65 results on '"Christian Witt"'
Search Results
2. A dramatic effect of oxygen on protection of human cells against γ-radiation by lycopene
- Author
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Christian Witt, Fritz Boehm, Ruth Edge, and Terence George Truscott
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0301 basic medicine ,Cell ,Biophysics ,chemistry.chemical_element ,Ascorbic Acid ,Biology ,Bioinformatics ,Biochemistry ,Oxygen ,03 medical and health sciences ,chemistry.chemical_compound ,Lycopene ,0302 clinical medicine ,Superoxides ,Structural Biology ,Genetics ,medicine ,Radiation damage ,Humans ,Vitamin E ,Vitamin A ,Molecular Biology ,Carotenoid ,chemistry.chemical_classification ,γ radiation ,Cell Death ,Hydroxyl Radical ,Spectrum Analysis ,Dose-Response Relationship, Radiation ,Cell Biology ,Carotenoids ,030104 developmental biology ,Membrane ,medicine.anatomical_structure ,chemistry ,Cytoprotection ,Gamma Rays ,030220 oncology & carcinogenesis ,Limiting oxygen concentration - Abstract
Reducing radiation damage is important and dietary antioxidants that can protect cells from such damage are of value. Dietary lycopene, a carotenoid found in tomatoes, protects human lymphoid cell membranes from damage by γ-radiation. We report that such protective effects are remarkably reduced as the oxygen concentration increases - near zero at 100% oxygen from fivefold protection at 20% oxygen and, dramatically, from 50-fold protection at 0% oxygen. Such huge differences imply that under higher oxygen concentrations lycopene could lead to improved cancer therapy using γ-radiation. The cells are not efficiently protected from the superoxide radical by lycopene. Noncellular studies suggest molecular mechanisms for the oxygen effect.
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- 2016
3. Erlotinib and bevacizumab versus cisplatin, gemcitabine and bevacizumab in unselected nonsquamous nonsmall cell lung cancer
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Christian Witt, Thomas Acker, Jens Kollmeier, Ernst Müller, Michael Schenk, Martin Wolf, Michael Thomas, Philipp A. Schnabel, Roland Penzel, Jürgen Fischer, Alexander Reuss, Michael Schröder, Cornelius Kortsik, Stefan Andreas, Michael von Eiff, Monika Serke, Matthias Villalobos, Niels Reinmuth, and Christian Grah
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Adult ,Male ,Pulmonary and Respiratory Medicine ,Oncology ,medicine.medical_specialty ,Lung Neoplasms ,Bevacizumab ,DNA Mutational Analysis ,Deoxycytidine ,Disease-Free Survival ,Erlotinib Hydrochloride ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Carcinoma, Non-Small-Cell Lung ,Internal medicine ,Antineoplastic Combined Chemotherapy Protocols ,medicine ,Humans ,Epidermal growth factor receptor ,Aged ,Proportional Hazards Models ,030304 developmental biology ,Aged, 80 and over ,Cisplatin ,0303 health sciences ,biology ,business.industry ,Combination chemotherapy ,Middle Aged ,Gemcitabine ,3. Good health ,ErbB Receptors ,Treatment Outcome ,chemistry ,030220 oncology & carcinogenesis ,Mutation ,Disease Progression ,biology.protein ,Female ,Erlotinib ,business ,medicine.drug - Abstract
Erlotinib with bevacizumab showed promising activity in recurrent nonsquamous (NS) nonsmall cell lung cancer (NSCLC). The INNOVATIONS study was designed to assess in first-line treatment of unselected cisplatin-eligible patients this combination compared to cisplatin, gemcitabine and bevacizumab.Stage IIIB/IV patients with NS-NSCLC were randomised on erlotinib (150 mg daily) and bevacizumab (15 mg·kg−1 on day 1, every 3 weeks) (EB) until progression, or cisplatin (80 mg·m−2 on day 1, every 3 weeks) and gemcitabine (1250 mg·m−2 on days 1 and 8, every 3 weeks) up to six cycles and bevacizumab (15 mg·kg−1 on day 1, every 3 weeks) (PGB) until progression.224 patients were randomised (EB n=111, PGB n=113). The response rate (12% versus 36%; pversus 6.9 months; hazard ratio (HR) 1.85, 95% CI 1.39–2.45; pversus 17.8 months; HR 1.41, 95% CI 1.01–1.97; p=0.04) clearly favoured PGB. In patients with epidermal growth factor receptor mutations (n=32), response rate, progression-free survival and overall survival were not superior with EB.Platinum-based combination chemotherapy remains the standard of care in first-line treatment of unselected NS-NSCLC. Molecular targeted approaches strongly mandate appropriate testing and patient selection.
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- 2015
4. Induction of Ankrd1 in Dilated Cardiomyopathy Correlates with the Heart Failure Progression
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Jens Fielitz, Giedrė Balčiūnaitė, Virginija Grabauskienė, Dainius Daunoravičus, Julius Bogomolovas, Siegfried Labeit, Daiva Bironaitė, Kathrin Brohm, Virginija Bukelskienė, Jelena Čelutkienė, and Christian Witt
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Adult ,Cardiomyopathy, Dilated ,Male ,ANKRD2 ,medicine.medical_specialty ,ANKRD1 ,Article Subject ,Heart disease ,Gene Expression ,Muscle Proteins ,lcsh:Medicine ,Biology ,Ligands ,General Biochemistry, Genetics and Molecular Biology ,Contractility ,Internal medicine ,Idiopathic dilated cardiomyopathy ,medicine ,Humans ,Connectin ,Ventricular remodeling ,Heart Failure ,General Immunology and Microbiology ,Myocardium ,lcsh:R ,Nuclear Proteins ,Dilated cardiomyopathy ,Atrial Remodeling ,General Medicine ,Middle Aged ,medicine.disease ,Repressor Proteins ,Cardiovascular and Metabolic Diseases ,Heart failure ,Disease Progression ,Cardiology ,Female ,Adiponectin ,Research Article - Abstract
Progression of idiopathic dilated cardiomyopathy (IDCM) is marked with extensive left ventricular remodeling whose clinical manifestations and molecular basis are poorly understood. We aimed to evaluate the clinical potential of titin ligands in monitoring progression of cardiac remodeling associated with end-stage IDCM. Expression patterns of 8 mechanoptotic machinery-associated titin ligands (ANKRD1,ANKRD2,TRIM63,TRIM55,NBR1,MLP,FHL2, andTCAP) were quantitated in endomyocardial biopsies from 25 patients with advanced IDCM. When comparing NYHA disease stages, elevatedANKRD1expression levels marked transition from NYHA < IV to NYHA IV.ANKRD1expression levels closely correlated with systolic strain depression and short E wave deceleration time, as determined by echocardiography. On molecular level, myocardialANKRD1and serum adiponectin correlated with lowBAX/BCL-2ratios, indicative of antiapoptotic tissue propensity observed during the worsening of heart failure. ANKRD1 is a potential marker for cardiac remodeling and disease progression in IDCM.ANKRD1expression correlated with reduced cardiac contractility and compliance. The association ofANKRD1with antiapoptotic response suggests its role as myocyte survival factor during late stage heart disease, warranting further studies on ANKRD1 during end-stage heart failure.
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- 2015
5. Clinical Impact of Rare and Compound Mutations of Epidermal Growth Factor Receptor in Patients With Non–Small-Cell Lung Cancer
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Robert Kromminga, Michael Hummel, Christian Witt, Juliane Martin, Hans-Wilhelm Pau, Frederick Klauschen, Stefan Moegling, Korinna Jöhrens, Joachim Gottschalk, Antje Tessmer, Dido Lenze, Annika Lehmann, Berndt Schmidt, and Christian Grohé
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Adult ,Male ,0301 basic medicine ,Pulmonary and Respiratory Medicine ,Cancer Research ,Lung Neoplasms ,Disease ,medicine.disease_cause ,03 medical and health sciences ,symbols.namesake ,0302 clinical medicine ,Carcinoma, Non-Small-Cell Lung ,medicine ,Humans ,Clinical significance ,Epidermal growth factor receptor ,Lung cancer ,Protein Kinase Inhibitors ,Gene ,Aged ,Aged, 80 and over ,Sanger sequencing ,Mutation ,biology ,business.industry ,High-Throughput Nucleotide Sequencing ,Middle Aged ,medicine.disease ,respiratory tract diseases ,ErbB Receptors ,Treatment Outcome ,030104 developmental biology ,Oncology ,Drug Resistance, Neoplasm ,030220 oncology & carcinogenesis ,Cancer research ,biology.protein ,symbols ,Female ,business ,Tyrosine kinase - Abstract
Background Standard therapy of advanced non–small-cell lung cancer harboring an activating mutation in the epidermal growth factor receptor (EGFR) gene is treatment with tyrosine kinase inhibitors (TKI). However, for rare and compound mutations of the EGFR gene, the clinical evidence of TKI therapy is still unclear. Patients and Methods A total of 2906 lung cancer samples were analyzed for EGFR mutations during routine analysis between 2010 and 2017. The samples have been investigated by Sanger sequencing and since 2014 by next-generation sequencing. Results We detected EGFR mutations in 408 specimens (14%). Among these, we found 41 samples with rare and 22 with compound mutations. In these 63 samples, 56 different rare EGFR mutations occurred. Information about the clinical outcome was available for 37. Among those with rare mutations, only one patient harboring the mutation p.G874D had disease that responded to first-generation TKI therapy. In contrast, the disease of all patients with compound mutations responded to first- or second-generation TKI therapy. Furthermore, we collected data on clinical relevance regarding TKI therapy from different databases and from an additional literature search, and only found data for 36 of the 56 detected rare mutations. Conclusion Information about the clinical outcome of patients with rare and compound EGFR mutations remains limited. At present, second- and third-generation TKIs are available, which may represent new treatment strategies for these patients. Therefore, it is becoming increasingly important to maintain databases concerning rare EGFR mutations.
- Published
- 2019
6. MuRF1 is a muscle fiber-type II associated factor and together with MuRF2 regulates type-II fiber trophicity and maintenance
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Henk Granzier, Christian Witt, Anselmo Sigari Moriscot, Siegfried Labeit, Stephanie Hirner, Igor L. Baptista, and Julius Bogomolovas
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Ubiquitin-Protein Ligases ,Muscle Proteins ,Biology ,Article ,Tripartite Motif Proteins ,Mice ,NFAT Pathway ,Tibialis anterior muscle ,Structural Biology ,Two-Hybrid System Techniques ,Myosin ,medicine ,Animals ,Mice, Knockout ,Denervation ,Soleus muscle ,Myosin Heavy Chains ,Ubiquitin ,Microfilament Proteins ,NFAT ,Muscle atrophy ,Cell biology ,Mice, Inbred C57BL ,Calcineurin ,Muscular Atrophy ,Biochemistry ,Muscle Fibers, Fast-Twitch ,medicine.symptom ,Carrier Proteins - Abstract
MuRF1 is a member of the RBCC (RING, B-box, coiled-coil) superfamily that has been proposed to act as an atrogin during muscle wasting. Here, we show that MuRF1 is preferentially induced in type-II muscle fibers after denervation. Fourteen days after denervation, MuRF1 protein was further elevated but remained preferentially expressed in type-II muscle fibers. Consistent with a fiber-type dependent function of MuRF1, the tibialis anterior muscle (rich in type-II muscle fibers) was considerably more protected in MuRF1-KO mice from muscle wasting when compared to soleus muscle with mixed fiber-types. We also determined fiber-type distributions in MuRF1/MuRF2 double-deficient KO (dKO) mice, because MuRF2 is a close homolog of MuRF1. MuRF1/MuRF2 dKO mice showed a profound loss of type-II fibers in soleus muscle. As a potential mechanism we identified the interaction of MuRF1/MuRF2 with myozenin-1, a calcineurin/NFAT regulator and a factor required for maintenance of type-II muscle fibers. MuRF1/MuRF2 dKO mice had lost myozenin-1 expression in tibialis anterior muscle, implicating MuRF1/MuRF2 as regulators of the calcineurin/NFAT pathway. In summary, our data suggest that expression of MuRF1 is required for remodeling of type-II fibers under pathophysiological stress states, whereas MuRF1 and MuRF2 together are required for maintenance of type-II fibers, possibly via the regulation of myozenin-1.
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- 2010
7. Site Specific Nutrient Management for Maize on Ultisols Lampung
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Andarias Makka Murni, Julie Mae Pasuquin, and Christian Witt
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Nutrient management ,Sowing ,Ultisol ,Biology ,engineering.material ,fertilizer ,Crop ,Nutrient ,Agronomy ,nutrient management ,Dry season ,yield response ,engineering ,Paddy field ,Fertilizer ,site specific ,lcsh:Science (General) ,Agronomic efficiency ,lcsh:Q1-390 - Abstract
Site Specific Nutrient Management for Maize on Ultisols Lampung (A M Murni, JM Pasuquin, and C Witt): Lampung is the third major maize producing province in Indonesia after East Java and Central Java. In Lampung maize is cultivated mainly in upland areas with ultisols and only some cultivated on paddy field as a secondary crop in the dry season. The average maize yield in Lampung is still 3.4 Mg ha-1 bellow yield potential of 7-10 Mg ha-1. To increase the productivity of maize through site-specific nutrient management (SSNM), on-farm trials were conducted in five locations in Lampung i.e. four locations in Central Lampung District (Sidowaras, Binjai Ngagung, Watu Agung and Balai Rejo) and one location in South Lampung District (Trimulyo, Tegineneng Sub District) during the 2004/2005, 2005/2006 and 2006/2007 rainy seasons. The experimental setup followed a standard protocol at all sites and included nutrient omission plots (PK, NK, NP) to estimate indigenous nutrient supplies, an NPK plot to measure yield response to fertilizer application, and a farmers’ fertilizer practice (FFP) plot in each farmer’s field. An SSNM treatment plot was included in the second and third seasons. Each of the above treatments was paralleled by a plot with improved crop management practice (ICM), i.e. higher planting density, addition of lime, and addition of magnesium. Results showed that yield response to fertilizer N, P and K application in these sites were: N = 2.3-4.1 Mg ha-1; P = 0.6-2.0 Mg ha-1; K = 0.3-2.4 Mg ha-1. Attainable yield in the three seasons on average ranged from 7.6 Mg ha-1 to 10.6 Mg ha-1. Yield in the SSNM treatment (with or without ICM) was significantly higher than the FFP indicating great opportunities for farmers to increase productivity and profitability with improved nutrient and crop management.
