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W A Clark

Publications and source records attributed to W A Clark.

At least 73 records · Page 4Linked to original sources
PubMed1984

Immunofluorescence analysis of the primordial myosin detectable in embryonic striated muscle.

Immunofluorescence analysis showed that the earliest myosin detectable in both the embryonic chicken heart and somitic myotome, the precursor to skeletal muscle, was strongly reactive with two different monoclonal antibodies specific for the heavy chain of cardiac ventricular myosin, but it showed no reactivity with affinity-purified polyclonal antibodies specific for the heavy chains of either fast-twitch or slow-tonic skeletal myosins. The heart remained reactive exclusively with the antibodies to cardiac myosin throughout development, while late embryonic (day 20) skeletal muscles were strongly reactive only with their homologous skeletal myosin antibodies. Our findings suggest that the primordial myosin heavy chain detectable in both forms of embryonic chicken striated muscle, the myotome and the heart, is immunologically distinct from myosins expressed in later embryonic as well as adult skeletal muscles, but it contains antigenic determinants similar to those present in cardiac ventricular myosin.

Animals↗
PubMed1984

Classification and characterization of cardiac isomyosins.

The structural properties of cardiac isomyosins from several species were compared using native gel electrophoresis, analysis of proteolytic digests, analysis of monoclonal antibody reactivity to specific proteolytic fragments on electroblots and S1 nuclease mapping with cDNA probes. The structure of specific regions of the myosin molecule was analyzed by reacting monoclonal antibodies with chymotryptic peptides of myosin separated by two-dimensional electrophoresis. The pattern of fragments reactive with antibody CCM-52 (epitope in LMM) was identical in all types of V3 isomyosin examined, and different in each type of V1 isomyosin. Peptides reactive with RCM-79 (epitope in HMM) were different from those reactive with CCM-52 and were also significantly different in each type of myosin examined. Thus, HC-alpha is structurally similar in the LMM portion of the molecule in all animals examined, while in the HMM region there are significant structural differences. HC-alpha differs from HC-beta, with structural differences in both LMM and HMM. We have also shown that atrial myosin HC and ventricular HC-alpha in the rabbit are indistinguishable both by RIA and peptide mapping analysis. The same conclusion was derived after analysis of the myosin HC mRNA expressed in rabbit atria and ventricles. Using cDNA probes specific for the alpha and beta myosin HC mRNA, we could not distinguish between the atrial myosin mRNA and ventricular HC alpha (V1 isomyosin) mRNA by S1 nuclease mapping experiments. Classification of different cardiac myosins is largely based on their mobility on native gel electrophoresis, immunological cross-reactivity, and ATPase activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Quantitation of myosin in muscle.

The amount of myosin per gram of cardiac and skeletal muscle was determined in sodium dodecyl sulfate-solubilized tissue homogenates by radioimmunoassay and by isotope dilution. In the rabbit ventricle, there was an average of 27 mg myosin/g wet wt of tissue. In chickens, the myosin content of typical "red" (anterior latissimus dorsi) and "white" (posterior latissimus dorsi) skeletal muscles was higher than that of ventricular muscle, averaging 36 and 48 mg/g of tissue, respectively. The stoichiometry of the heavy and light chains in cardiac myosin was also determined from the quantitative binding of 125I-labeled Coomassie blue to each subunit after separation of the subunits by sodium dodecyl sulfate-gel electrophoresis. With this procedure, we found that the combined light-chain subunits contributed 19% of the myosin mass. After adjustment for the light-chain contribution, the myosin heavy-chain content of the rabbit ventricle averaged 22 mg/g wet wt of tissue.

Animals↗
PubMed1983

Change in synthesis rates of alpha- and beta-myosin heavy chains in rabbit heart after treatment with thyroid hormone.

