropic activity in vivo, and failed to produce significant binding to PRLR but resulted in alterations in the effects on the hypothalamic and anterior pituitary function in transgenic mice. Apparently region(s) outside the domains examined are essential for lactogenic activity of hGH, and different portions of the GH molecule are responsible for its diverse actions in vivo. 35 refs.

  16. Lymphocyte GH-axis hormones in immunity.

    PubMed

    Weigent, Douglas A

    2013-01-01

    The production and utilization of common ligands and their receptors by cells of the immune and neuroendocrine systems constitutes a biochemical information circuit between and within the immune and neuroendocrine systems. The sharing of ligands and receptors allows the immune system to serve as the sixth sense notifying the nervous system of the presence of foreign entities. Within this framework, it is also clear that immune cell functions can be altered by neuroendocrine hormones and that cells of the immune system have the ability to produce neuroendocrine hormones. This review summarizes a part of this knowledge with particular emphasis on growth hormone (GH). The past two decades have uncovered a lot of detail about the actions of GH, acting through its receptor, at the molecular and cellular level and its influence on the immune system. The production and action of immune cell-derived GH is less well developed although its important role in immunity is also slowly emerging. Here we discuss the production of GH, GH-releasing hormone (GHRH) and insulin-like growth factor-1 (IGF-1) and their cognate receptors on cells of the immune system and their influence via endocrine/autocrine/paracrine and intracrine pathways on immune function. The intracellular mechanisms of action of immune cell-derived GH are still largely unexplored, and it is anticipated that further work in this particular area will establish an important role for this source of GH in normal physiology and in pathologic situations. PMID:24177252

  17. Immobilization of Glycoside Hydrolase Families GH1, GH13, and GH70: State of the Art and Perspectives.

    PubMed

    Graebin, Natália G; Schöffer, Jéssie da N; Andrades, Diandra de; Hertz, Plinho F; Ayub, Marco A Z; Rodrigues, Rafael C

    2016-01-01

    Glycoside hydrolases (GH) are enzymes capable to hydrolyze the glycosidic bond between two carbohydrates or even between a carbohydrate and a non-carbohydrate moiety. Because of the increasing interest for industrial applications of these enzymes, the immobilization of GH has become an important development in order to improve its activity, stability, as well as the possibility of its reuse in batch reactions and in continuous processes. In this review, we focus on the broad aspects of immobilization of enzymes from the specific GH families. A brief introduction on methods of enzyme immobilization is presented, discussing some advantages and drawbacks of this technology. We then review the state of the art of enzyme immobilization of families GH1, GH13, and GH70, with special attention on the enzymes β-glucosidase, α-amylase, cyclodextrin glycosyltransferase, and dextransucrase. In each case, the immobilization protocols are evaluated considering their positive and negative aspects. Finally, the perspectives on new immobilization methods are briefly presented. PMID:27548117

  18. GH-related and extra-endocrine actions of GH secretagogues in aging.

    PubMed

    Muller, Eugenio E; Rigamonti, Antonello E; Colonna, Vito De Gennaro; Locatelli, Vittorio; Berti, Ferruccio; Cella, Silvano G

    2002-01-01

    Growth hormone (GH) secretion declines with aging in mammals, including humans. Defective pituitary function does not play a major role in this event. Rather, age-related changes involving the function of specific hypothalamic peptides for GH regulation, GH-releasing hormone (GHRH) and somatostatin (SS), appear to be the fundamental factors. Experimental evidence indicates that GHRH synthesis is impaired with aging in the rat hypothalamus, and that a relative hyperfunction of the SS-ergic system is present. In the elderly, systemic, short-term administration of GHRH enhances GH secretion and increases the formation of the GH-dependent peptide IGF-1. In old dogs, a combination of GHRH and clonidine (CLO), an alpha(2)-adrenoceptor agonist, reportedly acting via GHRH stimulation and SS inhibition, is the most effective stimulus to rejuvenate the apulsatile GH secretion. Discovery of GH secretagogues (GHSs), a series of peptydil and non-peptydil synthetic molecules endowed with a strong GH-releasing activity, the cloning of a GHS receptor (GHS-R), present in the hypothalamus and the pituitary, the isolation of the endogenous ligand of GHS-R, ghrelin, a 28-amino acid peptide whose main source is the stomach, pose the issue for the physiologic role of the GHS/ghrelin system and forces revision of our current understanding of GH regulation in different GH deficiency (GHD) states, including aging. GHSs are very effective for enhancing GH secretion in old subjects, but long-term studies are needed to assess their safety and the real biological impact of GHS replacement therapy in aging. Therapeutic use of GHSs can also be envisaged to restore, via GH-independent mechanisms, functional, and structural age-related alterations, such as anorexia, sexual dysfunction, cardiovascular damage, bone loss. PMID:12392795

  19. In Vivo Differentiation of Mesenchymal Stem Cells into Insulin Producing Cells on Electrospun Poly-L-Lactide Acid Scaffolds Coated with Matricaria chamomilla L. Oil

    PubMed Central

    Fazili, Afsaneh; Gholami, Soghra; Minaie Zangi, Bagher; Seyedjafari, Ehsan; Gholami, Mahdi

    2016-01-01

    Objective This study examined the in vivo differentiation of mesenchymal stem cells (MSCs) into insulin producing cells (IPCs) on electrospun poly-L-lactide acid (PLLA) scaffolds coated with Matricaria chammomila L. (chamomile) oil. Materials and Methods In this interventional, experimental study adipose MSCs (AMSCs) were isolated from 12 adult male New Zealand white rabbits and characterized by flow cytometry. AMSCs were subsequently differentiated into osteogenic and adipogenic lines. Cells were seeded onto either a PLLA scaffold (control) or PLLA scaffold coated with chamomile oil (experimental). A total of 24 scaffolds were inserted into the pancreatic area of each rabbit and placement was confirmed by ultrasound. After 21 days, immunohistochemistry analysis of insulin-producing like cells on protein levels confirmed insulin expression of insulin producing cells (IPSCs). Real-time polymerase chain reaction (PCR) determined the expressions of genes related to pancreatic endocrine development and function. Results Fourier transform infrared spectroscopy (FTIR) results confirmed the existence of oil on the surface of the PLLA scaffold. The results showed a new peak at 2854 cm-1 for the aliphatic CH2 bond. Pdx1 expression was 0.051 ± 0.007 in the experimental group and 0.009 ± 0.002 in the control group. There was significantly increased insulin expression in the scaffold coated with chamomile oil (0.09 ± 0.001) compared to control group (0.063 ± 0.009, P≤0.05). Both groups expressed Ngn3 and Pdx1 specific markers and pancreatic tissue was observed at 21 days post transplantation. Conclusion The pancreatic region is an optimal site for differentiation of AMSCs to IPCs. Chamomile oil (as an antioxidant agent) can affect cell adhesion to the scaffold and increase cell differentiation. In addition, the oil may lead to increased blood glucose uptake in pathways in the muscles, liver and fatty tissue of a diabetic animal model by some probable molecular mechanisms

  20. T3 acutely increases GH mRNA translation rate and GH secretion in hypothyroid rats.

    PubMed

    Silva, F Goulart da; Giannocco, G; Luchessi, A D; Curi, R; Nunes, M T

    2010-04-12

    Cytoskeleton controls the stability of transcripts, by mechanisms that involve mRNAs and eEF1A attachment to it. Besides, it plays a key role in protein synthesis and secretion, which seems to be impaired in somatotrophs of hypothyroid rats, whose cytoskeleton is disarranged. This study investigated the: eEF1A and GH mRNA binding to cytoskeleton plus GH mRNA translation rate and GH secretion, in sham-operated and thyroidectomized rats treated with T3 or saline, and killed 30min thereafter. Thyroidectomy reduced: (a) pituitary F-actin content, and eEF1A plus GH mRNA binding to it; (b) GH mRNA recruitment to polysome; and (c) liver IGF-I mRNA expression, indicating that GH mRNA stability and translation rate, as well as GH secretion were impaired. T3 acutely reversed all these changes, which points toward a nongenomic action of T3 on cytoskeleton rearrangement, which might contribute to the increase on GH mRNA translation rate and GH secretion. PMID:20015464

  1. Functional characterization of GhSOC1 and GhMADS42 homologs from upland cotton (Gossypium hirsutum L.).

    PubMed

    Zhang, Xiaohong; Wei, Jianghui; Fan, Shuli; Song, Meizhen; Pang, Chaoyou; Wei, Hengling; Wang, Chengshe; Yu, Shuxun

    2016-01-01

    In Arabidopsis flowering pathway, MADS-box genes encode transcription factors, with their structures and functions highly conserved in many species. In our study, two MADS-box genes GhSOC1 and GhMADS42 (Gossypium hirsutum L.) were cloned from upland cotton CCRI36 and transformed into Arabidopsis. GhSOC1 was additionally transformed into upland cotton. Comparative analysis demonstrated sequence conservation between GhSOC1 and GhMADS42 and genes of other plant species. Tissue-specific expression analysis of GhSOC1 and GhMADS42 revealed spatiotemporal expression patterns involving high transcript levels in leaves, shoot apical buds, and flowers. In addition, overexpression of both GhSOC1 and GhMADS42 in Arabidopsis accelerated flowering, with GhMADS42 transgenic plants showing abnormal floral organ phenotypes. Overexpression of GhSOC1 in upland cotton also produced variations in floral organs. Furthermore, chromatin immunoprecipitation assay demonstrated that GhSOC1 could regulate GhMADS41 and GhMADS42, but not FLOWERING LOCUS T, by directly binding to the genes promoter. Finally, yeast two-hybrid and bimolecular fluorescence complementation approaches were undertaken to better understand the interaction of GhSOC1 and other MADS-box factors. These experiments showed that GhSOC1 can interact with APETALA1/FRUITFULL-like proteins in cotton. PMID:26566835

  2. Generation of Functional Insulin-Producing Cells From Mouse Embryonic Stem Cells Through 804G Cell-Derived Extracellular Matrix and Protein Transduction of Transcription Factors

    PubMed Central

    Kaitsuka, Taku; Noguchi, Hirofumi; Shiraki, Nobuaki; Kubo, Takuya; Wei, Fan-Yan; Hakim, Farzana; Kume, Shoen

    2014-01-01

    Embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications to regenerative medicine for diabetes; however, a useful and safe way to generate pancreatic β cells has not been developed. In this study, we tried to establish an effective method of differentiation through the protein transduction of three transcription factors (Pdx1, NeuroD, and MafA) important to pancreatic β cell development. The method poses no risk of unexpected genetic modifications in target cells. Transduction of the three proteins induced the differentiation of mouse ES and mouse iPS cells into insulin-producing cells. Furthermore, a laminin-5-rich extracellular matrix efficiently induced differentiation under feeder-free conditions. Cell differentiation was confirmed with the expression of the insulin 1 gene in addition to marker genes in pancreatic β cells, the differentiated cells secreted glucose-responsive C-peptide, and their transplantation restored normoglycemia in diabetic mice. Moreover, Pdx1 protein transduction had facilitative effects on differentiation into pancreatic endocrine progenitors from human iPS cells. These results suggest the direct delivery of recombinant proteins and treatment with laminin-5-rich extracellular matrix to be useful for the generation of insulin-producing cells. PMID:24292793

  3. In vitro generation of glucose-responsive insulin producing cells using lentiviral based pdx-1 gene transduction of mouse (C57BL/6) mesenchymal stem cells.

    PubMed

    Rahmati, Saman; Alijani, Najva; Kadivar, Mehdi

    2013-08-01

    The objective of this study was to evaluate the potential of this type of recombinant lentivirus to generate glucose-responsive insulin producing cells in vitro. All steps of cloning were confirmed using restriction digests. After the transduction, mesenchymal stem cells gradually began to change their morphology and showed differentiation into islet like structures. RT-PCR results confirmed the expression of insulin1, insulin2 and pdx-1 in differentiated cells. Dithizone staining of mouse MSCs showed the concentration of glucose in islet like structures. ELISA analysis validated the insulin secretion of islet like structures which in the high-glucose medium (25mmol/l) was 7.44 fold higher than that secreted in the low-glucose medium (5mmol/l). Our results demonstrated that mouse mesenchymal stem cells can be differentiated into effective glucose-responsive insulin producing cells through our new recombinant lentiviral transduction of pdx-1 gene in vitro. This new lentiviral vector could be suggested as an effective candidate for using in gene therapy of type-1 diabetes. PMID:23831626

  4. PDX-1 mRNA-induced reprogramming of mouse pancreas-derived mesenchymal stem cells into insulin-producing cells in vitro.

    PubMed

    Guo, Xing Rong; Wang, Xiao Li; Li, Man Chol; Yuan, Ya Hong; Chen, Yun; Zou, Dan Dan; Bian, Liu Jiao; Li, Dong Sheng

    2015-11-01

    Pancreatic islet transplantation has remained an effective therapy for type 1 diabetes since 2000. Its widespread use has been prohibited by the shortage of suitable donors. It is critical to explore an applicable alternative for β-cell replacement. This study was performed to generate insulin-producing cells (IPCs) from pancreas-derived mesenchymal stem cells (pMSCs). pMSCs were isolated from discarded pancreatic tissue in the filter liquor during islet isolation procedure in mice and ex vivo expanded in culture. IPCs were induced by transfection of pancreas and duodenal transcription factor 1 (PDX-1) mRNA in vitro. Some islet characteristics were identified on pMSC-derived IPCs in mRNA and protein levels. Our results demonstrated that mouse pMSCs can be transdifferentiated into effective glucose-responsive insulin-producing cells through transfecting synthetic modified PDX-1 mRNA in vitro. The study of PDX-1 mRNA-induced pMSC reprogramming may pave the way toward the development of a novel β-cell source for the treatment of diabetes. PMID:25345580

  5. Regulation of the growth hormone (GH) receptor and GH-binding protein mRNA

    SciTech Connect

    Kaji, Hidesuke; Ohashi, Shin-Ichirou; Abe, Hiromi; Chihara, Kazuo

    1994-12-31

    In fasting rats, a transient increase in growth hormone-binding protein (GHBP) mRNA levels was observed after 1 day, in muscle, heart, and liver, but not in fat tissues. The liver GH receptor (GHR) mRNA level was significantly increased after 1 day (but not after 5 days) of bovine GH (bGH) treatment in fed rats. Both the liver GHR mRNA level and the net increment of plasma IGF-I markedly decreased after 5 days of bGH administration in fasting rats. These findings suggest that GHR and GHBP mRNAs in the liver are expressed in a different way and that the expression of GHBP mRNA is regulated differently between tissues, at least in rats. The results also suggest that refractoriness to GH in a sustained fasting state might be beneficial in preventing anabolic effects of GH. In humans, GHR mRNA in lymphocytes, from subjects with either GH-deficiency or acromegaly, could be detected by the reverse transcription-polymerase chain reaction method. In one patient with partial GH insensitivity, a heterozygous missense mutation (P561T) was identified in the cytoplasmic domain of GHR. 15 refs., 4 figs.

  6. Hypophysectomy eliminates and growth hormone (GH) maintains the midpregnancy elevation in GH receptor and serum binding protein in the mouse

    SciTech Connect

    Sanchez-Jimenez, F.; Fielder, P.J.; Martinez, R.R.; Smith, W.C.; Talamantes, F. )

    1990-02-01

    ({sup 125}I)Iodomouse GH (({sup 125}I)iodo-mGH) binding to samples of serum and hepatic microsomal membranes was measured in hypophysectomized pregnant, sham-operated pregnant, intact pregnant, and intact adult virgin mice. Surgeries were carried out on day 11 of pregnancy, and the animals were killed on day 14. The binding of mGH to both serum and hepatic microsomal membranes of intact virgin mice was much lower than to those of intact pregnant mice. In hypophysectomized mice, the mGH-binding capacity of both serum and hepatic microsomes decreased to values similar to those of nonpregnant mice. No significant differences were observed between intact and sham-operated pregnant animals in the maternal serum mGH concentration, the serum GH-binding protein concentration, or the hepatic GH receptor concentration. GH receptor and binding protein-encoding mRNAs were also higher in intact and sham-operated pregnant mice than in virgin and hypophysectomized mice. Hypophysectomized mice were treated with 200 micrograms/day bovine GH, administered by osmotic minipump; after 3 days of treatment, a significant elevation of hepatic GH receptor and serum GH-binding protein levels was observed. These results demonstrate an up-regulation of hepatic GH receptors and serum GH-binding protein by GH during pregnancy in the mouse.

  7. Identification and characterization of HolGH15: the holin of Staphylococcus aureus bacteriophage GH15.

    PubMed

    Song, Jun; Xia, Feifei; Jiang, Haiyan; Li, Xinwei; Hu, Liyuan; Gong, Pengjuan; Lei, Liancheng; Feng, Xin; Sun, Changjiang; Gu, Jingmin; Han, Wenyu

    2016-05-01

    Holins are phage-encoded hydrophobic membrane proteins that spontaneously and non-specifically accumulate and form lesions in the cytoplasmic membrane. The ORF72 gene (also designated HolGH15) derived from the genome of the Staphylococcus aureus phage GH15 was predicted to encode a membrane protein. An analysis indicated that the protein encoded by HolGH15 potentially consisted of two hydrophobic transmembrane helices. This protein exhibited the structural characteristics of class II holins and belonged to the phage_holin_1 superfamily. Expression of HolGH15 in Escherichia coli BL21 cells resulted in growth retardation of the host cells, which was triggered prematurely by the addition of 2,4-dinitrophenol. The expression of HolGH15 caused morphological alterations in engineered E. coli cells, including loss of the cell wall and cytoplasmic membrane integrity and release of intracellular components, which were visualized by transmission electron microscopy. HolGH15 exerted efficient antibacterial activity at 37 °C and pH 5.2. Mutation analysis indicated that the two transmembrane domains of HolGH15 were indispensable for the activity of the full-length protein. HolGH15 showed a broad antibacterial range: it not only inhibited Staphylococcus aureus, but also demonstrated antibacterial activity against other species, including Listeria monocytogenes, Bacillus subtilis, Pseudomonas aeruginosa, Klebsiella pneumoniae and E. coli. At the minimal inhibitory concentration, HolGH15 evoked the release of cellular contents and resulted in the shrinkage and death of Staphylococcus aureus and Listeria monocytogenes cells. To the best of our knowledge, this study is the first report of a Staphylococcus aureus phage holin that exerts antibacterial activity against heterogeneous pathogens. PMID:26873847

  8. Moving one step closer to catching the GH cheats: The GH-2004 experience.

    PubMed

    Holt, Richard I G; Bassett, E Eryl; Erotokritou-Mulligan, Ioulietta; McHugh, Cathy; Cowan, David; Bartlett, Christiaan; Sönksen, Peter H

    2009-08-01

    Growth hormone is abused by athletes for its anabolic and lipolytic properties. The detection of GH abuse is challenging because it is an endogenous hormone whose concentration varies widely in any one day. The GH-2000 project proposed a test based on the measurement of IGF-I and type III pro-collagen (P-III-P). When the results of the GH-2000 project were presented to an expert workshop, the method was supported but it was felt that several issues needed to be resolved before the method could be adopted. The first was a potential effect of ethnicity as most subjects in the GH-2000 were white Europeans and the second was a possible effect of injury as P-III-P is a marker of soft tissue turnover. The GH-2004 project was conceived to address these concerns. The GH-2004 project has shown that while there are minor differences in IGF-I and P-III-P between ethnicities, these are small and do not affect the performance of the test. Injury leads to a small rise in P-III-P but again this is not of sufficient magnitude to affect the performance of the test. The GH-2004 project has provided further support for the marker approach as a means of detecting GH abuse in athletes. As WADA have not developed their own immunoassays, however, further work is needed to validate newer commercial assays measuring IGF-I and P-III-P to establish reliable conversion factors to the original GH-2000 units to allow the published formulae to be used. PMID:19467613

  9. Generation of Functional Insulin-Producing Cells from Neonatal Porcine Liver-Derived Cells by PDX1/VP16, BETA2/NeuroD and MafA

    PubMed Central

    Ham, Dong-Sik; Shin, Juyoung; Kim, Ji-Won; Park, Heon-Seok; Cho, Jae-Hyoung; Yoon, Kun-Ho

    2013-01-01

    Surrogate β-cells derived from stem cells are needed to cure type 1 diabetes, and neonatal liver cells may be an attractive alternative to stem cells for the generation of β-cells. In this study, we attempted to generate insulin-producing cells from neonatal porcine liver-derived cells using adenoviruses carrying three genes: pancreatic and duodenal homeobox factor1 (PDX1)/VP16, BETA2/NeuroD and v-maf musculo aponeurotic fibrosarcoma oncogene homolog A (MafA), which are all known to play critical roles in pancreatic development. Isolated neonatal porcine liver-derived cells were sequentially transduced with triple adenoviruses and grown in induction medium containing a high concentration of glucose, epidermal growth factors, nicotinamide and a low concentration of serum following the induction of aggregation for further maturation. We noted that the cells displayed a number of molecular characteristics of pancreatic β-cells, including expressing several transcription factors necessary for β-cell development and function. In addition, these cells synthesized and physiologically secreted insulin. Transplanting these differentiated cells into streptozotocin-induced immunodeficient diabetic mice led to the reversal of hyperglycemia, and more than 18% of the cells in the grafts expressed insulin at 6 weeks after transplantation. These data suggested that neonatal porcine liver-derived cells can be differentiated into functional insulin-producing cells under the culture conditions presented in this report and indicated that neonatal porcine liver-derived cells (NPLCs) might be useful as a potential source of cells for β-cell replacement therapy in efforts to cure type I diabetes. PMID:24260156

  10. 48 CFR Appendixes G-H to Chapter 7 - [Reserved

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false G Appendixes G-H to Chapter 7 Federal Acquisition Regulations System AGENCY FOR INTERNATIONAL DEVELOPMENT Appendixes G-H to Chapter 7...

  11. 48 CFR Appendixes G-H to Chapter 7 - [Reserved

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false G Appendixes G-H to Chapter 7 Federal Acquisition Regulations System AGENCY FOR INTERNATIONAL DEVELOPMENT Appendixes G-H to Chapter 7...

  12. 48 CFR Appendixes G-H to Chapter 7 - [Reserved

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false G Appendixes G-H to Chapter 7 Federal Acquisition Regulations System AGENCY FOR INTERNATIONAL DEVELOPMENT Appendixes G-H to Chapter 7...

  13. 48 CFR Appendixes G-H to Chapter 7 - [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false G Appendixes G-H to Chapter 7 Federal Acquisition Regulations System AGENCY FOR INTERNATIONAL DEVELOPMENT Appendixes G-H to Chapter 7...

  14. 48 CFR Appendixes G-H to Chapter 7 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false G Appendixes G-H to Chapter 7 Federal Acquisition Regulations System AGENCY FOR INTERNATIONAL DEVELOPMENT Appendixes G-H to Chapter 7...

  15. Effectiveness of GH isoform differential immunoassay for detecting rhGH doping on application of various growth factors.

    PubMed

    Okano, Masato; Nishitani, Yasunori; Sato, Mitsuhiko; Kageyama, Shinji

    2012-09-01

    The analytical method for detecting growth hormone (GH) doping, the so-called GH isoform differential immunoassay, is currently approved by the World Anti-Doping Agency (WADA). Anti-doping laboratories often face challenges by athletes' lawyers and need to have various types of scientific evidence against the claim that the adverse analytical finding (AAF) result was caused by excess ectopic or abnormal excretion. In this work, a population study of Japanese athletes (255 male and 256 female) and administration studies of recombinant human GH (rhGH) in Japanese females were conducted to confirm the applicability of GH isoform differential immunoassay. The present paper describes the effectiveness of the GH isoform differential immunoassay under abnormal excretion of endogenous GH as determined by administration studies of GH releasing hormone (GHRH(1-44)) and insulin-like growth factor-1 (IGF-1). No false positive findings were found in Japanese athletes. The GH isoform differential immunoassays could detect application of rhGH for approximately 12-24 h. The administration of GHRH(1-44) and IGF-1 as well as ghrelin receptor agonists did not affect the isoform ratio (no false positives). We conclude that the GH isoform differential immunoassay is a highly specific method for detecting rhGH doping. Subject-based profiling (i.e. athlete biological passport) very likely will represent a highly sensitive approach for detecting rhGH doping. PMID:22733714

  16. Graphene oxide inhibits hIAPP amyloid fibrillation and toxicity in insulin-producing NIT-1 cells.

    PubMed

    Nedumpully-Govindan, Praveen; Gurzov, Esteban N; Chen, Pengyu; Pilkington, Emily H; Stanley, William J; Litwak, Sara A; Davis, Thomas P; Ke, Pu Chun; Ding, Feng

    2016-01-01

    Human islet amyloid polypeptide (hIAPP or amylin) aggregation is directly associated with pancreatic β-cell death and subsequent insulin deficiency in type 2 diabetes (T2D). Since no cure is currently available for T2D, it is of great benefit to devise new anti-aggregation molecules, which protect β-cells against hIAPP aggregation-induced toxicity. Engineered nanoparticles have been recently exploited as anti-aggregation nanomedicines. In this work, we studied graphene oxide (GO) nanosheets for their potential for hIAPP aggregation inhibition by combining computational modeling, biophysical characterization and cell toxicity measurements. Using discrete molecular dynamics (DMD) simulations and in vitro studies, we showed that GO exhibited an inhibitory effect on hIAPP aggregation. DMD simulations indicated that the strong binding of hIAPP to GO nanosheets was driven by hydrogen bonding and aromatic stacking and that the strong peptide-GO binding efficiently inhibited hIAPP self-association and aggregation on the nanosheet surface. Secondary structural changes of hIAPP upon GO binding derived from DMD simulations were consistent with circular dichroism (CD) spectroscopy measurements. Transmission electron microscopy (TEM) images confirmed the reduction of hIAPP aggregation in the presence of GO. Furthermore, we carried out a cell toxicity assay and found that these nanosheets protected insulin-secreting NIT-1 pancreatic β-cells against hIAPP-induced toxicity. Our multidisciplinary study suggests that GO nanosheets have the potential to be utilized as an anti-aggregation nanomedicine itself in addition to a biosensor or delivery vehicle for the mitigation of T2D progression. PMID:26625841

  17. Evaluation of growth hormone (GH) action in mice: discovery of GH receptor antagonists and clinical indications.

    PubMed

    Kopchick, John J; List, Edward O; Kelder, Bruce; Gosney, Elahu S; Berryman, Darlene E

    2014-04-01

    The discovery of a growth hormone receptor antagonist (GHA) was initially established via expression of mutated GH genes in transgenic mice. Following this discovery, development of the compound resulted in a drug termed pegvisomant, which has been approved for use in patients with acromegaly. Pegvisomant treatment in a dose dependent manner results in normalization of IGF-1 levels in most patients. Thus, it is a very efficacious and safe drug. Since the GH/IGF-1 axis has been implicated in the progression of several types of cancers, many have suggested the use of pegvisomant as an anti-cancer therapeutic. In this manuscript, we will review the use of mouse strains that possess elevated or depressed levels of GH action for unraveling many of GH actions. Additionally, we will describe experiments in which the GHA was discovered, review results of pegvisomant's preclinical and clinical trials, and provide data suggesting pegvisomant's therapeutic value in selected types of cancer. PMID:24035867

  18. Determinants of GH-releasing hormone and GH-releasing peptide synergy in men

    PubMed Central

    Veldhuis, Johannes D.; Bowers, Cyril Y.

    2009-01-01

    Age, sex steroids, and abdominal-visceral fat (AVF) jointly affect pulsatile growth hormone (GH) secretion. Pulsatile GH secretion in turn is controlled by GH-releasing hormone (GHRH), GH-releasing peptide (GHRP), and somatostatin. Marked stimulation of pulsatile GH secretion is achieved via GHRH-GHRP synergy. Nonetheless, how key modulators of GH secretion, such as age, sex steroids, and body mass index, modify GHRH-GHRP synergy is not known. The present strategy was to 1) infuse GHRH and GHRP-2 simultaneously to evoke synergy and 2) downregulate the gonadal axis with leuprolide and then restore placebo (Pl) or testosterone (T) to clamp the sex steroid milieu. Forty-seven men [18–74 yr of age, T = 7–1,950 ng/dl, estradiol (E2) = 5–79 pg/ml, insulin-like growth factor (IGF)-I = 115–817 μg/l, AVF = 11–349 cm2] were studied. GHRH-GHRP synergy correlated negatively with age and AVF (both P < 0.001) and positively with IGF-I (P < 0.001) and IGF-binding protein (IGFBP)-3 (P = 0.031). Unstimulated basal (nonpulsatile) GH secretion correlated positively with T (P = 0.015) and E2 (P = 0.004) concentrations. Fasting pulsatile GH secretion varied negatively with age (P = 0.017) and positively with IGF-I (P = 0.002) and IGFBP-3 (P = 0.001). By stepwise forward-selection multivariate analyses, AVF, IGF-I, and IGFBP-3 together explained 60% of the variability in GHRH-GHRP synergy (P < 0.001), E2 accounted for 17% of the variability in basal GH secretion (P = 0.007), and IGF-I explained 20% of the variability in fasting pulsatile GH secretion (P = 0.002). In conclusion, a paradigm examining GHRH-GHRP synergy under a sex steroid clamp reveals highly selective control of basal, pulsatile, and synergistic peptide-driven GH secretion by AVF, E2, and IGF-I in healthy men. PMID:19240251

  19. Genetics of GHRH, GHRH-receptor, GH and GH-receptor: its impact on pharmacogenetics.

    PubMed

    Mullis, Primus-E

    2011-02-01

    When a child is not following the normal, predicted growth curve, an evaluation for underlying illnesses and central nervous system abnormalities is required and, appropriate consideration should be given to genetic defects causing GH deficiency (GHD). Because Insulin-like-Growth Factor-I (IGF-I) plays a pivotal role, GHD could also be considered as a form of IGF-I deficiency (IGFD). Although IGFD can develop at any level of the GHRH-GH-IGF axis, a differentiation should be made between GHD (absent to low GH in circulation) and IGFD (normal to high GH in circulation). The main focus of this review is on the GH-gene, the various gene alterations and their possible impact on the pituitary gland. However, although transcription factors regulating the pituitary gland development may cause multiple pituitary hormone deficiency they may present initially as GHD. These defects are discussed in various different chapters within this book, whereas, the impact of alterations of the GHRH-, GHRH-receptor- --as well as the GH-receptor (GHR) gene--will be discussed here. PMID:21396573

  20. Growth hormone (GH)–releasing hormone and GH secretagogues in normal aging: Fountain of Youth or Pool of Tantalus?

    PubMed Central

    Hersch, Elizabeth C; Merriam, George R

    2008-01-01

    Although growth hormone (GH) is primarily associated with linear growth in childhood, it continues to have important metabolic functions in adult life. Adult GH deficiency (AGHD) is a distinct clinical entity, and GH replacement in AGHD can improve body composition, strength, aerobic capacity, and mood, and may reduce vascular disease risk. While there are some hormone-related side effects, the balance of benefits and risks is generally favorable, and several countries have approved GH for clinical use in AGHD. GH secretion declines progressively and markedly with aging, and many age-related changes resemble those of partial AGHD. This suggests that replacing GH, or stimulating GH with GH-releasing hormone or a GH secretagogue could confer benefits in normal aging similar to those observed in AGHD – in particular, could reduce the loss of muscle mass, strength, and exercise capacity leading to frailty, thereby prolonging the ability to live independently. However, while most GH studies have shown body composition effects similar to those in AGHD, functional changes have been much less inconsistent, and older adults are more sensitive to GH side effects. Preliminary reports of improved cognition are encouraging, but the overall balance of benefits and risks of GH supplementation in normal aging remains uncertain. PMID:18488883

  1. GH safety workshop position paper: A critical appraisal of recombinant human GH therapy in children and adults

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant human Growth Hormone (rhGH) has been in use for 30 years, and over that time its safety and efficacy in children and adults has been subject to considerable scrutiny. In 2001, a statement from the GH Research Society (GRS) concluded that 'for approved indications, GH is safe'; however, t...

  2. The robustness of diagnostic tests for GH deficiency in adults.

    PubMed

    Andersen, Marianne

    2015-06-01

    Since the 1970s, GH treatment has been an important tool in paediatric endocrinology for the management of growth retardation. It is now accepted that adults with severe GH deficiency (GHD) demonstrate impaired physical and psychological well-being and may benefit from replacement therapy with recombinant human GH. There is, however, an ongoing debate on how to diagnose GHD, especially in adults. A GH response below the cut-off limit of a GH-stimulation test is required in most cases for establishing GHD in adults. No 'gold standard' GH-stimulation test exists, but some GH stimulation tests may be more robust to variations in patient characteristics such as age and gender, as well as to pre-test conditions like heat exposure due to a hot bath or bicycling. However, body mass index (BMI) is negatively associated with GH-responses to all available GH-stimulation tests and glucocorticoid treatment, including conventional substitution therapy, influences the GH-responses. Recently, the role of IGF-I measurements in the clinical decision making has been discussed. The aim of this review is to discuss the available GH-stimulation tests. In this author's opinion, tests which include growth-hormone-releasing hormone (GHRH) tend to be more potent and robust, especially the GHRH+arginine test which has been proven to be of clinical use. In contrast, the insulin tolerance test (ITT) and the glucagon test appear to have too many drawbacks. PMID:25900364

  3. Sequence polymorphisms at the growth hormone GH1/GH2-N and GH2-Z gene copies and their relationship with dairy traits in domestic sheep (Ovis aries).