- Published
- 2018
8. Nebulin Alters Cross-bridge Cycling Kinetics and Increases Thin Filament Activation
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Ranganath Mamidi, Coen A.C. Ottenheijm, Steven J. Ford, Carlos Hidalgo, Murali Chandra, Siegfried Labeit, Christian Witt, and Henk Granzier
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Myosin light-chain kinase ,biology ,Chemistry ,Skeletal muscle ,Cell Biology ,Biochemistry ,Troponin ,Sarcomere ,Nebulin ,medicine.anatomical_structure ,Myosin ,biology.protein ,medicine ,Biophysics ,medicine.symptom ,Molecular Biology ,Actin ,Muscle contraction - Abstract
Nebulin is a giant filamentous F-actin-binding protein (∼800 kDa) that binds along the thin filament of the skeletal muscle sarcomere. Nebulin is one of the least well understood major muscle proteins. Although nebulin is usually viewed as a structural protein, here we investigated whether nebulin plays a role in muscle contraction by using skinned muscle fiber bundles from a nebulin knock-out (NEB KO) mouse model. We measured force-pCa (−log[Ca2+]) and force-ATPase relations, as well as the rate of tension re-development (ktr) in tibialis cranialis muscle fibers. To rule out any alterations in troponin (Tn) isoform expression and/or status of Tn phosphorylation, we studied fiber bundles that had been reconstituted with bacterially expressed fast skeletal muscle recombinant Tn. We also performed a detailed analysis of myosin heavy chain, myosin light chain, and myosin light chain 2 phosphorylation, which showed no significant differences between wild type and NEB KO. Our mechanical studies revealed that NEB KO fibers had increased tension cost (5.9 versus 4.4 pmol millinewtons−1 mm−1 s−1) and reductions in ktr (4.7 versus 7.3 s−1), calcium sensitivity (pCa50 5.74 versus 5.90), and cooperativity of activation (nH 3.64 versus 4.38). Our findings indicate the following: 1) in skeletal muscle nebulin increases thin filament activation, and 2) through altering cross-bridge cycling kinetics, nebulin increases force and efficiency of contraction. These novel properties of nebulin add a new level of understanding of skeletal muscle function and provide a mechanism for the severe muscle weakness in patients with nebulin-based nemaline myopathy.
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- 2009
9. A novel method for sensitive and specific detection of DNA methylation biomarkers based on DNA restriction during PCR cycling
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Thomas Schlegel, Dimo Dietrich, Jörn Lewin, Anke Seegebarth, Stefanie Seemann, Christian Witt, Michael Fleischhacker, Bernd Schmidt, Nadja Flemming, Volker Liebenberg, and Christoph Kneip
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Lung Neoplasms ,Nerve Tissue Proteins ,Biology ,Polymerase Chain Reaction ,Sensitivity and Specificity ,General Biochemistry, Genetics and Molecular Biology ,law.invention ,chemistry.chemical_compound ,law ,Biomarkers, Tumor ,Humans ,Methylated DNA immunoprecipitation ,Polymerase chain reaction ,Homeodomain Proteins ,DNA Restriction Enzymes ,DNA, Neoplasm ,Methylation ,DNA Methylation ,chemistry ,Biochemistry ,DNA methylation ,Illumina Methylation Assay ,Biomarker (medicine) ,Cancer biomarkers ,Bronchoalveolar Lavage Fluid ,DNA ,Biotechnology - Abstract
DNA methylation is an important epigenetic mechanism involved in fundamental biological processes such as development, imprinting, and carcino-genesis. For these reasons, DNA methylation represents a valuable source for cancer biomarkers. Methods for the sensitive and specific detection of methylated DNA are a prerequisite for the implementation of DNA biomarkers into clinical routine when early detection based on the analysis of body fluids is desired. Here, a novel technique is presented for the detection of DNA methylation biomarkers, based on real-time PCR of bisulfite-treated template with enzymatic digestion of background DNA during amplification using the heat-stable enzyme Tsp509I. An assay for the lung cancer methylation biomarker BARHL2 was used to show clinical and analytical performance of the method in comparison with methylation-specific PCR technology. Both technologies showed comparable performance when analyzing technical DNA mixtures and bronchial lavage samples from 75 patients suspected of having lung cancer. The results demonstrate that the approach is useful for sensitive and specific detection of a few copies of methylated DNA in samples with a high background of unmethylated DNA, such as in clinical samples from body fluids.
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- 2009
10. Co-expression of p21Waf1/Cip1 in adenovirus vectors improves expression of a second transgene
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Christian Witt, A. Schumacher, C Woischwill, G Wolff, and S. Horvat
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Cyclin-Dependent Kinase Inhibitor p21 ,Regulation of gene expression ,Cell Cycle ,Genetic Vectors ,Alpha (ethology) ,Cell cycle ,Biology ,CDKN1A Gene ,medicine.disease_cause ,Molecular biology ,Adenoviridae ,Cell Line ,Gene Expression Regulation ,Cell culture ,alpha 1-Antitrypsin ,Gene expression ,Hepatocytes ,Genetics ,medicine ,Humans ,Molecular Medicine ,Transgenes ,Vector (molecular biology) ,Molecular Biology - Abstract
First-generation adenoviral (Ad) vectors are frequently used vectors for experimental and clinical gene transfer. Earlier it has been shown that parallel overexpression of the cell cycle regulator p21(Waf1/Cip1) (p21) or antiapoptotic bcl-2 from a second vector reduces cytotoxicity and improves transgene expression. Here, we investigate whether the co-expression of p21 and alpha(1)-antitrypsin from a single vector improves vector safety and alpha(1)-antitrypsin expression. Cell lines (A549 and HeLa) and primary cells (small airway epithelial cells and hepatocytes) were infected with adenovirus vectors transducing alpha(1)-antitrypsin with (AdCMV.p21-RSV.hAAT) or without (AdRSV.hAAT) p21. alpha(1)-Antitrypsin expression and cytotoxicity were analyzed using western blot/ELISA and LDH/ALT/AST assays, respectively. Cell cycle profiles were determined by flow cytometry. Co-expression of p21 strongly increased the alpha(1)-antitrypsin expression in all cell types and at all doses tested. No changes in ALT/AST from hepatocytes and only minor increases in the LDH release in A549 and HeLa were observed with either vector. Cell cycle profiles were also not affected adversely. Incorporation of p21 in Ad vectors together with a gene of interest improves the vector performance; such vectors will allow the application of lower doses and thereby reduce immunological side effects.
- Published
- 2009
11. Induction of MuRF1 Is Essential for TNF-α-Induced Loss of Muscle Function in Mice
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Stephan Gielen, Hermann-Josef Thierse, Gerhard Schuler, Christian Witt, Stephanie Hirner, Alexander Gasch, Norman Mangner, Volker Adams, Christian Krohne, Axel Linke, and Siegfried Labeit
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medicine.medical_specialty ,Ubiquitin-Protein Ligases ,Muscle Fibers, Skeletal ,Down-Regulation ,Muscle Proteins ,In Vitro Techniques ,Models, Biological ,Cell Line ,Proinflammatory cytokine ,Tripartite Motif Proteins ,Mice ,Peptide Elongation Factor 1 ,Atrophy ,Troponin T ,Western blot ,Structural Biology ,Internal medicine ,medicine ,Animals ,Immunoprecipitation ,Muscle, Skeletal ,Myopathy ,Molecular Biology ,biology ,medicine.diagnostic_test ,Tumor Necrosis Factor-alpha ,Ubiquitin ,Skeletal muscle ,medicine.disease ,Troponin ,Ubiquitin ligase ,Eukaryotic Initiation Factor-4E ,Endocrinology ,medicine.anatomical_structure ,Immunology ,biology.protein ,Tumor necrosis factor alpha ,medicine.symptom ,Muscle Contraction - Abstract
Humoral circulating inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) can impair skeletal muscle contractility. Furthermore, TNF-alpha expression correlates with elevated levels of atrogin-like muscle-specific ubiquitin E3 ligases, which are presumed to mediate muscle protein breakdown and atrophy. However, the casual relationships between MuRF1 and TNF-alpha and their relative contributions to muscle function impairment are not known.TNF-alpha or saline was injected into either C57Bl6 or MuRF1(-/-) mice. After 16-24 h, the expression of MuRF1 in skeletal muscle was quantified by quantitative reverse transcription-PCR and Western blot analysis. Muscle function was measured in an organ bath. To obtain a broader overview on potential alterations, two-dimensional gel electrophoresis was performed.Wild-type animals injected with TNF-alpha had higher MuRF1 mRNA expression (saline versus TNF-alpha: 56.6+/-12.1 versus 133.6+/-30.3 arbitrary units; p0.05) and protein expression (saline versus TNF-alpha: 0.38+/-0.11 versus 1.07+/-0.25 arbitrary units; p0.05) as compared to saline-injected littermates. Furthermore, TNF-alpha reduced force development at 150 Hz by 25% in C57Bl6 animals (saline versus TNF-alpha: 2412+/-120 versus 1799+/-114 g/cm(2); p0.05), but not in MuRF1(-/-) mice (saline versus TNF-alpha: 2424+/-198 versus 2431+/-180 g/cm(2); p=NS). Proteome analysis revealed a significant down-regulation of fast skeletal muscle troponin T in wild-type animals treated with TNF-alpha as compared to MuRF1(-/-) mice that received TNF-alpha.The results of this study demonstrate for the first time that TNF-alpha-induced reduction in skeletal muscle force development depends on the induction of the atrophy-related E3 ubiquitin ligase MuRF1. A link for the reduction in muscle force may be the TNF-alpha/MuRF1-mediated down-regulation of fast skeletal muscle troponin T.
- Published
- 2008
12. Titin expression in human articular cartilage and cultured chondrocytes : A novel component in articular cartilage biomechanical sensing?
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Markus Schwarz, Stephanie H. Witt, Andreas Buettner, Johannes Stoeve, Christian Witt, Hanns-Peter Scharf, Barbara Schneider-Wald, Stefan Milz, and Siegfried Labeit
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Adult ,Cartilage, Articular ,Pathology ,medicine.medical_specialty ,animal structures ,Adolescent ,Muscle Proteins ,macromolecular substances ,Osteoarthritis ,Chondrocyte ,Exon ,Chondrocytes ,Transcription (biology) ,Gene expression ,medicine ,Humans ,Connectin ,RNA, Messenger ,Cells, Cultured ,Aged ,Aged, 80 and over ,Pharmacology ,biology ,Chemistry ,Cartilage ,Infant, Newborn ,Cardiac muscle ,Infant ,General Medicine ,Middle Aged ,musculoskeletal system ,medicine.disease ,Biomechanical Phenomena ,Cell biology ,medicine.anatomical_structure ,cardiovascular system ,biology.protein ,Titin ,Protein Kinases ,tissues - Abstract
In striated muscle tissues, the giant protein titin acts as a biomechanically active filament system, coupling stress/strain to gene expression. The objective of the study is to show the existence of titin fragments in human articular cartilage, as in diarthodial joints, chondrocytes are also known to sense and respond to stretching. We have surveyed human cultured cartilage collected from adults with osteoarthritis (OA), without OA and from infants with a set of titin antibodies and primer pairs. Three different antibodies were used for immunolabelling, reacting with titin's N-terminal Z1-Z2 domains, its Novex III exon, and with its PEVK region. An antibody directed to a titin ligand was included, since in cardiac muscle, this has been shown to participate in the transmission of stretch dependent titin-based signals. Our results indicate that although at low levels, titin is expressed in cartilage. Primer pairs detected titin transcripts in cartilage, and consistent with this, antibodies directed to titin's Z-disc region and to its elastic region stained cartilage. Moreover, we also could detect transcription of the titin ligand CARP. Components of the stretch dependent signal machinery in muscle are also expressed in cartilage. Further studies are warranted to address if common stress/strain dependent signalling are conserved in muscle and cartilage tissues.
- Published
- 2008
13. Muscle RING-Finger Protein-1 (MuRF1) as a Connector of Muscle Energy Metabolism and Protein Synthesis
- Author
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Vladimir Rybin, Alexander Gasch, Stephanie H. Witt, Yasuko Ono, Hiroyuki Sorimachi, Shoji Hata, Koichi Ojima, Thomas Franz, Fujiko Kitamura, Stefanie Lerche, Tomoki Chiba, Christian Witt, Keiji Tanaka, Naoko Doi, Suguru Koyama, Siegfried Labeit, and Keiko Abe
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medicine.medical_specialty ,Ubiquitin-Protein Ligases ,Muscle Proteins ,Protein degradation ,Tripartite Motif Proteins ,Mice ,Ubiquitin ,Structural Biology ,Internal medicine ,medicine ,Protein biosynthesis ,Animals ,Humans ,Amino Acids ,Muscle, Skeletal ,Molecular Biology ,Mice, Knockout ,Polycomb Repressive Complex 1 ,biology ,Catabolism ,GTPase-Activating Proteins ,Ubiquitination ,Skeletal muscle ,Metabolism ,Muscle atrophy ,DNA-Binding Proteins ,Repressor Proteins ,Alcohol Oxidoreductases ,Muscular Atrophy ,Endocrinology ,medicine.anatomical_structure ,Protein Biosynthesis ,biology.protein ,Creatine kinase ,medicine.symptom ,Energy Metabolism - Abstract
During pathophysiological muscle wasting, a family of ubiquitin ligases, including muscle RING-finger protein-1 (MuRF1), has been proposed to trigger muscle protein degradation via ubiquitination. Here, we characterized skeletal muscles from wild-type (WT) and MuRF1 knockout (KO) mice under amino acid (AA) deprivation as a model for physiological protein degradation, where skeletal muscles altruistically waste themselves to provide AAs to other organs. When WT and MuRF1 KO mice were fed a diet lacking AA, MuRF1 KO mice were less susceptible to muscle wasting, for both myocardium and skeletal muscles. Under AA depletion, WT mice had reduced muscle protein synthesis, while MuRF1 KO mice maintained nonphysiologically elevated levels of skeletal muscle protein de novo synthesis. Consistent with a role of MuRF1 for muscle protein turnover during starvation, the concentrations of essential AAs, especially branched-chain AAs, in the blood plasma significantly decreased in MuRF1 KO mice under AA deprivation. To clarify the molecular roles of MuRF1 for muscle metabolism during wasting, we searched for MuRF1-associated proteins using pull-down assays and mass spectrometry. Muscle-type creatine kinase (M-CK), an essential enzyme for energy metabolism, was identified among the interacting proteins. Coexpression studies revealed that M-CK interacts with the central regions of MuRF1 including its B-box domain and that MuRF1 ubiquitinates M-CK, which triggers the degradation of M-CK via proteasomes. Consistent with MuRF1's role of adjusting CK activities in skeletal muscles by regulating its turnover in vivo, we found that CK levels were significantly higher in the MuRF1 KO mice than in WT mice. Glucocorticoid modulatory element binding protein-1 and 3-hydroxyisobutyrate dehydrogenase, previously identified as potential MuRF1-interacting proteins, were also ubiquitinated MuRF1-dependently. Taken together, these data suggest that, in a multifaceted manner, MuRF1 participates in the regulation of AA metabolism, including the control of free AAs and their supply to other organs under catabolic conditions, and in the regulation of ATP synthesis under metabolic-stress conditions where MuRF1 expression is induced.
