Myogenic defect in acetylcholinesterase regulation in muscular dystrophy of the chicken.

To determine whether inherited muscular dystrophy of the chicken is neurogenic or myogenic in origin, limb buds from homozygous normal and dystrophic chick embryos were exchanged prior to muscle differentiation and innervation. Biceps muscles of hatched chicks, in which muscle of the donor was innervated by nerves of the host, were analyzed for embryonic properties of muscle acetylcholinesterase and for fiber diameter, two distinctive markers for expression of the dystrophic gene. The results indicate that muscular dystrophy of the chicken is caused by an initial biochemical lesion in the limb and its muscle rather than in its innervating nerve.

Avian muscular dystrophy: functional and biochemical improvement with diphenylhydantoin.

Chicks affected with hereditary muscular dystrophy were injected twice daily with 20 milligrams of diphenylhydantoin per kilogram of body weight on days 1 to 40 after hatching. The righting ability of dystrophic chicks treated with diphenylhydantoin was improved compared to that of untreated dystrophic chicks, and acetylcholinesterase activity was reduced to normal levels in the posterior latissimus dorsi muscles.

Fractionation of skin tumor-initiating activity in coal liquids.

Narrow temperature range distillates from biologically active solvent refined coal-I and -II heavy-end coal liquids were fractionated according to chemical class and assayed for initiation of skin carcinogenesis in CD-1 mice. In addition, instrumental chemical analyses were performed on the distillates and their chemical fractions. Results showed that initiation activity in these complex fuel mixtures could be segregated both by boiling point and chemical class. Neutral polycyclic aromatic hydrocarbon fractions were the most active of the chemical classes, with some initiating activity being shown by nitrogen-containing polycyclic aromatic hydrocarbon. Aliphatic and hydroxylated polycyclic aromatic hydrocarbon fractions showed little or no initiating activity. For the two solvent refined coal-II distillates studied, initiating activity was substantially higher in the material boiling above 850 degrees F than in that boiling 800-850 degrees F, although both contained essentially the same concentrations of benzo[a]pyrene. These data indicate that the overall initiating activity of these complex mixtures is highly dependent on interactions of the many chemical carcinogens and that relative concentrations of known carcinogenic polycyclic aromatic hydrocarbons, such as benzo[a]pyrene and dimethylbenz[a]anthracene, are not the sole determinants of initiating activity.

Development of surrogate substrates for neuropathy target esterase.

Seventeen substrates were synthesized and their activities as surrogate substrates for Neuropathy Target Esterase were tested. Substrates investigated are carbon analogs of phenylvalerate (1) with oxygen and sulfur substituted at the alpha, beta and gamma positions. Phenol and thiophenol esters of these analogs constitute two series of compounds tested. The ratio of catalytic hydrolysis to background hydrolysis increased at lower pH values with all substrates tested including phenylvalerate (1). There was more than a 2.5-fold increase in specific activity with phenylthiopropylethanoate (6) at pH of 6.75 compared to phenylvalerate (1). Furthermore, a 19-fold decrease in Km is reported with compound 6. This and related compounds can be used as the basis of more sensitive assays for neuropathy target esterase. Thiophenyl esters in this series are sufficiently good substrates to hold promise in continuous assays.

Purine nucleotides modulate proliferation of brown fat preadipocytes.

The hypothesis that purine nucleotides and nucleosides affect brown fat preadipocyte proliferation was tested using isolated rat interscapular brown fat preadipocytes in culture. Daily addition of 100 microM adenosine triphosphate (ATP) (n = 4) to cultures enhanced the relative DNA content by 1.5-fold compared to control cultures (P < 0.05) measured using CyQUANT-GR fluorescence. Higher concentrations of ATP inhibited growth and 500 (n = 2) or 1000 microM ATP (n = 3) almost completely inhibited growth. ATP (100 microM) did not affect while 250-1000 microM ATP decreased protein content relative to control cultures. Adenosine (100 microM; n = 3) did not affect DNA or protein content, but 500 microM and 1000 microM adenosine suppressed brown adipocyte proliferation and inhibited protein synthesis. Cultured brown adipocytes quickly removed or degraded ATP in the culture media as determined by luciferin-luciferase bioluminescence, suggesting that the inhibitory effects of high ATP concentrations may result from its breakdown to adenosine. The results support the conclusion that ATP promotes and adenosine inhibits brown adipocyte proliferation.

