Evidence for a noncholinergic function of acetylcholinesterase during development of chicken retina as shown by fasciculin

Fasciculin 2 (FAS), an acetylcholinesterase (AChE) peripheral site ligand that inhibits mammalian AChE in the picomolar range and chicken AChE only at micromolar concentrations, was used in chick retinal cell cultures to evaluate the influence of AChE on neuronal development. The effects of other AChE inhibitors that bind the active and/or the peripheral site of the enzyme [paraoxon, eserine, or 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51)] were also studied. Morphological changes of cultured neurons were observed with the drugs used and in the different cell culture systems studied. Cell aggregates size decreased by more than 35% in diameter after 9 days of FAS treatment, mainly due to reduction in the presumptive plexiform area of the aggregates. Eserine showed no effect on the morphology of the aggregates, although it fully inhibited the activity of AChE. In dense stationary cell culture, cluster formation increased after 3 days and 6 days of FAS treatment. However, FAS, at concentrations in which changes of morphological parameters were observed, did not inhibit the AChE activity as measured histochemically. In contrast, paraoxon treatment produced a slight morphological alteration of the cultures, while a strong inhibition of enzyme activity caused by this agent was observed. BW284c51 showed a harmful, probably toxic effect, also causing a slight AChE inhibition. It is suggested that the effect of an anticholinesterase agent on the morphological modifications of cultured neurons is not necessarily associated with the intensity of the AChE inhibition, especially in the case of FAS. Moreover, most of the effects of AChE on culture morphology appear to be independent of the cholinolytic activity of the enzyme. The results obtained demonstrate that FAS is not toxic for the cells and suggest that regions of the AChE molecule related to the enzyme peripheral site are likely to be involved with the nonclassical role of AChE.     

Differentiation in cultures derived from embryonic chicken muscle: I. Muscle-specific enzyme changes before fusion in EGTA-synchronized cultures

It is known that myoblast fusion fails to occur in cultures containing EGTA (a calcium-specific chelator) but occurs very rapidly after EGTA medium is replaced with standard high-calcium medium. On the basis of a careful analysis of the time course of fusion in cultures switched from EGTA to standard medium, it is proposed that this method of synchronization be used routinely in studies of the timing of different processes during in vitro myogenesis. The kinetics of accumulation of total enzyme activity for creatine kinase and fructose diphosphate aldolase indicate that the increases characteristic of terminal muscle differentiation begin prior to the experimentally imposed onset of fusion in EGTA-synchronized cultures. Additionally, the accumulation of M-creatine kinase subunits, also typical for muscle differentiation, is shown by microcomplement fixation to begin before the switch from EGTA to standard medium. Creatine kinase isoenzyme patterns also show that the transition from B- to M-subunit-containing creatine kinases occurs in EGTA cultures not switched to standard medium. Like EGTA, 5-bromodeoxyuridine (BrdUrd) reversibly prevents myoblast fusion. By adding EGTA and BrdUrd in different sequences to muscle cell cultures, it is shown that they act at different stages in the course of in vitro myogenesis. Cells cultured in EGTA from 23 to 69 hr after plating fused very rapidly when switched to medium containing BrdUrd. In the reverse experiment, in which BrdUrd preceded EGTA, no fusion occurred. Parallel experiments with 5-fluorodeoxyuridine suggest that cell division is necessary to reverse the inhibitory effect of BrdUrd, but not that of EGTA; this is consistent with the observed kinetics of fusion after switching to standard medium. These data strongly support a model of myogenesis in vitro in which two processes (one BrdUrd-sensitive, the other EGTA-sensitive) occur sequentially. In the first process, myogenic cells give rise to cells capable of producing molecules necessary for (terminal) skeletal muscle differentiation, including both those required for cell fusion and specific isoenzymes. The second process, fusion itself, can occur in the presence of BrdUrd or in the absence of cell division.     

Changes in myosin isozymes during development of chicken breast muscle

The patterns of myosin isozymes in embryonic and adult chicken pectoralis muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light chains and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, the predominant isozyme component in embryonic pectoralis myosin could be clearly distinguished from adult myosin isozymes. SDS-polyacrylamide gel electrophoresis indicated that the light chain composition of embryonic myosin was also different from that of adult myosin. The pattern of peptide fragments produced by myosin digestion with a-chymotrypsin differed significantly between embryonic and adult skeletal myosin. These results suggest that myosin in the embryonic pectoralis muscle is different in both light and heavy chain composition from myosin in the same adult tissue.     

