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Biomedical subjects

I Pastan

Publications and source records attributed to I Pastan.

At least 631 records · Page 35Linked to original sources
PubMed1981

Accurate in vitro transcriptional initiation of the chick alpha 2 (Type I) collagen gene.

Chick genomic DNA containing the extreme 5' end of the alpha 2 (type I) collagen gene has been used as template in an in vitro HeLa cell transcription system. RNA polymerase II-dependent transcription initiates from a specific site on this DNA. The precise location of this site was determined by three types of experiments: sizing of in vitro-synthesized RNA runoff transcripts, comparing the sequence of the in vitro-made RNA transcripts with the structure of the DNA template, and identifying the first and second nucleotides of the in vitro-synthesized transcripts. Transcription was found to initiate 33 base pairs downstream from a canonical Goldberg-Hogness sequence (TATAAATA). This in vitro start site is the same as the initiation site of in vivo-synthesized collagen RNA.

Animals↗
PubMed1981

Glycogen synthase activity in Chinese hamster ovary cells. Studies with wild-type and mutant cells defective in cyclic AMP-dependent protein kinase.

Glycogen synthase (EC 2.4.1.11) activity was studied in cell extracts from wild-type Chinese hamster ovary (CHO) cells and three mutants resistant to cyclic AMP effects on cell shape and cell growth. Based on the capacity of crude extracts to phosphorylate exogenous histone, two of the mutants appeared to have altered cyclic AMP-dependent protein kinase (EC 2.7.1.37) and one of them had apparently normal amounts of kinase activity. Glycogen synthase activity was present in comparable amounts in wild-type and all three mutant strains in a presumably inactive phosphorylated form since activity was virtually completely dependent upon the presence of glucose 6-phosphate. The enzyme could be partially dephosphorylated by endogenous phosphatases and rephosphorylated by exogenous cyclic AMP-dependent protein kinase. Attempts to find culture conditions (e.g. glucose starvation) or cell treatment (e.g. insulin) which might activate glycogen synthase in intact cells were unsuccessful. since glycogen synthase activity present in CHO cells was independent of the level of cyclic AMP-dependent kinase, we conclude that cyclic AMP-dependent protein kinase does not play a critical role in regulating the state of phosphorylation of the synthase.

Animals↗
PubMed1981

Molecular characterization of human clathrin.

Clathrin extracted from coated vesicles at pH 8.0 sediments as a single boundary with 8.1S sedimentation constant (s020,w) of 8.1 +/- 0.1 S. Sedimentation equilibrium gave a molecular weight (Mr) of 610 000 +/- 30 000. The clathrin frictional ratio (pH 7.5) computed from s020,w and Mr is very large, i.e., 3.06 +/- 0.18. Analysis of the circular dichroic spectrum in the far-ultraviolet showed that about half of the peptide residues are in a alpha-helical conformation. The molecular weight of a preparation of clathrin purified to homogeneity on a Sepharose CL-4B column in 6 M guanidine hydrochloride was 170 000 +/- 26 000 by sedimentation equilibrium, which is in agreement with the values we and others obtained by sodium dodecyl sulfate gel electrophoresis. The 8.1S clathrin species may be regarded as the "native" promoter since (1) it is extracted from coated vesicles by an extremely mild procedure, (2) it is stable over considerable ranges of pH, temperature, and ionic strength, and (3) it readily polymerizes into characteristic closed lattice structures resembling those observed in coated vesicles in the electron microscope. The 8.1S clathrin molecule self-associates at pH 6.3 to form two very high molecular weight species with average sedimentation coefficients of 150 and 300 S. The sedimenting boundaries of both of these species have been analyzed to reveal their molecular heterogeneity. The two species observed by sedimentation velocity may correspond to the two sizes of coated vesicles previously reported to be present in some cells when observed by electron microscopy. Analysis of the sedimentation pattern in the ultracentrifuge also gives the amount of unreacted 8.1S clathrin from which the yield of polymerizable clathrin is obtainable. This methodology can therefore be employed to estimate the quality of the 8.1S preparation of clathrin and thereby affords an assay of its activity.

Circular Dichroism↗
PubMed1981

Regulation or procollagen messenger ribonucleic acid levels in Rous sarcoma virus transformed chick embryo fibroblasts.

