Troponin-I is the inhibitory protein of the regulatory troponin-tropomyosin complex in striated muscle responsible for sensitising the interaction between actin and myosin to changes in calcium ion concentration. In cardiac muscle, a tissue specific isoform of troponin-I is present which possesses an additional 26 -27 residues on the N-terminus when compared with the fast and slow skeletal isoforms [I]. Two adjacent phosphoserine residues have been identified in this sequence at positions 23 and 24 [2,31. Both of these residues are substrates for CAMP and cGMP dependent protein kinases as well as protein kinase C [4]. Phosphorylation of troponin-I at these site(s) occurs in the heart in-vivo and is correlated with change in calcium sensitivity of the actin-activated myosin M& ATPase and force production [S,61. Stimulation of the heart with D-adrenergic agents also results in increased phosphorylation of troponin-I [7]. Given the cardiac specific nature of these phosphorylation sites it is important to find probes that can be used to investigate the relationship between extent of phosphorylation and biochemical function. We have therefore produced a number of monoclonal antibodies to cardiac troponin-I with this aim in mind.A panel of monoclonal antibodies directed against human and canine cardiac troponin-I were produced by conventional hybridoma techniques. Antibodies were screened by enzyme-linked immunoassay (ELISA) at an early stage for cardiac specificity and only monoclonals showing no reactivity with fast or slow skeletal isoforms were investigated. Furthermore, monoclonals were also tested for species cross-reactivities by ELISA using purified troponin-I from human, canine, porcine, bovine and rat hearts. From an original seven cardiac specific monoclonal antibodies, four (20L?G, 19/C12, I W S and B4/J36) were found to be generally species cross-reactive and were selected for further study. methods for their ability to bind to cardiac troponin-I in the myofibril. The binding of fluorescent labelled antibodies to troponin-I inside damaged cardiac myocytes was investigated by fluorescence-activated cell sorting and binding to isolated cardiac myofibrils was studied by combined phase-contrast and indirect immunofluorescence. Myocytes prepared by collagenase perfusion of isolated rat hearts contained a mixture of live and necrotic cells. These were incubated with each monoclonal antibody which was labelled with a fluoroscein isothyocyanate labelled second antibody. Non-immune mouse I& acted as a control in each cell sorting experiment and nuclear propidium iodide uptake acted as an independent fluorescent marker of cell viability. Antibodies 20nG and 19/C12 showed significant specific binding to troponin-I. B4B6 showed no binding while I W S gave intermediate binding. When tested on isolated myofibrils, the above results were confirmed with the exception that 1O/FS showed no binding. This was probably a result of the conformational state of the cross-bridges in myofibrils which were prepared under rigor condi...
Basic fibroblast growth factor (bFGF) has been implicated in the development of the heart and in the proliferation of cardiac myocytes [ 1,2 and reviewed in 31. Mammalian ventricular myocytes lose their ability to undergo mitosis soon after birth and subsequent repair of damaged myocardium is limited to scar formation and hypertrophy of existing myocytes. While both ventricular and atrial myocytes proliferate in the foetal period, there is controversy as to whether atrial myocytes retain mitotic potential in the adult and whether this can be activated given an appropriate stimulus [2,4]. Atrial myocytes are less differentiated than their ventricular counterparts and in the adult retain certain protein phenotypes characteristic of the foetal atria 151.bFGF inhibits terminal differentiation of skeletal muscle cells [6] while in isolated neonatal cardiac myocytes, bFGF and insulin-like growth factors stimulate DNA synthesis and myocyte proliferation [2]. Higher levels of bFGF have been reported in adult atrial than ventricular tissue in the chicken, bovine, rat and sheep heart [2] although there is uncertainty as to the absolute levels present in heart tissue and their precise cellular location.In the present study we have determined the levels of bFGF in the developing and adult bovine atrium and ventricle using two independent immunoassays. The first which was developed in this laboratory, was a sensitive competitive enzyme-linked immunoassay