A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

Loyola University Chicago

The EzrA protein of Bacillus subtilis is a negative regulator for FtsZ (Z)-ring formation. It is able to modulate the frequency and position of Z-ring formation during cell division. The loss of this protein results in cells with multiple Z rings located at polar as well as medial sites; it also lowers the critical concentration of FtsZ required for ring formation (P. A. Levin, I. G. Kurster, and A. D. Grossman, Proc. Natl. Acad. Sci. USA 96:9642-9647, 1999). We have studied the regulation of ezrA expression during the growth of B. subtilis and its effects on the intracellular level of EzrA as well as the cell length of B. subtilis. With the aid of promoter probing, primer extension, in vitro transcription, and Western blotting analyses, two overlapping sigmaA-type promoters, P1 and P2, located about 100 bp upstream of the initiation codon of ezrA, have been identified. P1, supposed to be an extended -10 promoter, was responsible for most of the ezrA expression during the growth of B. subtilis. Disruption of this promoter reduced the intracellular level of EzrA very significantly compared with disruption of P2. Moreover, deletion of both promoters completely abolished EzrA in B. subtilis. More importantly, the cell length and percentage of filamentous cells of B. subtilis were significantly increased by disruption of the promoter(s). Thus, EzrA is required for efficient cell division during the growth of B. subtilis, despite serving as a negative regulator for Z-ring formation.

Transcription regulation of ezrA and its effect on cell division of Bacillus subtilis.

The aim of this study is to determine and identify the possible molecular mechanisms of anti-cancer effect of rhubarb under the physiologically achievable concentrations by using an ex vivo approach.

Suppression on metastasis by rhubarb through modulation on MMP-2 and uPA in human A549 lung adenocarcinoma: an ex vivo approach.

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament assembly protein that stabilizes sarcomeric structure and regulates cardiac function; however, the profile of cMyBP-C degradation after myocardial infarction (MI) is unknown. We hypothesized that cMyBP-C is sensitive to proteolysis and is specifically increased in the bloodstream post-MI in rats and humans. Under these circumstances, elevated levels of degraded cMyBP-C could be used as a diagnostic tool to confirm MI. To test this hypothesis, we first established that cMyBP-C dephosphorylation is directly associated with increased degradation of this myofilament protein, leading to its release in vitro. Using neonatal rat ventricular cardiomyocytes in vitro, we were able to correlate the induction of hypoxic stress with increased cMyBP-C dephosphorylation, degradation, and the specific release of N'-fragments. Next, to define the proteolytic pattern of cMyBP-C post-MI, the left anterior descending coronary artery was ligated in adult male rats. Degradation of cMyBP-C was confirmed by a reduction in total cMyBP-C and the presence of degradation products in the infarct tissue. Phosphorylation levels of cMyBP-C were greatly reduced in ischemic areas of the MI heart compared to non-ischemic regions and sham control hearts. Post-MI plasma samples from these rats, as well as humans, were assayed for cMyBP-C and its fragments by sandwich ELISA and immunoprecipitation analyses. Results showed significantly elevated levels of cMyBP-C in the plasma of all post-MI samples. Overall, this study suggests that cMyBP-C is an easily releasable myofilament protein that is dephosphorylated, degraded and released into the circulation post-MI. The presence of elevated levels of cMyBP-C in the blood provides a promising novel biomarker able to accurately rule in MI, thus aiding in the further assessment of ischemic heart disease.

Cardiac myosin binding protein-C is a potential diagnostic biomarker for myocardial infarction.

In the title compound, C(17)H(14)N(2)O(2), the hy-droxy-ethanimine group adopts an anti-periplanar conformation. In the crystal, mol-ecules are linked by O-Hâ‹¯N hydrogen bonds, forming zigzag chains running along the c axis.

(E)-2-({2-[(E)-(Hy-droxy-imino)-meth-yl]phen-oxy}meth-yl)-3-phenyl-acrylonitrile.

