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J L Lessard

Publications and source records attributed to J L Lessard.

At least 37 records · Page 2Linked to original sources
PubMed1988

Two monoclonal antibodies to actin: one muscle selective and one generally reactive.

Two IgG1, kappa monoclonal antibodies (Mab) against actin have been obtained from a fusion in which chicken gizzard actin was used as the immunogen. One Mab, designated B4, shows a preferential reactivity toward enteric smooth muscle actin but also cross-reacts with skeletal, cardiac, and aorta actins on the basis of immunoblots, ELISA assays, and indirect immunofluorescence. However, this antibody does not react with either cytoplasmic actin in any of these assay systems. A second Mab, designated C4, reacts with all six known vertebrate isoactins as well as Dictyostelium discoideum and Physarum polycephalum actins. Thus B4 Mab appears to react with an epitope that is at least partially shared among the muscle actins but not found in cytoplasmic actins, while C4 Mab binds to an antigenic determinant that has been highly conserved among the actins. The binding sites of both Mabs on skeletal actin overlap that of pancreatic DNase I. Both antibodies bind a SV8 proteolytic product comprising the amino-terminal two-thirds of the actin molecule, and their epitopes appear to overlap since C4 can compete for the binding of B4 to skeletal actin. Neither antibody is able to prevent actin polymerization.

Actins↗
PubMed1988

Unique isoactins in the brush border of rat intestinal epithelial cells.

The mammalian genome contains 20-30 genes encoding a family of actins. To date, however, only six proteins (four muscle and two nonmuscle isoforms) encoded by this multigene complex have been identified. We have isolated two actins from the brush border of rat intestinal epithelial cells that have isoelectric points and N-terminal peptides characteristic of the cytoplasmic beta- and gamma-actins. However, using a panel of actin-specific monoclonal antibodies, we show that these actins contain a set of epitopes that distinguishes them from any of the known cytoplasmic or muscle isoforms. These unique actins share features of both the nonmuscle and muscle isoforms, suggesting that they represent an intermediate in the evolution of the specialized muscle actins.

Actins↗
PubMed1988

Studies on cardiac myofibrillogenesis with antibodies to titin, actin, tropomyosin, and myosin.

Cardiac myofibrillogenesis was examined in cultured chick cardiac cells by immunofluorescence using antibodies against titin, actin, tropomyosin, and myosin. Primitive cardiomyocytes initially contained stress fiber-like structures (SFLS) that stained positively for alpha actin and/or muscle tropomyosin. In some cases the staining for muscle tropomyosin and alpha actin was disproportionate; this suggests that the synthesis and/or assembly of these two isoforms into the SFLS may not be stoichiometric. The alpha actin containing SFLS in these myocytes could be classified as either central or peripheral; central SFLS showed developing sarcomeric titin while peripheral SFLS had weak titin fluorescence and a more uniform stain distribution. Sarcomeric patterns of titin and myosin were present at multiple sites on these structures. A pair of titin staining bands was clearly associated with each developing A band even at the two or three sarcomere stage, although occasional examples of a titin band being associated with a half sarcomere were noted. The appearance of sarcomeric titin patterns coincided or preceded sarcomere periodicity of either alpha actin or muscle tropomyosin. The early appearance of titin in myofibrillogenesis suggests it may have a role in filament alignment during sarcomere assembly.

Actins↗
PubMed1988

The development expression of the rat alpha-vascular and gamma-enteric smooth muscle isoactins: isolation and characterization of a rat gamma-enteric actin cDNA.

