scholarly journals Subetta Treatment Increases Adiponectin Secretion by Mature Human AdipocytesIn Vitro

2013 ◽  
Vol 2013 ◽  
pp. 1-4 ◽  
Author(s):  
Jim Nicoll ◽  
Evgeniy A. Gorbunov ◽  
Sergey A. Tarasov ◽  
Oleg I. Epstein
Keyword(s):  
Insulin Receptor ◽  
Mechanism Of Action ◽  
Cannabinoid Receptor ◽  
Beta Subunit ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Reference Drug ◽  
Peripheral Tissues ◽  
Negative Controls

Purpose. To investigate the mechanism of action in peripheral tissues of novel complex drug containing release-active dilutions of antibodies to the beta subunit of the insulin receptor and antibodies to endothelial nitric oxide synthase (Subetta), which has shown efficacy in animal models of diabetes.Methods. Human mature adipocytes were incubated either with Subetta, with one of negative controls (placebo or vehicle), with one of nonspecific controls (release-active dilutions of antibodies to cannabinoid receptor type I or release-active dilutions of rabbit nonimmune serum), or with dimethyl sulfoxide (DMSO) at 37°C in a humidified incubator at 5% CO2for three days. Rosiglitazone was used as reference drug. Secretion of adiponectin was measured by quantitative enzyme-linked immunosorbent assay (ELISA).Results. Only Subetta significantly stimulates adiponectin production by mature human adipocytes. Nonspecific controls did not significantly affect adiponectin secretion, resulting in adiponectin levels comparable to background values of the negative controls and DMSO.Conclusion. Increasing adiponectin production in absence of insulin by Subetta probably via modulating effect on the beta subunit of the insulin receptor might serve as one of the mechanisms of the antidiabetic effect of this drug. Thesein vitroresults give first insight on possible mechanism of action of Subetta and serve as a background for further studies.

scholarly journals Studies on the autophosphorylation of the insulin receptor from human placenta. Analysis of the sites phosphorylated by two-dimensional peptide mapping

1988 ◽  
Vol 252 (2) ◽  
pp. 607-615 ◽  
Author(s):  
J M Tavaré ◽  
R M Denton
Keyword(s):  
Thin Layer ◽  
Insulin Receptor ◽  
Peptide Mapping ◽  
Beta Subunit ◽  
Two Dimensional ◽  
Intact Cells ◽  
Tyrosine Residues ◽  
Domain 3 ◽  
Receptor Beta

1. A partially purified preparation of human placental insulin receptors was incubated with [gamma-32P]ATP in the presence or absence of insulin. The 32P-labelled insulin-receptor beta-subunits were then isolated, cleaved with trypsin followed by protease V8 and the [32P]phosphopeptides generated were analysed by thin layer electrophoresis and chromatography. This approach revealed that insulin stimulates autophosphorylation of the insulin-receptor beta-subunit in vitro on at least seven tyrosine residues distributed among three distinct domains. 2. One domain (domain 2), containing tyrosine residues 1146, 1150 and 1151 was the most rapidly phosphorylated and could be recovered as mono-, di- and triphosphorylated peptides cleaved by trypsin at Arg-1143 and either Lys-1153 or Lys-1156. Multiple phosphorylation of this domain appears to partially inhibit the cleavage at Lys-1153 by trypsin. 3. In a second domain (domain 3) containing two phosphorylated tyrosine residues at positions 1316 and 1322 the tyrosines were phosphorylated more slowly than those in domain 2. This domain is close to the C-terminus of the beta-subunit polypeptide chain. 4. At least two further tyrosine residues appeared to be phosphorylated after those in domains 2 and 3. These residues probably residue within a domain lying in close proximity to the inner face of the plasma membrane containing tyrosines 953, 960 and 972, but conclusive evidence is still required. 5. The two-dimensional thin-layer analysis employed in this study to investigate insulin-receptor phosphorylation has several advantages over previous methods based on reverse-phase chromatography. It allows greater resolution of 32P-labelled tryptic peptides and, when coupled to radioautography, is considerably more sensitive. The approach can be readily adapted to study phosphorylation of the insulin receptor within intact cells.

