scholarly journals The Branch Point Enzyme of the Mevalonate Pathway for Protein Prenylation Is Overexpressed in theob/ob Mouse and Induced by Adipogenesis

2000 ◽  
Vol 20 (6) ◽  
pp. 2158-2166 ◽  
Author(s):  
David Vicent ◽  
Eleftheria Maratos-Flier ◽  
C. Ronald Kahn
Keyword(s):  
Skeletal Muscle ◽  
Western Blot ◽  
Branch Point ◽  
Northern Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Northern Blot Analysis ◽  
Geranylgeranyl Diphosphate ◽  
Protein Prenylation ◽  
Ggpp Synthase

ABSTRACT We have recently reported that skeletal muscle of theob/ob mouse, an animal model of genetic obesity with extreme insulin resistance, exhibits alterations in the expression of multiple genes. Analysis and cloning of a full-length cDNA of one of the overexpressed mRNAs revealed a 300-amino-acid protein that could be identified as the mouse geranylgeranyl diphosphate synthase (GGPP synthase) based on its homology to proteins cloned from yeast and fungus. GGPP synthase catalyzes the synthesis of all-trans-geranylgeranyl diphosphate (GGPP), an isoprenoid used for protein isoprenylation in animal cells, and is a branch point enzyme in the mevalonic acid pathway. Three mRNAs for GGPP synthase of 4.3, 3.2, and 1.7 kb were detected in Northern blot analysis. Western blot analysis of tissue homogenates using specific antipeptide antibodies revealed a single band of 34.8 kDa. Expression level of this protein in different tissues correlated with expression of the 4.3- and 3.2-kb mRNAs. GGPP synthase mRNA expression was increased 5- to 20-fold in skeletal muscle, liver, and fat of ob/obmice by Northern blot analysis. Western blot analysis also showed a twofold overexpression of the protein in muscle and fat but not in liver, where the dominant isoform is encoded by the 1.7-kb mRNA. Differentiation of 3T3-L1 fibroblasts into adipocytes induced GGPP synthase expression more than 20-fold. Using the immunoprecipitated protein, we found that mammalian GGPP synthase synthesizes not only GGPP but also its metabolic precursor farnesyl diphosphate. Thus, the expression of GGPP synthase is regulated in multiple tissues in obesity and is induced during adipocyte differentiation. Altered regulation in the synthesis of isoprenoids for protein prenylation in obesity might be a factor determining the ability of the cells to respond to hormonal stimulation requiring both Ras-related small GTPases and trimeric G protein-coupled receptors.

scholarly journals A Spore Coat Protein, CotS, of Bacillus subtilis Is Synthesized under the Regulation of ςKand GerE during Development and Is Located in the Inner Coat Layer of Spores

1998 ◽  
Vol 180 (11) ◽  
pp. 2968-2974 ◽  
Author(s):  
Hiromu Takamatsu ◽  
Yukari Chikahiro ◽  
Takeko Kodama ◽  
Hidekatsu Koide ◽  
Satoshi Kozuka ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Western Blot ◽  
Northern Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Northern Blot Analysis ◽  
Spore Coat ◽  
Consensus Sequences ◽  
Coat Layer ◽  
Spore Coat Protein

ABSTRACT The spore coat of Bacillus subtilis has a unique morphology and consists of polypeptides of different sizes, whose synthesis and assembly are precisely regulated by a cascade of transcription factors and regulatory proteins. We examined the factors that regulate cotS gene expression and CotS assembly into the coat layer of B. subtilis by Northern blot and Western blot analysis. Transcription of cotS mRNA was not detected in sporulating cells of ςK and gerE mutants by Northern blot analysis. By Western blot analysis using anti-CotS antibody, CotS was first detected in protein samples solubilized from wild-type cells at 5 h after the start of sporulation. CotS was not detected in the vegetative cells and spores of a gerEmutant or in the spores of mutants deficient in ςE, ςF, ςG, or ςK. CotS was detected in the sporangium but not in the spores of a cotEmutant. The sequence of the promoter region of cotS was similar to the consensus sequences for binding of ςK and GerE. These results demonstrate that ςK and GerE are required for cotS expression and that CotE is essential for the assembly of CotS in the coat. Immunoelectron microscopic observation using anti-CotS antibody revealed that CotS is located within the spore coat, in particular in the inner coats of dormant spores.

