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R Craig

Publications and source records attributed to R Craig.

At least 127 records · Page 7Linked to original sources
PubMed1983

Regulation of myosin filament assembly by light-chain phosphorylation.

Myosins isolated from vertebrate smooth muscles and non-muscle cells such as lymphocytes and platelets contain regulatory light chains (Mr = 20000), which are phosphorylated by a Ca2+-calmodulin-dependent kinase and dephosphorylated by a Ca2+-insensitive phosphatase. Phosphorylation of the regulatory light chains of these myosins in vitro regulates not only their interactions with actin but also their assembly into filaments. Under approximately physiological conditions (0.15 M NaCl, pH 7.0) stoichiometric levels of Mg-ATP disassemble these non-phosphorylated myosin filaments into species with sedimentation coefficients (So20,w) of approximately 11S. Hydrodynamic and electron microscope observations have indicated that this 11S species is a monomer with a folded conformation (Trybus et al., Proc. natn. Acad. Sci. U.S.A. 79, 6151 (1982)). Rotary shadowing reveals that the tails of disassembled gizzard and thymus myosins are folded twice at two hinge points to form a folded three-segment structure. Phosphorylation of the regulatory light chains of these myosins causes these folded 11S molecules to unfold into the conventional extended monomeric form (6S), which is able to assemble into filaments. Thus in vitro these myosin filaments can be assembled or disassembled by phosphorylation or dephosphorylation of their light chains. Whether these results have any relevance to the situation within living non-muscle and smooth muscle cells remains to be established.

Actins↗
PubMed1983

Electron microscopy and image analysis of myosin filaments from scallop striated muscle.

Thick filaments have been isolated from the striated adductor muscle of the scallop and examined by electron microscopy after negative staining. Many filaments appear intact, and reveal a centrally located bare-zone and a well-defined helical surface array of myosin crossbridges characterized by a 145 A axial period and prominent helical tracks of pitch 480 A. Heavy-metal shadowing shows that these helices are right-handed. A small perturbation of alternate crossbridge levels produces an axial period of 290 A, which is most prominent in a region on either side of the bare-zone. Image analysis reveals that the crossbridge array has 7-fold rotational symmetry, one of the possibilities suggested by earlier X-ray diffraction studies of native filaments in scallop muscle. A low-resolution three-dimensional reconstruction shows elongated surface projections ("crossbridges") that probably represent unresolved pairs of myosin heads. They run almost parallel to the filament surface, but are slewed slightly from the axis so that they lie along the right-handed helical tracks of pitch 480 A. The connection to the filament backbone probably occurs at the end of the crossbridges nearer the bare-zone; thus, their sense of tilt appears to be opposite to that of rigor attachment to actin. The 290 A period arises from a different distribution of crossbridge density at alternate levels; in addition, there are weak connections between the top of one crossbridge and the bottom of the next, 145 A away. The prominence of the 290 A period near the bare-zone suggests that anti-parallel molecular interactions are mainly responsible for this perturbation.

Animals↗
PubMed1982

Glycosaminoglycans produced by human synovial cell cultures.

Human synovial cells in culture are known to synthesize hyaluronic acid, but the production of sulfated glycosaminoglycans (GAG) has received less attention. Using 14C-glucosamine as a precursor, GAG content was studied in the medium, trypsin-solubilized pericellular layer, and cell residue fraction of cultured synovial cells derived from the synovial membranes of nonrheumatoid and rheumatoid joints. Over 90% of the total non-dialyzable counts appeared in the culture medium, for the most part in hyaluronic acid. The remaining nondialyzable counts were cell-associated, almost equally divided between the pericellular layer and cell residues. In these fractions, only part of the counts were in GAG susceptible to testicular hyaluronidase digestion, and GAG were significantly lower in the cell residue of the rheumatoid synovial cells compared to the nonrheumatoid cells. Analysis of the chondroitinase ABC and AC digestion products of these GAG indicated the presence of chondroitin-4 and -6 sulfates, and dermatan sulfate, but not heparan sulfate. Similar findings with respect to the identity of the GAG in nonrheumatoid and rheumatoid synovial cell culture media were obtained with 35SO4 as a precursor.

Arthritis, Rheumatoid↗
PubMed1981

Identification of fatty acid synthetase messenger RNA on free polyribosomes isolated from lactating rabbit mammary gland.

The synthesis of fatty acid synthetase on free polyribosomes from lactating rabbit mammary gland was demonstrated by using polyribosomes run-off techniques and immunochemical identification of products with synthetase antiserum. Several reproducible and discrete immunoprecipitable polypeptides were observed which were within the molecular-weight range of the synthetase subunit (235 000--252 000), as well as several of lower molecular weight.

Animals↗
PubMed1981

The influence of topology and glycosylation on the fate of heterologous secretory proteins made in Xenopus oocytes.

