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W Northemann

Publications and source records attributed to W Northemann.

At least 55 records · Page 3Linked to original sources
PubMed1989

Structure and negative transcriptional regulation by glucocorticoids of the acute-phase rat alpha 1-inhibitor III gene.

DNA clones representing the negative acute-phase gene coding for the plasma proteinase inhibitor alpha 1-inhibitor III were isolated from a rat genomic library. Structural analysis established the existence of at least four different members of the alpha 1-inhibitor III gene family. Partial DNA sequence analysis of the 5'-terminal regions was performed for the alpha 1-inhibitor III gene and the related alpha 1-inhibitor IV gene. The transcription start site of the alpha 1-inhibitor III gene was located by S1 mapping and primer extension. No stable alpha 1-inhibitor IV mRNA was detected in rat liver. In an experimentally induced acute-phase reaction, the transcription rate of the alpha 1-inhibitor III gene was reduced 12.7-fold at 6 h after stimulation. Four hours after injection of a high dose of dexamethasone into rats, the transcription rate of this gene was reduced 9-fold. Thus, glucocorticoids alone are capable of causing a strong transient down-regulation of the transcription of this gene in rats, independent of other inflammatory mediators. An inverted consensus glucocorticoid responsive element (5'GGACACAATAT3') shared with the glucocorticoid-regulated alpha 1-fetoprotein, alpha 2u-globulin, and alpha 1-acid glycoprotein genes was detected by computer-assisted sequence analysis in the promoter proximal 5'-flanking region of the alpha 1-inhibitor III gene.

Acute-Phase Proteins↗
PubMed1988

Structure and acute-phase regulation of the rat alpha 2-macroglobulin gene.

Seven genomic DNA clones representing the rat alpha 2-macroglobulin gene were isolated and characterized. The cloned sequence covered the entire gene (48 kilobases) plus 2 kilobases of 3'- and 13.7 kilobases of 5'-flanking sequences. A restriction cleavage map of the gene was produced, and the restriction cleavage pattern of genomic DNA suggested that the alpha 2-macroglobulin gene is a single-copy gene. A 7.7-kilobase fragment from the 5'-terminal region and a 250 base pair fragment from the 3'-terminal region of the gene were sequenced, and the 3' end of the gene was mapped. The sequenced 5'-terminal fragment contained 4.5 kilobases of 5'-flanking sequences plus the first three exons and two introns of the gene. Two transcription start sites, a minor and a major site, located 65 nucleotides apart, were defined by primer extension, S1 mapping, and RNaseH experiments. During an acute-phase response, transcription from both sites was induced in the liver, and over 90% of the transcripts originated from the major site. Very high concentrations of alpha 2-macroglobulin mRNA originating from both start sites were also found in the uterus but not in the liver of pregnant females. A glucocorticoid response element (GRE), a conserved consensus sequence for a potential glucocorticoid receptor DNA binding site, was found by computer search in the promoter-proximal 5'-flanking region of the alpha 2-macroglobulin gene.

Animals↗
PubMed1988

Sequence and acute phase regulation of rat alpha 1-inhibitor III messenger RNA.

cDNA clones coding for the plasma proteinase inhibitor alpha 1-inhibitor III were isolated from an acute phase rat liver library. The isolates could be divided into four groups with characteristic BamHI restriction fragment patterns. The identity of the prototype clone pRLA1I3/2J was established by comparison with the published amino acid sequence of the purified protein. It codes for a 1477-amino acid precursor polypeptide with a 24-residue signal peptide. The mature protein shares 58% overall sequence identity with rat alpha 2-macroglobulin and contains a typical internal thiolester sequence. Twenty-two of its twenty-three cysteinyl residues are conserved with alpha 2-macroglobulin implying similar tertiary structure. However, the prototype alpha 1-inhibitor III sequence differed significantly from the rat and human alpha 2-macroglobulin sequences in its bait region suggesting alpha 1-inhibitor III possesses proteinase inhibitory specificities different from those of alpha 2-macroglobulin. The variant alpha 1-inhibitor III clone pRLA1I3/2J from a second cDNA group also differed from the prototype in the bait region coding sequence, although both specify similar signal peptides and NH2 termini. The observation of variant cDNA classes suggests that acute phase rat livers produce a heterogeneous mixture of alpha 1-inhibitor III mRNA molecules. Evidence was obtained for the presence of at least four different alpha 1-inhibitor III-related genes in the rat genome. During the first 24 h of an acute phase response the abundance of hepatic alpha 1-inhibitor III mRNA was decreased 3-4-fold. This decrease was of the same order of magnitude as the reported reduction of the corresponding plasma protein concentration, suggesting that in the early phase of the acute inflammatory response the plasma concentration of this protein is mainly controlled through the abundance of its hepatic mRNA.

