scholarly journals Tetracycline-regulated secretion of human insulin in a transfected non-endocrine cell line

2003 ◽  
Vol 30 (3) ◽  
pp. 331-346 ◽  
Author(s):  
KT Scougall ◽  
CA Maltin ◽  
JA Shaw
Keyword(s):  
Insulin Secretion ◽  
Cell Line ◽  
Human Insulin ◽  
Endocrine Cell ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Host Cells ◽  
Cleavage Sites ◽  
Wild Type ◽  
Transfected Cells

Long-term constitutive secretion of insulin by implantation of ex vivo transfected cells such as fibroblasts or myoblasts or in situ by intramuscular injection of naked plasmid DNA provides a potential approach to gene therapy for diabetes mellitus. A mechanism for regulating insulin secretion will be necessary to realize the therapeutic potential of this approach. A second obstacle is the inability of non-endocrine host cells to fully process proinsulin. Therefore, alteration of the wild-type cDNA will be necessary to achieve processing of proinsulin by endogenous endoproteases within these cells. The cDNAs for beta-galactosidase (beta), human wild-type proinsulin (hppI1) and a mutated construct (hppI4), in which the dibasic PC2 and PC3 cleavage sites had been altered to form furin cleavage sites, were sub-cloned into four vectors (pCR3, pVR1012, pIRES, pTRE), including a tetracycline responsive plasmid (pTRE) that requires co-transfection with another plasmid encoding a transactivator (pTet-off) for transgene expression. Transient transfection of the COS-7 fibroblast cell line with these constructs was performed using DEAE-dextran and liposomes. Analysis of vector efficiencies revealed that pTRE/pTet-off>pIRES>pCR3>pVR1012. Further analysis demonstrated total pro/insulin secretion of 2.33 ng/10(6) cells/24 h with > or =25% processed to insulin in hppI-1.pTRE/pTet-off-transfected cells compared with 0.39 ng/10(6) cells/24 h and >70% processing in hppI-4.pTRE/pTet-off-transfected cells. In co-transfection studies with pTRE-hppI1/pTet-off and pTRE-hppI4/pTet-off constructs, pro/insulin secretion was inhibited to 65-66% and 36-38% of control (100%) in the presence of 0.01 and 0.1 microg/ml tetracycline respectively over a 24-h incubation period. Furthermore, reversal of tetracycline inhibition was demonstrated for pTRE-hppI1/pTet-off- and pTRE-hppI4/pTet-off-transfected cells. After a 48-h incubation with 1.0 microg/ml tetracycline, total pro/insulin levels were 10 and 14% compared with untreated cells respectively. On tetracycline removal, total proinsulin levels increased and were equivalent to untreated groups 72 h later. In conclusion, regulation of fully processed human insulin secretion has been achieved in a transiently transfected non-endocrine cell line.

scholarly journals Pancreatic beta-cells expressing the Arg64 variant of the beta(3)-adrenergic receptor exhibit abnormal insulin secretory activity

2001 ◽  
Vol 27 (2) ◽  
pp. 133-144 ◽  
Author(s):  
R Perfetti ◽  
H Hui ◽  
K Chamie ◽  
S Binder ◽  
M Seibert ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Beta Cells ◽  
Western Blotting ◽  
Adrenergic Receptor ◽  
Pancreatic Beta Cells ◽  
Secretory Activity ◽  
Host Cells ◽  
Human Pancreas ◽  
Dependent Manner ◽  
Wild Type

The Arg64 beta(3)-adrenergic receptor (beta(3)AR) variant is associated with an earlier age of onset of diabetes and lower levels of insulin secretion in humans. The aims of this study were to investigate whether beta(3)AR is expressed by islet cells, if receptor binding affects insulin secretion and, finally, if the beta(3)AR Arg64 variant induces abnormal insulin secretory activity. Human pancreas extracts were subjected to RT-PCR, Western blotting and immunostaining analyses. DNA sequencing and Western blotting demonstrated that the beta(3)AR gene is transcribed and translated in the human pancreas; immunostaining showed that it is expressed by the islets of Langerhans. Cultured rat beta-cells responded to human beta(3)AR agonists in a dose- and time-dependent manner. Transfection of cultured rat beta-cells with the wild-type human beta(3)AR produced an increased baseline and ligand-dependent insulin secretion compared with parental cells. On the other hand, cells transfected with the Arg64 variant of the beta(3)AR secreted less insulin, both spontaneously and after exposure to human beta(3)AR agonists. Furthermore, while transfection with the wild-type beta(3)AR preserved the glucose-dependent secretion of insulin, expression of the variant receptor rendered the host cells significantly less responsive to glucose. In summary, cells express the beta(3)AR, and its activation contributes to the regulation of insulin secretion. These findings may help explain the low levels of insulin secretion in response to an i.v. glucose tolerance test observed in humans carrying the Arg64 polymorphism.

