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N Riedel

Publications and source records attributed to N Riedel.

At least 19 recordsLinked to original sources
PubMed1991

Dioxin induces transforming growth factor-alpha in human keratinocytes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a widespread environmental toxicant, is a tumor promoter that induces hyperplasia in epithelial cells. Exposure of cultured human keratinocytes to TCDD, resulted in a time-dependent dioxin-specific Ah receptor-mediated release of transforming growth factor-alpha (TGF-alpha) into the culture medium. Cultures exposed to TCDD showed a rate of TGF-alpha secretion into the medium of about 30 fmol/ml/day, as well as a 3- to 6-fold increase in TGF-alpha mRNA expression. Increased production of TGF-alpha in human keratinocytes exposed to TCDD demonstrates a modulation of autocrine regulation in those cells. These results suggest that induction of TGF-alpha could be an important part of the mechanism of dioxin-mediated toxicity and tumor promotion.

Blotting, Northern↗
PubMed1991

SV40T-immortalized lung alveolar epithelial cells display post-transcriptional regulation of proliferation-related genes.

To study the regulation of proliferation of lung alveolar epithelial type 2 cells, we have established a cell line derived from neonatal type 2 cells by transfection with the SV40 large T antigen gene. We find that this cell line, designated SV40-T2, displays the same post-transcriptional control of expression of proliferation-related genes, including c-myc, ornithine decarboxylase, thymidine kinase, and histone, that we have previously described in primary isolates of type 2 cells (Clement et al., Proc. Natl. Acad. Sci. USA 87, 318-322, 1990). Both proliferating and nonproliferating SV40-T2 cells express these genes at high levels, but their translation products are only detected in proliferating cells. Using the histone gene as an example, we have found that regulation of expression occurs at the level of transcription and of mRNA turnover, as previously described in other mammalian systems. However, in addition, regulation of expression also occurs at the level of translation of the histone mRNA, because its protein product is not detectable in nonproliferating SV40-T2 cells. We have analyzed the steps which are potentially involved in this translational regulation of histone gene expression in SV40-T2 cells. In both proliferating and nonproliferating cells, histone mRNA was found to be efficiently transported from the nucleus to the cytoplasm and to associate with the translationally active heavy polysomal fractions. These results indicate that control of histone gene expression (and perhaps that of other proliferation-related genes) in lung epithelial cells may involve either rapid and selective degradation of histone protein or binding factor(s) which modulate translational efficiency of histone mRNA.

Animals↗
PubMed1990

Poly(A) binding proteins located at the inner surface of resealed nuclear envelopes.

We have used a photoreactive cross-linking reagent, poly(A/8-N3-A) (a poly(A) of average molecular mass of 100 kDa in which 5-10% of the A residues are replaced by 8-N3-A), to label poly(A) binding proteins of rat liver nuclear envelopes. This reagent was prepared by polymerizing a mixture of ADP and 8-N3-ADP with polynucleotide phosphorylase. The purified poly(A) was labeled in the 5'-position with a 32P group. In nuclear envelopes prepared by a low salt DNase I procedure, the poly(A/8-N3-A) labeled a protein-nucleic acid complex of approximately 270 kDa, which on degradation with RNase U2 or NaOH at pH 10 yielded two polypeptides of approximately 50 and 30 kDa. These photoreaction products were markedly decreased when resealed nuclear envelopes or non-nuclear envelope proteins were irradiated in the presence of poly(A/8-N3-A). The affinity labeling was intensified when resealed vesicles were made leaky by freezing or ultrasonication, suggesting that the poly(A) binding proteins are accessible from the nucleoplasmic but not the cytoplasmic face of the envelope. Moreover binding was specific for poly(A). Alternative reagents, random poly(A/8-N3-A,C,G,U) of about 100 kDa and poly(dA) (molecular mass between 350 and 515 kDa), showed a very low affinity for poly(A) recognition proteins in the low salt DNase I-treated nuclear envelopes; the 270-kDa band was labeled only weakly. The binding site was not protected by poly(A,C,G,U), weakly by poly(dA), and distinctly by poly(A).

Animals↗
PubMed1990

GTP-binding proteins in rat liver nuclear envelopes.