    PubMed

    Vacca, G M; Dettori, M L; Balia, F; Luridiana, S; Mura, M C; Carcangiu, V; Pazzola, M

    2013-09-01

    The purpose was to analyze the growth hormone GH1/GH2-N and GH2-Z gene copies and to assess their possible association with milk traits in Sarda sheep. Two hundred multiparous lactating ewes were monitored. The two gene copies were amplified separately and each was used as template for a nested PCR, to investigate single strand conformation polymorphism (SSCP) of the 5'UTR, exon-1, exon-5 and 3'UTR DNA regions. SSCP analysis revealed marked differences in the number of polymorphic patterns between the two genes. Sequencing revealed five nucleotide changes at the GH1/GH2-N gene. Five nucleotide changes occurred at the GH2-Z gene: one was located in exon-5 (c.556G > A) and resulted in a putative amino acid substitution G186S. All the nucleotide changes were copy-specific, except c.*30delT, which was common to both GH1/GH2-N and GH2-Z. Variability in the promoter regions of each gene might have consequences on the expression level, due to the involvement in potential transcription factor binding sites. Both gene copies influenced milk yield. A correlation with milk protein and casein content was also evidenced. These results may have implications that make them useful for future breeding strategies in dairy sheep breeding. PMID:23653010

  4. Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells into Insulin-Producing Cells: Evidence for Further Maturation In Vivo

    PubMed Central

    Gabr, Mahmoud M.; Zakaria, Mahmoud M.; Refaie, Ayman F.; Khater, Sherry M.; Ashamallah, Sylvia A.; Ismail, Amani M.; El-Halawani, Sawsan M.; Ghoneim, Mohamed A.

    2015-01-01

    The aim of this study was to provide evidence for further in vivo maturation of insulin-producing cells (IPCs) derived from human bone marrow-derived mesenchymal stem cells (HBM-MSCs). HBM-MSCs were obtained from three insulin-dependent type 2 diabetic volunteers. Following expansion, cells were differentiated according to a trichostatin-A/GLP protocol. One million cells were transplanted under the renal capsule of 29 diabetic nude mice. Blood glucose, serum human insulin and c-peptide levels, and glucose tolerance curves were determined. Mice were euthanized 1, 2, 4, or 12 weeks after transplantation. IPC-bearing kidneys were immunolabeled, number of IPCs was counted, and expression of relevant genes was determined. At the end of in vitro differentiation, all pancreatic endocrine genes were expressed, albeit at very low values. The percentage of IPCs among transplanted cells was small (≤3%). Diabetic animals became euglycemic 8 ± 3 days after transplantation. Thereafter, the percentage of IPCs reached a mean of ~18% at 4 weeks. Relative gene expression of insulin, glucagon, and somatostatin showed a parallel increase. The ability of the transplanted cells to induce euglycemia was due to their further maturation in the favorable in vivo microenvironment. Elucidation of the exact mechanism(s) involved requires further investigation. PMID:26064925

  5. In Vitro Differentiation of Human Umbilical Cord Blood CD133+Cells into Insulin Producing Cells in Co-Culture with Rat Pancreatic Mesenchymal Stem Cells

    PubMed Central

    Sahraneshin Samani, Fazel; Ebrahimi, Marzieh; Zandieh, Tahereh; Khoshchehreh, Reyhaneh; Baghaban Eslaminejad, Mohamadreza; Aghdami, Nasser; Baharvand, Hossein

    2015-01-01

    Objective Pancreatic stroma plays an important role in the induction of pancreatic cells by the use of close range signaling. In this respect, we presume that pancreatic mesenchymal cells (PMCs) as a fundamental factor of the stromal niche may have an effective role in differentiation of umbilical cord blood cluster of differentiation 133+ (UCB-CD133+) cells into newly-formed β-cells in vitro. Materials and Methods This study is an experimental research. The UCB-CD133+cells were purified by magnetic activated cell sorting (MACS) and differentiated into insulin producing cells (IPCs) in co-culture, both directly and indirectly with rat PMCs. Immunocytochemistry and enzyme linked immune sorbent assay (ELISA) were used to determine expression and production of insulin and C-peptide at the protein level. Results Our results demonstrated that UCB-CD133+differentiated into IPCs. Cells in islet-like clusters with (out) co-cultured with rat pancreatic stromal cells produced insulin and C-peptide and released them into the culture medium at the end of the induction protocol. However they did not respond well to glucose challenges. Conclusion Rat PMCs possibly affect differentiation of UCB-CD133+cells into IPCs by increasing the number of immature β-cells. PMID:26199900

  6. Association of expression levels of pluripotency/stem cell markers with the differentiation outcome of Wharton's jelly mesenchymal stem cells into insulin producing cells.

    PubMed

    Kassem, Dina H; Kamal, Mohamed M; El-Kholy, Abd El-Latif G; El-Mesallamy, Hala O

    2016-08-01

    Recently, there has been much attention towards generation of insulin producing cells (IPCs) from stem cells, especially from Wharton's jelly mesenchymal stem cells (WJ-MSCs). However, generation of mature IPCs remains a challenge. Assessment of generation of IPCs was usually done by examining β-cell markers, however, assessment of pluripotency/stem cell markers drew less attention. Therefore, the purpose of this study was to investigate the levels of pluripotency/stem cell markers during differentiation of WJ-MSCs into IPCs and the association of these levels with differentiation outcomes. WJ-MSCs were isolated, characterized then induced to differentiate into IPCs using three different protocols namely A, B and C. Differentiated IPCs were assessed by the expression of pluripotency/stem cell markers, together with β-cell markers using qRT-PCR, and functionally by measuring glucose stimulated insulin secretion. Differentiated cells from protocol A showed lowest expression of pluripotency/stem cell markers and relatively best GSIS. However, protocol B showed concomitant expression of pluripotency/stem cell and β-cell markers with relatively less insulin secretion as compared to protocol A. Protocol C failed to generate glucose-responsive IPCs. In conclusion, sustained expression of pluripotency/stem cell markers could be associated with the incomplete differentiation of WJ-MSCs into IPCs. A novel finding for which further investigations are warranted. PMID:27265786

  7. Insulin signaling, lifespan and stress resistance are modulated by metabotropic GABA receptors on insulin producing cells in the brain of Drosophila.

    PubMed

    Enell, Lina E; Kapan, Neval; Söderberg, Jeannette A E; Kahsai, Lily; Nässel, Dick R

    2010-01-01

    Insulin-like peptides (ILPs) regulate growth, reproduction, metabolic homeostasis, life span and stress resistance in worms, flies and mammals. A set of insulin producing cells (IPCs) in the Drosophila brain that express three ILPs (DILP2, 3 and 5) have been the main focus of interest in hormonal DILP signaling. Little is, however, known about factors that regulate DILP production and release by these IPCs. Here we show that the IPCs express the metabotropic GABA(B) receptor (GBR), but not the ionotropic GABA(A) receptor subunit RDL. Diminishing the GBR expression on these cells by targeted RNA interference abbreviates life span, decreases metabolic stress resistance and alters carbohydrate and lipid metabolism at stress, but not growth in Drosophila. A direct effect of diminishing GBR on IPCs is an increase in DILP immunofluorescence in these cells, an effect that is accentuated at starvation. Knockdown of irk3, possibly part of a G protein-activated inwardly rectifying K(+) channel that may link to GBRs, phenocopies GBR knockdown in starvation experiments. Our experiments suggest that the GBR is involved in inhibitory control of DILP production and release in adult flies at metabolic stress and that this receptor mediates a GABA signal from brain interneurons that may convey nutritional signals. This is the first demonstration of a neurotransmitter that inhibits insulin signaling in its regulation of metabolism, stress and life span in an invertebrate brain. PMID:21209905

  8. Marker vaccine potential of a foot-and-mouth disease virus with a partial VP1 G-H loop deletion.

    PubMed

    Fowler, V L; Knowles, N J; Paton, D J; Barnett, P V

    2010-04-26

    Previous work in cattle and pigs demonstrated that protection against foot-and-mouth disease (FMD) could be achieved following vaccination with chimeric foot-and-mouth disease virus (FMDV) vaccines, in which the VP1 G-H loop had been substituted with that from another serotype. This indicated that the VP1 G-H loop may not be essential for the protection of natural hosts against FMDV. If this could be substantiated there would be potential to develop FMD marker vaccines, characterised by the absence of this region. Here, we investigate the serological responses to vaccination with a virus with a partial VP1 G-H loop deletion in order to determine the likelihood of achieving protection and the potential of this virus as a marker vaccine. Inactivated, oil adjuvanted, vaccines, consisting of chemically inactivated virus with or without a partially deleted VP1 G-H loop, were used to immunise cattle. Serum was collected on days 0, 7, 14 and 21 and antibody titres calculated using the virus neutralisation test (VNT) to estimate the likelihood of protection. We predict a good likelihood that cattle vaccinated with a vaccine characterised by a partial VP1 G-H loop would be protected against challenge with the same virus containing the VP1 G-H loop. We also present evidence on the potential of such a construct to act as a marker vaccine, when used in conjunction with a novel serological test. PMID:20199761

  9. GH11 xylanases: Structure/function/properties relationships and applications.

    PubMed

    Paës, Gabriel; Berrin, Jean-Guy; Beaugrand, Johnny

    2012-01-01

    For technical, environmental and economical reasons, industrial demands for process-fitted enzymes have evolved drastically in the last decade. Therefore, continuous efforts are made in order to get insights into enzyme structure/function relationships to create improved biocatalysts. Xylanases are hemicellulolytic enzymes, which are responsible for the degradation of the heteroxylans constituting the lignocellulosic plant cell wall. Due to their variety, xylanases have been classified in glycoside hydrolase families GH5, GH8, GH10, GH11, GH30 and GH43 in the CAZy database. In this review, we focus on GH11 family, which is one of the best characterized GH families with bacterial and fungal members considered as true xylanases compared to the other families because of their high substrate specificity. Based on an exhaustive analysis of the sequences and 3D structures available so far, in relation with biochemical properties, we assess biochemical aspects of GH11 xylanases: structure, catalytic machinery, focus on their "thumb" loop of major importance in catalytic efficiency and substrate selectivity, inhibition, stability to pH and temperature. GH11 xylanases have for a long time been used as biotechnological tools in various industrial applications and represent in addition promising candidates for future other uses. PMID:22067746

  10. Comparative analysis of three hyperthermophilic GH1 and GH3 family members with industrial potential.

    PubMed

    Cota, Junio; Corrêa, Thamy L R; Damásio, André R L; Diogo, José A; Hoffmam, Zaira B; Garcia, Wanius; Oliveira, Leandro C; Prade, Rolf A; Squina, Fabio M

    2015-01-25

    Beta-glucosidases (BGLs) are enzymes of great potential for several industrial processes, since they catalyze the cleavage of glucosidic bonds in cellobiose and other short cellooligosaccharides. However, features such as good stability to temperature, pH, ions and chemicals are required characteristics for industrial applications. This work aimed to provide a comparative biochemical analysis of three thermostable BGLs from Pyrococcus furiosus and Thermotoga petrophila. The genes PfBgl1 (GH1 from P. furiosus), TpBgl1 (GH1 from T. petrophila) and TpBgl3 (GH3 from T. petrophila) were cloned and proteins were expressed in Escherichia coli. The purified enzymes are hyperthermophilic, showing highest activity at temperatures above 80°C at acidic (TpBgl3 and PfBgl1) and neutral (TpBgl1) pHs. The BGLs showed greatest stability to temperature mainly at pH 6.0. Activities using a set of different substrates suggested that TpBgl3 (GH3) is more specific than GH1 family members. In addition, the influence of six monosaccharides on BGL catalysis was assayed. While PfBgl1 and TpBgl3 seemed to be weakly inhibited by monosaccharides, TpBgl1 was activated, with xylose showing the strongest activation. Under the conditions tested, TpBgl1 showed the highest inhibition constant (Ki=1100.00mM) when compared with several BGLs previously characterized. The BGLs studied have potential for industrial use, specifically the enzymes belonging to the GH1 family, due to its broad substrate specificity and weak inhibition by glucose and other saccharides. PMID:25102284

  11. Exendin-4 enhances expression of Neurod1 and Glut2 in insulin-producing cells derived from mouse embryonic stem cells

    PubMed Central

    Zhao, Qiaoshi; Yang, Yuzhi; Hu, Jing; Shan, Zhiyan; Wu, Yanshuang

    2016-01-01

    Introduction Stem cells involved cell replacement therapies for type 1 diabetes mellitus is promising, yet time-consuming and inefficient. Exendin-4 is a glucagon-like peptide-1 (GLP-1) receptor agonist which has been reported to possess anti-apoptotic effects, thereby increasing β-cell mass and improving β-cell function. The present study aimed to investigate whether exendin-4 would enhance the differentiation of embryonic stem cells into insulin-secreting cells and improve the pancreatic differentiation strategy. Material and methods R1 embryonic stem cells were treated with different concentrations of exendin-4 and divided into three groups. In the high dosage group (group H), exendin-4 was added at the dosage of 10 nmol/l. In the low dosage group (group L), exendin-4 was added at the dosage of 0.1 nmol/l. Group C was a control. Expression of genes related to the β-cell phenotype and immunofluorescence staining of insulin and C-peptide were detected. Results Compared with groups L and C, group H had the highest mRNA expression levels of Isl1, Pdx1, Ngn3, and Insulin1 (p < 0.05). Neurod1 and Glut2 only emerged at the final stage of differentiation in group H. Immunofluorescence analysis revealed that exendin-4 upregulated the protein expression of insulin and C-peptide. Conclusions Exendin-4 remarkably facilitated Neurod1 and Glut2 gene transcription, and was able to induce differentiation of embryonic stem cells into endocrine and insulin-producing cells. PMID:26925137

  12. Promoting Long-Term Survival of Insulin-Producing Cell Grafts That Differentiate from Adipose Tissue-Derived Stem Cells to Cure Type 1 Diabetes

    PubMed Central

    Zhang, Shuzi; Dai, Hehua; Wan, Ni; Moore, Yolonda; Dai, Zhenhua

    2011-01-01

    Background Insulin-producing cell clusters (IPCCs) have recently been generated in vitro from adipose tissue-derived stem cells (ASCs) to circumvent islet shortage. However, it is unknown how long they can survive upon transplantation, whether they are eventually rejected by recipients, and how their long-term survival can be induced to permanently cure type 1 diabetes. IPCC graft survival is critical for their clinical application and this issue must be systematically addressed prior to their in-depth clinical trials. Methodology/Principal Findings Here we found that IPCC grafts that differentiated from murine ASCs in vitro, unlike their freshly isolated islet counterparts, did not survive long-term in syngeneic mice, suggesting that ASC-derived IPCCs have intrinsic survival disadvantage over freshly isolated islets. Indeed, β cells retrieved from IPCC syngrafts underwent faster apoptosis than their islet counterparts. However, blocking both Fas and TNF receptor death pathways inhibited their apoptosis and restored their long-term survival in syngeneic recipients. Furthermore, blocking CD40-CD154 costimulation and Fas/TNF signaling induced long-term IPCC allograft survival in overwhelming majority of recipients. Importantly, Fas-deficient IPCC allografts exhibited certain immune privilege and enjoyed long-term survival in diabetic NOD mice in the presence of CD28/CD40 joint blockade while their islet counterparts failed to do so. Conclusions/Significance Long-term survival of ASC-derived IPCC syngeneic grafts requires blocking Fas and TNF death pathways, whereas blocking both death pathways and CD28/CD40 costimulation is needed for long-term IPCC allograft survival in diabetic NOD mice. Our studies have important clinical implications for treating type 1 diabetes via ASC-derived IPCC transplantation. PMID:22216347

  13. Effects of rat growth hormone (rGH)-releasing factor and somatostatin on the release and synthesis of rGH in dispersed pituitary cells

    SciTech Connect

    Fukata, J.; Diamond, D.J.; Martin, J.B.

    1985-08-01

    The effects of rat hypothalamic GH-releasing factor (GRF) and somatostatin (SRIF) on the release and biosynthesis of rat GH were studied by RIA and quantitative immunoprecipitation using monolayer cultures of rat anterior pituitary cells. In kinetic studies, GRF stimulation of GH release appeared at the first sampling time (20-min incubation) and the effect began to diminish after 2-h incubation with GRF. On the other hand, total (cell plus medium) content of GH significantly increased only after 24-h incubation. To examine the GH-synthesizing effect of GRF more directly, newly synthesized GH labeled by (TVS)methionine during incubation with GRF was quantified by immunoprecipitation. The amount of immunoprecipitable GH increased significantly and specifically also only after 24-h incubation. When GH pools were labeled with (TVS)methionine under different schedules, the basal release of newly synthesized GH, which was labeled for 1 h immediately before chase incubation was lower during the first 15 min than stored GH which had been labeled earlier. Basal newly synthesized GH secretion exceeded stored GH secretion after 30 min. In this system, SRIF suppressed both the basal and stimulated release of GH but did not modify GH biosynthesis under either condition. Newly synthesized GH showed significant degradation during 24-h incubation; neither GRF nor SRIF affected the rate of GH degradation during the same incubation period.

  14. Proteomics analysis of cytokine-induced dysfunction and death in insulin-producing INS-1E cells: new insights into the pathways involved.

    PubMed

    D'Hertog, Wannes; Overbergh, Lut; Lage, Kasper; Ferreira, Gabriela Bonfim; Maris, Michael; Gysemans, Conny; Flamez, Daisy; Cardozo, Alessandra Kupper; Van den Bergh, Gert; Schoofs, Liliane; Arckens, Lut; Moreau, Yves; Hansen, Daniel Aaen; Eizirik, Decio Laks; Waelkens, Ettienne; Mathieu, Chantal

    2007-12-01

    Cytokines released by islet-infiltrating immune cells play a crucial role in beta-cell dysfunction and apoptotic cell death in the pathogenesis of type 1 diabetes and after islet transplantation. RNA studies revealed complex pathways of genes being activated or suppressed during this beta-cell attack. The aim of the present study was to analyze protein changes in insulin-producing INS-1E cells exposed to inflammatory cytokines in vitro using two-dimensional DIGE. Within two different pH ranges we observed 2214 +/- 164 (pH 4-7) and 1641 +/- 73 (pH 6-9) spots. Analysis at three different time points (1, 4, and 24 h of cytokine exposure) revealed that the major changes were taking place only after 24 h. At this time point 158 proteins were altered in expression (4.1%, n = 4, p < or = 0.01) by a combination of interleukin-1beta and interferon-gamma, whereas only 42 and 23 proteins were altered by either of the cytokines alone, giving rise to 199 distinct differentially expressed spots. Identification of 141 of these by MALDI-TOF/TOF revealed proteins playing a role in insulin secretion, cytoskeleton organization, and protein and RNA metabolism as well as proteins associated with endoplasmic reticulum and oxidative stress/defense. We investigated the interactions of these proteins and discovered a significant interaction network (p < 1.27e-05) containing 42 of the identified proteins. This network analysis suggests that proteins of different pathways act coordinately in a beta-cell dysfunction/apoptotic beta-cell death interactome. In addition the data suggest a central role for chaperones and proteins playing a role in RNA metabolism. As many of these identified proteins are regulated at the protein level or undergo post-translational modifications, a proteomics approach, as performed in this study, is required to provide adequate insight into the mechanisms leading to beta-cell dysfunction and apoptosis. The present findings may open new avenues for the understanding

  15. The Generation of Insulin Producing Cells from Human Mesenchymal Stem Cells by MiR-375 and Anti-MiR-9

    PubMed Central

    Jafarian, Arefeh; Taghikani, Mohammad; Abroun, Saeid; Allahverdi, Amir; Lamei, Maryam; Lakpour, Niknam; Soleimani, Masoud

    2015-01-01

    Background MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs that regulate gene expression at the post-transcriptional level. A number of studies have led to the notion that some miRNAs have key roles in control of pancreatic islet development and insulin secretion. Based on some studies on miRNAs pattern, the researchers in this paper investigated the pancreatic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) by up-regulation of miR-375 and down-regulation of miR-9 by lentiviruses containing miR-375 and anti-miR-9. Methodology After 21 days of induction, islet-like clusters containing insulin producing cells (IPCs) were confirmed by dithizone (DTZ) staining. The IPCs and β cell specific related genes and proteins were detected using qRT-PCR and immunofluorescence on days 7, 14 and 21 of differentiation. Glucose challenge test was performed at different concentrations of glucose so extracellular and intracellular insulin and C-peptide were assayed using ELISA kit. Although derived IPCs by miR-375 alone were capable to express insulin and other endocrine specific transcription factors, the cells lacked the machinery to respond to glucose. Conclusion It was found that over-expression of miR-375 led to a reduction in levels of Mtpn protein in derived IPCs, while treatment with anti-miR-9 following miR-375 over-expression had synergistic effects on MSCs differentiation and insulin secretion in a glucose-regulated manner. The researchers reported that silencing of miR-9 increased OC-2 protein in IPCs that may contribute to the observed glucose-regulated insulin secretion. Although the roles of miR-375 and miR-9 are well known in pancreatic development and insulin secretion, the use of these miRNAs in transdifferentiation was never demonstrated. These findings highlight miRNAs functions in stem cells differentiation and suggest that they could be used as therapeutic tools for gene-based therapy in diabetes mellitus. PMID:26047014

  16. Efficient and simple production of insulin-producing cells from embryonal carcinoma stem cells using mouse neonate pancreas extract, as a natural inducer.

    PubMed

    Ebrahimie, Marzieh; Esmaeili, Fariba; Cheraghi, Somayeh; Houshmand, Fariba; Shabani, Leila; Ebrahimie, Esmaeil

    2014-01-01

    An attractive approach to replace the destroyed insulin-producing cells (IPCs) is the generation of functional β cells from stem cells. Embryonal carcinoma (EC) stem cells are pluripotent cells which can differentiate into all cell types. The present study was carried out to establish a simple nonselective inductive culture system for generation of IPCs from P19 EC cells by 1-2 weeks old mouse pancreas extract (MPE). Since, mouse pancreatic islets undergo further remodeling and maturation for 2-3 weeks after birth, we hypothesized that the mouse neonatal MPE contains essential factors to induce in vitro differentiation of pancreatic lineages. Pluripotency of P19 cells were first confirmed by expression analysis of stem cell markers, Oct3/4, Sox-2 and Nanog. In order to induce differentiation, the cells were cultured in a medium supplemented by different concentrations of MPE (50, 100, 200 and 300 µg/ml). The results showed that P19 cells could differentiate into IPCs and form dithizone-positive cell clusters. The generated P19-derived IPCs were immunoreactive to proinsulin, insulin and insulin receptor beta. The expression of pancreatic β cell genes including, PDX-1, INS1 and INS2 were also confirmed. The peak response at the 100 µg/ml MPE used for investigation of EP300 and CREB1 gene expression. When stimulated with glucose, these cells synthesized and secreted insulin. Network analysis of the key transcription factors (PDX-1, EP300, CREB1) during the generation of IPCs resulted in introduction of novel regulatory candidates such as MIR17, and VEZF1 transcription factors, as well as MORN1, DKFZp761P0212, and WAC proteins. Altogether, we demonstrated the possibility of generating IPCs from undifferentiated EC cells, with the characteristics of pancreatic β cells. The derivation of pancreatic cells from EC cells which are ES cell siblings would provide a valuable experimental tool in study of pancreatic development and function as well as rapid production of

  17. Multiple low-dose streptozotocin-induced diabetes in the mouse. Evidence for stimulation of a cytotoxic cellular immune response against an insulin-producing beta cell line.

    PubMed Central

    McEvoy, R C; Andersson, J; Sandler, S; Hellerström, C

    1984-01-01

    Mice were examined for the presence of splenocytes specifically cytotoxic for a rat insulinoma cell line (RIN) during the induction of diabetes by streptozotocin (SZ) in multiple low doses (Multi-Strep). Cytotoxicity was quantitated by the release of 51Cr from damaged cells. A low but statistically significant level of cytolysis (5%) by splenocytes was first detectable on day 8 after the first dose of SZ. The cytotoxicity reached a maximum of approximately 9% on day 10 and slowly decreased thereafter, becoming undetectable 42 d after SZ was first given. The time course of the in vitro cytotoxic response correlated with the degree of insulitis demonstrable in the pancreata of the Multi-Strep mice. The degree of cytotoxicity after Multi-Strep was related to the number of effector splenocytes to which the target RIN cells were exposed and was comparable to that detectable after immunization by intraperitoneal injection of RIN cells in normal mice. The cytotoxicity was specific for insulin-producing cells; syngeneic, allogeneic, and xenogeneic lymphocytes and lymphoblasts, 3T3 cells, and a human keratinocyte cell line were not specifically lysed by the splenocytes of the Multi-Strep mice. This phenomenon was limited to the Multi-Strep mice. Splenocytes from mice made diabetic by a single, high dose of SZ exhibited a very low level of cytotoxicity against the RIN cells. The cytotoxic response was also quantitated in splenocytes from control and Multi-Strep mice (10 d after the first dose of SZ) before and after culture with mitomycin-treated RIN cells in the presence of T cell growth factor (TCGF). The cytotoxicity of the Multi-Strep splenocytes was enhanced more than fivefold after such culture, suggesting the proliferation of an effector cell that could be stimulated and supported in vitro by TCGF. These results support the hypothesis that cell-mediated anti-beta cell autoimmunity may play a role in the destruction of the beta cells in this animal model. The

  18. Suppression of growth hormone (GH) secretion by a selective GH-releasing hormone (GHRH) antagonist. Direct evidence for involvement of endogenous GHRH in the generation of GH pulses.

    PubMed Central

    Jaffe, C A; Friberg, R D; Barkan, A L

    1993-01-01

    To study the potential involvement of growth hormone-releasing hormone (GHRH) in the generation of growth hormone (GH) pulses in humans we have used a competitive antagonist to the GHRH receptor, (N-Ac-Tyr1,D-Arg2)GHRH(1-29)NH2(GHRH-Ant). Six healthy young men were given a bolus injection of GHRH-Ant 400 micrograms/kg body wt or vehicle at 2200 h and nocturnal GH concentrations were assessed by every 10-min blood sampling until 0800 h. Integrated total and pulsatile GH secretion were suppressed during GHRH-Ant treatment by 40 +/- 6 (SE) % and 75 +/- 5%, respectively. GHRH-Ant suppressed maximum (7.6 +/- 2.2 vs 1.8 +/- 0.5 micrograms/liter; P < 0.001) and mean (3.3 +/- 1.0 vs 1.1 +/- 0.2 micrograms/liter; P = 0.02) GH pulse amplitudes. There was no change in integrated nonpulsatile GH levels, pulse frequency, or interpulse GH concentration. GHRH-Ant 400 micrograms/kg also suppressed the GH responses to intravenous boluses of GHRH 0.33 micrograms/kg given 1, 6, 12, and 24 h later by 95, 81, 59, and 4%, respectively. In five healthy men, the responses to 10-fold larger GHRH boluses (3.3 micrograms/kg) were suppressed by 82 and 0%, 1 and 6 h after GHRH-Ant 400 micrograms/kg, respectively. These studies provide the first direct evidence that endogenous GHRH participates in the generation of spontaneous GH pulses in humans. PMID:8349808

  19. Growth hormone (GH) induces tyrosine-phosphorylated proteins in mouse L cells that express recombinant GH receptors.

    PubMed Central

    Wang, X; Xu, B; Souza, S C; Kopchick, J J

    1994-01-01

    Porcine and bovine GH receptor (GHR) cDNAs were stably expressed in mouse L cells, which normally do not possess detectable levels of mouse GHR. Expression of the GHR cDNAs resulted in specific binding of 125I-labeled GH by these cell lines. To study GHR-related signaling events in these cells, protein tyrosine phosphorylation was examined. In GH-treated cells, a tyrosine-phosphorylated protein with a molecular mass of approximately 95 kDa (pp95) was increased dramatically (approximately 100-fold) relative to non-GH-treated cells. The amount of pp95 within the cells after GH treatment was positively correlated with the number of GHRs on the cells. Tyrosine phosphorylation of pp95 could not be induced by prolactin, insulin, insulin-like growth factor I, interleukin 2, epidermal growth factor, platelet-derived growth factor, or fibroblast growth factor. Phosphorylation of pp95 was found to be a rapid event that could be observed 60 sec after GH treatment. Also, pp95 appears to exist as a complex of two proteins, i.e., pp95 and pp96. The GH-induced response by these cells may be of use in screening GH analogs for biological activity. Images PMID:7509070

  20. GH safety workshop position paper: a critical appraisal of recombinant human GH therapy in children and adults

    PubMed Central

    Allen, D B; Backeljauw, P; Bidlingmaier, M; Biller, B M K; Boguszewski, M; Burman, P; Butler, G; Chihara, K; Christiansen, J; Cianfarani, S; Clayton, P; Clemmons, D; Cohen, P; Darendeliler, F; Deal, C; Dunger, D; Erfurth, E M; Fuqua, J S; Grimberg, A; Haymond, M; Higham, C; Ho, K; Hoffman, A R; Hokken-Koelega, A; Johannsson, G; Juul, A; Kopchick, J; Lee, P; Pollak, M; Radovick, S; Robison, L; Rosenfeld, R; Ross, R J; Savendahl, L; Saenger, P; Toft Sorensen, H; Stochholm, K; Strasburger, C; Swerdlow, A; Thorner, M

    2015-01-01

    Recombinant human GH (rhGH) has been in use for 30 years, and over that time its safety and efficacy in children and adults has been subject to considerable scrutiny. In 2001, a statement from the GH Research Society (GRS) concluded that ‘for approved indications, GH is safe’; however, the statement highlighted a number of areas for on-going surveillance of long-term safety, including cancer risk, impact on glucose homeostasis, and use of high dose pharmacological rhGH treatment. Over the intervening years, there have been a number of publications addressing the safety of rhGH with regard to mortality, cancer and cardiovascular risk, and the need for long-term surveillance of the increasing number of adults who were treated with rhGH in childhood. Against this backdrop of interest in safety, the European Society of Paediatric Endocrinology (ESPE), the GRS, and the Pediatric Endocrine Society (PES) convened a meeting to reappraise the safety of rhGH. The ouput of the meeting is a concise position statement. PMID:26563978

  1. Considering GH replacement for GH-deficient adults with a previous history of cancer: a conundrum for the clinician.

    PubMed

    Yuen, Kevin C J; Heaney, Anthony P; Popovic, Vera

    2016-05-01

    Previous studies have shown that GH and IGF-I may enhance tumorigenesis, metastasis, and cell proliferation in humans and animals. Evidence supporting this notion is derived from animal model studies, epidemiological studies, experience from patients with acromegaly, molecular therapeutic manipulation of GH and IGF-I actions, and individuals with GH receptor and congenital IGF-I deficiencies. Prior exposure to radiation therapy, aging, family history of cancer, and individual susceptibility may also contribute to increase this risk. Therefore, the use of GH replacement in patients with a history of cancer raises hypothetical safety concerns for patients, caregivers, and providers. Studies of GH therapy in GH-deficient adults with hypopituitarism and childhood cancer survivors have not convincingly demonstrated an increased cancer risk. Conversely, the risk of occurrence of a second neoplasm (SN) in childhood cancer survivors may be increased, with meningiomas being the most common tumor; however, this risk appears to decline over time. In light of these findings, if GH replacement is to be considered in patients with a previous history of cancer, we propose this consideration to be based on each individual circumstance and that such therapy should only be initiated at least 2 years after cancer remission is achieved with the understanding that in some patients (particularly those with childhood cancers), GH may potentially increase the risk of SNs. In addition, close surveillance should be undertaken working closely with the patient's oncologist. More long-term data are thus needed to determine if GH replacement in GH-deficient adults with a history of cancer is associated with the development of de novo tumors and tumor recurrence. PMID:26732039

  2. Human growth hormone (GH)-releasing factor stimulates and somatostatin inhibits the release of rat GH variants.

    PubMed

    Yokoya, S; Friesen, H G

    1986-11-01

    Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) was used for the analysis of proteins secreted by male rat pituitary cells in monolayer culture in the presence of 10 nM human GH-releasing factor (hGRF) or 30 nM somatostatin (SRIF) or in the absence of these factors. More than 300 medium proteins were reproducibly detected either by fluorographic autoradiography or by silver staining. Immunoreactivity of each protein was examined after 2D PAGE followed by Western blotting and immunostaining with affinity-purified antirat GH (rGH) antibody. While there was a cluster of immunoreactive spots in the GH dimer range (40,000-50,000 mol wt), at least 16 medium proteins of mol wt 22,000 or less were also stained. Among these 16 proteins the release of 15 was stimulated and the release of 14 was inhibited by hGRF and SRIF, respectively. On the other hand, there were 3 proteins of approximate mol wt 16,000 whose secretion was regulated in a coordinate manner as rGH by the hypothalamic factors but which did not cross-react with anti-rGH antibodies. The increase or decrease in the radioactivity of each protein spot obtained from media after pituitary cells were incubated with [35S]methionine and hypothalamic factors was analyzed statistically. A pulse-chase study suggested that at least 7 of the hormonally regulated proteins, including rGH, were synthesized very rapidly. Finally, the 2D PAGE analysis of cell-free translation products of messenger RNA derived from male rat anterior pituitaries revealed the presence of about 40 rGH-immunoreactive proteins which included pre-GH. These data suggest that there are multiple forms of rGH-variants or rGH-related proteins. The biological significance(s) of all the rGH immunoreactive proteins and of the GRF- and SRIF-regulated pituitary proteins remains unclear. It is evident that a number of these proteins are synthesized and released rapidly by pituitary cells in culture. Furthermore, the presence of multiple genes for

  3. Expression of endogenous and exogenous growth hormone (GH) messenger (m) RNA in a GH-transgenic tilapia (Oreochromis niloticus).

