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D A Fischman

Publications and source records attributed to D A Fischman.

At least 55 records · Page 3Linked to original sources
PubMed1990

Differential distribution of subsets of myofibrillar proteins in cardiac nonstriated and striated myofibrils.

Cultured cardiac myocytes were stained with antibodies to sarcomeric alpha-actinin, troponin-I, alpha-actin, myosin heavy chain (MHC), titin, myomesin, C-protein, and vinculin. Attention was focused on the distribution of these proteins with respect to nonstriated myofibrils (NSMFs) and striated myofibrils (SMFs). In NSMFs, alpha-actinin is found as longitudinally aligned, irregular approximately 0.3-microns aggregates. Such aggregates are associated with alpha-actin, troponin-I, and titin. These I-Z-I-like complexes are also found as ectopic patches outside the domain of myofibrils in close apposition to the ventral surface of the cell. MHC is found outside of SMFs in the form of discrete fibrils. The temporal-spatial distribution and accumulation of the MHC-fibrils with respect to the I-Z-I-like complexes varies greatly along the length of the NSMFs. There are numerous instances of I-Z-I-like complexes without associated MHC-fibrils, and also cases of MHC-fibrils located many microns from I-Z-I-like complexes. The transition between the terminal approximately 1.7-microns sarcomere of any given SMF and its distal NSMF-tip is abrupt and is marked by a characteristic narrow alpha-actinin Z-band and vinculin positive adhesion plaque. A titin antibody T20, which localizes to an epitope at the Z-band in SMFs, precisely costains the 0.3-microns alpha-actinin aggregates in ectopic patches and NSMFs. Another titin antibody T1, which in SMFs localizes to an epitope at the A-I junction, typically does not stain ectopic patches and NSMFs. Where detectable, the T1-positive material is adjacent to rather than part of the 0.3-microns alpha-actinin aggregates. Myomesin and C-protein are found only in their characteristic sarcomeric locations (even in just perceptible SMFs). These A-band-associated proteins appear to be absent in ectopic patches and NSMFs.

Actinin↗
PubMed1989

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles.

The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations. Nuclear bag1 fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag2 fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with anti-neonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag1 fibers lacked staining for M-protein, whereas bag2 fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag1 fibers were less reactive and clear striations were not observed, in contrast to bag2 and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested. Taken together, the data show that in adult rat soleus, slow tonic and neonatal myosin heavy chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.

Animals↗
PubMed1989

Size and charge heterogeneity of C-protein isoforms in avian skeletal muscle. Expression of six different isoforms in chicken muscle.

C-protein is an abundant protein, of unknown function, found in the striated muscles of all vertebrates (Offer et al., 1973). Based on differences in size, charge, antigenicity and sarcomere distribution, at least three different isoforms of this protein have been identified (Callaway & Bechtel, 1981; Yamamoto & Moos, 1983; Reinach et al., 1982; Dhoot et al., 1985). These have been termed fast-, slow- and cardiac-type isoforms, relative to their distribution in adult striated muscles. Each of these isoforms appears to be expressed sequentially during the development of the chicken pectoralis muscle (Obinata et al., 1984; Obinata, 1985). To better characterize the various isoforms of C-protein, we have reexamined its in vivo expression during avian myogenesis using a combination of 1- and 2-dimensional gel electrophoresis, cell-free translation and immunoblotting procedures. In this manuscript we demonstrate for the first time that at least four major C-protein isoforms can be distinguished in adult chicken muscles. These include a fast-type isoform in the pectoralis (PECT) muscle (Cf), a slow-type isoform in the anterior latissimus dorsi (ALD) muscle (Cs3), a second slow-type isoform in the posterior latissimus dorsi (PLD) muscle (Cs4) and a cardiac-type in the ventricle (Cc). During embryonic development of the PECT muscle two additional isoforms can be resolved. These are both slow-type isoforms based on their reactivities with ALD66, a monoclonal antibody specific for adult slow-type C-protein. These latter isoforms have been termed Cs1 and Cs2. Several of the isoforms, particularly Cs1 ands Cs3, exhibit two or more spots of different charge but identical molecular weight on 2-D gels. This observation suggests the possibility that these isoforms are post-translationally modified and possibly phosphorylated. Our data show the C-protein family in avian striated muscles to be highly complex. Additional genetic analyses and primary sequence studies will be required to distinguish transcriptional from post-transcriptional variants.