Acute-Phase Proteins↗
PubMed1987

Modified nuclear processing of alpha 1-acid glycoprotein RNA during inflammation.

Rat alpha 1-acid glycoprotein is an acute phase reactant which shows a marked elevation in mRNA level following inflammatory induction. It has been proposed that both transcriptional and post-transcriptional mechanisms regulate the induction of the gene. We have studied the processing of the primary transcript of alpha 1-acid glycoprotein. The preferred pathway of intron removal was determined by Northern blot analysis and was found to be unaltered after inflammatory stimulation. The final nuclear precursor did exhibit size alterations, manifest as a quantitative shift from the final precursor at 6 h to a second progressively shorter form at 18 and 24 h. Deadenylation of nuclear RNA showed that the difference in size of the precursor is due to a change in poly(A) tail length, which occurs after the splicing out of the last intron. Nuclear run-on transcription assays measured a 2.5-fold increase in transcriptional activity, with a peak at 12 h. The highest level of cytoplasmic RNA with a long poly(A) tail, however, occurs before 12 h. Our data suggest that the reduction in poly(A) tail size is due to a rapid trimming of the tail in the nucleus and that this process is modified upon inflammatory induction.

Animals↗
PubMed1987

Sequence of rat liver alpha 2-macroglobulin and acute phase control of its messenger RNA.

Six alpha 2-macroglobulin cDNA clones were isolated from two liver cDNA libraries produced from rats undergoing acute inflammation. The coding sequence for rat alpha 2-macroglobulin including its 27-residue signal peptide and the 3' - and part of the 5' nontranslated regions were determined. The mature protein consisting of 1445 amino acids is coded for by a 4790 +/- 40 nucleotide messenger RNA. It contains a typical internal thiol ester region and 25 cysteine residues which are conserved between rat and human alpha 2-macroglobulin. Although the amino acid sequences of rat and human alpha 2-macroglobulin share 73% identity, two small divergent areas of 17 and 38 residues were found, corresponding to 29 and 11% identity, respectively. These areas are located in the bait region and, therefore, may confer specific proteinase recognition capabilities on rat alpha 2-macroglobulin. Following an inflammatory stimulation, rat alpha 2-macroglobulin mRNA levels increased 214-fold over control values and reached a maximum at 18 h. By 24 h the levels had decreased to less than 30% of the maximum value. Transcription rates from the alpha 2-macroglobulin gene as measured in nuclear run-on experiments showed a less than 3-fold increase in nuclei from acutely inflamed rats as compared to controls. These results suggest that the accummulation of alpha 2M mRNA is due to the combined effects of increased transcription rates and post-transcriptional processing.

Amino Acid Sequence↗
PubMed1986

Alpha 2-macroglobulin gene expression during rat development studied by in situ hybridization.

The sites of alpha 2-macroglobulin mRNA synthesis during rat development have been localized by in situ hybridization using a rat alpha 2-macroglobulin cDNA probe. Fetal liver was found to be the major site of alpha 2-macroglobulin mRNA synthesis. In addition, alpha 2-macroglobulin mRNA was detected in brain, spinal cord and eye. Alpha 2-Macroglobulin mRNA was quantitated by use of a sensitive RNAse protection assay. Maximal levels of alpha 2-macroglobulin mRNA were found in fetal livers shortly before birth. A rapid decline of alpha 2-macroglobulin mRNA occurred within 1 day after parturition. A similar time course, although at an approximately 20-fold lower level, was observed for alpha 2-macroglobulin mRNA in livers of pregnant rats. Alpha 2-Macroglobulin mRNA could also be detected in placenta. The levels were comparable to those found in maternal livers.

