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PubMed1991

Autoantibodies to nucleolin in systemic lupus erythematosus and other diseases.

The 110-kDa intracellular phosphoprotein (110K) described previously by this laboratory as a common IgM autoantigen in SLE and certain other systemic autoimmune disorders and viral infections is identified as nucleolin in the present investigation. Using rabbit antiserum to rat nucleolin as a probe, IgM autoantibody-reactive 110K co-migrated with human lymphocyte nucleolin in one- and two-dimensional immunoblots. Rabbit anti-nucleolin also specifically depleted autoreactive 110K from detergent lysates of human cells. Because nucleolin shares amino acid sequence similarity and/or forms dynamic particles with other prominent autoantigens, the present observation raises the possibility that the nucleolin/anti-nucleolin system may be of special significance for the development of humoral autoreactivity to nuclear Ag.

Antibody Specificity↗
PubMed1991

Increased stability of nucleolin in proliferating cells by inhibition of its self-cleaving activity.

Nucleolin is the major nucleolar phosphoprotein of exponentially growing eukaryotic cells and is presumably involved in pre-rRNA transcription and ribosome biogenesis. Monoclonal antibodies against nucleolin were selected by a differential dot-immunobinding assay. Nucleolin expression during T lymphocyte activation was monitored by the specific antibody. Results showed that nucleolin fluctuated in parallel to DNA synthesis. The intact 105-kDa nucleolin molecule was the major species in actively dividing cells, whereas the degraded forms were relatively abundant in nondividing cells. These results imply that stability of nucleolin molecule is cell proliferation-dependent. When affinity purified nucleolin containing undetectable contaminants was incubated at 37 degrees C, the majority of 105-kDa nucleolin was cleaved by 6 h and completely degraded within 24 h. This purified nucleolin was further separated from possible copurified protease, if any, on a reducing sodium dodecyl sulfate-polyacrylamide gel. After renaturation, the 105-kDa nucleolin immobilized in the gel was also cleaved at 37 degrees C. These data have confirmed that nucleolin protein autocatalyzes its own degradation. The self-cleaving activity of nucleolin was inhibited by nuclear extracts prepared from proliferating cells. Apparently, a proteolytic inhibitor(s) in the nuclei of proliferating cells stabilized the nucleolin molecule. It provides an unique regulatory mechanism for nucleolin expression.

Antibodies, Monoclonal↗
PubMed1991

Molecular cloning of matrin 3. A 125-kilodalton protein of the nuclear matrix contains an extensive acidic domain.

We report here the cloning and sequencing of matrin 3, an acidic internal matrix protein, from a rat insuloma cDNA library. The nucleotide sequence has a single open reading frame encoding a polypeptide of 845 amino acids. The Genbank and National Biomedical Research Foundation databases did not contain any sequences similar to that of matrin 3. The primary structure consists of 33% charged residues and is generally hydrophilic. The amino-terminal region (residues 1-120) is positively charged and contains a large number of amino acids with free hydroxyl groups (26 of the first 100 residues) as in the lamins and several non-lamin intermediate filament proteins. A highly acidic domain (approximately 170 amino acids) near the carboxyl terminus, in which 32% of the amino acid residues are acidic (Glu or Asp), is a characteristic found in other nuclear proteins (Earnshaw, W. C. (1987) J. Cell Biol. 105, 1479-1482). A putative nuclear targeting signal sequence (Ser-Lys-Lys-Lys-Leu-Lys-Lys-Val-Glu) is located in the middle of the highly acidic domain. The corresponding human deduced partial amino acid sequence is 96% identical to the rat sequence, indicating that matrin 3 is a highly conserved protein.

Amino Acid Sequence↗
PubMed1990

Identification of the CBP1 polypeptide in mitochondrial extracts from Saccharomyces cerevisiae.

CBP1 is a nuclearly encoded yeast protein required for stability of mitochondrial cytochrome b pre-mRNA. Previous studies have shown that CBP1 stabilizes the cytochrome b transcripts via interaction with the 5'-end. For the present study, both rabbit polyclonal and mouse monoclonal antibodies against CBP1 were prepared using a trpE-CBP1 fusion polypeptide as a source of antigen. CBP1 was undetectable in a crude mitochondrial fraction from a wild-type strain by Western blot assay, but a 66-kDa immunoreactive protein was detected in a more purified fraction. The 66-kDa protein was absent in the equivalent fraction from a strain with a deletion in CBP1. Assignment of Mr = 66,000 to the mature CBP1 polypeptide was verified by Western analysis of mitochondria from a strain which over-expresses CBP1. Mitochondrial localization was verified by transcribing CBP1 in vitro with T3 polymerase, translating the artificial mRNA in a rabbit reticulocyte system and importing 35S-CBP1 precursor polypeptides into isolated mitochondria. The mature protein product was 66 kDa, whereas the precursor protein migrated as if it were 68 kDa rather than 76 kDa as predicted from the sequence. Analysis of polypeptides truncated at the carboxyl terminus showed that CBP1 polypeptides migrate anomalously fast in the Laemmli system due to a property of the carboxyl two-thirds of the primary sequence, several sections of which are extremely basic.