Dystrophin in skeletal muscle I. Western blot analysis using a monoclonal antibody

1989 ◽  
Vol 94 (1-3) ◽  
pp. 125-136 ◽  
Author(s):  
Louise V.B. Nicholson ◽  
Keith Davison ◽  
Gavin Falkous ◽  
Colin Harwood ◽  
Elizabeth O'Donnell ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Monoclonal Antibody ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis

Abstract 255: Cytoprotective Effects of Erythropoietin and Erythropoietin-derivatives in Ischaemic Human Myotubes and the Role of Pi3k/akt Signalling Pathway

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Rebekah Sian Hwee Yu ◽  
Daryll Baker ◽  
David Abraham ◽  
Janice Tsui
Keyword(s):  
Skeletal Muscle ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Signalling Pathway ◽  
Critical Limb Ischaemia ◽  
Human Myotubes ◽  
Muscle Biopsies

Objectives Erythropoietin (Epo) has tissue-protective effects in response to injury, acting through the EpoR-βcR heteroreceptor. We have previously demonstrated the presence and interaction of the EpoR and βcR in human skeletal muscle. Here we aim to investigate the potential cytoprotective effects of Epo and an Epo-derivative (ARA-290) in a human in vitro model of skeletal muscle and establish a potential downstream signalling pathway utilised in protecting cells from apoptosis (including Jak-2, PI3k/Akt, NFkB). Methods Gastrocnemius muscle biopsies were obtained from patients with critical limb ischaemia and control samples were obtained from non-ischaemic patients. Human myoblasts were isolated from muscle biopsies, cultured, and allowed to differentiate into myotubes in order to investigate the cytoprotective effects of Epo and ARA-290 on myotubes subjected to simulated ischaemia. The PI3k inhibitors, LY294002 and wortmannin, were then used to determine the role of PI3k/Akt pathway in mediating cytoprotection. Following this, inhibitors against the upstreatm (Jak-2) and downstream (NFkB) molecules were also investigated. Western blot analysis, using the pro-apoptotic marker cleaved caspase-3 was performed and compared with levels of Akt and phosphorylated-Akt, using western blot analysis. Results Exogenous administration of Epo and ARA-290 were able to ameliorate the ischaemia-induced apoptosis on isolated human myotubes as shown by a significant reduction in cleaved caspase-3 expression. Addition of all inhibitors, to ARA-290 or Epo pre-treated cells, abolished the reduction in apoptosis. Conclusion The ability of ARA-290 to attenuate apoptosis in human myotubes undergoing ischaemic insult suggests a potential role in tissue protection in skeletal muscle injury. We propose that the PI3k/Akt signalling pathway is involved in mediating this cytoprotection.

Gender and transcriptional regulation of NO synthase and ET-1 in porcine aortic endothelial cells

1997 ◽  
Vol 273 (4) ◽  
pp. H1962-H1967 ◽  
Author(s):  
Xiaofang Wang ◽  
Dustan A. Barber ◽  
Debra A. Lewis ◽  
Christopher G. A. McGregor ◽  
Gary C. Sieck ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Western Blot ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Ovarian Hormones ◽  
Intact Male ◽  
Male And Female ◽  
Aortic Endothelial Cells

Experiments were designed to determine whether normal fluctuations in sex steroid hormones alter gene transcription for endothelial nitric oxide synthase (NOS) and preproendothelin-1 (prepro-ET-1). Aortic endothelial cells were removed from adult, gonadally intact male and female or ovariectomized Yorkshire pigs. Endothelial cells were prepared for Northern blot analysis, Western blot analysis or enzyme activity. Nitric oxide products (NOx) and endothelin-1 (ET-1) in plasma were measured by chemiluminescence and radioimmunoassay, respectively. Northern blot analysis identified single bands corresponding to endothelial NOS and prepro-ET-1. Quantification of the blots showed an increase in expression of mRNA for both endothelial NOS and prepro-ET-1 in ovariectomized pigs compared with gonadally intact male and female pigs. There were no differences in amount of endothelial NOS protein identified by Western blot analysis among groups. On the contrary, plasma concentrations of NOx were significantly decreased in ovariectomized pigs, and there were no differences either in the concentrations of ET-1 in the plasma or extracts from the coronary arteries. These results suggest that expression of endothelial NOS and prepro-ET-1 may be regulated at transcriptional level by ovarian hormones. In addition, the ovarian hormones may regulate production of these endothelium-derived factors at the posttranscriptional level.

