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L F Lemanski

Publications and source records attributed to L F Lemanski.

At least 73 records · Page 4Linked to original sources
PubMed1989

Immunofluorescent, immunogold, and electrophoretic studies for desmin in embryonic hearts of normal and cardiac mutant Mexican axolotls, Ambystoma mexicanum.

Recessive mutant gene c for "cardiac nonfunction" in axolotls results in an absence of normal heart contractions in affected embryos due to a failure of myofibril formation. In the present study, the intermediate filament protein, desmin, is compared in developing normal and mutant hearts by means of two-dimensional gel electrophoresis, immunofluorescent microscopy, and immunoelectron microscopy. Tissues were fixed in periodate-lysine-paraformaldehyde or paraformaldehyde-glutaraldehyde solutions and rapidly frozen or embedded in Lowicryl resin. Frozen sections stained with FITC-conjugated antibodies by an indirect approach revealed that desmin is localized in the I-band regions of adult cardiac myofibrils. In normal embryonic hearts at stage 32 (preheartbeat) desmin is localized as "spots" or amorphous collections in the cells. As development progresses to stage 35, staining for desmin in normal hearts becomes more intense with localization being most pronounced at the cell peripheries. By stage 41 most of the desmin in normal hearts is localized in the I band areas of the organized myofibrils and the staining of amorphous areas is much less prominent. During early development, the distribution of desmin in mutant hearts is similar to normal. However, while most of the desmin in normal organs at stage 41 is associated with myofibrils, the staining remains diffuse in mutants. Two-dimensional gel electrophoresis reveals comparable patterns for desmin from normal and mutant hearts. Immunogold staining shows desmin localization to be between the myofibrils and around the I-band regions in adult cardiac muscle and in stage 41 normal embryonic hearts. Immunogold staining confirms a diffuse distribution of desmin in mutant hearts.

Ambystoma mexicanum↗
PubMed1989

Studies of heart development in normal and cardiac lethal mutant axolotls: a review.

The morphology of developing hearts in axolotls, Ambystoma mexicanum, has been studied by scanning electron microscopy in order to provide a chronology of morphogenesis that can be correlated with ongoing biochemical and immunocytochemical studies. In addition to normal embryos we have studied homozygous recessive cardiac lethal mutant axolotls. The mutant myocardium undergoes aberrant sarcomere development and lacks a normal heartbeat. Morphogenesis of mutant hearts appears to be nearly normal with respect to myocardial cell shape changes, epicardial formation, and the distribution of extracellular matrix fibrils in the cardiac jelly. This suggests that the deficient arrangement of contractile proteins in mutant myocardial cells does not prevent the normal organization or function of cytoskeletal isoforms of these proteins in the developing myocardium and epicardium. The implications of biochemical and morphological investigations of axolotl hearts are considered in the context of the entire developmental history of the cardiogenic mesoderm.

Ambystoma↗
PubMed1988

Myocardial cell relationships during morphogenesis in normal and cardiac lethal mutant axolotls, Ambystoma mexicanum.

Sarcomere formation has been shown to be deficient in the myocardium of axolotl embryos homozygous for the recessive cardiac lethal gene c. We examined the developing hearts of normal and cardiac mutant embryos from tailbud stage 33 to posthatching stage 43 by scanning electron microscopy in order to determine whether that deficiency has any effect on heart morphogenesis. Specifically, we investigated the relationships of myocardial cells during the formation of the heart tube (stage 33), the initiation of dextral looping (stages 34-36), and the subsequent flexure of the elongating heart (stages 38-43). In addition, we compared the morphogenetic events in the axolotl to the published accounts of comparable stages in the chick embryo. In the axolotl (stage 33), changes in cell shape and orientation accompany the closure of the myocardial trough to form the tubular heart. The ventral mesocardium persists longer in the axolotl embryo than in the chick and appears to contribute to the asymmetry of dextral looping (stages 34-36) in two ways. First, as a persisting structure it places constraints on the simple elongation of the heart tube and the ability of the heart to bend. Second, after it is resorbed, the ventral myocardial cells that contributed to it are identifiable by their orientation, which is orthogonal to adjacent cells: a potential source of shearing effects. Cardiac lethal mutant embryos behave identically during these events, indicating that functional sarcomeres are not necessary to these processes. The absence of dynamic apical myocardial membrane changes, characteristic of the chick embryo (Hamburger and Hamilton stages 9-11), suggests that sudden hydration of the cardiac jelly is less likely to be a major factor in axolotl cardiac morphogenesis. Subsequent flexure (stages 38-43) of the axolotl heart is the same in normal and cardiac lethal mutant embryos as the myocardial tube lengthens within the confines of a pericardial cavity of fixed length. However, the cardiac mutant begins to exhibit abnormalities at this time. The lack of trabeculation (normally beginning at stage 37) in the mutant ventricle is evident at the same time as an increase in myocardial surface area, manifest in extra bends of the heart tube at stage 39. Nonbeating mutant hearts (stage 41) have an abnormally large diameter in the atrioventricular region, possibly the result of the accumulation of ascites fluid. In addition, mutant myocardial cells have a larger apical surface area compared to normals.