Antigens, Fungal↗
PubMed1990

Cleavage of spermidine as the first step in deoxyhypusine synthesis. The role of NAD.

The biosynthesis of deoxyhypusine (N-(4-aminobutyl)lysine) occurs by the transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in a precursor of eukaryotic translation initiation factor 4D (eIF-4D). Deoxyhypusine synthase, the enzyme that catalyzes this reaction, was purified approximately 700-fold from rat testis. The Km values for the substrates, spermidine, the eIF-4-D precursor protein, and NAD+, were estimated as approximately 1, 0.08, and 30 microM, respectively. After incubation of partially purified enzyme with [1,8-3H]spermidine, NAD+, and the eIF-4D precursor, equal amounts of radioactivity were found in free 1,3-diaminopropane and in protein-bound deoxyhypusine. However, when the protein substrate (eIF-4D precursor) was omitted, radioactivity was found in 1,3-diaminopropane and in delta 1-pyrroline in nearly equal quantities, providing evidence that the cleavage of spermidine occurs, albeit at a slower rate, in the absence of the eIF-4D precursor. That NAD+, which is required for this reaction, functions as the hydrogen acceptor was demonstrated by the fact that radioactivity from spermidine labeled with 3H at position 5 is found in NADH as well as in delta 1-pyrroline. Transfer of this hydrogen from spermidine to the re face of the nicotinamide ring of NAD+, as determined by the use of dehydrogenases of known stereospecificity, defines the first step of deoxyhypusine synthesis as a pro-R, or A, stereospecific dehydrogenation. Based on these findings, an enzyme mechanism involving imine intermediate formation is proposed.

Animals↗
PubMed1990

Cell-type-specific splicing factors and the regulation of alternative RNA splicing.

A very wide variety of biological processes are regulated by alternative splicing. By this means, a gene can be transcribed in several different tissues but in each tissue the RNA transcript is spliced in a particular way to produce a different mRNA and hence a different protein. It is now clear that alternative splicing is regulated by factors which are expressed in a tissue-specific manner and which are necessary for the splicing events to occur. This review will discuss the evidence for the existence of these factors, their nature, and the mechanisms by which they regulate splicing by interacting with sequences in the RNA.

Animals↗
PubMed1990

Calyculin A, an inhibitor of protein phosphatases, a potent tumor promoter on CD-1 mouse skin.

Calyculin A, isolated from a marine sponge, has a novel spiro ketal skeleton. Structurally unrelated to okadaic acid, calyculin A bound to the okadaic acid receptors in particulate and soluble fractions of mouse skin. The biochemical and tumor-promoting activities of calyculin A were studied with those of okadaic acid. Calyculin A inhibited the activity of protein phosphatases, which serve as the okadaic acid receptors. The effective dose of calyculin A for 50% inhibition was 0.3 nM, similar to that of okadaic acid. Like okadaic acid, calyculin A induced ornithine decarboxylase in mouse skin and hyperphosphorylation of a Mr 60,000 protein in human papilloma virus type 16-transformed human keratinocytes. A two-stage carcinogenesis experiment on mouse skin, initiated by 100 micrograms (390 nmol) of 7,12-dimethylbenz(a)anthracene and followed by 1 microgram (1.0 nmol) of calyculin A, revealed that calyculin A is an additional member of the okadaic acid class of tumor promoters. The percentages of tumor-bearing mice in the groups treated with DMBA plus calyculin A, and with DMBA followed by 1 microgram (1.2 nmol) of okadaic acid were 86.7 and 80.0%, respectively, in week 30. The mechanisms of action of calyculin A and okadaic acid, in addition to dinophysistoxin-1 (35-methylokadaic acid), are discussed. Calyculin A is the first tumor promoter to be screened by the okadaic acid receptor binding test.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1990

Binding properties of avian retroviral proteins. I. Preparation and basic characterization of ASLV NC(p12) and MA(p19).