Nuclear transport as well as reassembly of the nuclear envelope (NE) after completion of mitosis are processes that have been shown to require GTP and ATP. To study the presence and localization of GTP-binding proteins in the NE, we have combined complementary techniques of [alpha-32P]GTP binding to Western-blotted proteins and UV crosslinking of [alpha-32P]GTP with well-established procedures for NE subfractionation. GTP binding to blotted NE proteins revealed five low molecular mass GTP-binding proteins of 26, 25, 24.5, 24, and 23 kDa, and [alpha-32P]GTP photoaffinity labeling revealed major proteins with apparent molecular masses of 140, 53, 47, 33, and 31 kDa. All GTP-binding proteins appear to localize preferentially to the inner nuclear membrane, possibly to the interface between inner nuclear membrane and lamina. Despite the evolutionary conservation between the NE and the rough endoplasmic reticulum, the GTP-binding proteins identified differed between these two compartments. Most notably, the 68- and 30-kDa GTP-binding subunits of the signal recognition particle receptor, which photolabeled with [alpha-32P]GTP in the rough endoplasmic reticulum fraction, were totally excluded from the NE fraction. Conversely, a major 53-kDa photolabeled protein in the NE was absent from rough endoplasmic reticulum. Whereas Western-blotted NE proteins bound GTP specifically, all [alpha-32P]GTP photolabeled proteins could be blocked by competition with ATP, although with a competition profile that differed from that obtained with GTP. In comparative crosslinking studies with [alpha-32P]ATP, we have identified three specific ATP-binding proteins with molecular masses of 160, 78, and 74 kDa. The localization of GTP- and ATP-binding proteins within the NE appears appropriate for their involvement in nuclear transport and in the GTP-dependent fusion of nuclear membrane vesicles required for reassembly of the nucleus after mitosis.

Adenosine Triphosphate↗
PubMed1990

Growth-related gene expression in type 2 alveolar epithelial cells.

Our laboratory is studying mechanisms of growth control in alveolar type 2 cells. This highly differentiated cell is induced to proliferate in lungs of animals of all ages during various forms of growth and during the repair process after lung injury. Using type 2 (T2) cells isolated from adult and neonatal rat lungs and an SV40-T transfected T2 cell line, we have shown tha growth-arrested T2 cells constitutively express genes associated with G1 and S phase of the cell cycle, yet they do not efficiently translate the proteins encoded by these genes. This block of growth-related gene expression is post-transcriptional and appears to involve mechanisms that control translation, perhaps at the level of initiation. Furthermore, growth-arrested T2 cells initiate DNA synthesis; however, the cells do not complete the cell cycle, suggesting that they are arrested in a late stage, perhaps the G1/S border. Differential screening of a cDNA library of growth-arrested T2 cells with DNA from growing and growth-arrested T2 cells has identified four families of genes preferentially expressed in the growth-arrested cells. These genes, which are in the process of being characterized, may be responsible for the unusual type of growth arrest demonstrated by T2 cells.

Animals↗
PubMed1990

[3H]thymidine incorporation does not correlate with growth state in cultured alveolar type II cells.

Quantitative measurement of [3H]thymidine [( 3H]TdR) incorporation into cultured cells is widely used as an indicator of cell proliferation. The observation that adult type II cells are able to incorporate large amounts of [3H]TdR despite the fact that they are not proliferating raised the question of the meaning of [3H]TdR incorporation in these cells. Comparing different systems of proliferating and nonproliferating type II cells and lung fibroblasts, we show that nonproliferating type II cells are able to synthesize some thymidine nucleotides used as immediate precursors for DNA synthesis and that most of the radioactivity incorporated into acid-insoluble material in these cells is actually in DNA. We found that hydroxyurea inhibited [3H]TdR incorporation into DNA, suggesting that nonreplicating type II cells use thymidine for scheduled, i.e., replicative, rather than unscheduled, or repair, DNA synthesis. However, newly synthesized DNA does not appear to be in a stable form, available for replication. These studies demonstrate that, in culture, adult type II cells initiate but are unable to complete scheduled DNA synthesis. They also establish that [3H]TdR incorporation cannot be used as an indicator of cell proliferation in cultured type II cells.

Animals↗
PubMed1989

Interaction of a nuclear location signal with isolated nuclear envelopes and identification of signal-binding proteins by photoaffinity labeling.