    PubMed

    Caelers, Antje; Maclean, Norman; Hwang, Gyulin; Eppler, Elisabeth; Reinecke, Manfred

    2005-02-01

    We have previously produced transgenic fish from crosses between a wild-type female tilapia (Oreochromis niloticus) and a G transgenic male. This line of growth-enhanced tilapia carries a single copy of a chinook salmon (s) growth hormone (GH) gene spliced to an ocean pout antifreeze promoter (OPA-FPcsGH) co-ligated to a carp beta-actin/lacZ reporter gene construct, integrated into the tilapia genome. Because little is known about the expression sites of transgenes, we have characterised the gene expression patterns of sGH and tilapia (t)GH in transgenic tilapia using a newly established real-time PCR to measure the absolute mRNA amounts of both hormones. The sGH gene, which was expected to be expressed mainly in liver, was also found to be expressed in other organs, such as gills, heart, brain, skeletal muscle, kidney, spleen, intestine and testes. However, in pituitary no sGH mRNA but only tGH mRNA was found. Tilapia GH mRNA in wild-type pituitary amounted to 226 +/- 30 pg/microg total RNA but in transgenics only to 187 +/- 43 pg/microg total RNA. Liver exhibited the highest level of sGH mRNA (8.3 +/- 2.5 pg/microg total RNA) but the extrahepatic sites expressed considerable amounts of sGH mRNA ranging from 4.1 +/- 2.0 pg/microg total RNA in gills to 0.2 +/- 0.08 pg/microg total RNA in kidney. The widespread expression of the sGH gene is assumed to be due to the tissue specificity of the type III AFP gene promoter. It is assumed that our transgenic experiments, which in contrast to some other approaches caused no obvious organ abnormalities, mimick the GH expression during ontogeny. Because sGH mRNA is expressed both in liver and in extrahepatic sites it may not only promote secretion and release of liver-derived (endocrine) IGF-I leading to an overall growth enhancement but also stimulate IGF-I expression within the different organs in a paracrine/autocrine manner and, thus, further promote organ growth. PMID:15865052

  4. Action of three GH51 and one GH54 α-arabinofuranosidases on internally and terminally located arabinofuranosyl branches.

    PubMed

    Koutaniemi, Sanna; Tenkanen, Maija

    2016-07-10

    The action of three glycoside hydrolase family GH51 and one GH54 α-arabinofuranosidases (ABFs) was studied on polymeric arabinoxylan and arabinoxylooligosaccharides. The substrates covered all possible arabinofuranosyl (Araf) substituents, i.e., terminal and internal α(1→2) and α(1→3)-linked Araf monosubstitutions and disubstitutions. The GH51 ABFs removed nearly all mono- and disubstitutions from terminal non-reducing end xylopyranosyl (Xylp) residues, showing dual ABF-m/d activity. From internal Xylp, primarily monosubstitutions were removed, except after treatment with GH51 Aspergillus niger ABF. It degraded slowly internal disubstitutions as well, showing versatility in substrate specificity within GH51. GH54 Trichoderma reesei ABF core protein also presented dual ABF-m/d activity, slowly degrading Araf disubstitutions from both terminal and internal positions. Surprisingly, regioselectivity of the T. reesei ABF changed from α(1→3)-linked Araf on terminal Xylp to α(1→2)-linked Araf on internal Xylp on both mono- and disubstitutions. In conclusion, systematic analysis of natural substrates revealed interesting new details on the action T. reesei ABF and showed that the dual ABF-m/d activity could be more prevalent than previously thought, especially within GH51 ABFs. PMID:27142490

  5. Effect of biosynthetic methionyl growth hormone (GH) therapy on the immune function in GH-deficient children.

    PubMed

    Bozzola, M; Cisternino, M; Valtorta, A; Moretta, A; Biscaldi, I; Maghnie, M; De Amici, M; Schimpff, R M

    1989-01-01

    The ability of growth hormone (GH) to influence certain immune functions has been studied in 21 GH-deficient children aged 1.8-17.7 years, before and during therapy with biosynthetic methionyl-hGH (12 IU/m2) injected intramuscularly 3 times weekly. Blood was collected prior to GH treatment, then after 1 week, again at 3-6 months, and finally at 9-12 months of therapy. We studied (1) the distribution of the T lymphocyte subpopulations: T total (CD3), helper/inducer (CD4) and suppressor/cytotoxic (CD8) cells, using monoclonal antibodies (OKT3, OKT4, OKT8) and (2) the in vitro IgM production stimulated by pokeweed mitogen. Pretreatment CD3, CD4, CD8 values were within the normal range. They did not change after 1 week of GH therapy. Following 3-6 months of GH treatment, CD3 significantly increased (p less than 0.001), CD4 decreased (p less than 0.01), CD8 increased (p less than 0.001) and the CD4/CD8 ratio decreased (p less than 0.001). At 9-12 months of therapy, the percentages of the different groups of T cells was not significantly different from the pretreatment values. In vitro IgM production before and following 3-6 months of GH treatment was significantly lower (p less than 0.005) than that of 15 age-matched controls. At 9-12 months, GH therapy restored the in vitro IgM production. No variations in the levels of serum immunoglobulins were observed throughout the treatment period. These data suggest that GH plays a role in the development of the immune function in children. PMID:2571554

  6. TIMP3 Modulates GHR Abundance and GH Sensitivity.

    PubMed

    Zhang, Yue; Wang, Xiangdong; Loesch, Kimberly; May, Larry A; Davis, George E; Jiang, Jing; Frank, Stuart J

    2016-06-01

    GH receptor (GHR) binds GH at the cell surface via its extracellular domain and initiates intracellular signal transduction, resulting in important anabolic and metabolic actions. GH signaling is subject to dynamic regulation, which in part is exerted by modulation of cell surface GHR levels. Constitutive and inducible metalloprotease-mediated cleavage of GHR regulate GHR abundance and thereby modulate GH action. We previously demonstrated that GHR proteolysis is catalyzed by the TNF-α converting enzyme (TACE; ADAM17). Tissue inhibitors of metalloproteases-3 (TIMP3) is a natural specific inhibitor of TACE, although mechanisms underlying this inhibition are not yet fully understood. In the current study, we use two model cell lines to examine the relationships between cellular TACE, TIMP3 expression, GHR metalloproteolysis, and GH sensitivity. These two cell lines exhibited markedly different sensitivity to inducible GHR proteolysis, which correlated directly to their relative levels of mature TACE vs unprocessed TACE precursor and indirectly to their levels of cellular TIMP3. Our results implicate TIMP3 as a modulator of cell surface GHR abundance and the ability of GH to promote cellular signaling; these modulatory effects may be conferred by endogenous TIMP3 expression as well as exogenous TIMP3 exposure. Furthermore, our analysis suggests that TIMP3, in addition to regulating the activity of TACE, may also modulate the maturation of TACE, thereby affecting the abundance of the active form of the enzyme. PMID:27075707

  7. Therapeutic Efficacy and Safety of GH in Japanese Children with Down Syndrome Short Stature Accompanied by GH Deficiency

    PubMed Central

    Meguri, Kyoko; Inoue, Masaru; Narahara, Koji; Sato, Takahiro; Takata, Ami; Ohki, Nobuhiko; Ozono, Keiichi

    2013-01-01

    In this study, we investigated the effects of GH treatment in children with Down syndrome who had been diagnosed with GH deficiency (GHD). A total of 20 subjects were investigated in this study. Fourteen Down syndrome children (5 boys and 9 girls) with short stature due to GHD were treated with GH at Okayama Red Cross General Hospital, and 6 Down syndrome children (4 boys and 2 girls) with short stature due to GHD were registered in the Pfizer International Growth Database (KIGS). Height SD score (SDS) increased throughout the three-year GH treatment period. The overall mean height SDS increased from –3.5 at baseline to –2.5 after 3 yr of treatment. The mean change in height SDS during these 3 yr was 1.1. In addition, height assessment of SD score based on Down syndrome-specific growth data in the Japanese population revealed that the height SDS (Down syndrome) also increased across the 3-yr GH treatment period. The mean change in height SDS (Down syndrome) during these three years was 1.3. GH therapy was effective for Down syndrome short stature accompanied by GHD, and no new safety concerns were found in this study. PMID:24170963

  8. Molecular and SNP characterization of two genome specific transcription factor genes GhMyb8 and GhMyb10 in cotton species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two R2R3-Myb cDNAs (GhMyb8 and GhMyb10) and their corresponding genes were isolated and characterized from allotetraploid cotton (Gossypium hirsutum L. cv. DES119) fiber cells. Both GhMyb8 and GhMyb10 exhibit some conserved features shared in subgroup 4 of plant R2R3-MYB proteins, including the GIDx...

  9. The characterization of six auxin-induced tomato GH3 genes uncovers a member, SlGH3.4, strongly responsive to arbuscular mycorrhizal symbiosis.

    PubMed

    Liao, Dehua; Chen, Xiao; Chen, Aiqun; Wang, Huimin; Liu, Jianjian; Liu, Junli; Gu, Mian; Sun, Shubin; Xu, Guohua

    2015-04-01

    In plants, the GH3 gene family is widely considered to be involved in a broad range of plant physiological processes, through modulation of hormonal homeostasis. Multiple GH3 genes have been functionally characterized in several plant species; however, to date, limited works to study the GH3 genes in tomato have been reported. Here, we characterize the expression and regulatory profiles of six tomato GH3 genes, SlGH3.2, SlGH3.3, SlGH3.4, SlGH3.7, SlGH3.9 and SlGH3.15, in response to different phytohormone applications and arbuscular mycorrhizal (AM) fungal colonization. All six GH3 genes showed inducible responses to external IAA, and three members were significantly up-regulated in response to AM symbiosis. In particular, SlGH3.4, the transcripts of which were barely detectable under normal growth conditions, was strongly activated in the IAA-treated and AM fungal-colonized roots. A comparison of the SlGH3.4 expression in wild-type plants and M161, a mutant with a defect in AM symbiosis, confirmed that SlGH3.4 expression is highly correlated to mycorrhizal colonization. Histochemical staining demonstrated that a 2,258 bp SlGH3.4 promoter fragment could drive β-glucuronidase (GUS) expression strongly in root tips, steles and cortical cells of IAA-treated roots, but predominantly in the fungal-colonized cells of mycorrhizal roots. A truncated 654 bp promoter failed to direct GUS expression in IAA-treated roots, but maintained the symbiosis-induced activity in mycorrhizal roots. In summary, our results suggest that a mycorrhizal signaling pathway that is at least partially independent of the auxin signaling pathway has evolved for the co-regulation of the auxin- and mycorrhiza-activated GH3 genes in plants. PMID:25535196

  10. The role of GH in adipose tissue: lessons from adipose-specific GH receptor gene-disrupted mice.

    PubMed

    List, Edward O; Berryman, Darlene E; Funk, Kevin; Gosney, Elahu S; Jara, Adam; Kelder, Bruce; Wang, Xinyue; Kutz, Laura; Troike, Katie; Lozier, Nicholas; Mikula, Vincent; Lubbers, Ellen R; Zhang, Han; Vesel, Clare; Junnila, Riia K; Frank, Stuart J; Masternak, Michal M; Bartke, Andrzej; Kopchick, John J

    2013-03-01

    GH receptor (GHR) gene-disrupted mice (GHR-/-) have provided countless discoveries as to the numerous actions of GH. Many of these discoveries highlight the importance of GH in adipose tissue. For example GHR-/- mice are insulin sensitive yet obese with preferential enlargement of the sc adipose depot. GHR-/- mice also have elevated levels of leptin, resistin, and adiponectin, compared with controls leading some to suggest that GH may negatively regulate certain adipokines. To help clarify the role that GH exerts specifically on adipose tissue in vivo, we selectively disrupted GHR in adipose tissue to produce Fat GHR Knockout (FaGHRKO) mice. Surprisingly, FaGHRKOs shared only a few characteristics with global GHR-/- mice. Like the GHR-/- mice, FaGHRKO mice are obese with increased total body fat and increased adipocyte size. However, FaGHRKO mice have increases in all adipose depots with no improvements in measures of glucose homeostasis. Furthermore, resistin and adiponectin levels in FaGHRKO mice are similar to controls (or slightly decreased) unlike the increased levels found in GHR-/- mice, suggesting that GH does not regulate these adipokines directly in adipose tissue in vivo. Other features of FaGHRKO mice include decreased levels of adipsin, a near-normal GH/IGF-1 axis, and minimal changes to a large assortment of circulating factors that were measured such as IGF-binding proteins. In conclusion, specific removal of GHR in adipose tissue is sufficient to increase adipose tissue and decrease circulating adipsin. However, removal of GHR in adipose tissue alone is not sufficient to increase levels of resistin or adiponectin and does not alter glucose metabolism. PMID:23349524

  11. Cloning and characterization of the first GH10 and GH11 xylanases from Rhizopus oryzae.

    PubMed

    Xiao, Zhizhuang; Grosse, Stephan; Bergeron, Hélène; Lau, Peter C K

    2014-10-01

    The only available genome sequence for Rhizopus oryzae strain 99-880 was annotated to not encode any β-1,4-endoxylanase encoding genes of the glycoside hydrolase (GH) family 10 or 11. Here, we report the identification and cloning of two such members in R. oryzae strain NRRL 29086. Strain 29086 was one of several selected fungi grown on wheat or triticale bran and screened for xylanase activity among other hydrolytic actions. Its high activity (138 U/ml) in the culture supernatant led to the identification of two activity-stained proteins, designated Xyn-1 and Xyn-2 of respective molecular masses 32,000 and 22,000. These proteins were purified to electrophoretic homogeneity and characterized. The specific activities of Xyn-1 and Xyn-2 towards birchwood xylan were 605 and 7,710 U/mg, respectively. Kinetic data showed that the lower molecular weight Xyn-2 had a higher affinity (K m=3.2 ± 0.2 g/l) towards birchwood xylan than Xyn-1 by about 4-fold. The melting temperature (T m) of the two proteins, estimated to be in the range of 49.5-53.7 °C indicated that they are rather thermostable proteins. N-terminal and internal peptide sequences were obtained by chemical digestion of the purified xylanases to facilitate cloning, expression in Escherichia coli, and sequencing of the respective gene. The cloned Rhizopus xylanases were used to demonstrate release of xylose from flax shives-derived hemicellulose as model feedstock. Overall, this study expands the catalytic toolbox of GH10 and 11 family proteins that have applications in various industrial and bioproducts settings. PMID:24760228

  12. Estrogen priming effect on growth hormone (GH) provocative test: a useful tool for the diagnosis of GH deficiency.

    PubMed

    Martínez, A S; Domené, H M; Ropelato, M G; Jasper, H G; Pennisi, P A; Escobar, M E; Heinrich, J J

    2000-11-01

    We have studied the effect of estradiol (E2) on the GH-insulin-like growth factor (GH-IGF) axis in 15 prepubertal GH deficiency (GHD) children and 44 prepubertal or early pubertal children with idiopathic short stature (SS). All of them received a daily dose of micronized E2 (1 or 2 mg) or placebo, for 3 days, before a sequential arginine-clonidine test. In SS children, GH maximal responses were 17.8+/-10.9 on placebo and 27.9+/-14.5 microg/L on estrogen (P < 0.0001). The lower 95% confidence limits for GH maximal response changed from 3.7 microg/L (without E2) to 8.3 microg/L (on E2). In GHD children, no significant stimulatory effect of estrogen on GH levels was observed. After placebo, a cut-off limit of 3.7 microg/L (the lower 95% confidence interval limit) resulted in 73% sensitivity, 95% specificity, and an overall 90% diagnostic efficiency. After E2, a cut-off limit of 8.3 microg/L resulted in a sensitivity of 87%, a specificity of 98%, and a diagnostic efficiency of 95%. After placebo, 68% of SS showed normal IGF-I levels, and the mean did not change on E2 (13.7+/-6.3 vs. 14.3+/-6.8 nmol/L, not significant). In 93% of SS, IGF binding protein (IGFBP)-3 levels were normal during placebo. On E2, mean IGFBP-3 did not change (2.63+/-0.70 vs. 2.70+/-0.70 mg/L, not significant). In 14 of 15 GHD patients, IGF-I values were below normal on placebo, and the mean of the group did not change after E2. During placebo, 13 of 15 GHD children presented low IGFBP-3 values. During E2, there was a small significant increase in IGFBP-3 values (1.06+/-0.58 vs. 1.20+/-0.69 mg/L, P < 0.02). The highest diagnostic efficiencies for IGF-I and IGFBP-3 were observed during placebo (75% and 91%, respectively). We conclude that GH stimulation tests after E2 priming had the highest diagnostic efficiency. Our findings suggest that the effect of estrogen priming on GH stimulated levels, by reducing the number of false nonresponders, might be useful to better discriminate between normal and

  13. [GH10 Family of Glycoside Hydrolases: Structure and Evolutionary Connections].

    PubMed

    Naumoff, D G

    2016-01-01

    Evolutionary connections were analyzed for endo-β-xylanases, which possess the GH10 family catalytic domains. A homology search yielded thrice as many proteins as are available from the Carbohydrate-Active Enzymes (CAZy) database. Lateral gene transfer was shown to play an important role in evolution of bacterial proteins of the family, especially in the phyla Acidobacteria, Cyanobacteria, Planctomycetes, Spirochaetes, and Verrucomicrobia. In the case of Verrucomicrobia, 23 lateral transfers from organisms of other phyla were detected. Evolutionary relationships were observed between the GH10 family domains and domains with the TIM-barrel tertiary structure from several other glycosidase families. The GH39 family of glycoside hydrolases showed the closest relationship. Unclassified homologs were grouped into 12 novel families of putative glycoside hydrolases (GHL51-GHL62). PMID:27028821

  14. An efficient expression of Human Growth Hormone (hGH) in the milk of transgenic mice using rat {beta}-casein/hGH fusion genes

    SciTech Connect

    Lee, Chul-Sang; Yu, Dae-Yeul; Lee, Kyung-Kwang

    1996-03-01

    In order to produce human growth hormone (hGH) in the milk of transgenic mice, two expression vectors for hGH differing in their 3{prime} flanking sequences were constructed by placing the genomic sequences of hGH gene under the control of the rat {beta}-casein gene promotor. The 3{prime} flanking sequences of the expression constructs were derived from either the hGH gene (pBCN1GH) or the rat {beta}-casein gene (pBCN2GH). Transgenic lines bearing pBCN1GH expressed hGH more efficiently than those bearing pBCN2GH in the milk (19-5500 {mu}g/mL vs 0.7-2 {mu}g/mL). In particular, one of the BCN1GH lines expressed hGH as much as 5500 {plus_minus} 620 {mu}g/mL. Northern blot analysis showed that the transgene expression was specifically confined to the mammary gland and developmentally regulated like the endogeneous mouse {beta}-casein gene in the mammary gland. However, a low level of nonmammary expression was also detected with more sensitive assay methods. In conclusion, the rat {beta}-casein/hGH fusion gene could direct an efficient production of hGH in a highly tissue- and stage-specific manner in the transgenic mice and the 3{prime} flanking sequences of hGH gene had an important role for the efficient expression. 27 refs., 5 figs., 2 tabs.

  15. Internalization and synaptogenic effect of GH in retinal ganglion cells (RGCs).

    PubMed

    Fleming, Thomas; Martínez-Moreno, Carlos G; Mora, Janeth; Aizouki, Miray; Luna, Maricela; Arámburo, Carlos; Harvey, Steve

    2016-08-01

    In the chicken embryo, GH gene expression occurs in the neural retina and retinal GH promotes cell survival and induces axonal growth of retinal ganglion cells. Neuroretinal GH is therefore of functional importance before the appearance of somatotrophs and the onset of pituitary GH secretion to the peripheral plasma (at ED15-17). Endocrine actions of pituitary GH in the development and function of the chicken embryo eye are, however, unknown. This possibility has therefore been investigated in ED15 embryos and using the quail neuroretinal derived cell line (QNR/D). During this research, we studied for the first time, the coexistence of exogenous (endocrine) and local GH (autocrine/paracrine) in retinal ganglion cells (RGCs). In ovo systemic injections of Cy3-labeled GH demonstrated that GH in the embryo bloodstream was translocated into the neural retina and internalized into RGC's. Pituitary GH may therefore be functionally involved in retinal development during late embryogenesis. Cy3-labelled GH was similarly internalized into QNR/D cells after its addition into incubation media. The uptake of exogenous GH was by a receptor-mediated mechanism and maximal after 30-60min. The exogenous (endocrine) GH induced STAT5 phosphorylation and increased growth associated protein 43 (GAP43) and SNAP-25 immunoreactivity. Ex ovo intravitreal injections of Cy3-GH in ED12 embryos resulted in GH internalization and STAT5 activation. Interestingly, the CY3-labeled GH accumulated in perinuclear regions of the QNR/D cells, but was not found in the cytoplasm of neurite outgrowths, in which endogenous retinal GH is located. This suggests that exogenous (endocrine) and local (autocrine/paracrine) GH are both involved in retinal function in late embryogenesis but they co-exist in separate intracellular compartments within retinal ganglion cells. PMID:27036926

  16. 60 YEARS OF NEUROENDOCRINOLOGY: The hypothalamo-GH axis: the past 60 years.

    PubMed

    Murray, P G; Higham, C E; Clayton, P E

    2015-08-01

    At the time of the publication of Geoffrey Harris's monograph on 'Neural control of the pituitary gland' 60 years ago, the pituitary was recognised to produce a growth factor, and extracts administered to children with hypopituitarism could accelerate growth. Since then our understanding of the neuroendocrinology of the GH axis has included identification of the key central components of the GH axis: GH-releasing hormone and somatostatin (SST) in the 1970s and 1980s and ghrelin in the 1990s. Characterisation of the physiological control of the axis was significantly advanced by frequent blood sampling studies in the 1980s and 1990s; the pulsatile pattern of GH secretion and the factors that influenced the frequency and amplitude of the pulses have been defined. Over the same time, spontaneously occurring and targeted mutations in the GH axis in rodents combined with the recognition of genetic causes of familial hypopituitarism demonstrated the key factors controlling pituitary development. As the understanding of the control of GH secretion advanced, developments of treatments for GH axis disorders have evolved. Administration of pituitary-derived human GH was followed by the introduction of recombinant human GH in the 1980s, and, more recently, by long-acting GH preparations. For GH excess disorders, dopamine agonists were used first followed by SST analogues, and in 2005 the GH receptor blocker pegvisomant was introduced. This review will cover the evolution of these discoveries and build a picture of our current understanding of the hypothalamo-GH axis. PMID:26040485

  17. Health Alert: Adrenal Crisis Causes Death in Some People Who Were Treated with hGH

    MedlinePlus

    ... Alternate Language URL Health Alert: Adrenal Crisis Causes Death in Some People Who Were Treated with hGH ... individuals treated with hGH looked at causes of death among recipients and found some disturbing news. Many ...

  18. Effects of Huang Bai (Phellodendri Cortex) and Three Other Herbs on GnRH and GH Levels in GT1–7 and GH3 Cells

    PubMed Central

    Lee, Sun Haeng; Kwak, Sung Chul; Kim, Dong Kwan; Park, Sang Woug; Kim, Hyun Soo; Kim, Young-Sik; Lee, Donghun; Lee, Ju Won; Lee, Chang Gon; Lee, Hae Kyung; Cho, Sung-Min; Shin, Yu Jeong; Lee, Jin Yong; Kim, Hocheol; Chang, Gyu Tae

    2016-01-01

    The present study was to evaluate the effects of Huang Bai, Zhi Mu, Mai Ya, and Xia Ku Cao on hormone using the GT1–7 and GH3 cells. The GT1–7 and GH3 cell lines were incubated with DW; DMSO; and 30, 100, or 300 μg/mL of one of the four extract solutions in serum-free media for 24 hours. The MTT assay was performed to determine the cytotoxicity of the four herbs. The GT1–7 and GH3 cells were incubated in DW, estradiol (GT1–7 only), or noncytotoxic herb solutions in serum-free medium for 24 hours. A quantitative RT-PCR and western blot were performed to measure the GnRH expression in GT1–7 cells and GH expression in GH3 cells. Huang Bai, Zhi Mu, Xia Ku Cao, and Mai Ya inhibited the GnRH mRNA expression in GT1–7 cells, whereas Huang Bai enhanced GH mRNA expression in GH3 cells. Additionally, Xia Ku Cao inhibited GnRH protein expression in GT1–7 cells and Huang Bai promoted GH protein expression in GH3 cells. The findings suggest that Huang Bai can delay puberty by inhibiting GnRH synthesis in the hypothalamus while also accelerating growth by promoting GH synthesis and secretion in the pituitary. PMID:26925153

  19. Update in mortality in GH-treated patients.

    PubMed

    Erfurth, Eva Marie

    2013-11-01

    During GH therapy for 2.3-9.6 years, male adult-onset GH-deficient patients with a diagnosis of a nonfunctioning adenoma have no increased all-cause mortality. However, women with adult-onset GH deficiency (GHD) are still at slightly higher risk. This general improvement in mortality is due to a more contemporary regimen of cardiovascular drugs, a refinement of surgical procedures, besides the introduction of GH therapy improved hormone replacement regimens with lowered glucocorticoid replacement, updated approaches of sex steroids for women, and less use of cranial radiotherapy. The underlying disease is the most important predictor for mortality: eg, a craniopharyngioma, malignant causes of hypopituitarism, previous Cushing's disease, and the presence of diabetes insipidus/aggressive tumors. The main cause of increased mortality was cerebrovascular diseases and infectious/respiratory diseases in ACTH-deficient patients. Furthermore, there was a significant impact of young age at disease onset and of death from secondary brain tumors, with a higher risk after cranial radiotherapy. Reports on four cohorts of GH-treated childhood-onset GHD patients have been published. Two of them included only patients with idiopathic isolated GHD, neurosecretory dysfunction, idiopathic short stature, or being born short for gestational age. Increased mortality in circulatory disorders, ill-defined diseases, and bone cancer were recorded in one study, but not in the other smaller study, where suicide and accidents caused the majority of deaths. A third childhood-onset GHD cohort included patients with a background of malignant tumors, craniopharyngioma, pituitary adenomas, pituitary aplasia/hypoplasia, and trauma. An increase of all-cause mortality was recorded in both males and females. The fourth cohort included isolated GHD and idiopathic short stature (60%), but also diagnosis of chronic renal failure and Turner's syndrome. In these latter studies, an underlying serious

  20. Control of cotton fibre elongation by a homeodomain transcription factor GhHOX3

    PubMed Central

    Shan, Chun-Min; Shangguan, Xiao-Xia; Zhao, Bo; Zhang, Xiu-Fang; Chao, Lu-men; Yang, Chang-Qing; Wang, Ling-Jian; Zhu, Hua-Yu; Zeng, Yan-Da; Guo, Wang-Zhen; Zhou, Bao-Liang; Hu, Guan-Jing; Guan, Xue-Ying; Chen, Z. Jeffrey; Wendel, Jonathan F.; Zhang, Tian-Zhen; Chen, Xiao-Ya

    2014-01-01

    Cotton fibres are unusually long, single-celled epidermal seed trichomes and a model for plant cell growth, but little is known about the regulation of fibre cell elongation. Here we report that a homeodomain-leucine zipper (HD-ZIP) transcription factor, GhHOX3, controls cotton fibre elongation. GhHOX3 genes are localized to the 12th homoeologous chromosome set of allotetraploid cotton cultivars, associated with quantitative trait loci (QTLs) for fibre length. Silencing of GhHOX3 greatly reduces (>80%) fibre length, whereas its overexpression leads to longer fibre. Combined transcriptomic and biochemical analyses identify target genes of GhHOX3 that also contain the L1-box cis-element, including two cell wall loosening protein genes GhRDL1 and GhEXPA1. GhHOX3 interacts with GhHD1, another homeodomain protein, resulting in enhanced transcriptional activity, and with cotton DELLA, GhSLR1, repressor of the growth hormone gibberellin (GA). GhSLR1 interferes with the GhHOX3–GhHD1 interaction and represses target gene transcription. Our results uncover a novel mechanism whereby a homeodomain protein transduces GA signal to promote fibre cell elongation. PMID:25413731

  1. ARE THE METABOLIC EFFECTS OF GH AND IGF-I SEPARABLE?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    IGF-I mediates some, but not all of the metabolic actions of GH and it has both GH-like and insulin-like actions in vivo. GH and IGF-I both have a net anabolic effect in man enhancing whole body protein synthesis over a period of weeks and perhaps months. Both hormones favorably improve body compos...

