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Biomedical subjects

S Penman

Publications and source records attributed to S Penman.

At least 91 records · Page 5Linked to original sources
PubMed1980

The spatial distribution of polyribosomes in 3T3 cells and the associated assembly of proteins into the skeletal framework.

Acridine fluorescence reveals polyribosomes in fibroblasts and Triton-extracted skeletal frameworks; simultaneous phase-contrast images show cellular structure. Polyribosomes appear near nuclei of both intact fibroblasts and skeletal frameworks. Simultaneous autoradiography of cells so examined locates radioactive proteins. After synthesis, most proteins diffuse rapidly through the cytoplasm; intact cells autoradiographed afer a 35S pulse show grains throughout. In sharp contrast, extraction with Triton leaves only radioactive skeletal proteins, which, although released from ribosomes, are near polyribosomes after a pulse. After a chase, skeletal-associated radioactivity is found throughout the framework structure. However, skeletal proteins migrate only if protein synthesis continues. Emetine administered following a pulse block protein migration; skeletal framework radioactivity remains near polyribosomes. This also indicates limited exchange between skeletal framework and soluble cytoplasmic proteins. The fact that proteins insert themselves into the skeletal framework at or near their synthesis site, with limited subsequent exchange, appears to contradict current view of protein self-assembly.

Actins↗
PubMed1980

Protein synthesis requires cell-surface contact while nuclear events respond to cell shape in anchorage-dependent fibroblasts.

Anchorage-dependent mouse fibroblasts grow only if attached to and spread on a solid substrate. The suspension of cells in methyl-cellulose results in dramatic, coordinated inhibition of the major RNA and protein synthesis systems, and these systems are sequentially restored when cells are replated on a tissue culture dish surface. In the present report the effects on metabolism of cell reattachment are separated from those of subsequent spreading by controlling cell shape. Macromolecular metabolism is first strongly suppressed by long-term suspension culture. The cells are then replated in the presence of a variety of spreading inhibitors. The recovery of protein synthesis, which rapidly follows reattachment, does not require extensive cell spreading. Contact of a limited portion of the plasma membrane with the solid culture dish surface is apparently a sufficient signal by itself. A very different method of controlling cell shape is afforded by changing culture dish surface adhesivity. Suspended cells are replated on dishes precoated with thin layers of the hydrophilic hydrogen poly(2-hydroxyethyl methacrylate). The final mean cell diameter is then varied over wide limits. As before, protein synthesis recovery is unaffected. However, nuclear events such as DNA and rRNA synthesis and mRNA production are profoundly affected by cell shape. Thus, cell surface contact and cell shape give rise to distinctly different regulatory responses.

Animals↗
PubMed1980

Association of the src gene product of Rous sarcoma virus with cytoskeletal structures of chicken embryo fibroblasts.

We have prepared cytoskeletons from normal and Rous sarcoma virus-transformed cells by extraction with nonionic detergents in a buffered salt solution designed to preserve the structure as it exists in vivo. Virtually all of the phosphoprotein pp60src in the cell is bound to such cytoskeletons. Furthermore, when these cytoskeletons are incubated in situ with [gamma-32P]ATP, pp60src is phosphorylated. Labeling of other apparently transformation-specific cytoskeletons phosphoproteins is also observed. These results directly demonstrate an association between pp60src and elements of the cytoskeleton and suggest that pp60src may exert at least some of its effects as a consequence of its interaction with this cellular framework.

Adenosine Triphosphate↗
PubMed1979

The cytoskeletal framework and poliovirus metabolism.

