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Biomedical subjects

I Pastan

Publications and source records attributed to I Pastan.

At least 721 records · Page 40Linked to original sources
PubMed1977

Localization of serum-derived alpha 2 macroglobulin in cultured cells and decrease after Moloney sarcoma virus transformation.

NRK cells and many other cultured fibroblasts were found to contain the protease inhibitor, alpha 2 macroglobulin (alpha 2M). This alpha 2M is present as a result of uptake of alpha 2M from the calf serum in the culture medium. Some of this alpha 2M is released back into the medium. In radiolabeling experiments with 14C-amino acids, no radioactivity was detected in intracellular or extracellular alpha 2M. Fluorescence microscopy of fixed cells using rhodamine-labeled antibodies indicated that alpha 2M is present in vesicular organelles different from primary lysosomes. Fluorescence microscopy of living cells shows that rhodamine-labeled alpha 2M (rhodamine-alpha 2M) is taken up into similar structures. Of the many cell lines examined, Moloney sarcoma virus-transformed cells had the lowest amounts of alpha 2M. Some of the effects of serum on the behavior of cultured cells could be a consequence of inhibition of cellular proteases by alpha 2M.

Animals↗
PubMed1977

Role of cell surface carbohydrates and proteins in cell behavior: studies on the biochemical reversion of an N-acetylglucosamine-deficient fibroblast mutant.

AD6, a mutant derived from 3T3 Balb/c cells, is characterized by low adhesion to substratum, round shape, increase in surface microvilli, increase in agglutinability by concanavalin A, and loss of directional motility. These properties are often observed in transformed cells. However, the mutant has normal growth properties and anchorage-dependence of growth, and it does not form tumors. In AD6, the biosynthesis of complex carbohydrates and glycoproteins is impaired because of a block in the acetylation of GlcN-6-P. This defect is responsible for all the surface alterations because feeding of GlcNAc to AD6 cells corrects the defects in the synthesis of complex carbohydrates and the exposure of glycoproteins at the outer surface of the plasma membrane. Parallel to this biochemical reversion, there is full restoration of the altered biological properties. In contrast, GlcNAc has no effect on the morphologic features of two lines of transformed cells. Our results suggest that the carbohydrate portion of cell surface proteins has an important role in adhesion and related aspects of cell behavior. The fact that a defined alteration of the cell surface induces many properties often encountered in transformed cells, without affecting control of cell division, strongly suggests that these alterations in properties are not sufficient to account for the loss of growth regulation.

Acetylglucosamine↗
PubMed1977

Mobility and distribution of a cell surface glycoprotein and its interaction with other membrane components.

Fluorescence photobleaching recovery and immunofluorescence methods have been used to study the lateral mobility and topographical distribution of a major cell surface glycoprotein (CSP). Both endogenous CSP and fluorescent-labeled exogenous CSP bind to the cell surface in a fibrillar pattern and are immobile on the experimental time scale. Azide, vinblastine, and cytochalasin B do not alter the immobility and cell surface distribution of the CSP molecules. Therefore, oxidative phosphorylation and the cytoskeleton do not seem to be responsible for the properties of the bound glycoprotein. The presence of immobile CSP fibrils does not, however, impede the diffusion of a lipid probe, a ganglioside analogue, or various surface antigens. Therefore, the fibrils apparently do not form a "barrier" across the lipid phase of the plasma membrane. In contrast, concanavalin A binds to CSP and is largely immobile in regions rich in CSP. The presence of immobile concanavalin A receptors in areas or on cells lacking CSP indicates that other types of immobile concanavalin A receptors also exist.CSP does not bind to lipid bilayers composed of phosphatidylcholine or oxidized cholesterol. It does bind to dextran-coated bilayers as a diffuse distribution of mobile molecules that can patch after addition of antibodies to CSP. The latter result suggests that CSP molecules do not interact strongly with other CSP molecules under these conditions. Exogenous CSP binds to regions on the cell surface that already bear CSP. In view of the apparent weakness of CSP-CSP interactions on the lipid bilayer, it seems possible that the assembly of CSP fibrils is nucleated by cell surface components in addition to CSP.

Antigens↗
PubMed1977

Levels of translatable mRNAs for cell surface protein, collagen precursors, and two membrane proteins are altered in Rous sarcoma virus-transformed chick embryo fibroblasts.