- Published
- 2008
14. Myocardial expression of Murf-1 and MAFbx after induction of chronic heart failure: Effect on myocardial contractility
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Nicolle Kränkel, Sandra Erbs, Gerhard Schuler, Rainer Hambrecht, Christian Witt, Ursula Müller-Werdan, Ulrik Wisløff, Volker Adams, Siegfried Labeit, Christian Doring, and Axel Linke
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MAPK/ERK pathway ,medicine.medical_specialty ,Pyridines ,Physiology ,Ubiquitin-Protein Ligases ,medicine.medical_treatment ,Blotting, Western ,Gene Expression ,Muscle Proteins ,Biology ,Rats, Inbred WKY ,p38 Mitogen-Activated Protein Kinases ,Tripartite Motif Proteins ,Contractility ,Ventricular Dysfunction, Left ,Physiology (medical) ,Internal medicine ,Troponin I ,medicine ,Animals ,Myocyte ,RNA, Messenger ,Flavonoids ,Heart Failure ,Mitogen-Activated Protein Kinase 1 ,SKP Cullin F-Box Protein Ligases ,Tumor Necrosis Factor-alpha ,Myocardium ,Imidazoles ,medicine.disease ,Rats ,Endocrinology ,Cytokine ,Animals, Newborn ,Echocardiography ,Heart failure ,Models, Animal ,Circulatory system ,RNA Interference ,Tumor necrosis factor alpha ,Cardiology and Cardiovascular Medicine - Abstract
Objective: In chronic heart failure (CHF) the myocardial expression of the inflammatory cytokine tumor necrosis factor alpha (TNF-α), which is thought to contribute to myocardial remodeling, was found to be increased. However, it is unknown whether the E3-ubiquitin ligases MAFbx and Murf-1 are involved in this remodeling process and whether their expression is regulated by TNF-α. Methods: Rats underwent ligation of the left coronary artery to induce CHF or were sham-operated. The expression of MAFbx/Murf-1 and troponin I was analyzed by RT-PCR and Western blotting in the non-infarcted area of the left ventricle. In cell culture experiments the potency of TNF-α to stimulate Murf-1/MAFbx expression, the intracellular signaling pathway, and the involvement of the E3-ligases for the impairment of contractility were assessed. Results: In CHF the myocardial expression of TNF-α was elevated 3.1-fold as compared to control. This was associated with a 4.5-fold and 2.7-fold increase in MAFbx and Murf-1 expression, respectively. A positive correlation between TNF-α and the expression of MAFbx or Murf-1 was evident. In neonatal rat cardiomyocytes, TNF-α induced the expression of MAFbx through p38MAPK-dependent pathways, whereas the induction of Murf-1 required the activation of the p42/44 MAPK pathway. Exposure of cardiomyocytes to TNF-α resulted in troponin I ubiquitinylation, subsequent degradation, and a decline in contractility. This was completely abrogated by siRNAs against Murf-1/MAFbx. Conclusion: TNF-α, which is increasingly expressed in CHF, induces troponin I degradation through a MAFbx/Murf-1-dependent pathway. This was associated with an impairment of contractility and might be one mechanism involved in the adverse remodeling process in CHF.
- Published
- 2007
15. Normal cardiac contraction in mice lacking the proline-alanine rich region and C1 domain of cardiac myosin binding protein C
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Samantha P. Harris, Sabine J. van Dijk, and Christian Witt
- Subjects
Sarcomeres ,medicine.medical_specialty ,Contraction (grammar) ,Proline ,Systole ,Molecular Sequence Data ,chemistry.chemical_element ,Gene Expression ,Biology ,Calcium ,Sarcomere ,Article ,Mice ,Internal medicine ,Troponin I ,medicine ,Animals ,Humans ,Myocytes, Cardiac ,Adrenergic agonist ,Amino Acid Sequence ,Molecular Biology ,Isovolumetric contraction ,Sequence Deletion ,Mice, Knockout ,Alanine ,Myocardium ,Wild type ,Isoproterenol ,Stroke Volume ,Adrenergic beta-Agonists ,Myocardial Contraction ,Protein Structure, Tertiary ,Endocrinology ,chemistry ,Biochemistry ,Echocardiography ,Cardiology and Cardiovascular Medicine ,Carrier Proteins - Abstract
Cardiac myosin binding protein C (cMyBP-C) is an essential regulator of cross bridge cycling. Through mechanisms that are incompletely understood the N-terminal domains (NTDs) of cMyBP-C can activate contraction even in the absence of calcium and can also inhibit cross bridge kinetics in the presence of calcium. In vitro studies indicated that the proline-alanine rich (p/a) region and C1 domain are involved in these processes, although effects were greater using human proteins compared to murine proteins (Shaffer et al. J Biomed Biotechnol 2010, 2010: 789798). We hypothesized that the p/a and C1 region are critical for the timing of contraction. In this study we tested this hypothesis using a mouse model lacking the p/a and C1 region (p/a-C1(-/-) mice) to investigate the in vivo relevance of these regions on cardiac performance. Surprisingly, hearts of adult p/a-C1(-/-) mice functioned normally both on a cellular and whole organ level. Force measurements in permeabilized cardiomyocytes from adult p/a-C1(-/-) mice and wild type (Wt) littermate controls demonstrated similar rates of force redevelopment both at submaximal and maximal activation. Maximal and passive force and calcium sensitivity of force were comparable between groups as well. Echocardiograms showed normal isovolumetric contraction times, fractional shortening and ejection fraction, indicating proper systolic function in p/a-C1(-/-) mouse hearts. p/a-C1(-/-) mice showed a slight but significant reduction in isovolumetric relaxation time compared to Wt littermates, yet this difference disappeared in older mice (7-8months of age). Moreover, stroke volume was preserved in p/a-C1(-/-) mice, corroborating sufficient time for normal filling of the heart. Overall, the hearts of p/a-C1(-/-) mice showed no signs of dysfunction even after chronic stress with an adrenergic agonist. Together, these results indicate that the p/a region and the C1 domain of cMyBP-C are not critical for normal cardiac contraction in mice and that these domains have little if any impact on cross bridge kinetics in mice. These results thus contrast with in vitro studies utilizing proteins encoding the human p/a region and C1 domain. More detailed insight in how individual domains of cMyBP-C function and interact, across species and over the wide spectrum of conditions in which the heart has to function, will be essential to a better understanding of how cMyBP-C tunes cardiac contraction.
- Published
- 2015
16. Dimerization of the cardiac ankyrin protein CARP: Implications for MARP titin-based signaling
- Author
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Henk Granzier, Dietmar Labeit, Stephanie H. Witt, Christian Witt, and Siegfried Labeit
- Subjects
ANKRD2 ,ANKRD1 ,Physiology ,Protein subunit ,Muscle Proteins ,Transfection ,Biochemistry ,Desmin ,Two-Hybrid System Techniques ,Escherichia coli ,Humans ,Gene family ,Ankyrin ,Connectin ,Carp ,chemistry.chemical_classification ,biology ,Nuclear Proteins ,Cell Biology ,biology.organism_classification ,Molecular biology ,Peptide Fragments ,Recombinant Proteins ,Ankyrin Repeat ,Cell biology ,Molecular Weight ,Repressor Proteins ,chemistry ,biology.protein ,Titin ,Ankyrin repeat ,sense organs ,Dimerization ,Protein Kinases ,Protein Binding ,Signal Transduction - Abstract
Cardiac ankyrin repeat protein (CARP) and its two close homologs ankrd2 (Arpp) and DARP correspond to a conserved gene family of muscle ankyrin repeat proteins (MARPs). All three genes respond to a variety of stress/strain injury signals with their cytokine-like induction and can associate with the elastic region of titin/connectin. Recently, both CARP and ankrd2 were observed to be elevated in cardiac diseases as well as muscular dystrophies, implicating their joined signaling in muscle diseases. Here we show that CARP in the yeast two-hybrid system (YTH) interacts with itself and desmin. To further verify the YTH data and to investigate possible CARP subunit structure(s), we expressed CARP in E. coli. Expressed CARP has an apparent mobility of about 70 kDa on gel filtration, corresponding to a dimeric species. Yeast two-hybrid experiments using amino- and carboxyterminal deletion clones suggest that CARP, ankrd2, and DARP contain potential coiled-coil dimerization motifs within their unique aminoterminal domains that mediate the formation of homo-dimers. In contrast, we could not detect the formation of hetero-dimers between CARP, ankrd2, and DARP. Therefore, when CARP, ankrd2 and DARP are upregulated in disease/stress states, they are likely to be sorted into distinct structural protein complexes since CARP within the MARP family contains a unique aminoterminal dimerization motif.
- Published
- 2006
17. Anemia and Inflammation in COPD
- Author
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Wolfram Doehner, Darlington D Okonko, Soeren Hoernig, Stefan D. Anker, Matthias John, and Christian Witt
- Subjects
Male ,Pulmonary and Respiratory Medicine ,medicine.medical_specialty ,Anemia ,Vital Capacity ,Critical Care and Intensive Care Medicine ,Gastroenterology ,Body Mass Index ,Hemoglobins ,Pulmonary Disease, Chronic Obstructive ,Interquartile range ,Weight loss ,Forced Expiratory Volume ,hemic and lymphatic diseases ,Internal medicine ,Weight Loss ,medicine ,Humans ,Erythropoietin ,Inflammation ,COPD ,biology ,Interleukin-6 ,business.industry ,Interleukin-8 ,C-reactive protein ,Middle Aged ,medicine.disease ,Pathophysiology ,Interleukin-10 ,Immunology ,biology.protein ,Female ,Inflammation Mediators ,medicine.symptom ,Cardiology and Cardiovascular Medicine ,business ,Body mass index ,medicine.drug - Abstract
Anemia in patients with COPD and its pathophysiology is an understudied issue.In a group of 101 COPD patients (FEV(1) percentage of predicted, 37 +/- 2% [mean +/- SEM]; mean age, 61 +/- 1 years; 35% female gender), the prevalence of anemia and its relationship to body mass and weight loss, inflammatory parameters, and erythropoietin levels was determined. Data were compared to a control group (healthy persons with matched age) in order to identify potential factors that may influence the development of anemia in patients with COPD.Anemia was diagnosed in 13 patients (hemoglobin levels13.5 mg/dL in male patients and12.0 mg/dL in female patients), which represents a prevalence of 13%. Anemic COPD patients showed elevated erythropoietin levels (41.8 +/- 25.4 U/L vs 16.3 +/- 2.9 U/L) and an increased inflammatory response compared to nonanemic patients. A significant inverse correlation of hemoglobin vs erythropoietin (r = - 0.84, p0.01) was observed in anemic COPD patients, but not in the nonanemic group.Anemic COPD patients show high erythropoietin levels, which may indicate presence of erythropoietin resistance. The latter may be mediated through inflammatory mechanisms, which is typical for anemia of chronic illness.
- Published
- 2005
18. Induction and Myofibrillar Targeting of CARP, and Suppression of the Nkx2.5 Pathway in the MDM Mouse with Impaired Titin-based Signaling
- Author
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Hiroyuki Sorimachi, Yasuko Ono, Yiming Wu, Siegfried Labeit, Henk Granzier, Christian Witt, Mark McNabb, Carol C. Gregorio, Michael Gotthardt, Stephanie H. Witt, Dietmar Labeit, Markus Haak, and Eva Puschmann
- Subjects
ANKRD2 ,Macromolecular Substances ,Ubiquitin-Protein Ligases ,Molecular Sequence Data ,Muscle Proteins ,Mice, Inbred Strains ,Biology ,medicine.disease_cause ,Sarcomere ,Tripartite Motif Proteins ,Mice ,Myofibrils ,Structural Biology ,medicine ,Animals ,Connectin ,Amino Acid Sequence ,Muscular dystrophy ,Muscle, Skeletal ,Molecular Biology ,Homeodomain Proteins ,Mutation ,Myositis ,Cardiac muscle ,Nuclear Proteins ,Skeletal muscle ,Muscular Dystrophy, Animal ,medicine.disease ,Molecular biology ,Mice, Inbred C57BL ,Repressor Proteins ,medicine.anatomical_structure ,Gene Expression Regulation ,biology.protein ,Titin ,sense organs ,Myofibril ,Protein Kinases ,Signal Transduction - Abstract
Muscular dystrophy with myositis ( mdm ) is a recessive mouse mutation that is caused by a small deletion in the giant elastic muscle protein titin. Homozygous mdm / mdm mice develop a progressive muscular dystrophy, leading to death at ∼2 months of age. We surveyed the transcriptomes of skeletal muscles from 24 day old homozygous mdm / mdm and +/+ wild-type mice, an age when MDM animals have normal passive and active tensions and sarcomeric structure. Of the 12,488 genes surveyed (U74 affymetrix array), 75 genes were twofold to 30-fold differentially expressed, including CARP (cardiac ankyrin repeat protein), ankrd2/Arpp (a CARP-like protein) and MLP (muscle LIM protein), all of which associate with the titin filament system. The four genes most strongly affected (eightfold to 30-fold change) were all members of the CARP-regulated Nkx-2.5-dependent signal pathway, and CARP mRNA level was 30-fold elevated in MDM skeletal muscle tissues. The CARP protein overexpressed in MDM became associated with the I-band region of the sarcomere. The mdm mutation excises the C-terminal portion of titin's N2A region, abolishing its interaction with p94/calpain-3 protease. Thus, the composition of the titin N2A protein complex is altered in MDM by incorporation of CARP and loss of p94/calpain-3. These changes were absent from the following control tissues (1) cardiac muscles from homozygous mdm / mdm animals, (2) skeletal and cardiac muscle from heterozygous mdm /+ animals, and (3) dystrophic muscles from MDX mice. Thus, the altered composition of the titin N2A complex is specific for the titin-based skeletal muscular dystrophy in MDM.
- Published
- 2004
19. Detection of cell-free nucleic acids in bronchial lavage fluid supernatants from patients with lung cancer
- Author
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B. Jandrig, Christian Witt, Bernd Schmidt, E. Engel, Michael Fleischhacker, and T. Carstensen
- Subjects
Male ,Cancer Research ,Lung Neoplasms ,Biology ,law.invention ,chemistry.chemical_compound ,law ,Carcinoma, Non-Small-Cell Lung ,Gene expression ,medicine ,Humans ,RNA, Neoplasm ,Carcinoma, Small Cell ,Polymerase chain reaction ,Aged ,Messenger RNA ,medicine.diagnostic_test ,Reverse Transcriptase Polymerase Chain Reaction ,Smoking ,RNA ,DNA, Neoplasm ,Middle Aged ,Virology ,Molecular biology ,Reverse transcriptase ,Early Diagnosis ,Bronchoalveolar lavage ,Oncology ,chemistry ,Nucleic acid ,Female ,Bronchoalveolar Lavage Fluid ,DNA ,Microsatellite Repeats - Abstract
The aim of this study was to determine whether nucleic acids are detectable in cell-free bronchial lavage supernatants, and whether it is possible to find alterations in this DNA and RNA of genes known to be present in lung tumour cells. DNA was isolated from cell-free lavage supernatants from 30 and RNA from 25 lung cancer patients. The DNA was examined for microsatellite alterations (MA) and the RNA analysed for the expression of seven tumour-associated genes. Intact DNA and mRNA could be isolated from all cell-free bronchial lavage supernatants. MA were found in lavage supernatants of 12/30 patients and in lavage cells of 6/30 patients. Altogether alterations were found in 14/30 patients. Analyses of tumour-associated gene expression showed positive results, with at least one marker in the lavage supernatants of all 25 patients. Thus, we could demonstrate, for the first time, that it is possible to isolate intact DNA and RNA from cell-free bronchial lavage supernatants. Their quantity and quality is sufficient for further amplification by polymerase chain reaction (PCR)/reverse transcriptase (RT)-PCR. Altogether, tumour-associated changes were detected in DNA samples from 47% of the patients and in RNA samples from all of the patients analysed.