The cytoskeletal framework prepared by detergent lysis of suspension-grown HeLa cells is compared to the structure obtained from poliovirus-infected cells. This framework, which retains major features of cell morphology and carries the cellular polyribosomes as well as the major structural filaments, is profoundly reorganized following virus infection. This reorganization underlies, at least in part, the morphological changes termed the "cytoplasmic effect." These cytoskeletal changes appear related to the involvement of the framework with viral-specific metabolism. Extensive cytoskeleton alterations occur even when guanidine inhibits viral replication, and thus result from small amounts of early viral products. The normally spheroidal nucleus deforms, allowing a modified region of the cytoplasm to occupy a central position in the cell, and many membrane-enclosed vesicles peculiar to the infected cell are elaborated here. The skeleton preparation reveals that this region contains intermediate filaments arranged in a pattern unique to infected cells. Further changes occur when viral replication is permitted. The central region filaments become coated with darkly staining material which may be viral RNA. Numerous small particles appear on the filaments which resemble partially assembled virions. Mature virions, however, have no affinity for the cytoskeleton and appear to be free in the cytoplasm. Host cell messenger RNA, normally attached to the skeletal framework, is released in infected cells and is replaced by the viral-specific polyribosomes. The trabecular network which carries polyribosomes appears to be rearranged; the viral polyribosomes are located principally at the cell periphery and are excluded from the central region. The viral replication complex with its double-stranded RNA is also attached to the skeletal framework and may comprise the dark staining material coating the filaments of the central cell region.

Cell Nucleus↗
PubMed1979

Mechanisms of regulating tubulin synthesis in cultured mammalian cells.

Colchicine and nocadazole both depolymerize microtubules in cultured fibroblasts and lead to a rapid inhibition of tubulin synthesis. The level of translatable tubulin mRNA is greatly reduced in drug-treated cells as demonstrated by translation in a reticulocyte-derived in vitro protein synthesizing system. A model of tubulin synthesis regulation is proposed in which the elevated level of unpolymerized tubulin in drug-treated cells inhibits the formation of new tubulin mRNA and the preexisting message decays rapidly. In agreement with this model, tubulin message is found to be short-lived and has an approximately 2 hr half-life in cells treated with actinomycin D. Another prediction of the proposed model is that destabilization of microtubules without a concomitant increase in free tubulin will not inhibit tubulin synthesis. Vinblastine also disrupts microtubules but leads to the aggregation of tubulin into large paracrystals with an apparent decrease in the concentration of free tubulin. This drug does not inhibit tubulin production but rather leads to a measurable enhancement of tubulin synthesis.

Animals↗
PubMed1979

The outer boundary of the cytoskeleton: a lamina derived from plasma membrane proteins.

We prepared the cytoskeletal framework by gently extracting cells with Triton X-100. Lipids and soluble proteins were removed, leaving a complex meshlike structure which contains the cell nucleus and is composed of the major cell filament networks as well as the microtrabeculae with attached polyribosomes. The surface sheet or lamina covering this structure contains most of the cell surface proteins by the following criteria. Intact cells are labeled externally with radioiodine and then extracted with detergent. The iodinated poteins remain almost entirely with skeletal framework. A new major integral protein, the coat protein of Sindbis virus, is inserted into the plasma membrane of infected cells. This new protein is heavily iodinated and remains almost completely associated with the framework after extraction. Lectin binding and poliovirus binding sites are also retained after detergent extraction. Our results indicate that plasma membrane proteins form a sheet or lamina upon removal of lipids. This lamina reproduces even complex surface convolutions and appears to be supported by and intimately connected to the underlying skeleton. In this case, the surface lamina, and hence the plasma membrane of the original intact cell, might be viewed as a component of the cytoskeletal framework.

Animals↗
PubMed1979

Large species differences in the pattern of snPI RNA which can distinguish ape from human.

The snPI RNA species are a recently described set of molecules whose sizes range from 5S to 10S. They can be labeled in vitro in isolated nuclei and are apparently formed by an RNA polymerase I type of activity. However, in contrast to ribosomal precursor RNA, the usual polymerase I product, they are not found in the nucleolus but rather are located in the nucleoplasm. The snPI RNAs have been found in all mammalian cell types studied. The spectrum seen in gel electrophoresis is unique to each animal species studied but is essentially the same in different cell types within a species. The differences in snPI patterns are quite large between even closely related species and are clearly distinguishable in gorilla and human cells.