  2. Early pregnancy IGF-I and placental GH and risk of epithelial ovarian cancer: A nested case-control study

    PubMed Central

    Schock, Helena; Fortner, Renée T; Surcel, Heljä-Marja; Grankvist, Kjell; Pukkala, Eero; Lehtinen, Matti; Lundin, Eva

    2014-01-01

    Insulin-like growth factor-I (IGF-I) signaling may promote ovarian tumor development by exerting mitotic, anti-apoptotic, and pro-angiogenic effects. During pregnancy, maternal production of IGF-I is regulated by placental growth hormone (GH). Parity is an established protective factor for ovarian cancer, however, no prior study has evaluated placental GH and IGF-I in pregnancy and epithelial ovarian cancer (EOC). Prior prospective studies on the association between IGF-I and EOC in non-pregnant populations were inconclusive and did not address associations in subtypes of EOC. Among members of the Finnish Maternity Cohort and the Northern Sweden Maternity Cohort we identified 1,045 EOC cases, diagnosed after recruitment (1975-2008) and before March 2011, and 2,658 individually matched controls. Placental GH and IGF-I were measured in serum from the last pregnancy before EOC diagnosis or selection as control. We used conditional logistic regression to estimate odds ratios (OR) and 95% confidence intervals [CI] for tertiles and a doubling of hormone concentrations. Higher IGFI was associated with a non-significant decrease in risk for invasive (ORT3 vs. T1: 0.79 [0.62-1.02]; ptrend=0.07) and endometrioid tumors (ORT3 vs. T1: 0.55 [0.28-1.07]; ptrend=0.07). The protective association between higher IGF-I levels and risk of invasive EOC was stronger in analyses limited to women aged <55 years at diagnosis (ORT3 vs. T1: 0.74 [0.57-0.96]; ptrend=0.03). Our study provides the first data on placental GH and IGF-I in pregnancy and EOC risk overall and by subtype. Our data suggest higher IGF-I levels in pregnancy may be associated with lower risk of invasive and endometrioid EOC. PMID:25516257

  3. Early pregnancy IGF-I and placental GH and risk of epithelial ovarian cancer: A nested case-control study.

    PubMed

    Schock, Helena; Fortner, Renée T; Surcel, Heljä-Marja; Grankvist, Kjell; Pukkala, Eero; Lehtinen, Matti; Lundin, Eva

    2015-07-15

    Insulin-like growth factor-I (IGF-I) signaling may promote ovarian tumor development by exerting mitotic, antiapoptotic and proangiogenic effects. During pregnancy, maternal production of IGF-I is regulated by placental growth hormone (GH). Parity is an established protective factor for ovarian cancer, however, no prior study has evaluated placental GH and IGF-I in pregnancy and epithelial ovarian cancer (EOC). Prior prospective studies on the association between IGF-I and EOC in nonpregnant populations were inconclusive and did not address associations in subtypes of EOC. Among members of the Finnish Maternity Cohort and the Northern Sweden Maternity Cohort, we identified 1,045 EOC cases, diagnosed after recruitment (1975-2008) and before March 2011 and 2,658 individually matched controls. Placental GH and IGF-I were measured in serum from the last pregnancy before EOC diagnosis or selection as control. We used conditional logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for tertiles and a doubling of hormone concentrations. Higher IGF-I was associated with a nonsignificant decrease in risk for invasive [ORT3 vs. T1 : 0.79 (0.62-1.02); ptrend  = 0.07] and endometrioid tumors [ORT3 vs. T1 : 0.55 (0.28-1.07); ptrend  = 0.07]. The protective association between higher IGF-I levels and risk of invasive EOC was stronger in analyses limited to women aged <55 years at diagnosis [ORT3 vs. T1 : 0.74 (0.57-0.96); ptrend  = 0.03]. Our study provides the first data on placental GH and IGF-I in pregnancy and EOC risk overall and by subtype. Our data suggest higher IGF-I levels in pregnancy may be associated with lower risk of invasive and endometrioid EOC. PMID:25516257

  4. Structural-Functional Analysis Reveals a Specific Domain Organization in Family GH20 Hexosaminidases

    PubMed Central

    Val-Cid, Cristina; Biarnés, Xevi; Faijes, Magda; Planas, Antoni

    2015-01-01

    Hexosaminidases are involved in important biological processes catalyzing the hydrolysis of N-acetyl-hexosaminyl residues in glycosaminoglycans and glycoconjugates. The GH20 enzymes present diverse domain organizations for which we propose two minimal model architectures: Model A containing at least a non-catalytic GH20b domain and the catalytic one (GH20) always accompanied with an extra α-helix (GH20b-GH20-α), and Model B with only the catalytic GH20 domain. The large Bifidobacterium bifidum lacto-N-biosidase was used as a model protein to evaluate the minimal functional unit due to its interest and structural complexity. By expressing different truncated forms of this enzyme, we show that Model A architectures cannot be reduced to Model B. In particular, there are two structural requirements general to GH20 enzymes with Model A architecture. First, the non-catalytic domain GH20b at the N-terminus of the catalytic GH20 domain is required for expression and seems to stabilize it. Second, the substrate-binding cavity at the GH20 domain always involves a remote element provided by a long loop from the catalytic domain itself or, when this loop is short, by an element from another domain of the multidomain structure or from the dimeric partner. Particularly, the lacto-N-biosidase requires GH20b and the lectin-like domain at the N- and C-termini of the catalytic GH20 domain to be fully soluble and functional. The lectin domain provides this remote element to the active site. We demonstrate restoration of activity of the inactive GH20b-GH20-α construct (model A architecture) by a complementation assay with the lectin-like domain. The engineering of minimal functional units of multidomain GH20 enzymes must consider these structural requirements. PMID:26024355

  5. Structure-function relationships of family GH70 glucansucrase and 4,6-α-glucanotransferase enzymes, and their evolutionary relationships with family GH13 enzymes.

    PubMed

    Meng, Xiangfeng; Gangoiti, Joana; Bai, Yuxiang; Pijning, Tjaard; Van Leeuwen, Sander S; Dijkhuizen, Lubbert

    2016-07-01

    Lactic acid bacteria (LAB) are known to produce large amounts of α-glucan exopolysaccharides. Family GH70 glucansucrase (GS) enzymes catalyze the synthesis of these α-glucans from sucrose. The elucidation of the crystal structures of representative GS enzymes has advanced our understanding of their reaction mechanism, especially structural features determining their linkage specificity. In addition, with the increase of genome sequencing, more and more GS enzymes are identified and characterized. Together, such knowledge may promote the synthesis of α-glucans with desired structures and properties from sucrose. In the meantime, two new GH70 subfamilies (GTFB- and GTFC-like) have been identified as 4,6-α-glucanotransferases (4,6-α-GTs) that represent novel evolutionary intermediates between the family GH13 and "classical GH70 enzymes". These enzymes are not active on sucrose; instead, they use (α1 → 4) glucans (i.e. malto-oligosaccharides and starch) as substrates to synthesize novel α-glucans by introducing linear chains of (α1 → 6) linkages. All these GH70 enzymes are very interesting biocatalysts and hold strong potential for applications in the food, medicine and cosmetic industries. In this review, we summarize the microbiological distribution and the structure-function relationships of family GH70 enzymes, introduce the two newly identified GH70 subfamilies, and discuss evolutionary relationships between family GH70 and GH13 enzymes. PMID:27155661

  6. Sequential Exercise in Triathletes: Variations in GH and Water Loss

    PubMed Central

    Galy, Olivier; Chamari, Karim; Peyreigne, Christelle; Mercier, Jacques; Hue, Olivier

    2014-01-01

    Growth hormone (GH) may stimulate water loss during exercise by activating sweating. This study investigated GH secretion and water loss during sequential cycling and running, taking postural changes into account. The two exercise segments had similar durations and were performed at the same relative intensity to determine their respective contributions to water loss and the plasma volume variation noted in such trials. Eight elite triathletes first performed an incremental cycle test to assess maximal oxygen consumption. Then, the triathletes performed one of two trials in randomized order: constant submaximal cycling followed by treadmill running (C1-R2) or an inversed succession of running followed by cycling (R1-C2). Each segment of both trials was performed for 20 minutes at ∼75% of maximal oxygen consumption. The second trial, reversing the segment order of the first trial, took place two weeks later. During cycling, the triathletes used their own bicycles equipped with a profiled handlebar. Blood sampling (for GH concentrations, plasma viscosity and plasma volume variation) was conducted at rest and after each segment while water loss was estimated from the post- and pre-measures. GH increases were significantly lower in R2 than C2 (72.2±50.1 vs. 164.0±157 ng.ml−1.min−1, respectively; P<0.05). Water loss was significantly lower after C1-R2 than R1-C2 (1105±163 and 1235±153 ml, respectively; P<0.05). Plasma volume variation was significantly negative in C1 and R1 (−6.15±2.0 and −3.16±5.0%, respectively; P<0.05), not significant in C2, and significantly positive for seven subjects in R2 (4.05±3.1%). We concluded that the lower GH increases in R2 may have contributed to the smaller reduction in plasma volume by reducing sweating. Moreover, this lower GH response could be explained by the postural change during the transition from cycling to running. We recommend to pay particular attention to their hydration status during R1 which could limit a

  7. Sequential exercise in triathletes: variations in GH and water loss.

    PubMed

    Galy, Olivier; Chamari, Karim; Peyreigne, Christelle; Mercier, Jacques; Hue, Olivier

    2014-01-01

    Growth hormone (GH) may stimulate water loss during exercise by activating sweating. This study investigated GH secretion and water loss during sequential cycling and running, taking postural changes into account. The two exercise segments had similar durations and were performed at the same relative intensity to determine their respective contributions to water loss and the plasma volume variation noted in such trials. Eight elite triathletes first performed an incremental cycle test to assess maximal oxygen consumption. Then, the triathletes performed one of two trials in randomized order: constant submaximal cycling followed by treadmill running (C1-R2) or an inversed succession of running followed by cycling (R1-C2). Each segment of both trials was performed for 20 minutes at ∼75% of maximal oxygen consumption. The second trial, reversing the segment order of the first trial, took place two weeks later. During cycling, the triathletes used their own bicycles equipped with a profiled handlebar. Blood sampling (for GH concentrations, plasma viscosity and plasma volume variation) was conducted at rest and after each segment while water loss was estimated from the post- and pre-measures. GH increases were significantly lower in R2 than C2 (72.2±50.1 vs. 164.0±157 ng x ml(-1) x min(-1), respectively; P<0.05). Water loss was significantly lower after C1-R2 than R1-C2 (1105±163 and 1235±153 ml, respectively; P<0.05). Plasma volume variation was significantly negative in C1 and R1 (-6.15±2.0 and -3.16±5.0%, respectively; P<0.05), not significant in C2, and significantly positive for seven subjects in R2 (4.05±3.1%). We concluded that the lower GH increases in R2 may have contributed to the smaller reduction in plasma volume by reducing sweating. Moreover, this lower GH response could be explained by the postural change during the transition from cycling to running. We recommend to pay particular attention to their hydration status during R1 which could limit a

  8. Structure and regulation of expression of the mouse GH receptor.

    PubMed

    Talamantes, F; Ortiz, R

    2002-10-01

    GH-binding protein (GHBP) in the mouse consists of a ligand-binding domain, which is identical to the extracellular portion of the GH receptor (GHR), and a hydrophilic C-terminal domain, in place of the transmembrane and intracellular domains of the GHR. The two proteins are encoded by separate mRNAs which are derived from a single gene by alternative splicing. Determination of the gestational profiles of GHR and GHBP mRNA expression in mouse liver and placenta shows that in the liver, the 1.4 kb mRNA corresponding to the mouse GHBP increases approximately 20-fold between non-pregnant and late pregnant mice, whereas the relative increase in the expression of the 4.2 kb mouse GHR was 8-fold. The rise in the steady-state levels of both mRNAs began on day 9 of gestation. Mouse GHBP mRNA levels continue to rise until day 15 of pregnancy, while GHR mRNA abundance reaches a plateau by day 13. By elucidating the temporal changes in GHR and GHBP mRNA abundance during pregnancy and lactation in multiple maternal tissues and by assessing the ontogeny of these mRNAs in fetal and early postnatal mouse liver, our studies have demonstrated that the alternative splicing of mouse GHR/GHBP mRNA precursor is regulated in a tissue-, developmental stage- and physiological state-specific manner. In vitro studies using hepatocytes in culture have begun to elucidate the hormonal factor(s) involved in the gestation control of the expression of GHR and GHBP. Treatment of hepatocytes with GH or estradiol (E2) alone did not have any effect on the cellular concentrations of GHBP and GHR. However, the combination of E2 and GH up-regulated the cellular concentrations of GHBP and GHR 2- to 3-fold. GHBP and GHR mRNA concentrations were also up-regulated 2- to 3-fold. ICI 182-780, a competitive inhibitor of E2 for the estrogen receptor (ER), at different concentrations inhibited the E2- and GH-induced stimulation of GHBP and GHR. Furthermore, ER concentrations increased 5- to 7-fold in hepatocytes

  9. The GH/IGF-1 System in Critical Illness

    PubMed Central

    Elijah, Itoro; Branski, Ludwik K.; Finnerty, Celeste F.; Herndon, David N.

    2013-01-01

    The Growth Hormone and Insulin-like Growth Factor-1 axis plays a pivotal role in critical illness, with a derangement leading to profound changes in metabolism. Protein wasting with skeletal muscle loss, delayed wound healing, and impaired recovery of organ systems are some of the most feared consequences. The use of human recombinant Growth Hormone (rhGH) and Insulin-like Growth Factor-1 (IGF-1) - alone and in combination - has been studied extensively in preclinical and clinical trials. This article reviews the current knowlegde and clinical practice of the use of rhGh and IGF-1 in critically ill patients, with a special focus on the trauma and burns patient population. PMID:21925076

  10. Enhanced spontaneous locomotor activity in bovine GH transgenic mice involves peripheral mechanisms.

    PubMed

    Bohlooly-Y, M; Olsson, B; Gritli-Linde, A; Brusehed, O; Isaksson, O G; Ohlsson, C; Söderpalm, B; Törnell, J; Ola, B

    2001-10-01

    Clinical and experimental studies indicate a role for GH in mechanisms related to anhedonia/hedonia, psychic energy, and reward. Recently we showed that transgenic mice with general overexpression of bovine GH display increased spontaneous locomotor activity. In the present study, we investigated whether this behavioral change is owing to a direct action of GH in the central nervous system or to peripheral GH actions. A transgenic construct, containing the glial fibrillary acidic protein promoter directing specific expression of bovine GH to the central nervous system, was designed. The central nervous system-specific expression of bovine GH in the glial fibrillary acidic protein-bovine GH transgenic mice was confirmed, but no effect on spontaneous locomotor activity was observed. Serum bovine GH levels were increased in glial fibrillary acidic protein-bovine GH transgenic mice but clearly lower than in transgenic mice with general overexpression of bovine GH. In contrast to the transgenic mice with general overexpression of bovine GH, glial fibrillary acidic protein-bovine GH mice did not display any difference in serum IGF-I levels. The levels of free T(3) and the conversion of the free T(4) to free T(3) were only increased in transgenic mice with general overexpression of bovine GH, but serum corticosterone levels were similarly increased in both transgenic models. These results suggest that free T(3) and/or IGF-I, affecting dopamine and serotonin systems in the central nervous system, may mediate the enhanced locomotor activity observed in transgenic mice with general overexpression of bovine GH. PMID:11564723

  11. Molecular evolution of GH in primates: characterisation of the GH genes from slow loris and marmoset defines an episode of rapid evolutionary change.

    PubMed

    Wallis, O C; Zhang, Y P; Wallis, M

    2001-06-01

    Pituitary growth hormone (GH), like several other protein hormones, shows an unusual episodic pattern of molecular evolution in which sustained bursts of rapid change are imposed on long periods of very slow evolution (near-stasis). A marked period of rapid change occurred in the evolution of GH in primates or a primate ancestor, and gave rise to the species specificity that is characteristic of human GH. We have defined more precisely the position of this burst by cloning and sequencing the GH genes for a prosimian, the slow loris (Nycticebus pygmaeus) and a New World monkey, marmoset (Callithrix jacchus). Slow loris GH is very similar in sequence to pig GH, demonstrating that the period of rapid change occurred during primate evolution, after the separation of lines leading to prosimians and higher primates. The putative marmoset GH is similar in sequence to human GH, demonstrating that the accelerated evolution occurred before divergence of New World monkeys and Old World monkeys/apes. The burst of change was confined largely to coding sequence for mature GH, and is not marked in other components of the gene sequence including signal peptide, 5' upstream region and introns. A number of factors support the idea that this episode of rapid change was due to positive adaptive selection. Thus (1) there is no apparent loss of function of GH in man compared with non-primates, (2) after the episode of rapid change the rate of evolution fell towards the slow basal level that is seen for most mammalian GHs, (3) the accelerated rate of substitution for the exons of the GH gene significantly exceeds that for introns, and (4) the amino acids contributing to the hydrophobic core of GH are strongly conserved when higher primate and other GH sequences are compared, and for coding sequences other than that coding for hydrophobic core residues the rate of substitution for non-synonymous sites (K(A)) is significantly greater than that for synonymous sites (K(S)). In slow loris, as

  12. Prolonged retention after aggregation into secretory granules of human R183H-growth hormone (GH), a mutant that causes autosomal dominant GH deficiency type II.

    PubMed

    Zhu, Yong Lian; Conway-Campbell, Becky; Waters, Michael J; Dannies, Priscilla S

    2002-11-01

    Human R183H-GH causes autosomal dominant GH deficiency type II. Because we show here that the mutant hormone is fully bioactive, we have sought to locate an impairment in its progress through the secretory pathway as assessed by pulse chase experiments. Newly synthesized wild-type and R183H-GH were stable when expressed transiently in AtT20 cells, and both formed equivalent amounts of Lubrol-insoluble aggregates within 40 min after synthesis. There was no evidence for intermolecular disulfide bond formation in aggregates of wild-type hormone or the R183H mutant. Both wild-type and R183H-GH were packaged into secretory granules, assessed by the ability of 1 mM BaCl2 to stimulate release and by immunocytochemistry. The mutant differed from wild-type hormone in its retention in the cells after packaging into secretory granules; 50% more R183H-GH than wild-type aggregates were retained in AtT20 cells 120 min after synthesis, and stimulated release of R183H-GH or a mixture of R183H-GH and wild-type that had been retained in the cell was reduced. The longer retention of R183H-GH aggregates indicates that a single point mutation in a protein contained in secretory granules affects the rate of secretory granule release. PMID:12399418

  13. A novel NAP member GhNAP is involved in leaf senescence in Gossypium hirsutum

    PubMed Central

    Fan, Kai; Bibi, Noreen; Gan, Susheng; Li, Feng; Yuan, Shuna; Ni, Mi; Wang, Ming; Shen, Hao; Wang, Xuede

    2015-01-01

    Premature leaf senescence has a negative influence on the yield and quality of cotton, and several genes have been found to regulate leaf senescence. Howeer, many underlying transcription factors are yet to be identified. In this study, a NAP-like transcription factor (GhNAP) was isolated from Gossypium hirsutum. GhNAP has the typical NAC structure and a conserved novel subdomain in its divergent transcription activation region (TAR). GhNAP was demonstrated to be a nuclear protein, and it showed transcriptional activation activity in yeast. Furthermore, the expression of GhNAP was closely associated with leaf senescence. GhNAP could rescue the delayed-senescence phenotype of the atnap null mutant. Overexpression of GhNAP could cause precocious senescence in Arabidopsis. However, down-regulation of GhNAP delayed leaf senescence in cotton, and affected cotton yield and its fibre quality. Moreover, the expression of GhNAP can be induced by abscisic acid (ABA), and the delayed leaf senescence phenotype in GhNAPi plants might be caused by the decreased ABA level and reduced expression level of ABA-responsive genes. All of the results suggested that GhNAP could regulate the leaf senescence via the ABA-mediated pathways and was further related to the yield and quality in cotton. PMID:25991739

  14. Low doses of estradiol partly inhibit release of GH in sheep without affecting basal levels.

    PubMed

    Hudmon, A; Davenport, G; Coleman, E S; Sartin, J L

    2009-10-01

    Estradiol increases basal growth hormone (GH) concentrations in sheep and cattle. This study sought to determine the effects of estradiol on GH-releasing hormone (GRH)-stimulated GH release in sheep. Growth hormone secretory characteristics, the GH response to GRH, and steady-state GH mRNA concentrations were determined in castrated male lambs treated with 2 different doses of estradiol 17-beta for a 28-d experimental period. Although no differences between treatments in mean GH, basal GH, or GH pulse number were observed after 28 d of estradiol treatment, GH pulse amplitude was greater (P < 0.05) in the 2.00-cm implant-treated animals than in the control and 0.75-cm implant group. The effect of estradiol treatment on GRH-stimulated GH release revealed differences between the control and estradiol-treated animals (P < 0.05). The 15-min GH responses to 0.075 microg/kg hGRH in the control, 0.75-cm, and 2.00-cm implant groups, respectively, were 76 +/- 10, 22.6 +/- 2.1, and 43.6 +/- 15.0 ng/mL. Growth hormone mRNA content was determined for pituitary glands from the different treatment groups, and no differences in steady-state GH mRNA levels were observed. There were no differences in the mean plasma concentrations of IGF-I, cortisol, T(3), or T(4) from weekly samples. Growth hormone release from cultured ovine pituitary cells from control sheep was not affected by estradiol after 72 h or in a subsequent 3-h incubation with estradiol combined with GRH. These data suggest that estradiol has differing actions on basal and GRH-stimulated GH concentrations in plasma, but the increase in pulse amplitude does not represent an increased pituitary sensitivity to GRH. PMID:19616401

  15. Excessive Growth Hormone Expression in Male GH Transgenic Mice Adversely Alters Bone Architecture and Mechanical Strength

    PubMed Central

    Lim, S. V.; Marenzana, M.; Hopkinson, M.; List, E. O.; Kopchick, J. J.; Pereira, M.; Javaheri, B.; Roux, J. P.; Chavassieux, P.; Korbonits, M.

    2015-01-01

    Patients with acromegaly have a higher prevalence of vertebral fractures despite normal bone mineral density (BMD), suggesting that GH overexpression has adverse effects on skeletal architecture and strength. We used giant bovine GH (bGH) transgenic mice to analyze the effects of high serum GH levels on BMD, architecture, and mechanical strength. Five-month-old hemizygous male bGH mice were compared with age- and sex-matched nontransgenic littermates controls (NT; n=16/group). Bone architecture and BMD were analyzed in tibia and lumbar vertebrae using microcomputed tomography. Femora were tested to failure using three-point bending and bone cellular activity determined by bone histomorphometry. bGH transgenic mice displayed significant increases in body weight and bone lengths. bGH tibia showed decreases in trabecular bone volume fraction, thickness, and number compared with NT ones, whereas trabecular pattern factor and structure model index were significantly increased, indicating deterioration in bone structure. Although cortical tissue perimeter was increased in transgenic mice, cortical thickness was reduced. bGH mice showed similar trabecular BMD but reduced trabecular thickness in lumbar vertebra relative to controls. Cortical BMD and thickness were significantly reduced in bGH lumbar vertebra. Mechanical testing of femora confirmed that bGH femora have decreased intrinsic mechanical properties compared with NT ones. Bone turnover is increased in favor of bone resorption in bGH tibia and vertebra compared with controls, and serum PTH levels is also enhanced in bGH mice. These data collectively suggest that high serum GH levels negatively affect bone architecture and quality at multiple skeletal sites. PMID:25646711

  16. Neuroprotection by GH against excitotoxic-induced cell death in retinal ganglion cells.

    PubMed

    Martínez-Moreno, Carlos G; Ávila-Mendoza, José; Wu, Yilun; Arellanes-Licea, Elvira Del Carmen; Louie, Marcela; Luna, Maricela; Arámburo, Carlos; Harvey, Steve

    2016-08-01

    Retinal growth hormone (GH) has been shown to promote cell survival in retinal ganglion cells (RGCs) during developmental waves of apoptosis during chicken embryonic development. The possibility that it might also against excitotoxicity-induced cell death was therefore examined in the present study, which utilized quail-derived QNR/D cells as an in vitro RGC model. QNR/D cell death was induced by glutamate in the presence of BSO (buthionine sulfoxamide) (an enhancer of oxidative stress), but this was significantly reduced (P<0.01) in the presence of exogenous recombinant chicken GH (rcGH). Similarly, QNR/D cells that had been prior transfected with a GH plasmid to overexpress secreted and non-secreted GH. This treatment reduced the number of TUNEL-labeled cells and blocked their release of lactate dehydrogenase (LDH). In a further experiment with dissected neuroretinal explants from ED (embryonic day) 10 embryos, rcGH treatment of the explants also reduced (P<0.01) the number of glutamate-BSO-induced apoptotic cells and blocked the explant release of LDH. This neuroprotective action was likely mediated by increased STAT5 phosphorylation and increased bcl-2 production, as induced by exogenous rcGH treatment and the media from GH-overexpressing QNR/D cells. As rcGH treatment and GH-overexpression cells also increased the content of IGF-1 and IGF-1 mRNA this neuroprotective action of GH is likely to be mediated, at least partially, through an IGF-1 mechanism. This possibility is supported by the fact that the siRNA knockdown of GH or IGF-1 significantly reduced QNR/D cell viability, as did the immunoneutralization of IGF-1. GH is therefore neuroprotective against excitotoxicity-induced RGC cell death by anti-apoptotic actions involving IGF-1 stimulation. PMID:27129619

  17. Construction of GH16 β-glucanase mini-cellulosomes to improve the nutritive value of barley-based diets for broilers.

    PubMed

    Costa, Mónica; Fernandes, Vania O; Ribeiro, Teresa; Serrano, Luís; Cardoso, Vânia; Santos, Helena; Lordelo, Madalena; Ferreira, Luís M A; Fontes, Carlos M G A

    2014-07-30

    Anaerobic cellulolytic bacteria organize a comprehensive range of cellulases and hemicellulases in high molecular weight multienzyme complexes termed cellulosomes. Integration of cellulosomal components occurs via highly ordered protein-protein interactions between cohesins and dockerins. This paper reports the production of mini-cellulosomes containing one (GH16-1C) or three (GH16-3C) copies of Clostridium thermocellum glucanase 16A (CtGlc16A). Barley β-1,3-1,4-glucans are known to be antinutritive for monogastric animals, particularly for poultry. GH16-1C and GH16-3C were used to supplement barley-based diets for broilers. The data revealed that the two mini-cellulosomes effectively improved the nutritive value of barley-based diets for broilers. Analysis of mini-cellulosome molecular integrity revealed that linker sequences separating protein domains in scaffoldins and cellulosomal catalytic units are highly susceptible to proteolytic attack in vivo. The data suggest that linker protection could result in further improvements in enzyme efficacy to improve the nutritive value of barley-based diets for monogastric animals. PMID:25010714

  18. GH, IGF-I and binding proteins in altered nutritional states.

    PubMed

    Ross, R J

    2000-06-01

    Nutritional state has profound actions at all levels of the GH/IGF-I axis and growth hormone has potent effects on nutritional state. The growth promoting effects of GH on linear height in children has long been recognised and, more recently, the important action of GH in maintaining adult body composition have been appreciated. The strong anabolic actions of GH have made it a drug of abuse among athletes and, of interest, clinicians, as a potent anti-catabolic therapy. In this review we consider the effects of nutrition on the GH/IGF-I axis and then discuss its potential use as an anti-catabolic agent. We also note the recent study in critically ill patients which has been associated with a poor outcome using high dose GH therapy. PMID:10997619

  19. The R3-MYB Gene GhCPC Negatively Regulates Cotton Fiber Elongation

    PubMed Central

    Liu, Bingliang; Zhu, Yichao; Zhang, Tianzhen

    2015-01-01

    Cotton (Gossypium spp.) fibers are single-cell trichomes that arise from the outer epidermal layer of seed coat. Here, we isolated a R3-MYB gene GhCPC, identified by cDNA microarray analysis. The only conserved R3 motif and different expression between TM-1 and fuzzless-lintless mutants suggested that it might be a negative regulator in fiber development. Transgenic evidence showed that GhCPC overexpression not only delayed fiber initiation but also led to significant decreases in fiber length. Interestingly, Yeast two-hybrid analysis revealed an interaction complex, in which GhCPC and GhTTG1/4 separately interacted with GhMYC1. In transgenic plants, Q-PCR analysis showed that GhHOX3 (GL2) and GhRDL1 were significantly down regulated in −1–5 DPA ovules and fibers. In addition, Yeast one-hybrid analysis demonstrated that GhMYC1 could bind to the E-box cis-elements and the promoter of GhHOX3. These results suggested that GhHOX3 (GL2) might be downstream gene of the regulatory complex. Also, overexpression of GhCPC in tobacco led to differential loss of pigmentation. Taken together, the results suggested that GhCPC might negatively regulate cotton fiber initiation and early elongation by a potential CPC-MYC1-TTG1/4 complex. Although the fibers were shorter in transgenic cotton lines than in the wild type, no significant difference was detected in stem or leaf trichomes, even in cotton mutants (five naked seed or fuzzless), suggesting that fiber and trichome development might be regulated by two sets of genes sharing a similar model. PMID:25646816

  20. GH/IGF-I Transgene Expression on Muscle Homeostasis

    NASA Technical Reports Server (NTRS)

    Schwartz, Robert J.

    1999-01-01

    We propose to test the hypothesis that the growth hormone/ insulin like growth factor-I axis through autocrine/paracrine mechanisms may provide long term muscle homeostasis under conditions of prolonged weightlessness. As a key alternative to hormone replacement therapy, ectopic production of hGH, growth hormone releasing hormone (GHRH), and IGF-I will be studied for its potential on muscle mass impact in transgenic mice under simulated microgravity. Expression of either hGH or IGF-I would provide a chronic source of a growth-promoting protein whose biosynthesis or secretion is shut down in space. Muscle expression of the IGF-I transgene has demonstrated about a 20% increase in hind limb muscle mass over control nontransgenic litter mates. These recent experiments, also establish the utility of hind-limb suspension in mice as a workable model to study atrophy in weight bearing muscles. Thus, transgenic mice will be used in hind-limb suspension models to determine the role of GH/IGF-I on maintenance of muscle mass and whether concentric exercises might act in synergy with hormone treatment. As a means to engineer and ensure long-term protein production that would be workable in humans, gene therapy technology will be used by to monitor muscle mass preservation during hind-limb suspension, after direct intramuscular injection of a genetically engineered muscle-specific vector expressing GHRH. Effects of this gene-based therapy will be assessed in both fast twitch (medial gastrocnemius) and slow twitch muscle (soleus). End-points include muscle size, ultrastructure, fiber type, and contractile function, in normal animals, hind limb suspension, and reambutation.

  1. Tripartite neuroendocrine activation of the human growth hormone (GH) axis in women by continuous 24-hour GH-releasing peptide infusion: pulsatile, entropic, and nyctohemeral mechanisms.

    PubMed

    Shah, N; Evans, W S; Bowers, C Y; Veldhuis, J D

    1999-06-01

    Despite the discovery of potent GH-releasing peptides (GHRPs) more than 15 yr ago and the recent cloning of human, rat, and pig GHRP receptors in the hypothalamus and pituitary gland, the neuroregulatory mechanisms of action of GHRP agonists on the human hypothalamo-somatotroph unit are not well delineated. To gain such clinical insights, we evaluated the ultradian (pulsatile), entropic (pattern orderliness), and nyctohemeral GH secretory responses during continuous 24-h i.v. infusion of saline vs. the most potent clinically available hexapeptide, GHRP-2 (1 microg/kg x h) in estrogen-unreplaced (mean serum estradiol, 12 +/- 2.4 pg/mL) postmenopausal women (n = 7) in a paired, randomized design. Blood was sampled every 10 min for 24 h during infusions and was assayed by ultrasensitive GH chemiluminescence assay. Pulsatile GH secretion was quantitated by deconvolution analysis, orderliness of GH release patterns by the approximate entropy statistic, and 24-h GH rhythmicity by cosinor analysis. Statistical analysis revealed that GHRP-2 elicited a 7.7-fold increase in (24-h) mean serum (+/-SEM) GH concentrations, viz. from 0.32 +/- 0.042 (saline) to 2.4 +/- 0.34 microg/L (GHRP-2; P = 0.0006). This occurred via markedly stimulated pulsatile GH release, namely a 7.1-fold augmentation of GH secretory burst mass: 0.87 +/- 0.18 (control) vs. 6.3 +/- 1.3 microg/L (GHRP-2; P = 0.0038). Enhanced GH pulse mass reflected a commensurate 10-fold (P = 0.023) rise in GH secretory burst amplitude [maximal GH secretory rate (micrograms per L/min) attained within a secretory pulse] with no prolongation in event duration. GH burst frequency, interpulse interval, and calculated GH half-life were all invariant of GHRP-2 treatment. Concurrently, as detected in the ultrasensitive GH assay, GHRP-2 augmented deconvolution-estimated interpulse (basal) GH secretion by 4.5-fold (P = 0.025). The approximate entropy of 24-h serum GH concentration profiles rose significantly during GHRP-2 infusion

  2. Preclinical and Clinical In Vitro In Vivo Correlation of an hGH Dextran Microsphere Formulation

    PubMed Central

    de Vrueh, R.; Gresnigt, M. G.; Hoogerbrugge, C. M.; van Buul-Offers, S. C.; de Leede, L. G. J.; Sterkman, L. G. W.; Crommelin, D. J. A.; Hennink, W. E.; Verrijk, R.

    2007-01-01

    Purpose To investigate the in vitro in vivo correlation of a sustained release formulation for human growth hormone (hGH) based on hydroxyethyl methacrylated dextran (dex-HEMA) microspheres in Pit-1 deficient Snell dwarf mice and in healthy human volunteers. Materials and Methods A hGH-loaded microsphere formulation was developed and tested in Snell dwarf mice (pharmacodynamic study) and in healthy human volunteers (pharmacokinetic study). Results Single subcutaneous administration of the microspheres in mice resulted in a good correlation between hGH released in vitro and in vivo effects for the hGH-loaded microsphere formulation similar to daily injected hGH indicating a retained bioactivity. Testing the microspheres in healthy volunteers showed an increase (over 7–8 days) in hGH serum concentrations (peak concentrations: 1–2.5 ng/ml). A good in vitro in vivo correlation was obtained between the measured and calculated (from in vitro release data) hGH serum concentrations. Moreover, an increased serum concentration of biomarkers (insulin-like growth factor-I (IGF-I), IGF binding protein-3 (IGFBP-3) was found again indicating that bioactive hGH was released from the microspheres. Conclusions Good in vitro in vivo correlations were obtained for hGH-loaded dex-HEMA microspheres, which is an important advantage in predicting the effect of the controlled drug delivery product in a clinical situations. PMID:17929148

  3. Growth hormone activity in mitochondria depends on GH receptor Box 1 and involves caveolar pathway targeting

    SciTech Connect

    Perret-Vivancos, Cecile; Abbate, Aude; Ardail, Dominique; Raccurt, Mireille; Usson, Yves; Lobie, Peter E.; Morel, Gerard . E-mail: gerard.morel@univ-lyon1.fr

    2006-02-01

    Growth hormone (GH) binding to its receptor (GHR) initiates GH-dependent signal transduction and internalization pathways to generate the biological effects. The precise role and way of action of GH on mitochondrial function are not yet fully understood. We show here that GH can stimulate cellular oxygen consumption in CHO cells transfected with cDNA coding for the full-length GHR. By using different GHR cDNA constructs, we succeeded in determining the different parts of the GHR implicated in the mitochondrial response to GH. Polarography and two-photon excitation fluorescence microscopy analysis showed that the Box 1 of the GHR intracellular domain was required for an activation of the mitochondrial respiration in response to a GH exposure. However, confocal laser scanning microscopy demonstrated that cells lacking the GHR Box 1 could efficiently internalize the hormone. We demonstrated that internalization mediated either by clathrin-coated pits or by caveolae was able to regulate GH mitochondrial effect: these two pathways are both essential to obtain the GH stimulatory action on mitochondrial function. Moreover, electron microscopic and biochemical approaches allowed us to identify the caveolar pathway as essential for targeting GH and GHR to mitochondria.

