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A Colman

Publications and source records attributed to A Colman.

90 records · Page 5Linked to original sources
PubMed1981

Synthesis of androgen-dependent secretory proteins by rat seminal vesicles.

Androgenic steroids control the synthesis and secretion of several proteins by the seminal vesicles of the male rat. Prominent among them are 2 basic proteins, S and F, previously used as markers of androgen action. These proteins are not found among translation products of a wheat-germ protein-synthesising system supplied with mRNA from seminal vesicles of normal rats. Instead, higher molecular weight forms, S' and F', are formed which are nonetheless related antigenically to S and F respectively. Injected into Xenopus Laevis oocytes, seminal vesicle mRNA does direct synthesis and secretion of polypeptides S and F. Partial peptide analysis confirms that S' and F' have extensive amino acid sequence homology with S and F respectively. We conclude that S' and F' are precursor forms of the secreted proteins and thus at least 2 abundant proteins of this male accessory tissue are secreted by a mechanism common to a wide number of secreted proteins.

Androgens↗
PubMed1981

Fate of secretory proteins trapped in oocytes of Xenopus laevis by disruption of the cytoskeleton or by imbalanced subunit synthesis.

The effects of imbalanced subunit synthesis, temperature, colchicine, and cytochalasin on the secretion from Xenopus laevis oocytes of a variety of avian and mammalian proteins were investigated; these proteins were encoded by microinjected messenger RNA. Cytochalasin and colchicine together severely reduced secretion in a temperature-independent manner, the exact reduction varying among the different proteins. In contrast cytochalasin alone had no effect, whereas colchicine alone caused a smaller, temperature-dependent reduction. The synthesis and subcellular compartmentation of these proteins were unaffected by the drug treatments; however, the proteins did not accumulate in the drug-treated oocytes but were degraded. The rate of degradation of each protein was similar to its rate of exocytosis from untreated oocytes. A similar result was obtained without recourse to drugs by studying the fate of immunoglobulin light chains trapped in oocytes by a deficiency in heavy chain synthesis. These results are discussed in terms of the disruptive effects, as revealed by electron microscopy, of the drug treatments on the cytoskeleton of the oocyte.

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↗
PubMed1981

The secretion of proteins in vitro from Xenopus oocytes and their accessory cells: a biochemical and morphological study.

Protein secretion by Xenopus laevis oocytes and their surrounding follicular cells in vitro has been investigated using two-dimensional gel electrophoresis. Viable oocytes, devoid of follicle layers, were prepared by treatment with collagenase; they retain in full their capacity to synthesize, sequester and export secretory proteins following microinjection with heterologous messenger RNA. Both RNA-injected and normal cells export a large number of endogenous oocyte proteins and, as with heterologous secretory translation products, these proteins are found within the oocyte in a vesicle fraction. Electron microscopy indicates that secretion involves exocytotic release of cortical vesicle contents. The follicular cells themselves also seem to contribute a number of proteins to the incubation medium surrounding isolated oocytes, but the presence of follicle layers is not required for the export of endogenous oocyte proteins.

Animals↗
PubMed1980

Multiple ribosomal gene sites revealed by in situ hybridization of Xenopus rDNA to Triturus lampbrush chromosomes.

A variety of 3H-labelled ribosomal gene probes were hybridized in situ to the nascent transcripts of lampbrush chromosomes from the crested newt, Triturus cristatus carnifex. The probes were from Xenopus laevis and included rDNA isolated by CsCl gradient centrifugation, recombinant plasmids and purified restriction fragments of rDNA. All the probes gave essentially the same result. About 10-15 loop pairs were distinctly labelled in each preparation, almost all of them located on the heteromorphic arms (HTAs) of chromosome 1. Ribosomal gene probes were also hybridized in situ to the DNA of denatured mitotic chromosomes from some of the individuals used to provide lampbrush preparations. Minor, scattered sites of hybridization were found in the HTAs, but the main clusters of ribosomal genes were found on chromosomes 6 and/or 9, in agreement with previous determinations of nucleolus organizer position in this species. However, the nucleolus organizers were not sites of labelled loops in lampbrush transcript hybridizations.--We have incubated isolated lampbrush-stage nuclei in media containing alpha-amanitin and labelled RNA precursors. Although extrachromosomal nucleolar genes incorporated label, supposedly due to transcription by RNA polymerase I, no lampbrush loops were labelled.--It appears that in T. c. carnifex there are ribosomal gene sequences at the main nucleolus organizers and at a number of sites scattered along the HTAs. The ribosomal genes at the nucleolus organizers are not extended in the form of actively transcribing loops unlike the ribosomal sequences on the HTAs, which are heavily labelled in transcript hybridization. The ribosomal sequences on the HTAs appear not to be transcribed by the same RNA polymerase that transcribes the ribosomal genes of extrachromosomal nucleoli.