- Published
- 2004
20. The Muscle Ankyrin Repeat Proteins: CARP, ankrd2/Arpp and DARP as a Family of Titin Filament-based Stress Response Molecules
- Author
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Christian Witt, Melanie K. Miller, Carol C. Gregorio, Siegfried Labeit, Kaori Watanabe, Charles Trombitas, Abigail S. McElhinny, Dietmar Labeit, Marie Louise Bang, and Henk Granzier
- Subjects
ANKRD2 ,ANKRD1 ,animal structures ,Amino Acid Motifs ,Molecular Sequence Data ,Muscle Proteins ,Muscle hypertrophy ,Structural Biology ,Humans ,Myocyte ,Connectin ,Amino Acid Sequence ,Muscle, Skeletal ,Molecular Biology ,Conserved Sequence ,Base Sequence ,biology ,Myocardium ,Nuclear Proteins ,musculoskeletal system ,Molecular biology ,Repressor Proteins ,Actinin, alpha 2 ,biology.protein ,Titin ,Ankyrin repeat ,Myofibril ,Protein Kinases ,tissues - Abstract
CARP, ankrd-2/Arpp, and DARP, are three members of a conserved gene family, referred to here as MARPs (muscle ankyrin repeat proteins). The expression of MARPs is induced upon injury and hypertrophy (CARP), stretch or denervation (ankrd2/Arpp), and during recovery following starvation (DARP), suggesting that they are involved in muscle stress response pathways. Here, we show that MARP family members contain within their ankyrin repeat region a binding site for the myofibrillar elastic protein titin. Within the myofibril, MARPs, myopalladin, and the calpain protease p94 appear to be components of a titin N2A-based signaling complex. Ultrastructural studies demonstrated that all three endogenous MARP proteins co-localize with I-band titin N2A epitopes in adult heart muscle tissues. In cultured fetal rat cardiac myocytes, passive stretch induced differential distribution patterns of CARP and DARP: staining for both proteins was increased in the nucleus and at the I-band region of myofibrils, while DARP staining also increased at intercalated discs. We speculate that the myofibrillar MARPs are regulated by stretch, and that this links titin-N2A-based myofibrillar stress/strain signals to a MARP-based regulation of muscle gene expression.
- Published
- 2003
21. Soil Fertility and Indigenous Nutrient Supply in Irrigated Rice Domains of Asia
- Author
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V. T. K. Thoa, T. T. Son, M. Babu, Guanghuo Wang, P. Muthukrishnan, G. C. Simbahan, M. A. A. Adviento, S. Abdulrachman, N. V. Chien, R. Nagarajan, A. Dobermann, C. V. Phung, P. Stalin, Christian Witt, H. C. Gines, V. Ravi, and P. S. Tan
- Subjects
biology ,Growing season ,engineering.material ,Oryza ,biology.organism_classification ,Crop ,Nutrient ,Agronomy ,Soil water ,engineering ,Fertilizer ,Soil fertility ,Agronomy and Crop Science ,Agroecology - Abstract
Knowledge-intensive approaches have been proposed to manage the variability in indigenous nutrient supplies (IS) in irrigated rice (Oryza saliva L.) systems. On-farm experiments were conduced at 155 locations in seven domains of Asia to quantify the variability of soil properties, grain yield, and nutrient uptake in N, P, and K omission plots (0-N, 0-P, and 0-K, respectively). Except for pH, coefficients of variation of soil properties within a domain ranged from 17 to 43%. Similar ranges were measured for grain yield and plant nutrient uptake in nutrient omission plots, which served as crop-based estimates of indigenous N, P, and K supply. Soil properties showed little association with plant nutrient uptake or grain yield in nutrient omission plots. Mean grain yields in nutrient omission plots increased in the order 0-N (3.9 Mg ha - 1 ) < 0-K (5.1 Mg ha - 1 ) ≤ 0-P (5.2 Mg ha - 1 ). Soils, climate, and crop management caused large variability of IS among irrigated rice domains, years, growing seasons, and fields within a domain. Grain yield and nutrient uptake in omission plots were mostly higher in high-yielding than in low-yielding climatic seasons. No changes in indigenous N supply occurred for periods of 4 to 6 yr in the same seasons. Grain yields in nutrient omission plots were strongly correlated with each other and also with the yield in the fertilized farmers' fields. Fertilizer recommendations should be fine-tuned to spatial domains with relatively uniform agroecological characteristics, cropping practices, and socioeconomic conditions. Within such domains, season-specific management of the IS variability can include field-specific approaches.
- Published
- 2003
22. Estimating Indigenous Nutrient Supplies for Site‐Specific Nutrient Management in Irrigated Rice
- Author
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V. T. K. Thoa, Guanghuo Wang, A. Dobermann, S. Abdulrachman, T. T. Son, G. C. Simbahan, P. S. Tan, V. Ravi, P. Stalin, P. Muthukrishnan, V. Bartolome, Christian Witt, N. V. Chien, C. V. Phung, M. A. A. Adviento, M. Babu, H. C. Gines, and R. Nagarajan
- Subjects
biology ,Soil test ,Nutrient management ,engineering.material ,Oryza ,biology.organism_classification ,Nutrient ,Agronomy ,Yield (wine) ,engineering ,Grain yield ,Fertilizer ,Agronomy and Crop Science ,Mathematics - Abstract
Nutrient supplies from indigenous sources (IS) can be estimated by measuring plant nutrient uptake in nutrient omission plots. Onfarm experiments were conducted in irrigated rice (Oryza saliva L.) domains of Asia to evaluate relationships of plant N, P, and K uptake with soil tests or grain yield measured in N, P, and K omission (0-N, 0-P, and 0-K, respectively) plots and to develop guidelines for the use of omission plots in site-specific management. Relationships between grain yield or nutrient accumulation and soil tests were scattered. Only 17% of the variation in plant N uptake in 0-N plots was explained by total soil organic C. Extractable Olsen P explained 34% of plant P uptake in 0-P plots, whereas 1 M ammonium acetate K showed no common relationship with plant K uptake in 0-K plots. With good calibration, indigenous supply of N (INS), P (IPS), and K (IKS) can be estimated from grain yields in omission plots with a precision of about ′5 to 10 kg N ha - 1 , ′2 to 3 kg P ha - 1 , and ′10 to 20 kg K ha - 1 , respectively. Sampling requirements for estimating domain-specific IS values depend on the homogeneity of the domain of interest. For irrigated rice domains of about 100 to 200 km 2 , grain yield in omission plots should be measured in at least one high-yielding season in about 10 farms to estimate the domain mean INS, IPS, and IKS. Future research should focus on developing geospatial techniques for delineating fertilizer recommendation domains based on biophysical and socioeconomic characteristics that determine yield potential, IS, and response to fertilizer.
- Published
- 2003
23. The Complete Mouse Nebulin Gene Sequence and the Identification of Cardiac Nebulin
- Author
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Parker B. Antin, Norbert Huebner, Steven T. Kazmierski, Siegfried Labeit, Christian Witt, Abigail S. McElhinny, and Carol C. Gregorio
- Subjects
Blotting, Western ,Molecular Sequence Data ,Muscle Proteins ,Sarcomere ,Protein filament ,Mice ,Xenopus laevis ,Exon ,Nebulin ,Structural Biology ,medicine ,Animals ,Protein Isoforms ,Amino Acid Sequence ,Muscle, Skeletal ,Molecular Biology ,In Situ Hybridization ,Actin ,Models, Genetic ,Sequence Homology, Amino Acid ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,Myocardium ,Skeletal muscle ,Heart ,Exons ,Molecular biology ,Introns ,Protein Structure, Tertiary ,Rats ,medicine.anatomical_structure ,Microscopy, Fluorescence ,Nebulette ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Myofibril - Abstract
Nebulin is a giant (M(r) 750-850kDa), modular sarcomeric protein proposed to regulate the assembly, and to specify the precise lengths of actin (thin) filaments in vertebrate skeletal muscles. Nebulin's potential role as a molecular template is based on its structural and biochemical properties. Its central approximately 700kDa portion associates with actin along the entire length of the thin filament, its N-terminal region extends to thin filament pointed ends, and approximately 80kDa of its C-terminal region integrates within the Z-line lattice. Here, we determined the exon/intron organization of the entire mouse nebulin gene, which contains 165 exons in a 202kb segment. We identified 16 novel exons, 15 of which encode nebulin-repeat motifs (12 from its central region and 3 from its Z-line region). One novel exon shares high sequence homology to the 20 residue repeats of the tight-junction protein, ZO-1. RT-PCR analyses revealed that all 16 novel exons are expressed in mouse skeletal muscle. Surprisingly, we also amplified mRNA transcripts from mouse and human heart cDNA using primers designed along the entire length of nebulin. The expression of cardiac-specific nebulin transcripts was confirmed by in situ hybridization in fetal rat cardiomyocytes and in embryonic Xenopus laevis (frog) heart. On the protein level, antibodies specific for skeletal muscle nebulin's N and C-terminal regions stained isolated rat cardiac myofibrils at the pointed and barbed ends of thin filaments, respectively. These data indicate a conserved molecular layout of the nebulin filament systems in both cardiac and skeletal myofibrils. We propose that thin filament length regulation in cardiac and skeletal muscles may share conserved nebulin-based mechanisms, and that nebulin isoform diversity may contribute to thin filament length differences in cardiac and skeletal muscle.
- Published
- 2003
24. Distribution of Respiratory Mucin Proteins in Human Nasal Mucosa
- Author
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Christian Peiser, K. Fan Chung, Paul R. Eynott, John Matthias, David A. Groneberg, Werner Heppt, Axel Fischer, Q. Thai Dinh, Ingemar Carlstedt, and Christian Witt
- Subjects
Pathology ,medicine.medical_specialty ,Mucous membrane of nose ,Mucin 5AC ,Biology ,Fluorescence ,medicine ,Humans ,RNA, Messenger ,Respiratory system ,Respiratory Tract Infections ,chemistry.chemical_classification ,Reverse Transcriptase Polymerase Chain Reaction ,Tumor Necrosis Factor-alpha ,Mucin ,Mucins ,respiratory system ,Immunohistochemistry ,Mucin-5B ,Mucus ,Epithelium ,Staining ,Nasal Mucosa ,medicine.anatomical_structure ,Otorhinolaryngology ,chemistry ,Acute Disease ,Goblet Cells ,Glycoprotein ,Interleukin-1 ,Respiratory tract - Abstract
Objectives/Hypothesis: The upper respiratory tract is involved in many acute and chronic respiratory tract diseases that present with the symptom of mucus hypersecretion. Mucin genes that encode for the backbone of glycoproteins contribute to the viscoelastic property of airway mucus. We examined the cellular expression and distribution of two major respiratory mucus-forming glycoproteins, MUC5AC and MUC5B, in normal human nasal tissues. Methods: Immunohistochemical analysis using polyclonal antibodies against the mucins MUC5AC and MUC5B was performed in normal human nasal tissues. Results: An abundant staining of submucosal mucus gland and epithelial goblet cells for MUC5B was found. hnmunohistochemical analysis of MUC5AC showed staining of surface epithelium goblet cells, whereas there was no staining of glandular cells. Comparison of the expression to lower airways revealed a similar pattern of expression of both mucins. Conclusions: The data in the present study demonstrated the localization of the two major respiratory mucin proteins in human nasal mucosa with a similar distribution of expression of MUC5AC and MUC5B in normal upper and lower airways. Mucin protein expression parallels that of mucin messenger RNA expression. (Less)
- Published
- 2003
25. Using Leaf Color Charts to Estimate Leaf Nitrogen Status of Rice
- Author
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Woon-Ho Yang, Roland J. Buresh, Shaobing Peng, Arnel L. Sanico, Christian Witt, and Jianliang Huang
- Subjects
Oryza sativa ,biology ,food and beverages ,chemistry.chemical_element ,Oryza ,biology.organism_classification ,Nitrogen ,Horticulture ,chemistry ,Agronomy ,Dry weight ,Color chart ,Dry season ,Botany ,Poaceae ,Cultivar ,Agronomy and Crop Science ,Mathematics - Abstract
Leaf color charts (LCCs) have substituted for chlorophyll meter (SPAD) to estimate leaf N status of rice (Oryza saliva L.) and to properly time N fertilizer application. The objectives of this study were to (i) compare three different types of LCC in estimating leaf N status, (ii) determine if specific leaf weight (SLW) affects the estimation of dry weight-based N concentration (N dw ) by LCC, and (iii) determine the relationship between LCC scores and SPAD values. Two field experiments were conducted in the Philippines with different N rates and cultivars grown during 2000 wet season and 2001 dry season. The LCC score and SPAD reading were taken on uppermost fully expanded leaves at three growth stages, and SLW was calculated as the ratio of dry weight to leaf area. Nitrogen content was determined by micro-Kjeldahl procedure and expressed as N dw and leaf area-based N content (N a ). There was a linear relationship between LCC scores and N dw at each growth stage (R 2 range of 0.25-0.97) and across growth stages (R 2 range of 0.46-0.62). Adjusting LCC scores for SLW (LCC/SLW) greatly improved the prediction of N dw across growth stages (R 2 range of 0.84-0.92), suggesting that leaf thickness affects LCC scores. Leaf color chart estimated N, better than N dw across growth stages. Leaf color chart scores were closely related to SPAD values (R 2 range of 0.62-0.98). Strong correlations existed among the scores of the three types of LCC (r range of 0.93-0.99). They are all suitable for use by rice farmers in timing N topdressing.