The ventricles of rabbit heart contain three isomyosins (V1, V2, and V3) whose proportions change during development, and after treatment with thyroid hormone. The V1 isomyosin accumulates rapidly after administration of thyroid hormone and we have shown that this results from marked change in the synthesis rates of the heavy chains of both V1 (HC alpha) and V3 (HC beta). The fractional rates of synthesis were calculated from the specific radioactivities of leucine in plasma and in HC alpha and HC beta after continuous infusion with [3H] leucine for 1 h. The V1 and V3 isomyosins were separated by affinity chromatography with monoclonal antibodies and the heavy chains were then isolated by sodium dodecyl sulfate-electrophoresis. In 32- to 35-day-old euthyroid rabbits, the fractional rates of synthesis of the two heavy chains were not significantly different with a combined mean rate of 0.14 day-1. Administration of 3,5,3',5'-tetraiodothyronine (200 micrograms/kg/day) caused a 3-fold increase in the synthesis rate of HC alpha and a comparable decrease in that of HC beta. The fractional synthesis rate of atrial myosin heavy chain also changed when 3,5,3',5'-tetraiodothyronine was administered, increasing from 0.15 day-1 to an average of 0.20 day-1 after 1 day of treatment. This was not accompanied by any change in the heavy chain type as determined by radioimmunoassay.

Animals↗
PubMed1983

Analysis of population cytokinetics of chick myocardial cells in tissue culture.

The growth of embryonic chick cardiac myocytes and fibroblasts in tissue culture was evaluated by the kinetics of nuclear labeling during continuous exposure to [3H]thymidine. The fraction of mitotically active cells, the mean intermitotic period and the population doubling times were determined in each cell type during 3 weeks in culture. After 24 hr in culture, 90% of the muscle cells were mitotically active with minimal population doubling times of 65 hr. By 17 days in culture only 5% of the myocytes continued to divide with population doubling times greater than 3000 hr. Primarily, the lengthening of doubling times was due to a withdrawal of cells from the mitotic cycle and much less to a lengthening of the intermitotic period. Growth of cardiac muscle cells from embryonic hearts from 4 to 10 days of development was also compared. Muscle cells from younger hearts displayed greater mitotic activity than those from older hearts at equivalent times in culture.

Animals↗
PubMed1983

Patterns of cellular injury in myocardial ischemia determined by monoclonal antimyosin.

The development of cellular injury in the rat left ventricle resulting from left coronary artery occlusion was examined by immunofluorescence after intravenous injection of monoclonal antimyosin. Cardiac muscle cells that bound antimyosin during ischemia were localized by staining sections with fluorescein-conjugated anti-mouse IgG. Fluorescent staining was detectable within the ischemic region of the left ventricle 3 hr after occlusion and injection of antimyosin. After 6 hr of ischemia, the highly irregular margin of the ischemic zone was clearly outlined by fluorescent cells. At 3-6 hr after occlusion, marked heterogeneity in cellular staining was observed in the epicardial half of the ischemic area, with intensely fluorescent cells intermixed with cells of markedly lower fluorescence. By 24 hr, a homogeneous pattern of staining was observed throughout the ischemic zone. In nonischemic regions of the heart and in rats treated for 24 hr with antimyosin without occlusion, there were only background levels of staining. We conclude that: (i) visualization of ischemic cells via antimyosin provides a sensitive means for examining developing patterns of injury; (ii) the heterogeneity of staining during early ischemia may reflect variation in cellular resistance to deprivation; and (iii) the pattern of fluorescence at the margin of the occluded region indicates that the "border zone" is composed of interdigitating ischemic and nonischemic tissues.

Animals↗
PubMed1983

Myosin isoenzymes in normal and hypertrophied human ventricular myocardium.