Animals↗
PubMed1980

Post-translational events in the production of human lymphoblastoid interferon.

Newly synthesized interferon and its mRNA were membrane-associated in a human lymphoblastoid cell (Namalwa) that had been induced with Sendai virus. Treatment with zinc, which acts as an inhibitor of proteolytic cleavage, prevented interferon production. When cytoskeletal function was disrupted by adding both colchicine and cytochalasin B to the induced Namalwa cells, secretion of interferon was inhibited. It is concluded that after translation of the interferon mRNA, the newly synthesized interferon polypeptide is discharged into the lumen of the endoplasmic reticulum, and undergoes a proteolytic cleavage before it is secreted by a process involving the cytoskeleton.

Burkitt Lymphoma↗
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

Export of proteins from oocytes of Xenopus laevis.

When human lymphoblastoid mRNA was microinjected into X. laevis oocytes, titers of interferon rapidly reached a maximum inside the oocyte while accumulation of interferon continued in the incubation medium for at least 45 hr. If interferon protein was injected into oocytes it was rapidly inactivated. Significantly, newly synthesized interferon but not injected interferon was found to be membrane-associated. Further experiments involving the co-injection of mRNAs coding for secretory proteins (guinea pig milk proteins and human interferon) and nonsecretory proteins (rabbit globin) revealed that only the secretory proteins were exported from the oocyte. Moreover, different proteins were exported at different rates. A distinct subclass of newly synthesized oocyte proteins of unknown function also accumulated in the incubation medium. Since the information encoded in the messenger RNAs of secretory proteins is sufficient to specify synthesis, compartmentation and secretion of these proteins, the oocyte may provide a complete system for the analysis of the secretory process.

Animals↗
PubMed1979

Characterization of interferon messenger RNA from human lymphoblastoid cells.

After treatment with Sendai virus, Namalwa cells form large amounts of interferon. RNA extracted from treated whole cells or from their polysomes was injected into Xenopus laevis oocytes and the interferon formed was titrated. The results show that the amount of interferon mRNA was maximal by 9 h after treatment of the cells with Sendai virus and then declined. Sucrose gradient centrifugation of the mRNA gave substantial purification and showed that its size was 12 S.

Animals↗
PubMed1978

Rapid purification of plasmid DNAs by hydroxyapatite chromatography.

A method is described for the rapid preparation of plasmid DNAs of molecular weight up to 14 X 10(6). This method involves the chromatography, at room temperature, of bacterial cleared lysates on hydroxyapatite in the presence of high concentrations of phosphate and urea. All detectable protein and RNA contamination of plasmid DNA is removed by this procedure and the conformation of the plasmid DNA is unaffected. Less than 0.5% chromosomal DNA is present in the purified preparation and even this can be removed if necessary by a simple extention of the procedure to include a heat-denaturation step. The method is extremely rapid and amenable to large-scale plasmid preparation; 5 mg ColE1 DNA have been purified within 40 min. The yield of plasmid DNA is similar to that obtained with the conventional dye-centrifugation technique, however the purity is greater.

Chromatography↗
PubMed1976

31P nuclear-magnetic-resonance studies on the developing embryos of Xenopus laevis.