Decreased muscle insulin receptor kinase correlates with insulin resistance in normoglycemic Pima Indians

1997 ◽  
Vol 273 (2) ◽  
pp. E276-E283 ◽  
Author(s):  
J. F. Youngren ◽  
I. D. Goldfine ◽  
R. E. Pratley
Keyword(s):  
Insulin Resistance ◽  
Insulin Receptor ◽  
Body Fat ◽  
Insulin Action ◽  
Enzyme Linked Immunosorbent Assay ◽  
Percent Body Fat ◽  
Receptor Function ◽  
Pima Indians ◽  
Insulin Receptor Tyrosine Kinase

Defects in insulin receptor tyrosine kinase activity are present in insulin-resistant non-insulin-dependent diabetes mellitus patients and certain nondiabetic individuals, both lean and obese. However, the relationship between insulin receptor function, insulin action, and obesity is unclear. To address this issue, we have employed a new and highly sensitive enzyme-linked immunosorbent assay to measure in vitro insulin-stimulated autophosphorylation of immunocaptured muscle insulin receptors in a group of 25 normoglycemic Pima Indians. Insulin action, determined during two-step euglycemic insulin clamps, varied widely in these subjects. Maximal in vitro insulin stimulation of insulin receptor autophosphorylation strongly correlated with both low (Mlow)- and high (Mhigh)-dose insulin-stimulated glucose disposal (r = 0.62 and 0.51, P < 0.002 and 0.011, respectively). Insulin receptor autophosphorylation was inversely related to percent body fat (r = -0.52, P < 0.009). After control for percent body fat, receptor autophosphorylation remained correlated with Mlow (partial r = 0.49, P < 0.025). These data therefore suggest that defects in insulin receptor function are major contributors to insulin resistance in both lean and obese normoglycemic Pima Indians.

Effect of MMP-13 degraded SPARC on type I collagen and its biomarker potential in colorectal cancer.

2018 ◽  
Vol 36 (4_suppl) ◽  
pp. 717-717
Author(s):  
Stephanie Nina Kehlet ◽  
Henrik Harling ◽  
Lars N Jørgensen ◽  
Morten A Karsdal ◽  
Nicholas Willumsen
Keyword(s):  
Colorectal Cancer ◽  
Type I Collagen ◽  
Collagen Degradation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Matricellular Protein ◽  
Elisa Assay ◽  
Type I ◽  
Healthy Controls ◽  
Collagen Deposition

717 Background: Increased collagen deposition and remodeling of the extracellular matrix has been shown to play a role in the pathology of gastrointestinal cancer (GC). The matricellular protein SPARC (secreted proteome acidic and rich in cysteine) binds collagens and hereby regulates collagen fibrillogenesis. Matrix metalloproteinase (MMP) mediated cleavage of SPARC, increases the affinity for collagens up to 20-fold. SPARC has been shown to be overexpressed in GC patients and associated with GC cell invasion and metastasis. Increased expression and cleavage of SPARC might therefore be implicated in GC pathology by increasing collagen deposition. Here, we present the development and validation of a competitive enzyme-linked immunosorbent assay (ELISA) quantifying a specific MMP-13 generated fragment of SPARC - a cleavage site involved in increased collagen affinity. The biomarker potential of this fragment was examined in serum from colorectal cancer (CRC) patients and healthy controls. Moreover, we evaluated the ability of cleaved SPARC to prevent type I collagen degradation in vitro. Methods: A monoclonal antibody was raised against a MMP-13-generated neo-epitope of SPARC and a competitive ELISA assay (SPARC-M) was developed and technically validated. Serum levels were assessed in CRC patients (n=50) and healthy controls (n=30). The ability of cleaved SPARC to prevent collagen degradation was investigated using an ELISA assay measuring type I collagen degradation by MMP-9. Results: SPARC-M was technically robust and specific for SPARC cleaved by MMP-13. The fragment was elevated in CRC patients when compared to healthy controls (p=0.0097). When MMP-13 degraded SPARC was incubated with type I collagen and MMP-9, type I collagen degradation was completely inhibited suggesting that SPARC increases collagen deposition by preventing collagen degradation. Conclusions: SPARC-M was significantly elevated in CRC patients compared to healthy controls suggesting biomarker potential. Biologically, cleaved SPARC may prevent type I collagen degradation hereby leading to a pro-tumorigenic environment. Larger clinical studies are needed to validate the clinical use of this biomarker in GC.

scholarly journals Expression of activin A and its receptors in human pheochromocytomas

