i) Human cardiac cDNA libraries were constructed and clones encoding human cardiac troponin I (cTnI) isolated. ii) Both the entire <i>cTNI</i> cDNA and a 5'-portion, were expressed in <i>Escherichia coli</i> as fusion products with β-galactosidase. The full-length cDNA was also expressed unfused. iii) The murine monoclonal antibody 29Mu is specific for human and baboon cTnI whereas the 31Mu antibody reacts with cTnI from a range of species. These antibodies might be useful in the imaging of necrotic cardiac tissue. In this study, 31Mu was found to bind to all prepared forms of cTnI antigen, in enzyme-linked immunosorbant assays (ELISAs) and, where tested, in Western blots. Thus, its epitope is localised towards the N-terminus of cTnI. 29Mu bound to the bacterially-produced unfused cTnI but not to the fusion polypeptides or crude bovine cTn. Its ability to bind to human cardiac extracts was related to the method of their preparation, indicating that the epitope of 29Mu shows greater conformational dependency than that of 31Mu. iv) cDNAs encoding the variable domains of 29Mu and 31Mu were cloned and chimaeric antibodies, comprising murine variable and human constant regions produced. Humanised antibodies, in which only the antigen-binding sites were of murine origin, were also produced. Such recombinant antibodies would be expected to exhibit reduced immunogenicity in man. v) Neither the chimaeric nor humanised antibody versions of 29Mu bound cTnI. Chimaerised 31Mu reacted with all forms of cTnI but did not show complete equivalence to 31Mu. An antibody containing the humanised 31 kappa chain and the chimaeric heavy chain was reactive to all forms cTnI in ELISAs but its efficiency of binding, relative to that of the chimaeric antibody, was dependent upon the antigen source. Humanised heavy chains were produced utilising two different human frameworks and the framework, showing closer homology to the 31Mu variable domain, supported antigen binding with fewer murine residue substitutions. However, both successful humanised 31 antibodies showed some cross-reactivity.

Background: Hyperthyroidism has substantial effects on the circulatory system. The cardiac biomarkers NT-proBNP and troponin I (cTNI) have proven useful in identifying cats with myocardial disease but have not been as extensively investigated in hyperthyroidism. Hypothesis: Plasma NT-proBNP and cTNI concentrations are higher in cats with primary cardiac disease than in cats with hyperthyroidism and higher in cats with hyperthyroidism than in healthy control cats. Animals: Twenty-three hyperthyroid cats, 19 cats with HCM without congestive heart failure, and 19 euthyroid, normotensive healthy cats eight years of age or older. Fourteen of the hyperthyroid cats were re-evaluated three months after administration of 131I. Methods: A complete history, physical examination, complete blood count, serum biochemistries, urinalysis, blood pressure measurement, serum T4 concentration, plasma concentrations of NT-proBNP and cTNI, and echocardiogram was prospectively obtained from each cat. Results: Hyperthyroid and HCM cats had plasma NT-proBNP and cTNI concentrations that were significantly greater than healthy older cats, but there was no significant difference between hyperthyroid and HCM cats with respect to concentration of either biomarker. In hyperthyroid cats that were re-evaluated three months after 131I treatment, plasma NT-proBNP and cTNI concentrations as well as ventricular wall thickness decreased. Conclusions and Clinical Relevance: Although there may be a role for NT-proBNP in monitoring the cardiac response to treatment of hyperthyroidism, neither NT-proBNP nor cTNI can be used to distinguish hyperthyroid cats from cats with HCM. Therefore, the thyroid status of older cats should be ascertained prior to interpreting results of cardiac biomarker testing. / Master of Science

Matrix metalloproteinase2 (MMP2) was recently revealed to have targets and actions within the cardiac myocyte. In ischemia/reperfusion (I/R) injury, MMP2 is activated and degrades troponin I (TnI) and actinin. The regulation of intracellular MMP2 activity is relatively unknown and is thus the subject of this thesis. The localization of MMP2 in caveolae of endothelial cells suggests that caveolin1 (Cav1) may play a role in regulating MMP2. Whether Cav1 is responsible for regulating MMP2 in the heart is unknown. A Cav1 knockout mouse model was used to explore the role Cav1 may play in the regulation of MMP2 activity. The initial studies found that MMP2 and Cav1 were colocalized in cardiomyocytes and that MMP2 activity in Cav1/ hearts was markedly enhanced. Additionally, the caveolin scaffolding domain inhibited MMP2 activity in a concentrationdependent manner. To explore whether increased MMP2 in Cav1/ hearts translates to impaired cardiac function, Cav1+/+ and Cav1/ isolated working hearts were physiologically challenged with increasing increments of left atrial preload followed by increasing concentrations of isoproterenol. Cav1/ hearts show similar or better cardiac function compared to Cav1+/+ hearts following preload challenge or adrenergic stimulation in vitro, and this appears unrelated to changes in MMP2. Though the function of Cav1/ hearts appears similar to that of Cav1+/+ hearts during physiological situations, whether this is the case during I/R injury is not known. Cav1+/+ and Cav1/ isolated working mouse hearts exposed to global, noflow ischemia showed no functional differences. However, Cav1/ hearts had significantly higher levels of both TnI and actinin following I/R than Cav1+/+ hearts. Posttranslational modifications of the intracellular MMP2 substrates could alter susceptibility to MMP2 proteolysis. Isolated working mouse hearts were exposed to isoproterenol and/or I/R injury to examine the phosphorylation status of TnI. Isoproterenol and I/R both result in the phosphorylation of TnI, however, isoproterenol lead to a more highly phosphorylated form of TnI than that observed in hearts exposed I/R alone. These and subsequent studies will further reveal the molecular mechanisms that underlie the complex interactions between Cav1 and MMP2. This may eventually lead to a novel avenue of therapeutic intervention for heart diseases.