Transformation of chick embryo fibroblasts by Rous sarcoma virus results in decreased amounts of a major cell surface protein and of collagen. To determine the mechanism accounting for the decreased production of these proteins, we have measured the relative amounts of functional mRNAs for these and other transformation-sensitive proteins. Total cellular RNAs extracted from normal cells and from cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus were translated in a cell-free system derived from wheat germ. Analysis of the in vitro translation products of RNAs from normal and transformed chick embryo fibroblasts shows a 5-fold reduction in the translatable mRNA for cell surface protein and a 10-fold reduction in translatable mRNA for two collagen precursors. In addition, increases in functional mRNA are observed for myosin and for two membrane polypeptides with molecular weights of 95,000 and 78,000; the latter two proteins increase on transformation, but the increases are in large part secondary to the depletion of glucose from the medium of transformed cells. Our data suggest that some of the major cellular changes induced by oncogenic viruses are due to changes in the activity of specific cellular genes.

Actins↗
PubMed1977

Glucose depletion accounts for the induction of two transformation-sensitive membrane proteinsin Rous sarcoma virus-transformed chick embryo fibroblasts.

Chick embryo fibroblasts transformed by Rous sarcoma virus have an increased content of two membrane proteins of molecular weights 78,000 and 95,000. The increased content of the 95,000-dalton protein and the principal increase in the content of the 78,000-dalton protein are not an early consequence of cell transformation but instead are secondary to the rapid depletion of glucose from the growth medium of transformed cells. When glucose is maintained at high levels in the growth medium of transformed cells, the synthesis of the 95,000-dalton protein is arrested and that of the 78,000-dalton protein is markedly suppressed. Upon removal of glucose from the growth medium of normal cells, these proteins increase to levels comparable to those of transformed cells. Because the amount of these two proteins is influenced by the presence or absence of glucose, we suggest they be referred to as "glucose-regulated proteins." GRP-78 and GRP-95. These proteins may have an important role in regulating the utilization of glucose in cultured cells.

Animals↗
PubMed1977

Quantitation of a transformation-sensitive, adhesive cell surface glycoprotein. Decrease of several untransformed permanent cell lines.

We have quantitated the transformation-sensitive, cell surface LETS glycoprotein on many untransformed cell types. By SDS-polyacrylamide gel electrophoresis, this trypsin-sensitive iodinatable glycoprotein comprises 1-3% of total cellular protein of the seven early passage cell types tested. In contrast, it constitutes less than 0.15% of the protein in four of six continuous cell lines. This decrease is reflected in alterations both in [14C]glucosamine labeling and in the immunofluorescent staining of early passage vs. these four permanent cell lines. These results help to clarify previous experiments in which CSP, a purified LETS protein, partially restored a fibroblastic phenotype to cells transformed by tumor viruses. These findings also indicate that a major decrease in this cell surface glycoprotein can occur in the establishment of a continuous cell line without resulting in cellular transformation.

Animals↗
PubMed1976

The purification and quantitation of myosin from cultured cells.

Myosin has been purified from the following cultured cell lines: normal rat kidney fibroblast (NRK), HeLa-Rhino (HeLa), human choriocarcinoma, human acute lymphoblastic leukemia, rat hepatoma (HTC), monkey kidney (VERO), pigmented mouse melanoma, Y-1 rat adrenal cortex, and growth hormone-secreting GH-1. Myosin constitutes 0.5-5.4% of the protein of these cells. It was not detected in washed human erythrocytes or in two types of mouse plasmacytoma cells. Two methods for the purification of myosin from cultured cells have been employed. With Method I highly purified myosin was prepared by Sepharose 4B and DEAE-cellulose chromatography from 10(10) L-929 cells as well as from mouse uterus. Those myosins have similar molecular and subunit weights as well as ATPase activity but are immunologically distinct. Method II involving ultracentrifugation and Sepharose 4B chromatography, is suitable for the production of moderately pure myosin in good yield from as few as 5-10(7) cells (five 100-mm Petrie dishes).

Adenosine Triphosphatases↗
PubMed1976

Purification and properties of filamin, and actin binding protein from chicken gizzard.