- Published
- 2003
26. Adenovirus-mediated E2F-1 gene transfer in nonsmall-cell lung cancer induces cell growth arrest and apoptosis
- Author
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Hartmut Kuhn, Christian Gessner, Uta Liebers, Christian Witt, Gerhard Wolff, Joachim Schauer, I. Kovesdi, and A. Schumacher
- Subjects
Pulmonary and Respiratory Medicine ,Lung Neoplasms ,Genetic enhancement ,Genetic Vectors ,Apoptosis ,Cell Cycle Proteins ,Mice, SCID ,Biology ,Adenoviridae ,Flow cytometry ,Mice ,Carcinoma, Non-Small-Cell Lung ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,E2F1 ,Lung cancer ,Severe combined immunodeficiency ,medicine.diagnostic_test ,Cell growth ,Cell Cycle ,Gene Transfer Techniques ,medicine.disease ,E2F Transcription Factors ,DNA-Binding Proteins ,Cell culture ,Cancer research ,Cell Division ,E2F1 Transcription Factor ,Transcription Factors - Abstract
Since overexpression of E2F-1 has been shown to induce apoptosis, the ability of adenovirus-mediated transfer of E2F-1 to inhibit tumour growth in nonsmall-cell lung cancer cell lines was investigated. Three cell lines with various genomic status were infected with AdE2F. Cell proliferation and viability were determined by trypan blue exclusion. Apoptosis induction was assessed by flow cytometry and poly-adenosine diphosphate-ribose-polymerase cleavage assay. In vivo, the effect of E2F-1 on tumour growth was determined in severe combined immunodeficiency (SCID) mice. The current experiments showed that overexpression of E2F-1 suppressed tumour cell growth. The population of apoptotic cells was dramatically increased 96 h after infection with AdE2F. Inhibition of cell growth and induction of apoptosis was not dependent on genomic status. Moreover, treatment of implanted tumours in SCID mice with AdE2F inhibited tumour growth. These data suggest that adenovirus-mediated E2F-1 gene therapy may be effective in the treatment of nonsmall-cell lung cancer.
- Published
- 2002
27. Infection of cells with replication deficient adenovirus induces cell cycle alterations and leads to downregulation of E2F-1
- Author
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Uta Liebers, Hartmut Kuhn, Christian Witt, Axel Schumacher, Leonid Karawajew, V. Ruppert, Gerhard Wolff, and Christian Gessner
- Subjects
p53 ,Cyclin-Dependent Kinase Inhibitor p21 ,Ultraviolet Rays ,E2F-1 ,Genetic Vectors ,Down-Regulation ,Apoptosis ,Cell Cycle Proteins ,Adenovirus vector ,Biology ,Retinoblastoma Protein ,Adenoviridae ,Viral vector ,Downregulation and upregulation ,Cyclins ,Tumor Cells, Cultured ,Humans ,RNA, Messenger ,Vector (molecular biology) ,E2F ,Molecular Biology ,Gene ,S phase ,Cell Cycle ,Cell Biology ,Cell cycle ,Molecular biology ,E2F Transcription Factors ,Cell biology ,DNA-Binding Proteins ,pRb ,Tumor Suppressor Protein p53 ,biological phenomena, cell phenomena, and immunity ,E2F1 Transcription Factor ,Transcription Factors - Abstract
Gene products of recombinant replication-deficient adenovirus vectors of the first generation (Ad vector) can induce cell cycle dysregulation and apoptosis after infection in eukaryotic cells. The mechanisms underlying this complex process are largely unknown. Therefore, we investigated the regulation of the pRb/E2F-1 complex, which controls transition from G(0)/G(1) to S phase of the cell cycle. As Ad vector infection results in a decrease in the number of cells in G(0)/G(1) phase of the cell cycle, we observed a decline of the pRb protein level and, surprisingly, also a decrease of the E2F-1 protein and mRNA level in infected cell lines. Furthermore, in contrast to the reduction of cells in the G(0)/G(1) phase we observed increased protein levels of p53 and p21 proteins. However, as experiments in p53 deficient cell lines indicated, the decrease of pRb and E2F-1 is independent of p53 and p21 expression. Moreover, results obtained with Rb deficient cell lines indicated that the reduced E2F-1 expression is independent of pRb. These results suggest that Ad vector-induced cell cycle dysregulation is associated with a specific downregulation of E2F-1 independent of Rb and p53 genomic status of cells.
- Published
- 2002
28. BAX and p16INK4Aare independent positive prognostic markers for advanced tumour stage of nonsmall cell lung cancer
- Author
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Uta Liebers, Christian Gessner, P. Stiehl, Hartmut Kuhn, Christian Witt, Gerhard Wolff, and Joachim Schauer
- Subjects
Male ,Pulmonary and Respiratory Medicine ,Oncology ,medicine.medical_specialty ,Pathology ,Lung Neoplasms ,Polymerase Chain Reaction ,Frameshift mutation ,Bcl-2-associated X protein ,Carcinoma, Non-Small-Cell Lung ,Proto-Oncogene Proteins ,Internal medicine ,Biomarkers, Tumor ,Carcinoma ,Humans ,Medicine ,Prospective cohort study ,neoplasms ,Survival rate ,Cyclin-Dependent Kinase Inhibitor p16 ,Aged ,Retrospective Studies ,bcl-2-Associated X Protein ,Aged, 80 and over ,biology ,business.industry ,Respiratory disease ,DNA, Neoplasm ,Middle Aged ,Prognosis ,medicine.disease ,Immunohistochemistry ,Survival Rate ,Proto-Oncogene Proteins c-bcl-2 ,Apoptosis ,Lymphatic Metastasis ,biology.protein ,Female ,Tumor Suppressor Protein p53 ,business - Abstract
Clinical studies suggest prognostic relevance of p16INK4A in nonsmall cell lung cancer (NSCLC) while conflicting results for p53 have been published. However, the importance of the apoptosis regulating gene BAX, a downstream regulator of p53, on the prognosis of NSCLC is unknown. The present study investigated the prognostic relevance of BAX with respect to the status of p53 and P16INK4A in 61 patients with advanced NSCLC. Protein expression of BAX, p53 and p16INK4A was investigated retrospectively by immunohistochemistry. Tumour deoxyribonucleic acid (DNA) was screened for p53 mutations by single strand-conformation polymorphism polymerase chain reaction (PCR) and BAX frameshift mutations by fragment length analysis. Patients with positive BAX protein expression had a significantly longer median survival (14 months) than those patients without BAX expression (6 months, p=0.0004). In contrast, p53 status did not influence prognosis. Patients with p161NK4A negative tumours had a significantly shorter survival (4 months) than those with p16INK41 protein expression (15 months, p=0.0001). Furthermore, the loss of p16INK4A protein expression correlated strongly with the pressure of distant and advanced lymph-node metastases. The best survival was seen in a subgroup of 20 patients with positive p16INK4A expression and intact BAX (p=0.0002). The results of the present study suggest that the loss of BAX and p16INK4A expression are independent markers for poor prognosis in nonsmall cell lung cancer. The study suggests that multimarker analysis of genes involved in apoptosis may be useful for determining individual therapy and for identifying targets for gene-replacement therapy. This should be assessed in a prospective study with a larger cohort of patients.
- Published
- 2002
29. Gene transfer into solid tumours—is a special application device beneficial?
- Author
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Uta Liebers, W Arnold, Christian Witt, Hartmut Kuhn, Bernd Schmidt, and Gerhard Wolff
- Subjects
Cancer Research ,Lung Neoplasms ,Tumor suppressor gene ,Genetic enhancement ,Genetic Vectors ,Biology ,Transfection ,Polymerase Chain Reaction ,Adenoviridae ,law.invention ,Viral vector ,Mice ,Transduction (genetics) ,law ,Carcinoma, Non-Small-Cell Lung ,Tumor Cells, Cultured ,Animals ,Humans ,Transgenes ,Gene ,Polymerase chain reaction ,Reporter gene ,Genetic transfer ,Gene Transfer Techniques ,Equipment Design ,Genetic Therapy ,Molecular biology ,Mice, Inbred C57BL ,Oncology ,Needles ,DNA, Viral ,Neoplasm Transplantation - Abstract
The replacement of inactivated tumour suppressor genes is a promising approach in cancer therapy. The aim of this study was to evaluate the influence of technical determinants on the efficiency of adenoviral-mediated gene transfer into solid tumours. Therefore, we compared the efficacy of two different injection needle types, a conventional needle and a modified needle characterised by perforations at the side of the shaft in vivo. The total amount of adenoviral vector DNA and the activity of the transferred reporter gene were quantitatively analysed by polymerase chain reaction (PCR) specific for the E4 region of the Ad vector genome and the beta-galactosidase assay, respectively. The levels of adenoviral DNA, as well as the total beta-galactosidase activity, varied widely, but were not significantly different for the two groups. These results suggest, that the efficiency of intratumoral gene transfer cannot be improved by the design of the application device.
- Published
- 2001
30. Titin–Actin Interaction in Mouse Myocardium: Passive Tension Modulation and Its Regulation by Calcium/S100A1
- Author
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Rob Yamasaki, Siegfried Labeit, M. Berri, Karoly Trombitás, Christian Witt, Yiming Wu, Miklós S.Z. Kellermayer, Marion L. Greaser, Dietmar Labeit, Mark McNabb, and Henk Granzier
- Subjects
Biophysics ,Muscle Proteins ,Motility ,macromolecular substances ,Biology ,Mice ,Calcium-binding protein ,medicine ,Animals ,Myocyte ,Connectin ,Actin ,Binding Sites ,Myocardium ,Calcium-Binding Proteins ,Osmolar Concentration ,S100 Proteins ,Cardiac muscle ,Skeletal muscle ,Actins ,Recombinant Proteins ,Protein Structure, Tertiary ,medicine.anatomical_structure ,Biochemistry ,biology.protein ,Calcium ,Titin ,medicine.symptom ,Protein Kinases ,Research Article ,Muscle Contraction ,Muscle contraction - Abstract
Passive tension in striated muscles derives primarily from the extension of the giant protein titin. However, several studies have suggested that, in cardiac muscle, interactions between titin and actin might also contribute to passive tension. We expressed recombinant fragments representing the subdomains of the extensible region of cardiac N2B titin (tandem-Ig segments, the N2B splice element, and the PEVK domain), and assayed them for binding to F-actin. The PEVK fragment bound F-actin, but no binding was detected for the other fragments. Comparison with a skeletal muscle PEVK fragment revealed that only the cardiac PEVK binds actin at physiological ionic strengths. The significance of PEVK–actin interaction was investigated using in vitro motility and single-myocyte mechanics. As F-actin slid relative to titin in the motility assay, a dynamic interaction between the PEVK domain and F-actin retarded filament sliding. Myocyte results suggest that a similar interaction makes a significant contribution to the passive tension. We also investigated the effect of calcium on PEVK–actin interaction. Although calcium alone had no effect, S100A1, a soluble calcium-binding protein found at high concentrations in the myocardium, inhibited PEVK–actin interaction in a calcium-dependent manner. Gel overlay analysis revealed that S100A1 bound the PEVK region in vitro in a calcium-dependent manner, and S100A1 binding was observed at several sites along titin’s extensible region in situ, including the PEVK domain. In vitro motility results indicate that S100A1–PEVK interaction reduces the force that arises as F-actin slides relative to the PEVK domain, and we speculate that S100A1 may provide a mechanism to free the thin filament from titin and reduce titin-based tension before active contraction.
- Published
- 2001
31. Identification of muscle specific ring finger proteins as potential regulators of the titin kinase domain
- Author
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Christian Witt, Hiroyuki Sorimachi, Siegfried Labeit, Carol C. Gregorio, Eiichi Kimura, Marie Louise Bang, Thomas Centner, Henk Granzier, Abigail S. McElhinny, Katarina Pelin, Karoly Trombitás, and Junko Yano
- Subjects
Sarcomeres ,animal structures ,Molecular Sequence Data ,Fluorescent Antibody Technique ,Muscle Proteins ,Obscurin ,Biology ,Mice ,Structural Biology ,Two-Hybrid System Techniques ,Ring finger ,medicine ,Animals ,Humans ,Myotilin ,Connectin ,Amino Acid Sequence ,RNA, Messenger ,Kinase activity ,Microscopy, Immunoelectron ,Molecular Biology ,Phylogeny ,Sequence Homology, Amino Acid ,Gene Expression Profiling ,Muscles ,Zinc Fingers ,Physical Chromosome Mapping ,musculoskeletal system ,Protein Structure, Tertiary ,Rats ,Cell biology ,Actinin, alpha 2 ,medicine.anatomical_structure ,Protein kinase domain ,Biochemistry ,Organ Specificity ,biology.protein ,Titin ,Myofibril ,Dimerization ,Protein Kinases ,Sequence Alignment ,tissues ,Protein Binding - Abstract
The giant myofibrillar protein titin contains within its C-terminal region a serine-threonine kinase of unknown function. We have identified a novel muscle specific RING finger protein, referred to as MURF-1, that binds in vitro to the titin repeats A168/A169 adjacent to the titin kinase domain. In myofibrils, MURF-1 is present within the periphery of the M-line lattice in close proximity to titin's catalytic kinase domain, within the Z-line lattice, and also in soluble form within the cytoplasm. Yeast two-hybrid screens with MURF-1 as a bait identified two other highly homologous MURF proteins, MURF-2 and MURF-3. MURF-1,2,3 proteins are encoded by distinct genes, share highly conserved N-terminal RING domains and in vitro form dimers/heterodimers by shared coiled-coil motifs. Of the MURF family, only MURF-1 interacts with titin repeats A168/A169, whereas MURF-3 has been reported to affect microtubule stability. Association of MURF-1 with M-line titin may potentially modulate titin's kinase activity similar to other known kinase-associated proteins, whereas differential expression and heterodimerization of MURF1, 2 and 3 may link together titin kinase and microtubule-dependent signal pathways in striated muscles.