Biosynthesis of tropomyosin and troponin during development of the chicken skeletal muscle

The level of functional mRNA coding for myofibrillar proteins was studied during development of the chicken skeletal muscle. RNA isolated from the developing chicken muscle directed protein synthesis in a wheat germ cell-free system. By means of polyacrylamide gel electrophoresis and immunological analysis, tropomyosin subunits and troponin components were identified among the cell-free translation products. The mRNA activities for alpha- and beta-subunit of tropomyosin were prominent in the embryonic breast muscle as well as in the embryonic leg muscle. At the early post-embryonic stage, the mRNA activity for beta-subunit disappeared from the breast muscle, while those for alpha- and beta-subunit were detectable in the leg muscle. Troponin-C and troponin-I synthesized in vitro in response to the muscle RNA formed a binary complex in the presence of calcium ion. Despite the observed difference in molecular weight between troponin-Ts in the breast and leg muscle, RNA preparations from the two muscles encoded identical troponin-Ts whose molecular weights were indistinguishable from that of troponin-T present in the breast muscle of adult chicken. It is suggested from these results that the biosynthesis of tropomyosin is regulated at the pre-translational level during the development of the chicken skeletal muscle, whereas post-translational (or co-translational) events may produce the tissue-specific form of troponin-T.     

Precocious in vitro development of satellite cells from dystrophic chicken muscle

Satellite cells, liberated from pectoral muscle of juvenile dystrophic chickens by sequential treatment with collagenase, hyaluronidase, and trypsin and preplated to remove fibroblasts and cultured on gelatin proliferated rapidly, fused and formed confluent muscle cultures within 6 d in vitro with minimal contamination by fibroblasts. When identical isolation and culturing techniques were applied to muscle from age-matched normal chickens proliferation and differentiation were slower, contamination with fibroblasts was much greater, and only a small number of myotubes were formed. After injection of the myotoxic anesthetic marcaine into normal pectoral muscle for 5 consecutive days, myotube formation was accelerated in satellite cell cultures, but the rate of differentiation was not as rapid as that occurring in cells from dystrophic muscle.     

Precocious in vitro development of satellite cells from dystrophic chicken muscle

Summary Satellite cells, liberated from pectoral muscle of juvenile dystrophic chickens by sequential treatment with collagenase, hyaluronidase, and trypsin and preplated to remove fibroblasts and cultured on gelatin proliferated rapidly, fused and formed confluent muscle cultures within 6 d in vitro with minimal contamination by fibroblasts. When identical isolation and culturing techniques were applied to muscle from age-mateched normal chickens proliferation and differentiation were slower, contamination with fibroblasts was much greater, and only a small number of myotubes were formed. After injection of the myotoxic anesthetic marcaine into normal pectoral muscle for 5 consecutive days, myotube formation was accelerated in satellite cell cultures, but the rate of differentiation was not as rapid as that occurring in cells from dystrophic muscle. This research was supported by a grant from the Muscular Dystrophy Association of Canada.     

Monoclonal antibodies against chicken brain acetylcholinesterase. Their use in immunopurification and immunochemistry to demonstrate allelic variants of the enzyme

Acetylcholinesterase (AChE) from 1-day chicken brain was enriched over 2000-fold by affinity chromatography using N-methylacridinium-Sepharose. This preparation was used to prepare monoclonal antibodies (mAb) directed against AChE, of which two were extensively characterised for further application. Both mAbs bound to the enzyme from the chicken with high affinity (Kd approximately 8 X 10(-10) M) and one mAb, in addition, recognised AChE from quail brain and muscle. Neither mAb cross-reacted with mammalian or fish AChE. Both mAbs recognised AChE in the endplate region of adult chicken skeletal muscle and bound with equal affinity to the three major oligomeric forms found in early ambryonic muscle. One mAb was used to immunopurify chicken brain AChE to homogeneity (over 12000-fold enrichment), with nearly complete recovery of the enzyme and without detectable proteolytic breakdown. The other mAb recognised AChE after immunoblotting and was used to screen crude brain extracts from individual chickens for allelic variations. Evidence is presented to show that two allelic forms occur, represented in SDS-PAGE by a doublet polypeptide of Mr approximately 110,000, this pattern is maintained after deglycosylation of the N-linked oligosaccharides. This variation was found throughout development and in both the brain and the muscle of individuals. We conclude that the gene encoding the catalytic subunit of chicken AChE is polymorphic with either one or two equally active alleles being expressed.     