Filamin, a protein recently identified in chicken gizzard (Wang, K., Ash, F., and Singer, S. J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 4483-4486), has been purified free of other components and its molecular properties have been examined. Filamin has a sedimentation constant (S020,w) of 8.86 S and a partial specific volume of 0.734 ml/g. Sedimentation equilibrium experiments give a value of 498,000 for the molecular weight of native filamin. From these data a frictional ratio of 2.32 has been calculated. On sodium dodecyl sulfate gel electrophoresis, filamin migrates as a single protein band with an estimated molecular weight of 240,000. Filamin is a soluble protein and under a variety of conditions tested does not by itself form filaments or precipitate from solution. However, filamin binds to rabbit skeletal muscle F-actin, and the complex is readily sedimented by centrifugation to yield a gelatinous pellet containing actin and filamin. These results indicate that filamin is a dimeric protein with a moderate degree of asymmetry that binds to actin. The results also suggest that the distribution of filamin in cells is derived from its interaction with polymerized actin.

Actins↗
PubMed1976

Stimulation of guanylate cyclase of fibroblasts by free fatty acids.

The membranous guanylate cyclase of Balb 3T3 fibroblasts was stimulated by a fraction of calf serum extracted by ether. Stimulation was observed with Mg2+ as the only bivalent cation in the presence of Lubrol PX. The activator co-chromatographed with free fatty acids, and several of these were found to stimulate guanylate cyclase. Among the saturated fatty acids, myristic acid had the highest activity. Stimulating activity diminished as the hydrocarbon chain of the fatty acid was lengthened or shortened. Introduction of an unsaturated bond enhanced the activation by the longer fatty acids. This pattern of specificity is similar to that observed for the effect of fatty acids on many other membranous functions. Under appropriate conditions fatty acids were found to stimulate guanylate cyclase activity in the absence of Lubrol PX. The relationship among the effects of Mg2+, Mn2+, Lubrol PX, and fatty acids on enzyme activity was examined. On the basis of these studies, it appears that fatty acids stimulate the enzyme by a mechanism different from nonionic detergents or Mn2+.

Adenylyl Cyclases↗
PubMed1976

Guanylate cyclase and cyclic guanosine 3':5'-monophosphate phosphodiesterase activities and cyclic guanosine 3':5'-monophosphate levels in normal and transformed fibroblasts in culture.

To investigate the role of guanosine 3':5'-monophosphate (cyclic GMP) in cultured cells we have measured guanylate cyclase and cyclic GMP phosphodiesterase activities and cyclic GMP levels in normal and transformed fibroblastic cells. Guanylate cyclase activity is found almost exclusively in the particulate fraction of normal rat kidney (NRK) and BALB 3T3 cells. Enzyme activity is stimulated 3- to 10-fold by treatment with the detergent Lubrol PX. However, enhancement of guanylate cyclase by fibroblast growth factor could not be demonstrated under a variety of assay conditions. In both NRK and BALB 3T3 cells guanylate cyclase activity is low during logarithmic growth and increases as the cells crowd together and growth slows. Guanylate cyclase activity is undetectable in homogenates of NRK cells transformed by the Kirsten sarcoma virus (KNRK cells) either in the presence or absence of Lubrol PX. Guanylate cyclase activity is also greatly decreased in NRK cells transformed by Moloney, Schmidt-Ruppin, or Harvey viruses. BALB 3T3 cells transformed by RNA viruses (Kirsten, Harvey, or Moloney), by a DNA virus (SV40), by methylcholanthrene, or spontaneously, all have diminished but readily detectable guanylate cyclase activity. Cyclic GMP phosphodiesterase activity is found predominately in the soluble fraction of NRK cells. This activity increases slightly as NRK cells enter the stationary growth phase. Cyclic GMP phosphodiesterase activity is undetectable in two clones of KNRK cells under a variety of assay conditions, and is decreased relative to the level present in NRK cells in a third KNRK clone. However, both Moloney- and Schmidt-Ruppin-transformed NRK cells have a phosphodiesterase activity similar to that found in NRK cells. Boiled supernatant from both NRK and KNRK cells is observed to appreciably enhance the activity of activator-deficient phosphodiesterase from bovine heart. This result indicates that the absence of cyclic GMP phosphodiesterase activity in KNRK cells is not due to a loss of the phosphodiesterase activator. The intracellular concentration of cyclic GMP is found to be very low in transformed NRK cells when compared to levels measured in confluent NRK cells. The low levels of cyclic GMP in transformed NRK cells reflect the greatly decreased guanylate cyclase activity observed in these cells. These results do not appear to support the suggestion that cyclic GMP promotes the growth of fibroblastic cells.