Cardiac myosin binding protein-C (cMyBP-C) plays a role in sarcomeric structure and stability, as well as modulating heart muscle contraction. The 150 kDa full-length (FL) cMyBP-C has been shown to undergo proteolytic cleavage during ischemia-reperfusion injury, producing an N-terminal 40 kDa fragment (mass 29 kDa) that is predominantly associated with post-ischemic contractile dysfunction. Thus far, the pathogenic properties of such truncated cMyBP-C proteins have not been elucidated. In the present study, we hypothesized that the presence of these 40 kDa fragments is toxic to cardiomyocytes, compared to the 110 kDa C-terminal fragment and FL cMyBP-C. To test this hypothesis, we infected neonatal rat ventricular cardiomyocytes and adult rabbit ventricular cardiomyocytes with adenoviruses expressing the FL, 110 and 40 kDa fragments of cMyBP-C, and measured cytotoxicity, Ca(2+) transients, contractility, and protein-protein interactions. Here we show that expression of 40 kDa fragments in neonatal rat ventricular cardiomyocytes significantly increases LDH release and caspase 3 activity, significantly reduces cell viability, and impairs Ca(2+) handling. Adult cardiomyocytes expressing 40 kDa fragments exhibited similar impairment of Ca(2+) handling along with a significant reduction of sarcomere length shortening, relaxation velocity, and contraction velocity. Pull-down assays using recombinant proteins showed that the 40 kDa fragment binds significantly to sarcomeric actin, comparable to C0-C2 domains. In addition, we discovered several acetylation sites within the 40 kDa fragment that could potentially affect actomyosin function. Altogether, our data demonstrate that the 40 kDa cleavage fragments of cMyBP-C are toxic to cardiomyocytes and significantly impair contractility and Ca(2+) handling via inhibition of actomyosin function. By elucidating the deleterious effects of endogenously expressed cMyBP-C N-terminal fragments on sarcomere function, these data contribute to the understanding of contractile dysfunction following myocardial injury.

Pathogenic properties of the N-terminal region of cardiac myosin binding protein-C in vitro.

Earlier studies have shown that cardiac myosin binding protein-C (cMyBP-C) is easily releasable into the circulation following myocardial infarction (MI) in animal models and patients. However, since its release kinetics has not been clearly demonstrated, no parameters are available to judge its efficacy as a bona fide biomarker of MI in patients with MI. To make this assessment, plasma levels of cMyBP-C and six known biomarkers of MI were determined by sandwich enzyme-linked immunosorbent assay in patients with MI who had before and after Percutaneous Transcoronary Angioplasty (PTCA), as well as healthy controls. Compared to healthy controls (22.3 Â± 2.4 ng/mL (n=54)), plasma levels of cMyBP-C were significantly increased in patients with MI (105.1 Â± 8.8 ng/mL (n=65), P<0.001). Out of 65 patients, 24 had very high levels of plasma cMyBP-C (116.5 Â± 13.3 ng/mL), indicating high probability of MI. Importantly, cMyBP-C levels were significantly decreased in patients (n=40) at 12 hours post-PTCA (41.2 Â± 9.3 ng/mL, P<0.001), compared to the patients with MI. Receiver operating characteristic analysis revealed that a plasma cMyBP-C reading of 68.1 ng/mL provided a sensitivity of 66.2% and a specificity of 100%. Also, myoglobin, carbonic anhydrase and creatine kinase-MB levels were significantly increased in MI patients who also had higher cMyBP-C levels. In contrast, levels of cardiac troponin I, glycogen phosphorylase and heart-type fatty acid binding protein were not significantly changed in the samples, indicating the importance of evaluating the differences in release kinetics of these biomarkers in the context of accurate diagnosis. Our findings suggest that circulating cMyBP-C is a sensitive and cardiac-specific biomarker with potential utility for the accurate diagnosis of MI.

Suresh Govindan

Department of Cell and Molecular Physiology

Loyola University Chicago

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Suresh Govindan

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell and Molecular Physiology

Loyola University Chicago

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Department of Cell and Molecular Physiology