M-protein in chicken cardiac muscle.

Chicken cardiac muscle myofibrils lack a visible M-line. Antibodies against chicken breast muscle M-protein, an M-line component with M_r = 165 000, were used to demonstrate the presence of a similar protein in chicken heart muscle. The immunoreplica technique showed the heart protein to have about the same molecular weight as the breast muscle M-protein on polyacrylamide slab gels in the presence of sodium dodecyl sulfate (SDS). Positive staining within the H-zone was observed when the indirect immunofluorescence technique was used to localize the M-protein in isolated heart myofibrils. This result was confirmed by electron microscopic investigations on longitudinal sections of antibody-incubated heart muscle fiber bundles showing the antibody against M-protein to be bound within a region corresponding to the M-line region of breast muscle myofibrils.     

Acetylcholinesterase activity in developing skeletal muscle cells

Acetylcholinesterase activity has been demonstrated biochemically and cytochemically in developing chick embryo skeletal muscle cells growing in culture. The enzyme shows the same pattern of drug sensitivity as that of adult skeletal muscle acetylcholinesterase and in present in cultured myogenic cells before the time of cell fusion, the formation of myotubes, and the subsequent increase in rate of myosin synthesis. Myogenic cell fusion is accompanied, however, by a large increase in activity of acetylcholinesterase. The enzyme activity is restricted in these cultures to myogenic cells. Neighboring fibroblasts show no cytochemical responses when challenged with techniques showing intense activity in myoblasts and myotubes. In addition, evidence is presented which strongly suggests that acetylcholinesterase activity in dividing myogenic cells is not constant over the cell cycle.     

The influence of magnesium on calcium-activated, vascular smooth muscle actomyosin ATPase activity

Histamine activation of adenylyl cyclase activity in sonicated enriched rat gastric parietal cells showed a time, temperature, and concentration dependence upon guanine diphosphoimide (Gpp(NH)p). Enzyme activation was first order with Gpp(NH)p alone or Gpp(NH)p plus histamine. The K_a for Gpp(NH)p was ~2 μm and was not influenced by histamine. GTP and GDP were inactive alone or with histamine and were competitive with Gpp(NH)p, showing apparent K_i's of near 0.4 and 0.3 μm, respectively. In the presence of Gpp(NH)p, parietal cell adenylyl cyclase was activated by histamine with an EC₅₀ of 24 μm, the most potent in a series of histamine analogs, further substantiating an H₂-receptor classification for this response. H₂-Receptor antagonists were competitive inhibitors with submicromolar K_i's. Preincubation of parietal cells with histamine and Gpp(NH)p resulted in adenylyl cyclase activity up to 15 times the basal level. The activated state was retained after washing the cells free of histamine and Gpp(NH)p and was not reversed by the subsequent addition of either histamine, cimetidine, or GTP. The other gastric acid secretagogues, pentagastrin and carbamylcholine, were without effect upon histamine activation or the activated state of adenylyl cyclase. These results describe a level of control of histamine-sensitive adenylyl cyclase that requires consideration in the activation of the parietal cell H₂-receptor system by histamine to modulate acid secretion.     

Purification of lipoamide dehydrogenase from Ascaris muscle mitochondria and its relationship to NADH:NAD+ transhydrogenase activity

Lipoamide dehydrogenase (NADH:lipoamide oxidoreductase EC 1.6.4.3) has been isolated from Ascaris suum muscle mitochondria. This activity has been purified to apparent homogeneity from both the pyruvate dehydrogenase complex and from 150,000g mitochondrial supernatants which were devoid of pyruvate dehydrogenase complex activity. The enzymes from both sources exhibited similar kinetic, catalytic, and regulatory properties and appear to be identical as judged by polyacrylamide gel electrophoresis. The native enzyme acts as a dimer, containing 2 mol of FAD, and has a subunit molecular weight of 54,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel chromatography. The enzyme also possesses substantial NADH:NAD⁺ transhydrogenase activity. Heat denaturation and differential solubilization experiments imply that the transhydrogenase activity previously reported is, in fact, associated with the lipoamide dehydrogenase moiety of the Ascaris pyruvate dehydrogenase complex. Whether or not this activity functions physiologically in hydride ion translocation, as previously suggested, remains to be demonstrated.     