Ambystoma↗
PubMed1988

Three-dimensional immunogold localization of alpha-actinin within the cytoskeletal networks of cultured cardiac muscle and nonmuscle cells.

The ultrastructural distribution of alpha-actinin was studied in cultured hamster heart cells by immunogold replica electron microscopy. This technique enabled us to localize alpha-actinin within the cytoskeletal networks at high resolution and in three dimensions. Colloidal gold, indicating the presence of alpha-actinin, was localized on the Z bands of nascent myofibrils in myocytes and on stress fiber bundles in nonmuscle cells. alpha-Actinin staining was also seen on stellate foci, where cytoskeletal filaments converged along the inner myocyte cell membranes. Intermediate filaments were associated with Z bands of myofibrils, stress fibers, and subplasmalemmal actin networks at the specific points where alpha-actinin was localized on these structures. Heavy meromyosin treatment prior to immunostaining confirmed that the thin filaments contained actin. These results suggest that alpha-actinin serves to interlink these various cytoskeletal elements. In addition, this protein may be involved in the initial phases of filament organization during myofibrillogenesis along the inner surface of the myocyte plasma membrane.

Actinin↗
PubMed1987

Induction of myofibrillogenesis in cardiac lethal mutant axolotl hearts rescued by RNA derived from normal endoderm.

A strain of axolotl, Ambystoma mexicanum, that carries the cardiac lethal or c gene presents an excellent model system in which to study inductive interactions during heart development. Embryos homozygous for gene c contain hearts that fail to beat and do not form sarcomeric myofibrils even though muscle proteins are present. Although they can survive for approximately three weeks, mutant embryos inevitably die due to lack of circulation. Embryonic axolotl hearts can be maintained easily in organ culture using only Holtfreter's solution as a culture medium. Mutant hearts can be induced to differentiate in vitro into functional cardiac muscle containing sarcomeric myofibrils by coculturing the mutant heart tube with anterior endoderm from a normal embryo. The induction of muscle differentiation can also be mediated through organ culture of mutant heart tubes in medium 'conditioned' by normal anterior endoderm. Ribonuclease was shown to abolish the ability of endoderm-conditioned medium to induce cardiac muscle differentiation. The addition of RNA extracted from normal early embryonic anterior endoderm to organ cultures of mutant hearts stimulated the differentiation of these tissues into contractile cardiac muscle containing well-organized sarcomeric myofibrils, while RNA extracted from early embryonic liver or neural tube did not induce either muscular contraction or myofibrillogenesis. Thus, RNA from anterior endoderm of normal embryos induces myofibrillogenesis and the development of contractile activity in mutant hearts, thereby correcting the genetic defect.

Ambystoma↗
PubMed1986

Immunoelectron microscopic localization of alpha-actinin and actin in embryonic hamster heart cells.