The nuclear envelope (NE) separates the two major compartments of eukaryotic cells, the nucleus and the cytoplasm. Recent studies suggest that the uptake of nuclear proteins into the nucleus is initiated by binding of nuclear location signals (NLSs) contained within these proteins to receptors in the NE, followed by translocation through the nuclear pore complex. To examine the binding step without interference from intranuclear events, we have used a system consisting of (i) purified rat liver NEs fixed onto glass slides and (ii) the prototype simian virus 40 large T antigen (SV40 T) NLS conjugated to nonnuclear carrier proteins, and we have visualized the receptor-ligand interaction by indirect immunofluorescence. In this system, incubation of isolated NEs with the wild-type SV40 T NLS conjugate with carrier proteins resulted in binding that was signal sequence-dependent, could be competitively blocked with excess conjugated and unconjugated wild-type peptide, did not require ATP, and was not affected by the transport-inhibiting lectin wheat germ agglutinin. In contrast, only minimal binding was observed with a mutant SV40 T NLS conjugate. These results are consistent with those obtained in other, more complex in vitro systems and suggest that binding of the SV40 T NLS is receptor-mediated. Binding is largely abolished by extraction of the NE with the nonionic detergent Triton X-100, suggesting that the receptor is soluble in detergent. We find in the Triton X-100 supernatant four major NLS-binding proteins with apparent molecular masses of 76, 67, 59, and 58 kDa by photoaffinity labeling with a highly specific crosslinker, azido-NLS. The reduced complexity of the system described here should be useful for the functional study of other potential NLSs for the identification and isolation of their binding sites and for the screening of antibodies raised against these binding sites.

Affinity Labels↗
PubMed1988

Transduction of endogenous envelope genes by feline leukaemia virus in vitro.

Feline leukaemia viruses (FeLV) are exogenous retroviruses that can be detected in most cats with leukaemia, aplastic anaemia, myeloproliferative diseases and fatal immunosuppression. FeLV isolates have been divided into three subgroups, based on the viral envelope-determined properties of interference and host range in vitro. FeLV-A is present in all natural isolates and is generally minimally pathogenic. FeLV-B is found with FeLV-A in isolates from approximately 40% of natural infections and in a higher percentage of cats with lymphoma. Following the fundamental observations of genetic reassortment of avian retroviruses with endogenous viral genes and the origination of lymphomagenic viruses during the ontogeny of AKR mice, we show here that transfection of feline cells with FeLV-A DNA results in its recombination with endogenous FeLV-related sequences to produce viruses with the structural and host range properties of FeLV-B. Thus in vitro propagation of a retrovirus may result in the generation of variants with very different properties.

Animals↗
PubMed1988

Pathogenic and host range determinants of the feline aplastic anemia retrovirus.

Feline leukemia virus (FeLV) C-Sarma (or FSC) is a prototype of subgroup C FeLVs, which induce fatal aplastic anemia in outbred specific-pathogen-free (SPF) cats. FeLV C isolates also possess an extended host range in vitro, including an ability, unique among FeLVs, to replicate in guinea pig cells. To identify the viral determinants responsible for the pathogenicity and host range of FSC we constructed a series of proviral DNAs by exchanging gene fragments between FSC and FeLV-61E (or F6A), the latter of which is minimally pathogenic and whose host range in vitro is restricted to feline cells. Transfer of an 886-base-pair (bp) fragment of FSC, encompassing the codons for 73 amino acids at the 3' end of pol (the integrase/endonuclease gene) and the codons for 241 amino acids of the N-terminal portion of env [the extracellular glycoprotein (gp70) gene], into the F6A genome was sufficient to confer onto chimeric viruses the ability to induce fatal aplastic anemia in SPF cats. In contrast, no chimera lacking this sequence induced disease. When assayed in vitro, all chimeric viruses containing the 886-bp fragment of FSC acquired the ability to replicate in heterologous cells, including dog and guinea pig cells. Thus, the pathogenic and the host range determinants of the feline aplastic anemia retrovirus colocalize to a 3' pol-5' env region of the FSC genome and likely reside within a region encoding 241 amino acid residues of the N terminus of the extracellular glycoprotein.