Using SP-Sephadex column chromatography we isolated from an avian retrovirus, AMV(MAV), nucleic acid-binding proteins ASLV NC(p12) and MA(p19). As shown by several criteria, namely SDS-PAGE, PR(p15) protease activity, and nucleic acid binding assay with the use of both ss and ds DNAs, our NC(p12) and MA(p19) isolates are virtually pure proteins mutually not cross-contaminated. Rabbit anti-NC(p12) and anti-MA(p19) sera which we prepared did not cross-react mutually. We conclude that both NC(p12) and MA(p19) and antibodies against them are adequately pure preparations for investigating their nucleic acid binding specificities towards AMV(MAV) genomic RNA and MAV-1 proviral DNA using electron microscopy supported by computer analysis of electron micrographs.

Animals↗
PubMed1990

Binding properties of avian retroviral proteins. II. Binding of protein ASLV NC(p12) to viral RNA and proviral DNA.

Binding of the major avian retroviral nucleocapsid protein ASLV NC(p12) to the MAV-1 (myeloblastosis-associated virus) proviral dsDNA and viral ssRNA was analysed using electron microscopy. Specificity of the binding was estimated by special computer programs. The NC(p12) protein bound to MAV-1 proviral dsDNA (clone pAT153--MAV-1), but specificity of this binding was not found by computer evaluation. NC(p12) also bound to nondenatured 70S viral RNA at a rate of 25 +/- 3 molecules per RNA molecule. When this RNA was denatured either before or after the complexing, it showed no binding affinity for the protein. This result implies that preserved secondary structure of the viral RNA was required for the binding.

Animals↗
PubMed1990

Modulation of rat Kupffer cells on high density lipoprotein receptors on hepatocytes.

The present study found that conditioned media from Kupffer cells preincubated with acetylated LDL or acetylated LDL and zymosan increased the number of HDL receptors on hepatocytes, using the method of conditioned media transfer. This indicated that the transferable factors produced by Kupffer cells modulate HDL receptors on hepatocytes.

Animals↗
PubMed1990

The ANB1 locus of Saccharomyces cerevisiae encodes the protein synthesis initiation factor eIF-4D.

The Saccharomyces cerevisiae anaerobic gene (ANB1) is negatively regulated both by oxygen and heme. We have shown recently that an upstream repressor site located in the 5'-flanking region of this gene controls its expression (Mehta, K.D., and Smith, M. (1989) J. Biol. Chem. 265, 8670-8675). In this paper, we present the complete genomic sequence of the ANB1 locus of S. cerevisiae. The ANB1 locus encodes a protein of 157 residues with an Mr of 17, 134. The deduced amino acid sequence of the ANB1 gene product shows strikingly extensive sequence and structural homology (63.5% identical residues and an additional 15% conservative substitutions) to the 154-amino-acid-long human and rabbit eukaryotic translation initiator factor (eIF)-4D. Factor eIF-4D is the only known mammalian protein that undergoes a unique post-translational modification of Lys-50 to the amino acid hypusine, and interestingly the same lysine is also present in the ANB1 gene product. Results presented provide strong evidence that the ANB1 locus that encodes a transcript, tr-2, and a second locus encoding a transcript, tr-1, together encode two forms of yeast eIF-4D. Interestingly, heme regulates both the loci in an opposite manner; as a result it can dictate the isoform available under conditions of high and low oxygen tension. The ROX1 locus of S. cerevisiae is known to regulate CYC1, COXVb, and ANB1 genes at the transcriptional level; the ROX1 locus thus regulates all known anaerobically expressed genes that are involved in different cellular functions such as respiration and protein synthesis.

Amino Acid Sequence↗
PubMed1990

Characterization of unr; a gene closely linked to N-ras.

The mammalian N-ras gene is believed to play a role in cellular proliferation, differentiation, and transformation. While investigating N-ras, we isolated cDNA's that originate from a closely linked upstream gene. RNase protection assays reveal that this gene, unr, is transcribed in the same direction as N-ras and that its 3' end is located just 130 base pairs away from the point at which N-ras transcription begins. The close spatial relationship between the two genes is conserved in all species from which the N-ras gene has been isolated. An open reading frame, potentially encoding a 798 amino acid protein, is contained within the unr cDNA. Neither the primary protein structure nor the nucleic acid sequence of unr is homologous to any other known gene, including N-ras. Unr transcripts are detected in mouse, rat and human cells, and Southern analysis indicates that the unr locus found immediately upstream of the N-ras gene is transcriptionaly active in the mouse since only a single copy of unr is detected in this species. Unr produces multiple transcripts that differ in their 3' ends and are apparently created through the differential use of multiple polyadenylation sites located in the 3' untranslated region of the gene. Both unr and N-ras are expressed in all tissues examined. In the testis, both genes are developmentally regulated, with an increase in expression occurring upon testicular maturation. Thus the two genes may be coordinately regulated, at least in certain circumstances. Our findings suggest that a thorough analysis of the relationship that exists between the two genes could potentially provide insights into the regulation and/or function of N-ras.

Amino Acid Sequence↗