- Published
- 2001
32. Series of Exon-Skipping Events in the Elastic Spring Region of Titin as the Structural Basis for Myofibrillar Elastic Diversity
- Author
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Henk Granzier, Karoly Trombitás, Alexandra Freiburg, Olivier Cazorla, Thomas Centner, Bernhard Kolmerer, Francoise Fougerousse, Christian Witt, Jaques S. Beckmann, Siegfried Labeit, Carol C. Gregorio, Wolfgang Hell, Physiologie & médecine expérimentale du Cœur et des Muscles [U 1046] (PhyMedExp), Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Généthon
- Subjects
MESH: Connectin ,Transcription, Genetic ,Swine ,Physiology ,MESH: Rabbits ,Muscle Proteins ,MESH: Amino Acid Sequence ,MESH: Base Sequence ,MESH: Protein Isoforms ,MESH: Myofibrils ,Exon ,0302 clinical medicine ,Myofibrils ,Protein Isoforms ,Myotilin ,MESH: Animals ,Connectin ,MESH: Swine ,MESH: Muscle, Skeletal ,0303 health sciences ,Genome ,biology ,Exons ,Anatomy ,musculoskeletal system ,Cell biology ,Actinin, alpha 2 ,embryonic structures ,cardiovascular system ,Titin ,Rabbits ,Cardiology and Cardiovascular Medicine ,tissues ,Gene isoform ,MESH: Myocardium ,animal structures ,MESH: Rats ,Molecular Sequence Data ,Obscurin ,MESH: Muscle Proteins ,03 medical and health sciences ,[SDV.MHEP.CSC]Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system ,[SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,Animals ,Humans ,MESH: Genome ,Amino Acid Sequence ,[PHYS.MECA.BIOM]Physics [physics]/Mechanics [physics]/Biomechanics [physics.med-ph] ,Muscle, Skeletal ,MESH: Protein Kinases ,030304 developmental biology ,MESH: Humans ,MESH: Molecular Sequence Data ,Base Sequence ,MESH: Transcription, Genetic ,Myocardium ,Elasticity ,Exon skipping ,Rats ,MESH: Elasticity ,biology.protein ,MESH: Exons ,Myofibril ,Protein Kinases ,030217 neurology & neurosurgery - Abstract
Abstract —Titins are megadalton-sized filamentous polypeptides of vertebrate striated muscle. The I-band region of titin underlies the myofibrillar passive tension response to stretch. Here, we show how titins with highly diverse I-band structures and elastic properties are expressed from a single gene. The differentially expressed tandem-Ig, PEVK, and N2B spring elements of titin are coded by 158 exons, which are contained within a 106-kb genomic segment and are all subject to tissue-specific skipping events. In ventricular heart muscle, exons 101 kb apart are joined, leading to the exclusion of 155 exons and the expression of a 2.97-MDa cardiac titin N2B isoform. The atria of mammalian hearts also express larger titins by the exclusion of 90 to 100 exons (cardiac N2BA titin with 3.3 MDa). In the soleus and psoas skeletal muscles, different exon-skipping pathways produce titin transcripts that code for 3.7- and 3.35-MDa titin isoforms, respectively. Mechanical and structural studies indicate that the exon-skipping pathways modulate the fractional extensions of the tandem Ig and PEVK segments, thereby influencing myofibrillar elasticity. Within the mammalian heart, expression of different levels of N2B and N2BA titins likely contributes to the elastic diversity of atrial and ventricular myofibrils.
- Published
- 2000
33. Dynamics of soil microbial biomass and nitrogen availability in a flooded rice soil amended with different C and N sources
- Author
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C. C. Galicia, U. Biker, Johannes C. G. Ottow, and Christian Witt
- Subjects
chemistry.chemical_classification ,Crop residue ,biology ,food and beverages ,Soil Science ,Soil classification ,Straw ,biology.organism_classification ,Microbiology ,Green manure ,chemistry ,Agronomy ,Sesbania rostrata ,Paddy field ,Organic matter ,Energy source ,Agronomy and Crop Science - Abstract
A greenhouse experiment was conducted to compare effects of different C and N sources applied to a flooded soil on soil microbial biomass (SMB) C and N, extractable soil organic N (NORG), and NH4+-N in relation to plant N accumulation of rice (Oryza sativa L.). In addition to a control without inputs (CON), four treatments were imposed receiving: prilled urea (PU), rice straw (RS), RS and PU (RS+PU), or Sesbania rostrata as green manure (SES). Treatments were arranged according to a completely randomized design with four replicates and further consisted of pots with and without transplanted rice. While plant effects on the SMB were relatively small, the application of organic N sources resulted in a rapid increase in SMB until 10 days after transplanting (DAT) followed by a gradual decline until 73 DAT. Plant N accumulation data in these treatments clearly indicated that the SMB underwent a transition from a sink to a source of plant-available soil N during the period of crop growth. Seasonal variation of the SMB was small in treatments without amendment of organic material (CON, PU) presumably due to a lack of available C as energy source. Extractable NORG was significantly affected by soil planting status and organic N source amendment, but represented only a small N pool with little temporal variation despite an assumed rapid turnover. Among the three treatments receiving the same amount of N from different sources, the recovery efficiency of applied N was 58% for PU and 28% for both RS+PU and SES treatments at 73 DAT. The N uptake of rice, however, was not driven by N availability alone, as most evident in the RS+PU treatment. We assume that root physiological functions were impeded after application of organic N sources.
- Published
- 2000
34. I-Band Titin in Cardiac Muscle Is a Three-Element Molecular Spring and Is Critical for Maintaining Thin Filament Structure
- Author
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Thomas Centner, Wolfgang A. Linke, Christian Witt, Diane E. Rudy, Mathias Gautel, Carol C. Gregorio, and Siegfried Labeit
- Subjects
Sarcomeres ,Protein Folding ,Recombinant Fusion Proteins ,Immunoelectron microscopy ,Muscle Proteins ,Obscurin ,Myosins ,connectin ,Transfection ,Models, Biological ,Sarcomere ,Antibodies ,Epitopes ,03 medical and health sciences ,0302 clinical medicine ,Myofibrils ,Myosin ,Animals ,Protein Isoforms ,Myocyte ,Microscopy, Immunoelectron ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,biology ,Molecular Motor Proteins ,Myocardium ,heart muscle ,Cell Biology ,Molecular biology ,Actins ,Elasticity ,Actinin, alpha 2 ,Actin Cytoskeleton ,cardiovascular system ,biology.protein ,Biophysics ,Original Article ,Titin ,Rabbits ,sarcomere ,Myofibril ,Chickens ,Protein Kinases ,030217 neurology & neurosurgery - Abstract
In cardiac muscle, the giant protein titin exists in different length isoforms expressed in the molecule's I-band region. Both isoforms, termed N2-A and N2-B, comprise stretches of Ig-like modules separated by the PEVK domain. Central I-band titin also contains isoform-specific Ig-motifs and nonmodular sequences, notably a longer insertion in N2-B. We investigated the elastic behavior of the I-band isoforms by using single-myofibril mechanics, immunofluorescence microscopy, and immunoelectron microscopy of rabbit cardiac sarcomeres stained with sequence-assigned antibodies. Moreover, we overexpressed constructs from the N2-B region in chick cardiac cells to search for possible structural properties of this cardiac-specific segment.We found that cardiac titin contains three distinct elastic elements: poly-Ig regions, the PEVK domain, and the N2-B sequence insertion, which extends ∼60 nm at high physiological stretch. Recruitment of all three elements allows cardiac titin to extend fully reversibly at physiological sarcomere lengths, without the need to unfold Ig domains. Overexpressing the entire N2-B region or its NH2 terminus in cardiac myocytes greatly disrupted thin filament, but not thick filament structure. Our results strongly suggest that the NH2-terminal N2-B domains are necessary to stabilize thin filament integrity. N2-B–titin emerges as a unique region critical for both reversible extensibility and structural maintenance of cardiac myofibrils.
- Published
- 1999
35. Degradation and protein release properties of microspheres prepared from biodegradable poly(lactide-co-glycolide) and ABA triblock copolymers: influence of buffer media on polymer erosion and bovine serum albumin release
- Author
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Karsten Mäder, Beate Bittner, Thomas Kissel, and Christian Witt
- Subjects
Time Factors ,Polymers ,Serum albumin ,Pharmaceutical Science ,Buffers ,In Vitro Techniques ,Polyethylene Glycols ,chemistry.chemical_compound ,Polymer chemistry ,medicine ,Copolymer ,Animals ,Particle Size ,Bovine serum albumin ,Polyglactin 910 ,chemistry.chemical_classification ,Ethylene oxide ,biology ,Electron Spin Resonance Spectroscopy ,Serum Albumin, Bovine ,Polymer ,Hydrogen-Ion Concentration ,Microspheres ,Biodegradation, Environmental ,chemistry ,Chemical engineering ,Ionic strength ,Delayed-Action Preparations ,biology.protein ,Cattle ,Emulsions ,Swelling ,medicine.symptom ,Drug carrier - Abstract
The aim of the present study was to investigate the influence of the chemical insertion of poly(ethylene oxide), PEO, into a poly(lactide-co-glycolide), PLG, backbone on the mechanisms of in vitro degradation and erosion of the polymer. For this purpose microspheres prepared by a modified W/O/W double emulsion technique using ABA triblock copolymers, consisting of PLG A-blocks attached to central PEO B-blocks were compared with microspheres prepared from PLG. Due to their molecular architecture the ABA triblock copolymers differed in their erosion and degradation behavior from PLG. Degradation occurred faster in the ABA polymers by cleavage of ester bonds inside the polymer backbone. Even erosion was shown to start immediately after incubation in different buffer media. By varying pH and ionic strength of the buffer it was found that both mass loss and molecular weight decay were accelerated in alkaline and acidic pH in the case of the ABA triblock copolymers. Although the pH of the medium had a moderate influence on the degradation of PLG, the molecular weight decay was not accompanied by a mass loss during the observation time. In a second set of experiments we prepared bovine serum albumin, BSA, loaded microspheres from both polymers. The release of BSA from ABA microspheres under in vitro conditions parallels the faster swelling and erosion rates. This could be confirmed by electron paramagnetic resonance, EPR, measurements with spin labeled albumin where an influx of buffer medium into the ABA microspheres was already observed within a few minutes. In contrast, PLG microspheres revealed a burst release without any erosion. The current study shows that the environmental conditions affected the degradation and erosion of the pure polymer microspheres in the same way as the release of the model protein. This leads to the conclusion that the more favorable degradation profile of the ABA triblock copolymers was responsible for the improvement of the release profile.
- Published
- 1999
36. A rapid protocol for cardiac troponin T gene mutation detection in familial hypertrophic cardiomyopathy
- Author
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Rainer Dietz, Karl-Josef Osterziel, Ludwig Thierfelder, Brenda Gerull, and Christian Witt
- Subjects
Genetics ,Mutation ,Polymorphism, Genetic ,Troponin T ,Genetic Linkage ,TNNT2 ,DNA Mutational Analysis ,Cardiomyopathy, Hypertrophic ,Biology ,Gene mutation ,medicine.disease_cause ,Troponin ,Molecular biology ,Exon ,genomic DNA ,medicine ,biology.protein ,Humans ,Genetic Testing ,Gene ,Genetics (clinical) ,Repetitive Sequences, Nucleic Acid - Abstract
Mutations in the human cardiac troponin T gene (TNNT2) are associated with familial hypertrophic cardiomyopathy (FHC) linked to chromosome 1q3 (CMH2). Mutation analyses of TNNT2 have been restricted to RNA-based screening methods because only the TNNT2 cDNA sequence was known. We characterized the genomic structure of 15 TNNT2 exons spliced into the adult isoform. A protocol for rapid mutation detection based on direct sequencing of large PCR-amplified genomic DNA fragments revealed a known TNNT2 mutation (Phe110Ile) in one of 30 FHC probands. Three polymorphic short tandem repeat elements (D1S477, D1S2622, and D1S1723), useful for FHC pedigree analyses at CMH2, were shown to be physically tightly linked to TNNT2.
- Published
- 1998
37. Improved Method for Isolating Cell-Free DNA
- Author
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Christian Witt, Sabine Weickmann, Bernd Schmidt, and Michael Fleischhacker
- Subjects
Biochemistry (medical) ,Clinical Biochemistry ,Cancer ,Improved method ,Biology ,medicine.disease ,Molecular biology ,DNA extraction ,law.invention ,chemistry.chemical_compound ,Biochemistry ,chemistry ,Cell-free fetal DNA ,law ,Nucleic acid ,medicine ,Free circulating dna ,DNA ,Polymerase chain reaction - Abstract
After Mandel and Metais (1) had originally described the existence of free circulating nucleic acids in plasma/serum in humans, Stroun et al. (2)(3) demonstrated that part of the free circulating DNA in cancer patients is of tumor origin (4)(5)(6). The quantity of DNA that can be isolated from human plasma/serum is very low and is frequently the limiting factor when a larger marker panel is to be used for genetic characterization of the DNA. In most of the recently published studies, commercially available columns were used for isolation of cell-free plasma/serum DNA, which made DNA isolation fast and allowed it to be standardized. Mandel and Metais (1) found ∼5.4 μg/mL total nucleic acids in the plasma in 10 healthy controls, and Stroun et al. (3) isolated between 150 ng/mL and 12 μg/mL DNA from the plasma of cancer patients. Much less DNA can be isolated with the aforementioned columns, however; we therefore wondered whether there are ways …
- Published
- 2005
38. In vivo and in vitro investigations of heterozygous nebulin knock-out mice disclose a mild skeletal muscle phenotype
- Author
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Patrick J. Cozzone, J.M. de Winter, K. Brohm, Coen A.C. Ottenheijm, Y. Le Fur, Marc Jubeau, N. Gretz, Benoît Giannesini, Siegfried Labeit, C. Kohl, Julien Gondin, Charlotte Gineste, Emilie Pecchi, Christophe Vilmen, David Bendahan, Henk Granzier, Christian Witt, Ger J.M. Stienen, Physics of Living Systems, Physiology, and ICaR - Heartfailure and pulmonary arterial hypertension
- Subjects
Male ,Heterozygote ,Magnetic Resonance Spectroscopy ,Gene Expression ,Muscle Proteins ,In Vitro Techniques ,Myopathies, Nemaline ,Severity of Illness Index ,Nebulin ,Mice ,Nemaline myopathy ,SDG 3 - Good Health and Well-being ,Gene expression ,medicine ,Animals ,Muscle Strength ,Muscle, Skeletal ,Genetics (clinical) ,Mice, Knockout ,Muscle Weakness ,biology ,Skeletal muscle ,Muscle weakness ,medicine.disease ,Congenital myopathy ,Phenotype ,Magnetic Resonance Imaging ,Cell biology ,Disease Models, Animal ,medicine.anatomical_structure ,Neurology ,Biochemistry ,Pediatrics, Perinatology and Child Health ,Knockout mouse ,Mutation ,biology.protein ,Neurology (clinical) ,medicine.symptom - Abstract
Nemaline myopathy is the most common congenital skeletal muscle disease, and mutations in the nebulin gene account for 50% of all cases. Recent studies suggest that the disease severity might be related to the nebulin expression levels. Considering that mutations in the nebulin gene are typically recessive, one would expect that a single functional nebulin allele would maintain nebulin protein expression which would result in preserved skeletal muscle function. We investigated skeletal muscle function of heterozygous nebulin knock-out (i.e., nebulin(+/-)) mice using a multidisciplinary approach including protein and gene expression analysis and combined in vivo and in vitro force measurements. Skeletal muscle anatomy and energy metabolism were studied strictly non-invasively using magnetic resonance imaging and 31P-magnetic resonance spectroscopy. Maximal force production was reduced by around 16% in isolated muscle of nebulin(+/-) mice while in vivo force generating capacity was preserved. Muscle weakness was associated with a shift toward a slower proteomic phenotype, but was not related to nebulin protein deficiency or to an impaired energy metabolism. Further studies would be warranted in order to determine the mechanisms leading to a mild skeletal muscle phenotype resulting from the expression of a single nebulin allele.