Complete sequence of a type-I microfibrillar wool keratin gene.

We have isolated and sequenced the gene encoding a type-I microfibrillar wool keratin (47.6 kDa) together with about 500 bp of flanking DNA at each end. The primary transcript is 5180 bp in length, with seven exons. The positions of the introns are highly conserved in regions of the gene coding for the alpha-helical protein domains common to all intermediate filaments. The 5'-flanking region contains sequences similar to published consensus sequences from a variety of other keratins, including the high-sulfur wool matrix proteins. Further upstream, this region also contains artiodactyl-specific middle-repetitive DNA of the 'C-A3' family and intron II displays regions similar to a recently described sequence known as an Art2 element.

Regulation of acetylcholinesterase in cultured muscle by chemical agents and electrical stimulation.

Cultures of 11 day old chick embryo pectoral muscle were used to study the effects of direct electrical stimulation and neurochemicals such as acetylcholine, acetyl-beta-methylcholine, tetrodotoxin, and d-tubocurarine on the acetylcholinesterase levels of muscle. The results suggest that excitation-contraction is an important factor in regulation of muscle acetylcholinesterase. Tetrodotoxin, acetylcholine and its analog acetyl-beta-methylcholine increased acetylcholinesterase levels and reduced spontaneous contractions. D-tubocurarine blocked the increase in acetylcholinesterase and the decrease in spontaneous contractions caused by acetyl-beta-methylcholine. Electrical stimulation decreased acetylcholinesterase and increased muscle contractions in normal and in diisopropylfluorophosphate treated cultures. Tetrodotoxin also affected the morphology of the muscle cells, as if it adversely affected normal growth and differentiation. Electrical stimulation did not increase muscle creatine kinase.

Regulation of acetylcholinesterase in chick muscle cultures after treatment with diisopropylphosphorofluoridate: ribonucleic acid and protein synthesis.

Previous studies have established that short treatments with diisoprophylphosphorofluoridate of chick embryo muscle cultures irreversibly inhibit the enzyme acetylcholinesterase and that the enzyme's levels in the cells rapidly recover due to synthesis of new protein. In addition, it has been shown that acetylcholine, acetyl-beta-methylcholine and choline increase the acetylcholinesterase content of the cultures. In the experiments presented here, actinomycin D and cycloheximide were used to study the relationships between the synthesis of ribonucleic acid and of protein, the recovery of acetylcholinesterase levels after diisopropylphosphorofluoridate treatment and the increase of acetylcholinesterase levels evoked by choline and its esters. Both recovery of acetylcholinesterase and the increase in its activity with acetyl-beta-methylcholine occurred in the absence of ribonucleic acid synthesis but required protein synthesis. The results suggest that transcription of desoxyribonucleic acid and a change in the degradation rate of acetylcholinesterase are not involved. An additional finding was that the level of newly synthesized acetylcholinesterase activity continued to increase for a short period of time after synthesis of protein had ceased, as if some previously synthesized protein was being transformed into active enzyme.

Ultrastructural localization of acetylcholinesterase in cultured cells. III. DFP treated embryo muscle.

Brief treatment with 10(-4)M diisopropylfluorophosphate (DEP) irreversibly inactivates acetylcholinesterase (E.C.3.1.1.7; acetylcholine hydrolase) (AChE) activity in 10 day old chick embryonic muscle cultures. Electron microscopic cytochemistry was employed to follow the distribution of new AChE during recovery from DEP treatment. In normal 10 day cultures of embryo pectoralis muscles AChE is localized in the nuclear envelope, perinuclear sarcoplasm, sarcotubular system, subsurface vesicles and bound outside the cells. Immediately after DFP treatment AChE activity is absent in large myotubes. Within 15 min, activity is randomly present in small amounts in the sarcotubular system and nuclear envelope. There is a dramatic increase in activitv in the nuclear envelope during the 1st hr of recovery, and connections between the nuclear envelope and sarcotubular system are often seen. The next few hr of recovery show increased AChE activity. By 4 hr activity approaches that of controls. Six to 8 hr after treatment, AChE activity can be detected spectrophotometrically in the medium and can be seen bound outside the cells with the electron microscope. The spatial and temporal patterns of AChE activity demonstrate that the recovery of AChE and its mobilization and release from DFP-treated cells are not governed solely by the levels attained by the enzyme in the cultured embryo muscle.