We tested the hypothesis that hypertrophy of the human heart is associated with the redistribution of ventricular isomyosins. Human cardiac myosin was isolated from autopsy samples of left ventricular free wall of patients with cardiac hypertrophy and of fetal, young, and adult subjects without heart disease. The following parameters were studied: electrophoretic migration in denaturing and non-denaturing conditions; immunological cross-reactivities with three different types of antibodies; and early phosphate burst size and steady state ATPase activities stimulated by K+-EDTA, Ca++, Mg++, and actin. The antibodies were chosen for their ability to recognize selectively the rat V1 and V3 cardiac isomyosins. The first type was a monoclonal antibody, CCM-52, prepared against embryonic chick cardiac myosin, the second was an anti-beef atrial myosin, and the third was an anti-rat V1 myosin. CCM-52 reacted with a greater affinity with rat V3 than with rat V1, and was a probe of mammalian V3. Anti-beef atrial myosin and anti-rat V1 myosin both recognized specifically beef atrial and rat V1 myosins, and were thus considered as probes of mammalian V1. Under non-denaturing conditions, human myosins migrated as rat V3 isomyosin; under denaturing conditions, no difference was observed in any of the electrophoretic parameters between all samples tested, except for the fetal hearts which contained a fetal type of light chain. The immunological studies indicated that human myosins were composed mostly of a V3 type (HV3), but contained also some V1 isomyosin. A technique was developed to quantify the amount of human VI isomyosin which was found to range from almost 0 to 15% of total myosin, and to vary from one heart to the other, regardless of the origin of the heart. Enzymatic studies showed no significant difference between normal, hypertrophied, and fetal hearts in any of the activities tested. However, there was a significant correlation between Ca++-stimulated ATPase activities and HV1 amount (at 0.05 M KCl, n = 18, r2 equal 0.49, P less than 0.01; at 0.5 M KCl, n = 18, r 2 = 0.5, P less than 0.01). These data demonstrate the heterogeneity of human ventricular myosin, which appears to be composed, as in other mammalian species, of V1 and V3 isoforms of different ATPase activities (V1 greater than V3). However it seems that V1 to V3 shifts do not appear to be of physiological significance in the adaptation of human heart to chronic mechanical overloads.

Actins↗
PubMed1983

Studies on fat digestion, absorption, and transport in the suckling rat. IV. In vivo rates of triacylglycerol secretion by intestine and liver.

The rate of triacylglycerol entry into plasma in 14- to 15-day-old suckling rats was measured using the Triton WR1339 method. When the pups were left with the mother and allowed to nurse continually, the rate was 31.8 +/- 2.3 mumol/hr per rat. If the pups were removed from the mother with full stomachs and kept in a warm box at 30-32 degrees C, the rate was 12.5 +/- 1.5 mumol/hr per rat. The rate of triacylglycerol output from the intestine measured in lymph duct-cannulated animals (kept in a warm box at 30-32 degrees C) was 14.9 +/- 2.7 mumol/hr per rat. Triton treatment of lymph duct-cannulated animals caused only a small increase in plasma triacylglycerol concentration (less than 2 mumol/hr per rat). These data suggest that nearly 100% of plasma triacylglycerol arises from intestinal input in the suckling rat.

Animal Population Groups↗
PubMed1983

Lipoprotein metabolism in the suckling rat: characterization of plasma and lymphatic lipoproteins.

Suckling rat plasma contains (in mg/dl): chylomicrons (85 +/- 12); VLDL (50 +/- 6); LDL (200 +/- 23); HDL1 (125 +/- 20); and HDL2 (220 +/- 10), while lymph contains (in mg/dl): chylomicrons (9650 +/- 850) and VLDL (4570 +/- 435) and smaller amounts of LDL and HDL. The lipid composition of plasma and lymph lipoproteins are similar to those reported for adults, except that LDL and HDL1 have a somewhat higher lipid content. The apoprotein compositions of plasma lipoproteins are similar to those of adult lipoproteins except for the LDL fraction, which contains appreciable quantities of apoproteins other than apoB. Although the LDL fraction was homogeneous by analytical ultracentrifugation and electrophoresis, the apoprotein composition suggests the presence of another class of lipoproteins, perhaps a lipid-rich HDL1. The lipoproteins of lymph showed low levels of apoproteins E and C. The triacylglycerols in chylomicrons and VLDL of both lymph and plasma are rich in medium-chain-length fatty acids, whereas those in LDL and HDL have little or none. Phospholipids in all lipoproteins lack medium-chain-length fatty acids. The cholesteryl esters of the high density lipoproteins are enriched in arachidonic acid, whereas those in chylomicrons, VLDL, and LDL are enriched in linoleic acid, suggesting little or no exchange of cholesteryl esters between these classes of lipoproteins. The fatty acid composition of phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine were relatively constant in all lipoprotein fractions, suggesting ready exchange of these phospholipids. However, the fatty acid composition of phosphatidylethanolamine in plasma chylomicrons and VLDL differed from that in plasma LDL, HDL1, and HDL2. LDL, HDL1, and HDL2 were characterized by analytical ultracentrifugation and shown to have properties similar to that reported for adult lipoproteins. The much higher concentration of triacylglycerol-rich lipoproteins in lymph, compared to plasma, suggests rapid clearance of these lipoproteins from the circulation.