Anemia, Aplastic↗
PubMed1988

Strong sequence conservation among horizontally transmissible, minimally pathogenic feline leukemia viruses.

We report the first complete nucleotide sequence (8,440 base pairs) of a biologically active feline leukemia virus (FeLV), designated FeLV-61E (or F6A), and the molecular cloning, biological activity, and env-long terminal repeat (LTR) sequence of another FeLV isolate, FeLV-3281 (or F3A). F6A corresponds to the non-disease-specific common-form component of the immunodeficiency disease-inducing strain of FeLV, FeLV-FAIDS, and was isolated from tissue DNA of a cat following experimental transmission of naturally occurring feline acquired immunodeficiency syndrome. F3A clones were derived from a subgroup-A-virus-producing feline tumor cell line. Both are unusual relative to other molecularly cloned FeLVs studied to date in their ability to induce viremia in weanling (8-week-old) cats and in their failure to induce acute disease. The F6A provirus is organized into 5'-LTR-gag-pol-env-LTR-3' regions; the gag and pol open reading frames are separated by an amber codon, and env is in a different reading frame. The deduced extracellular glycoproteins of F6A, F3A, and the Glasgow-1 subgroup A isolate of FeLV (M. Stewart, M. Warnock, A. Wheeler, N. Wilkie, J. Mullins, D. Onions, and J. Neil, J. Virol. 58:825-834, 1986) are 98% homologous, despite having been isolated from naturally infected cats 6 to 13 years apart and from widely different geographic locations. As a group, their envelope gene sequences differ markedly from those of the disease-associated subgroup B and acutely pathogenic subgroup C viruses. Thus, F6A and F3A correspond to members of a highly conserved family and represent prototypes of the horizontally transmitted, minimally pathogenic FeLV present in all naturally occurring infections.

Amino Acid Sequence↗
PubMed1987

The genome organization of STLV-3 is similar to that of the AIDS virus except for a truncated transmembrane protein.

Nucleotide sequence analysis of the 3' portion of the genome of simian T-lymphotropic virus type 3 from African green monkeys (STLV-3agm) reveals that it has the same general genome structure as the human immunodeficiency virus (HIV-1), the etiologic agent of AIDS. Short segments of strong amino acid homology and similar predicted protein structure characterize the tat and art/trs open reading frames (orf) of both viruses. Strong conservation of 3' orf and of another, cs-orf, for which no protein product has been identified suggests that they both encode proteins important to the life cycle of these viruses. The extracellular glycoproteins of STLV-3 and HIV-1 share a similar backbone structure and 50%-55% amino acid homology in constant domains of the HIV-1 protein. The most evident departure in structural organization is truncation of the transmembrane glycoproteins in two STLV-3agm clones and a biologically active, noncytopathic clone of HTLV-4.

Amino Acid Sequence↗
PubMed1987

A subset of yeast snRNA's contains functional binding sites for the highly conserved Sm antigen.

Autoimmune sera of the Sm specificity react with the major class of small nuclear RNA (snRNA)-containing ribonucleoprotein particles (snRNP's) from organisms as evolutionarily divergent as insects and dinoflagellates but have been reported not to recognize snRNP's from yeast. The Sm antigen is thought to bind to a conserved snRNA motif that includes the sequence A(U3-6)G. The hypothesis was tested that yeast also contains functional analogues of Sm snRNA's, but that the Sm binding site in the RNA is more strictly conserved than the Sm antigenic determinant. After microinjection of labeled yeast snRNA's into Xenopus eggs or oocytes, two snRNA's from Saccharomyces cerevisiae become strongly immunoprecipitable with human auto-antibodies known as anti-Sm. These each contain the sequence A(U5-6)G, are essential for viability, and are constituents of the spliceosome. At least six other yeast snRNA's do not become immunoprecipitable and lack this sequence; these non-Sm snRNA's are all dispensable.

Animals↗
PubMed1987

Preparation and characterization of nuclear-envelope vesicles from rat liver nuclei.