scholarly journals Streptococcus pneumoniae-Induced Inhibition of Rat Ependymal Cilia Is Attenuated by Antipneumolysin Antibody

2004 ◽  
Vol 72 (11) ◽  
pp. 6694-6698 ◽  
Author(s):  
Robert A. Hirst ◽  
Bashir J. Mohammed ◽  
Timothy J. Mitchell ◽  
Peter W. Andrew ◽  
Christopher O'Callaghan
Keyword(s):  
Streptococcus Pneumoniae ◽  
Therapeutic Potential ◽  
Ependymal Cell ◽  
Ex Vivo ◽  
Beat Frequency ◽  
Ciliary Beat Frequency ◽  
Ependymal Cells ◽  
Wild Type ◽  
Ependymal Cilia ◽  
The Brain

ABSTRACT Ciliated ependymal cells line the ventricular surfaces and aqueducts of the brain. In ex vivo experiments, pneumolysin caused rapid inhibition of the ependymal ciliary beat frequency and caused ependymal cell disruption. Wild-type pneumococci and pneumococci deficient in pneumolysin caused ciliary slowing, but penicillin lysis of wild-type, not pneumolysin-deficient, pneumococci increased the extent of ciliary inhibition. This effect was abolished by antipneumolysin antibody. Ependymal ciliary stasis by purified pneumolysin was also blocked by the addition of antipneumolysin monoclonal antibodies. These data show that antibiotic lysis of Streptococcus pneumoniae can be detrimental to the ciliated ependyma and that antipneumolysin antibody may have a therapeutic potential.

scholarly journals Adequate connexin-mediated coupling is required for proper insulin production.

1995 ◽  
Vol 131 (6) ◽  
pp. 1561-1572 ◽  
Author(s):  
C Vozzi ◽  
S Ullrich ◽  
A Charollais ◽  
J Philippe ◽  
L Orci ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Gap Junctions ◽  
Beta Cells ◽  
Stable Transfection ◽  
Insulin Production ◽  
Wild Type ◽  
Transfected Cells ◽  
Insulin Producing Cells ◽  
And Storage ◽  
Rat Insulinoma

To assess whether connexin (Cx) expression contributes to insulin secretion, we have investigated normal and tumoral insulin-producing cells for connexins, gap junctions, and coupling. We have found that the glucose-sensitive cells of pancreatic islets and of a rat insulinoma are functionally coupled by gap junctions made of Cx43. In contrast, cells of several lines secreting insulin abnormally do not express Cx43, gap junctions, and coupling. After correction of these defects by stable transfection of Cx43 cDNA, cells expressing modest levels of Cx43 and coupling, as observed in native beta-cells, showed an expression of the insulin gene and an insulin content that were markedly elevated, compared with those observed in both wild-type (uncoupled) cells and in transfected cells overexpressing Cx43. These findings indicate that adequate levels of Cx-mediated coupling are required for proper insulin production and storage.

scholarly journals Analysis of Novel Histone Methylase MLL Function for Glucose Metabolism in Mouse Pancreas

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A316-A317
Author(s):  
Satoshi Yoshino ◽  
Emi Ishida ◽  
Kazuhiko Horiguchi ◽  
Shunichi Matsumoto ◽  
Yasuyo Nakajima ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Glucose Metabolism ◽  
Cell Line ◽  
Knockout Mice ◽  
Tunel Staining ◽  
Type I ◽  
Wild Type ◽  
Chromosome 11 ◽  
Glucose Stimulation ◽  
Knockdown Cell