Ultrastructural localization of acetylcholinesterase in cultured cells. II. Cycloheximide-treated embryo muscle.

The acetylcholinesterase activity (AChE) of cultured chick embryo muscle fibers that remains after the cells have been treated with the protein synthesis inhibitor cycloheximide was examined with cytochemical stains and the electron microscope. AChE activity that decreased rapidly after addition of the inhibitor was associated with enzyme within the cells, and AChE activity that was relatively insensitive to the inhibitor was associated with AChE outside of the cells. The results support the view that there are at least two fractions of AChE in developing muscle fibers, one intracellular and labile, the other extracelullar and stable.

Ultrastructural localization of acetylcholinesterase in cultured cells. I. Embryo muscle.

Several techniques were employed to examine the localization of acetylcholinesterase (EC 3.1.1.7, AChE) in cultured chick embryonic skeletal muscle. Glutaraldehyde produced the best cellular preservation but less enzyme activity was lost when the cells were fixed in paraformaldehyde. Two staining methods were examined: in one (Karnovsky MJ, Roots L: J Histochem Cytochem 12:219, 1964) potassium ferricyanide was added with the primary reactants, and in the other (Tsuji S: Histochemistry 42:99, 1974) the potassium ferricyanide was added at the end of the staining procedure. Localizations of AChE were similar with both stains; activity was present in the nuclear envelope, the perinuclear sarcoplasm, the sarcoplasmic reticulum, subsurface vesicles and bound outside the cells. /owever, a granular artifact was found with the method of Karnovsky and Roots that did not appear with the method of Tsuji. The localization of AChE are consistent with kinetic data that AchE binds, moves and is released from cultured muscle fibers.

Absorption of 125I-labelled sheep growth hormone in single proximal tubules of the rat kidney.

Techniques of kidney micropuncture and electron microscope autoradiography have been used to study the uptake of 125I-labelled sheep growth hormone (GH) in rat renal proximal tubules. After microperfusion of a proximal tubule with 125I-labelled GH, the transport of label by the tubular epithelium was studied autoradiographically at selected times up to 1 h. The sequential transfer of labelled material from tubule to microvilli, then to small and large apical vacuoles and finally to lysosomes followed the pattern of absorption that has been described for other proteins. Evidence of lysosomal degradation of the transported protein was obtained from studies in vitro; lysosomes isolated from the renal cortex rapidly converted 125I-labelled GH to products of lower molecular weight. In addition to the absorptive pathway through the intracellular vacuolar apparatus is appeared that there was also an alternative pathway, less well defined, whereby GH could be absorbed without being degraded.

Recovery of acetylcholinesterase forms in quail muscle cultures after intoxication with diisopropylfluorophosphate.

Studies of recovery of acetylcholinesterase (AChE, EC 3.1.1.7) after inhibition by organophosphates (OPs) have been hampered by the low number of in vitro systems with large collagen-tailed forms of AChE characteristic of motor end plates. Pectoral muscle cultures from Japanese quail with high levels of a large 20S form of AChE were used to study recovery of cells from acute treatment with diisopropylfluorophosphate (DFP), an irreversible AChE inhibitor. Low molecular weight AChE forms were synthesized more rapidly than the large 20S form following a 15-min treatment with 10(-4)M DFP. Most of the activities of the small forms, but only part of the activity of the 20S form, disappeared within 48 hr after cycloheximide was added to block protein synthesis. To the contrary, virtually all the activity of the 20S AChE that was newly synthesized after DFP treatment was lost within 24 hr after cycloheximide treatment. The results were generally consistent with the idea that the 20S AChE form is assembled inside the cell near to its surface and then is released to bind to its outside.

Multiple molecular forms and lectin interactions of organophosphate-sensitive plasma and liver esterases during development of the chick.

