Embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.

It has been demonstrated that embryonic chicken gizzard smooth muscle contains a unique embryonic myosin light chain of 23,000 mol wt, called L23 (Katoh, N., and S. Kubo, 1978, Biochem. Biophys. Acta, 535:401-411; Takano-Ohmuro, H., T. Obinata, T. Mikawa, and T. Masaki, 1983, J. Biochem. (Tokyo), 93:903-908). When we examined myosins in developing chicken ventricular and pectoralis muscles by two-dimensional gel electrophoresis, the myosin light chain (Le) that completely comigrates with L23 was detected in both striated muscles at early developmental stages. Two monoclonal antibodies, MT-53f and MT-185d, were applied to characterize the embryonic light chain Le of striated muscles. Both monoclonal antibodies were raised to fast skeletal muscle myosin light chains; the former antibody is specific to fast muscle myosin light chains 1 and 3, whereas the latter recognizes not only fast muscle myosin light chains but also the embryonic smooth muscle light chain L23. The immunoblots combined with both one- and two-dimensional gel electrophoresis showed that Le reacts with MT-185d but not with MT-53f. These results strongly indicate that Le is identical to L23 and that embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.

Mammalian ribosomal protein: analysis by electrophoresis on polyacrylamide gel.

Ribosomal protein from five mammalian tissues when analyzed by discontinuous electrophoresis on polyacrylamide gel at pH 4.5 yielded 24 bands. Densitometric tracings indicated that the patterns of the basic ribosomal proteins from the several tissues were qualitatively similar. Protein from Escherichia coli ribosomes analyzed at pH 4.5 gave 29 bands, and the pattern was different from that of mammalian ribosomal protein. No distinct band was found when mammalian ribosomal protein was analyzed at pH 8.3 (acidic proteins). Ribosomal protein from Escherichia coli gave eight bands at pH 8.3. Thus, the structure of the genes responsible for synthesis of ribosomal protein in several mammalian tissues is the same, and different genes direct synthesis of ribosomal protein in bacteria.

Selective crystallization of horse isoferritins.

Various precipitating agents were examined in order to crystallize horse heart and spleen ferritins. Cadmium sulfate induced the crystallization of the spleen ferritin, while 2-methyl-2,4-pentanediol and poly(ethylene glycol) only induced that of the heart ferritin. Isoelectric focusing analysis showed that the crystals grown from cadmium sulfate contained only the more acidic isoferritins, and those grown from methyl pentanediol only the less acidic isoferritins. Heart ferritin crystallizes in a cubic space group, as previously reported for spleen ferritin crystals grown from cadmium sulfate.

Bovine aorta actin. Development of an improved purification procedure and comparison of polymerization properties with actins from other types of muscle.

Crude actin extracts from acetone-dried powder of the muscle layer of bovine aorta contain an actin-modulating protein which promotes nucleation of actin monomers and decreases the average length of actin filaments in a Ca2+-dependent manner. This observation has allowed the development of an improved purification procedure for aorta actin which increases the yield 2- to 3-times. The actin obtained with this procedure consists of 77% alpha- and 23% gamma-isoelectric species. Pure aorta actin is indistinguishable from actins from skeletal, cardiac and chicken-gizzard smooth muscle in its polymerization rate, critical concentration, and reduced viscosity when polymerized with KCl at 25 degrees C. It differs from sarcomeric actins, but not from chicken-gizzard smooth muscle actin, in the temperature dependence of polymerization equilibria in KCl. This difference correlates with the amino acid replacements Val-17----Cys-17 and Thr-89----Ser-89, supporting a conclusion drawn from other studies that the N-terminal portion of actin polypeptide chain contains sites important for polymerization.

Purification and characterization of the activated mineralocorticoid receptor from rat myocardium.