Animals↗
PubMed1989

Starvation leads to decreased levels of mRNA for myofibrillar proteins.

Malnutrition is a common complicating factor in surgical illness. To investigate the cellular changes and mechanisms responsible for the protein wasting associated with nutritional deprivation, Sprague-Dawley rats were subjected to total protein-calorie starvation for 3 (n = 12) or 5 days (n = 12) and compared to freely fed animals monitored for 3 (n = 8) or 5 (n = 8) days. Gastrocnemius protein and RNA content and levels of mRNA coding for the myofibrillar proteins myosin heavy chain, myosin light chain, and alpha-actin were measured. Starvation resulted in a significant decrease in gastrocnemius mass and protein content, and was associated with decreases in mRNA levels for the three myofibrillar proteins assayed. We conclude that changes in mRNA levels for these proteins likely contribute to the loss of peripheral protein which occurs during total nutritional deprivation. In addition, the changes in mRNA levels for these three structural proteins appear to be coordinate, suggesting that transcription of no single myofibrillar protein is rate-limiting in the regulation of skeletal muscle protein content.

Actins↗
PubMed1989

Recombinant growth hormone enhances muscle myosin heavy-chain mRNA accumulation and amino acid accrual in humans.

A potentially lethal complication of trauma, malignancy, and infection is a progressive erosion of muscle protein mass that is not readily reversed by nutritional support. Growth hormone is capable of improving total body nitrogen balance, but its role in myofibrillar protein synthesis in humans is unknown. The acute, in situ muscle protein response to an infusion of methionyl human growth hormone was investigated in the limbs of nutritionally depleted subjects during a period of intravenous refeeding. A 6-hr methionyl growth hormone infusion achieved steady-state serum levels comparable to normal physiologic peaks and was associated with a significant increase in limb amino acid uptake, without a change in body amino acid oxidation. Myosin heavy-chain mRNA levels, measured by quantitative dot blot hybridization, were also significantly elevated after growth hormone administration. The data indicate that methionyl growth hormone can induce intracellular amino acid accrual and increased levels of myofibrillar protein mRNA during hospitalized nutritional support and suggest growth hormone to be a potential therapy of lean body wasting.

Adult↗
PubMed1989

Differential response of myofibrillar and cytoskeletal proteins in cells treated with phorbol myristate acetate.

Muscle-specific and nonmuscle contractile protein isoforms responded in opposite ways to 12-o-tetradecanoyl phorbol-13-acetate (TPA). Loss of Z band density was observed in day-4-5 cultured chick myotubes after 2 h in the phorbol ester, TPA. By 5-10 h, most I-Z-I complexes were selectively deleted from the myofibril, although the A bands remained intact and longitudinally aligned. The deletion of I-Z-I complexes was inversely related to the appearance of numerous cortical, alpha-actinin containing bodies (CABs), transitory structures approximately 3.0 microns in diameter. Each CAB consisted of a filamentous core that costained with antibodies to alpha-actin and sarcomeric alpha-actinin. In turn each CAB was encaged by a discontinuous rim that costained with antibodies to vinculin and talin. Vimentin and desmin intermediate filaments and most cell organelles were excluded from the membrane-free CABs. These curious bodies disappeared over the next 10 h so that in 30-h myosacs all alpha-actin and sarcomeric alpha-actinin structures had been eliminated. On the other hand vinculin and talin adhesion plaques remained prominent even in 72-h myosacs. Disruption of the A bands was first initiated after 15-20 h in TPA (e.g., 15-20-h myosacs). Thick filaments of apparently normal length and structure were progressively released from A segments, and by 40 h all A bands had been broken down into enormous numbers of randomly dispersed, but still intact single thick filaments. This breakdown correlated with the formation of amorphous cytoplasmic aggregates which invariably colocalized antibodies to myosin heavy chain, MLC 1-3, myomesin, and C protein. Complete elimination of all immunoreactive thick filament proteins required 60-72 h of TPA exposure. The elimination of the thick filament-associated proteins did not involve the participation of vinculin or talin. In contrast to its effects on myofibrils, TPA did not induce the disassembly of the contractile proteins in stress fibers and microfilaments either in myosacs or in fibroblastic cells. Similarly, TPA, which rapidly induces the translocation of vinculin and talin to ectopic sites in many types of immortalized cells, had no gross effect on the adhesion plaques of myosacs, primary fibroblastic cells, or presumptive myoblasts. Clearly, the response to TPA of contractile protein and some cytoskeletal isoforms not only varies among phenotypes, but even within the domains of a given myotube the myofibrils respond one way, the stress fibers/microfilaments another.