AnkyrinG is associated with the postsynaptic membrane and the sarcoplasmic reticulum in the skeletal muscle fiber

1998 ◽  
Vol 111 (15) ◽  
pp. 2197-2207 ◽  
Author(s):  
E. Kordeli ◽  
M.A. Ludosky ◽  
C. Deprette ◽  
T. Frappier ◽  
J. Cartaud
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Postsynaptic Membrane ◽  
Skeletal Muscle Fiber ◽  
Membrane Domains ◽  
Gene Products ◽  
Rat Skeletal Muscle

Ankyrins are a multi-gene family of peripheral proteins that link ion channels and cell adhesion molecules to the spectrin-based skeleton in specialized membrane domains. In the mammalian skeletal myofiber, ankyrins were immunolocalized in several membrane domains, namely the costameres, the postsynaptic membrane and the triads. Ank1 and Ank3 transcripts were previously detected in skeletal muscle by northern blot analysis. However, the ankyrin isoforms associated with these domains were not identified, with the exception of an unconventional Ank1 gene product that was recently localized at discrete sites of the sarcoplasmic reticulum. Here we study the expression and subcellular distribution of the Ank3 gene products, the ankyrinsG, in the rat skeletal muscle fiber. Northern blot analysis of rat skeletal muscle mRNAs using domain-specific Ank3 cDNA probes revealed two transcripts of 8.0 kb and 5.6 kb containing the spectrin-binding and C-terminal, but not the serine-rich, domains. Reverse transcriptase PCR analysis of rat skeletal muscle total RNA confirmed the presence of Ank3 transcripts that lacked the serine-rich and tail domains, a major insert of 7813 bp at the junction of the spectrin-binding and C-terminal domains that was previously identified in brain Ank3 transcripts. Immunoblot analysis of total skeletal muscle homogenates using ankyrinG-specific antibodies revealed one major 100 kDa ankyrinG polypeptide. Immunofluorescence labeling of rat diaphragm cryosections showed that ankyrin(s)G are selectively associated with (1) the depths of the postsynaptic membrane folds, where the voltage-dependent sodium channel and N-CAM accumulate, and (2) the sarcoplasmic reticulum, as confirmed by codistribution with the sarcoplasmic reticulum Ca2+-ATPase (SERCA 1). At variance with ankyrin(s)G, ankyrin(s)R (ank1 gene products) accumulate at the sarcolemma and at sarcoplasmic structures, in register with A-bands. Both ankyrin isoforms codistributed over Z-lines and at the postsynaptic membrane. These data extend the notion that ankyrins are differentially localized within myofibers, and point to a role of the ankyrinG family in the organization of the sarcoplasmic reticulum and the postsynaptic membrane.

scholarly journals Clinical and Experimental Results on Cardiac Troponin Expression in Duchenne Muscular Dystrophy

2001 ◽  
Vol 47 (3) ◽  
pp. 451-458 ◽  
Author(s):  
Angelika Hammerer-Lercher ◽  
Petra Erlacher ◽  
Reginald Bittner ◽  
Rudolf Korinthenberg ◽  
Daniela Skladal ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Western Blot ◽  
Blot Analysis ◽  
Cardiac Troponin ◽  
Western Blot Analysis ◽  
Clinical Evidence ◽  
Mdx Mouse ◽  
Muscle Biopsies

Abstract Background: Because of controversial earlier studies, the purpose of this study was to provide novel experimental and additional clinical data regarding the possible reexpression of cardiac troponin T (cTnT) in regenerating skeletal muscle in Duchenne muscular dystrophy (DMD). Methods: Plasma from 14 patients (mean age, 7.5 years; range, 5.7–19.4 years) with DMD was investigated for creatine kinase (CK), the CK MB isoenzyme (CKMB), cTnT and cardiac troponin I (cTnI), and myoglobin. cTnT concentrations were measured by an ELISA (second-generation assay; Roche) using the ES 300 Analyzer. cTnI, myoglobin, and CKMB were measured by an ELISA using the ACCESS System (Beckman Diagnostics). Troponin isoform expression was studied by Western blot analysis in remnants of skeletal muscle biopsies of three patients with DMD and in an animal model of DMD (mdx mice; n = 6). Results: There was no relation of cTnT and cTnI to clinical evidence for cardiac failure. cTnI concentrations remained below the upper reference limit in all patients. cTnT was increased (median, 0.11 μg/L; range, 0.06–0.16 μg/L) in 50% of patients. The only significant correlation was found for CK (median, 3938 U/L; range, 2763–5030 U/L) with age (median, 7.5 years; range, 6.8–10.9 years; r = −0.762; P = 0.042). Western blot analysis of human or mouse homogenized muscle specimens showed no evidence for cardiac TnT and cTnI expression, despite strong signals for skeletal muscle troponin isoforms. Conclusions: We found no evidence for cTnT reexpression in human early-stage DMD and in mdx mouse skeletal muscle biopsies. Discrepancies of cTnT and cTnI in plasma samples of DMD patients were found, but neither cTnT nor cTnI plasma concentrations were related with other clinical evidence for cardiac involvement.