Cardiac cytosol from adrenalectomized rats was radiolabelled with 10 nM tritiated RU 26752, R 5020 or aldosterone, to saturate the mineralocorticoid receptor (MCR) in the presence of 1 microM RU 38486 to block the glucocorticoid and progestin receptors. Free steroids were removed by charcoal treatment and the radiolabelled cytosol was passed through a phosphocellulose column. The MCR peak in the phosphocellulose eluate was activated at 25 degrees C for 45 min, adsorbed onto the DNA-cellulose and finally extracted once each with buffers containing 1 M potassium chloride or 25 mM magnesium chloride. The pooled DNA-cellulose extracts, reequilibrated with 10 nM [3H]RU 26752, were resolved as a single, homogeneous band of 78 kDa upon polyacrylamide gel electrophoresis. Ion-exchange analysis of the purified MCR on DEAE-cellulose-52 revealed a single peak in the 0.017 M sodium phosphate region with both RU 26752 and R 5020, but aldosterone dissociated during this procedure. Molecular filtration on Ultrogel AcA-44 columns revealed a major 145 kDa peak, with some smaller components of 40 and 80 kDa. These hydrodynamic properties of the purified MCR are at variance with those of the native receptor in crude myocardial cytosol, and suggest that some post-translational modifications in vivo may be required for the expression of MCR-mediated responses.

Melittin induces HII phase formation in cardiolipin model membranes.

The interaction of melittin with bovine heart cardiolipin model membranes was investigated via binding assays, 31P-NMR, freeze-fracture electron microscopy, small angle X-ray diffraction and fluorescence based fusion assays. A strong binding (Kd less than 10(-7) M) appeared to be accompanied by the formation of large structures, resulting from a fusion process of extremely fast initial rate. As the melittin content is increased, bilayer structure is gradually lost and from a cardiolipin to melittin ratio of about 6 the lipid starts to organize itself in an hexagonal HII phase. At lower temperatures (T less than 40 degrees C) the coexistence of another structure is observed, characterized by a broad isotropic 31P-NMR signal and giving rise to sharp X-ray reflections, most probably a cubic phase, as suggested also be freeze-fracture images, showing orderly stacked particles. The results are discussed in relation to contrasting observations on the structural changes induced by melittin in the zwitterionic phospholipid system of dipalmitoylphosphatidylcholine (Dufourcq. J. et al. (1986) Biochim. Biophys. Acta 859, 33-48). The biological relevance of the observations with respect to the process of protein insertion into membranes is indicated.

Lipid specific penetration of melittin into phospholipid model membranes.

The relative depth of penetration of melittin into egg phosphatidylcholine and bovine heart cardiolipin model membranes was investigated using fluorescence spectroscopy techniques. The tryptophan intrinsic fluorescence shift suggests a more hydrophobic surrounding of this residue in cardiolipin, while the accessibility for charged and uncharged aqueous quenchers is decreased in the cardiolipin system when compared with the phosphatidylcholine-bound situation. A lipid incorporated hydrophobic, collisional quencher and a resonance energy transfer acceptor on the other hand are more effective in quenching the tryptophan fluorescence of cardiolipin bound melittin. The combination of these results is interpreted as prove of a deeper positioning of the tryptophan containing part of the peptide molecule in the cardiolipin system in comparison with the situation in phosphatidylcholine. Models that take this difference into account are presented, which try to explain the opposite effect of melittin binding to the two lipid systems with respect to supramolecular structure, as reported in the preceding article (Batenburg, A.M., Hibbeln, J.C.L., Verkleij, A.J. and De Kruijff, B. (1987) Biochim. Biophys. Acta 903, 142-154).

Beta adrenoceptor density and adenylate cyclase response in right atrial and left ventricular myocardium of patients with mitral valve disease.

Beta adrenoceptor density, measured by radioligand binding techniques, is reportedly much higher in atrial than in ventricular myocardium of patients with mitral valve disease. In the present study adenylate cyclase activity, both basal and in response to beta adrenergic agonists and sodium fluoride, in biopsy preparations from these same patients was significantly lower in atrial than in ventricular tissue when stimulated with either isoproterenol, noradrenaline, isoproterenol combined with terbutaline, or sodium fluoride. Terbutaline stimulated and basal adenylate cyclase activity was not significantly different in the two cardiac regions. The ratios of receptor density to isoproterenol stimulated and to sodium fluoride stimulated adenylate cyclase activity were 4-5 times higher in atrial than in ventricular biopsy specimens. Thus ventricular beta receptors, although present in comparatively low concentrations, are coupled to considerably more catalytic moieties of the receptor-adenylate cyclase complex than their atrial counterparts. The reason for this is probably a relative lack of coupling proteins (N components) in atrial tissue. A weak positive correlation between receptor density and isoproterenol stimulated adenylate cyclase activity was found in atrial but not in ventricular tissue. This may indicate individual variation in receptor-adenylate cyclase coupling. Furthermore, no correlation was found between atrial and ventricular values for any variable. One reason for this may be the different haemodynamic stresses in the two cardiac chambers.