Liver and plasma acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase activities of the chick embryo and adult chickens were separated by sucrose density gradient sedimentation and further differentiated by their lectin affinities and organophosphate sensitivities. Changes in plasma cholinesterases during development indicated a characteristic shift in tetrameric (G4) isoforms from a slightly larger G4 AChE in the embryo to G4 BChE in the adult. These changes were not reflected in isoform patterns of liver homogenates, however. Interestingly, the time course of an increase in plasma BChE activity corresponded to the time course of a decrease in liver BChE activity, as if this enzyme was being mobilized and released. The distribution of liver esterases included both monomeric (G1) and G4 BChE and a large p-nitrophenylacetate (p-NPA) esterase activity that was separated into two main peaks by density gradient ultracentrifugation. The effects of organophosphate inhibitors indicated that the two liver p-NPA esterase activities may be regarded as carboxylesterases; however, these enzymes showed very different sensitivities to paraoxon and diisopropylfluorophosphate (DFP), with IC50 values differing by 3 and 4 orders of magnitude. Lectin affinity studies with multiple esterase forms suggested a heterogeneous group of glycoproteins that were packaged at different sites in the liver cell and were consistent with the presence of an intracellular precursor form to plasma BChE.

Characterization of neuropathy target esterase using trifluoromethyl ketones.

Neuropathy target esterase (NTE) is a membrane-bound carboxylesterase activity which is proposed as the target site in nerve tissue for initiation of organophosphate-induced delayed neuropathy. This activity is identified as phenyl valerate hydrolysis which is resistant to treatment with paraxon and sensitive to co-incubation with paraxon and mipafox. NTE preparations were obtained, which did not contain paraxon-sensitive or mipafox-resistant hydrolases, by selective reconstitution of detergent-solubilized NTE from chick embryo brain into asolectin vesicles during gel filtration. The topography of the catalytic site of NTE was then examined by investigating the inhibition of NTE by a series of 3-alkylthio- and 3-arylthio-1,1.1-trifluoro-propan-2-ones. These trifluoromethyl ketones were found to be rapidly reversible, competitive inhibitors of NTE with I50 values 1.3 x 10(-4) M to 4.9 x 10(-8) M. Correlation of I50 values with octanol/water partition coefficients (P), in the range of log P = 1.5 to 5.9. indicated that the optimal lipophilicity for NTE substrates and inhibitors is in the range of log P = 3.0 to 3.4. Electrophilic substitution at the meta position of aromatic rings increased the inhibitory capacity of these inhibitors, whereas substitution at the ortho position reduced inhibitory capacity. These results indicate both that a large hydrophobic pocket is closely associated with the catalytic residue of NTE, and that affinity for the active site is affected by steric and electronic parameters.

Increasing uptake and bioactivation with development positively modulate diazinon toxicity in early life stage medaka (Oryzias latipes).

Diazinon, an organophosphate pesticide, becomes biotransformed to a more potent oxon metabolite that inhibits acetylcholinesterase (AChE). Early life stages (els) of medaka, Oryzias latipes, were used to determine how development of this teleost affects sensitivity to diazinon. With developmental progression, from day of fertilization to 7-day-old larvae, we found that the 96-h LC50 and AChE IC50 values decreased, indicating greater host sensitivity to diazinon upon continued development. We then examined changes in AChE activity, its inhibition by the active metabolite diazoxon, and uptake and bioactivation of the compound. AChE activity remained low during much of development but increased rapidly just prior to hatch. In addition, in vitro incubation of tissue homogenates from embryos or larvae showed no differences in the sensitivity of AChE to diazoxon. Uptake studies with 14C-diazinon revealed greater body burdens of 14C as medaka developed. In addition, AChE IC50 values determined by in vivo exposure to diazoxon were greater in larvae than in embryos. Because diazinon is bioactivated by the P450 enzyme system, two P450 inhibitors were used in vivo to explore the role of metabolism in sensitivity. When exposure to diazinon occurred in the presence of increasing amounts of piperonyl butoxide (PBO), AChE inhibition decreased in a dose-response fashion and 2.0 x 10(-5) M PBO alleviated any difference in inhibition between larvae and embryos. However, PBO did not alter total 14C uptake when exposed simultaneously with 14C-diazinon, nor did it affect AChE inhibition using diazoxon. Controls ruled out differential effects of PBO on uptake and inhibition. In addition, a second general P450 inhibitor, 1-aminobenzotriazole, also decreased AChE inhibition. Finally, using exogenous acetylcholinesterase as a trap for the oxon metabolite, larval microsomes displayed greater bioactivation of diazinon than did a microsomal preparation from embryos. Taken together, results suggest that uptake and bioactivation are working to enhance diazinon sensitivity in this developmental model of a teleost fish.