Animals↗
PubMed1982

Immunochemical analysis of fibronectin using monoclonal antibodies.

Cloned hybrid cell lines secreting antibodies directed against human plasma fibronectin were prepared according to the methods of Kohler and Milstein (Kohler, G. and Milstein, C. (1975) Nature (London) 256, 495-497 and (1976) Eur. J. Immunol. 6, 511-519). The specificity of each monoclonal antibody for fibronectin was established from autoradiograms of radioimmunoprecipitates following SDS-polyacrylamide gel electrophoresis. The monoclonal antibodies were reactive with both native and SDS-denatured fibronectin. Ascites fluids obtained from infected isogenic mice precipitated 85-95% of the 125I-labelled fibronectin radioactivity in indirect radioimmunoprecipitation tests. Localization of specific epitopes to restricted regions of the fibronectin molecule was carried out by monitoring monoclonal antibody binding to proteolytic fragments. Of the five monoclonal antibodies analyzed in this study, three recognized determinants which resided in the terminal 35 kDa region of the fibronectin monomer. Furthermore, these epitopes were localized to fragments as small as 20 kDa. Competition studies carried out using plasma fibronectins isolated from different species revealed that three monoclonal antibodies recognized sites which were relatively conserved, while two monoclonal antibodies recognized epitopic sequences which were unique to the human protein. The corresponding anti-fibronectin serum also demonstrated discriminatory capabilities. Immunofluorescent analysis of human fibroblasts grown in vitro demonstrated that all the monoclonal antibodies tested were reactive with pericellular fibronectin.

Animals↗
PubMed1982

Species correlations between cardiac isomyosins. A comparison of electrophoretic and immunological properties.

Structural relationships between cardiac isomyosins were analyzed in 10 species using native-gel electrophoresis and radioimmunoassay. In the rat and rabbit, three types of ventricular isomyosin, V1, V2, and V3, were identified by electrophoresis. Monoclonal antibodies specific for the heavy chains of either type V1 or type V3 isomyosin in the rat and rabbit were used for comparison of immunological relationships between atrial and ventricular myosins in other species. Normal guinea pig ventricular myosin reacted with both anti-V2 and anti-V3 antibodies, but only a single myosin band was detected in this species by electrophoresis. When thyrotoxic cardiac hypertrophy was induced in guinea pigs, there was a decrease in myosin reactivity with the anti-V3 antibody and an increase in anti-V1 reactivity. This change in immunological reactivity indicated a change in proportions of two cardiac isomyosins in the guinea pig ventricle even though no myosin heterogeneity was detected by electrophoresis. In six other species including Xenopus, chicken, dog, pig, beef, and human, only a single band of myosin was detected by electrophoresis, and each myosin reacted only with the anti-V3 antibody. In the mouse, three types of ventricular myosin were also detected by electrophoresis. However, unlike V1 isomyosin of the rat and rabbit, mouse V1 isomyosin reacted equally with both anti-V1 and anti-V3 antibodies. In conclusion, we have identified highly conserved epitopes in cardiac myosin, which were found to specifically occur on either the high Ca2+-ATPase type V1 isomyosin or the lower ATPase type V3 ventricular isomyosin in most of the species examined.

Animals↗
PubMed1982

Isolation and characterization of two molecular variants of myosin heavy chain from rabbit ventricle. Change in their content during normal growth and after treatment with thyroid hormone.