We describe a procedure for the preparation of sealed nuclear-envelope vesicles from rat liver nuclei. These vesicles are strikingly similar in their polypeptide composition when compared with those of nuclear envelopes prepared conventionally using deoxyribonuclease I. Subfractionation analysis by means of extraction with high salt and urea show that the components of the nuclear envelope, e.g. the pore-complex/lamina fraction, are present. The residual DNA content is only 1.5%, and typical preparations consist of about 80% vesicles, with the vesicular character of these envelopes shown by microscopic and biochemical studies. The vesicles can be obtained in high yield, are tight and stable for at least two days and are enriched in a nucleoside triphosphatase thought to be involved in nucleocytoplasmic transport processes. Because the vesicles are largely free of components of the nuclear interior, but retain properties of intact nuclei, we believe that they are a valuable model system to study nucleocytoplasmic transport. Although in transport studies with isolated nuclei interference from intranuclear events has to be considered, the nuclear-envelope vesicles provide the possibility of studying translocation alone. Furthermore, the less complex nature of these vesicles compared with whole nuclei should facilitate investigation of the components involved in the regulation of nuclear transport processes.

Animals↗
PubMed1987

Nuclear-envelope vesicles as a model system to study nucleocytoplasmic transport. Specific uptake of nuclear proteins.

In the preceding paper [Riedel & Fasold (1987) Biochem. J. 241, 203-212] we have described a procedure for the preparation of nuclear-envelope vesicles (NE vesicles) from rat liver nuclei. These vesicles, which are largely free of components of the nuclear interior, were employed in an assay system in vitro to study protein translocation across the NE. We found that nuclear proteins such as histones, high-mobility-group proteins and acidic chromosomal proteins are specifically taken up and accumulated in the NE vesicles, whereas there is little or no affinity for non-nuclear proteins like immunoglobulin, myoglobin and cytochrome c. The kinetics of histone uptake into the NE vesicles are similar to those obtained for whole rat liver nuclei, and comparative studies with non-vesicular NEs prepared by deoxyribonuclease I-treatment (DNAase-NEs) indicate that the NE of the vesicles affects the uptake kinetics and increases the capacity for nuclear proteins. The uptake of histones into NE vesicles, but not the binding to DNAase-NEs, can be stimulated by GTP and GDP. Furthermore, we found that even very large molecules can be entrapped in the vesicles during their preparation. These results indicate that the NE vesicles might provide a useful system in vitro with which to investigate the structures and mechanisms involved in protein translocation across the NE.

Animals↗
PubMed1987

Permeability measurements with closed vesicles from rat liver nuclear envelopes.

Closed nuclear envelope ghosts in the physiological orientation were prepared from rat liver and nuclei as previously described. Here we report transport measurements of various proteins and ribonucleic acids across the envelope of these vesicles. Histones were accumulated rapidly in the ghosts, in contrast to other, nonnuclear, proteins. Triton X-100 removal of the external nuclear membrane from loaded vesicles, as well as comparative studies with open vesicles, excluded the effects of external adsorption. The exchange rate of histones across the nuclear envelope is strongly depressed in the presence of GTP and GDP. The vesicles contain the translocation mechanism for poly(A)-containing RNA. The translocation of poly(A), messenger RNA, and ribosomal RNA was investigated after entrapment of these nucleic acids during the preparation of vesicles. Our data show that the complete export of only poly(A)-containing RNA from the vesicles is enhanced in the presence of 2 mM ATP. This RNA, as well as poly(A), is transported unidirectionally.

Animals↗
PubMed1986

Small nuclear RNAs from Saccharomyces cerevisiae: unexpected diversity in abundance, size, and molecular complexity.

Previous work showed that the simple eukaryote Saccharomyces cerevisiae contains a group of RNAs with the general structural properties predicted for small nuclear RNAs (snRNAs), including possession of the characteristic trimethylguanosine 5'-terminal cap. It was also demonstrated that, unlike their metazoan counterparts, the yeast snRNAs are present in low abundance (200-500 molecules per haploid cell). We have now used antibody directed against the 5' cap to investigate the total set size of snRNAs in this organism. We present evidence that the number of distinct yeast snRNAs is on the order of several dozen, that the length of the capped RNAs can exceed 1000 nucleotides, and that the relative abundance of a subset of these RNAs is 1/5th to 1/20th that of the class of snRNAs described previously. These findings suggest that the six highly abundant species of snRNAs (U1-U6) typically reported in metazoans may represent a serious underestimation of the total diversity of snRNAs in eukaryotes.

Cloning, Molecular↗