Aging↗
PubMed1986

Levels of messenger ribonucleic acids for plasma proteins in rat liver during acute experimental inflammation.

The levels of mRNA for plasma proteins and for metallothionein in rat liver during the acute-phase response were studied by hybridization to specific cDNA probes. The mRNA for alpha 2-macroglobulin, the beta-chain of fibrinogen, alpha 1-acid glycoprotein (so-called acute-phase reactants) reached a maximum level between 18 and 36 h after inducing an acute inflammation. The level of mRNA for metallothionein-I peaked earlier, after 12 h. The mRNA for transferrin showed a delayed increase with a broad maximum for its relative level after 36-60 h. The mRNA levels for albumin and alpha 2u-globulin (so-called negative acute-phase reactants) decreased, reaching a minimum of 25% of the normal level after 36 h (albumin) and after 72 h (alpha 2u-globulin). The ratios of the rates of incorporation of leucine into the proteins over the levels of their mRNA in liver changed only a little, indicating that the rates of synthesis of plasma proteins in the liver are regulated at the mRNA level during the acute-phase response to inflammation.

Animals↗
PubMed1985

Molecular cloning of cDNA sequences for rat alpha 2-macroglobulin and measurement of its transcription during experimental inflammation.

Poly(A)+ RNA enriched in alpha 2-macroglobulin (alpha 2M) mRNA isolated from livers of rats 18 h after injection of turpentine was used for the synthesis of double-stranded cDNA. The double-stranded cDNA was inserted into the PstI site of the plasmid pBR322 by oligo(dG)-oligo(dC)-tailing technique. Clones containing sequences complementary to alpha 2M mRNA were selected by differential colony hybridization using 32P-labeled poly(A)+ RNA and [32P]cDNA from livers of control and turpentine-treated rats and subsequent hybrid-selected translation. The isolated p alpha 2M1 clone had an insert of 657 base pairs. DNA sequence analysis revealed a homology of about 80% to human alpha 2M. Northern analysis showed that the alpha 2M mRNA from rat liver is about 5600 bases in length. The alpha 2M cDNA was used to measure the in vitro transcription of the alpha 2M gene in isolated nuclei. A 4-fold increase in alpha 2M gene activity was found 14 h after turpentine administration. We conclude that alpha 2M transcription is induced during inflammation.

Amino Acid Sequence↗
PubMed1984

Induction of rat alpha 2-macroglobulin in vivo and in hepatocyte primary cultures: synergistic action of glucocorticoids and a Kupffer cell-derived factor.

Turpentine injection into rats elicits enhanced secretion of acute phase proteins including alpha 2-macroglobulin (alpha 2M). Hypophysectomized rats, however, do not respond in this way unless dexamethasone is given together with turpentine. On the other hand, dexamethasone injection alone did not result in an induction of alpha 2M synthesis. When a medium of Kupffer cell cultures was added to hepatocytes, a dose-dependent stimulation of alpha 2M synthesis of up to 4-fold after 10-12 h was observed. However, the presence of low concentrations (10(-9)M) of dexamethasone was essential for the stimulatory effect. We conclude that the acute phase induction of alpha 2M in hepatocytes requires the synergistic action of glucocorticoids and a non-dialysable factor secreted by Kupffer cells.

Animals↗
PubMed1983

Cell-free synthesis of rat alpha 2-macroglobulin and induction of its mRNA during experimental inflammation.