We have prepared monoclonal antibodies specific for cardiac myosin heavy chain. These antibodies were used for the separation and characterization of the molecular variants of myosin heavy chain present in the rabbit heart. Two molecular forms of myosin heavy chain, HC alpha and HC beta, were isolated from the euthyroid rabbit heart by affinity chromatography. Their reactivity with our antibodies indicated that the primary structures of HC alpha and HC beta differ in at least four and share at least two antigenic determinants. Differences in the primary structure of HC alpha and HC beta were confirmed by analysis of the peptides produced by limited chymotryptic digestion of the two heavy chains. Thirteen peptide differences were consistently found. The HC alpha and HC beta variants are shown by immunologic analysis and in chymotryptic peptide profiles to be identical with the predominant forms of myosin heavy chain synthesized in the hearts of hyperthyroid and adult euthyroid rabbits, respectively. During development and maturation of the euthyroid rabbit heart, HC alpha comprises approximately 50% of the ventricular myosin between birth and 4 weeks of age; it diminishes to 20-30% by 8 weeks and to 10-20% by 12 weeks of age. Cardiac myosin from a 1-year-old rabbit is composed almost entirely of HC beta. Cardiac myosin from embryonic animals at 20 days gestation contained 20% HC alpha. These results show that HC alpha occurs normally in the euthyroid rabbit heart and that the relative proportions of HC alpha and HC beta depend on both the developmental stage and the thyroid state of the animal.

Aging↗
PubMed1981

Assessment of fractional rates of protein synthesis in cardiac muscle cultures after equilibrium labeling.

Protein synthesis, accumulation, and breakdown were examined in growing cultures of contractile embryonic chick heart cells, and the fractional synthetic rates of several individual proteins were compared. Fractional rates of protein synthesis were evaluated with a procedure that combined equilibrium and pulse labeling for determination of specific radioactivities of the precursor in the medium and in proteins isolated by electrophoresis. Kinetic analysis during equilibration of the precursor specific radioactivity with that in labeled proteins indicated that the specific radioactivity of leucine in the culture medium closely approximated that of the immediate reaction precursor for protein synthesis. Protein accumulation and breakdown were evaluated by standard growth and decay kinetics. Fractional protein synthesis rates determined during either short pulse labeling or during continuous labeling to equilibrium were found to be in agreement with independent measurements of protein accumulation and breakdown. We also determined fractional synthetic rates of proteins isolated from cultured heart cells on single and two-dimensional electrophoresis and found the following order of fractional synthesis rates: fibronectin greater than or equal to alpha-actinin greater than myosin heavy chain = tropomyosin = myosin light chains greater than 55,000-dalton proteins (desmin and tubulin) greater than or equal to actin. The half-lives of these proteins ranged from T 1/2 = 1.1 days for fibronectin and 2.0 days for myosin heavy chain to 4.7 days for actin.

Actins↗
PubMed1981

Public policy and residential mobility in Dutch cities.

"The role of government policy in the housing market is examined for its impact on residential mobility in medium-sized Dutch cities. Mobility rates for these cities are regressed against in-migration rates, age and family composition variables, housing stock characteristics and measures of policy. There is little relationship between in-migration and intra-urban movement. After controlling for demographic and housing stock characteristics, the policy measures (the provision of subsidies for new construction and renewal subsidies) do explain a significant proportion of the variation in mobility rates across cities." It is also found that "when the cities are grouped by mobility rate, the policy measures account for important proportions of the variance in two of the three groups of cities. For the other group of cities housing stock characteristics are important explanatory variables."

Demography↗
PubMed1980

Dimethyl sulfide in milk of lactating dairy cows fed various sulfur compounds.

Mature dairy cows in late lactation were given capsules containing 26, 40, 60, or 80 g of D,L-methionine; 32, 49, 69, or 90 g of methionine hydroxy analog; or 1, 2, or 4 ml of dimethyl sulfide. Animals used as controls received capsules containing water. Milk was collected at hourly intervals for 5 h and analyzed for dimethyl sulfide content by gas chromatography. All experimental cows were managed and fed (mainly corn silage plus concentrate with some hay and pasture) with the rest of the herd but were limited to water and clover hay during the 5-h experimental periods. Administration of 60 or 80 g of D,L-methionine or 1, 2, or 4 ml of dimethyl sulfide resulted in the appearance of dimethyl sulfide in the milk. The concentration of dimethyl sulfide in milk from cows receiving orally administered dimethyl sulfide (2 or 4 ml) was greatest at 1 h. When D,L-methionine (60 or 80 g) was given, dimethyl sulfide concentrations were lower and peaked later in the sampling period. The maximum concentration of dimethyl sulfide in the milk was 4.5 ppb when 4 ml of dimethyl sulfide were administered orally. No dimethyl sulfide was in the milk for any amount of methionine hydroxy analog, for 26 or 40 g of D,L-methionine, or for control capsules.

Animals↗