名古屋大学博士学位論文 学位の種類:博士(医療技術学) (課程) 学位授与年月日:平成20年3月25日

Troponin I (TnI), like many striated muscle contractile proteins, consists of multiple isoforms encoded by a multigene family whose members are differentially expressed in the different striated muscle cell types. Two TnI genes, TnIfast and TnIslow, are expressed in skeletal muscle the former in fast muscle fibers, the latter in slow fibers. The tissue- and fiber-type-specificities of the TnI fast and slow genes are driven by transcriptional enhancer elements: a Slow Upstream Regulatory Element (SURE) upstream of the TnIslow gene and a fast Intronic Regulatory Element (IRE) within the first intron of the TnIfast gene. Within the 144 bp IRE, there are 4 known cis elements, and the aim of this work was to initiate the studies to map the element(s) that are chiefly responsible for directing the fast-fiber-specificity of IRE-driven gene expression. This was approached by making IRE end-deletion constructs lacking either the left-most or right-most IRE cis-element. These IRE derivatives were coupled to a reporter gene consisting of a minimal (enhancer-dependent) TnIfast promoter linked to E. coli beta-galactosidase coding sequences. The transcriptional activity of these constructs was first evaluated in cell culture transfection experiments, and then by in vivo gene transfer into adult mouse skeletal muscles. The conclusion of these experiments was that fast-fiber-specificity of IRE-driven gene expression resides in the left-most 30 bp of the IRE, a region including an E-box binding site for myogenic regulatory factors of the MyoD family.

No description available.

国立情報学研究所で電子化したコンテンツを使用している。

Thesis (M.Sc.). / Written for the Dept. of Biology. Title from title page of PDF (viewed 2008/01/14). Includes bibliographical references.

No description available.

Es wurden Studien zur biochemischen Evaluierung des akuten Koronarsyndroms unter drei Aspekten unternommen: (1) Die klinische Anwendung der biochemischen Marker zur Diagnosesicherung und Risikostratifizierung bei Patienten mit akutem Koronarsyndrom, (2) die Bedeutung kardialer Troponine unter besonderen physiologischen und pathophysiologischen Bedingungen und (3) die tierexperimentelle Evaluierung passagerer Ischämie mit der Fragestellung, ob eine Erhöhung kardialer Troponine im Plasma bei reversibler Myokardschädigung auftreten kann. Die klinische Anwendung der kardialen Marker kann zuverlässig nach aktuellen Richtlinien erfolgen und sollte immer auch die Messung eines kardialen Troponins beinhalten. Troponin-Testsysteme sollten in klinischen Studien hinsichtlich des diagnostischen und prognostischen Nutzens evaluiert worden sein. Die in dieser Arbeit untersuchten Systeme wiesen nachweisbare Unterschiede auf, die jedoch für den klinischen Einsatz nicht bedeutsam sind. Geringe Troponin T-Erhöhungen bei Patienten mit akutem Koronarsyndrom und eher geringer oder atypischer Symptomatik haben eine eindeutige prognostische Aussagekraft und ergänzen damit signifikant die klinische Einschätzung und das EKG. Kardiale Troponine können bei herzgesunden Probanden unter extremer körperlicher Leistung gering erhöht sein. Die prognostische Bedeutung dieser Befunde ist unklar. Kardiales Troponin kann bei kardial asymptomatischen Patienten mit Niereninsuffizienz ohne sichere prognostische Bedeutung erhöht sein. Tierexperimentell ergeben sich Hinweise darauf, daß es bei reversibler Ischämie im Sinne eines "continuous release" zur Freisetzung von kardialem Troponin bzw. zumindest von Degradationsprodukten desselben kommen kann. Die zukünftige Entwicklung von Richtlinien zum Einsatz biochemischer Marker wird entscheidend davon abhängen, ob auf den erhobenen Befunden therapeutische Strategien mit nachgewiesenem Nutzen im Sinne einer "evidence based medicine" aufbauen. / Three studies with respect to the biochemical evaluation of acute coronary syndromes were undertaken : (1) The clinical application of biochemical markers for diagnosis and risk stratification in patients with acute coronary syndroms. (2) The value of cardiac troponins under different physiological and pathophysiological conditions. (3) Experimental transient myocardial ischemia in an animal model with respect to the question, whether elevation of cardiac troponins in plasma perhaps occur after reversible myocardial damage. The clinical appilication of cardiac markers is sufficiently possible following actual guidelines and should include cardiac troponin measurement. The troponin test-system has to be evaluated in clinical studies with respect to its diagnostic and prognostic properties. In this study significant differences between two cardiac troponin I test-systems could be shown. The differences were below clinical relevance. Mild to moderate elevations of cardiac troponin T in patients with acute coronary syndromes and low grade Braunwald class angina are of prognostic value and add on information obtained by history and ECG. Cardiac troponins may be found elevated in apparently healthy athletes after exhaustive exercise. The prognostic significance of these findings remains unclear. Cardiac troponins are frequently elevated in renal insufficiency patients without cardiac symptoms. These elevations had no prognostic value in our study. The experimental data suggest that troponins are released in reversible myocardial damage during transient ischemia. This adds evidence on the continuous release hypothesis of cardiac troponins and degradation products. The future development of guidelines for the use of cardiac markers in daily routine will strictly depend on therapeutic consequences which base on the analytical results in the sense of evidence based medicine.