Animals↗
PubMed1978

Altered translatability of messenger RNA from suspended anchorage-dependent fibroblasts: reversal upon cell attachment to a surface.

Anchorage-dependent cells, when forced into suspension culture, display a repertoire of dramatic, coordinated regulatory phenomena. Message production promptly decreases 5 gold but the cells maintain a constant amount of poly(A)+ by means of a concomitant stabilization of mRNA against decay. Protein synthesis shuts down much later and the mRNA is stored in a nonfunctioning state. In this study, the inactive mRNA is extracted from suspended cells and shown to have aberrant translation properties. Well defined polypeptides are apparently no longer synthesized when this mRNA directs protein formation in either reticulocyte or wheat germ-derived heterologous translation systems. Rather, shortened peptides are formed by this mRNA and these become smaller as mRNA is used from cells suspended for longer periods of time. Very few focused spots are formed when the aberrant polypeptides are analyzed in two-dimensional electrophoresis. The sedimentation properties of suspended cell mRNA and the size of poly(A) are unchanged from control monolayer cells. Cross-hybridization of cDNA transcribed from a control cell message population with suspended cell mRNA shows that all sequences are present in normal concentrations. While most identifiable spots disappear from the two-dimensional gel electropherograms of the protein products produced by suspended cell mRNA, a few polypeptides are still synthesized in relatively normal amounts. Conserved polypeptides are found in products of both the reticulocyte and wheat germ systems, but they are different products in each case. The lesion in the suspended cell mRNA does not seem to be at the 5' termini, since synthesis of the shortened peptides is fully sensitive to inhibition of pm7G. Cells that contain extensively modified message can resume protein synthesis when allowed to reattach to a solid substrate. There is an apparent remodification of mRNA to normal translatability within a few hours of cell reattachment, since mRNA from recovering cells quickly resumes directing relatively normal patterns of polypeptide synthesis in vitro. The restoration of normal message function occurs even when new message formation is blocked with actinomycin. Cells recovering after reattachment synthesize supranormal amounts of a few major proteins involved with cell structure, as shown in these studies by an increased amount of translatable sequences which encode these proteins. The most apparent enhanced message is that coding for actin. mRNA from recovering cells produces in vitro several times more actin relative to other proteins than does control cell mRNA. The enhancement of actin mRNA is not seen in the message population of cells that reattach in the presence of actinomycin. The results suggest a morphologically related induction of gene expression.

Actins↗
PubMed1978

Heterogeneous nuclear RNA-protein fibers in chromatin-depleted nuclei.

The heterogeneous nuclear RNA-protein (hnRNP) fibers in HeLa cell nuclei are visualized by a nuclear subfractionation technique which removes 96% of the chromatin in a single step and 99% in a two-step elution but leaves the bulk of the hnRNA complexed with the remnant nuclear structure or lamina. Both steady-state and newly synthesized (approximately 15-s label) hnRNA are associated with the remnant nuclei to about the same extent. This association does not appear to depend on the presence of chromatin and exists in addition to any possible association of hnRNP with chromatin itself. Electron microscopy of partially purified nuclear hnRNA complexes shows that the hnRNP fibers form a ribonucleoprotein network throughout the nucleus, whose integrity is dependent on the RNA. Autoradiography confirms that hnRNA is a constituent of the fibers. The RNA network visualized in these remnant nuclei may be similar to RNA networks seen in intact cells. The hnRNA molecules appear to be associated with the nuclear lamina, at least in part, by unusual hnRNA sequences. More than half of the recovered poly(A) and double-stranded hnRNA regions remains associated with the nuclear structures or the laminae after digestion with RNase and elution with 0.4 M ammonium sulfate. In contrast, the majority of oligo(A), another ribonuclease resistant segment, is released together with most of the partially digested but still acid-precipitable single-stranded hnRNA and the hnRNP proteins not eluted by the ammonium sulfate alone. These special RNA regions appear to be tightly bound and may serve as points of attachment of the hnRNA to nuclear substructures. It is suggested that hnRNA metabolism does not take place in a soluble nucleoplasmic compartment but on organized structures firmly bound to the nuclear structure.