Purine metabolism and urate biosynthesis in isolated chicken hepatocytes

The metabolism of some purine compounds to urate and their effects on de novo urate synthesis in chicken hepatocytes were investigated. The purines, listed in descending order of rates of catabolism to urate, were hypoxanthine, xanthine, inosine, guanosine, guanine, IMP, GMP, adenosine, AMP, and adenine. During a 1-h incubation period, conversion to urate accounted for more than 80% of the total quantities of guanine, guanosine, and inosine metabolized, but only 42% of the adenosine and 23% of the adenine metabolism. Adenine, adenosine, and AMP inhibited de novo urate synthesis [( 14C]formate incorporation into urate), whereas the other purines, especially guanine, guanosine, and GMP, stimulated de novo urate synthesis. When hepatocytes were incubated with glutamine and adenosine, AMP, guanine, guanosine, or GMP, the rates of de novo urate synthesis were lower than the additive effects of glutamine and the purine in separate incubations. Increasing phosphate concentrations had no effect on urate synthesis in the absence of added purines but, in combination with adenosine, AMP, guanosine, or GMP, increased urate synthesis. These results indicate that the ratio of adenine to guanine nucleotides and the interaction between substrates and purine nucleotides are involved in the regulation of urate biosynthesis in chicken liver.     

Remyelination in the chicken sciatic nerve

Remyelination in the chicken sciatic nerve occurring after the injection of diphtheria toxin was studied. The rates of fast axonal transport and conduction velocities were measured sixty days after the injection of the toxin. Fast axonal transport rates were found to have returned to normal in the remyelinated nerves, but conduction velocity was markedly reduced even though the birds appeared to walk normally. The remyelinated nerve fibres had on histological examination relatively thin myelin sheaths. Of greater interest was the number of Schmidt-Lanterman clefts observed in both the control and remyelinated nerves when viewed in the electron microscope.     

Abnormal collagen synthesis in skeletal muscle of dystrophic chicken

Specific molecular properties of skeletal muscle collagens from normal and dystrophic chickens have been compared. When dystrophy develops in skeletal muscle tissue there was an increase in the amount of total collagen and an increased proportion of Type III collagen in the tissue. The results from the cross-link study as well as the analysis of the solubility of collagen showed that skeletal muscle of dystrophic chicken produces more immature collagen fibers compared to normal chicken. These findings strongly indicate an important role of collagen in the pathogenesis of the extensive connective tissue prolipheration characteristic of muscular dystrophies.     

Regional differences in the expression of myosin light chains and tropomyosin subunits during development of chicken breast muscle

Types of myosin light chains and tropomyosins present in various regions and at different developmental stages of embryonic and posthatched chicken breast muscle (pectoralis major) have been characterized by two-dimensional gel electrophoresis. In the embryonic muscle all areas appear to accumulate both slow and fast forms of mysoin light chains in addition to α and β forms of tropomyosin. During development regional differences in myosin and tropomyosin expression become apparent. Slow myosin subunits become gradually restricted to areas of the anterior region of the muscle and finally become localized to a small red strip found on its anterior deep surface. This red region is characterized by the presence of slow and fast myosin light chains, α-fast, α-slow, and β-tropomyosin. In all other areas of the muscle examined only fast myosin light chains, β-tropomyosin and the α-fast form of tropomyosin, are found. In addition, β-tropomyosin also gradually becomes lost in the posterior regions of the developing breast muscle. In the adult, the red strip area represents less than 1% of the total pectoralis major mass and of the myosin extracted from this area approximately 15% was present as an isozyme that comigrated on nondenaturing gels with myosin from a slow muscle (anterior latissimus dorsi). The red region accumulates therefore fast as well as slow muscle myosin. Thus while the adult chicken pectoralis major is over 99% fast white muscle, the embryonic muscle displays a significant and changing capacity to accumulate both fast and slow muscle peptides.     