The concentrations of nucleoside triphosphate, inorganic phosphate and the yolk proteins, phosvitin and lipovitellin, have been monitored in living embryos of Xenopus laevis by 31P nuclear magnetic resonance (NMR) spectroscopy. The nucleoside triphosphate levels remain relatively constant at about 3.5-4.5 nmol/embryo at least until the 'spontaneous movement' stage of development. By the swimming tadpole stage an inorganic phosphate resonance representing about 30 nmol/embryo becomes evident in the NMR spectrum. Computer manipulation also shows such a resonance, although smaller, to be present at a somewhat earlier developmental stage; these findings are confirmed biochemically. The major contribution to the NMR spectrum of oocytes, unfertilized eggs and early embryos is the yolk phosphoprotein resonance. On isolation of the yolk from the embryos it is possible to quantify the contribution to the NMR spectrum from the lipid-phosphate and protein-phosphate moieties of the yolk proteins. During development, as the yolk is used up, it is found that the protein-phosphate resonance disappears at a greater rate than the lipid-phosphate peak. The total phosphorus content of the embryo (approximately 200 nmol/embryo) is shown biochemically to remain constant during development; however, the total amount of phosphorus observed by NMR decreases by about 40% during development. From the resonance positions of their alpha, beta and gamma phosphate groups it is deduced that the nucleoside triphosphate molecules are liganded in vivo to a divalent cation which is not manganese, but could be either magnesium or calcium. From the position of the inorganic phosphate resonance it is deduced that the internal pH of embryos where this resonance is evident is 6.8 +/- 0.2.

Animals↗
PubMed1975

Transcription of DNAs of known sequence after injection into eggs and oocytes of Xenopus laevis.

1. When the synthetic polynucleotide, poly[d(A-T) - d(A-T)] is injected into the eggs and oocytes of Xenopus laevis, a stimulation of RNA synthesis results. Analysis of extracted RNA by high-voltage electrophoresis, shows that this stimulation of RNA synthesis is due to the production of poly[r(A-U)] transcripts. The rate of poly[r(A-U)] synthesis is calculated to be at least ten-fold greater in eggs than in oocytes. The amount of poly[r(A-U)] detectable in injected eggs has reached a maximum by 1.5 h after injection; in oocytes, however, poly[r(A-U)] continues to accumulate between the third and 16th hour after injection. The transcripts range in length from less than 80 nucleotides up to over 2000 nucleotides long. The co-injection of poly[d(A-T)] - d(A-T)] and alpha-amanitin into oocytes, has shown that the synthesis of poly[r(A-U)] is approximately 90% inhibited at a concentration of alpha-amanitin which has no effect on the capacity of the oocyte to synthesize ribosomal and 4-S RNA; thus the nucleoplasmic RNA polymerases IIa and/or IIb, are implicated as playing a major role in poly[r(A-U)] synthesis in oocytes. 2. When poly(dG) - poly(d-C), poly(dA), poly(dA) - poly(dT) and poly[d(I-C) - d(I-C)] are individually injected into eggs only poly[d(I-C) - d(I-C)] is transcribed as efficiently as poly[d(A-T) - d(A-T)]. 3. When calf thymus native or denatured DNA, polyoma, T2, T4 and phiX DNAs are individually injected into eggs only the injection of calf thymus native DNA causes a detectable stimulation of RNA synthesis. 4. The activities of crude preparations of egg and oocyte RNA polymerases are tested in vitro with different DNA templates. In contrast to the situation in vivo, it is found that poly[d(A-T1 - d(A-T)] is as efficiently transcribed in vitro by oocyte polymerase as by egg polymerase. Additionally poly[d(A-T) - d(A-T)] is transcribed ten-fold more efficiently in vitro than calf thymus native DNA. When poly(dA) - poly(dT), poly(dA), phiX, T2, and calf thymus denatured DNA are tested in vitro, only calf thymus denatured DNA is transcribed to a significant extent. The above results are discussed in relation to the known synthetic activities of Xenopus eggs and oocytes.

Animals↗
PubMed1973

Attitudes of obstetric and gynecologic residents toward abortion.

As a follow-up to Wolf's study of attitudes of obstetrical housestaff toward therapeutic abortion,(5) the attitudes of 48 obstetrical residents in the San Francisco Bay area were evaluated by questionnaire and structured interview. Specific issues studied were: (1) Willingness to perform therapeutic abortion, (2) impact of therapeutic abortion on Resident Training Program, (3) attitudes toward different operational procedures, and (4) preferred physician-patient relationship with therapeutic abortion patients. Findings suggest that although there is recognition and acceptance by most residents of the social need for therapeutic abortion, considerable ambivalence persists.

Abortion, Therapeutic↗