  4. Genetic and protein biomarkers in blood for the improved detection of GH abuse.

    PubMed

    Ferro, P; Ventura, R; Pérez-Mañá, C; Farré, M; Segura, J

    2016-09-01

    Human Growth Hormone (hGH, somatotropin) is one of the relevant forbidden substances to be detected in sport drug testing. Since the appearance of recombinant hGH (rhGH) in the 80's, its expansion and availability through the black market have increased, so the detection of its abuse continues to be a challenge at present. New techniques or biomarkers that are robust, reliable, sensitive and allowing a large detection time window are welcome. rhGH produces an increase of insulin-like growth factor 1 (IGF-1). FN1 (fibronectin 1) and RAB31 (member of RAS oncogene family) genes have been suggested as two potential biomarkers for IGF-1 abuse. Following this line, in the present study some genetic and proteomic approaches have been performed with fourteen healthy male subjects treated with rhGH (which produces increase of IGF-1 concentrations) to study FN1 gene, FN1 protein, RAB31 gene and RAB31 protein as potential biomarkers for rhGH abuse. The results showed that both, RAB31 and FN1 genes and FN1 protein could be potential biomarkers for rhGH administration. Preliminary assessments of gender, age, acute sport activities and GHRP-2 (pralmorelin, a rhGH releasing peptide) influence suggest they are not relevant confounding factors. Thus, the selected markers present high sensitivity and a larger detection window for rhGH detection than IGF-1 itself. PMID:27243825

  5. Osmoregulatory actions of the GH/IGF axis in non-salmonid teleosts

    USGS Publications Warehouse

    Mancera, J.M.; McCormick, S.D.

    1998-01-01

    Salmonid fishes provided the first findings on the influence of the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis on osmoregulation in teleost fishes. Recent studies on non-salmonid species, however, indicate that this physiological action of the GH/IGF-I axis is not restricted to salmonids or anadromous fishes. GH-producing cells in the pituitary of fish acclimated to different salinities show different degrees of activation depending on the species studied. Plasma GH levels either increase or do not change after transfer of fish from freshwater to seawater. Treatment with GH or IGF-I increases salinity tolerance and/or increases gill Na+,K+-ATPase activity of killifish (Fundulus heteroclitus), tilapia (Oreochromis mossambicus and Oreochromis niloticus) and striped bass (Morone saxatilis). As in salmonids, a positive interaction between GH and cortisol for improving hypoosmoregulatory capacity has been described in tilapia (O. mossambicus). Research on the osmoregulatory role of the GH/IGF-I axis is derived from a small number of teleost species. The study of more species with different osmoregulary patterns will be necessary to fully clarify the osmoregulatory role of GH/IGF-I axis in fish. The available data does suggest, however, that the influence of the GH/IGF-I axis on osmoregulation may be a common feature of euryhalinity in teleosts. Copyright (C) 1998 Elsevier Science Inc.

  6. Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus

    SciTech Connect

    Tong, Junsen; Yang, Huiseon; Eom, Soo Hyun; Chun, ChangJu; Im, Young Jun

    2014-09-12

    Graphical abstract: - Highlights: • The crystal structure of GKAP homology domain 1 (GH1) was determined. • GKAP GH1 is a three-helix bundle connected by short flexible loops. • The predicted helix α4 associates weakly with the helix α3, suggesting dynamic nature of the GH1 domain. - Abstract: Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domain of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering.

  7. Pulsatile characteristics of spontaneous growth hormone (GH) concentration profiles in boys evaluated by an ultrasensitive immunoradiometric assay: Evidence for ultradian periodicity of GH secretion

    SciTech Connect

    Goji, Katsumi )

    1993-03-01

    To investigate underlying ultradian periodicities in spontaneous circulating GH concentrations, blood samples were drawn from 15 normal short boys every 20 min over a 24-h period, and plasma GH concentrations were measured using an ultrasensitive immunoradiometric assay. The limit of detection for the GH assay was 0.01 [mu]g/L. The GH time series were analyzed using the Cluster program, Ultra program, cosinor analysis, and autocorrelation analysis. Plasma GH concentrations in 1,095 samples derived from 15 normal short boys were all within the detectable range of the assay and ranged from 0.07-52.2 [mu]g/L. Thirty-six percent of the GH values in the 1,095 samples from 15 normal short boys were below 1 [mu]g/L, and 82% of them occurred during the diurnal awakening period. Cluster analysis disclosed a total of 176 peaks in 15 normal short boys, with a mean [+-] SEM number of significant GH peaks of 12.1 [+-] 0.5/24 h. Twelve percent of the 176 peaks were below 1 [mu]g/L, and 95% of them occurred during the diurnal awakening period. In addition, Cluster analysis disclosed 161 interpulse intervals in total, with a mean [+-] SEM interval of 116.5 [+-] 4.3 min. The GH interpulse interval did not show a significant 24-h rhythm, whereas the GH peak height increased significantly at night. An independent discrete peak detection in program, Ultra, identified 12.6 [+-] 0.5 GH peaks/24 h. This result was in good agreement with that from analysis by the Cluster program (P = NS). Autocorrelation analysis revealed that GH time series were significantly autocorrelated in 9 of the 15 boys, with maximal autocorrelation coefficients at 115.5 min, on the average. The mean autocorrelation coefficient for a group of 15 normal short boys was significantly positive at a 100-min lag. These findings suggest that there could be a regularly occurring periodicity of approximately 100-120 min in the human GH time series. 18 refs., 4 figs., 1 tab.

  8. Divalent metal activation of a GH43 β-xylosidase.

    PubMed

    Lee, Charles C; Braker, Jay D; Grigorescu, Arabela A; Wagschal, Kurt; Jordan, Douglas B

    2013-02-01

    Depolymerization of xylan, a major fraction of lignocellulosic biomass, releases xylose which can be converted into transportation fuels and chemical feedstocks. A requisite enzyme for the breakdown of xylan is β-xylosidase. A gene encoding the 324-amino acid β-xylosidase, RS223-BX, was cloned from an anaerobic mixed microbial culture. This glycoside hydrolase belongs to family 43. Unlike other GH43 enzymes, RS223-BX can be strongly activated by exogenously supplied Ca(2+), Co(2+), Fe(2+), Mg(2+), Mn(2+) and Ni(2+) (e.g., 28-fold by Mg(2+)) and it is inhibited by Cu(2+) or Zn(2+). Sedimentation equilibrium centrifugation experiments indicated that the divalent metal cations mediate multimerization of the enzyme from a dimeric to a tetrameric state, which have equal catalytic activity on an active-site basis. Compared to the determined active sites of other GH43 β-xylosidases, the predicted active site of RS223-BX contains two additional amino acids with carboxylated side chains that provide potential sites for divalent metal cations to reside. Thus, the divalent metal cations likely occupy the active site and participate in the catalytic mechanism. RS223-BX accepts as substrate xylobiose, arabinobiose, 4-nitrophenyl-β-D-xylopyranoside, and 4-nitrophenyl-α-L-arabinofuranoside. Additionally, the enzyme has good pH and temperature stabilities and a large K(i) for D-glucose (1.3 M), favorable properties for performance in saccharification reactors. PMID:23273276

  9. A case of GH deficiency and beta-thalassemia.

    PubMed

    Smacchia, M P; Mercuri, V; Antonetti, L; Bassotti, G; D'Amico, T; Pietrobono, D; Gargiulo, P

    2012-06-01

    A 23-year-old male patient, who suffers from beta-thalassemia major, came to us for an endocrine-metabolic evaluation. Medical history showed a diagnosis of heart disease with heart failure since the age of 16, type 1 diabetes mellitus diagnosed at the age of 18, treated with an intensive insulin therapy with a poor glycometabolic control. Patient performed regular blood transfusions and iron chelation with deferasirox. An echocardiogram revealed an enlarged left ventricle. Patient had undergone a comprehensive study of buoyancy both basal and hormone-stimulated and it was therefore carried out a diagnosis of GH deficiency and hypogonadotropic hypogonadism. A recombinant GH replacement therapy was then prescribed. After six months of therapy, the patient reported a net improvement of asthenic symptoms. Physical examination showed a reduction in abdominal adiposity in waist and an increase of 5 cm in stature. Laboratory tests showed an amelioration of glycometabolic control, such as to justify a reduction in daily insulin dose. The stature observed was thought appropriate to begin the administration of testosterone. Moreover, the cardiological framework showed a reduction of left ventricular dilatation, good ventricular motility, global minimum persistent tricuspid but not mitral regurgitation and no alteration on ECG. PMID:22691893

  10. Identification of novel GH-regulated genes in C2C12 cells.

    PubMed

    Resmini, E; Morte, B; Sorianello, E; Gallardo, E; de Luna, N; Illa, I; Zorzano, A; Bernal, J; Webb, S M

    2011-12-01

    Growth hormone (GH) is the main regulator of longitudinal growth before puberty, and treatment with human recombinant (rh) GH can increase muscle strength. Nevertheless, molecular mechanisms responsible remain mostly unknown. Many physiological effects of GH require hormone-mediated changes in gene expression. In an attempt to gain insight into the mechanism of GH action in muscle cells we evaluated the effects of rhGH on gene expression profile in a murine skeletal muscle cell line C2C12. The objective of the work was to identify changes in gene expression in the murine skeletal muscle cell line C2C12 after rGH treatment using microarray assays. C2C12 murine skeletal muscle cell cultures were differentiated during 4 days. After 16 h growing in serum-free medium, C2C12 myotubes were stimulated during 6 h with 500 ng/ml rhGH. Four independent sets of experiments were performed to identify GH-regulated genes. Total RNA was isolated and subjected to analysis. To validate changes candidate genes were analyzed by real-time quantitative polymerase chain reaction. One hundred and fifty-four differentially expressed genes were identified; 90 upregulated and 64 downregulated. Many had not been previously identified as GH-responsive. Real-time PCR in biological replicates confirmed the effect of rGH on 15 genes: Cish, Serpina3g, Socs2, Bmp4, Tnfrsf11b, Rgs2, Tgfbr3, Ugdh, Npy1r, Gbp6, Tgfbi, Tgtp, Btc, Clec3b, and Bcl6. This study shows modifications in the gene expression profile of the C2C12 cell line after rhGH exposure. In vitro and gene function analysis revealed genes involved in skeletal and muscle system as well as cardiovascular system development and function. PMID:22072432

  11. Expression and functional characterization of intrafollicular GH-IGF system in the zebrafish ovary.

    PubMed

    Zhou, Rui; Yu, Susana Man Ying; Ge, Wei

    2016-06-01

    The somatotrophic axis plays important roles in influencing reproduction. All key members of this axis including growth hormone (GH, gh), GH receptors (ghra and ghrb), insulin-like growth factors (IGFs, igf1, igf2 and igf3) and IGF receptors (igf1ra and igf1rb) were detected in the zebrafish ovary. GH was exclusively expressed in the full-grown oocytes, while its receptors were detectable in both the follicle cells and oocytes. The IGFs and their receptors were all expressed in both compartments except igf3, which was expressed in the follicle cells only. During folliculogenesis, there was a sharp decrease of gh expression at follicle activation; however, the expression of its receptors increased significantly. The expression profiles of igf1, igf2a, and igf2b were similar to that of fshr, whereas igf3 expression was close to lhcgr, suggesting differential roles for different forms of IGFs in follicle development. To examine if the ovarian GH-IGF system is regulated by gonadotropins (e.g., hCG) and GH, we performed in vitro experiments using cultured zebrafish follicle cells. The expression of igf1 and igf1ra, but not others, was down-regulated by hCG (LH analog), whereas recombinant zebrafish GH stimulated igf1 expression. In addition, GH also increased the expression of activin βA subunit (inhbaa). In agreement with this, the stimulatory effect of GH but not IGF-I on oocyte maturation could be abolished by follistatin. In conclusion, the present study revealed an intrafollicular network involving GH-IGF mini-axis in the zebrafish ovary; however, it might not work in the same way as that of the systemic somatotrophic axis. PMID:26654745

  12. Autocrine/paracrine proliferative effect of ovarian GH and IGF-I in chicken granulosa cell cultures.

    PubMed

    Ahumada-Solórzano, S Marisela; Martínez-Moreno, Carlos G; Carranza, Martha; Ávila-Mendoza, José; Luna-Acosta, José Luis; Harvey, Steve; Luna, Maricela; Arámburo, Carlos

    2016-08-01

    It is known that growth hormone (GH) and its receptor (GHR) are expressed in granulosa cells (GC) and thecal cells during the follicular development in the hen ovary, which suggests GH is involved in autocrine/paracrine actions in the female reproductive system. In this work, we show that the knockdown of local ovarian GH with a specific cGH siRNA in GC cultures significantly decreased both cGH mRNA expression and GH secretion to the media, and also reduced their proliferative rate. Thus, we analyzed the effect of ovarian GH and recombinant chicken GH (rcGH) on the proliferation of pre-hierarchical GCs in primary cultures. Incubation of GCs with either rcGH or conditioned media, containing predominantly a 15-kDa GH isoform, showed that both significantly increased proliferation as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, proliferating cell nuclear antigen (PCNA) quantification and ((3)H)-thymidine incorporation ((3)H-T) assays in a dose response fashion. Both, locally produced GH and rcGH also induced the phosphorylation of Erk1/2 in GC cultures. Furthermore, GH increased IGF-I synthesis and its release into the GC culture incubation media. These results suggest that GH may act through local IGF-I to induce GC proliferation, since IGF-I immunoneutralization completely abolished the GH-induced proliferative effect. These data suggest that GH and IGF-I may play a role as autocrine/paracrine regulators during the follicular development in the hen ovary at the pre-hierarchical stage. PMID:27174747

  13. Growth hormone protects human lymphocytes from irradiation-induced cell death

    PubMed Central

    Lempereur, Laurence; Brambilla, Daria; Maria Scoto, Giovanna; D'Alcamo, Maria; Goffin, Vincent; Crosta, Lucia; Palmucci, Tullio; Rampello, Liborio; Bernardini, Renato; Cantarella, Giuseppina

    2003-01-01

    Undesired effects of cancer radiotherapy mainly affect the hematopoietic system. Growth hormone (GH) participates in both hematopoiesis and modulation of the immune response. We report both r-hGH cell death prevention and restoration of secretory capacities of irradiated human peripheral blood lymphocytes (PBL) in vitro. r-hGH induced cell survival and increased proliferation of irradiated cells. Western blot analysis indicated that these effects of GH were paralleled by increased expression of the antiapoptotic protein Bcl-2. r-hGH restored mitogen-stimulated release of IL-2 by PBL. Preincubation of irradiated lymphocytes with the growth hormone receptor (GHR) antagonists B2036 and G120 K abrogated r-hGH-dependent IL-2 release. These results demonstrate that r-hGH protects irradiated PBL from death in a specific, receptor-mediated manner. Such effect of r-hGH on PBL involves activation of the antiapoptotic gene bcl-2 and prevention of cell death, associated with preserved functional cell capacity. Finally, potential use of GH as an immunopotentiating agent could be envisioned during radiation therapy of cancer. PMID:12721095

  14. Induction of chronic growth hormone deficiency by anti-GH serum

    NASA Technical Reports Server (NTRS)

    Grindeland, R. E.; Smith, A. T.; Ellis, S.; Evans, E. S.

    1974-01-01

    The observations reported indicate that the growth rate of neonatal rats can be specifically inhibited for at least 78 days following the administration of antisera against growth hormone (GH) for only four days after birth. The inhibition can be correlated with a marked deficit of tibial growth promoting activity in the pituitary but not with the plasma concentrations of immuno-reactive GH.

  15. Effects of fulvestrant on biological activity and Wnt expression in rat GH3 cells☆

    PubMed Central

    Bai, Jiwei; Wang, Yan; Li, Chuzhong; Zhang, Yazhuo

    2012-01-01

    The present study investigated the influence of anti-estrogen treatment (fulvestrant) on pituitary adenoma cell line GH3 biological activity, the estrogen receptor α pathway, the WnT pathway, and mechanisms of decreased Wnt inhibitory factor-1 expression in GH3 cells. Results showed that fulvestrant suppressed GH3 cell proliferation and reduced hormone secretion in a dose-dependent manner. Estrogen receptor α and Wnt4 expression decreased, but Wnt inhibitory factor-1 expression increased in a dose-dependent manner following fulvestrant treatment, and β-catenin expression remained unchanged. Inhibitors of DNA methylation and histone modification upregulated Wnt inhibitory factor-1 expression. Results suggested that fulvestrant suppressed biological activity of GH3 cells via the estrogen receptor α and Wnt pathways. These results suggested that decreased Wnt inhibitory factor-1 expression in GH3 cells played a role in epigenetic mechanisms. Anti-estrogen therapies could provide novel treatments for growth hormone adenomas. PMID:25806070

  16. Effects of Double Transgenesis of Somatotrophic Axis (GH/GHR) on Skeletal Muscle Growth of Zebrafish (Danio rerio).

    PubMed

    Silva, Ana Cecilia Gomes; Almeida, Daniela Volcan; Nornberg, Bruna Felix; Figueiredo, Marcio Azevedo; Romano, Luis Alberto; Marins, Luis Fernando

    2015-12-01

    Transgenic fish for growth hormone (GH) has been considered as a potential technological improvement in aquaculture. In this study, a double-transgenic zebrafish was used to evaluate the effect of GH and its receptor (GHR) on muscle growth. Double transgenics reached the same length of GH transgenic, but with significantly less weight, featuring an unbalanced growth. The condition factor of GH/GHR-transgenic fish was lower than the other genotypes. Histological analysis showed a decrease in the percentage of thick muscle fibers in GH/GHR genotype of ∼ 80% in comparison to GH-transgenic line. The analysis of gene expression showed a significant decrease in genes related to muscle growth in GH/GHR genotype. It seems that concomitant overexpression of GH and GHR resulted in a strong decrease of the somatotrophic axis intracellular signaling by diminishing its principal transcription factor signal transducer and activator of transcription 5.1 (STAT5.1). PMID:26574627

  17. Enzymes for Pancreatic Islet Isolation Impact Chemokine-Production and Polarization of Insulin-Producing β-Cells with Reduced Functional Survival of Immunoisolated Rat Islet-Allografts as a Consequence

    PubMed Central

    de Vos, Paul; Smink, Alexandra M.; Paredes, Genaro; Lakey, Jonathan R. T.; Kuipers, Jeroen; Giepmans, Ben N. G.; de Haan, Bart J.; Faas, Marijke M.

    2016-01-01

    The primary aim of this study was to determine whether normal variations in enzyme-activities of collagenases applied for rat-islet isolation impact longevity of encapsulated islet grafts. Also we studied the functional and immunological properties of rat islets isolated with different enzyme preparations to determine whether this impacts these parameters. Rat-islets were isolated from the pancreas with two different collagenases with commonly accepted collagenase, neutral protease, and clostripain activities. Islets had a similar and acceptable glucose-induced insulin-release profile but a profound statistical significant difference in production of the chemokines IP-10 and Gro-α. The islets were studied with nanotomy which is an EM-based technology for unbiased study of ultrastructural features of islets such as cell-cell contacts, endocrine-cell condition, ER stress, mitochondrial conditions, and cell polarization. The islet-batch with higher chemokine-production had a lower amount of polarized insulin-producing β-cells. All islets had more intercellular spaces and less interconnected areas with tight cell-cell junctions when compared to islets in the pancreas. Islet-graft function was studied by implanting encapsulated and free islet grafts in rat recipients. Alginate-based encapsulated grafts isolated with the enzyme-lot inducing higher chemokine production and lower polarization survived for a two-fold shorter period of time. The lower survival-time of the encapsulated grafts was correlated with a higher influx of inflammatory cells at 7 days after implantation. Islets from the same two batches transplanted as free unencapsulated-graft, did not show any difference in survival or function in vivo. Lack of insight in factors contributing to the current lab-to-lab variation in longevity of encapsulated islet-grafts is considered to be a threat for clinical application. Our data suggest that seemingly minor variations in activity of enzymes applied for islet

  18. Elevated systolic blood pressure in male GH transgenic mice is age dependent.

    PubMed

    Jara, Adam; Benner, Chance M; Sim, Don; Liu, Xingbo; List, Edward O; Householder, Lara A; Berryman, Darlene E; Kopchick, John J

    2014-03-01

    Acromegaly is associated with an increased incidence of cardiovascular disease. Transgenic mice expressing bovine GH (bGH) gene have previously been used to examine the effects of chronic GH stimulation on cardiovascular function. Results concerning systolic blood pressure (SBP) in bGH mice are conflicting. We hypothesized that these discrepancies may be the result of the various ages of the mice used in previous studies. In the current study, SBP was assessed monthly in male bGH mice from 3-12 months of age. Factors known to alter blood pressure were assessed during this time and included: levels of brain natriuretic peptide (BNP) and glucose homeostasis markers, and renal levels of angiotensin-converting enzyme 2 and endothelial nitric oxide synthase. Beginning at 6 months of age bGH had increased SBP compared with wild-type controls, which remained elevated through 12 months of age. Despite having increased blood pressure and cardiac BNP mRNA, bGH mice had decreased circulating levels of BNP. Additionally, bGH mice had an age-dependent decline in insulin levels. For example, they were hyperinsulinemic at 3 months, but by 11 months of age were hypoinsulinemic relative to wild-type controls. This decrease in insulin was accompanied by improved glucose tolerance at 11 months. Finally, both angiotensin-converting enzyme 2 and endothelial nitric oxide synthase expression were severely depressed in kidneys of 11-month-old bGH mice. These results indicate that elevated SBP in bGH mice is dependent on age, independent of insulin resistance, and related to alterations in both the natriuretic peptide and renin-angiotensin systems. PMID:24424040

  19. GH and Pituitary Hormone Alterations After Traumatic Brain Injury.

    PubMed

    Karaca, Züleyha; Tanrıverdi, Fatih; Ünlühızarcı, Kürşad; Kelestimur, Fahrettin

    2016-01-01

    Traumatic brain injury (TBI) is a crucially important public health problem around the world, which gives rise to increased mortality and is the leading cause of physical and psychological disability in young adults, in particular. Pituitary dysfunction due to TBI was first described 95years ago. However, until recently, only a few papers have been published in the literature and for this reason, TBI-induced hypopituitarism has been neglected for a long time. Recent studies have revealed that TBI is one of the leading causes of hypopituitarism. TBI which causes hypopituitarism may be characterized by a single head injury such as from a traffic accident or by chronic repetitive head trauma as seen in combative sports including boxing, kickboxing, and football. Vascular damage, hypoxic insult, direct trauma, genetic predisposition, autoimmunity, and neuroinflammatory changes may have a role in the development of hypopituitarism after TBI. Because of the exceptional structure of the hypothalamo-pituitary vasculature and the special anatomic location of anterior pituitary cells, GH is the most commonly lost hormone after TBI, and the frequency of isolated GHD is considerably high. TBI-induced pituitary dysfunction remains undiagnosed and therefore untreated in most patients because of the nonspecific and subtle clinical manifestations of hypopituitarism. Treatment of TBI-induced hypopituitarism depends on the deficient anterior pituitary hormones. GH replacement therapy has some beneficial effects on metabolic parameters and neurocognitive dysfunction. Patients with TBI without neuroendocrine changes and those with TBI-induced hypopituitarism share the same clinical manifestations, such as attention deficits, impulsion impairment, depression, sleep abnormalities, and cognitive disorders. For this reason, TBI-induced hypopituitarism may be neglected in TBI victims and it would be expected that underlying hypopituitarism would aggravate the clinical picture of TBI itself

  20. Molecular Engineering of Fungal GH5 and GH26 Beta-(1,4)-Mannanases toward Improvement of Enzyme Activity

    PubMed Central

    Couturier, Marie; Féliu, Julia; Bozonnet, Sophie; Roussel, Alain; Berrin, Jean-Guy

    2013-01-01

    Microbial mannanases are biotechnologically important enzymes since they target the hydrolysis of hemicellulosic polysaccharides of softwood biomass into simple molecules like manno-oligosaccharides and mannose. In this study, we have implemented a strategy of molecular engineering in the yeast Yarrowia lipolytica to improve the specific activity of two fungal endo-mannanases, PaMan5A and PaMan26A, which belong to the glycoside hydrolase (GH) families GH5 and GH26, respectively. Following random mutagenesis and two steps of high-throughput enzymatic screening, we identified several PaMan5A and PaMan26A mutants that displayed improved kinetic constants for the hydrolysis of galactomannan. Examination of the three-dimensional structures of PaMan5A and PaMan26A revealed which of the mutated residues are potentially important for enzyme function. Among them, the PaMan5A-G311S single mutant, which displayed an impressive 8.2-fold increase in kcat/KM due to a significant decrease of KM, is located within the core of the enzyme. The PaMan5A-K139R/Y223H double mutant revealed modification of hydrolysis products probably in relation to an amino-acid substitution located nearby one of the positive subsites. The PaMan26A-P140L/D416G double mutant yielded a 30% increase in kcat/KM compared to the parental enzyme. It displayed a mutation in the linker region (P140L) that may confer more flexibility to the linker and another mutation (D416G) located at the entrance of the catalytic cleft that may promote the entrance of the substrate into the active site. Taken together, these results show that the directed evolution strategy implemented in this study was very pertinent since a straightforward round of random mutagenesis yielded significantly improved variants, in terms of catalytic efiiciency (kcat/KM). PMID:24278180

  1. Cello-oligosaccharide oxidation reveals differences between two lytic polysaccharide monooxygenases (family GH61) from Podospora anserina.

    PubMed

    Bey, Mathieu; Zhou, Simeng; Poidevin, Laetitia; Henrissat, Bernard; Coutinho, Pedro M; Berrin, Jean-Guy; Sigoillot, Jean-Claude

    2013-01-01

    The genome of the coprophilic ascomycete Podospora anserina encodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserina GH61A [PaGH61A] and PaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced in Pichia pastoris. Synergistic cooperation between PaGH61A or PaGH61B with the cellobiose dehydrogenase (CDH) of Pycnoporus cinnabarinus on cellulose resulted in the formation of oxidized and nonoxidized cello-oligosaccharides. A striking difference between PaGH61A and PaGH61B was observed through the identification of the products, among which were doubly and triply oxidized cellodextrins, which were released only by the combination of PaGH61B with CDH. The mass spectrometry fragmentation patterns of these oxidized products could be consistent with oxidation at the C-6 position with a geminal diol group. The different properties of PaGH61A and PaGH61B and their effect on the interaction with CDH are discussed in regard to the proposed in vivo function of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis. PMID:23124232

  2. Cello-Oligosaccharide Oxidation Reveals Differences between Two Lytic Polysaccharide Monooxygenases (Family GH61) from Podospora anserina

    PubMed Central

    Bey, Mathieu; Zhou, Simeng; Poidevin, Laetitia; Henrissat, Bernard; Coutinho, Pedro M.; Sigoillot, Jean-Claude

    2013-01-01

    The genome of the coprophilic ascomycete Podospora anserina encodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserina GH61A [PaGH61A] and PaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced in Pichia pastoris. Synergistic cooperation between PaGH61A or PaGH61B with the cellobiose dehydrogenase (CDH) of Pycnoporus cinnabarinus on cellulose resulted in the formation of oxidized and nonoxidized cello-oligosaccharides. A striking difference between PaGH61A and PaGH61B was observed through the identification of the products, among which were doubly and triply oxidized cellodextrins, which were released only by the combination of PaGH61B with CDH. The mass spectrometry fragmentation patterns of these oxidized products could be consistent with oxidation at the C-6 position with a geminal diol group. The different properties of PaGH61A and PaGH61B and their effect on the interaction with CDH are discussed in regard to the proposed in vivo function of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis. PMID:23124232

  3. GH binding to liver in young and old female rats: relation to somatomedin-C secretion

    SciTech Connect

    Takahashi, S.; Meites, J.

    1987-11-01

    Age-related changes in binding of /sup 125/I-bovine GH to liver membrane fractions were measured in female Long-Evans rats 2, 6, 12, and 20 months of age. Specific GH binding did not change between 2 and 6 months of age but increased significantly at 12 and 20 months of age. Scatchard analyses showed that the plots were curvilinear and consisted of high- and low-affinity binding sites. The age-related increases in binding sites were mainly due to an increase in number of low-affinity binding sites. Serum somatomedim-C (SM-C) levels in 20-month-old rats were about half those in the 6-month-old rats. Twice daily injections of ovine GH (2 mg/kg body wt) for 7 days depressed liver GH binding and increased serum SM-C levels in 19-month-old female rats, but had no effect on GH binding in 2-month-old female rats. These results suggest that the increase in liver GH binding sites and the decrease in SM-C secretion are associated with our previously reported decrease in GH secretion in old female rats.

  4. Specific expression of a beta-tubulin gene (GhTub1) in developing cotton fibers.

    PubMed

    Li, Yuanli; Sun, Jie; Li, Chunhong; Zhu, Yongqing; Xia, Guixian

    2003-06-01

    A cDNA library was constructed using poly (A)(+) RNA isolated from -1-15 DPA fibers of upland cotton (Gossypium hirsutum). The cDNA encoding a beta-tubulin isoform (designated as GhTub1) was identified through EST search. Northern blot analysis using 3'-UTR of the cDNA as a gene-specific probe was performed to investigate the expression levels of GhTub1 in various organs and in the developing fibers. The results showed that GhTub1 gene was specifically expressed in cotton fiber cells. During fiber development, GhTub1 transcripts accumulated highly at the stage of cell rapid elongation with the highest expression appearing at the time when fiber expansion reaches the peak rate. To probe the in vivo function of GhTub1, its cDNA was cloned in the yeast expression vector pREP1 and transformed into the fission yeast Schizosaccharomyces pombe. Overexpression of GhTub1 in yeast cells caused severe changes in the cell morphology. These results suggest that GhTub1 may play a role in the polar elongation of cotton fibers. To our knowledge, this is the first report on the fiber-specific transcript accumulation of a cotton beta-tubulin gene. PMID:18763138

  5. Enhanced lignocellulosic biomass hydrolysis by oxidative lytic polysaccharide monooxygenases (LPMOs) GH61 from Gloeophyllum trabeum.