Enzymatic assays of L-ornithine and L-delta 1-pyrroline-5-carboxylate in tissues, and orniathine-load test in human subjects

The efficiencies of estimates obtained from the direct linear plot (A. Cornish-Bowden and R. Eisenthal, 1978, Biochem. Biophys, Acta, 523, 268) are shown to be dependent on the spacing of substrate concentrations. When substrate values are harmonically spaced, the direct linear plot should not be used. The nonparametric confidence limits based on the direct linear plot are accurate in their confidence coefficient, but their efficiencies are shown to be dependent on substrate spacing. Harmonic spacing is, in general, a more efficient experimental design for estimating K_m than arithmetic spacing when the appropriate estimation methods are used. If assumptions about the error structure cannot be made, the best procedure for estimating K_m is to have harmonic spacing of substrate values and use weighted least squares for estimation. The most accurate and precise estimation of enzyme kinetic parameters requires knowledge of the error structure and utilization of the appropriate nonlinear regression.     

Mutagenesis and anti-mutagenesis in Salmonella: influence of ethionine and caffeine on yields of mutations induced by 2-aminopurine and 9-aminoacridine

Ethionine, the ethyl analogue of methionine, slightly reduced the yield of reversions of the hisC3076 frameshift marker induced by 9-aminoacridine (9AA) in an excision-proficient strain of Salmonella typhimurium, but completely abolished mutagenesis genesis by 9AA in the excision-deficient uvrB-deletion strain TA1537. No toxic effects of ethionine were apparent in either the excision-proficient or the excision-deficient strain. Because of the differential effects of ethionine on mutagenesis in the two strains, it seemed possible that an ethionine-sensitive step in the process(es) leading to fixation of 9AA-induced mutations might be compensated for by the uvrA,B,C⁺ excision-repair system. To further test this possibility, we used caffeine (a compound known to significantly reduce the efficacy of the excision-repair process) as a co-treatment with ethionine for cells of an excision-proficient strain exposed to 9AA. Treatment with caffeine alone or ethionine alone had very little effect on reversion yield, whereas co-treatment with the two agents abolished 9AA mutagenesis. It appeared, therefore, that either the caffeine-sensitive pathway or the ethionine-sensitive pathway needed to be functioning if 9AA-induced reversions of the hisC3076 marker were to be detected. Addition of methionine to cells of the excision-deficient strain exposed to 9AA restored their ability to be mutated by 9AA, however. In a base-pair substitution back-mutation system, ethionine slightly enhanced the yields of revertants of the trpE8 marker induced by 2-aminopurine (2AP) in both an excision-proficient strain (at all 2AP dose levels tested) and an excision-deficient strain (only at the lower dose levels). In the excision-deficient strain, doses of 2AP above 300 μg/plate were highly toxic when ethionine was also present. It was for this reason that no 2AP-induced revertants were recovered at the higher 2AP concentrations. Treatment of the trpE8 strain with methionine also enhanced the yield of 2AP-induced revertants of this marker.     

The effect of thyroxine on transparency and PAPS synthesis in the avian cornea

The normal onset of developing corneal transparency, which begins on Day 14 of chick embryogenesis, can be accelerated by the in vivo application of exogenous thyroxine, as originally demonstrated by Coulombre and Coulombre (1964, Exp. Eye Res.3, 105–114). When thyroxine (5 μg) is injected at Day 6, measurements of ³⁵SO^2?₄ incorporation by corneal homogenates indicate that synthesis of 3′-adenosine phosphosulfate (APS) in the cornea is increased at Day 8, but not that of 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Injection of hormone at Day 9 results in a precocious increase in corneal transparency at Day 12 and a corresponding increase in the synthesis of APS and PAPS.     