Abstract Background) Myeloid / Lymphoid or Mixed-lineage leukemia gene (MLL) is translocated to chromosome 11 long arm q23 region (11q23) and the MLL fusion gene expressed as a result of translocation reconstruction plays an important role in MLL-related leukemia development. It has also been reported that MLL and MLL protein play an important role in tumor development as a Menin-binding protein in Multiple Endocrine Neoplasia Type I (MEN1). More recently, normal MLL protein has been shown to have histone H3 lysine 4-methylation (H3K4-HMT) activity and to be an epigenetic transcriptional regulator. In addition, the function of MLL protein as a histone methylase has been reported in the gene region involved in metabolism regions. Here, we analyzed the involvement of MLL in glucose metabolism in the pancreas using MLL knockout mice. Methods:) Glucose metabolism in MLL knockout mice and the function of MLL in cultured cells were analyzed. Result) Since the homozygotes of MLL knockout mice are embryonic lethal, we analyzed them using Heterozygous mice. MLL heterozygous mice showed significantly weight loss compared to the wild type mice. MLL heterozygous mice showed no difference in food intake compared to wild type mice. IPGTT showed impaired glucose tolerance in MLL heterozygous mice. However, ITT showed no insulin resistance and decreased insulin secretion during glucose loading. In GSIS tests, Islets isolated from heterozygous mice pancreas have been observed to decrease insulin secretion in the response to glucose stimulation. In comprehensive gene analysis using Microarray analysis of mRNA extracted from mice islet, the gene expression changes related insulin secretion and apoptosis have been revealed in MLL heterozygous mice. Histological search showed no decrease in β-cell number, and immunohistological search showed no difference in insulin, glucagon, and TUNEL staining between heterozygous and wild type mice. And also, MLL knockdown was performed in a cultured cell line. Insulin secretion was decreased to glucose stimulation in MLL knockdown cell line same as in MLL knockout mice. In addition, RNA microarrays were performed to these cell lines, several same genes that have confirmed in MLL mouse islets were observed in MLL knockdown cell. In conclusion, MLL knockout mice showed decreased insulin secretion. It was suggested that MLL may be involved in insulin secretion in islets.

Evi1 Promotes Leukemogenesis by Anti-Apoptotic Rather Than Differentiation-Blocking Effects in Murine MLL-AF9 Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 593-593
Author(s):  
Ashish Kumar ◽  
Quanzhi Li ◽  
Wendy A Hudson ◽  
Thien N Sam ◽  
Weili Chen ◽  
...  
Keyword(s):  
Cell Line ◽  
Propidium Iodide ◽  
High Efficiency ◽  
Therapeutic Potential ◽  
Rank Test ◽  
Dependent Manner ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Knock Down ◽  
Control Virus

Abstract In a search for molecular targets in MLL -leukemias, we found that transformed hematopoietic stem cells in knock-in MLL-AF9 mice expressed high levels of Evi1 compared to their wild type counterparts (Chen et al, Cancer Cell, 2008). About 50% of human AMLs with MLL rearrangements also express Evi1 and Evi1-expressing human leukemias are associated with an aggressive disease. The current experiments were designed to study mechanisms in which Evi1 is important for the pathogenesis of leukemia. To investigate its role in MLL-AF9 leukemia, we studied the effect of Evi1 knock-down in a cell line derived from a leukemic MLL-AF9 knock-in mouse. This cell line (4166) displays a myelo-monocytic phenotype, similar to human MLL-AF9 leukemias and induces leukemia with high efficiency when transplanted in wild type mice. To knock down Evi1, we screened multiple shRNA constructs cloned into a lentivirus expression vector (The RNAi Consortium/Open Biosystems). Of five shRNAs screened, two clones (E95 and E97) consistently inhibited cell growth in a dose dependent manner at multiplicities of infection (MOI) of 10 or greater. In one representative experiment, at 5 days post transduction at an MOI of 100, growth of E95 transduced 4166 cells was decreased by >80% compared to control-virus transduced cells. Evi1 mRNA was decreased in E95 transduced cells by >70% compared to controls at 48 hours. When cultured in semi-solid methylcellulose media for 7 days, E95 transduced cells formed significantly fewer colonies than control virus transduced cells (457.8 ± 24.7 and 847.8 ± 92.9 colonies per 1000 cells respectively, p<0.02). Surprisingly however, there was no change in the proportions of dense vs. loose colonies indicating that loss of Evi1 did not result in differentiation. Morphologic analysis also showed no change in morphology of E95 transduced cells. Further confirming a lack of differentiation-inhibiting effects of Evi1, quantitative RT-PCR assays showed no change in expression of genes associated with myeloid differentiation (integrin alpha-L, integrin beta-5, lysozyme, Csf-1). In Evi1 knock-down cells, analysis of DNA content by flow cytometry at 48 hours showed no change in the proportions of cells in the G1/G0, S and G2/M phases of the cell cycle. In contrast to lack of an effect on differentiation, there was an increase in apoptosis with Evi1 knock-down as evidenced by an increase in the proportion of cells stained with Annexin V and Propidium iodide (51% vs. 29%) or a pan activated-caspase-marker and Propidium iodide (41% vs. 15%, E95 vs. control virus transduced cells respectively). To investigate the role of Evi1 in leukemia in vivo, 105 cells transduced with either E95 or control virus were injected into irradiated wild type mice. Establishment of leukemia was confirmed by leukocytosis, splenomegaly and evidence of malignant infiltrates in the spleen by morphology and FACS. Eight of nine mice that received control virus transduced cells died of leukemia by day 170 while all those given E95 transduced cells were alive. Leukemia development was significantly delayed in the animals receiving E95 transduced cells (p<0.001, log rank test) with 44% of the animals being long term survivors. Overall, our results suggest that contrary to the view that leukemic-oncogenes induce self-renewal coupled to a block in differentiation, in murine MLL-AF9 leukemia Evi1 inhibits pro-apoptotic pathways and promotes cell-survival without a differentiation-block. Additionally, given the critical requirement of this transcription factor in the survival of leukemia cells, targeting Evi1 may have therapeutic potential to treat leukemia.