2000 ◽  
Vol 165 (2) ◽  
pp. 503-508 ◽  
Author(s):  
J Liu ◽  
P Heikkila ◽  
AI Kahri ◽  
R Voutilainen
Keyword(s):  
Protein Kinase ◽  
Mrna Expression ◽  
Kinase Inhibitor ◽  
Receptor Gene ◽  
Primary Cultures ◽  
Activin A ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Pheochromocytoma Cells

Activin A (a homodimer of two activin betaA subunits) has been shown to induce the neuronal differentiation of rat pheochromocytoma PC12 cells. We studied activin A and its receptor gene expression in human pheochromocytomas in vivo and in vitro to clarify the potential involvement of activin A in the pathophysiology of these tumors. We first screened 20 pheochromocytomas and nine normal adrenal tissues for activin betaA mRNA expression. Northern blots hybridized with specific oligonucleotide probes detected weak signals for activin betaA transcripts in pheochromocytomas. Both type I and type II activin receptor (ActR-I, ActR-IB and ActR-II) mRNA expression was also detectable in the pheochromocytoma tissues. In primary cultures of pheochromocytoma cells, expression of activin betaA mRNA was readily detectable by Northern blotting, and secretion of activin A into the conditioned medium was confirmed by an enzyme-linked immunosorbent assay. The expression of activin betaA mRNA and secretion of activin A were induced by (Bu)(2)cAMP after 1 and 3 days of treatment (all P<0.05). A protein kinase inhibitor, staurosporine, inhibited the basal and (Bu)(2)cAMP-induced accumulation of activin betaA mRNA (P<0.05). In addition, induction of chromaffin phenotype by dexamethasone also inhibited the basal and (Bu)(2)cAMP-induced expression of activin A at both mRNA and protein levels (all P<0.05). In contrast, the expression of ActR-I and ActR-IB mRNAs was not affected by these agents in cultured pheochromocytoma cells. In summary, activin betaA subunit and activin receptors are expressed in human pheochromocytomas. Production of activin A in cultured pheochromocytoma cells is induced through the protein kinase A pathway, but reduced during chromaffin differentiation. Therefore, activin A may function as a local neurotrophic factor via an auto/paracrine manner in human pheochromocytomas.

Glycated calmodulin from platelets as an index of glycemic control

1993 ◽  
Vol 39 (5) ◽  
pp. 815-819 ◽  
Author(s):  
A Muruganandam ◽  
G J Romsa ◽  
R J Thibert ◽  
R M Cheung ◽  
T F Draisey ◽  
...  
Keyword(s):  
Glycemic Control ◽  
Time Window ◽  
Human Subjects ◽  
Enzyme Linked Immunosorbent Assay ◽  
Poor Correlation ◽  
Type I ◽  
Clinical Role ◽  
Short Time Window ◽  
Short Time

Abstract In an effort to test whether a significant fraction of calmodulin would become glycated within the life span of the platelet (10-14 days), we monitored the kinetics of calmodulin glycation in vitro. Under the conditions we used, the fraction of glycated calmodulin reached a maximum (approximately 21%) within 10 days. We then extended the studies to human subjects. The intraplatelet concentrations of calmodulin and glycated calmodulin from age-matched type I diabetic subjects were monitored by a combination of m-aminophenylboronate affinity chromatography and enzyme-linked immunosorbent assay. The results indicate that the concentrations of total intraplatelet calmodulin (nonglycated plus glycated) were not dependent on the glycemic state of the subjects. Data from control and diabetic subjects showed a poor correlation between the concentrations of glycohemoglobin and of glycated calmodulin. However, a better correlation was obtained when glycated calmodulin concentrations were compared with those of serum fructosamine. The fraction of glycated calmodulin in the control population (7.71% +/- 0.75%) was significantly (P &lt; 0.05) different from that of the diabetic population (21.6% +/- 1.26%). Given that the clinical role of the fructosamine assay remains controversial, estimation of glycated calmodulin in platelets might be useful as a short time-window index of glycemic control.

scholarly journals In Vitro Activity and Mechanism of Action of Methylenecyclopropane Analogs of Nucleosides against Herpesvirus Replication