    PubMed

    Jung, Sera; Song, Younho; Kim, Ho Myeong; Bae, Hyeun-Jong

    2015-09-01

    Lignocellulose is a renewable resource that is extremely abundant, and the complete enzymatic hydrolysis of lignocellulose requires a cocktail containing a variety of enzyme groups that act synergistically. The hydrolysis efficiency can be improved by introducing glycoside hydrolase 61 (GH61), a new enzyme that belongs to the auxiliary activity family 9 (AA9). GH61was isolated from Gloeophyllum trabeum and cleaves the glycosidic bonds on the cellulose surface via oxidation of various carbons. In this study, we investigated the properties of GH61. GtGH61 alone did not exhibit any notable activity, but the synergistic activity of GtGH61 with xylanase (GtXyl10G) or cellulase (GtCel5B) showed efficient bioconversion rates of 56 and 174% in pretreated kenaf (Hibiscus cannabinus L.) and oak (Quercus spp.), respectively. Furthermore, the GtGH61 activity was strongly accelerated in the presence of cobalt Co(2+). Enzyme cocktails (GtXyl10G, GtCel5B, and GtGH61) increased the amount of sugar released by 7 and 6% for pretreated oak and kenaf, respectively, and the addition of Co(2+) stimulated bioconversion by 12 and 11% in pretreated oak and kenaf, respectively. PMID:26138398

  6. Prokaryotic overexpression of TEV-rhGH and characterization of its polyclonal antibody.

    PubMed

    Murad, Hossam; Ali, Bouthina; Makeya, Rima; Abbady, Abdul Qader

    2014-05-25

    Recombinant protein technology represents one of the best solutions to achieve rapid, efficient, and cost-effective protein expression and purification of therapeutic proteins. Growth hormone (GH) is an excellent example of these proteins used in the therapy of hormone deficiencies. In this work, a plasmid, pRSET-TEV-rhGH, has been constructed to overexpress recombinant human GH (rhGH) by cloning its gene downstream of an N-terminal 6 × His-tagged polypeptide (43 aa) in the T7 promoter-plasmid pRSET. This polypeptide was cleavable by means of the integrated recognition site for the tobaccos etch virus (TEV) protease, resulting in an rhGH protein at an exact length and sequence. After IPTG induction, this plasmid effectively expressed TEV-rhGH protein (27 kDa) in the cytoplasm of Escherichia coli, which accumulated in the form of inclusion bodies. The 6 × His-tagged protein, with a yield of ~150 mg/L of culture, was purified from the cell extract using metal affinity chromatography, as shown after SDS-PAGE blue staining, and was confirmed by immunoblotting using specific commercial monoclonal antibodies. In order to detect TEV-rhGH, in ELISA and immunoblotting, specific polyclonal antibody, with high titer (~10⁻⁵ fold dilution), was produced in a rabbit and purified using affinity chromatography. Preliminary tests have proved that TEV-rhGH protein and its specific purified IgG antibody could provide valuable tools for rhGH productive and diagnostic purposes. PMID:24534464

  7. Does the GH/IGF-1 axis contribute to skeletal sexual dimorphism? Evidence from mouse studies.

    PubMed

    Liu, Zhongbo; Mohan, Subburaman; Yakar, Shoshana

    2016-04-01

    The contribution of the gonadotropic axis to skeletal sexual dimorphism (SSD) was clarified in recent years. Studies with animal models of estrogen receptor (ER) or androgen receptor (AR) null mice, as well as mice with bone cell-specific ablation of ER or AR, revealed that both hormones play major roles in skeletal acquisition, and that estrogen regulates skeletal accrual in both sexes. The growth hormone (GH) and its downstream effector, the insulin-like growth factor-1 (IGF-1) are also major determinants of peak bone mass during puberty and young adulthood, and play important roles in maintaining bone integrity during aging. A few studies in both humans and animal models suggest that in addition to the differences in sex steroid actions on bone, sex-specific effects of GH and IGF-1 play essential roles in SSD. However, the contributions of the somatotropic (GH/IGF-1) axis to SSD are controversial and data is difficult to interpret. GH/IGF-1 are pleotropic hormones that act in an endocrine and autocrine/paracrine fashion on multiple tissues, affecting body composition as well as metabolism. Thus, understanding the contribution of the somatotropic axis to SSD requires the use of mouse models that will differentiate between these two modes of action. Elucidation of the relative contribution of GH/IGF-1 axis to SSD is significant because GH is approved for the treatment of normal children with short stature and children with congenital growth disorders. Thus, if the GH/IGF-1 axis determines SSD, treatment with GH may be tailored according to sex. In the following review, we give an overview of the roles of sex steroids in determining SSD and how they may interact with the GH/IGF-1 axis in bone. We summarize several mouse models with impaired somatotropic axis and speculate on the possible contribution of that axis to SSD. PMID:26843472

  8. Mechanisms Involved in Glucocorticoid Induction of Pituitary GH Expression During Embryonic Development

    PubMed Central

    Ellestad, Laura E.; Puckett, Stefanie A.

    2015-01-01

    Glucocorticoid hormones are involved in functional differentiation of GH-producing somatotrophs. Glucocorticoid treatment prematurely induces GH expression in mammals and birds in a process requiring protein synthesis and Rat sarcoma (Ras) signaling. The objective of this study was to investigate mechanisms through which glucocorticoids initiate GH expression during embryogenesis, taking advantage of the unique properties of chicken embryos as a developmental model. We determined that stimulation of GH expression occurred through transcriptional activation of GH, rather than enhancement of mRNA stability, and this process requires histone deacetylase activity. Through pharmacological inhibition, we identified the ERK1/2 pathway as a likely downstream Ras effector necessary for glucocorticoid stimulation of GH. However, we also found that chronic activation of ERK1/2 activity with a constitutively active mutant or stimulatory ligand reduced initiation of GH expression by glucocorticoid treatment. Corticosterone treatment of cultured embryonic pituitary cells increased ERK1/2 activity in an apparent cyclical manner, with a rapid increase within 5 minutes, followed by a reduction to near-basal levels at 3 hours, and a subsequent increase again at 6 hours. Therefore, we conclude that ERK1/2 signaling must be strictly controlled for maximal glucocorticoid induction of GH to occur. These results are the first in any species to demonstrate that Ras- and ERK1/2-mediated transcriptional events requiring histone deacetylase activity are involved in glucocorticoid induction of pituitary GH during embryonic development. This report increases our understanding of the molecular mechanisms underlying glucocorticoid recruitment of somatotrophs during embryogenesis and should provide insight into glucocorticoid-induced developmental changes in other tissues and cell types. PMID:25560830

  9. Altered pituitary growth hormone (GH) regulation in streptozotocin-diabetic rats: a combined defect of hypothalamic somatostatin and GH-releasing factor.

    PubMed

    Olchovsky, D; Bruno, J F; Wood, T L; Gelato, M C; Leidy, J W; Gilbert, J M; Berelowitz, M

    1990-01-01

    Diabetes mellitus in the rat is associated with loss of pulsatile GH secretion. An interplay between hypothalamic GH-releasing factor (GRF) and inhibitory factor [somatostatin (SRIF)] secretion is thought to account for episodic pituitary GH release. An increase in SRIF tone/action or a decrease in GRF release/response in diabetic rats could account for the suppressed GH levels. Pituitaries from streptozotocin-diabetic rats contained less GH than controls (15.9 +/- 2.5 vs. 29.5 +/- 4.6 micrograms/mg; P less than 0.05) despite normal somatotrope representation, as demonstrated using immunofluorescence studies. Basal GH secretion from monolayer culture of dispersed anterior pituitary (AP) cells from diabetic rats was proportionately decreased (150 +/- 10 vs. 103 +/- 10 ng/10(5) cells; P less than 0.005). GRF (10(-11)-10(-8) M)-induced release of GH from AP cells was decreased in diabetic rats (maximum response to 10(-8) M GRF, 401 +/- 60 vs. 618 +/- 41 ng/10(5) cells; P less than 0.01); however, sensitivity to GRF was unchanged (EC50, 79 +/- 41 vs. 128 +/- 67 pM). By contrast, SRIF (10(-7)-10(-10)-induced inhibition of GRF (10(-8) M)-mediated GH release was impaired in AP cells of diabetic rats compared to that in controls (IC50, 112 +/- 33 vs. 55 +/- 31 pM; P less than 0.05) associated with a decrease in AP plasma membrane SRIF receptor concentration (63.4 +/- 15.6 vs. 160.3 +/- 13.7 fmol/mg protein; P less than 0.05), with no change in affinity. These findings are consistent with chronic exposure to increased hypothalamic SRIF influence. GH synthesis has been shown to be independent of SRIF regulation; however, insulin-like growth factor-I and GRF inhibit and stimulate GH synthesis, respectively. In diabetic rats insulin-like growth factor-I levels were decreased, appropriate to low GH status, in serum (290 +/- 66 vs. 1662 +/- 92 ng/ml; P less than 0.001) and hypothalamus (6.8 +/- 1.0 vs. 13.0 +/- 0.4 pg/mg wet wt; P less than 0.001) and, thus, did not seem to

  10. GH16 and GH81 family β-(1,3)-glucanases in Aspergillus fumigatus are essential for conidial cell wall morphogenesis.

    PubMed

    Mouyna, Isabelle; Aimanianda, Vishukumar; Hartl, Lukas; Prevost, Marie-Christine; Sismeiro, Odile; Dillies, Marie-Agnès; Jagla, Bernd; Legendre, Rachel; Coppee, Jean-Yves; Latgé, Jean-Paul

    2016-09-01

    The fungal cell wall is a rigid structure because of fibrillar and branched β-(1,3)-glucan linked to chitin. Softening of the cell wall is an essential phenomenon during fungal morphogenesis, wherein rigid cell wall structures are cleaved by glycosylhydrolases. During the search for glycosylhydrolases acting on β-(1,3)-glucan, we identified seven genes in the Aspergillus fumigatus genome coding for potential endo-β-(1,3)-glucanase. ENG1 (previously characterized and named ENGL1, Mouyna et al., ), belongs to the Glycoside-Hydrolase 81 (GH81) family, while ENG2 to ENG7, to GH16 family. ENG1 and four GH16 genes (ENG2-5) were expressed in the resting conidia as well as during germination, suggesting an essential role during A. fumigatus morphogenesis. Here, we report the effect of sequential deletion of AfENG2-5 (GH16) followed by AfENG1 (GH81) deletion in the Δeng2,3,4,5 mutant. The Δeng1,2,3,4,5 mutant showed conidial defects, with linear chains of conidia unable to separate while the germination rate was not affected. These results show, for the first time in a filamentous fungus, that endo β-(1,3)-glucanases are essential for proper conidial cell wall assembly and thus segregation of conidia during conidiation. PMID:27306610

  11. Benefits of long-term GH therapy in Prader-Willi syndrome: a 4-year study.

    PubMed

    Carrel, Aaron L; Myers, Susan E; Whitman, Barbara Y; Allen, David B

    2002-04-01

    Obesity, poor growth, and hypotonia in children with Prader-Willi syndrome (PWS) are accompanied by abnormal body composition resembling a GH-deficient state. Hypothalamic dysfunction in PWS includes decreased GH secretion, suggesting a possible therapeutic role for GH treatment. While short-term benefits of treatment with GH have been shown, whether these beneficial effects are dose dependent and persist or wane with prolonged therapy remains uncertain. Effects of 24 additional months of GH treatment at varying doses (0.3, 1.0, and 1.5 mg/m(2).d) on growth, body composition, strength and agility, pulmonary function, resting energy expenditure (REE), and fat utilization were assessed in 46 children with PWS, who had previously been treated with GH therapy (1 mg/m(2).d) for 12-24 months. Percent body fat, lean muscle mass, and bone mineral density (BMD) were measured by dual x-ray absorptiometry. Indirect calorimetry was used to determine REE and to calculate respiratory quotient. A modified Bruininks-Oseretski test of physical performance evaluated strength and agility. During months 24-48 of GH therapy, continued beneficial effects on body composition (decrease in fat mass and increase in lean body mass), growth velocity, and REE occurred with GH therapy doses of 1.0 and 1.5 mg/m(2).d (P < 0.05), but not with 0.3 mg/m(2).d. BMD continued to improve at all doses of GH (P < 0.05). Prior improvements in strength and agility that occurred during the initial 24 months were sustained but did not improve further during the additional 24 months regardless of dose. Salutary and sustained GH-induced changes in growth, body composition, BMD, and physical function in children with PWS can be achieved with daily administration of GH doses > or =1 mg/m(2). Lower doses of GH, (0.3 mg/m(2).d) effective in improving body composition in GHD adults, do not appear to be effective in children with PWS at sustaining improvement in body composition. PMID:11932286

  12. pKa Modulation of the Acid/Base Catalyst within GH32 and GH68: A Role in Substrate/Inhibitor Specificity?

    PubMed Central

    Yuan, Shuguang; Le Roy, Katrien; Venken, Tom; Lammens, Willem; Van den Ende, Wim; De Maeyer, Marc

    2012-01-01

    Glycoside hydrolases of families 32 (GH32) and 68 (GH68) belong to clan GH-J, containing hydrolytic enzymes (sucrose/fructans as donor substrates) and fructosyltransferases (sucrose/fructans as donor and acceptor substrates). In GH32 members, some of the sugar substrates can also function as inhibitors, this regulatory aspect further adding to the complexity in enzyme functionalities within this family. Although 3D structural information becomes increasingly available within this clan and huge progress has been made on structure-function relationships, it is not clear why some sugars bind as inhibitors without being catalyzed. Conserved aspartate and glutamate residues are well known to act as nucleophile and acid/bases within this clan. Based on the available 3D structures of enzymes and enzyme-ligand complexes as well as docking simulations, we calculated the pKa of the acid-base before and after substrate binding. The obtained results strongly suggest that most GH-J members show an acid-base catalyst that is not sufficiently protonated before ligand entrance, while the acid-base can be fully protonated when a substrate, but not an inhibitor, enters the catalytic pocket. This provides a new mechanistic insight aiming at understanding the complex substrate and inhibitor specificities observed within the GH-J clan. Moreover, besides the effect of substrate entrance on its own, we strongly suggest that a highly conserved arginine residue (in the RDP motif) rather than the previously proposed Tyr motif (not conserved) provides the proton to increase the pKa of the acid-base catalyst. PMID:22662155

  13. Dense calcification in a GH-secreting pituitary macroadenoma

    PubMed Central

    Ibrahim, Ramez; Kalhan, Atul; Lammie, Alistair; Kotonya, Christine; Nannapanenni, Ravindra; Rees, Aled

    2014-01-01

    Summary A 30-year-old female presented with a history of secondary amenorrhoea, acromegalic features and progressive visual deterioration. She had elevated serum IGF1 levels and unsuppressed GH levels after an oral glucose tolerance test. Magnetic resonance imaging revealed a heterogeneously enhancing space-occupying lesion with atypical extensive calcification within the sellar and suprasellar areas. Owing to the extent of calcification, the tumour was a surgical challenge. Postoperatively, there was clinical, radiological and biochemical evidence of residual disease, which required treatment with a somatostatin analogue and radiotherapy. Mutational analysis of the aryl hydrocarbon receptor-interacting protein (AIP) gene was negative. This case confirms the relatively rare occurrence of calcification within a pituitary macroadenoma and its associated management problems. The presentation, biochemical, radiological and pathological findings are discussed in the context of the relevant literature. Learning points Calcification of pituitary tumours is relatively rare.Recognising calcification in pituitary adenomas on preoperative imaging is important in surgical decision-making.Gross total resection can be difficult to achieve in the presence of extensive calcification and dictates further management and follow-up to achieve disease control. PMID:24683483

  14. Substrate-specific transcription of the enigmatic GH61 family of the pathogenic white-rot fungus Heterobasidion irregulare during growth on lignocellulose.

    PubMed

    Yakovlev, Igor; Vaaje-Kolstad, Gustav; Hietala, Ari M; Stefańczyk, Emil; Solheim, Halvor; Fossdal, Carl Gunnar

    2012-08-01

    The GH61 represents the most enigmatic Glycoside Hydrolase family (GH) regarding enzymatic activity and importance in cellulose degradation. Heterobasidion irregulare is a necrotizing pathogen and white-rot fungus that causes enormous damages in conifer forests. The genome of H. irregulare allowed identification of ten HiGH61 genes. qRT-PCR analysis separate the HiGH61 members into two groups; one that show up regulation on lignocellulosic substrates (HiGH61A, HiGH61B, HiGH61D, HiGH61G, HiGH61H, and HiGH61I) and a second showing either down-regulation or constitutive expression (HiGH61C, HiGH61E, HiGH61F, and HiGH61J). HiGH61H showed up to 17,000-fold increase on spruce heartwood suggesting a pivotal role in cellulose decomposition during saprotrophic growth. Sequence analysis of these genes reveals that all GH61s except HiGH61G possess the conserved metal-binding motif essential for activity. The sequences also divide into groups having either an insert near the N terminus or an insert near the second catalytic histidine, which may represent extensions of the substrate-binding surface. Three of the HiGH61s encode cellulose-binding modules (CBM1). Interestingly, HiGH61H and HiGH61I having CBM1s are up-regulated on pure cellulose. There was a common substrate-specific induction patterns of the HiGH61s with several reference cellulolytic and hemicellulolytic GHs, this taken together with their low transcript levels on media lacking lignocellulose, reflect the concerted nature of cell wall polymer degradation. PMID:22718248

  15. Structural Basis for Prereceptor Modulation of Plant Hormones by GH3 Proteins

    SciTech Connect

    Westfall, Corey S.; Zubieta, Chloe; Herrmann, Jonathan; Kapp, Ulrike; Nanao, Max H.; Jez, Joseph M.

    2013-04-08

    Acyl acid amido synthetases of the GH3 family act as critical prereceptor modulators of plant hormone action; however, the molecular basis for their hormone selectivity is unclear. Here, we report the crystal structures of benzoate-specific Arabidopsis thaliana AtGH3.12/PBS3 and jasmonic acid-specific AtGH3.11/JAR1. These structures, combined with biochemical analysis, define features for the conjugation of amino acids to diverse acyl acid substrates and highlight the importance of conformational changes in the carboxyl-terminal domain for catalysis. We also identify residues forming the acyl acid binding site across the GH3 family and residues critical for amino acid recognition. Our results demonstrate how a highly adaptable three-dimensional scaffold is used for the evolution of promiscuous activity across an enzyme family for modulation of plant signaling molecules.

  16. Effects of dietary genistein on GH/IGF-I axis of Nile tilapia Oreochromis niloticus

    NASA Astrophysics Data System (ADS)

    Chen, Dong; Wang, Wei; Ru, Shaoguo

    2016-01-01

    There is considerable concern that isoflavones, such as genistein in fish feed composed of soybean protein, aff ects somatic growth in fish. Our previous works demonstrated that 30 and 300 μg/g dietary genistein had no significant eff ect on growth performance in Nile tilapia (Oreochromis niloticus), but the higher level of genistein (3 000 μg/g) significantly depressed growth. This study was conducted to further examine the eff ects of dietary genistein on the endocrine disruption on growth hormone/insulin-like growth factor-I (GH/IGF-I) axis in Nile tilapia (O. niloticus). Juvenile fish were fed by hand twice daily to satiation with one of four isonitrogenous and isoenergetic diets, each containing either 0, 30, 300 or 3 000 μg/g genistein. Following an 8-week feeding period, plasma GH and IGF-I levels were investigated by radioimmunoassay and gene expression levels of gh, ghrelin, gnrhs, ghr, npy, npyrs, pacap, ghrs, igf-I, igf-Ir, and igfbp3 were examined by real-time PCR. The results show that no significant change in plasma GH and IGF-I levels in fish fed with diets containing 30 μg/g and 300 μg/g genistein. mRNA expression of genes along the GH/IGF-I axis remained unaff ected, except for igf-Ir, which was stimulated by the 300 μg/g genistein diet. While in fish fed the 3 000 μg/g genistein diet, the plasma GH and IGF-I levels decreased, and mRNA expression of gh, ghr2, npyr1, igf-I, and igf-Ir were also significantly depressed. In contrast, npy and igfbp3 mRNA expression were enhanced. This study provides convincing evidence for growth impediment by genistein by disturbing the GH/IGF-I axis in Nile tilapia O. niloticus.

  17. Transcriptome analysis of the mammary gland from GH transgenic goats during involution.

    PubMed

    Lin, Jian; Bao, Ze Kun; Zhang, Qiang; Hu, Wei Wei; Yu, Qing Hua; Yang, Qian

    2015-07-10

    Mammary glands are organs for milk production in female mammals. Growth hormone (GH) is known to affect the growth and development of the mammary gland, as well as to increase milk production in dairy goats. This study performed a comprehensive expression profiling of genes expressed in the mammary gland of early involution GH transgenic (n=4) and non-transgenic goats (n=4) by RNA sequencing. RNA was extracted from mammary gland tissues collected at day 3 of involution. Gene expression analysis was conducted by Illumina RNA sequencing and sequence reads were assembled and analyzed using TopHat. FPKM (fragments per kilobase of exon per million) values were analyzed for differentially expressed genes using the Cufflinks package. Gene ontology analysis of differentially expressed genes was categorized using agriGO, while KEGG pathway analysis was performed with the online KEGG automatic annotation server. Our results revealed that 75% of NCBI goat annotated genes were expressed during early involution. A total of 18,323 genes were expressed during early involution in GH transgenic goats, compared with 18,196 expressed genes during early involution of non-transgenic goats. In these expressed genes, the majority (17,589) were ubiquitously expressed in GH transgenic and non-transgenic goats. However, there were 745 differentially expressed genes, 421 of which were upregulated and 324 were downregulated in GH transgenic goats. GO and KEGG pathway analysis showed that these genes were involved in mammary gland physiology, including cell adhesion molecules, ECM-receptor interaction, Jak-STAT signaling pathway, and fat metabolism. Our results demonstrated that the GH receptor was strongly affected in GH transgenic goats, which may activate the IGF-1/Stat3 signaling pathway. Overall, our study provided a global view of the transcriptome during involution of GH transgenic and non-transgenic goats, which increases our understanding of the biology of involution in the goat. PMID

  18. Effects of dietary genistein on GH/IGF-I axis of Nile tilapia Oreochromis niloticus

    NASA Astrophysics Data System (ADS)

    Chen, Dong; Wang, Wei; Ru, Shaoguo

    2016-09-01

    There is considerable concern that isoflavones, such as genistein in fish feed composed of soybean protein, aff ects somatic growth in fish. Our previous works demonstrated that 30 and 300 μg/g dietary genistein had no significant eff ect on growth performance in Nile tilapia ( Oreochromis niloticus), but the higher level of genistein (3 000 μg/g) significantly depressed growth. This study was conducted to further examine the eff ects of dietary genistein on the endocrine disruption on growth hormone/insulin-like growth factor-I (GH/IGF-I) axis in Nile tilapia ( O. niloticus). Juvenile fish were fed by hand twice daily to satiation with one of four isonitrogenous and isoenergetic diets, each containing either 0, 30, 300 or 3 000 μg/g genistein. Following an 8-week feeding period, plasma GH and IGF-I levels were investigated by radioimmunoassay and gene expression levels of gh, ghrelin, gnrhs, ghr, npy, npyrs, pacap, ghrs, i gf-I, igf-Ir, and igfbp3 were examined by real-time PCR. The results show that no significant change in plasma GH and IGF-I levels in fish fed with diets containing 30 μg/g and 300 μg/g genistein. mRNA expression of genes along the GH/IGF-I axis remained unaff ected, except for igf-Ir, which was stimulated by the 300 μg/g genistein diet. While in fish fed the 3 000 μg/g genistein diet, the plasma GH and IGF-I levels decreased, and mRNA expression of gh, ghr2, npyr1, igf-I, and igf-Ir were also significantly depressed. In contrast, npy and igfbp3 mRNA expression were enhanced. This study provides convincing evidence for growth impediment by genistein by disturbing the GH/IGF-I axis in Nile tilapia O. niloticus.

  19. Adiponectin in mice with altered GH action: links to insulin sensitivity and longevity?

    PubMed

    Lubbers, Ellen R; List, Edward O; Jara, Adam; Sackman-Sala, Lucila; Cordoba-Chacon, Jose; Gahete, Manuel D; Kineman, Rhonda D; Boparai, Ravneet; Bartke, Andrzej; Kopchick, John J; Berryman, Darlene E

    2013-03-01

    Adiponectin is positively correlated with longevity and negatively correlated with many obesity-related diseases. While there are several circulating forms of adiponectin, the high-molecular-weight (HMW) version has been suggested to have the predominant bioactivity. Adiponectin gene expression and cognate serum protein levels are of particular interest in mice with altered GH signaling as these mice exhibit extremes in obesity that are positively associated with insulin sensitivity and lifespan as opposed to the typical negative association of these factors. While a few studies have reported total adiponectin levels in young adult mice with altered GH signaling, much remains unresolved, including changes in adiponectin levels with advancing age, proportion of total adiponectin in the HMW form, adipose depot of origin, and differential effects of GH vs IGF1. Therefore, the purpose of this study was to address these issues using assorted mouse lines with altered GH signaling. Our results show that adiponectin is generally negatively associated with GH activity, regardless of age. Further, the amount of HMW adiponectin is consistently linked with the level of total adiponectin and not necessarily with previously reported lifespan or insulin sensitivity of these mice. Interestingly, circulating adiponectin levels correlated strongly with inguinal fat mass, implying that the effects of GH on adiponectin are depot specific. Interestingly, rbGH, but not IGF1, decreased circulating total and HMW adiponectin levels. Taken together, these results fill important gaps in the literature related to GH and adiponectin and question the frequently reported associations of total and HMW adiponectin with insulin sensitivity and longevity. PMID:23261955

  20. Development of a Transnasal Delivery System for Recombinant Human Growth Hormone (rhGH): Effects of the Concentration and Molecular Weight of Poly-L-arginine on the Nasal Absorption of rhGH in Rats.

    PubMed

    Kawashima, Ryo; Uchida, Masaki; Yamaki, Tsutomu; Ohtake, Kazuo; Hatanaka, Tomomi; Uchida, Hiroyuki; Ueda, Hideo; Kobayashi, Jun; Morimoto, Yasunori; Natsume, Hideshi

    2016-01-01

    A novel system for delivering recombinant human growth hormone (rhGH) that is noninvasive and has a simple method of administration is strongly desired to improve the compliance of children. The aim of this study was to investigate the potential for the intranasal (i.n.) co-administration of rhGH with poly-L-arginine (PLA) as a novel delivery system by evaluating the effects of the concentration and molecular weight of PLA on the nasal absorption of rhGH. The influence of the formation of insoluble aggregates and a soluble complex in the dosage formulation on nasal rhGH absorption was also evaluated by size-exclusion chromatography and ultrafiltration. PLA enhanced the nasal absorption of rhGH at each concentration and molecular weight examined. Nasal rhGH absorption increased dramatically when the PLA concentration was 1.0 % (w/v) due to the improved solubility of rhGH in the formulation. A delay in rhGH absorption was observed when the molecular weight of PLA was increased. This appeared to be because the increase in molecular weight caused the formation of a soluble complex. It seems that the PLA concentration affects the absorption-enhancing effect on rhGH, while the molecular weight of PLA affects the time when the maximum plasma rhGH concentration was reached (Tmax) of rhGH after i.n. administration, mainly because of the interactions among rhGH, PLA, and additives. Therefore, the transnasal rhGH delivery system using PLA is considered to be a promising alternative to subcutaneous (s.c.) injection if these interactions are sufficiently controlled. PMID:26725528

  1. Dynamic Analysis of GH Receptor Conformational Changes by Split Luciferase Complementation

    PubMed Central

    Liu, Ying; Berry, Philip A.; Zhang, Yue; Jiang, Jing; Lobie, Peter E.; Paulmurugan, Ramasamy; Langenheim, John F.; Chen, Wen Y.; Zinn, Kurt R.

    2014-01-01

    The transmembrane GH receptor (GHR) exists at least in part as a preformed homodimer on the cell surface. Structural and biochemical studies suggest that GH binds GHR in a 1:2 stoichiometry to effect acute GHR conformational changes that trigger the activation of the receptor-associated tyrosine kinase, Janus kinase 2 (JAK2), and downstream signaling. Despite information about GHR-GHR association derived from elegant fluorescence resonance energy transfer/bioluminescence resonance energy transfer studies, an assessment of the dynamics of GH-induced GHR conformational changes has been lacking. To this end, we used a split luciferase complementation assay that allowed detection in living cells of specific ligand-independent GHR-GHR interaction. Furthermore, GH treatment acutely augmented complementation of enzyme activity between GHRs fused, respectively, to N- and C-terminal fragments of firefly luciferase. Analysis of the temporal pattern of GH-induced complementation changes, pharmacological manipulation, genetic alteration of JAK2 levels, and truncation of the GHR intracellular domain (ICD) tail suggested that GH acutely enhances proximity of the GHR homodimer partners independent of the presence of JAK2, phosphorylation of GHR-luciferase chimeras, or an intact ICD. However, subsequent reduction of complementation requires JAK2 kinase activity and the ICD tail. This conclusion is in contrast to existing models of the GHR activation process. PMID:25188449

  2. Identification and characterization of the GhHsp20 gene family in Gossypium hirsutum.

    PubMed

    Ma, Wei; Zhao, Ting; Li, Jie; Liu, Bingliang; Fang, Lei; Hu, Yan; Zhang, Tianzhen

    2016-01-01

    In higher plants, Heat Shock Protein 20 (Hsp20) plays crucial roles in growth, development and responses to abiotic stresses. In this study, 94 GhHsp20 genes were identified in G. hirsutum, and these genes were phylogenetically clustered into 14 subfamilies. Out of these, 73 paralogous gene pairs remained in conserved positions on segmental duplicated blocks and only 14 genes clustered into seven tandem duplication event regions. Transcriptome analysis showed that 82 GhHsp20 genes were expressed in at least one tested tissues, indicating that the GhHsp20 genes were involved in physiological and developmental processes of cotton. Further, expression profiles under abiotic stress exhibited that two-thirds of the GhHsp20 genes were responsive to heat stress, while 15 genes were induced by multiple stresses. In addition, qRT-PCR confirmed that 16 GhHsp20 genes were hot-induced, and eight genes were up-regulated under multiple abiotic stresses and stress-related phytohormone treatments. Taken together, our results presented here would be helpful in laying the foundation for understanding the complex mechanisms of GhHsp20 mediated developmental processes and abiotic stress signaling transduction pathways in cotton. PMID:27580529

  3. Dwarfism and increased adiposity in the gh1 mutant zebrafish vizzini.

    PubMed

    McMenamin, Sarah K; Minchin, James E N; Gordon, Tiffany N; Rawls, John F; Parichy, David M

    2013-04-01

    Somatic growth and adipogenesis are closely associated with the development of obesity in humans. In this study, we identify a zebrafish mutant, vizzini, that exhibits both a severe defect in somatic growth and increased accumulation of adipose tissue. Positional cloning of vizzini revealed a premature stop codon in gh1. Although the effects of GH are largely through igfs in mammals, we found no decrease in the expression of igf transcripts in gh1 mutants during larval development. As development progressed, however, we found overall growth to be progressively retarded and the attainment of specific developmental stages to occur at abnormally small body sizes relative to wild type. Moreover, both subcutaneous (sc) and visceral adipose tissues underwent precocious development in vizzini mutants, and at maturity, the sizes of different fat deposits were greatly expanded relative to wild type. In vivo confocal imaging of sc adipose tissue (SAT) expansion revealed that vizzini mutants exhibit extreme enlargement of adipocyte lipid droplets without a corresponding increase in lipid droplet number. These findings suggest that GH1 signaling restricts SAT hypertrophy in zebrafish. Finally, nutrient deprivation of vizzini mutants revealed that SAT mobilization was greatly diminished during caloric restriction, further implicating GH1 signaling in adipose tissue homeostasis. Overall, the zebrafish gh1 mutant, vizzini, exhibits decreased somatic growth, increased adipose tissue accumulation, and disrupted adipose plasticity after nutrient deprivation and represents a novel model to investigate the in vivo dynamics of vertebrate obesity. PMID:23456361

  4. Identification and characterization of the GhHsp20 gene family in Gossypium hirsutum

    PubMed Central

    Ma, Wei; Zhao, Ting; Li, Jie; Liu, Bingliang; Fang, Lei; Hu, Yan; Zhang, Tianzhen

    2016-01-01

    In higher plants, Heat Shock Protein 20 (Hsp20) plays crucial roles in growth, development and responses to abiotic stresses. In this study, 94 GhHsp20 genes were identified in G. hirsutum, and these genes were phylogenetically clustered into 14 subfamilies. Out of these, 73 paralogous gene pairs remained in conserved positions on segmental duplicated blocks and only 14 genes clustered into seven tandem duplication event regions. Transcriptome analysis showed that 82 GhHsp20 genes were expressed in at least one tested tissues, indicating that the GhHsp20 genes were involved in physiological and developmental processes of cotton. Further, expression profiles under abiotic stress exhibited that two-thirds of the GhHsp20 genes were responsive to heat stress, while 15 genes were induced by multiple stresses. In addition, qRT-PCR confirmed that 16 GhHsp20 genes were hot-induced, and eight genes were up-regulated under multiple abiotic stresses and stress-related phytohormone treatments. Taken together, our results presented here would be helpful in laying the foundation for understanding the complex mechanisms of GhHsp20 mediated developmental processes and abiotic stress signaling transduction pathways in cotton. PMID:27580529

  5. Evolutionary history of the GH3 family of acyl adenylases in rosids.

    PubMed

    Okrent, Rachel A; Wildermuth, Mary C

    2011-08-01

    GH3 amino acid conjugases have been identified in many plant and bacterial species. The evolution of GH3 genes in plant species is explored using the sequenced rosids Arabidopsis, papaya, poplar, and grape. Analysis of the sequenced non-rosid eudicots monkey flower and columbine, the monocots maize and rice, as well as spikemoss and moss is included to provide further insight into the origin of GH3 clades. Comparison of co-linear genes in regions surrounding GH3 genes between species helps reconstruct the evolutionary history of the family. Combining analysis of synteny with phylogenetics, gene expression and functional data redefines the Group III GH3 genes, of which AtGH3.12/PBS3, a regulator of stress-induced salicylic acid metabolism and plant defense, is a member. Contrary to previous reports that restrict PBS3 to Arabidopsis and its close relatives, PBS3 syntelogs are identified in poplar, grape, columbine, maize and rice suggesting descent from a common ancestral chromosome dating to before the eudicot/monocot split. In addition, the clade containing PBS3 has undergone a unique expansion in Arabidopsis, with expression patterns for these genes consistent with specialized and evolving stress-responsive functions. PMID:21594748

  6. GhWRKY68 Reduces Resistance to Salt and Drought in Transgenic Nicotiana benthamiana

    PubMed Central

    Jia, Haihong; Wang, Chen; Wang, Fang; Liu, Shuchang; Li, Guilin; Guo, Xingqi

    2015-01-01

    The WRKY transcription factors modulate numerous physiological processes, including plant growth, development and responses to various environmental stresses. Currently, our understanding of the functions of the majority of the WRKY family members and their possible roles in signalling crosstalk is limited. In particular, very few WRKYs have been identified and characterised from an economically important crop, cotton. In this study, we characterised a novel group IIc WRKY gene, GhWRKY68, which is induced by different abiotic stresses and multiple defence-related signalling molecules. The β-glucuronidase activity driven by the GhWRKY68 promoter was enhanced after exposure to drought, salt, abscisic acid (ABA) and H2O2. The overexpression of GhWRKY68 in Nicotiana benthamiana reduced resistance to drought and salt and affected several physiological indices. GhWRKY68 may mediate salt and drought responses by modulating ABA content and enhancing the transcript levels of ABA-responsive genes. GhWRKY68-overexpressing plants exhibited reduced tolerance to oxidative stress after drought and salt stress treatments, which correlated with the accumulation of reactive oxygen species (ROS), reduced enzyme activities, elevated malondialdehyde (MDA) content and altered ROS-related gene expression. These results indicate that GhWRKY68 is a transcription factor that responds to drought and salt stresses by regulating ABA signalling and modulating cellular ROS. PMID:25793865

  7. Sequence and Bioinformatic Analysis of Family 1 Glycoside Hydrolase (GH) 1 Gene from the Oomycete Pythium myriotylum Drechsler.