Molecular forms of acetylcholinesterase in the slow muscle and the fast muscle of the chicken]

Five molecular forms of AChE are present in the slow (ALD) and twitch (PLD) muscles of the chick. These forms have 4 S, 7 S, 11 S, 15 S and 20 S sedimentation coefficient in sucrose gradient. The heaviest forms, the 20 S and 15 S of AChE are absent in uninnervated muscles and present in innervated muscles. In innervated muscles, the 20 S and 15 S AChE are present in both nerve-free segments and end-plates zones. The 20 S and 15 S which are not specifically associated with the end-plate zones in the chick could be considered as a biochemical "marker" of neuromuscular interactions.     

Ganglioside changes in the chicken optic lobes and cerebrum during embryonic development

Summary The developmental profiles of 15 different gangliosides of the optic lobes and cerebrum of the chicken were followed from the 6 th day of incubation to hatching and correlated to morphological development. Five of these gangliosides appearing in both structures between the sixth and tenth day, have not been reported previously in higher vertebrates. Three chromatographed on TLC-plates similarly to G_T3, G_T2, and G_T1c gangliosides, which have been demonstrated in fish brain. One fraction moved just below G_Q1b and is suggested, to contain G_Q1c. These novel gangliosides, which are possibly related to a recently proposed separate and probably phylogenetically older biosynthetic pathway, contained up to 20% of total ganglioside sialic acid. The fifth novel fraction, containing up to 16% of total ganglioside-sialic acid, moved below the penta-sialoganglioside G_P1 and is suggested to contain hexa-sialogangliosides.There were two main changes in ganglioside synthesis, which were identical in both structures.The first occurred from the sixth to the eleventh day, parallel to decreased proliferation, maximal cell migration and neuroblast differentiation, G_D3 and G_D2 decreased rapidly in favour of G_Q1b, G_P1, and to the novel fractions, described above.The second occurred from the eleventh to the eighteenth day, parallel to increased growth and arborization of dendrites and axons as well as functional establishment of synaptic contacts, there was a sharp rise in the amount of G_D1b, G_T1b, and G_D1a. Concomitantly the novel gangliosides decreased. At hatching G_D1a was the predominant ganglioside. G_M3, G_M2, and G_M1 were always minor fractions, each accounting for less than 4% of total ganglioside-sialic acid. G_M4 was never detected, indicating neglegible myelinisation until hatching.Abbreviations NeuAc N-Acetylneuraminic acid - G_M4 I³NeuAc-GalCer - G_M3 II³NeuAc-LacCer - G_M2 II³NeuAc-G_gOse₃Cer - G_M1 II³NeuAc-G_gOse₄Cer - G_D3 II³(NeuAc)₂LacCer - G_D1a IV³NeuAc, II³NeuAc G_gOse₄Cer - G_T3 II³ (NeuAc)₃LacCer - G_D2 II³(NeuAc)₂G_gOse₃Cer - G_D1b II³(NeuAc)₂G_gOse₃Cer - G_D1b II³(NeuAc)₂G_gOse₄Cer - G_T2 II³(NeuAc)₃G_gOse₃Cer - G_T1b IV³NeuAc, II³(NeuAc)₂G_gOse₄Cer - G_T1c II³(NeuAc)₃G_gOse₄Cer - G_Q1b IV³(NeuAc)₂ II³(NeuAc)₂G_gOse₄Cer - G_Q1c IV³NeuAc, II³(NeuAc)₃G_gOse₄Cer - G_P1 IV³(NeuAc)₂, II³(NeuAc)₃G_gOse₄Cer - G_H(?) IV₃(NeuAc)₃, II³(NeuAc)₃G_gOse₄Cer     

Nonuniform volume changes during muscle contraction.

We measured dynamic changes in volume during contraction of live, intact frog skeletal muscle fibers through a high-speed, intensified, digital-imaging microscope. Optical cross-sections along the axis of resting cells were scanned and compared with sections during the plateau of isometric tetanic contractions. Contraction caused an increase in volume of the central third of a cell when axial force was maximum and constant and the central segment was stationary or lengthened slightly. But changes were unequal along a cell and not predicted by a cell's resting area or shape (circularity). Rapid local adjustments in the cytoskeletal evidently keep forces in equilibrium during contraction of living skeletal muscle. These results also show that optical signals may be distorted by nonuniform volume changes during contraction.     

Auto-ubiquitination of ubiquitin-activating enzymes from chicken breast muscle.