scholarly journals Human T-Lymphotropic Virus Type 1 Mitochondrion-Localizing Protein p13II Is Required for Viral Infectivity In Vivo

2006 ◽  
Vol 80 (7) ◽  
pp. 3469-3476 ◽  
Author(s):  
Hajime Hiraragi ◽  
Seung-Jae Kim ◽  
Andrew J. Phipps ◽  
Micol Silic-Benussi ◽  
Vincenzo Ciminale ◽  
...  
Keyword(s):  
Virus Infection ◽  
Cell Line ◽  
Virus Type ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Start Codon ◽  
Wild Type ◽  
T Lymphotropic Virus

ABSTRACT Human T-lymphotropic virus type 1 (HTLV-1), the etiological agent of adult T-cell leukemia, encodes unique regulatory and accessory proteins in the pX region of the provirus, including the open reading frame II product p13II. p13II localizes to mitochondria, binds farnesyl pyrophosphate synthetase, an enzyme involved in posttranslational farnesylation of Ras, and alters Ras-dependent cell signaling and control of apoptosis. The role of p13II in virus infection in vivo remains undetermined. Herein, we analyzed the functional significance of p13II in HTLV-1 infection. We compared the infectivity of a human B-cell line that harbors an infectious molecular clone of HTLV-1 with a selective mutation that prevents the translation of p13II (729.ACH.p13) to the infectivity of a wild-type HTLV-1-expressing cell line (729.ACH). 729.ACH and 729.ACH.p13 producer lines had comparable infectivities for cultured rabbit peripheral blood mononuclear cells (PBMC), and the fidelity of the start codon mutation in ACH.p13 was maintained after PBMC passage. In contrast, zero of six rabbits inoculated with 729.ACH.p13 cells failed to establish viral infection, whereas six of six rabbits inoculated with wild-type HTLV-1-expressing cells (729.ACH) were infected as measured by antibody responses, proviral load, and HTLV-1 p19 matrix antigen production from ex vivo-cultured PBMC. Our data are the first to indicate that the HTLV-1 mitochondrion-localizing protein p13II has an essential biological role during the early phase of virus infection in vivo.

scholarly journals Red Light Irradiation In Vivo Upregulates DJ-1 in the Retinal Ganglion Cell Layer and Protects against Axotomy-Related Dendritic Pruning

2021 ◽  
Vol 22 (16) ◽  
pp. 8380
Author(s):  
Kathy Beirne ◽  
Thomas J. Freeman ◽  
Malgorzata Rozanowska ◽  
Marcela Votruba
Keyword(s):  
Therapeutic Potential ◽  
Ganglion Cells ◽  
Ex Vivo ◽  
Red Light ◽  
Retinal Ganglion ◽  
Wild Type ◽  
Sholl Analysis ◽  
Retinal Explants ◽  
Oxidative Insult