Actin Cytoskeleton↗
PubMed1989

Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes.

Successive stages in the disassembly of myofibrils and the subsequent assembly of new myofibrils have been studied in cultures of dissociated chick cardiac myocytes. The myofibrils in trypsinized and dispersed myocytes are sequentially disassembled during the first 3 d of culture. They split longitudinally and then assemble into transitory polygons. Multiples of single sarcomeres, the cardiac polygons, are analogous to the transitory polygonal configurations assumed by stress fibers in spreading fibroblasts. They differ from their counterparts in fibroblasts in that they consist of muscle alpha-actinin vertices and muscle myosin heavy chain struts, rather than of the nonmuscle contractile protein isoforms of stress fiber polygons. EM sections reveal the vertices and struts in cardiac polygons to be typical Z and A bands. Most cardiac polygons are eliminated by day 5 of culture. Concurrent with the disassembly and elimination of the original myofibrils new myofibrils are rapidly assembled elsewhere in the same myocyte. Without exception both distal tips of each nascent myofibril terminate in adhesion plaques. The morphology and composition of the adhesion plaques capping each end of each myofibril are similar to those of the termini of stress fibers in fibroblasts. However, whereas the adhesion complexes involving stress fibers in fibroblasts consist of vinculin/nonmuscle alpha-actinin/beta- and gamma-actins, the analogous structures in myocytes involving myofibrils consist of vinculin/muscle alpha-actinin/alpha-actin. The addition of 1.7-2.0 microns sarcomeres to the distal tips of an elongating myofibril, irrespective of whether the myofibril consists of 1, 10, or several hundred tandem sarcomeres, occurs while the myofibril appears to remain linked to its respective adhesion plaques. The adhesion plaques in vitro are the equivalent of the in vivo intercalated discs, both in terms of their molecular composition and with respect to their functioning as initiating sites for the assembly of new sarcomeres. How 1.7-2.0 microns nascent sarcomeres can be added distally during elongation while the tips of the myofibrils remain inserted into submembranous adhesion plaques is unknown.

Actinin↗
PubMed1989

Cachectin/TNF or IL-1 alpha induces cachexia with redistribution of body proteins.

Macrophage secretory products are suspected to participate in the severe lean tissue wasting related to chronic illness. The protein metabolic effects of chronic, 7-day cachectin/tumor necrosis factor (cachectin) or interleukin 1 alpha (IL-1 alpha) administration in vivo were studied in male Wistar rats that were 1) freely fed, 2) pair fed, 3) total protein and calorie starved, 4) twice daily lipopolysaccharide (LPS) administered, 5) twice daily cachectin administered, and 6) twice daily IL-1 alpha administered. LPS, cachectin, or IL-1 alpha administration produced anorexia; weight loss in these groups was comparable to respective pair-fed animals. However, LPS, cachectin, or IL-1 alpha accelerated peripheral protein wasting while preserving liver protein content, unlike the pattern in the pair-fed or starved animals in which loss of liver proteins and relative preservation of skeletal muscle protein were observed. The decrease in skeletal muscle protein content in LPS- or cytokine-treated animals was associated with coordinate decreases in muscle mRNA levels for the myofibrillar proteins myosin heavy chain, myosin light chain, actin, and in the 18S and 28S subunits of ribosomal RNA. We conclude that chronic exposure to the cytokines, IL-1 alpha or cachectin, can simulate those body and muscle protein changes seen in experimental LPS administration or chronic disease and markedly differ from the pattern of protein redistribution due to caloric restriction.