Messenger-like RNA synthesis and DNA chromosomal puffs in the salivary glands of Rhynchosciara americana

RNA synthesis was studied mainly in the proximal sections of Rhynchosciara salivary glands in late fourth instar at two typical periods of development. These are characterized either by the absence or presence of the so-called “DNA puffs” in the salivary gland chromosomes. It was found that simultaneously with the appearance of the DNA puffs there is a great increase in the synthesis of all RNA species. The greatest increase was found to take place in the rate of synthesis of messenger-like RNA. Four main classes of messenger-like RNA were detected, having mobilities corresponding to 33, 23, 16, and 14 S RNA. There is a correlation between the abundance of the 16 S messenger-like RNA and the degree of opening of the B-2 DNA puff. This species might therefore be transcribed from this puff.     

Mutagenicity of non-K-region diols and diol-epoxides of benz(a)anthracene and benzo(a)pyrene in S. typhimurium TA 100

When incubated with a 9,000 x g rat-liver supernatant, benzo(a)pyrene 7,8-diol and benz(a)anthracene 8,9-diol were more active than the parent hydrocarbons in inducing his⁺ revertant colonies of S. typhimurium TA 100. Benzo(a) pyrene 9,10-diol was less active than benzo(a)pyrene; the K-region diols, benz(a)anthracene 5,6-diol and benzo(a)pyrene 4,5-diol, were inactive. None of the diols was active when the cofactors for the microsomal mono-oxygenase were omitted. The diol-epoxides benzo(a)pyrene 7,8-diol 9,10-oxide, benz(a)anthracene 8,9-diol 10,11-oxide and 7-methylbenz(a)anthracene 8,9-diol 10,11-oxide and the K-region epoxides, benzo(a)pyrene 4,5-oxide and benz(a)anthracene 5,6-oxide, were mutagenic without further metabolism.     

Relations between enzymatic and association reactions in the development of bovine fibrin clot structure

Development of fibrin clot structure was examined at pH 7.0, Γ/2 0.15, and 29 °C as a function of thrombin and fibrinogen concentrations. Parameters for the release of Apeptides, to give $\text{?}{}_{\mathrm{<mtext>A</mtext>}}$ were evaluated. Characteristics of time dependencies of development of turbidity, 90 ° light scattering, network, and compactible network were established. Mean mass/length ratios of fibrin in developing and mature networks were determined. Relationships between results combined with an inferred dependence of lateral interaction on release of B-peptides are used to disclose a model in which a protofibril network is formed first and the intrinsic length of this network (i.e., length exclusive of overlap or loose ends) determines network length, thus mean mass/length ratio, at maturity. Statements regarding initial protofibril network are: (i) A dominant group of $\text{?}{}_{\mathrm{<mtext>A</mtext>}}$-protofibrils appears first. With decreasing rate of production of $\text{?}{}_{\mathrm{<mtext>A</mtext>}}$ their average length increases and number decreases. (ii) Slower release of B-peptides produces $\text{?}{}_{\mathrm{<mtext>AB</mtext>}}$ whose fraction $\text{θ}{}_{\mathrm{<mtext>AB</mtext>}}\text{=}\text{?}{}_{\mathrm{<mtext>AB</mtext>}}\text{(?}{}_{\mathrm{A}}\text{+?}{}_{\mathrm{AB}}$) determines the occurrence of protofibril regions capable of contributing to a lateral interaction sufficiently stable for the formation of network. (iii) When dominant protofibrils attain a minimum combination of average length, number concentration, and frequency of occurrence of capable regions, an initial protofibril network is rapidly generated. (iv) Capable regions near protofibril ends are preferentially involved in initial network formation. (v) The initial network mesh size is large compared to average concomitant free protofibril length. (vi) With B-peptide release dependent on prior A-peptide release, protofibrils in the initial network have the highest capable region frequency, and this is maintained as lateral interaction progresses. Then, fibrin which is free at initial network formation and fibrin which is produced subsequently interact mainly to increase the mean mass/length ratio of initial network elements.     

Collagen in developing chick muscle in vivo and in vitro

Because of the known role of collagen in chick skeletal muscle differentiation the collagen synthesized by embryonic chick muscle was studied. The major collagen synthesized by this muscle was found to be type I collagen. In addition, the effectiveness of types I, II, III and IV collagens in promoting myoblast fusion in vitro was compared. These collagens were found to be equally effective as in vitro substrates.     