Myofibrillogenesis in developing cardiac cells of the Syrian hamster from early embryonic stages through newborn was studied by electron microscopy, immunofluorescence microscopy and immunoelectron microscopy. alpha-Actinin and actin were localized at light and electron microscopic levels in embryonic heart cells which had been fixed in a periodate-lysine-paraformaldehyde or a glutaraldehyde-formaldehyde mixture, and embedded in Lowicryl K4M. Indirect staining methods were used for immunofluorescence staining of thick sections and immunoferritin staining of thin sections. The earliest evidence of myofibrillogenesis in embryonic myocardial cells was the presence of many randomly arranged thin (6 nm) filaments and a few scattered thick filaments (15 nm) near the plasma membrane. alpha-Actinin was detected in a semi-continuous, diffuse layer in some portions of the cell just beneath the plasma membrane in association with the filamentous collections. Later in development, alpha-actinin coalesced into Z-plaques at the membrane as the filaments arranged into parallel arrays. Actin was localized in the thin filaments as expected. In later stages of development, alpha-actinin was observed at the Z-lines and intercalated discs of the mature myofibrils while actin was localized at both the I-band and Z-line. Our results suggest that myofibrillogenesis is initiated at the plasma membrane and that Z-plaques are precursors of myofibrillar Z-bands and may serve as organizing centers for myofibrillogenesis in developing cardiomyocytes.

Actinin↗
PubMed1985

Immunoelectron microscopic localization of alpha-actinin on Lowicryl-embedded thin-sectioned tissues.

A procedure has been developed for the immunoelectron microscopic localization of intracellular antigens on thin-sectioned tissues. The tissues were fixed in a periodate-lysine-paraformaldehyde solution or a formaldehyde-glutaraldehyde combination and embedded in the acrylate-methacrylate mixture, Lowicryl K4M (Polaron), which was polymerized under ultraviolet irradiation at -35 degrees C. Thin sections were mounted on gold grids, immunostained using an indirect method with ferritin-labeled antibodies, and, optionally, counterstained with osmium tetroxide and/or lead citrate and uranyl acetate. The procedure provided good morphologic preservation of the cell architecture in adult and embryonic heart, and skeletal and smooth muscle tissue, as well as nonmuscle cells. At the same time it retained the antigenicities of several contractile proteins, including myosin, tropomyosin, actin, and alpha-actinin. The method has advantages over en bloc staining techniques in that the problem of antibody penetration into the cells is eliminated and careful controls can be performed on adjacent sections. This technique will be useful for localizing, at the ultrastructural level, contractile and other selected proteins in a variety of muscle and non-muscle cells. Details of the new protocol and a description of the results of using antibody against the contractile protein, alpha-actinin, are given.

Acrylic Resins↗
PubMed1984

Anti-troponin-T monoclonal antibody crossreacts with all muscle types.

Monoclonal antibodies to troponin-T were produced by the hybridoma technique. Culture supernatants were initially screened using an enzyme-linked immunoabsorbent assay (ELISA). Positive clones were subcloned twice and further characterized. One of these, 7/H3:C9:D10, produced antibodies against troponin-T; immunoblotting experiments indicated its specificity for only troponin-T when challenged with a variety of striated muscle myofibrillar proteins. Indirect immunofluorescence staining with the antibody shows specific I-band staining in both adult and embryonic skeletal and cardiac muscle of various vertebrate species. In addition, intense but diffuse cytoplasmic staining was seen in chicken gizzard smooth muscle. Our results suggest that troponin-T contains an antigenic determinant that is common to both striated and smooth muscle.

Animals↗
PubMed1984

Accumulation and localization of troponin-T in developing hearts of Ambystoma mexicanum.

Troponin-T (Tn-T) expression in developing hearts of axolotls, Ambystoma mexicanum, was studied with the use of polyclonal and monoclonal antibodies and SDS-polyacrylamide gel electrophoresis. In precontractile hearts (stage 32/33), Tn-T was present in addition to myosin, actin and tropomyosin as evidenced by the presence of the protein bands in SDS-gels and by indirect immunofluorescence. Tn-T was localized in amorphous collections at the peripheries of these precontractile cells. Hearts of normal and cardiac lethal mutant siblings were also analysed for Tn-T expression. No detectable differences in the quantity of protein present was observed by gel electrophoresis or by indirect immuno-fluorescence. The most striking difference concerned the localization of the protein. In normal hearts, Tn-T was primarily localized in the I-bands of organized myofibrils; however, in mutant cells the Tn-T was localized in amorphous collections at the cell peripheries suggesting a reduction of myofibrillar organization in these cells. No differences were observed in the contractile protein composition between normal and mutant embryonic hearts by gel electrophoresis experiments.