Protection by oral pretreatment with taurine against the negative inotropic effects of low-calcium medium on isolated perfused chick heart.

Chicks were given taurine by mouth using a cannula pushed down into the oesophagus. This treatment had protected the heart, when subsequently removed, against the decline of contractile force in the perfused heart brought about by low-calcium media. A small but statistically significant increase in taurine concentration occurred in the ventricular tissue of such pretreated hearts. In contrast, pretreatment with taurine did not affect ventricular calcium level. Addition of taurine (3 to 100 mmol . litre-1) into the perfusing solution did not alter the cyclic AMP level in the control perfused hearts, and failed to induce slow channel action potentials in hearts whose fast Na+ channels had been inactivated by high K+ (25 mmol . litre-1). However, taurine perfusion produced a significant positive inotropic action which became stable after 5 to 10 min of exposure.

The use of monoclonal antibodies for the species and tissues distribution of phospholamban.

Monoclonal antibodies have been raised against canine phospholamban. Two antibodies have been used in the study of phospholamban distribution in tissues, and in different animal species. The antibodies recognized the three different forms of phospholamban: non-phosphorylated, phosphorylated, and dissociated forms. A survey of nine rabbit tissues revealed that phospholamban is a cardiac muscle specific protein. Phospholamban from different mammalian hearts were found to be identical with respect to molecular weight and antigenicity.

Immunoelectron microscopic localization of alpha-actinin on Lowicryl-embedded thin-sectioned tissues.

A procedure has been developed for the immunoelectron microscopic localization of intracellular antigens on thin-sectioned tissues. The tissues were fixed in a periodate-lysine-paraformaldehyde solution or a formaldehyde-glutaraldehyde combination and embedded in the acrylate-methacrylate mixture, Lowicryl K4M (Polaron), which was polymerized under ultraviolet irradiation at -35 degrees C. Thin sections were mounted on gold grids, immunostained using an indirect method with ferritin-labeled antibodies, and, optionally, counterstained with osmium tetroxide and/or lead citrate and uranyl acetate. The procedure provided good morphologic preservation of the cell architecture in adult and embryonic heart, and skeletal and smooth muscle tissue, as well as nonmuscle cells. At the same time it retained the antigenicities of several contractile proteins, including myosin, tropomyosin, actin, and alpha-actinin. The method has advantages over en bloc staining techniques in that the problem of antibody penetration into the cells is eliminated and careful controls can be performed on adjacent sections. This technique will be useful for localizing, at the ultrastructural level, contractile and other selected proteins in a variety of muscle and non-muscle cells. Details of the new protocol and a description of the results of using antibody against the contractile protein, alpha-actinin, are given.

Liquid ventilation of primates.

Three adult monkeys were anesthetized with ketamine and ventilated with fluorocarbon liquid [perfluoro bis (1, 4-isopropoxy) butane (Caroxin-D)] at 1 atmosphere on two separate occasions. During five runs, liquid ventilation was continued for 60 minutes. The sixth run was continued for ten minutes. Arterial blood gas levels during and after liquid ventilation were adequate for survival. Three years after the first period of liquid ventilation, the animals were killed. Approximately 0.001 mg of fluorocarbon per gram of tissue was present in the kidney, liver, brain, spleen, muscle, and heart. Fat contained approximately seven to nine times this amount, and the lung and pulmonary lymph nodes contained approximately 1,000 times this amount. In no case was it clinically evident that the monkeys had undergone periods of liquid ventilation. We conclude that primates can be ventilated successfully with liquid fluorocarbon on at least two separate occasions and can return to breathing air without obvious deleterious effects, but fluorocarbon is retained in small amounts for at least three years.

The earliest form of C-protein expressed during striated muscle development is immunologically the same as cardiac-type C-protein.