The synthesis and deployment of filamin in chicken skeletal muscle

During myogenesis in vitro the actin-binding protein filamin is present in myoblasts and early fused cells and is associated with α-actinin-containing filament bundles, as judged by double immunofluorescence using antibodies specific for these two proteins. Approximately one day after cell fusion, yet before the development of a-actinin-containing Z line striations, filamin disappears from the cells. Later in myogenesis, several days after the appearance of α-actinin-containing Z line striations, filamin reappears and accumulates in the cells. Double immunofluorescence with antibodies to filamin and vimentin (or desmin) reveals that the newly appearing filamin localizes now to the myofibril Z line and is visible there shortly before vimentin or desmin becomes associated with the Z line. Immunofluorescent localization of filamin in isolated chicken skeletal myofibrils and Z disc sheets indicates that filamin has the same distribution as desmin and vimentin; it surrounds each myofibril Z disc and forms honeycomb-like networks within each Z plane of the muscle fiber. Filamin may thus be involved in the transition of desmin and vimentin to the Z disc. Analysis of whole-cell extracts by SDS-polyacrylamide gel electrophoresis and by immunoautoradiography shows that filamin is present in myoblasts and in myotubes early after cell fusion. Concomitant with the absence of filamin fluorescence during the subsequent few days of myogenesis, the quantity of filamin is markedly reduced. During this time, metabolic pulse-labeling with ³⁵S-methionine reveals that the synthetic rate of filamin is also markedly reduced. As filamin fluorescence appears at the Z line, the quantity of filamin and its synthetic rate both increase. The removal of filamin from the cells suggests that filamin either may not be required, or may actually interfere with a necessary process, during the early stages of sarcomere morphogenesis. These results also indicate that the periphery of the Z disc is assembled in at least two distinct steps during myogenesis.     

pp60c-src Kinase is in chick and human embryonic tissues

The normal cellular protein pp60c-src is a tyrosine-specific protein kinase that is homologous to the transforming protein of Rous sarcoma virus (RSV) but its function is unknown. The expression of pp60c-src in chick and human embryonic tissues was monitored by the immune complex protein kinase assay, Western transfer analysis, and immunocytochemical staining at the light microscope level. pp60c-src kinase was expressed in the head and trunk regions of the chick embryo at all stages of development examined; however, expression increased significantly during the major period of organogenesis (Hamburger and Hamilton stages 21 to 32). Western transfer analysis showed that the amount of pp60c-src protein increased in parallel with the increase in kinase activity. Highest levels of pp60c-src kinase were present in the neural tube, brain, and heart of the stage 32 chick embryo. Lower levels of activity were found in eye, limb bud, and liver. Immunocytochemical staining of the neural tube region and heart of the chick confirmed the results of biochemical analysis and showed immunoreactive pp60c-src distributed throughout the neural tube and heart. The distribution of pp60c-src kinase in human fetal tissues was similar to that in the chick embryo; elevated levels of pp60c-src kinase were present in cerebral cortex, spinal cord, and heart, but all other tissues examined expressed some pp60c-src kinase. The results of our studies suggest that pp60c-src plays a fundamental role in an aspect of cellular metabolism that is particularly important in electrogenic tissues.     

Embryonic-like myosin heavy chains in regenerating chicken muscle

An antibody to chicken ventricular myosin was found to cross-react by enzyme immunoassay with myosin heavy chains from embryonic chicken pectorials, but not with adult skeletal myosins. This antibody, which was previously shown to label cultured muscle cells from embryonic pectoralis (Cantini et al., J cell biol 85 (1981) 903), was used to investigate by indirect immunofluorescence the reactivity of chicken skeletal muscle cells differentiating in vivo during embryonic development and muscle regeneration. Muscle fibers in 11-day old chick embryonic pectoralis and anterior latissimus dorsi muscles showed a differential reactivity with this antibody. Labelled fibers progressively decreasgd in number during subsequent stages and disappeared completely around hatching. Only rare small muscle fibers, some of which had the shape and location typical of satellite elements, were labelled in adult chicken muscle. A cold injury was produced with dry ice in the fast pectoralis and the slow anterior latissimys dorsi muscles of young chickens. Two days after injury a number of labelled cells was first seen in the intermediate region between the outer necrotic area and the underlying uninjured muscle. These muscle cells rapidly increased in number and size, thin myotubes were seen after 3 days and by 4–5 days a superficial layer of brightly stained newly formed muscle fibers was observed at the site of the injury. Between one and two weeks after the lesion the intensity of staining of regenerated fibers progressively decreased as their size further increased. These findings indicate that an embryonic type of myosin heavy chain is transitorily expressed during muscle regeneration.     