Using cloned cDNAs for pro-alpha 1 and pro-alpha 2 collagen messenger ribonucleic acid (mRNA), we have investigated the regulation of collagen mRNA levels in Rous sarcoma virus (RSV) transformed chick embryo fibroblasts (CEF). We find that both pro-alpha 1 and pro-alpha 2 mRNA levels are decreased approximately 10-fold in CEF transformed by either the Bryan high-titer strain or the Schmidt-Ruppin strain of RSV. Using temperature-sensitive mutants in the transforming gene src, we also investigated the rate of change in the levels of the two mRNA species. We employed mutants of both the Bryan high-titre strain (BHTa) and the Schmidt-Ruppin strain (ts68). With both mutants the results were similar. Upon shift from the permissive temperature (35 degrees C) to the non-permissive temperature (41 degrees C), collagen mRNA synthesis, did not increase until more than 5 h had passed, suggesting that action of src on collagen gene expression is indirect. Upon shift from 41 to 35 degrees C, collagen mRNA levels fell with a half-life of 10 h. Whether this fall reflects the half-life of procollagen mRNA or an effect of src on procollagen RNA stability is unclear. Both pro-alpha 1 and pro-alpha 2 mRNA levels were coordinately controlled.

Animals↗
PubMed1981

Binding and internalization of 125I-alpha 2-macroglobulin by cultured fibroblasts.

The binding and internalization of 125I-labeled alpha 2-macroglobulin (125I-alpha 2M) was studied in cultured fibroblasts. Two classes of binding sites were detected on cell surfaces. One class corresponds to the previously described, high affinity and low capacity sites. The other class of binding sites may mediate uptake of high physiological blood levels of 125I-alpha 2M and has not been described previously. At 0 degrees C, this lower affinity class saturates at approximately 1,000 micrograms/ml and has a capacity of approximately 600,000 sites/cell. The lower affinity class accounts for the vast majority of cellular receptors for alpha 2M. An assay employing pepsin at pH 4 was developed to distinguish between surface-bound and internalized 125I-alpha 2M. Cellular uptake of 125I-alpha 2M at 37 degrees C has a component which saturates between 200 and 1,000 micrograms/ml and the rate of internalization of this component was approximately 1,700,000 molecules/cell/h. One mM Ca2+ was required for cell uptake of 125I-alpha 2M at 37 degrees C. Ca2+ was also required for binding at 0 degrees C to both low and high affinity classes of binding sites for 125I-alpha 2M. The transglutaminase inhibitors bacitracin, monodansylcadaverine, and N-benzyloxycarboxyl-5-diazo-4-oxonorvaline paranitrophenyl ester all inhibited cellular internalization of 125I-alpha 2M at 37 degrees C. Each of these three compounds selectively reduced 125I-alpha 2M binding to the high affinity, low capacity component at 0 degrees C. Based on the current binding studies and previous studies using electron microscopy which showed that bacitracin and other transglutaminase inhibitors block clustering of alpha 2M-receptor complexes in coated pits, we suggest that the inhibitors block the accumulation of occupied lower affinity alpha 2M receptors in coated pits where they acquire a higher apparent affinity.

Binding Sites↗
PubMed1981

Decrease in the levels of nuclear RNA precursors for alpha 2 collagen in Rous sarcoma virus transformed fibroblasts.

We have examined the levels of type I alpha 2 collagen RNA precursors, containing both intron and exon sequences in nuclear RNA preparations of chick embryo fibroblasts (CEF) and of CEF transformed by the Schmidt-Rupin strain of Rous sarcoma virus (RSV). We have used two different fragments of chick alpha 2 collagen genomic DNA as hybridization probes in S1 mapping experiments. Each of these DNA probes contains an entire intron. Our results indicate that the levels of the primary transcript of alpha 2 collagen RNA are much lower in RSV-CEF than in CEF. They suggest, but do not prove that the effect of the transforming protein p60src on the synthesis of alpha 2 collagen is mediated by a transcriptional control mechanism.

Animals↗
PubMed1981

Clathrin and coated vesicle proteins Immunological characterization.