A soluble ubiquitin-depleted fraction from chicken skeletal muscle (fraction II), when incubated at neutral pH for several hours with 125I-ubiquitin and ATP, formed small amounts of a ubiquitin derivative (Mr 115,000) of the ubiquitin-activating enzyme E1 as well as certain similarly modified E2 species (Mr 37,000, 34,000 and 24,000). Treatment of such mixtures with NaOH during the incubations, even at early times, greatly enhanced the appearance of these entities; up to two-thirds of the thiolesters of ubiquitin bound to these proteins before alkali treatment were thus converted. The bonds involved had properties compatible with their being peptidic in nature, suggesting that auto-ubiquitination had occurred in each case. The protease inhibitor and alkylating agent tosyl-lysylchloromethane ('TLCK'), when preincubated at 50 microM with fraction II for 2 h at 37 degrees C before the addition of 125I-ubiquitin and ATP, promoted the subsequent auto-ubiquitination of E1 and inhibited its adenylate-forming and thiolester-transferring activities. The findings have a bearing on the physiological substrate- and site-specificity of ubiquitin-conjugating reactions.     

A hybrid static optimisation method to estimate muscle forces during muscle co-activation

The general static optimisation (GSO) process is one of various muscle force estimation methods due to its low computational requirements. However, it can show biased muscle force estimation under muscle co-contraction. In the present study, we introduced a novel hybrid static optimisation (HSO) method to estimate reasonable muscle forces during muscle co-activation movements using more specific equality constraints, i.e. agonist and antagonist muscle moments predicted from a new correlation coefficient approach. The new method was evaluated for heel-rise movements. We found that the proposed method improved the potential of antagonist muscle force estimation in comparison to the GSO solutions. The proposed HSO method could be applied in biomechanics and rehabilitation, for example.     

The proteome of chicken skeletal muscle: changes in soluble protein expression during growth in a layer strain

The whole animal, and the pectoralis muscle in particular, grows at a greatly enhanced rate in chickens selected for meat production (broilers) when compared to those selected for egg production (layers). As part of an ongoing study to analyse muscle protein dynamics under conditions of rapid growth, we have embarked upon a preliminary characterisation of the proteome of layer chicken pectoralis muscle, at specified time-points from 1 to 27 days after hatching. Soluble extracts of muscle homogenates were separated by two-dimensional (2-D) gel electrophoresis and selected spots were analysed by in-gel tryptic digestion and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Of 90 spots, 51 gave mass spectra that matched to existing chicken proteins present in on-line databases, 12 matched equivalent proteins from non-avian species and 11 yielded good quality spectra but were unable to be matched against existing databases. For many of these proteins, growth over 27 days elicited dramatic changes in relative expression levels. Chicken skeletal muscle offers an excellent system for developmental proteomics.     

Skeletal muscle satellite cells appear during late chicken embryogenesis.

The emergence of avian satellite cells during development has been studied using markers that distinguish adult from fetal cells. Previous studies by us have shown that myogenic cultures from fetal (Embryonic Day 10) and adult 12-16 weeks) chicken pectoralis muscle (PM) each regulate expression of the embryonic isoform of fast myosin heavy chain (MHC) differently. In fetal cultures, embryonic MHC is coexpressed with a ventricular MHC in both myocytes (differentiated myoblasts) and myotubes. In contrast, myocytes and newly formed myotubes in adult cultures express ventricular but not embryonic MHC. In the current study, the appearance of myocytes and myotubes which express ventricular but not embryonic MHC was used to determine when adult myoblasts first emerge during avian development. By examining patterns of MHC expression in mass and clonal cultures prepared from embryonic and posthatch chicken skeletal muscle using double-label immunofluorescence with isoform-specific monoclonal antibodies, we show that a significant number of myocytes and myotubes which stain for ventricular but not embryonic MHC are first seen in cultures derived from PM during fetal development (Embryonic Day 18) and comprise the majority, if not all, of the myoblasts present at hatching and beyond. These results suggest that adult type myoblasts become dominant in late embryogenesis. We also show that satellite cell cultures derived from adult slow muscle give results similar to those of cultures derived from adult fast muscle. Cultures derived from Embryonic Day 10 hindlimb form myocytes and myotubes that coexpress ventricular and embryonic MHCs in a manner similar to cells of the Embryonic Day 10 PM. Thus, adult and fetal expression patterns of ventricular and embryonic MHCs are correlated with developmental age but not muscle fiber type.