employing a polyclonal rabbit antibody raised against recombinant bovine bFGF. The assay which employed a goat anti-rabbit peroxidase conjugate was able to detect bFGF in tissue extracts down to levels of 0.1 ng/ml. The second assay was a dioimmunoassay (Amersham International plc. ) employing 125-iodine labelled recombinant bovine bFGF and a rabbit antisemm. Bound and unbound bFGF were separated by a donkey anti-rabbit semm coated onto magnetizable polymer particles. This assay was also sensitive to levels of 0.1 ng/ml.The antibodies utilised showed less than 0.01% cross reactivity in their respective assay systems with bovine recombinant acidic fibroblast growth factor.Bovine hearts from foetal animals aged 11 weeks to 40 weeks gestation (term), from neonates (4 weeks old) and adults were dissected into atria and ventricles immediately after death, frozen in liquid nitrogen and stored at -7OoC until assay. As the extraction procedure may influence the final recoveries of bFGF, two different extraction methods, one at acidic pH [ 71 and one at neutral pH [8] were employed. Table 1. bFGF levels in bovine atrium and ventricle Each value rmresents the mean of three determinations. bFGF (udkn muscle1 Age Atrium Ventricle 1 1 weeks gestation 62 1Foetal levels in both atrium and ventricle which ranged from 329 -650 Hg/kg were consistently higher than in the adult (182 -275 pg/kg; Table 1 ) . Levels in the foetal atria were also 20 -100% higher than in the foetal ventricle throughout gestation and in the neonate although this was reversed in the adult. There did not appear to be an...
Four major inorganic cations --Na +, K--, Mg 2+ and Ca 2+ contribute mainly to the regulation of activity of muscle cells. The aim of the present comparative study was fo reveal the principal factors which determine the great variety of the cationic contents in different muscles of various animais. Functionally distinguished muscles of 70 species of marine, freshwater and terrestrial animals of 6 types of metazoans were investigated. The analysis of this muscle variability in regulation to the intracellular cati, onic contents has confirmed the qualitative heterogeneity of the muscle fibre populations investigated. The data obtained have permitted a subdivision of the latter into some definite groups, depending on the ionic composition of the extracellular fluids (environmental factor) as well as on the direction and the level of the functional specialization of muscles (inherent factor). In general a linear reciprocal relationship between [K+]i and [Na+]i in different skeletal muscles of various species was observed. In the saine organism an acceleration of a contractile response of the muscles is associated with an increase of a cellular selectivity of K § as compared to Na+; (SK/Na) -V c = A + B/SK/N,~. The character of this relation (the value of B) is species specific and reflects the level of development of a locomotory activity of the animais. At the same time the results obtained enable us to draw the conclusion that the trends in the cationic parameters in muscles do not coincide with the general course of the animal evolution. Itis demonstrated that the interrelation between functional (contractile) properties of skeletal muscles and cationic distribution patterns can be used as an 'ionic testing' method in medical-biological practice for diagnosing the physiological state of muscles. The results are discussed in terms of the physiological significance of inorganic cations involvement in the intracellular information transmission.
Hypoxanthine is a central metabolite in ATP metabolism and its salvage to IMP is catalysed by hypoxanthine phosphoribosyltransferase (HPRT: EC 2.4.2.8.).Since deficiency of this enzyme in man causes profound neurological dysfunction and testicular atrophy, a regular supply of substrate should be available. However, only ATP depletion has been proven to produce a marked increase in hypoxanthine output (Harkness et al., 1983), but ATP depletion rarely occurs in human tissues under normal conditions. This study was set up to investigate hypoxanthine output under conditions of ATP turnover rather than depletion. This was achieved by exercising at low-tomoderate levels of ATP use and examining hypoxanthine, xanthine and urate output.Exercise was performed for 2 min on a bicycle ergometer by ten normal volunteers (five men and five women) at five different work rates on five consecutive days of one week.
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