Biomedical subjects

R H Singer

Publications and source records attributed to R H Singer.

54 records · Page 3Linked to original sources

PubMed1982

Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog.

The chicken muscle tissue culture system has been used for visualizing actin gene expression after in situ hybridization. Cell differentiation is morphologically distinguishable in this system as the myoblasts fuse into myotubes. This differentiation involves the production of large amounts of actin required for myofibrils. The presence of actin mRNA has been observed in cells preserved with ethanol and paraformaldehyde by hybridizing a recombinant plasmid into which a biotinated analog of dUTP was incorporated by nick-translation. The biotin was then detected by using an anti-biotin antibody and a rhodamine-conjugated second antibody. Alternatively, avidin conjugated to rhodamine or avidin complexed to biotinated peroxidase has been used for mRNA detection. The procedure described preserved morphological detail yet is compatible with hybridization conditions and reveals the disposition of actin mRNA during gene expression.

Actins↗

PubMed1981

Increased turnover of proteins from the sarcoplasmic reticulum of dystrophic chicken muscle cells in tissue culture.

Chicken myoblasts were cultured from the pectoralis muscles of dystrophic and normal 11-day-old embryos. Cells were allowed to grow to fusion (differentiation) and exposed to [35S]methionine for a short period. Subsequently, the decay of labeled proteins in the presence of cycloheximide was measured for various cellular fractions as well as individual proteins isolated from the sarcoplasmic reticulum and separated by gel electrophoresis. Some dystrophic material showed an increased decay when compared to normal material. The most significant (p less than 0.005) difference was found in a Mr = 65,000 component of the sarcoplasmic reticulum. This same component accumulates label at an accelerated rate in the presence of the protease inhibitor leupeptin. Increased turnover of this protein, possibly calsequestrin, may be a manifestation of the genetic disease.

Animals↗

PubMed1981

Sea urchin tube feet: unique structures that allow a cytological and molecular approach to the study of actin and its gene expression.

Actin is the major extractable protein component from the tube feet of four different species of sea urchin: Arbacia punctulata, Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, and Diadema setosum. Actin made up as much as 60% of the total Coomassie Blue-staining material after SDS polyacrylamide gel electrophoresis and densitometer analysis. Two-dimensional gel electrophoresis resolved two, and possible three, species of actin for each sea urchin of which the dominant component was analogous to the beta form in vertebrates. In a cell-free system from rabbit reticulocytes, total RNA from tube feet stimulated the synthesis of one protein that represented 80% of the total methionine incorporation, migrated with the properties characteristic of actin in a two-dimensional gel system, and on proteolysis yielded fragments identical to purified rabbit actin. The mRNAs from the tube feet of two divergent species of sea urchin, Arbacia punctulata and Strongylocentrotus purpuratus, synthesized actins differing by less than 0.02 pH unit for each isospecies 90% of the DNA copied from tube foot RNA by reverse transcriptase represented a highly abundant sequence class judged by copy DNA(cDNA)-RNA excess hybridization. At least two-thirds of this class represented a low-complexity component, with a Rot1/2 about three times that expected for actin messenger RNA. The remarkable degree of conservation of the actin protein is reflected in concomitant conservation of the protein-coding nucleotide sequences of the messenger RNA, which has allowed the use of a cDNA probe to isolate actin sequences from a human phage library.

Actins↗

PubMed1980

Intracellular filament bundles in whole mounts of chick and human myoblasts extracted with triton X-100.

The method of Triton X-100 extraction and critical point drying of whole mounts of cultured chick and human myoblasts was used to study the presence of intracellular bundles of filaments within these cells. Observation by means of transmission and scanning electron microscopy demonstrated a complex system of filament bundles which appeared morphologically and spatially heterogeneous. Most obvious were long dense bundles of cables traversing along the ventral surface of developing myoblasts, presumably the 'stress fibers' seen in light microscopy. Other bundle types occurred which were composed of loose aggregates of filaments coursing through the remnant cell body. A prominent accumulation of filaments was also seen at the lateral edges of these myoblasts. These lateral edge cables were thicker and denser than any other type of filament bundle observed in the myoblasts. Reaction of unextracted myoblasts directly to human antiplatelet myosin conjugated to rhodamine demonstrated that the most intense reaction also occurred along the lateral edges of both human and chick myoblasts. During development of chick myoblasts the filament bundles became oriented parallel to the cell axis giving the cell a fusiform morphology. It is possible that the various filament bundle structures and their differing structural and spatial dispositions could be related to functional differences among the diverse population of intracellular bundles of filaments.

Animals↗

PubMed1979

Electron microscopic visualization of the filamentous reticulum in whole cultured presumptive chick myoblasts.