    PubMed

    Nair, R Aswati; Geethu, C; Sangwan, Amit; Pillai, P Padmesh

    2015-06-01

    The oomycetous phytopathogen Pythium myriotylum secretes cellulases for growth/nutrition of the necrotroph. Cellulases are multi-enzyme system classified into different glycoside hydrolase (GH) families. The present study deals with identification and characterization of GH gene sequence from P. myriotylum by a PCR strategy using consensus primers. Cloning of the full-length gene sequence using genome walker strategy resulted in identification of 1230-bp P. myriotylum GH gene sequence, designated as PmGH1. Analysis revealed that PmGH1 encodes a predicted cytoplasmic 421 amino acid protein with an apparent molecular weight of 46.77 kDa and a theoretical pI of 8.11. Tertiary structure of the deduced amino acid sequence showed typical (α/β)8 barrel folding of family 1 GHs. Sequence characterization of PmGH1 identified the conserved active site residues, viz., Glu 181 and Glu 399, that function as acid-base catalyst and catalytically active nucleophile, respectively. Binding sites for N-acetyl-D-glucosamine (NAG) were revealed in the PmGH1 3D structure with Glu181 and Glu399 positioned on either side to form a catalytic pair. Phylogenetic analysis indicated a closer affiliation of PmGH1 with sequences of GH1 family. Results presented are first attempts providing novel insights into the evolutionary and functional perspectives of the identified P. myriotylum GH. PMID:25877398

  8. Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird’s-Nest Fern

    PubMed Central

    Tsay, Tung-Tsuan; Tsai, Isheng J.; Chen, Peichen J.

    2016-01-01

    Five Aphelenchoides besseyi isolates collected from bird’s-nest ferns or rice possess different parasitic capacities in bird’s-nest fern. Two different glycoside hydrolase (GH) 45 genes were identified in the fern isolates, and only one was found in the rice isolates. A Abe GH5-1 gene containing an SCP-like family domain was found only in the fern isolates. Abe GH5-1 gene has five introns suggesting a eukaryotic origin. A maximum likelihood phylogeny revealed that Abe GH5-1 is part of the nematode monophyletic group that can be clearly distinguished from those of other eukaryotic and bacterial GH5 sequences with high bootstrap support values. The fern A. besseyi isolates were the first parasitic plant nematode found to possess both GH5 and GH45 genes. Surveying the genome of the five A. besseyi isolates by Southern blotting using an 834 bp probe targeting the GH5 domain suggests the presence of at least two copies in the fern-origin isolates but none in the rice-origin isolates. The in situ hybridization shows that the Abe GH5-1 gene is expressed in the nematode ovary and testis. Our study provides insights into the diversity of GH in isolates of plant parasitic nematodes of different host origins. PMID:27391812

  9. Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird's-Nest Fern.

    PubMed

    Wu, Guan-Long; Kuo, Tzu-Hao; Tsay, Tung-Tsuan; Tsai, Isheng J; Chen, Peichen J

    2016-01-01

    Five Aphelenchoides besseyi isolates collected from bird's-nest ferns or rice possess different parasitic capacities in bird's-nest fern. Two different glycoside hydrolase (GH) 45 genes were identified in the fern isolates, and only one was found in the rice isolates. A Abe GH5-1 gene containing an SCP-like family domain was found only in the fern isolates. Abe GH5-1 gene has five introns suggesting a eukaryotic origin. A maximum likelihood phylogeny revealed that Abe GH5-1 is part of the nematode monophyletic group that can be clearly distinguished from those of other eukaryotic and bacterial GH5 sequences with high bootstrap support values. The fern A. besseyi isolates were the first parasitic plant nematode found to possess both GH5 and GH45 genes. Surveying the genome of the five A. besseyi isolates by Southern blotting using an 834 bp probe targeting the GH5 domain suggests the presence of at least two copies in the fern-origin isolates but none in the rice-origin isolates. The in situ hybridization shows that the Abe GH5-1 gene is expressed in the nematode ovary and testis. Our study provides insights into the diversity of GH in isolates of plant parasitic nematodes of different host origins. PMID:27391812

  10. Liver status in congenital, untreated, isolated GH deficiency

    PubMed Central

    Diniz, Rachel D C A; Souza, Renata M; Salvatori, Roberto; Franca, Alex; Gomes-Santos, Elenilde; Ferrão, Thiago O; Oliveira, Carla R P; Santana, João A M; Pereira, Francisco A; Barbosa, Rita A A; Souza, Anita H O; Pereira, Rossana M C; Oliveira-Santos, Alécia A; Silva, Allysson M P; Santana-Júnior, Francisco J; Valença, Eugênia H O; Campos, Viviane C; Aguiar-Oliveira, Manuel H

    2014-01-01

    Nonalcoholic fatty liver disease (NAFLD) is known to be associated with insulin resistance, atherosclerosis, and low serum IGF1 levels. We have described a large cohort of patients with isolated GH deficiency (IGHD) due to the c.57+1G→A mutation in the GHRH receptor gene. These subjects have increased insulin sensitivity (IS), delayed atherosclerosis, and normal longevity. We hypothesized that, despite visceral obesity, NAFLD would be absent or mild due to the increased IS. To assess the prevalence and severity of NAFLD in adult subjects with lifetime, congenital, untreated IGHD, we studied 22 IGHD adults and 25 controls (COs) matched for age and sex. NAFLD was assessed by a comprehensive liver function panel, and ultrasonographic pattern (hyperechogenic pattern, HP) coded as follows: 0, absent; 1, mild; 2, moderate; and 3, severe. Compared with COs, IGHD individual had lower serum IGF1 (P<0.0001), higher total cholesterol (P=0.027), lower prothrombin time (P=0.017), and similar activated partial thromboplastin time and fibrinogen values. Alanine transaminase (ALT) values were similar in the two groups, but aspartate transaminase was higher in IGHD (P=0.013). However, more IGHD subjects (7/22) than COs (3/23) had ALT above the upper limit of normal (P=0.044). The prevalence of NAFLD was higher in IGHD than COs (61 vs 29%, P=0.032), and the HP score was higher in IGHD than COs (P=0.041), but severe NAFLD was not observed in any IGHD (or CO) individual. Liver HP score is increased in lifetime, untreated, congenital IGHD, but the increase in transaminases is mild, suggesting a lack of advanced forms of NAFLD. PMID:25117570

  11. Optimization of a GO2/GH2 Impinging Injector Element

    NASA Technical Reports Server (NTRS)

    Tucker, P. Kevin; Shyy, Wei; Vaidyanathan, Rajkumar

    2001-01-01

    An injector optimization methodology, method i, is used to investigate optimal design points for a gaseous oxygen/gaseous hydrogen (GO2/GH2) impinging injector element. The unlike impinging element, a fuel-oxidizer- fuel (F-O-F) triplet, is optimized in terms of design variables such as fuel pressure drop, (Delta)P(sub f), oxidizer pressure drop, (Delta)P(sub o), combustor length, L(sub comb), and impingement half-angle, alpha, for a given mixture ratio and chamber pressure. Dependent variables such as energy release efficiency, ERE, wall heat flux, Q(sub w), injector heat flux, Q(sub inj), relative combustor weight, W(sub rel), and relative injector cost, C(sub rel), are calculated and then correlated with the design variables. An empirical design methodology is used to generate these responses for 163 combinations of input variables. Method i is then used to generate response surfaces for each dependent variable. Desirability functions based on dependent variable constraints are created and used to facilitate development of composite response surfaces representing some, or all, of the five dependent variables in terms of the input variables. Three examples illustrating the utility and flexibility of method i are discussed in detail. First, joint response surfaces are constructed by sequentially adding dependent variables. Optimum designs are identified after addition of each variable and the effect each variable has on the design is shown. This stepwise demonstration also highlights the importance of including variables such as weight and cost early in the design process. Secondly, using the composite response surface which includes all five dependent variables, unequal weights are assigned to emphasize certain variables relative to others. Here, method i is used to enable objective trade studies on design issues such as component life and thrust to weight ratio. Finally, specific variable weights are further increased to illustrate the high marginal cost of

  12. Growth responses following a single intra-muscular hGH plasmid administration compared to daily injections of hGH in dwarf mice.

    PubMed

    Higuti, Eliza; Cecchi, Claudia R; Oliveira, Nelio A J; Vieira, Daniel P; Jensen, Thomas G; Jorge, Alexander A L; Bartolini, Paolo; Peroni, Cibele N

    2012-12-01

    In previous work, sustained levels of circulating human growth hormone (hGH) and a highly significant weight increase were observed after electrotransfer of naked plasmid DNA (hGH-DNA) into the muscle of immunodeficient dwarf mice (lit/scid). In the present study, the efficacy of this in vivo gene therapy strategy is compared to daily injections (5 μg/twice a day) of recombinant hGH (r-hGH) protein, as assessed on the basis of several growth parameters. The slopes of the two growth curves were found to be similar (P > 0.05): 0.095 g/mouse/d for protein and 0.094 g/mouse/d for DNA injection. In contrast, the weight increases averaged 35.5% (P < 0.001) and 23.1% (P < 0.01) for protein and DNA administration, respectively, a difference possibly related to the electroporation methodology. The nose-to-tail linear growth increases were 15% and 9.6% for the protein and DNA treatments, respectively, but mouse insulin-like growth factor I (mIGF-I) showed a greater increase over the control with DNA (5- to 7-fold) than with protein (3- to 4-fold) administration. The weight increases of several organs and tissues (kidneys, spleen, liver, heart, quadriceps and gastrocnemius muscles) were 1.3- to 4.6-fold greater for protein than for DNA administration, which gave a generally more proportional growth. Glucose levels were apparently unaffected, suggesting the absence of effects on glucose tolerance. A gene transfer strategy based on a single hGH-DNA administration thus appears to be comparable to repeated hormone injections for promoting growth and may represent a feasible alternative for the treatment of growth hormone deficiency. PMID:22974419

  13. Biochemical and kinetic characterization of GH43 β-D-xylosidase/α-L-arabinofuranosidase and GH30 α-L-arabinofuranosidase/β-D -xylosidase from rumen metagenome.

    PubMed

    Zhou, Jungang; Bao, Lei; Chang, Lei; Zhou, Yufei; Lu, Hong

    2012-01-01

    The present study focuses on characterization of two hemicellulases, RuXyn1 and RuXyn2, from rumen bacterial metagenome and their capabilities for degradation of xylans. Glycosyl hydrolase (GH) family 43 β-D -xylosidase/α-L -arabinofuranosidase RuXyn1 can hydrolyze p-nitrophenyl-β-D -xylopyranoside (pNPX), p-nitrophenyl-α-L -arabinofuranoside (pNPA), and xylo-oligosaccharide substrates, while GH30 1,5-α-L -arabinofuranosidase/β-D -xylosidase RuXyn2, the first α-L -arabinofuranosidase assigned to this GH family, shows activities towards 1,5-α-L -arabinobiose and pNPX substrates but no activity for pNPA. Kinetic analysis for aryl-glycosides revealed that RuXyn2 had higher catalytic efficiency than RuXyn1 toward pNPX substrate. RuXyn1 shows high synergism with endoxylanase, elevating by 73% the reducing sugars released from brichwood xylans, and converted most intermediate xylo-oligosaccharide hydrolysate into xylose. The high xylose conversion capability of RuXyn1 suggests it has potential applications in enzymatic production of xylose and improvement of hemicellulose saccharification for production of biofuels. RuXyn2 shows no obviously synergistic effect in the endoxylanase-coupled assay for enzymatic saccharification of xylan. Further cosmid DNA sequencing revealed a neighboring putative GH43 α-L -arabinofuranosidase RuAra1 and two putative GH3 β-xylosidase/arabinosidases, RuXyn3 and RuXyn5, downstream of RuXyn2, indicating that this hemicellulase gene cluster may be responsible for production of end-product, xylose and arabinose, from hemicellulose biomass. PMID:21720773

  14. Short-Term, Low-Dose GH Therapy Improves Insulin Sensitivity Without Modifying Cortisol Metabolism and Ectopic Fat Accumulation in Adults With GH Deficiency

    PubMed Central

    Roberts, Charles T.; Frystyk, Jan; Rooney, William D.; Pollaro, James R.; Klopfenstein, Bethany J.; Purnell, Jonathan Q.

    2014-01-01

    Context: Low-dose GH (LGH) therapy has been reported to improve insulin sensitivity in GH-deficient adults; however, the mechanism is unclear. Hypothesis: Effects of LGH therapy on insulin sensitivity are mediated through changes in cortisol metabolism and ectopic fat accumulation. Design and Setting: This was a double-blind, placebo-controlled, parallel, 3-month study. Participants and Intervention: Seventeen GH-deficient adults were randomized to receive either daily LGH or placebo injections. Fasting blood samples were collected at baseline, and months 1 and 3, whereas hyperinsulinemic-euglycemic clamps, magnetic resonance spectroscopy scans, 24-hour cortisol production rates (CPRs), and sc abdominal fat biopsies were performed at baseline and month 3. Main Outcome Measures: Clamp glucose infusion rate, intramyocellular, extramyocellular, and intrahepatic lipid content, 24-hour CPRs, adipocyte size, and adipocyte 11β-hydroxysteroid dehydrogenase activity in adults with GH deficiency were evaluated. Results: At month 1, LGH did not alter fasting levels of glucose, insulin, C-peptide, free fatty acid, adiponectin, total IGF-1, IGF-1 bioactivity, IGF-2, IGF binding protein (IGFBP)-2, or IGF-1 to IGFBP-3 molar ratio. At month 3, LGH increased clamp glucose infusion rates (P < .01) and IGF-1 to IGFBP-3 molar ratio (P < .05), but fasting glucose, insulin, C-peptide, free fatty acid, adiponectin, IGF-1 bioactivity, IGF-2, IGFBP-2, 24-hour CPRs, adipocyte size, adipocyte 11β-hydroxysteroid dehydrogenase activity, intrahepatic lipid, extramyocellular, or intramyocellular were unchanged. In the placebo group, all within-group parameters from months 1 and 3 compared with baseline were unchanged. Conclusions: Short-term LGH therapy improves insulin sensitivity without inducing basal lipolysis and had no effect on cortisol metabolism and ectopic fat accumulation in GH-deficient adults. This may reflect an LGH-induced increase in IGF-1 to IGFBP-3 molar ratio exerting

  15. First Structural Insights into α-l-Arabinofuranosidases from the Two GH62 Glycoside Hydrolase Subfamilies*

    PubMed Central

    Siguier, Béatrice; Haon, Mireille; Nahoum, Virginie; Marcellin, Marlène; Burlet-Schiltz, Odile; Coutinho, Pedro M.; Henrissat, Bernard; Mourey, Lionel; O'Donohue, Michael J.; Berrin, Jean-Guy; Tranier, Samuel; Dumon, Claire

    2014-01-01

    α-l-Arabinofuranosidases are glycoside hydrolases that specifically hydrolyze non-reducing residues from arabinose-containing polysaccharides. In the case of arabinoxylans, which are the main components of hemicellulose, they are part of microbial xylanolytic systems and are necessary for complete breakdown of arabinoxylans. Glycoside hydrolase family 62 (GH62) is currently a small family of α-l-arabinofuranosidases that contains only bacterial and fungal members. Little is known about the GH62 mechanism of action, because only a few members have been biochemically characterized and no three-dimensional structure is available. Here, we present the first crystal structures of two fungal GH62 α-l-arabinofuranosidases from the basidiomycete Ustilago maydis (UmAbf62A) and ascomycete Podospora anserina (PaAbf62A). Both enzymes are able to efficiently remove the α-l-arabinosyl substituents from arabinoxylan. The overall three-dimensional structure of UmAbf62A and PaAbf62A reveals a five-bladed β-propeller fold that confirms their predicted classification into clan GH-F together with GH43 α-l-arabinofuranosidases. Crystallographic structures of the complexes with arabinose and cellotriose reveal the important role of subsites +1 and +2 for sugar binding. Intriguingly, we observed that PaAbf62A was inhibited by cello-oligosaccharides and displayed binding affinity to cellulose although no activity was observed on a range of cellulosic substrates. Bioinformatic analyses showed that UmAbf62A and PaAbf62A belong to two distinct subfamilies within the GH62 family. The results presented here provide a framework to better investigate the structure-function relationships within the GH62 family. PMID:24394409

  16. Cloning and expression of a novel chicken sulfotransferase cDNA regulated by GH.

    PubMed

    Cao, H; Agarwal, S K; Burnside, J

    1999-03-01

    We have used mRNA differential display to compare gene expression in normal and GH receptor-deficient dwarf chickens, and report here the characterization of one differentially expressed gene, which shows significant sequence identity to the sulfotransferase gene family. Partial cDNA clones were isolated from a chicken liver cDNA library and an additional sequence was obtained using 5' rapid amplification of cDNA ends. A complete cDNA probe hybridizes to three transcripts (2.4, 2.0 and 1.45 kb) on Northern blots of chicken liver RNA, which differ in the length of the 3' untranslated region. All three transcripts are expressed at higher levels in normal vs dwarf chickens, as expected for a GH-regulated gene. The expression of this sulfotransferase mRNA was also detected in skeletal muscle, but not other tissues. The administration of GH to chickens increased the hepatic expression within 1 h, suggesting this sulfotransferase could be directly regulated by GH. Sulfotransferase activity, using estradiol or corticosterone as substrate, is detected in cells transfected with an expression vector containing the full-length cDNA. The sequence of this sulfotransferase does not show significant similarity with any subfamily of the sulfotransferases and its endogenous substrate is presently unknown. However, we speculate that GH activation of sulfotransferase activity could play a role in reducing concentrations of growth-antagonistic steroid hormones in GH target tissues. These results demonstrate the usefulness of differential display in this model system to identify genes that play a role in mediating GH action. PMID:10076195

  17. Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5)

    PubMed Central

    2012-01-01

    Background The large Glycoside Hydrolase family 5 (GH5) groups together a wide range of enzymes acting on β-linked oligo- and polysaccharides, and glycoconjugates from a large spectrum of organisms. The long and complex evolution of this family of enzymes and its broad sequence diversity limits functional prediction. With the objective of improving the differentiation of enzyme specificities in a knowledge-based context, and to obtain new evolutionary insights, we present here a new, robust subfamily classification of family GH5. Results About 80% of the current sequences were assigned into 51 subfamilies in a global analysis of all publicly available GH5 sequences and associated biochemical data. Examination of subfamilies with catalytically-active members revealed that one third are monospecific (containing a single enzyme activity), although new functions may be discovered with biochemical characterization in the future. Furthermore, twenty subfamilies presently have no characterization whatsoever and many others have only limited structural and biochemical data. Mapping of functional knowledge onto the GH5 phylogenetic tree revealed that the sequence space of this historical and industrially important family is far from well dispersed, highlighting targets in need of further study. The analysis also uncovered a number of GH5 proteins which have lost their catalytic machinery, indicating evolution towards novel functions. Conclusion Overall, the subfamily division of GH5 provides an actively curated resource for large-scale protein sequence annotation for glycogenomics; the subfamily assignments are openly accessible via the Carbohydrate-Active Enzyme database at http://www.cazy.org/GH5.html. PMID:22992189

  18. Growth hormone (GH) treatment acts on the endocrine and autocrine/paracrine GH/IGF-axis and on TNF-α expression in bony fish pituitary and immune organs.

    PubMed

    Shved, N; Berishvili, G; Mazel, P; Baroiller, J-F; Eppler, Elisabeth

    2011-12-01

    There exist indications that the growth hormone (GH)/insulin-like growth factor (IGF) axis may play a role in fish immune regulation, and that interactions occur via tumour necrosis factor (TNF)-α at least in mammals, but no systematic data exist on potential changes in GH, IGF-I, IGF-II, GH receptor (GHR) and TNF-α expression after GH treatment. Thus, we investigated in the Nile tilapia the influence of GH injections by real-time qPCR at different levels of the GH/IGF-axis (brain, pituitary, peripheral organs) with special emphasis on the immune organs head kidney and spleen. Endocrine IGF-I served as positive control for GH treatment efficiency. Basal TNF-α gene expression was detected in all organs investigated with the expression being most pronounced in brain. Two consecutive intraperitoneal injections of bream GH elevated liver IGF-I mRNA and plasma IGF-I concentration. Also liver IGF-II mRNA and TNF-α were increased while the GHR was downregulated. In brain, no change occurred in the expression levels of all genes investigated. GH gene expression was exclusively detected in the pituitary where the GH injections elevated both GH and IGF-I gene expression. In the head kidney, GH upregulated IGF-I mRNA to an even higher extent than liver IGF-I while IGF-II and GHR gene expressions were not affected. Also in the spleen, no change occurred in GHR mRNA, however, IGF-I and IGF-II mRNAs were increased. In correlation, in situ hybridisation showed a markedly higher amount of IGF-I mRNA in head kidney and spleen after GH injection. In both immune tissues, TNF-α gene expression showed a trend to decrease after GH treatment. The stimulation of IGF-I and also partially of IGF-II expression in the fish immune organs by GH indicates a local role of the IGFs in immune organ regulation while the differential changes in TNF-α support the in mammals postulated interactions with the GH/IGF-axis which demand for further investigations. PMID:21903170

  19. Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

    PubMed Central

    Olinto, S.C.F.; Adrião, M.G.; Castro-Barbosa, T.; Goulart-Silva, F.; Nunes, M.T.

    2012-01-01

    The amino acid arginine (Arg) is a recognized secretagogue of growth hormone (GH), and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (∼250 g) were removed, divided into two halves, pooled (three hemi-pituitaries) and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM), the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM) and a cyclic guanosine monophosphate (cGMP) analogue (8-Br-cGMP, 1 mM) increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM) blunted the effect of SNP, and the combined treatment with Arg and L-NAME (an NO synthase (NOS) inhibitor, 55 mM) abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM) also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression. PMID:22641416

  20. Brain Recovery after a Plane Crash: Treatment with Growth Hormone (GH) and Neurorehabilitation: A Case Report

    PubMed Central

    Devesa, Jesús; Díaz-Getino, Gustavo; Rey, Pablo; García-Cancela, José; Loures, Iria; Nogueiras, Sonia; Hurtado de Mendoza, Alba; Salgado, Lucía; González, Mónica; Pablos, Tamara; Devesa, Pablo

    2015-01-01

    The aim of this study is to describe the results obtained after growth hormone (GH) treatment and neurorehabilitation in a young man that suffered a very grave traumatic brain injury (TBI) after a plane crash. Methods: Fifteen months after the accident, the patient was treated with GH, 1 mg/day, at three-month intervals, followed by one-month resting, together with daily neurorehabilitation. Blood analysis at admission showed that no pituitary deficits existed. At admission, the patient presented: spastic tetraplegia, dysarthria, dysphagia, very severe cognitive deficits and joint deformities. Computerized tomography scanners (CT-Scans) revealed the practical loss of the right brain hemisphere and important injuries in the left one. Clinical and blood analysis assessments were performed every three months for three years. Feet surgery was needed because of irreducible equinovarus. Results: Clinical and kinesitherapy assessments revealed a prompt improvement in cognitive functions, dysarthria and dysphagia disappeared and three years later the patient was able to live a practically normal life, walking alone and coming back to his studies. No adverse effects were observed during and after GH administration. Conclusions: These results, together with previous results from our group, indicate that GH treatment is safe and effective for helping neurorehabilitation in TBI patients, once the acute phase is resolved, regardless of whether or not they have GH-deficiency (GHD). PMID:26703581

  1. Identification of GH10 xylanases in strains 2 and Mz5 of Pseudobutyrivibrio xylanivorans.

    PubMed

    Grilli, Diego J; Kopečný, Jan; Mrázek, Jakub; Marinšek-Logar, Romana; Paez Lama, Sebastián; Escudero, Miguel Sosa; Arenas, Graciela N

    2014-11-01

    Genes encoding glycosyl hydrolase family 11 (GH11) xylanases and xylanases have been identified from Pseudobutyrivibrio xylanivorans. In contrast, little is known about the diversity and distribution of the GH10 xylanase in strains of P. xylanivorans. Xylanase and associated activities of P. xylanivorans have been characterized in detail in the type strain, Mz5. The aim of the present study was to identify GH10 xylanase genes in strains 2 and Mz5 of P. xylanivorans. In addition, we evaluated degradation and utilization of xylan by P. xylanivorans 2 isolated from rumen of Creole goats. After a 12-h culture, P. xylanivorans 2 was able to utilize up to 53% of the total pentose content present in birchwood xylan (BWX) and to utilize up to 62% of a ethanol-acetic acid-soluble fraction prepared from BWX. This is the first report describing the presence of GH10 xylanase-encoding genes in P. xylanivorans. Strain 2 and Mz5 contained xylanases which were related to GH10 xylanase of Butyrivibrio sp. Identifying xylanase-encoding genes and activity of these enzymes are a step toward understanding possible functional role of P. xylanivorans in the rumen ecosystem and contribute to providing an improved choice of enzymes for improving fiber digestion in ruminant animals, agricultural biomass utilization for biofuel production, and other industries. PMID:24942109

  2. A mechanism of glucose tolerance and stimulation of GH1 β-glucosidases

    PubMed Central

    Yang, Yang; Zhang, Xinxin; Yin, Qiang; Fang, Wei; Fang, Zemin; Wang, Xiaotang; Zhang, Xuecheng; Xiao, Yazhong

    2015-01-01

    β-Glucosidases are enzymes that hydrolyze β-glycosidic bonds to release non-reducing terminal glucosyl residues from glycosides and oligosaccharides, and thus have significant application potential in industries. However, most β-glucosidases are feedback inhibited by the glucose product, which restricts their application. Remarkably, some β-glucosidases of the glycoside hydrolase (GH) 1 family are tolerant to or even stimulated by glucose. Elucidation of the mechanisms of glucose tolerance and stimulation of the GH1 β-glucosidases will be crucial to improve their application through enzyme engineering. In this study, by comparing the primary and tertiary structures of two GH1 β-glucosidases with distinct glucose dependence, some putative glucose-dependence relevant sites were mutated to investigate their exact roles. Both biochemical and structural characterization of the mutants suggested that some sites at the entrance and middle of the substrate channel regulate the effects of glucose, and the relative binding affinity/preference of these sites to glucose modulates the glucose dependence. A mechanism was therefore proposed to interpret the glucose dependence of GH1 β-glucosidases. This research provides fresh insight into our current understanding of the properties and mechanisms of GH1 β-glycosidases and related enzymes that modulate their activity via feedback control mechanism. PMID:26603650

  3. In silico Identification and Taxonomic Distribution of Plant Class C GH9 Endoglucanases

    PubMed Central

    Kundu, Siddhartha; Sharma, Rita

    2016-01-01

    The glycoside hydrolase 9 superfamily, mainly comprising the endoglucanases, is represented in all three domains of life. The current division of GH9 enzymes, into three subclasses, namely A, B, and C, is centered on parameters derived from sequence information alone. However, this classification is ambiguous, and is limited by the paralogous ancestry of classes B and C endoglucanases, and paucity of biochemical and structural data. Here, we extend this classification schema to putative GH9 endoglucanases present in green plants, with an emphasis on identifying novel members of the class C subset. These enzymes cleave the β(1 → 4) linkage between non-terminal adjacent D-glucopyranose residues, in both, amorphous and crystalline regions of cellulose. We utilized non redundant plant GH9 enzymes with characterized molecular data, as the training set to construct Hidden Markov Models (HMMs) and train an Artificial Neural Network (ANN). The parameters that were used for predicting dominant enzyme function, were derived from this training set, and subsequently refined on 147 sequences with available expression data. Our knowledge-based approach, can ascribe differential endoglucanase activity (A, B, or C) to a query sequence with high confidence, and was used to construct a local repository of class C GH9 endoglucanases (GH9C = 241) from 32 sequenced green plants. PMID:27570528

  4. A mechanism of glucose tolerance and stimulation of GH1 β-glucosidases.

    PubMed

    Yang, Yang; Zhang, Xinxin; Yin, Qiang; Fang, Wei; Fang, Zemin; Wang, Xiaotang; Zhang, Xuecheng; Xiao, Yazhong

    2015-01-01

    β-Glucosidases are enzymes that hydrolyze β-glycosidic bonds to release non-reducing terminal glucosyl residues from glycosides and oligosaccharides, and thus have significant application potential in industries. However, most β-glucosidases are feedback inhibited by the glucose product, which restricts their application. Remarkably, some β-glucosidases of the glycoside hydrolase (GH) 1 family are tolerant to or even stimulated by glucose. Elucidation of the mechanisms of glucose tolerance and stimulation of the GH1 β-glucosidases will be crucial to improve their application through enzyme engineering. In this study, by comparing the primary and tertiary structures of two GH1 β-glucosidases with distinct glucose dependence, some putative glucose-dependence relevant sites were mutated to investigate their exact roles. Both biochemical and structural characterization of the mutants suggested that some sites at the entrance and middle of the substrate channel regulate the effects of glucose, and the relative binding affinity/preference of these sites to glucose modulates the glucose dependence. A mechanism was therefore proposed to interpret the glucose dependence of GH1 β-glucosidases. This research provides fresh insight into our current understanding of the properties and mechanisms of GH1 β-glycosidases and related enzymes that modulate their activity via feedback control mechanism. PMID:26603650

  5. Pharmacological activation of the GABAergic system does not affect GH and PRL release in acromegaly.

    PubMed

    Orio, F; Iovino, M; Monteleone, P; Agrusta, M; Steardo, L; Lombardi, G

    1988-11-01

    An extensive hypothalamic neurotransmitter impairment has been proposed in acromegaly. However, at the moment, the hypothalamic GABAergic system has been little investigated in this disorder. Since GABA has been shown to modulate growth hormone (GH) and prolactin (PRL) secretion in human subjects, it seemed reasonable to investigate hypothalamic GABAergic functioning through the assessment of basal GH and PRL responses to pharmacological activation of this system. 800 mg of sodium valproate (SV), a drug with GABA facilitating properties, were administered orally to 7 acromegalic patients and 9 healthy volunteers. Blood samples were collected before and after the drug administration for the measurement of plasma GH and PRL levels. SV induced a clear-cut rise in basal GH and a decrease in basal PRL in healthy subjects, but it did not induce any change in the basal levels of these hormones in acromegalics. These results suggest that the response of GH and PRL to SV in acromegaly is qualitatively different from normal controls. PMID:2850985

  6. In silico Identification and Taxonomic Distribution of Plant Class C GH9 Endoglucanases.

    PubMed

    Kundu, Siddhartha; Sharma, Rita

    2016-01-01

    The glycoside hydrolase 9 superfamily, mainly comprising the endoglucanases, is represented in all three domains of life. The current division of GH9 enzymes, into three subclasses, namely A, B, and C, is centered on parameters derived from sequence information alone. However, this classification is ambiguous, and is limited by the paralogous ancestry of classes B and C endoglucanases, and paucity of biochemical and structural data. Here, we extend this classification schema to putative GH9 endoglucanases present in green plants, with an emphasis on identifying novel members of the class C subset. These enzymes cleave the β(1 → 4) linkage between non-terminal adjacent D-glucopyranose residues, in both, amorphous and crystalline regions of cellulose. We utilized non redundant plant GH9 enzymes with characterized molecular data, as the training set to construct Hidden Markov Models (HMMs) and train an Artificial Neural Network (ANN). The parameters that were used for predicting dominant enzyme function, were derived from this training set, and subsequently refined on 147 sequences with available expression data. Our knowledge-based approach, can ascribe differential endoglucanase activity (A, B, or C) to a query sequence with high confidence, and was used to construct a local repository of class C GH9 endoglucanases (GH9C = 241) from 32 sequenced green plants. PMID:27570528

  7. Expression and Characterization of Hyperthermostable Exo-polygalacturonase TtGH28 from Thermotoga thermophilus.

    PubMed

    Wagschal, Kurt; Rose Stoller, J; Chan, Victor J; Lee, Charles C; Grigorescu, Arabela A; Jordan, Douglas B

    2016-07-01

    D-galacturonic acid is a potential platform chemical comprising the principal component of pectin in the citrus processing waste stream. Several enzyme activities are required for the enzymatic production of galacturonic acid from pectin, including exo- and endo-polygalacturonases. The gene TtGH28 encoding a putative GH28 polygalacturonase from Pseudothermotoga thermarum DSM 5069 (Theth_0397, NCBI# AEH50492.1) was synthesized, expressed in Escherichia coli, and characterized. Alignment of the amino acid sequence of gene product TtGH28 with other GH28 proteins whose structures and details of their catalytic mechanism have been elucidated shows that three catalytic Asp residues and several other key active site residues are strictly conserved. Purified TtGH28 was dimeric and hyperthermostable, with K t (0.5)  = 86.3 °C. Kinetic parameters for activity on digalacturonic acid, trigalacturonic acid, and polygalacturonic acid were obtained. No substrate inhibition was observed for polygalacturonate, while the K si values for the oligogalacturonides were in the low mM range, and K i for product galacturonic acid was in the low μM range. Kinetic modeling of the progress of reaction showed that the enzyme is both fully exo- and fully non-processional. PMID:27209035

  8. Long-term effects of growth hormone (GH) replacement therapy on hematopoiesis in a large cohort of children with GH deficiency.