Cell Nucleolus↗
PubMed1978

Messenger RNA abundance and lifetime: a correlation in Drosophila cells but not in HeLa.

Messenger RNA in eukaryotes is comprised of several abundance classes. Also, the decay of these unstable molecules shows at least two very different lifetimes. Two independent techniques are used here to examine the relation between message abundance and lifetime in cell lines from very different organisms. The methods give consistent results for each cell line; however, the two cell types show very different results. In brosophile cells, slowly decaying sequences fall in the abundant class while scarce sequences turn over rapidly. In contrast, in HeLa cells the abundant and scarce message classes are each comprised of long- and short-lived molecules.

Cell Line↗
PubMed1977

The relative amounts of the cytoplasmic RNA species in normal, transformed and senescent cultured cell lines.

We have examined the relative quantities of 18S and 28S rRNA, 4S RNA and poly (A) + mRNA in the following cultured cells: the mouse fibroblast lines 3T3 and 3T6 in the resting (contact inhibited) and growing (sparse) states, 3T3 clones transformed with SV40 (SV3T3) and with both SV40 and polyoma SV-Py 3T3), hamster lung fibrobalsts (v79), human cervical carcinoma cells (HeLa), and human diploid fibroblasts at early and late passage. The relative quantities of the RNA species were determined by labeling the cells to equilibrium with 32PO4 and measuring the amount of label in each RNA species. The ratio of mRNA to rRNA varied form 1.1% to 2.7% in the different cell lines, the more rapidly growing cell lines usually giving a higher ratio. In cells experiencing growth limitation either by contact inhibition or due to senescence, the ratio of mRNA to rRNA was about 30% lower than in the corresponding cells in the growing state. In most cell lines the ratio of 4S RNA to 18S rRNA was between 0.8 and 1.2, but in seescent fibroblasts, this ratio increased to greater than 1.7. Senescent fibroblasts also contained much more total RNA per unit of DNA than the same cells at early passage or than 3T6 or 3T3 cells.

Animals↗
PubMed1977

Coordinate regulation of protein synthesis and messenger RNA content during growth arrest of suspension Chinese hamster ovary cells.

We have found Chinese Hamster Ovary cells, cultured in suspension, are subject to growth control by serum. When suspended in medium containing 0.5% serum the cells becomes reversibly arrested in the beginning of the G1 phase of the cell cycle and can be maintained in this viable, nonproliferating state for several days. This system was used to examine the regulation of protein synthesis with growth rate. In particular, the experiments addressed the question whether mRNA content is the principal controlling factor determining the rate of protein synthesis. The rate of leucine incorporation in resting cells in low serum is 2-to 2.5-fold lower than that of cells growing in 10% serum. The steady-state number of cytoplasmic poly A (+) RNA molecules shows a proportional decrease, consistent with it being a determining factor controlling the rate of protein synthesis. Furthermore, the rate of production of poly A (+) and poly A (-) RNA appears to be regulated coordinately. Regulation of the rate of initiation of translation would result in fewer ribosomes bound per active message and/or a lower proportion of total mRNA's being active. Our measurements indicate that the fraction of cytoplasmic poly A (+) mRNA in polyribosomes and the relative degree of loading of each active poly A(+) mRNA with ribosomes is the same in resting and growing cells. Thus these cells resemble 3T6 and translational control does not appear to be an important part of the change in protein synthetic rate with teh state of growth.

Animals↗