We have isolated and characterized two cDNA clones from whole rat stomach, pRV alpha A-19 and pRE gamma A-11, which are specific for the alpha-vascular and gamma-enteric smooth muscle isoactins, respectively. The rat gamma-enteric smooth muscle actin contains a single amino acid substitution of a proline for a glutamine at position 359 of the mature peptide when compared with the chicken gizzard gamma-actin sequence (J. Vandekerckhove and K. Weber, FEBS Lett. 102:219, 1979). Sequence comparisons of the 5' and 3' untranslated (UT) regions of the two smooth muscle actin cDNAs demonstrate that these regions contain no apparent sequence similarities. Additional comparisons of the 5' UT regions of the two smooth muscle actin cDNAs to all other known actin sequences reveal no apparent sequence similarities for the rat gamma-enteric isoactin within the 15 base pairs of sequence currently available, while the rat alpha-vascular isoactin contains two separate sequences which are similar to sequences within the 5' UT regions of the human and chicken alpha-vascular actin genes. A similar comparison of the 3' UT regions of the two smooth muscle actins demonstrates that the alpha-vascular isoactins do not contain the high degree of cross-species sequence conservation observed for the other isoactins and that the gamma-enteric isoactin contains an inverted sequence of 52 nucleotides which is similar to a sequence found within the 3' UT regions of the human, chicken, and rat beta-cytoplasmic isoactins. These observations complicate the apparent cross-species conservation of isotype specificity of these domains previously observed for the other actin isoforms. Northern blot analysis of day 15 rat embryos and newborn, day 19 postbirth, and adult rats demonstrates that the day 15 rat embryo displays low to undetectable levels of smooth muscle isoactin mRNA expression. By birth, the stomach and small intestine show dramatic increases in alpha-vascular and gamma-enteric actin expression. These initially high levels of expression decrease through day 19 to adulthood. In the adult rat, the uterus and aorta differ in their content of smooth muscle isoactin mRNA. These results demonstrate that the gamma-enteric and alpha-vascular isoactin mRNAs are coexpressed to various degrees in tissues which contain smooth muscle.

Actins↗
PubMed1987

Fetal expression of muscle-specific isoactins in multiple organs of the Wistar-Kyoto rat.

Actin, a cytoskeletal and contractile protein, is expressed in six different isoforms that exhibit striking specificity. No studies have considered the muscle-specific actin expression in multiple organ systems in the intact fetus. Using a monoclonal antibody (B4) which reacts specifically with the isoactins of the smooth and skeletal muscle our immunohistochemical study examined whole fetal body sections to follow the development of actin expression throughout the last third of gestation in the Wistar-Kyoto rat. B4 staining was exclusively localized to muscle, confirming its high specificity and its usefulness for studying the ontogeny of muscle-specific isoactins. At 15 days of gestation, B4 staining was detected in the heart, the thoracic aorta and the skeletal muscle of the chest wall. The distribution and intensity of staining in the heart were initially higher than in the aorta or skeletal muscle and remained unchanged throughout the remainder of gestation, suggesting that the maturation of cardiac actin expression is well developed, although not fully completed before birth. Expression of muscle-specific actins in skeletal muscle was age-dependent and correlated with the maturational changes of muscle cell precursors. B4 staining in the fetal kidney was not apparent until day 20 of gestation and was localized to the inner cortical vessels. in association with the most mature nephrons, suggesting a centrifugal maturation of the intrarenal vasculature. The intensity of B4 staining in most tissues including bronchi, bowel, diaphragm, chest wall muscle and peripheral and pulmonary arteries increased by the end of gestation.

Actins↗
PubMed1987

Sequential disassembly of myofibrils induced by myristate acetate in cultured myotubes.

The phorbol ester TPA induces the sequential disassembly of myofibrils. First the alpha-actin thin filaments are disrupted and then, hours later, the myosin heavy chain (MHC) thick filaments. TPA does not induce the disassembly of the beta- and gamma-actin thin filaments of stress fibers in presumptive myoblasts or fibroblasts, nor does it block the reemergence of stress fibers in 72-h myosacs that have been depleted of all myofibrillar molecules. There are differences in where, when, and how myofibrillar alpha-actin and MHC are degraded and eliminated from TPA-myosacs. Though the anisodiametric myotubes have begun to retract into isodiametric myosacs after 5 h in TPA, staining with anti-MHC reveals normal tandem A bands. In contrast, staining with mAb to muscle actin fails to reveal tandem I bands. Instead, both mAb to muscle actin and rhophalloidin brilliantly stain numerous disk-like bodies approximately 3.0 micron in diameter. These muscle actin bodies do not fuse with one another, nor do they costain with anti-MHC. All muscle actin bodies and/or molecules disappear in 36-h myosacs. The collapse of A bands is first initiated in 10-h myosacs. Their loss correlates with the appearance of immense, amorphous MHC patches. MHC patches range from a few micrometers to over 60 micron in size. They do not costain with antimuscle actin or rho-phalloidin. While diminishing in number and fluorescence intensity, MHC aggregates are present in 30% of the 72-h myosacs. Myosacs removed from TPA rapidly elongate, and after 48 h display normal newly assembled myofibrils. TPA reversibly blocks incorporation of [35S]methionine into myofibrillar alpha-actin, MHC, myosin light chains 1 and 2, the tropomyosins, and troponin C. It does not block the synthesis of beta- or gamma-actins, the nonmyofibrillar MHC or light chains, tubulin, vimentin, desmin, or most household molecules.