    PubMed

    Esposito, Andrea; Capalbo, Donatella; De Martino, Lucia; Rezzuto, Martina; Di Mase, Raffaella; Pignata, Claudio; Salerno, Mariacarolina

    2016-07-01

    The aim of our prospective case-control study was to evaluate long-term effects of GH replacement therapy on erythrocytes parameters, leukocytes, and platelets numbers in a large cohort of children with isolated GH deficiency (GHD). Hemoglobin (Hb) concentration, hematocrit (Hct), mean corpuscular volume, mean corpuscular hemoglobin, red cell distribution width, number of erythrocytes, leukocytes, neutrophils, lymphocytes, monocytes and platelets, ferritin, and C-reactive protein were evaluated in 85 children with isolated GHD (10.20 ± 3.50 years) before and annually during the first 5 years of GH replacement therapy and in 85 healthy children age and sex comparable to patients during 5 years of follow-up. Compared with controls, GHD children at study entry showed lower Hb (-1.18 ± 0.87 vs. -0.40 ± 0.90 SDS, p < 0.0001), red cells number (-0.24 ± 0.81 vs. 0.25 ± 1.14 SDS, p < 0.0001), and Hct (-1.18 ± 0.86 vs. -0.68 ± 0.99 SDS, p < 0.0001). Twelve GHD patients (14 %) showed a normocytic anemia. GH therapy was associated with a significant increase in Hb, Hct, and red cells number which became all comparable to controls within the first 2 years of treatment. Moreover, hemoglobin levels normalized in all anemic GHD patients after 5 years of therapy. No difference between patients and controls was found in leukocytes and platelets numbers neither at baseline nor during the study. GHD in childhood is associated with an impairment of erythropoiesis which causes a normocytic anemia in a considerable percentage of patients. GH replacement therapy exerts a beneficial effect leading to a significant increase of erythrocytes parameters and recovery from anemia. Neither GHD nor GH replacement treatment exerts effects on leukocytes or platelets numbers. PMID:26511947

  9. An evolutionary analysis of the GH57 amylopullulanases based on the DOMON_glucodextranase_like domains.

    PubMed

    Jiao, Yu-Liang; Wang, Shu-Jun; Lv, Ming-Sheng; Fang, Yao-Wei; Liu, Shu

    2013-03-01

    Thermostable amylopullulanase (TAPU) is valuable in starch saccharification industry for its capability to catalyze both α-1,4 and α-1,6 glucosidic bonds under the industrial starch liquefication condition. The majority of TAPUs belong to glycoside hydrolase family 57 (GH57). In this study, we performed a phylogenetic analysis of GH57 amylopullulanase (APU) based on the highly conserved DOMON_glucodextranase_like (DDL) domain and classified APUs according to their multidomain architectures, phylogenetic analysis and enzymatic characters. This study revealed that amylopullulanase, pullulanase, andα-amylase had passed through a long joint evolution process, in which DDL played an important role. The phylogenetic analysis of DDL domain showed that the GH57 APU is directly sharing a common ancestor with pullulanase, and the DDL domains in some species undergo evolution scenarios such as domain duplication and recombination. PMID:22733591

  10. Direct stimulation of bone mass by increased GH signalling in the osteoblasts of Socs2−/− mice

    PubMed Central

    Dobie, R; MacRae, V E; Huesa, C; van't Hof, R; Ahmed, S F; Farquharson, C

    2014-01-01

    The suppressor of cytokine signalling (Socs2 −/−)-knockout mouse is characterised by an overgrowth phenotype due to enhanced GH signalling. The objective of this study was to define the Socs2 −/− bone phenotype and determine whether GH promotes bone mass via IGF1-dependent mechanisms. Despite no elevation in systemic IGF1 levels, increased body weight in 4-week-old Socs2 −/− mice following GH treatment was associated with increased cortical bone area (Ct.Ar) (P<0.01). Furthermore, detailed bone analysis of male and female juvenile and adult Socs2 −/− mice revealed an altered cortical and trabecular phenotype consistent with the known anabolic effects of GH. Indeed, male Socs2 −/− mice had increased Ct.Ar (P<0.05) and thickness associated with increased strength. Despite this, there was no elevation in hepatic Igf1 expression, suggesting that the anabolic bone phenotype was the result of increased local GH action. Mechanistic studies showed that in osteoblasts and bone of Socs2 −/− mice, STAT5 phosphorylation was significantly increased in response to GH. Conversely, overexpression of SOCS2 decreased GH-induced STAT5 signalling. Although an increase in Igf1 expression was observed in Socs2 −/− osteoblasts following GH, it was not evident in vivo. Igf1 expression levels were not elevated in response to GH in 4-week-old mice and no alterations in expression was observed in bone samples of 6-week-old Socs2 −/− mice. These studies emphasise the critical role of SOCS2 in controlling the local GH anabolic bone effects. We provide compelling evidence implicating SOCS2 in the regulation of GH osteoblast signalling and ultimately bone accrual, which maybe via mechanisms that are independent of IGF1 production in vivo. PMID:25074853

  11. Male Bovine GH Transgenic Mice Have Decreased Adiposity With an Adipose Depot-Specific Increase in Immune Cell Populations

    PubMed Central

    Benencia, Fabian; Harshman, Stephanie; Duran-Ortiz, Silvana; Lubbers, Ellen R.; List, Edward O.; Householder, Lara; Al-Naeeli, Mawadda; Liang, Xiaoyu; Welch, Lonnie; Kopchick, John J.

    2015-01-01

    White adipose tissue (WAT) is composed of mature adipocytes and a stromal vascular fraction (SVF), which contains a variety of cells, including immune cells that vary among the different WAT depots. Growth hormone (GH) impacts immune function and adiposity in an adipose depot-specific manner. However, its effects on WAT immune cell populations remain unstudied. Bovine GH transgenic (bGH) mice are commonly used to study the in vivo effects of GH. These giant mice have an excess of GH action, impaired glucose metabolism, decreased adiposity, increased lean mass, and a shortened lifespan. Therefore, the purpose of this study was to characterize the WAT depot-specific differences in immune cell populations in the presence of excess GH in vivo. Three WAT depots were assessed: inguinal (sc), epididymal (EPI), and mesenteric (MES). Subcutaneous and MES bGH WAT depots showed a significantly higher number of total SVF cells, yet only MES bGH WAT had higher leukocyte counts compared with control samples. By means of flow cytometry analysis of the SVF, we detected greater macrophage and regulatory T-cell infiltration in sc and MES bGH WAT depots compared with controls. However, no differences were observed in the EPI WAT depot. RNA-sequencing confirmed significant alterations in pathways related to T-cell infiltration and activation in the sc depot with fewer significant changes in the EPI bGH WAT depot. These findings collectively point to a previously unrecognized role for GH in influencing the distribution of WAT immune cell populations in a depot-specific manner. PMID:25521584

  12. Characterization of African bat henipavirus GH-M74a glycoproteins.

    PubMed

    Weis, Michael; Behner, Laura; Hoffmann, Markus; Krüger, Nadine; Herrler, Georg; Drosten, Christian; Drexler, Jan Felix; Dietzel, Erik; Maisner, Andrea

    2014-03-01

    In recent years, novel henipavirus-related sequences have been identified in bats in Africa. To evaluate the potential of African bat henipaviruses to spread in non-bat mammalian cells, we compared the biological functions of the surface glycoproteins G and F of the prototype African henipavirus GH-M74a with those of the glycoproteins of Nipah virus (NiV), a well-characterized pathogenic member of the henipavirus genus. Glycoproteins are central determinants for virus tropism, as efficient binding of henipavirus G proteins to cellular ephrin receptors and functional expression of fusion-competent F proteins are indispensable prerequisites for virus entry and cell-to-cell spread. In this study, we analysed the ability of the GH-M74a G and F proteins to cause cell-to-cell fusion in mammalian cell types readily permissive to NiV or Hendra virus infections. Except for limited syncytium formation in a bat cell line derived from Hypsignathus monstrosus, HypNi/1.1 cells, we did not observe any fusion. The highly restricted fusion activity was predominantly due to the F protein. Whilst GH-M74a G protein was found to interact with the main henipavirus receptor ephrin-B2 and induced syncytia upon co-expression with heterotypic NiV F protein, GH-M74a F protein did not cause evident fusion in the presence of heterotypic NiV G protein. Pulse-chase and surface biotinylation analyses revealed delayed F cleavage kinetics with a reduced expression of cleaved and fusion-active GH-M74a F protein on the cell surface. Thus, the F protein of GH-M74a showed a functional defect that is most likely caused by impaired trafficking leading to less efficient proteolytic activation and surface expression. PMID:24296468

  13. Arterial pulse wave velocity, inflammatory markers, pathological GH and IGF states, cardiovascular and cerebrovascular disease

    PubMed Central

    Graham, Michael R; Evans, Peter; Davies, Bruce; Baker, Julien S

    2008-01-01

    Blood pressure (BP) measurements provide information regarding risk factors associated with cardiovascular disease, but only in a specific artery. Arterial stiffness (AS) can be determined by measurement of arterial pulse wave velocity (APWV). Separate from any role as a surrogate marker, AS is an important determinant of pulse pressure, left ventricular function and coronary artery perfusion pressure. Proximal elastic arteries and peripheral muscular arteries respond differently to aging and to medication. Endogenous human growth hormone (hGH), secreted by the anterior pituitary, peaks during early adulthood, declining at 14% per decade. Levels of insulin-like growth factor-I (IGF-I) are at their peak during late adolescence and decline throughout adulthood, mirror imaging GH. Arterial endothelial dysfunction, an accepted cause of increased APWV in GH deficiency (GHD) is reversed by recombinant human (rh) GH therapy, favorably influencing the risk for atherogenesis. APWV is a noninvasive method for measuring atherosclerotic and hypertensive vascular changes increases with age and atherosclerosis leading to increased systolic blood pressure and increased left ventricular hypertrophy. Aerobic exercise training increases arterial compliance and reduces systolic blood pressure. Whole body arterial compliance is lowered in strength-trained individuals. Homocysteine and C-reactive protein are two inflammatory markers directly linked with arterial endothelial dysfunction. Reviews of GH in the somatopause have not been favorable and side effects of treatment have marred its use except in classical GHD. Is it possible that we should be assessing the combined effects of therapy with rhGH and rhIGF-I? Only multiple intervention studies will provide the answer. PMID:19337549

  14. Effects of Hypergravity Rearing on Growth Hormone (GH) Secretion In Preweanling Rats

    NASA Technical Reports Server (NTRS)

    Baer, L. A.; Chowdhury, J. H.; Wade, C. E.; Ronca, A. E.; Dalton, Bonnie (Technical Monitor)

    2002-01-01

    We previously reported that rat pups reared at 1.5-g, 1.75 or 2.0-g hypergravity weigh 6-15% less than 1.0-g controls. To account for these findings. we measured the lactational hormones, prolaction (Prl) and oxytocin (OT), in the pups' mothers. Gravity related differences in Prl were not observed whereas OT of lactating dams was significantly reduced relative to controls. Milk transfer from dam to pup was not impaired in hypergravity-reared litters tested at 1-g. Together, these findings suggest that impaired lactation and milk transfer do not account for reduced body masses of postnatal rats reared in hypergravity. In the present study, we analyzed growth hormone (GH) secretion and maternal licking in pups reared in hypergravity and in 1.0-g controls. Recent reports using dwarfing phenotypes in mouse mutants have provided evidence for postnatal dependence on GH and insulin-like growth factors (IGFs). Beginning on Gestational day (G)11 of the rats' 22 day pregnancy, rat dams and their litters were exposed to either 1.5-g, 1.75-g or 2.0-g. On Postnatal day (P)10, we measured plasma GH using enzyme immunoassay (EIA). Contrary to our hypothesis, GH was significantly elevated in pups reared at 2.0-g relative to 1.0-g controls. Pup-oriented behaviors of the hypergravity dams were also changed, possibly accounting for the increase in pup GH. GH alone does not appear to play a role in reduced body weights of hypergravity-reared pups.

  15. Fatty Liver Index Associates with Relative Sarcopenia and GH/ IGF- 1 Status in Obese Subjects

    PubMed Central

    Gnessi, Lucio; Mariani, Stefania; Lenzi, Andrea; Donini, Lorenzo Maria

    2016-01-01

    Introduction Recently the association between hepatic steatosis and sarcopenia has been described. GH/IGF-1 axis has been postulated to play a role in linking fatty liver and low muscle mass. The aim of our study was to explore the association between fatty liver index, sarcopenic obesity, insulin sensitivity, and GH/IGF-1 status. Methods 427 subjects [age: 45.65±13.94 years, BMI: 36.92±6.43 kg/m2] were enrolled. Participants were divided into three groups: fatty liver index (FLI) <20, 20≥FLI<60, and FLI≥60. Body composition was assessed by DXA. The truncal fat mass (TrFM) to appendicular skeletal muscle (ASM) ratio was used as an indicator of sarcopenic obesity. ISI-Matsuda index was used. Results BMI, fat mass, and the TrFM/ASM ratio were higher in subjects with FLI≥60. GH, IGF-1 and ISI-Matsuda were lower in the high FLI group (all p<0.05). A significantly positive correlation between FLI and TrFM/ ASM ratio (r = 0.221, p<0.001) was found, whereas FLI levels were negatively correlated with ISI- Matsuda (r = -0.335, p<0.001), GH (r = -0.200, p = 0.006), and IGF- 1 levels (r = -0.157, p = 0.028). Stepwise linear regression analysis showed that GH levels were significantly negatively correlated with FLI, while the TrFM/ ASM ratio was positively associated with FLI, after adjustment for age, BMI, total fat mass, truncal fat mass, fat- free mass, and ISI- Matsuda. Conclusions Impairment of GH/IGF-1 axis seems to be associated to the risk of the development of sarcopenic obesity and ectopic fat deposition in the liver. Metabolic and hormonal derangements as determinants of ectopic fat deposition and body composition deserve to be evaluated in obese subjects. PMID:26741958

  16. Phylogenetic analysis of β-xylanase SRXL1 of Sporisorium reilianum and its relationship with families (GH10 and GH11) of Ascomycetes and Basidiomycetes

    PubMed Central

    Álvarez-Cervantes, Jorge; Díaz-Godínez, Gerardo; Mercado-Flores, Yuridia; Gupta, Vijai Kumar; Anducho-Reyes, Miguel Angel

    2016-01-01

    In this paper, the amino acid sequence of the β-xylanase SRXL1 of Sporisorium reilianum, which is a pathogenic fungus of maize was used as a model protein to find its phylogenetic relationship with other xylanases of Ascomycetes and Basidiomycetes and the information obtained allowed to establish a hypothesis of monophyly and of biological role. 84 amino acid sequences of β-xylanase obtained from the GenBank database was used. Groupings analysis of higher-level in the Pfam database allowed to determine that the proteins under study were classified into the GH10 and GH11 families, based on the regions of highly conserved amino acids, 233–318 and 180–193 respectively, where glutamate residues are responsible for the catalysis. PMID:27040368

  17. Three-dimensional structure of RBcel1, a metagenome-derived psychrotolerant family GH5 endoglucanase

    PubMed Central

    Delsaute, Maud; Berlemont, Renaud; Dehareng, Dominique; Van Elder, Dany; Galleni, Moreno; Bauvois, Cédric

    2013-01-01

    RBcel1 is an endoglucanase belonging to glycoside hydrolase family 5 subfamily 5 (GH5_5) that was recently identified from a soil metagenome library from the Antarctic. Unlike its closest structural homologue (Cel5A from Thermoascus aurantiacus), this enzyme was reported to be able to catalyze transglycosylation reactions and has putatively been implicated in the bacterial cellulose-synthesis process. Here, the structure of RBcel1 at 1.4 Å resolution, solved by molecular replacement, is reported. The structure and putative substrate-binding site are described and compared with those of other GH5_5 subfamily members. PMID:23908022

  18. [Advances in studying microbial GH13 starch debranching enzyme--a review].

    PubMed

    Duan, Xuguo; Wu, Jing

    2013-07-01

    Pullulanase and isoamylase belong to the GH13 family (glycoside hydrolase family 13) with similar sequence, catalytic mechanism and three-dimensional fold ((beta/alpha)8-barrel structure). Starch debranching enzymes can hydrolyze the alpha-1,6-glucosidic bonds at the branch sites of starch, and improve raw material utilization and production efficiency in the starch industry. In this review, the substrate specificity, protein structure, advances and new trends in the study of microbial GH13 starch debranching enzyme were systematically introduced. In addition, some opinions on the research status and prospect for starch debranching enzyme were discussed. PMID:24195371

  19. Distinct Roles for Carbohydrate-Binding Modules of Glycoside Hydrolase 10 (GH10) and GH11 Xylanases from Caldicellulosiruptor sp. Strain F32 in Thermostability and Catalytic Efficiency

    PubMed Central

    Meng, Dong-Dong; Ying, Yu; Chen, Xiao-Hua; Lu, Ming; Ning, Kang; Wang, Lu-Shan

    2015-01-01

    Xylanases are crucial for lignocellulosic biomass deconstruction and generally contain noncatalytic carbohydrate-binding modules (CBMs) accessing recalcitrant polymers. Understanding how multimodular enzymes assemble can benefit protein engineering by aiming at accommodating various environmental conditions. Two multimodular xylanases, XynA and XynB, which belong to glycoside hydrolase families 11 (GH11) and GH10, respectively, have been identified from Caldicellulosiruptor sp. strain F32. In this study, both xylanases and their truncated mutants were overexpressed in Escherichia coli, purified, and characterized. GH11 XynATM1 lacking CBM exhibited a considerable improvement in specific activity (215.8 U nmol−1 versus 94.7 U nmol−1) and thermal stability (half-life of 48 h versus 5.5 h at 75°C) compared with those of XynA. However, GH10 XynB showed higher enzyme activity and thermostability than its truncated mutant without CBM. Site-directed mutagenesis of N-terminal amino acids resulted in a mutant, XynATM1-M, with 50% residual activity improvement at 75°C for 48 h, revealing that the disordered region influenced protein thermostability negatively. The thermal stability of both xylanases and their truncated mutants were consistent with their melting temperature (Tm), which was determined by using differential scanning calorimetry. Through homology modeling and cross-linking analysis, we demonstrated that for XynB, the resistance against thermoinactivation generally was enhanced through improving both domain properties and interdomain interactions, whereas for XynA, no interdomain interactions were observed. Optimized intramolecular interactions can accelerate thermostability, which provided microbes a powerful evolutionary strategy to assemble catalysts that are adapted to various ecological conditions. PMID:25576604

  20. Exclusion of growth hormone (GH)-releasing hormone gene mutations in familial isolated GH deficiency by linkage and single strand conformation analysis

    SciTech Connect

    Perez Jurado, L.A.; Francke, U.; Phillips, J.A. III

    1994-03-01

    The molecular basis and the locus responsible for most familial cases of isolated GH deficiency (IGHD) are still unknown. The GH-releasing hormone (GHRH) gene has been evaluated as a possible candidate in 23 unrelated families with IGHD, 14 of whom were classified as having autosomal recessive IGHD type IB and 9 of whom had autosomal dominant IGHD type II. Three highly polymorphic microsatellites (dinucleotide repeats), mapped close to GHRH on chromosome 20 by previous linkage studies, were analyzed as markers for the GHRH locus. All available family members were genotyped for D20S44 [no recombination with GHRH at a LOD (logarithm of the odds) score of 3.6]. Noninformative families were also genotyped for D20S45 and/or D20S54 (located at {approximately} 1 and 3 centiMorgan of genetic distance from GHRH, respectively). Twenty families were informative for linkage analysis with 1 or more of these markers. They found at least 1 obligate recombinant with discordance between phenotype and genotype in 19 of the 23 families (83%). There is only a very small chance (1-3% or less) that the discordances observed are due to recombination between the GHRH locus and the marker tested. Concordant segregation was seen in only 1 type IB family (4%). When probands from this and the 3 noninformative families were screened for sequence variants in the 5 exons of the GHRH gene by single strand conformation analysis, no abnormal patterns were observed. They conclude that mutations responsible for IGHD are not within or near the structural gene for GHRH on chromosome 20 in the 23 families studied. As linkage to the GH-1 gene has also been previously excluded in 65% of these families, mutations in a locus or loci unlinked to GH-1 and GHRH must be responsible for the majority of these IGHD families. 31 refs., 4 figs., 1 tab.

  1. GH, IGF-I and GH receptors mRNA expression in response to growth impairment following a food deprivation period in individually housed cichlid fish Cichlasoma dimerus.

    PubMed

    Delgadin, Tomás Horacio; Pérez Sirkin, Daniela Irina; Di Yorio, María Paula; Arranz, Silvia Eda; Vissio, Paula Gabriela

    2015-02-01

    Cichlasoma dimerus is a social cichlid fish capable of growing at high rates under laboratory conditions, but knowledge on somatic growth regulation is still unclear. Growth hormone (GH)/insulin-like growth factor I (IGF-I) axis is the key regulator of somatic growth in vertebrates. Two types of growth hormone receptors have been described in teleost fish, named GH receptor type 1 (GHR1) and type 2 (GHR2). In addition, isoforms of these receptors lacking part of the intracellular region have been described. The aim of this study was to evaluate the somatic growth, liver histology and changes in the GH/IGF-I axis after 4 weeks of food deprivation in C. dimerus. Four-week fasted fish showed reductions in specific growth rates in body weight (p < 0.001) and standard length (p < 0.001). Additionally, the hepatosomatic index (p < 0.001) and hepatocyte area (p < 0.001) decreased in fasted fish, while no changes in glucose levels were detected in plasma. The starvation protocol failed to induce changes in GH mRNA levels in the pituitary and IGF-I mRNA levels in liver. In contrast, IGF-I mRNA levels in muscle decreased in fasted fish (p = 0.002). On the other hand, GHR2 (detected with primer sets designed over the extracellular and intracellular region) was upregulated by starvation both in liver and muscle (p < 0.05), while GHR1 remained unchanged. These results show that a fasting period reduced somatic growth both in length and body weight concomitantly with alterations on liver and muscle GHR2 and muscle IGF-I mRNA expression. PMID:25351458

  2. Lack of association of progestin-induced cystic endometrial hyperplasia with GH gene expression in the canine uterus.

    PubMed

    Kooistra, H S; Okkens, A C; Mol, J A; van Garderen, E; Kirpensteijn, J; Rijnberk, A

    1997-01-01

    Growth hormone (GH) is produced in progestin-induced hyperplastic ductular mammary epithelia in dogs. Progestins also induce the development of cystic endometrial hyperplasia (CEH) in this species. The study reported here investigated whether GH gene expression could also be demonstrated in progestin-induced hyperplastic epithelium in the canine uterus. Eight beagle bitches were treated with 10 mg medroxyprogesterone acetate (MPA) kg-1 body mass s.c. at intervals of 3 weeks, for a total of five times in four dogs (group I) and for a total of 13 times in the other four dogs (group II). Blood samples were collected twice during each 3 week period for measurement of plasma concentrations of GH, insulin-like growth factor I (IGF-I) and IGF-II. At the end of the series of injections uterine tissue was obtained by ovario-hysterectomy. Histological examination confirmed that CEH was present in all uteri after MPA treatment; the changes in the dogs of group I were less marked than those in group II. Immunohistochemical examination of the uterine tissues showed that immunoreactive(i) GH was present in a number of uteri with CEH. iGH was usually located in the cytoplasm of glandular epithelial cells. However, reverse transcriptase PCR using GH-specific primers failed to demonstrate mRNA encoding GH in the uterine tissue of all dogs. It is concluded that local production of GH is not involved in progestin-induced hyperplasia of uterine epithelial cells in dogs. PMID:9404306

  3. Overexpression of a cotton annexin gene, GhAnn1, enhances drought and salt stress tolerance in transgenic cotton.

    PubMed

    Zhang, Feng; Li, Shufen; Yang, Shuming; Wang, Like; Guo, Wangzhen

    2015-01-01

    Plant annexins are members of a diverse, multigene protein family that has been associated with a variety of cellular processes and responses to abiotic stresses. GhAnn1, which encodes a putative annexin protein, was isolated from a cotton (Gossypium hirsutum L. acc 7235) cDNA library. Tissue-specific expression showed that GhAnn1 is expressed at differential levels in all tissues examined and strongly induced by various phytohormones and abiotic stress. In vivo and in vitro subcellular localization suggested that GhAnn1 is located in the plasma membrane. In response to drought and salt stress, transgenic cotton plants overexpressing GhAnn1 showed significantly higher germination rates, longer roots, and more vigorous growth than wild-type plants. In addition, plants overexpressing GhAnn1 had higher total chlorophyll content, lower lipid peroxidation levels, increased peroxidase activities, and higher levels of proline and soluble sugars, all of which contributed to increased salt and drought stress tolerance. However, transgenic cotton plants in which the expression of GhAnn1 was suppressed showed the opposite results compared to the overexpressing plants. These findings demonstrated that GhAnn1 plays an important role in the abiotic stress response, and that overexpression of GhAnn1 in transgenic cotton improves salt and drought tolerance. PMID:25330941

  4. Quality of Life and Psychological Well-Being in GH-Treated, Adult PWS Patients: A Longitudinal Study

    ERIC Educational Resources Information Center

    Bertella, L.; Mori, I.; Grugni, G.; Pignatti, R.; Ceriani, F.; Molinari, E.; Ceccarelli, A.; Sartorio, A.; Vettor, R.; Semenza, C.

    2007-01-01

    Background: Prader-Willi syndrome (PWS) is a congenital alteration of chromosome pair 15. It is characterized by short stature, muscular hypotonia, hyperphagia, obesity, behavioural and emotional disturbances, hypogonadism and partial Growth Hormone (GH) deficiency. The aim of this study was to assess the long-term effect of GH treatment on the…

  5. Increased linear bone growth by GH in the absence of SOCS2 is independent of IGF‐1

    PubMed Central

    Dobie, Ross; Ahmed, Syed F.; Staines, Katherine A.; Pass, Chloe; Jasim, Seema; MacRae, Vicky E.

    2015-01-01

    Growth hormone (GH) signaling is essential for postnatal linear bone growth, but the relative importance of GHs actions on the liver and/or growth plate cartilage remains unclear. The importance of liver derived insulin like‐growth factor‐1 (IGF‐1) for endochondral growth has recently been challenged. Here, we investigate linear growth in Suppressor of Cytokine Signaling‐2 (SOCS2) knockout mice, which have enhanced growth despite normal systemic GH/IGF‐1 levels. Wild‐type embryonic ex vivo metatarsals failed to exhibit increased linear growth in response to GH, but displayed increased Socs2 transcript levels (P < 0.01). In the absence of SOCS2, GH treatment enhanced metatarsal linear growth over a 12 day period. Despite this increase, IGF‐1 transcript and protein levels were not increased in response to GH. In accordance with these data, IGF‐1 levels were unchanged in GH‐challenged postnatal Socs2‐/‐ conditioned medium despite metatarsals showing enhanced linear growth. Growth‐plate Igf1 mRNA levels were not elevated in juvenile Socs2‐/‐ mice. GH did however elevate IGF‐binding protein 3 levels in conditioned medium from GH challenged metatarsals and this was more apparent in Socs2‐/‐ metatarsals. GH did not enhance the growth of Socs2‐/‐ metatarsals when the IGF receptor was inhibited, suggesting that IGF receptor mediated mechanisms are required. IGF‐2 may be responsible as IGF‐2 promoted metatarsal growth and Igf2 expression was elevated in Socs2‐/‐ (but not WT) metatarsals in response to GH. These studies emphasise the critical importance of SOCS2 in regulating GHs ability to promote bone growth. Also, GH appears to act directly on the metatarsals of Socs2‐/‐ mice, promoting growth via a mechanism that is independent of IGF‐1. J. Cell. Physiol. 9999: 2796–2806, 2015. © 2015 Wiley Periodicals, Inc. PMID:25833299

  6. IGF-I modulation of GH and LH secretion in the pig

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three experiments (EXP) were conducted to determine the role of IGF-I in modulating GH and LH secretion. In EXP I, prepuberal gilts, 65 ± 6 kg body weight (BW) and 140 days of age received intracerebroventricular (ICV) injections of saline (n = 4), 25µg (n = 4) or 75µg (n = 4) IGF-I and jugular blo...

  7. GH1-family 6-P-β-glucosidases from human microbiome lactic acid bacteria

    PubMed Central

    Michalska, Karolina; Tan, Kemin; Li, Hui; Hatzos-Skintges, Catherine; Bearden, Jessica; Babnigg, Gyorgy; Joachimiak, Andrzej

    2013-01-01

    In lactic acid bacteria and other bacteria, carbohydrate uptake is mostly governed by phosphoenolpyruvate-dependent phosphotransferase systems (PTSs). PTS-dependent translocation through the cell membrane is coupled with phosphorylation of the incoming sugar. After translocation through the bacterial membrane, the β-glycosidic bond in 6′-­P-­β-glucoside is cleaved, releasing 6-P-β-glucose and the respective aglycon. This reaction is catalyzed by 6-P-β-glucosidases, which belong to two glycoside hydrolase (GH) families: GH1 and GH4. Here, the high-resolution crystal structures of GH1 6-P-β-glucosidases from Lactobacillus plantarum (LpPbg1) and Streptococcus mutans (SmBgl) and their complexes with ligands are reported. Both enzymes show hydrolytic activity towards 6′-P-β-glucosides. The LpPbg1 structure has been determined in an apo form as well as in a complex with phosphate and a glucose molecule corresponding to the aglycon molecule. The S. mutans homolog contains a sulfate ion in the phosphate-dedicated subcavity. SmBgl was also crystallized in the presence of the reaction product 6-P-β-glucose. For a mutated variant of the S. mutans enzyme (E375Q), the structure of a 6′-P-salicin complex has also been determined. The presence of natural ligands enabled the definition of the structural elements that are responsible for substrate recognition during catalysis. PMID:23519420

  8. Interaction of Pseudomonas putida ATCC 12633 and Bacteriophage gh-1 in Berea Sandstone Rock.

    PubMed

    Chang, P L; Yen, T F

    1985-12-01

    Measurements of the passage of Pseudomonas putida ATCC 12633 and a phage-resistant mutant through Berea sandstone rock were made. When bacteriophage gh-1 was adsorbed within the rock matrix, a reduction in the passage of the susceptible but not the resistant cells through the rock was observed. PMID:16346956

  9. Interaction of Pseudomonas putida ATCC 12633 and Bacteriophage gh-1 in Berea Sandstone Rock

    PubMed Central

    Chang, Philip Lee; Yen, Teh Fu

    1985-01-01

    Measurements of the passage of Pseudomonas putida ATCC 12633 and a phage-resistant mutant through Berea sandstone rock were made. When bacteriophage gh-1 was adsorbed within the rock matrix, a reduction in the passage of the susceptible but not the resistant cells through the rock was observed. PMID:16346956

  10. Biochemical characterization of a GH43 ß-xylosidase from Bacteroides ovatus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Divalent-metal-activated, glycoside hydrolase (GH43) ß-xylosidases have been found to have high kcat/Km for xylooligosaccharides and may demonstrate high efficacy in industrial reactors digesting hemicellulose. By searching an amino acid database, we found an enzyme that is 81% identical in amino ac...