Properties of muscles from chickens with inherited muscular dystrophy.

Inherited muscular dystrophy of the chicken is an abnormality affecting the normal development and function of fast-twitch skeletal muscles. Several different strains of dystrophic chickens have been developed by selection for high lipid content in the pectoralis muscle and early onset of the disorder or by outcrossing the original New Hampshire stock into an inbred White Leghorn breed. The purpose of this study was to determine whether fast-twitch dystrophic muscles differ in expressed properties within the same bird and to examine the differences in gene expression between dystrophic New Hampshire and White Leghorn breeds. The biochemical and physiological properties examined were lactate dehydrogenase and acetylcholinesterase activities, total lipid content, muscle fiber diameter and electromyographic insertion activity. Results showed that fiber diameter and lipid levels were different in muscles within individual birds of two dystrophic lines and that the dystrophic gene causes rapid fiber atrophy and high lipid content in the White Leghorn breed. In addition, differences in lactate dehydrogenase activity and electromyographic patterns were found between two dystrophic lines. The results suggest that the expressed properties differ within each muscle of the dystrophic bird and that the expression of the dystrophic genes is dependent upon the nature of the genetic background of the breed.

Appearance of acetylcholinesterase and creatine kinase in plasma of normal chickens after denervation.

Evidence that acetylcholinesterase (AChE) activity is released from normal chick embryonic muscle fibers and from muscles of chickens with inherited muscular dystrophy suggested that denervated chick muscles, which have AChE properties similar to dystrophic muscles, would also release AChE. Bilateral denervation of the breast and wing muscles of normal chickens was followed by the appearance of AChE activity, distinguished from plasma cholinesterase by differential substrate hydrolysis, inhibitor sensitivity, and electrophoretic migration. Plasma creatine kinase (CK) activity was also elevated after denervation.

Incidence of acetylcholinesterase in the sarcoplasm of human and chicken muscles.

Fifty-nine biopsies of human muscle, 53 of them abnormal, 6 normal, were studied for the histochemical localization of acetylcholinesterase (AChE) using frozen sections and light microscopy. In addition to AChE which was found at the myoneural and myotendon junction, specific staining was found around the periphery of many fibers from normal and abnormal muscles. Moreover, AChE activity was found to be high in the sarcoplasm of more than 10% of the fibers from 28 biopsies of abnormal muscle including cases of hemiplegia, spinal cord injury, denervation and neuropathy, infantile spinal muscle atrophy, Duchenne, limb-girdle and facioscapulohumeral dystrophies, Schwartz-Jampel syndrome and a myasthenic syndrome. Of the muscles from experimental animals examined, only the Rhesus monkey exhibited AChE around the periphery of the fibers, and only the dystrophic chicken and not the dystrophic mouse or hamster, showed extensive sarcoplasmic AChE. Histograms of muscle fiber diameters indicated that AChE in the sarcoplasm was associated with fibers of all sizes, depending on the nature of the disorder examined. Fibers containing AChE were smaller than unstained fibers in dystrophic chicken muscle. The results suggest that in the human, sarcoplasmic AChE is reversibly repressed during muscle maturation and that its mode of regulation by motor neurons is similar to that found in the chicken.

The application of mass spectrometry to the study of the pineal gland.

This paper presents briefly the basic principles of operation for the mass spectrometer (MS) and gas chromatography-mass spectromtery (GCMS) systems as well as discuss some of the analytical and assay methods for indole compounds which utilize GCMS. Using 5-methoxytryptophol as an example, the development of such an assay method is examined in detail. Finally, some of the most recent methodological and instrumental developments in mass spectrometry, including field disorption mass spectrometry (FDMS), negative ion chemical ionization mass spectrometry, and isotope dilution techniques are discussed in view of the contributions they may be expected to make to the study of pineal chemistry.