Cell Line↗
PubMed1976

Cell surface protein decreases microvilli and ruffles on transformed mouse and chick cells.

Transformation of cultured fibroblasts usually results in a decrease in a high molecular weight cell surface glycoprotein (LETS protein) and often in increased numbers of surface microvilli and ruffles. We have isolated such a major cell surface glycoprotein from chick embryo fibroblasts; this protein, CSP, is decreased after transformation. Treatment of a mouse tumor cell line (SV1), L929 cells, and transformed chick fibroblasts with CSP results in a decrease in the number of microvilli and marginal ruffles, accompanied by restoration of a more normal morphology.

Actins↗
PubMed1976

Cell surface myosin in cultured fibroblasts.

The surface components of L-929 mouse fibroblast cells in monolayer culture have been labeled with 125I by lactoperoxidase-catalyzed iodination procedure. One of the membrane proteins iodinated has been shown to be myosin as follows: it has the same electrophoretic mobility (molecular weight 200,000 daltons) as myosin heavy chain on sodium dodecylsulfate polyacrylamide gels and the labeled myosin is specifically precipitated by fibroblast myosin antiserum from a preparation of purified plasma membrane that have been solubilized by treatment with 1% Triton X-100. One other 125I-labeled membranes protein (molecular weight 210, 000 daltons) is precipitated along with myosin; the latter does not combine directly with antimyosin antibody. This was determined by reacting polyacrylamide gels containing the separated membrane proteins with fibroblast myosin antiserum; myosin was the only membrane protein reacting with the antibody as determined by two separate methods. Membrane myosin is not labeled when the cells are grown in 14C-D-glucosamine or treated with galactose oxidase and potassium borotritide. Thus membrane myosin is probably not a glycoprotein.

Cell Membrane↗
PubMed1976

Insulin receptors in normal and transformed fibroblasts: relationship to growth and transformation.

The insulin receptors in normal and transformed lines of mouse Balb/3t3 fibroblasts have been studied. In the normal fibroblasts, the binding of insulin was low in growing cells and increased 2-9 fold in confluent stationary cells. Insulin binding was increased whether growth arrest was due to contact inhibition of growth or serum starvation. When serum-starved cells were stimulated to grow by the addition of fresh serum, insulin binding declined. In cells transformed by simian virus 40, Kirsten, Moloney, and Harvey sarcoma viruses, methylcholanthrene, X rays, or spontaneously, the binding was low, in the same range as growing normal cells. In simian virus 40-transformed cells, insulin binding increased 4 fold as the cells reached higher densities in culture. No relationship to changes in cell size was found. The differences in binding were due to changes in the concentration of the receptors, without changes in their affinity for the hormone.

Blood↗
PubMed1976

Mutants of Balb/c 3T3 fibroblasts defective in adhesiveness to substratum: evidence for alteration in cell surface proteins.

Two mutants of Balb/c 3T3 cells defective in adhesiveness to substratum have been isolated. After exposure to a mutagen a cell population was treated with prostaglandin E1 and methylisobutylxanthine to elevate the cells 3':5'-cyclic AMP levels and adhesion to the substratum; then, loosley adherent cells were harvested by gentle detachment. After four cycles of selection two clones were isolated. Both are characterized by a round morphology and a decreased adhesion to substratum. The defect in adhesion appears to be responsible for the change in cell shape, since the mutant cells become flattened and look similar to wild-type cells when treated with dibutyryl cyclic AMP even though their adhesiveness remains decreased. Further, the surface of both mutants is chemically altered. Lactoperoxidase-catalyzed iodination of the cell surface shows that two iodinated polypeptides (molecular weight, Mr, 90,000 and 135-140,000) present in wild-type cells are not detected on the surface of the mutants, and another iodinated band (110-120,000 Mr) is markedly decreased. In addition, one mutant is also missing the high-molecular-weight (230,000) cell surface protein that is sensitive to transformation. No changes have been found in the content of cyclic AMP or free or polymerized tubulin. The exponential growth rate and saturation density are not altered in these adhesion--eficient cells. This last result suggests that modulation of adhesion to substratum per se does not play an important role in the growth control of Balb/c 3T3 cells in vitro.

Cell Adhesion↗