Retinal ganglion cells (RGCs) undergo dendritic pruning in a variety of neurodegenerative diseases, including glaucoma and autosomal dominant optic atrophy (ADOA). Axotomising RGCs by severing the optic nerve generates an acute model of RGC dendropathy, which can be utilized to assess the therapeutic potential of treatments for RGC degeneration. Photobiomodulation (PBM) with red light provided neuroprotection to RGCs when administered ex vivo to wild-type retinal explants. In the current study, we used aged (13–15-month-old) wild-type and heterozygous B6;C3-Opa1Q285STOP (Opa1+/−) mice, a model of ADOA exhibiting RGC dendropathy. These mice were pre-treated with 4 J/cm2 of 670 nm light for five consecutive days before the eyes were enucleated and the retinas flat-mounted into explant cultures for 0-, 8- or 16-h ex vivo. RGCs were imaged by confocal microscopy, and their dendritic architecture was quantified by Sholl analysis. In vivo 670 nm light pretreatment inhibited the RGC dendropathy observed in untreated wild-type retinas over 16 h ex vivo and inhibited dendropathy in ON-center RGCs in wild-type but not Opa1+/− retinas. Immunohistochemistry revealed that aged Opa1+/− RGCs exhibited increased nitrosative damage alongside significantly lower activation of NF-κB and upregulation of DJ-1. PBM restored NF-κB activation in Opa1+/− RGCs and enhanced DJ-1 expression in both genotypes, indicating a potential molecular mechanism priming the retina to resist future oxidative insult. These data support the potential of PBM as a treatment for diseases involving RGC degeneration.

scholarly journals Processing of mutated proinsulin with tetrabasic cleavage sites to bioactive insulin in the non-endocrine cell line, COS-7

FEBS Letters ◽  
1992 ◽  
Vol 311 (1) ◽  
pp. 55-59 ◽  
Author(s):  
Masahiko Yanagita ◽  
Kazuhisa Nakayama ◽  
Toshiyuki Takeuchi
Keyword(s):  
Cell Line ◽  
Endocrine Cell ◽  
Cleavage Sites

scholarly journals Role of Cys41 in the N-terminal domain of lumican in ex vivo collagen fibrillogenesis by cultured corneal stromal cells

2003 ◽  
Vol 369 (3) ◽  
pp. 461-468 ◽  
Author(s):  
Eric C. CARLSON ◽  
Kazuhisa MAMIYA ◽  
Chia-Yang LIU ◽  
Robert L. GENDRON ◽  
David E. BIRK ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Ex Vivo ◽  
Mutant Cell ◽  
Dendritic Morphology ◽  
Wild Type ◽  
Mutant Cell Line ◽  
Stable Transformants ◽  
Collagen Fibrillogenesis

The keratan sulphate proteoglycan lumican regulates collagen fibrillogenesis to maintain the integrity and function of connective tissues such as cornea. We examined the role of a highly conserved cysteine-containing domain proximal to the N-terminus of lumican in collagen fibrillogenesis using site-specific mutagenesis to prepare plasmid DNA encoding wild-type murine lumican (Cys37-Xaa3-Cys41-Xaa-Cys-Xaa9-Cys) and a Cys→Ser (C/S) mutant (Cys37-Xaa3-Ser41-Xaa-Cys-Xaa9-Cys). cDNAs were cloned into the pSecTag2A vector, and cultures of MK/T-1 cells (an immortalized cell line from mouse keratocytes) were transfected with the cDNAs. Stable transformants were selected and cloned in the presence of Zeocin. All stable transformants maintained a dendritic morphology and growth rate similar to those of parental MK/T-1 cells. Western blot analysis with anti-lumican antibody detected a 42kDa lumican protein secreted into the culture medium of both wild-type and C/S mutant lumican cell lines. Ultrastructural analyses by transmission electron microscopy showed both cell lines to form a multi-layered stroma ex vivo, but the matrix assembled by the two cell lines differed. Compared with the mutant cell line, the wild-type cells assembled a more organized matrix with regions containing orthogonal collagen fibrils. In addition, the fibrils in the extracellular matrix formed by the mutant cell line exhibited alterations in fibril packing and structure. Immunostaining analysed by confocal microscopy showed a further difference in this matrix, with the marked occurrence of lumican and collagen I co-localization in the lumican wild-type cells, but a lack thereof in the lumican C/S mutant cells. The results indicate that the cysteine-rich domain of lumican is important in collagen fibrillogenesis and stromal matrix assembly.

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