Focal elevation of liver microsomal epoxide hydrolase in early preneoplastic stages and its behaviour in the further course of hepatocarcinogenesis

Treatment of rats with N-nitrosomorpholine (NNM) for 7 weeks led to a focal increase in liver microsomal epoxide hydrolase (EH) as early as 2 weeks after withdrawal of the carcinogen. This treatment also leads to hyperplastic nodules and liver tumors, but much later. At the same early time point, ATPase activity was decreased in the same islands. Most of these areas already had increased γ-glutamyltranspeptidase activity. The increase in EH at this early time point was more distinct than the decrease in ATPase which has thus far been considered a suitable marker of the earliest stages in hepatocarcinogenesis. The focal increase in EH was also observed in all benign hepatomas, but not in any of the hepatocellular carcinomas investigated so far.     

Regulation of glycolytic flux in an energetically controlled cell-free system: the effects of adenine nucleotide ratios, inorganic phosphate, pH, and citrate

A soluble extract from rat skeletal muscles has been used with purified mitochondrial ATPase (F₁) to develop steady states with respect to glycolytic flux, the concentrations of glycolytic intermediates and inorganic phosphate, and the concentrations and ratios of adenine nucleotides. Incubations were carried out in media resembling the ionic composition in the cell cytoplasm, in an attempt to evaluate the quantitative contributions of various effectors to the overall control mechanism under simulated in vivo conditions. The primary control reaction of glycolytic flux under the conditions studied could be identified with phosphofructokinase, followed by secondary control of the reaction catalyzed by hexokinase. Glycolytic flux was increased with increasing pH over the range 6.6–7.6, both in the absence and presence of ATPase. Without other added effectors, the glycolyzing extract maintained an ATP/ADP ratio of about 50 in the pH range 7.0–7.6, and phosphofructokinase was incompletely suppressed. Addition of increasing amounts of ATPase markedly stimulated glycolytic flux coincident with lowered steady-state ATP/ADP ratios, and decreased accumulation of hexose monophosphates. Control of flux by the ATP/ADP ratio (and simultaneously altered AMP concentration) was less effective if pH (7.3 to 7.6) or phosphate concentration (2 to 20 mm) was increased. Flux through phosphofructokinase was controlled principally when the ATP/ADP ratios were varied in the range between > 50 and 15. The inhibitory effect of citrate was evaluated. Suppression of glycolytic flux and accumulation of hexose monophosphates were dependent on incubation conditions. If the pH was 7.3 or less, and the phosphate concentration low (2 mm), flux through phosphofructokinase was significantly suppressed even at citrate concentrations less than 50 μm. Simultaneous decrease in the steady-state ATP/ADP ratio and elevation of AMP was ineffective in reversing this inhibition. At higher pH and, more dramatically, when the phosphate concentration was increased, sensitivity to citrate inhibition was markedly diminished. These data, taken together with studies of respiratory control with isolated mitochondria (21., 24.), J. Biol. Chem.250, 2275–2282) strongly suggest that adenine nucleotide control of both glycolysis and respiration is exerted when the ratio of free nucleotides (not protein bound) in the cytosol is in the range of 15 to > 50. The data further suggest that citrate plays an important role in the regulation of glycolysis in muscle when the ATP/ADP ratio is high (and the phosphate concentration is correspondingly low), but that this inhibition is overcome by liberation of inorganic phosphate during muscle contraction.