Actins↗
PubMed1983

Immunofluorescent studies for myosin, actin, tropomyosin and alpha-actinin in cultured cardiomyopathic hamster heart cells.

Primary cultures of cardiac myocytes from newborn normal and genetically cardiomyopathic (strain UM-X7.1) hamsters were analyzed by electron microscopy and immunofluorescent staining for myosin, actin, tropomyosin, and alpha-actinin. Antibody staining of these contractile proteins demonstrates that both normal and cardiomyopathic (CM) myocytes contain prominent myofibrils after 3 days in culture, although the CM myofibrils are disarrayed and not aligned as those in normal cells. The disarray becomes even more pronounced in CM cells after 5 days in culture. The immunofluorescent staining patterns of individual myofibrils in normal and CM cells were similar for myosin, actin, and tropomyosin. However, alpha-actinin staining reveals that the CM myofibrils have abnormally wide and irregularly shaped Z bands. Electron microscopy confirms the irregular Z-band appearance as well as the myofibril disarray. Thus, CM cardiomyocytes clearly show an aberrant pattern of myofibril structure and organization in culture.

Actinin↗
PubMed1983

An analysis of contractile proteins in developing chick heart by SDS polyacrylamide gel electrophoresis and electron microscopy.

Chick heart development was studied using transmission electron microscopy and SDS-polyacrylamide gel electrophoresis in combination with densitometry. Myosin heavy chain, alpha-actinin, actin and tropomyosin accumulations were analysed in developing hearts from preheartbeat stage 9 (Hamburger-Hamilton staging series) through 2 days after hatching. At the preheartbeat stage, electron microscopy revealed a significant number of thin filaments scattered throughout the cytoplasm of the myoblasts; however, very few thick filaments were seen. There was no obvious association between the two filament types. SDS-polyacrylamide tube gels of heart muscle homogenates demonstrated the presence of all five proteins in hearts at the preheartbeat stage. Further analyses of the proteins by gel densitometry indicated that both actin and myosin accumulated rapidly during heart development while alpha-actinin and tropomyosin levels remained relatively static. Our results show that detectable quantities of myosin heavy chain, alpha-actinin, actin and tropomyosin accumulate in myocardial cells prior to the appearance of myofibrils and initiation of the contractile function.

Actinin↗
PubMed1982

A radioimmunoassay method for quantification of alpha-tropomyosin in heart homogenates.

A new, extremely sensitive, solid phase radioimmunoassay has been developed to quantify tropomyosin levels in heart tissue homogenates. Specific antibody was coupled to Sepharose 4B and saturation levels of [125I]tropomyosin bound. Release of radiolabel into the supernatant portion occurred when heart homogenates, authentic tropomyosin or tropomyosin in the presence of homogenate were added to these immunobeads. Quantification of the amount of tropomyosin was based on the level of release effected by standard tropomyosin with and without homogenate. The assay was determined to be highly specific and sensitive for tropomyosin. Picomolar quantities of the protein were readily detectable. Linearity extended well over the range of 5-200 ng tropomyosin in the homogenate. The method can be applied to other proteins for quantification during embryonic development.

Amino Acids↗
PubMed1982

Quantification of tropomyosin by radioimmunoassay in developing hearts of cardiac mutant axolotls, Ambystoma mexicanum.

Recessive mutant gene c in axolotls results in a failure of embryonic heart function. Earlier morphological studies showed that the mutant myocardial cells lack organized sarcomeric myofibrils. Electrophoresis and immunofluorescent studies suggested that the mutant heart cells contain substantial amounts of actin, myosin and alpha-actinin; however, tropomyosin appeared deficient. In the present study, we employed a newly developed extremely sensitive solid-phase radioimmunoassay method to quantitate very accurately the tropomyosin levels in normal and mutant sibling hearts at early (stage 35), intermediate (stage 39) and late (stage 41) developmental stages. Our results demonstrate that cardiac mutant hearts contain significantly lower than normal quantities of antigenically detectable tropomyosin at all of the developmental stages examined. This insufficiency of tropomyosin in mutant hearts may be a primary cause at the cell level for their failure to form organized myofibrils.

Ambystoma↗