Actins↗
PubMed1987

Alteration of the protein composition in the haemolymph of American cockroaches immunized with soluble proteins.

Previous in vivo experiments demonstrating the existence of humoral immunity in cockroaches prompted us to investigate the composition and number of proteins that are newly induced or increased in quantity in the haemolymph of immune cockroaches compared to saline-injected control animals. SDS-PAGE analysis showed that six different haemolymph proteins of molecular weights 220,000, 162,000, 115,000, 102,000, 95,000 and 45,000 were induced in response to immunization by honeybee toxoid. However, only the 115,000 and 102,000 MW proteins consistently increased in quantity in the haemolymph of females immunized with various other soluble protein antigens, such as phospholipase A2 toxoid and inactivated creatine phosphokinase. Other experiments showed that the 115,000 MW band was not induced in immunized male cockroaches. Therefore, only the 102,000 MW protein was consistently enhanced in all samples of immune haemolymph from animals injected with various antigens. In addition, this protein was found in precipitates recovered from assays where immune haemolymph proteins were precipitated by the antigen. Gel filtration chromatography showed that this induced haemolymph protein is large, and bears an apparent native molecular weight of 600,000 +/- 80,000. This suggests that the 600,000 MW protein may consist of multiple subunits of 102,000.

Animals↗
PubMed1986

Tubulin: a factor necessary for the synthesis of both Sendai virus and vesicular stomatitis virus RNAs.

Tubulin acts as a positive transcription factor for in vitro RNA synthesis by two different negative-strand viruses: Sendai virus, a paramyxovirus; vesicular stomatitis virus (VSV), a rhabdovirus. A monoclonal antibody directed against beta-tubulin completely inhibited not only mRNA synthesis and RNA replication catalyzed in vitro by extracts of cells infected with either virus but also mRNA synthesis by detergent-disrupted purified virions. The synthesis of both a leader-like RNA and the NP mRNA directed by detergent-disrupted purified Sendai virions was shown to be totally dependent on the addition of purified tubulin. The addition of purified tubulin, although not required, also stimulated mRNA synthesis directed by detergent-disrupted VSV virions 2- to 7-fold. Finally, there appears to be an association between tubulin and the L protein of VSV, since both monoclonal and polyclonal anti-tubulin antisera specifically immunoprecipitated not only tubulin but also the L protein of two different VSV serotypes from the soluble protein fraction of infected cells.

Animals↗
PubMed1986

Immunolocalization of the gamma isoform of nonmuscle actin in cultured cells.

In many vertebrate nonmuscle cells, the microfilament subunit protein, actin, exists as two isoforms, called beta and gamma, whose sequences differ only in their amino-terminal regions. We have prepared a peptide antibody specifically reactive with the amino-terminal sequence of gamma actin. This antibody reacted with nonmuscle actin as determined by Western blots of SDS gels, and reacted with the gamma, but not the beta, nonmuscle actin isoform as shown by Western blots of isoelectric focusing gels. In immunofluorescence experiments, the gamma peptide antibody stained microfilament bundles, ruffled edges, and the contractile ring of a variety of cultured cells, including mouse L cells, which have previously been reported to contain only the beta actin isoform (Sakiyama, S., S. Fujimura, and H. Sakiyama, 1981, J. Biol. Chem., 256:31-33). Double immunofluorescence experiments using the gamma peptide antibody and an antibody reactive with all actin isoforms revealed no differences in isoform localization. Thus, at the level of resolution of light microscopy, we have detected the gamma actin isoform in all microfilament-containing structures in cultured cells, and have observed no subcellular sorting of the nonmuscle actin isoforms.

Actin Cytoskeleton↗
PubMed1982

Isolation and characterization of monoclonal antibodies to (Na+ + K+)-ATPase.