This present study describes an experimental approach whereby subcellular 3-dimensional filamentous structures present within whole cells can be examined, using a conventional transmission electron microscope. This procedure uses cells which have been cultured on carbon-coated titanium grids, and treated with Triton X-100 to extract the soluble cytoplasm. Subsequent fixation and critical-point drying allows filamentous proteins to be easily visualized, due to the increase in contrast produced by removal of the ground cytoplasm. The high resolution obtainable in these preparations permitted an initial classification and description of the filamentous reticulum within cultured presumptive myoblasts. This reticulum is a continuum of filaments and cables, all elements of which appear to be interconnected. These morphological findings were then correlated with the biochemical identification of detergent-insoluble proteins, of which only actin, myosin, and, perhaps, intermediate filament and LETS protein are the major elements.

Animals↗

PubMed1978

Ca2+ binding, ATP-dependent Ca2+ transport, and total tissue Ca2+ in embryonic and adult avian dystrophic pectoralis.

Avian muscular dystrophy is an autosomal recessive genetic disease characterized by early hypertrophy and loss of function of pectoralis major. The disease is progressive, ultimately resulting in atrophy and heavy lipid deposition. Previous investigators have noted a decrease in the ability of the dystrophic sarcoplasmic reticulum to concentrate Ca2+. More recently, other investigators have shown an abnormal calcium uptake in avian dystrophic sarcoplasmic reticulum. They indicated, using freeze-fracture techniques, that a 90 A particle of the vesicle membrane exhibited a decreased population and suggested that they might be the ATPase involved in calcium transport. Our studies confirm earlier observations of a decreased rate of Ca2+ uptake and Ca2+ binding capacity of dystrophic fragmented sarcoplasmic reticulum vesicles which are isolated from both embryonic and adult pectoralis. These observations correlate in turn with a 75% drop in the Ca:ATP transport efficiency of the dystrophic sarcoplasmic reticulum determined by measuring the rate of 32Pi liberation from gamma-ATP32 during active calcium transport by the isolated sarcoplasmic reticulum SR. In addition, we have found a quantitative deficiency in a 65,000 dalton component of the dystrophic fragmented SR at the time of myoblast fusion by measuring 35S-Methionine incorporation into the SR, coupled to high resolution polyacrylamide gel electrophoresis and radioautography. Analysis of total tissue calcium by atomic absorption spectroscopy revealed a decrease in the total calcium content of dystrophic muscle.

Adenosine Triphosphate↗

PubMed1978

Stability of polyadenylated RNA in differentiating myogenic cells.

Three independent methods of measurement showed that cytoplasmic polyadenylated RNA from the differentiating myogenic cell line L8 consists of two main populations with regard to stability, one with a half-life of less than 4 h and the other with a half-life of 17--54 h. Similar results were obtained in the presence and absence of actinomycin D. During the fusion of mononucleated myoblasts into multinucleated fibers, there was an increase in both the steady-state pool of the more stable polyadenylated RNA and the proportion of stable polyadenylated RNA synthesized in pulse labelling.

Animals↗

PubMed1978

The capacity of polyadenylated RNA from myogenic cells treated with actinomycin D to direct protein synthesis in a cell-free system.

Cytoplasmic polyadenylated RNA of myogenic cells was shown to decay with biphasic kinetics, suggesting the existence of two main populations of mRNA with respect to stability. In the present study, the stability of mRNA extracted from actinomycin-D-treated cultures of a myogenic cell line was tested by its capacity to direct protein synthesis in the wheat germ cell-free system. The products were analyzed by dodecylsulphate/polyacrylamide gel electrophoresis. All major radioactive bands found in gels used for analyzing the products of the cell-free system directed by polyadenylated RNA extracted from untreated cultures were also found in similar gels containing products of RNA extracted after many hours of application of actinomycin D. The capacity to code for specific protein bands decays with a half-life ranging between 11 and 40 h. No fast-decaying translatable mRNA could be detected by this method. Instead, it was found that during the first 4--6 h following application of actinomycin D, the capacity of RNA to stimulate incorporation of amino acids into total acid-insoluble material increased by 20--30%. The synthesis of specific products increased by up to 100%. The possibility that the fast-decaying polyadenylated RNA or part of it is nontranslatable RNA is discussed.

Cell Differentiation↗

PubMed1977

Synthesis and secretion of a high molecular weight form of nerve growth factor by skeletal muscle cells in culture.

Rat skeletal muscle cells and a cloned myogenic cell line synthesize and secrete in culture a molecule that is immunologically and biologically indistinguishable from the active form of nerve growth factor (NGF) from mouse submandibular gland. This protein can be detected in medium conditioned by muscle cells both before and after fusion and in the soluble fraction of muscle cell homogenates. Chromatographic data also reveal that the molecular properties of muscle cell NGF differ from those of the growth factor purified from mouse submandibular glands. Muscle cell NGF has a molecular weight between 140,000 and 160,000, whereas purified mouse gland NGF has a molecular weight of 26,000. The biologic function of muscle cell NGF is not known, although it could be that it plays some role relating to the association of nerves and muscle in vivo.

Animals↗

PubMed1975