A monoclonal antibody (C-315) specific for cardiac-type C-protein was prepared and, in combination with other antibodies specific for fast and slow skeletal muscle C-proteins, it was used to investigate the expression of C-protein isoforms in developing striated muscle cells in vivo and in vitro. During embryonic development of skeletal muscles, a C-protein recognized by C-315 appeared first but only transiently, it being replaced subsequently by two other isoforms recognized by the antibodies to slow and fast skeletal muscle C-proteins in a fiber-type specific manner as previously demonstrated (Obinata et al. (1984) Develop. Biol. 101, 116-124). In contrast, only cardiac-type C-protein was detected in cardiac muscle throughout the developmental stages. When myogenesis in vitro was monitored using the same antibodies, C-315 binding appeared first in multinucleated myotubes as in vivo which was followed by the sequential expression of two other C-protein variants. The reactivity of C-315 as well as that of anti-slow and anti-fast skeletal C-protein antibodies persisted during muscle development in culture. Thus, this study demonstrates that the earliest form of C-protein expressed in striated muscles may either be a cardiac-type isoform or a unique embryonic protein containing an epitope in common with the adult cardiac-type protein, and that transitions of C-protein isoform expression characteristic of each fiber-type occur during muscle development in vivo but not in vitro.

Rapid and high-yield purification of porcine heart tissue-type plasminogen activator by heparin-sepharose choromatography.

A rapid and high-yield procedure for the purification of single polypeptide tissue-type plasminogen activator (t-PA) from porcine heart tissue has been developed. Delipidated heart tissue was extracted with 0.45 M potassium acetate. The extract was fractionated with ammonium sulfate and purified by a combination of affinity chromatography on heparin-Sepharose CL-6B and gel filtration on Toyopearl HW-55S. The final product had a specific activity of 220,000 IU/mg protein and gave a single protein band (apparent molecular weight; 67,000) in SDS-polyacrylamide gels in the presence or absence of a reducing agent. The increase in specific activity was 3,200-fold, most of which was achieved in the step of heparin-Sepharose chromatography. The yield calculated from the active ammonium sulfate precipitate was about 90% and 500 micrograms or more of the purified enzyme was obtained from 1 kg wet tissue. This procedure may also be useful for the large-scale production of highly purified t-PA from other tissues or tissue culture cells.

Propranolol as xanthine oxidase inhibitor: implications for antioxidant activity.

Propranolol is the beta-blocker most widely used in the management of cardiovascular disorders. It has been proposed that propranolol may act as a "chain-breaking" antioxidant. We have directly examined the ability of propranolol to inhibit superoxide-dependent, iron-promoted cardiac membrane phospholipid peroxidation, with xanthine oxidase (XOD) as a physiologically-recognized, enzymatic superoxide generator. Our results demonstrate that propranolol not only protects cardiac-membrane lipid from peroxidative damage, but also acts as a simple, reversible XOD inhibitor, noncompetitive with xanthine substrate. Propranolol, at effective antiperoxidant and XOD-inhibitory concentrations, cannot scavenge superoxide radical. The antiperoxidative profile of propranolol resembles that of the known XOD inhibitor allopurinol, although allopurinol, a tight-binding substrate-analog competitive with xanthine, inhibits XOD in a manner mechanistically very different from that of propranolol. Furthermore, the antiperoxidative profiles of both propranolol and allopurinol do not resemble those of chain-breaking antioxidants such as alpha-tocopherol. These data, along with the tendency of propranolol to concentrate in myocardial membranes and cytosol, suggest that the observed antioxidant action of propranolol, as a consequence of XOD inhibition, could play a pharmacologic role in propranolol's cardioprotective effects.

The distribution of mercury in various tissues of guinea-pigs after application of dental amalgam fillings (a pilot study).

Mercury accumulation in brain, kidney, liver and heart following insertion of amalgam in the teeth of guinea-pigs has been studied. During the accelerated wear of the amalgam in these gnawing animals, a significant mercury accumulation in the above tissues was demonstrated. Finely diffused and abraded amalgam must not be ignored as a source of absorbable mercury.

Ageing processes in collagens from different tissues of rats.

It has been demonstrated by solubility data, that the differences in cross-linking and degree of polymerisation in connective tissue from different organs of a single animal are much more pronounced than the increase which occurs with advancing age. Judged from the cross-linking analysis one can therefore conclude that in a single animal there are connective tissues which differ in their degree of maturity. It is therefore conceivable to suggest that the level of cross-linking should not be taken as the measure of the biological age of the animal.