Four stable hybridoma cell lines secreting antibodies specific to the membrane (Na+ + K+)-dependent ATPase isolated from lamb kidney medulla have been produced by fusing mouse myeloma cells with spleen cells from immunized mice. These cell lines produce IgG gamma 1 heavy chain and kappa light chain antibodies which are directed against the catalytic or alpha-subunit of the (Na+ + K+)-ATPase enzyme. Binding studies, using antibodies that were produced by growing hybridomas in vivo and purified by affinity column chromatography, suggest a somewhat higher affinity of these antibodies for the isolated alpha-subunit than for the 'native' holoenzyme. In addition, these monoclonal antibodies show no reactivity with either the glycoprotein (beta) subunit of the lamb enzyme nor the (Na+ + K+)-ATPase from rat kidney, an ouabain-insensitive organ. Cotitration binding experiments have shown that the antibodies from two cell lines originally isolated independently from the same culture plate well population of fused cells bind to the same determinant site and are probably the same antibody. Cotitration and competition binding studies with two other antibodies have revealed two additional distinct antibody binding sites which appear to have little overlap with the first site. One of the three different antibodies isolated caused a partial inhibition of the (Na+ + K+)-ATPase activity. This antibody appears to be directed against a specific functionally important site of the alpha-subunit and is a competitive inhibitor of ATP binding. Under optimum conditions of ATPase activity, this inhibitory effect is not altered by the presence of the other two antibodies.

Animals↗
PubMed1982

Actin distribution patterns in the mouse neural tube during neurulation.

With the use of antibodies to actin and indirect immunofluorescent techniques regions of increased actin concentration were demonstrated first in basal and later in apical areas of mouse neuroepithelial cells. These patterns of staining corresponded to shape changes observed in cranial neural folds as they initially elevated from the neural plate and later moved toward the midline.

Actins↗
PubMed1980

Palate morphogenesis. I. Immunological and ultrastructural analyses of mouse palate.

Midpalate was analyzed for the presence of nonmuscle contractile systems. The results indicate that increased amounts of actin and myosin are present in cells of regions 2 and 3. A localization of the contractile proteins in cellular projections (filopodia) and in the peripheral cytoplasm of the cell body was confirmed by indirect immunofluorescence studies, using antibodies directed against smooth muscle myosin and against skeletal muscle actin. Specificity of the immunofluorescence reactions was ascertained by immunoabsorption studies using purified myosin and actin. Electron microscopic observations of the mesenchymal cells in region 2 revealed 70A microfilaments along the cell periphery and packed in fliopodia-like projections which course between the cells. These cells, which surround a small ossification center, show no orientation, but extend up to the cranial base perichondrium and down into the shelf between the tongue side epithelium and the ossification center. The cells and projections are attached to each other by adherens and tight-like junctions, forming a putative cohesive contractile network. Putative contractile cells in region 3 are strikingly aligned perpendicular to the oral epithelium and extend one-third of the distance into the shelf. Projections from region 3 cells are contiguous with basement membrane material of the oral epithelium. Axonal bundles and single axons were commonly observed coursing through regions 2 and 3, often seen in close association with the mesenchymal cells. Both clear and dense-core vesicles were found in the axons and cells of these regions. The possible role of these putative nonmuscle contractile cells in palate morphogenesis is discussed.

Actins↗
PubMed1978

Characteristics of the microvillus brush border of human placenta: insulin receptor localization in brush border membranes.

Insulin receptor characteristics were examined in purified brush border membrane from the syncytiotrophoblast of the normal human placenta and quantified during membrane preparation. Insulin receptor concentration was enriched 10- to 15-fold in this preparation, and insulin receptor specific activity followed closely the enrichment values for microvillus plasma membrane markers, alkaline phosphatase, Ca2+- and Mg2+-ATPase, and 5'-nucleotidase during cell fractionation. Insulin receptor concentrations and marker enzyme analyses were compared in whole homogenate, mitochondrial, microsomal, and microvillus fractions, and these fractions were characterized by SDS-gel electrophoresis. Microvillus insulin receptor interactions were dependent on time, [125I]iodoinsulin concentration, protein, and unlabeled hormone concentrations. Competition studies with porcine insulin and [125I]iodoinsulin for this receptor revealed a curvilinear Scatchard plot. Insulinase was demonstrated at 37 C but was minimal at 24 C in the microvillus fraction. Electron microscopy of the microvillus membrane preparation revealed its composition to be mainly spherical closed membrane vesicles and brush border fragments. Sodium dodecyl sulfate polyacrylamide and isoelectric focusing gels of membrane fractions were compared. Actin was tentatively identified as a major microvillus membrane protein and was further fractionated: beta-Actin and gamma-actin were present in approximately equal concentrations. The localization of the insulin receptor in the microvillus brush border of the human placenta suggests that this receptor interacts with maternal, rather than fetal insulin.

Cell Fractionation↗