- Published
- 2013
39. Dynamic distribution of muscle-specific calpain in mice has a key role in physical-stress adaptation and is impaired in muscular dystrophy
- Author
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Koichi Suzuki, Atsu Aiba, Harumi Nakao, Yasuko Ono, Fujiko Kitamura, Kazuki Nakao, Michiko Sorimachi, Keiko Abe, Tatsuya Maeda, Hidenori Suzuki, Naoko Doi, Henk Granzier, Siegfried Labeit, Shoji Hata, Yukiko Kawabata, Koichi Ojima, Coen A.C. Ottenheijm, Christian Witt, Hiroyuki Sorimachi, Hiroyuki Kawahara, Noriko Toyama-Sorimachi, Julius Bogomolovas, Physiology, and ICaR - Heartfailure and pulmonary arterial hypertension
- Subjects
Aging ,medicine.medical_treatment ,Muscle Proteins ,Biology ,Sarcomere ,Pathogenesis ,Mice ,Downregulation and upregulation ,Myofibrils ,Mutant protein ,Physical Conditioning, Animal ,medicine ,Animals ,Regeneration ,Muscular dystrophy ,Muscle, Skeletal ,Protease ,Calpain ,Gene Expression Profiling ,Nuclear Proteins ,General Medicine ,Anatomy ,medicine.disease ,Adaptation, Physiological ,Cell biology ,Mice, Inbred C57BL ,Repressor Proteins ,Muscular Dystrophies, Limb-Girdle ,biology.protein ,Stress, Mechanical ,Myofibril ,Research Article - Abstract
Limb-girdle muscular dystrophy type 2A (LGMD2A) is a genetic disease that is caused by mutations in the calpain 3 gene (CAPN3), which encodes the skeletal muscle-specific calpain, calpain 3 (also known as p94). However, the precise mechanism by which p94 functions in the pathogenesis of this disease remains unclear. Here, using p94 knockin mice (termed herein p94KI mice) in which endogenous p94 was replaced with a proteolytically inactive but structurally intact p94:C129S mutant protein, we have demonstrated that stretch-dependent p94 distribution in sarcomeres plays a crucial role in the pathogenesis of LGMD2A. The p94KI mice developed a progressive muscular dystrophy, which was exacerbated by exercise. The exercise-induced muscle degeneration in p94KI mice was associated with an inefficient redistribution of p94:C129S in stretched sarcomeres. Furthermore, the p94KI mice showed impaired adaptation to physical stress, which was accompanied by compromised upregulation of muscle ankyrin-repeat protein-2 and hsp upon exercise. These findings indicate that the stretch-induced dynamic redistribution of p94 is dependent on its protease activity and essential to protect muscle from degeneration, particularly under conditions of physical stress. Furthermore, our data provide direct evidence that loss of p94 protease activity can result in LGMD2A and molecular insight into how this could occur.
- Published
- 2010
40. Modulation of Muscle Atrophy, Fatigue and MLC Phosphorylation by MuRF1 as Indicated by Hindlimb Suspension Studies on MuRF1-KO Mice
- Author
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Jeong Jung, Henk Granzier, Siegfried Labeit, Christine H. Kohl, Christian Witt, and Dittmar Labeit
- Subjects
medicine.medical_specialty ,Myosin Light Chains ,Article Subject ,Ubiquitin-Protein Ligases ,Health, Toxicology and Mutagenesis ,lcsh:Biotechnology ,Muscle Fibers, Skeletal ,Muscle Proteins ,lcsh:Medicine ,Biology ,lcsh:Chemical technology ,lcsh:Technology ,Tripartite Motif Proteins ,Mice ,Atrophy ,Internal medicine ,lcsh:TP248.13-248.65 ,Genetics ,medicine ,Animals ,Myocyte ,lcsh:TP1-1185 ,Phosphorylation ,Muscle, Skeletal ,Molecular Biology ,Mice, Knockout ,Soleus muscle ,Denervation ,Muscle fatigue ,lcsh:T ,lcsh:R ,Organ Size ,General Medicine ,Anatomy ,medicine.disease ,Muscle atrophy ,Muscular Atrophy ,Endocrinology ,Hindlimb Suspension ,Muscle Fatigue ,Molecular Medicine ,Electrophoresis, Polyacrylamide Gel ,medicine.symptom ,ITGA7 ,Muscle Contraction ,Research Article ,Biotechnology ,Muscle contraction - Abstract
MuRF1 is a member of the TRIM/RBCC superfamily, a gene family that encompasses a large variety of proteins, all sharing the conserved TRIM (TripartiteMotive) sequential array ofRING,B-box, and coiled-coil domains. Within this family, MuRF1(also named TRIM63) is a specialized member that contributes to the development of muscle atrophy and sarcopenia. Here we studied MuRF1's role in muscle atrophy during muscle unloading induced by hindlimb suspension. Consistent with previous studies, we found that MuRF1 inactivation leads to an attenuated muscle atrophy response. The amount of protection was higher as compared to the denervation model, and within the 10 day-suspension period the soleus muscle was spared from atrophy in MuRF1-KO mice. Contractility studies on hindlimb suspended muscle tissues suggested that MuRF1's functions extend beyond muscle trophicity and implicate MuRF1 in muscle fatigue and MLC phosphorylation control: soleus muscle from MuRF1-KO mice fatigued significantly faster and in addition showed a reduced posttetanic twitch potentiation. Thus the present work further established the role of MuRF1 in muscle atrophy and for the first time shows that MuRF1 plays a role in muscle fatigue and twitch potentiation.
- Published
- 2010
41. Thin filament length dysregulation contributes to muscle weakness in nemaline myopathy patients with nebulin deficiency
- Author
-
Henk Granzier, Siegfried Labeit, Ger J.M. Stienen, Alan H. Beggs, Christian Witt, Coen A.C. Ottenheijm, Physiology, and ICaR - Heartfailure and pulmonary arterial hypertension
- Subjects
Male ,Sarcomeres ,Adolescent ,Muscle Proteins ,Myopathies, Nemaline ,Sarcomere ,Nebulin ,Mice ,Nemaline myopathy ,Myofibrils ,Genetics ,medicine ,Animals ,Humans ,Child ,Muscle, Skeletal ,Molecular Biology ,Genetics (clinical) ,Mice, Knockout ,Muscle Weakness ,biology ,Skeletal muscle ,Muscle weakness ,Infant ,General Medicine ,Anatomy ,Articles ,medicine.disease ,Congenital myopathy ,Cell biology ,medicine.anatomical_structure ,Child, Preschool ,biology.protein ,Female ,medicine.symptom ,Myofibril ,Muscle contraction ,Muscle Contraction - Abstract
Nemaline myopathy (NM) is the most common non-dystrophic congenital myopathy. Clinically the most important feature of NM is muscle weakness; however, the mechanisms underlying this weakness are poorly understood. Here, we studied the muscular phenotype of NM patients with a well-defined nebulin mutation (NM-NEB), using a multidisciplinary approach to study thin filament length regulation and muscle contractile performance. SDS-PAGE and western blotting revealed greatly reduced nebulin levels in skeletal muscle of NM-NEB patients, with the most prominent reduction at nebulin's N-terminal end. Muscle mechanical studies indicated approximately 60% reduced force generating capacity of NM-NEB muscle and a leftward-shift of the force-sarcomere length relation in NM-NEB muscle fibers. This indicates that the mechanism for the force reduction is likely to include shorter and non-uniform thin filament lengths in NM-NEB muscle compared with control muscle. Immunofluorescence confocal microscopy and electron microscopy studies indicated that average thin filament length is reduced from approximately 1.3 microm in control muscle to approximately 0.75 microm in NM-NEB muscle. Thus, the present study is the first to show a distinct genotype-functional phenotype correlation in patients with NM due to a nebulin mutation, and provides evidence for the notion that dysregulated thin filament length contributes to muscle weakness in NM patients with nebulin mutations. Furthermore, a striking similarity between the contractile and structural phenotypes of nebulin-deficient mouse muscle and human NM-NEB muscle was observed, indicating that the nebulin knockout model is well suited for elucidating the functional basis of muscle weakness in NM and for the development of treatment strategies.
- Published
- 2009
42. Integrity of cell-free plasma DNA in patients with lung cancer and nonmalignant lung disease
- Author
-
Bernd Schmidt, Sabine Weickmann, Christian Witt, and Michael Fleischhacker
- Subjects
Lung Diseases ,Male ,Pathology ,medicine.medical_specialty ,Lung Neoplasms ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Diagnosis, Differential ,chemistry.chemical_compound ,History and Philosophy of Science ,medicine ,Biomarkers, Tumor ,Humans ,In patient ,Lung cancer ,Aged ,Dna integrity ,Aged, 80 and over ,General Neuroscience ,Plasma dna ,DNA ,DNA, Neoplasm ,respiratory system ,Middle Aged ,medicine.disease ,Real-time polymerase chain reaction ,Cell-free fetal DNA ,chemistry ,Lung disease ,Female ,Bronchoalveolar Lavage Fluid - Abstract
In several papers an increased quantity of cell-free plasma DNA as well as the presence of long DNA fragments in cell-free plasma and serum has been described. We isolated cell-free DNA from plasma, serum, and bronchial lavage supernatants from 33 lung cancer patients and 27 patients with a benign lung disease. The DNA was amplified by real-time PCR, and the quantity as well as the DNA integrity was determined. We did not find significant differences between the patient populations. Our results led us to conclude that this method is not useful in a diagnostic setting and is not able to differentiate between lung cancer patients and patients with a benign lung disease.
- Published
- 2008
43. Sarcoplasmic reticulum calcium uptake and speed of relaxation are depressed in nebulin-free skeletal muscle
- Author
-
Muthu Periasamy, Siegfried Labeit, Gopal J. Babu, Christian Witt, Peter Vangheluwe, Frank Wuytack, Henk Granzier, Coen A.C. Ottenheijm, Chi Fong, and Physiology
- Subjects
SERCA ,Muscle Relaxation ,Proteolipids ,Muscle Proteins ,In Vitro Techniques ,Calsequestrin ,Biochemistry ,Research Communications ,Nebulin ,Mice ,Myofibrils ,Microsomes ,Genetics ,medicine ,Myocyte ,Animals ,Muscle, Skeletal ,Molecular Biology ,Mice, Knockout ,Ion Transport ,biology ,Chemistry ,Skeletal muscle ,Electric Stimulation ,Cell biology ,Phospholamban ,Up-Regulation ,Sarcolipin ,Kinetics ,Sarcoplasmic Reticulum ,medicine.anatomical_structure ,Muscle relaxation ,biology.protein ,Calcium ,Biotechnology - Abstract
Previous work suggested that altered Ca2+ homeostasis might contribute to dysfunction of nebulin-free muscle, as gene expression analysis revealed that the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-inhibitor sarcolipin (SLN) is up-regulated >70-fold in nebulin knockout mice, and here we tested this proposal. We investigated SLN protein expression in nebulin-free and wild-type skeletal muscle, as well as expression of other Ca2+-handling proteins. Ca2+ uptake capacity was determined in isolated sarcoplasmic reticulum vesicles and in intact myofibers by measuring Ca2+ transients. Muscle contractile performance was determined in skinned muscle activated with exogenous Ca2+, as well as in electrically stimulated intact muscle. We found profound up-regulation of SLN protein in nebulin-free skeletal muscle, whereas expression of other Ca2+-handling proteins was not (calsequestrin and phospholamban) or was minimally (SERCA) affected. Speed of Ca2+ uptake was >3-fold decreased in sarcoplasmic reticulum vesicles isolated from nebulin-free muscle as well as in nebulin-free intact myofibers. Ca2+-activated stress in skinned muscle and stress produced by intact nebulin-free muscle were reduced to a similar extent compared with wild type. Half-relaxation time was significantly longer in nebulin-free compared with wild-type muscle. Thus, the present study demonstrates for the first time that nebulin might also be involved in physiological Ca2+ handling of the SR-myofibrillar system.—Ottenheijm, C. A. C., Fong, C., Vangheluwe, P., Wuytack, F., Babu, G. J., Periasamy, M., Witt, C. C., Labeit, S., Granzier, H. Sarcoplasmic reticulum calcium uptake and speed of relaxation are depressed in nebulin-free skeletal muscle.
- Published
- 2008
44. Nebulin regulates thin filament length, contractility, and Z-disk structure in vivo
- Author
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Henk Granzier, Christoph Burkart, Christian Witt, Yiming Wu, Dietmar Labeit, Mark McNabb, and Siegfried Labeit
- Subjects
Sarcomeres ,Molecular Sequence Data ,Muscle Fibers, Skeletal ,Muscle Proteins ,macromolecular substances ,Myopathies, Nemaline ,Sarcomere ,General Biochemistry, Genetics and Molecular Biology ,Article ,Protein filament ,Nebulin ,Mice ,medicine ,Animals ,Muscle thin filament assembly ,Amino Acid Sequence ,Muscle, Skeletal ,Molecular Biology ,Actin ,Mice, Knockout ,General Immunology and Microbiology ,biology ,General Neuroscience ,Biochemistry ,Nebulette ,biology.protein ,Biophysics ,Titin ,Calcium ,sense organs ,medicine.symptom ,Muscle contraction ,Muscle Contraction - Abstract
The precise assembly of the highly organized filament systems found in muscle is critically important for its function. It has been hypothesized that nebulin, a giant filamentous protein extending along the entire length of the thin filament, provides a blueprint for muscle thin filament assembly. To test this hypothesis, we generated a KO mouse model to investigate nebulin functions in vivo. Nebulin KO mice assemble thin filaments of reduced lengths and approximately 15% of their Z-disks are abnormally wide. Our data demonstrate that nebulin functions in vivo as a molecular ruler by specifying pointed- and barbed-end thin filament capping. Consistent with the shorter thin filament length of nebulin deficient mice, maximal active tension was significantly reduced in KO animals. Phenotypically, the murine model recapitulates human nemaline myopathy (NM), that is, the formation of nemaline rods combined with severe skeletal muscle weakness. The myopathic changes in the nebulin KO model include depressed contractility, loss of myopalladin from the Z-disk, and dysregulation of genes involved in calcium homeostasis and glycogen metabolism; features potentially relevant for understanding human NM.