Animals↗
PubMed1988

Posttranslational incorporation of contractile proteins into myofibrils in a cell-free system.

The incorporation of newly synthesized protein into myofibrils has been examined in a cell-free system. Myofibrils were added to a reticulocyte lysate after the in vitro translation of muscle-specific poly(A)+RNA. Only a small number of the many synthesized proteins were found to associate with the exogenously added myofibrils. These proteins were all identified as sarcomeric components and had subunit mobilities (Mr) of 200, 140, 95, 86, 43, 38, 35, 25, 23, 20, and 18 kD. The association was rapid (t1/2 less than 15 min) and, for most of the proteins, relatively temperature insensitive. Except for a 43-kD polypeptide, tentatively identified as beta-actin, none of the proteins encoded by brain poly(A)+RNA associated with the myofibrils. When filaments made from purified myosin or actin were used as the "capture" substrates, only thick or thin filament proteins, respectively, were incorporated. Incorporation was substantially reduced when cross-linked myosin filaments were used. These results are compatible with a model in which proteins of the sarcomere are in kinetic equilibrium with homologous proteins in a soluble pool.

Animals↗
PubMed1987

Immunochemical analysis of protein isoforms in thick myofilaments of regenerating skeletal muscle.

The expression of myosin heavy chain (MHC) and C-protein isoforms has been examined immunocytochemically in regenerating skeletal muscles of adult chickens. Two, five, and eight days after focal freeze injury to the anterior latissimus dorsi (ALD) and posterior latissimus dorsi (PLD) muscles, cryostat sections of injured and control tissues were reacted with a series of monoclonal antibodies previously shown to specifically bind MHC or C-protein isoforms in adult or embryonic muscles. We observed that during the course of regeneration in each of these muscles there was a reproducible sequence of antigenic changes consistent with differential isoform expression for these two proteins. These isoform switches appear to be tissue specific; i.e., the isoforms of MHC and C-protein which are expressed during the regeneration of a "slow" muscle (ALD) differ from those which are synthesized in a regenerating "fast" muscle (PLD). Evidence has been obtained for the transient expression of a "fast-type" MHC and C-protein during ALD regeneration. Furthermore, during early stages of PLD regeneration this muscle contains MHCs which antigenically resemble those found in the pectoralis muscle at embryonic and early posthatch stages of development. Both regenerating muscles express an isoform of C-protein which appears immunochemically identical to that normally expressed in embryonic and adult cardiac muscle. These results support the concept that isoform transitions in regenerating skeletal muscles qualitatively resemble those found in developing muscles but differences may exist in temporal and tissue-specific patterns of gene expression.

Actin Cytoskeleton↗
PubMed1986

Electron microscopy of C-protein molecules from chicken skeletal muscle.

C-protein from chicken pectoralis muscle has been purified by sequential DEAE-Sephadex and hydroxyapatite chromatography and examined by transmission electron microscopy after spraying in glycerol onto mica and replicating by rotary shadowing with platinum. The most frequently observed particles were of three forms: rod-shaped, U-shaped and V-shaped. Within a size range of 15-40 nm these three groups accounted for 70% of over 800 particles categorized and measured. The remaining particles could not be classified. Since the relative abundance of each of these three forms was well in excess of any of the contaminating proteins detectable by SDS-polyacrylamide gel electrophoresis, we conclude that these variant forms represent C-protein molecules in differing conformations and/or deformations. Particles were observed which were intermediate between rod-shaped and tightly curved U-shaped forms, and between rod and acutely angled V-shaped forms. These results are compatible with a molecular model of a 32 nm X 3 nm flexible, rod-shaped C-protein monomer similar to one previously proposed from hydrodynamic studies and extend recent observations on the ultrastructure of cardiac C-protein. Infrequently, a discontinuously larger V-shaped form was seen, possibly representing a C-protein dimer.

Animals↗
PubMed1986

Myosin heavy chain expression in embryonic cardiac cell cultures.