2005 ◽  
Vol 49 (3) ◽  
pp. 1039-1045 ◽  
Author(s):  
Earl R. Kern ◽  
Nicole L. Kushner ◽  
Caroll B. Hartline ◽  
Stephanie L. Williams-Aziz ◽  
Emma A. Harden ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Human Herpesvirus ◽  
Murine Cytomegalovirus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Herpesvirus Type ◽  
Varicella Zoster ◽  
Barr Virus ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT We have reported previously that methylenecyclopropane analogs of nucleosides have excellent activity against certain members of the herpesvirus family. A second generation, the 2,2-bis-hydroxymethyl derivatives, were synthesized, and 18 compounds were tested for activity in vitro against herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), human and murine cytomegalovirus (HCMV and MCMV), varicella-zoster virus (VZV), and Epstein-Barr virus (EBV). Selected analogs were also evaluated against human herpesvirus type 6 (HHV-6) and HHV-8. None of the 18 compounds had activity against HSV-1 or HSV-2, but four were active against VZV by plaque reduction (PR) assay at 50% effective concentration (EC50) levels of ≤50 μM. Six of the 18 compounds were active against HCMV by cytopathic effect or PR assays with EC50s of 0.5 to 44 μM, and all were active against MCMV by PR (0.3 to 54 μM). Four of the compounds were active against EBV by enzyme-linked immunosorbent assay (<0.3 to 4.4 μM). Four compounds with CMV activity were also active against HHV-6A and HHV-6B (0.7 to 28 μM), and three compounds were active against HHV-8 (5.5 to 16 μM). One of these, ZSM-I-62, had particularly good activity against CMV, HHV-6, and HHV-8, with EC50s of 0.7 to 8 μM. Toxicity was evaluated in adherent and nonadherent cells, and minimal cytotoxicity was observed. Mechanism of action studies with HCMV suggested that these compounds are phosphorylated by the ppUL97 phosphotransferase and are potent inhibitors of viral DNA synthesis. These results indicate that at least one of these compounds may have potential for use in treating CMV and other herpesvirus infections in humans.

scholarly journals An Mr 180000 protein is an endogenous substrate for the insulin-receptor-associated tyrosine kinase in human placenta

1987 ◽  
Vol 243 (3) ◽  
pp. 797-801 ◽  
Author(s):  
F Machicao ◽  
H Häring ◽  
M F White ◽  
J M Carrascosa ◽  
B Obermaier ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Human Placenta ◽  
Insulin Binding ◽  
Specific Protein ◽  
Beta Subunit ◽  
Phosphorylated Proteins ◽  
Endogenous Substrate ◽  
32P Incorporation ◽  
Insulin Receptor Kinase

The beta-subunit of the insulin receptor contains a tyrosine-specific protein kinase. Insulin binding activates this kinase and causes phosphorylation of the beta-subunit of the insulin receptor. It is believed that phosphorylation of other proteins might transmit the insulin signal from the receptor to the cell. In the present study we used a polyclonal anti-phosphotyrosine antibody to detect other proteins that become tyrosine phosphorylated upon insulin stimulation. Glycoproteins from human placenta membranes were enriched by wheat germ agglutinin chromatography and phosphorylation was studied with [gamma-32P]ATP and insulin in vitro. Phosphorylated proteins were immunoprecipitated by antibodies against the insulin receptor and by serum containing the anti-phosphotyrosine antibody. Beside the insulin-stimulated phosphorylation of the 95 kDa beta-subunit of the insulin receptor, an insulin-stimulated phosphorylation of a 180 kDa protein was found. The phosphorylation of both proteins occurred only on tyrosine residues. Insulin increased 32P incorporation into the 180 kDa band 2.7-fold (S.E.M. +/- 0.3, n = 5). The 180 kDa protein was not precipitated by antibodies against the insulin receptor. H.p.l.c. chromatograms of tryptic fragments of the phosphorylated 180 kDa protein and of the beta-subunit of the insulin receptor revealed different patterns for both proteins. Insulin-stimulated phosphorylation of the 180 kDa protein was also detectable in unfractionated detergent-solubilized membranes. The phosphorylation of the 180 kDa protein was stimulated by insulin with the same dose-response curve as the phosphorylation of the beta-subunit, suggesting that this protein might be another endogenous substrate of the insulin receptor kinase.

scholarly journals RNA-Containing Immune Complexes Formed by Anti-Melanoma Differentiation Associated Gene 5 Autoantibody Are Potent Inducers of IFN-α

2021 ◽  
Vol 12 ◽  
Author(s):  
Kaiwen Wang ◽  
Jiangfeng Zhao ◽  
Wanlong Wu ◽  
Wenwen Xu ◽  
Shuhui Sun ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Dependent Manner ◽  
Type I Ifn ◽  
Cytoplasmic Rna ◽  
Healthy Donors ◽  
Rna Immunoprecipitation ◽  
High Level