Secretory proteins made in Xenopus laevis oocytes under the direction of heterologous messenger RNA are modified, topologically segregated and exported. Thus the oocyte may serve as a useful surrogate secretory system and we have studied some of the factors governing access to the export pathway. Unglycosylated chicken ovalbumin, synthesized and trapped in the cytosol, is not secreted but glycosylated ovalbumin, found sequestered within vesicles, is exported from oocytes. However, ovalbumin, which is transferred across the endoplasmic reticulum in the presence of tunicamycin and which is indistinguishable by immunoprecipitation, by two-dimensional gel electrophoresis and by concanavalin-A--Sepharose binding from the cytosolic form, is still secreted. Guinea-pig milk proteins and human interferon are also exported from tunicamycin-treated frog cells. These observations demonstrate that access to the endoplasmic reticulum but not glycosylation is a mandatory intermediate step in secretion, and emphasize the advantages of the oocyte as a surrogate system for the study of the later events in the gene expression pathway.

Animals↗
PubMed1980

Glucose metabolism in uremia.

Formation of CO2 from uniformly labeled 14C-glucose was measured in liver slices from uremic and normal rats. Both CO2 formation and lactate concentration were decreased in the uremic liver slices suggesting an inhibition of glucose oxidation. In addition, a net loss of glucose from the medium in the uremic preparation and a net gain in the normal controls suggested that there was increased nonoxidative utilization in the uremic liver. Such changes could not be explained by differences in glucose availability consequent to alterations in glycogen degradation. The most likely explanation is diversion of glucose into other biosynthetic pathways such as the synthesis of amino acids. In this regard, synthesis of glutamine appeared to be enhanced in uremia. Thus, products of carbohydrate metabolism may provide a potential mechanism for disposition of ammonia and synthesis of amino acids in uremia.

Animals↗
PubMed1980

The Xenopus oocyte as a surrogate secretory system. The specificity of protein export.

Combining messenger RNA from one kind of secretory cell with the cytoplasm of another such cell can reveal the nature and specificity of protein export mechanisms. We show that messenger RNAs from secretory cells of chickens, rats, mice, frogs, guinea-pigs, locusts and barley plants, when injected into Xenopus oocytes, direct the synthesis and export of proteins. Chicken ovalbumin, Xenopus albumin, mouse thyroid-stimulating hormone, locust vitellin and guinea-pig milk proteins were identified using specific antibodies, whilst chicken lysozyme and ovomucoid, rat albumin, Xenopus vitellogenin and rat seminal vesicle basic proteins were identified provisionally from their molecular weights. Certain endogenous proteins are sequestered and secreted although most oocyte proteins are not exported. Similarly the major polyoma viral protein and the simian virus 40 and polyoma tumour antigens are retained within the oocyte. Radioactive proteins exported by oocytes programmed with chicken oviduct or Xenopus liver RNA are not re-exported in detectable amounts when injected into fresh oocytes, nor is there secretion of chicken oviduct or guinea-pig mammary gland primary translation products prepared using wheat germ extracts. Thus the export of secretory proteins from oocytes cannot be explained by leakage and may require a cotranslational event. The secretory system of the oocyte is neither cell-type nor species-specific yet is highly selective. We suggest that the oocyte can be used as a general surrogate system for the study of gene expression, from transcription through translation to the final subcellular or extracellular destination of the processed protein.

Animals↗
PubMed1979

Sequestration and turnover of guinea-pig milk proteins and chicken ovalbumin in Xenopus oocytes.

The stability and distribution of proteins within the living cell can be studied using Xenopus laevis oocytes. Microinjection of messenger RNAs and secretory proteins, followed by cell fractionation, shows that transfer of ovalbumin and milk proteins across intracellular membranes of the oocyte only occurs during their synthesis. Thus milk protein primary translation products, made in the wheat germ cell-free system, when injected into oocytes remain in the cytosol and are not recovered within membrane vesicles. Such miscompartmentalized primary milk proteins are rapidly degraded (t 1/2 0.6 +/- 0.1 h). In contrast, processed milk proteins, extracted from oocytes injected with mammary gland RNA, are relatively stable when introduced into the cytosolic compartment (t 1/2 alpha-lactalbumin 20 +/- 8 h, casein A 6 h, casein B 4 h, casein C 8.3 h). The primary ovalbumin product is also stable (t 1/2 22 +/- 9 h). Indirect evidence that rapid degradation of miscompartmentalized milk protein primary translation products may occur in vivo was obtained by the injection of massive amounts of ovalbumin and milk protein mRNA. Under these conditions there is no accumulation of primary milk protein translation products, but a polypeptide resembling the unglycosylated ovalbium wheat germ primary product can be detected in the cytosol. Only the glyclosylated forms of ovalbumin are found in the oocyte membrane vesicle fraction. We discuss the roles played by the presence of detachable signal sequences and the absence of secondary modifications in determining the rate of degradation of primary translation products within the cytosol.

Animals↗