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Biomedical subjects

R H Singer

Publications and source records attributed to R H Singer.

108 records · Page 6Linked to original sources
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↗
PubMedDate not supplied

Actin-containing microprocesses in the fusion of cultured chick myoblasts.

Scanning electron microscopic studies of myoblasts from 11- to 13-day-old chick embroyonic breast muscle cultured on collagen-coated glass coverslips showed six stages of development into multinucleated myotubes: (1) growth of flattened, spread-out cells for 20-30 hr following initiation of monolayer cultures; (2) extension of microprocesses (1-150 microM) from cells that have become spindle shaped; (3) contact and adherence of microprocesses from adjacent cells; (4) thickening of fused processes; (5) approximation of the cells; and (6) coalescence of the cells to form a spindle-shaped myotube. When the calcium-ion concentration in the growth medium was lowered--either by increasing the concentration of ethylene-glycol-bis(aminoethyl ether)N,N'-tetraacetate (EGTA)or by decreasing the cconcentration of free calciumion used--the number of microprocesses present on the cells was reduced. Presumably, however, these microprocesses could still fuse together, provided that the calciumion concentration was greater than 160 microM. Indirect immunofluorescence assay with actin-specific antibody indicated that actin is a major component of the myoblasts' microprocesses. Cytochalasin B (5 microgram/ml) caused the microprocesses to retract within 15 min and the myoblasts to round up and detach from the glass substrate. This was presumably caused by the action of the drug on actin filaments.

Actins↗
PubMedDate not supplied

Localization of pre-mRNA splicing in mammalian nuclei.

In mammalian nuclei, precursor messenger RNA splicing factors are distributed non-uniformly. Antibodies directed against structural polypeptides of small nuclear ribonucleoprotein particles (snRNPs) and some non-snRNP splicing factors have shown that these components are concentrated in about 20-50 nuclear 'speckles'. These and other non-homogeneous distributions have been proposed to indicate nuclear 'compartments' that are distinct from the sites of transcription and in which RNA processing occurs. We have tested this idea using a new approach. Previous structural and biochemical data have shown that splicing can occur in association with transcription. Nascent RNA of specific genes can be detected by in situ hybridization as intense spots of nuclear stain which map to the sites of transcription. Here we identify active pre-mRNA splicing sites by localizing the nascent spliced mRNA of specific genes. We find that splicing occurs at the sites of transcription, which are not coincident with intranuclear speckles. We conclude that the nucleus is not compartmentalized with respect to transcription and pre-mRNA splicing.

Actins↗
PubMedDate not supplied

Ultrastructural distribution of poly (A)+ RNA during Trypanosoma cruzi-cardiomyocyte interaction in vitro: a quantitative analysis of the total mRNA content by in situ hybridization.

Ultrastructural in situ hybridization was used to visualize the spatial distribution of poly (A)+ RNA and quantitate its relative amount within different cellular compartments of cardiomyocytes after T. cruzi infection. The amount of poly (A)+ RNA remained about the same up to 24 h post-infection. In contrast, its content was reduced 65% after 72 h of interaction, showing a marked decrease in the cell cytoplasm. This decline in poly (A)+ RNA level in host cell cytoplasm was concomitant with intracellular proliferation of T. cruzi amastigotes. Thus, T. cruzi may affect host cell cytoplasmic mRNA stability, associated with the parasite's intracellular multiplication.

Animals↗
PubMedDate not supplied

Trypanosoma cruzi infection affects actin mRNA regulation in heart muscle cells.

We have previously described alterations in the cytoskeletal organization of heart muscle cells (HMC) infected with Trypanosoma cruzi in vitro. Our aim was to investigate whether these changes also affect the regulation of the actin mRNAs during HMC differentiation. Northern blot analysis revealed that alpha-cardiac actin mRNA levels increased during cell differentiation while beta-actin mRNA levels declined. Nonmuscle cells displayed beta-actin mRNA signal localized at the cell periphery, while alpha-cardiac actin mRNA had a perinuclear distribution in myocytes. Trypanosoma cruzi-infected cells showed 50% reduction in alpha-cardiac actin mRNA expression after 72 h of infection. In contrast, beta-actin mRNA levels increased approximately 79% after 48 h of infection. In addition, in situ beta-actin mRNA was delocalized from the periphery into the perinuclear region. These observations support the hypothesis that Trypanosoma cruzi affects actin mRNA regulation and localization through its effect on the cytoskeleton of heart muscle cells.

Actins↗
PubMedDate not supplied

The modified McBride bunionectomy utilizing the adductor tendon transfer.

A modification of the McBride bunionectomy utilizing the adductor tendon transfer is described. It includes transposition of the adductor hallucis tendon into the medial capsular flap via a pulley suture, and derotation of the sesamoidal apparatus. This is accomplished by torque placed on the medial capsular flap. It is transferred to the medial sesamoid through the tibial sesamoid ligament and then to the fibular sesamoid by the intersesamoidal ligament. The contracted fibular sesamoid ligament is sacrificed. The procedure helps to maintain reduction of an increased intermetatarsal angle, with hypermobile first ray. Another advantage is the ability to perform a concomitant metatarsal neck osteotomy, when necessary. This was not possible in the original McBride bunionectomy in which the adductor tendon was transposed to the head of the first metatarsal through a drill hole. Short-term results have been encouraging.

Adult↗