Antisera to a purified extract of bovine brain coated vesicles have been prepared. The extract contained clathrin (greater than 90%) and polypeptides of 35,000 (less than 5%) and 38,000 (less than 5%) molecular weight. Antibodies to clathrin were affinity purified on a homogeneous clathrin-Sepharose column and demonstrated to be monospecific by sensitive immunoprecipitation and immunofluorescence experiments. These antibodies give a fluorescence pattern consistent with coated pit localization in cultured cells from diverse species indicating extensive cross-reaction, and, thus, conservation of the clathrin antigen. Antibodies to the lower molecular weight proteins (LMWP) present in the clathrin preparation were also affinity purified on a column containing these polypeptides but devoid of clathrin. These antibodies cross-reacted completely and immunoprecipitated only clathrin from 3T3 cell extracts. Although the molecular weights and isoelectric points of the LMWP presented are quite similar to tropomyosins, these immunological results and limited protease digestion data indicate that tropomyosin and the LMWP are not related. Rather, the latter may be breakdown products of clathrin.

Animals↗
PubMed1981

Binding and mobility of the cell surface receptors for 3,3',5-triiodo-L-thyronine.

A fluorescent derivative of the thyroid hormone 3,3'-triiodo-L-thyronine binds to cultured mouse fibroblasts; such binding is saturable. Video intensification fluorescence microscopy indicates that binding occurs at the plasma membrane. Diffusion coefficients, obtained by fluorescence photobleaching recovery, are consistent with binding to a protein receptor on the cell surface.

Animals↗
PubMed1981

Microinjection of anticlathrin antibodies into fibroblasts does not interfere with the receptor-mediated endocytosis of alpha2-macroglobulin.

Affinity-purified antibodies prepared against the major coat protein of brain coated vesicles, clathrin, were microinjected into cultured fibroblasts, and their intracellular distribution was followed by immunofluorescence microscopy and ultrastructural immunocytochemistry. Microinjected anticlathrin antibodies were concentrated on coated regions of the plasma membrane and the GERL apparatus. When an excess of anticlathrin antibodies was injected into the cytosol, coated pits on the plasma membrane were covered by anticlathrin antibody but still functioned to cluster an internalize alpha2-macroglobulin. These results are discussed in terms of the role of clathrin in the pathway of receptor-mediated endocytosis. Our data indicate that in cultured fibroblasts coated pits are stable elements permanently attached to the plasma membrane.

Animals↗
PubMed1981

Structure of the promoter for chicken alpha 2 type I collagen gene.

The chicken alpha 2 type I collagen gene is 38 kilobases long and its coding information is subdivided into more than 50 exons. In the current study, we used primer extension and S1 nuclease mapping to determine the sequence of the 5' end of alpha 2 collagen mRNA and to locate the start site for transcription of the alpha 2 collagen gene. The DNA sequence around the start site for transcription shows a typical Goldberg-Hogness sequence, 5' T-A-T-A-A-A-T 3', between -33 and -26 and a 5' G-C-C-C-A-T-T 3' sequence ("CAT" box) between -84 and -78. Three AUGs are found in the initial portion of the mRNA, the first from +54 to +56, the second from +117 to +119, and the third from +134 to +136. The first two AUGs are followed by short coding sequences that could specify a hexapeptide a tetrapeptide, respectively. Only the third AUG is followed by an open reading frame coding for a sequence that presents considerable homology with the previously determined amino acid sequence of prepro alpha 1 collagen. In the promoter region sequence there are several extensive dyads of symmetry. Three of these inverted repeats which precede the start site for transcription overlap each other and may have a role in the developmental regulation of this gene.

Animals↗
PubMed1981

alpha 2-macroglobulin adsorbed to colloidal gold: a new probe in the study of receptor-mediated endocytosis.

alpha 2-Macroglobulin (alpha 2 M) was adsorbed to colloidal gold and used as a new tool in the study of receptor-mediated endocytosis. alpha 2 M-gold is easy to prepare and is clearly visualized at the electron microscope level. When cells were incubated with alpha 2 M-gold at 0 degrees C, gold was visualized both diffusely over the cell surface and concentrated in coated pits. After cells to which alpha 2 M-gold had been bound at 0 degrees C were warmed, the gold was rapidly internalized into uncoated vesicles, previously termed receptosomes. After 30 min of incubation or longer, gold was found in small lysosomes and, later, in large lysosomes and very small vesicles in the region of the Golgi complex. This pattern of localization is similar to that previously described, using peroxidase-labeled anti-alpha 2 M antibodies. By incubating cells with both alpha 2 M-gold and vesicular stomatitis virus (VSV), we studied the internalization of these two markers simultaneously. VSV and alpha 2 M-gold rapidly clustered in the same coated pits and were internalized in the same receptosomes. Proteins and hormones adsorbed to gold may be useful in the study of receptor-mediated endocytosis.

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