ObjectiveAnti-melanoma differentiation-associated gene 5 (MDA5) autoantibody is a distinctive serology hallmark of dermatomyositis (DM). As an autoantigen, MDA5 is a cytoplasmic RNA recognition receptor. The aim of this study was to address the question of whether the RNA-containing immune complex (IC) formed by MDA5 and anti-MDA5 could activate type I interferon (IFN) response.MethodPatients with anti-MDA5+ DM (n = 217), anti-MDA5− DM (n = 68), anti-synthase syndrome (ASyS, n = 57), systemic lupus erythematosus (SLE, n = 245), rheumatoid arthritis (RA, n = 89), and systemic sclerosis (SSc, n = 30) and healthy donors (HD, n = 94) were enrolled in our studies. Anti-MDA5 antibody was detected by line blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and Western blotting. Cytokine profiling was determined by multiplex flow cytometry, and IFN-α was further measured by ELISA. Type I IFN-inducible genes were detected by quantitative PCR (qPCR). RNA–IC binding was analyzed by RNA immunoprecipitation. Plasmacytoid dendritic cells (pDCs) derived from healthy donors were cultivated and stimulated with MDA5 ICs with or without RNase and Toll-like receptor 7 (TLR-7) agonist. The interaction between MDA5 ICs and TLR7 was evaluated by immunoprecipitation and confocal microscopy.ResultsAccording to our in-house ELISA, the presence of anti-MDA5 antibody in 76.1% of DM patients, along with 14.3% of SLE patients who had a lower titer yet positive anti-MDA5 antibody, was related to the high level of peripheral IFN-α. ICs formed by MDA5 and anti-MDA5 were potent inducers of IFN-α via TLR-7 in an RNA-dependent manner in vitro.ConclusionOur data provided evidence of the mechanistic relevance between the anti-MDA5 antibody and type I IFN pathway.

scholarly journals The effect of anti-inflammatory and antifibrotic agents on fibroblasts obtained from arthrofibrotic tissue

2018 ◽  
Vol 7 (3) ◽  
pp. 213-222 ◽  
Author(s):  
X. Tang ◽  
S. Teng ◽  
M. Petri ◽  
C. Krettek ◽  
C. Liu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Rabbit Model ◽  
Flexion Contracture ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Procollagen Type ◽  
Antifibrotic Agents ◽  
Anti Inflammatory

Objectives The aims of this study were to determine whether the administration of anti-inflammatory and antifibrotic agents affect the proliferation, viability, and expression of markers involved in the fibrotic development of the fibroblasts obtained from arthrofibrotic tissue in vitro, and to evaluate the effect of the agents on arthrofibrosis prevention in vivo. Methods Dexamethasone, diclofenac, and decorin, in different concentrations, were employed to treat fibroblasts from arthrofibrotic tissue (AFib). Cell proliferation was measured by DNA quantitation, and viability was analyzed by Live/Dead staining. The levels of procollagen type I N-terminal propeptide (PINP) and procollagen type III N-terminal propeptide (PIIINP) were evaluated with enzyme-linked immunosorbent assay (ELISA) kits. In addition, the expressions of fibrotic markers were detected by real-time polymerase chain reaction (PCR). Fibroblasts isolated from healthy tissue (Fib) served as control. Further, a rabbit model of joint contracture was used to evaluate the antifibrotic effect of the three different agents. Results Dexamethasone maintained the viability and promoted the proliferation of AFib. Diclofenac decreased the viability and inhibited the cell proliferation during the first week of cultivation. However, decorin inhibited AFib proliferation and downregulated the expressions of fibrotic markers. Additionally, decorin could improve the flexion contracture angle and inhibit the deposition of interstitial matrix components in the rabbit joint model. Conclusion Decorin decreased the expression of myofibroblast markers in AFib, inhibited the proliferation of AFib, and prevented the initial procedure of arthrofibrosis in vivo, suggesting that decorin could be a promising treatment to inhibit the development of arthrofibrosis. Cite this article: X. Tang, S. Teng, M. Petri, C. Krettek, C. Liu, M. Jagodzinski. The effect of anti-inflammatory and antifibrotic agents on fibroblasts obtained from arthrofibrotic tissue: An in vitro and in vivo study. Bone Joint Res 2018;7:213–222. DOI: 10.1302/2046-3758.73.BJR-2017-0219.R2.

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