Chick embryonic heart cell isolates and monolayer cultures were prepared from atria and ventricles at selected stages of cardiac development. The cardiac myocytes were assayed for myosin heavy chain (MHC) content using monoclonal antibodies (McAbs) specific in the heart for atrial (B-1), ventricular (ALD-19), or conductive system (ALD-58) isoforms. Using immunofluorescence microscopy or radioimmunoassay, MHC accumulation was measured before plating and at 48 hr or 7 days in culture. Reproducible changes in MHC antigenicity were observed by 7 days in both atrial and ventricular cultures. The changes were stage dependent and tissue specific but generally resulted in a decreased reactivity with the tissue specific MHC McAbs. In addition, the isoform recognized by ALD-58, characteristic of the conductive system cells in vivo, was never present in cultured myocytes. These results indicate that MHC isoforms produced in vivo may be replaced in monolayer cultures by an isoform(s) not recognized by our tissue specific MHC McAbs. This suggests that the intrinsic program of cardiac myogenesis, within cardiac myocytes, may not be sufficient to establish and maintain differential expression of tissue specific MHC in monolayer cell culture.

Animals↗
PubMed1986

Dynamic exchange of myosin molecules between thick filaments.

To examine thick filament assembly and myosin exchange, a fluorescence energy transfer assay has been established. Assembly-competent myosin molecules labeled with the sulfhydryl-specific fluorochromes 5-(2-[(iodoacetyl)-amino]ethyl)aminonaphthalene-1-sulfonic acids (IAEDANS) or 5-iodoacetamidofluorescein (IAF) were prepared. Using IAEDANS-labeled myosin as fluorescence donor and IAF-labeled myosin as acceptor, thick filament formation was followed by the decrease in donor fluorescence at 0.1 M KCl/10 mM potassium phosphate, pH 6.9. The critical concentration of myosin--i.e., that concentration that remained unassembled at equilibrium with fully formed filaments--was 40 nM. In FET and 125I-labeled myosin incorporation assays, extensive exchange of myosin between thick filaments was observed. The presence of a critical concentration and the measurements of extensive exchange suggest a dynamic equilibrium between fully polymerized myosin and a small pool of soluble myosin.

Actin Cytoskeleton↗
PubMed1985

Analysis of the metal-induced conformational change in myosin with a monoclonal antibody to light chain two.

A monoclonal antibody capable of detecting a conformational change in myosin light chain two (LC2) was characterized in detail. The antibody was shown to bind only to myosin LC2 when tested against fast skeletal myosin (chicken pectoralis muscle). With cardiac or slow muscle myosins, the antibody exclusively recognized their first light chains (LC1). Staining of myofibrils by the monoclonal antibody could be observed only after their irreversible denaturation by acetone or ethanol, or after incubation of the myofibrils in divalent metal chelators. This latter effect was shown to be fully reversible. The metal effect was independent of ionic strength although the affinity of the antibody for myosin was depressed at high salt concentrations. Similar metal effects were detected in the binding of antibody to cardiac or slow myosins. Neither the metal nor the ionic strength-related inhibition of antibody binding were detected with denatured myosin. The antibody binding site overlaps one of the alpha-chymotryptic sites in LC2 protected by divalent metals. Electron microscopic observations of myosin-antibody complexes demonstrated that the antibody binding site is located near the head-rod junction of myosin. Since the binding site of this monoclonal antibody has been mapped by recombinant DNA methods to the junction of the first alpha-helical domain with the calcium binding site of LC2, the location of the calcium binding site must also be located near the head-tail junction of myosin. A model for conformational changes at the myosin head-tail junction is proposed to account for the metal-induced blockage of antibody binding and the inhibition of alpha-chymotryptic digestion of LC2.

Animals↗
PubMed1985

Recombinant DNA approach for defining the primary structure of monoclonal antibody epitopes. The analysis of a conformation-specific antibody to myosin light chain 2.