- Published
- 2006
45. Expression of distinct classes of titin isoforms in striated and smooth muscles by alternative splicing, and their conserved interaction with filamins
- Author
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Henk Granzier, Dietmar Labeit, Christoph Burkart, Chi Fong, Sunshine Lahmers, Stephanie H. Witt, Christian Witt, Siegfried Labeit, and Mark McNabb
- Subjects
Adult ,Myofilament ,animal structures ,Transcription, Genetic ,Swine ,Filamins ,Blotting, Western ,Molecular Sequence Data ,Muscle Proteins ,Obscurin ,macromolecular substances ,Contractile Proteins ,Structural Biology ,Myosin ,medicine ,Myotilin ,Animals ,Humans ,Protein Isoforms ,Actinin ,Connectin ,Amino Acid Sequence ,Muscle, Skeletal ,Molecular Biology ,Aorta ,Cells, Cultured ,biology ,Microfilament Proteins ,Skeletal muscle ,Gene Expression Regulation, Developmental ,Muscle, Smooth ,Exons ,musculoskeletal system ,Molecular biology ,Protein Structure, Tertiary ,Actinin, alpha 2 ,Alternative Splicing ,Protein Transport ,medicine.anatomical_structure ,embryonic structures ,cardiovascular system ,biology.protein ,Titin ,Cattle ,Myofibril ,Protein Kinases ,Protein Binding - Abstract
While the role of titin as a sarcomeric protein is well established, its potential functional role(s) in smooth muscles and non-muscle tissues are controversial. We used a titin exon array to search for which part(s) of the human titin transcriptional unit encompassing 363 exons is(are) expressed in non-striated muscle tissues. Expression profiling of adult smooth muscle tissues (aorta, bladder, carotid, stomach) identified alternatively spliced titin isoforms, encompassing 80 to about 100 exons. These exons code for parts of the titin Z-disk, I-band and A-band regions, allowing the truncated smooth muscle titin isoform to link Z-disks/dense bodies together with thick filaments. Consistent with the array data, Western blot studies detected the expression of approximately 1 MDa smooth muscle titin in adult smooth muscles, reacting with selected Z-disc, I-band, and A-band titin antibodies. Immunofluorescence with these antibodies located smooth muscle titin in the cytoplasm of cultured human aortic smooth muscle cells and in the tunica media of intact adult bovine aorta. Real time PCR studies suggested that smooth muscle titins are expressed from a promoter located 35 kb or more upstream of the transcription initiation site used for striated muscle titin, driving expression of a bi-cistronic mRNA, coding 5' for the anonymous gene FL39502, followed 3' by titin, respectively. Our work showed that smooth muscle and striated muscle titins share in their conserved amino-terminal regions binding sites for alpha-actinin and filamins: Yeast two-hybrid screens using Z2-Zis1 titin baits identified prey clones coding for alpha-actinin-1 and filamin-A from smooth muscle, and alpha-actinin-2/3, filamin-C, and nebulin from skeletal muscle cDNA libraries, respectively. This suggests that the titin Z2-Zis1 domain can link filamins and alpha-actinin together in the periphery of the Z-line/dense bodies in a fashion that is conserved in smooth and striated muscles.
- Published
- 2006
46. MURF-1 and MURF-2 target a specific subset of myofibrillar proteins redundantly: towards understanding MURF-dependent muscle ubiquitination
- Author
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Christian Witt, Stephanie H. Witt, Henk Granzier, and Siegfried Labeit
- Subjects
Myosin light-chain kinase ,Ubiquitin-Protein Ligases ,Molecular Sequence Data ,Muscle Proteins ,Tripartite Motif Proteins ,Nebulin ,Mice ,Myofibrils ,Structural Biology ,medicine ,Myotilin ,Animals ,Amino Acid Sequence ,Muscle, Skeletal ,Molecular Biology ,biology ,Ubiquitin ,Myocardium ,Skeletal muscle ,musculoskeletal system ,Ubiquitin ligase ,medicine.anatomical_structure ,Biochemistry ,biology.protein ,Titin ,Titin binding ,Myofibril - Abstract
MURF-1, MURF-2 and MURF-3 are a specific class of RING finger proteins that are expressed in striated muscle tissues. MURF-1 has been suggested to act as an ubiquitin ligase, thereby controlling proteasome-dependent degradation of muscle proteins. Here, we performed yeast two-hybrid (YTH) screens of skeletal muscle cDNA libraries with MURF-1 baits to identify potential myocellular targets of MURF-1-dependent ubiquitination. This identified eight myofibrillar proteins as binding partners of MURF-1: titin, nebulin, the nebulin-related protein NRAP, troponin-I (TnI), troponin-T (TnT), myosin light chain 2 (MLC-2), myotilin and T-cap. YTH mating studies with MURF-1,2,3 baits indicated that these eight myofibrillar proteins are all targeted redundantly by both MURF-1 and MURF-2. Western blot studies on cardiac tissues from wild-type and MURF-1-deficient mice suggested that titin and nebulin were ubiquitinated at similar levels, and MLC-2 and TnI at reduced levels in MURF-1 KO mice. Mapping of the TnI and titin binding sites on MURF-1 peptide scans demonstrated their binding to motifs highly conserved between MURF-1 and MURF-2. Our data are consistent with a model in which MURF-1 and MURF-2 together target a specific set of myofibrillar proteins redundantly, most likely to control their ubiquitination-dependent degradation. Finally, our YTH screens identified the interaction of MURF-1 with 11 enzymes required for ATP/energy production in muscle including the mitochondrial ATP synthase and cytoplasmic creatine kinase. These data raise the possibility that MURF-1 may coordinately regulate the energy metabolism of mitochondrial and cytoplasmic compartments.
- Published
- 2005
47. Expression of tyrosine hydroxylase and neuropeptide tyrosine in mouse sympathetic airway-specific neurons under normal situation and allergic airway inflammation
- Author
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Nelly Frossard, Q. T. Dinh, Christian Witt, Burghard F. Klapp, Liliana Cifuentes, David A. Groneberg, Christian Peiser, and Axel Fischer
- Subjects
medicine.medical_specialty ,Allergy ,Sympathetic nervous system ,Sympathetic Nervous System ,Tyrosine 3-Monooxygenase ,Immunology ,Neuropeptide ,Biology ,chemistry.chemical_compound ,Mice ,Norepinephrine ,Internal medicine ,Tachykinins ,medicine ,Hypersensitivity ,Immunology and Allergy ,Animals ,Neuropeptide Y ,Neurotransmitter ,Lung ,Neurons ,Mice, Inbred BALB C ,Tyrosine hydroxylase ,respiratory system ,Allergens ,medicine.disease ,Immunohistochemistry ,Autonomic nervous system ,Endocrinology ,medicine.anatomical_structure ,chemistry ,Models, Animal ,Catecholamine ,Female ,Neuron ,medicine.drug - Abstract
Summary Background The traditional neurotransmitter catecholamine and the neuropeptide tyrosine in sympathetic airway nerves have been proposed to be involved in the pathogenesis of airway diseases. Objective The aim of the present study was to investigate the effect of allergic airway inflammation on the expression of catecholamine enzyme tyrosine hydroxylase (TH), neuropeptide tyrosine (NPY) and tachykinins in mouse sympathetic airway ganglia. Methods Using neuronal tracing in combination with immunohistochemistry, the present study was designed to characterize TH, NPY and tachykinin profiles of superior cervical (SCG) and stellate ganglia after allergen challenge. Results The vast majority of fast blue-labelled SCG neurons (allergen: 97.5±1.22% (mean±SEM) vs. controls: 94.5±1.48%, P=0.18) and stellate neurons (allergen: 95.3±1.01% vs. controls: 93.6±1.33%, P=0.34) were immunoreactive for TH. Of the TH immunoreactive and fast blue-labelled SCG neurons, 52.0±1.01% allergen vs. 51.2±3.58% controls (P=0.83) and stellate neurons, 57.3%±0.97 allergen vs. 56.4±1.65% controls (P=0.64) were positive for TH only but not NPY, whereas 45.3±1.05% allergen vs. 43.3±1.18% controls (P=0.47) of fast blue-labelled SCG neurons and 37.9±0.86% allergen vs. 37.1±1.24% controls (P=0.62) of fast blue-labelled stellate neurons were immunoreactive for both TH and NPY immunoreactivities. There was a trend of an increase, but not significant one, in the percentage of TH-/NPY-immunoreactive and fast blue-labelled neurons in allergen-treated animals in comparison with the controls. Tachykinins, however, were not expressed by sympathetic neurons and were also not induced in sympathetic neurons after allergen challenge. Conclusion The present study indicates that allergic airway inflammation does not alter the expression of noradrenalin and NPY in sympathetic ganglia and also shows that sympathetic neurons do not respond to allergic airway inflammation with tachykinins induction. However, a participation of catecholamine and NPY in the pathogenesis of allergic airway inflammation cannot be excluded in the present study as a higher neurotransmitter output per neuron following allergen challenge could be possible.
- Published
- 2005
48. Transcriptional down-regulation of neurotrophin-3 in chronic obstructive pulmonary disease
- Author
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David A. Groneberg, K. Fan Chung, Christian Peiser, Christian Witt, Paul R. Eynott, Axel Fischer, Reinhard Erbes, and Pia Welker
- Subjects
Pathology ,medicine.medical_specialty ,Transcription, Genetic ,Clinical Biochemistry ,Down-Regulation ,Bronchi ,Neurotrophin-3 ,Biology ,Biochemistry ,Pulmonary Disease, Chronic Obstructive ,Neurotrophin 3 ,medicine ,Transcriptional regulation ,Humans ,RNA, Messenger ,Molecular Biology ,Regulation of gene expression ,COPD ,Lung ,medicine.disease ,respiratory tract diseases ,medicine.anatomical_structure ,Nerve growth factor ,Gene Expression Regulation ,Immunology ,biology.protein ,Immunohistochemistry ,Neurotrophin - Abstract
Chronic obstructive pulmonary disease (COPD) leads to progressive development of airflow limitation and is characterised by cough, mucus hypersecretion and inflammatory changes. These characteristic features of the disease may be modulated by neural mediators such as neurotrophins (NT). Here we examined the expression and transcriptional regulation of neurotrophins in bronchial biopsies of COPD patients and compared the data to control biopsies. Histology revealed characteristic changes in the COPD tissues, including remodelling of the epithelial lining. RT-PCR demonstrated the mRNA expression of neurotrophins in all biopsies. Immunohistochemistry confirmed the expression of different proteins. To assess changes in the transcriptional expression level, quantitative real-time PCR was carried out and revealed differential mRNA expression of neurotrophins, with marked down-regulation of NT-3 mRNA expression and constant levels of nerve growth factor (NGF), brain-derived nerve factor (BDNF), and NT-4/5 mRNA expression. The present data on neurotrophin-specific transcriptional down-regulation of NT-3 in human COPD indicate a pathophysiological role for neurotrophins in COPD.
- Published
- 2005
49. Detection of cell-free DNA in bronchial lavage fluid supernatants of patients with lung cancer
- Author
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Burkhard Jandrig, Christian Witt, T. Carstensen, Michael Fleischhacker, Bernd Schmidt, and E. Engel
- Subjects
Male ,Lung Neoplasms ,Loss of Heterozygosity ,Biology ,Polymerase Chain Reaction ,General Biochemistry, Genetics and Molecular Biology ,law.invention ,Loss of heterozygosity ,chemistry.chemical_compound ,History and Philosophy of Science ,law ,Carcinoma, Non-Small-Cell Lung ,Carcinoma ,medicine ,Biomarkers, Tumor ,Humans ,Carcinoma, Small Cell ,Lung cancer ,Polymerase chain reaction ,Aged ,General Neuroscience ,DNA, Neoplasm ,Middle Aged ,medicine.disease ,Molecular biology ,chemistry ,Cell-free fetal DNA ,Nucleic acid ,Leukocytes, Mononuclear ,Microsatellite ,Feasibility Studies ,Female ,Bronchoalveolar Lavage Fluid ,DNA ,Microsatellite Repeats - Abstract
Recently, it was shown that it is possible to isolate free circulating DNA from plasma/serum of patients with benign and malignant diseases. In addition, several groups were able to detect tumor-associated alterations in these nucleic acids. We wondered whether any nucleic acids are detectable in cell-free bronchial lavage supernatants, which until now have been discarded after cell harvest. Additionally, we wanted to find out if it is possible to detect tumor-associated alterations in these DNA molecules. DNA was isolated from cell-free lavage supernatants from 30 lung cancer patients, and the DNA was examined for microsatellite alterations. Intact DNA could be isolated from all cell-free bronchial lavage supernatants. Microsatellite alterations were found in lavage supernatants of 12 of 30 patients and in lavage cells of 6 of 30 patients. Altogether, alterations were found in 14 of 30 patients. Thus, we could demonstrate for the first time that it is possible to isolate intact DNA from cell-free bronchial lavage supernatants. Their quantity and quality are sufficient for further amplification via polymerase chain reaction. Altogether, tumor-associated changes were detected in the DNA of 47% of the patients that were analyzed.
- Published
- 2004
50. Muscle-specific RING finger-2 (MURF-2) is important for microtubule, intermediate filament and sarcomeric M-line maintenance in striated muscle development
- Author
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Siegfried Labeit, Carol C. Gregorio, Cynthia N. Perry, Christian Witt, and Abigail S. McElhinny
- Subjects
Sarcomeres ,Cytoplasm ,Time Factors ,Ubiquitin-Protein Ligases ,Blotting, Western ,Cell Culture Techniques ,Intermediate Filaments ,Down-Regulation ,Muscle Proteins ,Obscurin ,Chick Embryo ,Transfection ,Microtubules ,Desmin ,Tripartite Motif Proteins ,Myoblast fusion ,Intermediate Filament Proteins ,Myocyte ,Animals ,Vimentin ,Connectin ,Myocytes, Cardiac ,RNA, Messenger ,Intermediate filament ,Cytoskeleton ,Fluorescent Antibody Technique, Indirect ,Muscle, Skeletal ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,Muscles ,Gene Expression Regulation, Developmental ,Proteins ,Cell Biology ,Oligonucleotides, Antisense ,Cell biology ,Protein Structure, Tertiary ,biology.protein ,Titin ,Myofibril ,Chickens ,Protein Kinases ,Muscle Contraction ,Signal Transduction - Abstract
The efficient functioning of striated muscle is dependent upon the structure of several cytoskeletal networks including myofibrils, microtubules, and intermediate filaments. However, little is known about how these networks function together during muscle differentiation and maintenance. In vitro studies suggest that members of the muscle-specific RING finger protein family (MURF-1, 2, and 3) act as cytoskeletal adaptors and signaling molecules by associating with myofibril components (including the giant protein, titin), microtubules and/or nuclear factors. We investigated the role of MURF-2, the least-characterized family member, in primary cultures of embryonic chick skeletal and cardiac myocytes. MURF-2 is detected as two species (approximately 55 kDa and approximately 60 kDa) in embryonic muscle, which are down-regulated in adult muscle. Although predominantly located diffusely in the cytoplasm, MURF-2 also colocalizes with a sub-group of microtubules and the M-line region of titin. Reducing MURF-2 levels in cardiac myocytes using antisense oligonucleotides perturbed the structure of stable microtubule populations, the intermediate filament proteins desmin and vimentin, and the sarcomeric M-line region. In contrast, other sarcomeric regions and dynamic microtubules remained unaffected. MURF-2 knock-down studies in skeletal myoblasts also delayed myoblast fusion and myofibrillogenesis. Furthermore, contractile activity was also affected. We speculate that some of the roles of MURF-2 are modulated via titin-based mechanisms.
- Published
- 2004
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