scholarly journals In Vivo Evaluation of Novel Geranylgeranyl Diphosphate Synthase Inhibitors

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 215-215 ◽  
Author(s):  
Staci L. Haney ◽  
Yashpal S. Chhonker ◽  
Michelle L. Varney ◽  
Geoffrey A. Talmon ◽  
Daryl J. Murry ◽  
...  
Keyword(s):  
Single Dose ◽  
Western Blot ◽  
Blot Analysis ◽  
Half Life ◽  
Western Blot Analysis ◽  
Weekly Schedule ◽  
Geranylgeranyl Diphosphate Synthase ◽  
Geranylgeranyl Diphosphate

Abstract The enzyme geranylgeranyl diphosphate synthase (GGDPS) synthesizes the 20-carbon isoprenoid geranylgeranyl diphosphate which is used in protein geranylgeranylation reactions. Our work has demonstrated that GGDPS inhibitors (GGDPSIs) represent a novel therapeutic strategy for multiple myeloma (MM) by disrupting Rab protein geranylgeranylation. Treatment of MM cells with GGDPSI results in disruption of monoclonal protein trafficking, leading to induction of the unfolded protein response pathway (UPR) and apoptosis. We have previously reported preclinical studies with a lead GGDPSI, VSW1198 (a mixture of homogeranyl/homoneryl triazole bisphosphonates), demonstrating the agent's metabolic stability, prolonged half-life (plasma elimination half-life of 47.7 (±7.4) hrs), systemic distribution and confirmed in vivo disruption of geranylgeranylation (Haney et al., Invest New Drugs, 2018). Additional structure-function efforts have led to the development of the α-methylated derivatives RAM2093 (homogeranyl) and RAM2061 (homoneryl). Intriguingly the addition of the α-methyl group abrogates the effects of the olefin stereochemistry on inhibitor potency such that the individual isomers display near identical ability to disrupt protein geranylgeranylation in enzyme and cell assays (Matthiesen et al., Bioorg Med Chem, 2018). As little is known regarding the impact of olefin stereochemistry on the pharmacokinetic (PK)/pharmacodynamic (PD) properties of drugs, we pursued additional in vitro and in vivo studies of RAM2093 and RAM2061 and investigated the efficacy of the GGDPSIs in a mouse MM xenograft model. In MM cell lines, qRT-PCR and western blot analysis showed that both isomers induce activation of UPR/apoptotic markers in a dose-dependent manner and with similar potency. Single dose testing in CD-1 mice identified a maximum tolerated dose of 0.5 mg/kg IV for RAM2061 and 0.3 mg/kg for RAM2093. Liver toxicity was the primary barrier to dose escalation with both compounds. Analysis of blood samples showed elevated liver transaminase levels with normal bilirubin/alkaline phosphatase levels and histopathological examination confirmed evidence of hepatocyte injury at higher doses. Multi-dose schedules of 0.1 mg/kg twice a week (two weeks on, one week off, two weeks on) for RAM2061 and RAM2093, as well as 0.2 mg/kg (RAM2061) and 0.15 mg/kg (RAM2093) weekly x four weeks were tested. All dosing schedules were tolerated with the exception of the twice-weekly schedule for RAM2093. Consistent with the findings from our single dose testing, both multi-dose schedules induced transient elevation of hepatic transaminases. No loss of weight was observed and creatinine and CBC results were within normal limits for both single and multi-dose injected animals. Importantly, western blot analysis of mouse tissues collected from both RAM2061 and RAM2093 multi-dose-treated mice showed accumulation of unmodified Rap1a in the liver, kidney and spleen, indicating in vivo disruption of protein geranylgeranylation was achieved. Furthermore, using MM.1S MM cell flank xenographs in NOD-SCID mice, we observed a significant reduction in tumor growth in mice treated with either VSW1198 or RAM2061 relative to vehicle control. Lastly, PK/biodistribution studies with RAM2093 and RAM2061 were performed following a single dose of 0.3 mg/kg IV. Both compounds were detectable in plasma and liver samples for up to seven days post-injection demonstrating prolonged half-life and tissue distribution. The full PK parameters for both compounds will be presented and studies measuring drug levels in other tissues are ongoing. Taken together, these data suggest that RAM2061 and RAM2093 have equivalent anti-MM activity in vitro, but that RAM2061 is better tolerated in vivo. Whether this is a consequence of different PK properties vs. differences in tissue uptake or metabolism will be determined. These studies also confirm the in vivo efficacy of our novel GGDPSIs and support further development of these agents for the treatment of MM. Figure. Figure. Disclosures Holstein: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Prolactin and growth hormone mRNA and protein characterization in SMtTW rat pituitary tumours