A monoclonal antibody (MF5), capable of recognizing a divalent cation-induced conformational change in myosin light chain 2 (LC2f), has been used to screen a cDNA library constructed in the expression vector lambda gt11. A clone has been isolated that contains the whole coding sequence of this myosin subunit. The light chain was synthesized as a fusion peptide linked to beta-galactosidase by ten amino acids encoded in the 5' untranslated region of its mRNA. Seven imperfect repeats were identified in the 3' untranslated region of the mRNA. The amino acids conferring specificity on the MF 5 epitope were established by first determining the nucleotide sequence of shorter subclones that expressed the epitope and then eliminating those amino acid residues shared by cardiac myosin LC2, which was unreactive with this antibody. The epitope, which becomes accessible to MF 5 upon removal of bound divalent cations, resides at the junction between the first alpha-helical domain and the metal binding site. Theoretically, this approach can be used to define the primary structure of most protein epitopes.

Amino Acid Sequence↗
PubMed1985

Axial arrangement of the myosin rod in vertebrate thick filaments: immunoelectron microscopy with a monoclonal antibody to light meromyosin.

A monoclonal antibody, MF20, which has been shown previously to bind the myosin heavy chain of vertebrate striated muscle, has been proven to bind the light meromyosin (LMM) fragment by solid phase radioimmune assay with alpha-chymotryptic digests of purified myosin. Epitope mapping by electron microscopy of rotary-shadowed, myosin-antibody complexes has localized the antibody binding site to LMM at a point approximately 92 nm from the C-terminus of the myosin heavy chain. Since this epitope in native thick filaments is accessible to monoclonal antibodies, we used this antibody as a high affinity ligand to analyze the packing of LMM along the backbone of the thick filament. By immunofluorescence microscopy, MF20 was shown to bind along the entire A-band of chicken pectoralis myofibrils, although the epitope accessibility was greater near the ends than at the center of the A-bands. Thin-section, transmission electron microscopy of myofibrils decorated with MF20 revealed 50 regularly spaced, cross-striations in each half A-band, with a repeat distance of approximately 13 nm. These were numbered consecutively, 1-50, from the A-band to the last stripe, approximately 68 nm from the filament tips. These same striations could be visualized by negative staining of native thick filaments labeled with MF20. All 50 striations were of a consecutive, uninterrupted repeat which approximated the 14-15-nm axial translation of cross-bridges. Each half M-region contained five MF20 striations (approximately 13 nm apart) with a distance between stripes 1 and 1', on each half of the bare zone, of approximately 18 nm. This is compatible with a packing model with full, antiparallel overlap of the myosin rods in the bare zone region. Differences in the spacings measured with negatively stained myofilaments and thin-sectioned myofibrils have been shown to arise from specimen shrinkage in the fixed and embedded preparations. These observations provide strong support for Huxley's original proposal for myosin packing in thick filaments of vertebrate muscle (Huxley, H. E., 1963, J. Mol. Biol., 7:281-308) and, for the first time, directly demonstrate that the 14-15-nm axial translation of LMM in the thick filament backbone corresponds to the cross-bridge repeat detected with x-ray diffraction of living muscle.

Animals↗
PubMed1985

Monoclonal antibodies to desmin: evidence for stage-dependent intermediate filament immunoreactivity during cardiac and skeletal muscle development.

Monoclonal antibodies reactive with desmin (D3 and D76) have been generated and their specificities validated by immunoblots, RIAs, and immunocytochemistry. No cross-reaction with other IFPs has been observed. The McAbs recognized different epitopes but both reside in the amino-terminal rod domain of desmin. Whereas McAb D3 produces a staining pattern characteristic of desmin throughout the development of cardiac and skeletal muscles, McAb D76 was selectively unreactive with certain regions of early (three days in ovo) embryonic cardiac anlage, with cultured cardiac myocytes derived from 7-day-old embryos, and with skeletal myotubes in early stages of myogenesis in vitro. Positive reactivity of D76 was seen at stages of myofibrillogenesis when the sarcomeres assume lateral alignment. Evidence was presented that differential reactivity of D76 did not result from the biosynthesis of a new desmin isoform or the post-translational modification of an existing protein. We suggest that the appearance of D76 immunoreactivity during striated muscle development represents an unmasking of the epitope by some IF-associated protein. Since this transition during skeletal muscle differentiation occurs during lateral alignment of the myofibrils, this antibody may serve as a useful probe for exploring this reorganization of the contractile apparatus during myogenesis and muscle regeneration.

Animals↗