1995 ◽  
Vol 15 (3) ◽  
pp. 233-243 ◽  
Author(s):  
M Delhase ◽  
F Rajas ◽  
P Verdood ◽  
C Remy ◽  
P Chevallier ◽  
...  
Keyword(s):  
Western Blot ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Biological Significance ◽  
Protein Characterization ◽  
Pituitary Tumours ◽  
Rat Pituitary ◽  
Normal Rat

ABSTRACT We have combined different techniques to analyse passages of five different rat spontaneous pituitary tumours (SMtTW) that were transplanted under the kidney capsule. These tumours were secreting prolactin (PRL), GH or both hormones. RIA, immunocytochemistry (ICC) and Western blot analysis were applied to characterize the hormone(s) stored (ICC and Western blot) and secreted (RIA). mRNA content was analysed by PCR, Northern blot analysis and in situ hybridization. The data point not only to the reliability of the techniques used at both protein and RNA levels for each tumour studied but also to the complementarity of some techniques. For example, whereas Northern blot analysis demonstrates the presence and size of hormone mRNA, in situ hybridization indicates the percentage of cells expressing a given hormone mRNA and allows the presence of one population (or more) of cells in a given tumour to be identified. Moreover, the tumours were compared with normal rat pituitary. Although the PRL and GH mRNAs were identical in size, the amount of mRNA was lower in the tumours. At the protein level, the PRL and GH variants exhibited a different pattern of expression in tumours compared with the normal rat pituitary. The biological significance of these differences is discussed.

Localization of the ANG II type 2 receptor in the microcirculation of skeletal muscle

1998 ◽  
Vol 275 (4) ◽  
pp. H1395-H1403 ◽  
Author(s):  
Elizabeth H. Nora ◽  
Diane H. Munzenmaier ◽  
Feona M. Hansen-Smith ◽  
Julian H. Lombard ◽  
Andrew S. Greene
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Immunohistochemical Analysis ◽  
Wide Distribution ◽  
Ang Ii ◽  
Skeletal Muscle Microcirculation

Only functional studies have suggested the presence of the ANG II type 2 (AT2) receptor in the microcirculation. To determine the distribution of this receptor in the rat skeletal muscle microcirculation, a polyclonal rabbit anti-rat antiserum was developed and used for immunohistochemistry and Western blot analysis. The antiserum was prepared against a highly specific and antigenic AT2-receptor synthetic peptide and was validated by competition and sensitivity assays. Western blot analysis demonstrated a prominent, single band at ∼40 kDa in cremaster and soleus muscle. Immunohistochemical analysis revealed a wide distribution of AT2 receptors throughout the skeletal muscle microcirculation in large and small microvessels. Microanatomic studies displayed an endothelial localization of the AT2 receptor, whereas dual labeling with smooth muscle α-actin also showed colocalization of the AT2 receptor with vascular smooth muscle cells. Other cells associated with the microvessels also stained positive for AT2 receptors. Briefly, this study confirms previous functional data and localizes the AT2 receptor to the microcirculation. These studies demonstrate that the AT2 receptor is present on a variety of vascular cell types and that it is situated in a fashion that would allow it to directly oppose ANG II type 1 receptor actions.

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