Poly(A)-rich RNA was isolated from the livers of acutely inflamed rats by extraction with guanidinium HCl and oligo(dT)-cellulose chromatography. After translation in a recticulocyte lysate and immunoprecipitation with a specific antiserum to alpha 2-macroglobulin a polypeptide with an apparent molecular weight of 162000 could be detected. The cell-free synthesis of alpha 2-macroglobulin was stimulated 8-fold by the addition of RNase inhibitor. Full-length alpha 2-macroglobulin polypeptide chains appeared after 35 min in the presence of 1.85 mM Mg2+ and 100 mM K+. A nucleotide number of about 5100 was estimated for alpha 2-macroglobulin by means of sucrose gradient centrifugation of poly(A)-rich RNA followed by translation in vitro and immunoprecipitation of alpha 2-macroglobulin. In normal liver alpha 2-macroglobulin mRNA represented about 0.0007% of total translatable RNA. Acute inflammation generated by intramuscular injection of turpentine led to a 66-fold increase in translatable alpha 2-macroglobulin mRNA after 18 h, followed by a rapid decrease. In accordance to the induction of alpha 2-macroglobulin mRNA serum concentrations of alpha 2-macroglobulin increased to about 2 mg/ml. Unlike alpha 2-macroglobulin mRNA serum alpha 2-macroglobulin levels remained unchanged up to 60 h.

Animals↗
PubMed1983

Messenger RNA activities of four acute phase proteins during inflammation.

Poly(A)+ RNA isolated from the livers of normal rats and of rats suffering from an acute inflammation was translated in a cell-free translation system from rabbit reticulocytes. The translation products were immunoprecipitated with specific antisera against alpha 1-acid glycoprotein, alpha 2-macroglobulin, transferrin, alpha 1-proteinase inhibitor and albumin. 15 to 21 h after intramuscular injection of turpentine 73-, 66-, 2.8-, and 2-fold increases in translatable mRNAs for alpha 1-acid glycoprotein, alpha 2-macroglobulin, transferrin and alpha 1-proteinase inhibitor, respectively, were observed. For albumin a decrease in translatable mRNA to about 30% of controls was measured.

Animals↗
PubMed1983

Synthesis of rat-liver lactate dehydrogenase and characterization of its mRNA.

Rat liver lactate dehydrogenase (LDH) has been synthesized in hepatocytes and in a cell-free translation system. The subunit synthesized in both systems displayed the same electrophoretic mobility upon sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis. Sequence analysis of the subunits translated in vitro and synthesized in vivo indicated N-acetyl-alanyl-alanine for both N termini. Thus, the newly synthesized subunit does not exhibit an amino-terminal extension. The mRNA for the lactate dehydrogenase subunit was exclusively found in free polysomes. A size of 2120 +/- 240 nucleotides was estimated for the mRNA. Only about 50% of these nucleotides are needed to code for the polypeptide chain of the enzyme.

Amino Acid Sequence↗
PubMed1983

Small cytoplasmic RNAs from rat liver mRNP particles. Studies on their structure and function.

Free cytoplasmic 40S mRNP particles from rat liver were treated with EDTA and separated into two populations of RNP particles with sedimentation maxima of 20S and 35S, respectively. A characteristic set of distinct scRNAs is found for 20S and 35S RNP particles. The sequences of two of the most abundant scRNAs from 20S RNP particles with chain lengths of 104 (alpha 1-RNA) and 124 (beta 1-RNA) nucleotides, respectively, are presented. alpha 1-RNA shows a high sequence homology to the 3'-end of 18S rRNA. Since alpha 1-RNA carries a cap, it cannot be a degradation product of 18S rRNA. The beta 1-RNA is strongly post-transcriptionally modified, but uncapped. When the individual scRNAs of 20S and 35S RNP particles isolated from preparative polyacrylamide gels were assayed for their capability to inhibit in vitro protein synthesis, several potent translational inhibitory RNAs were detected. Particularly, the scRNAs of 147,203 and 263 nucleotide length associated with the 35S RNP particles turned out to be strong inhibitors of protein synthesis.

Animals↗
PubMed1982

Cell-free synthesis of a larger-molecular-weight precursor of cytochrome c oxidase subunit V from rat liver and the distribution of its mRNA between free and membrane-bound polysomes.

Poly(A)-rich RNA from phenol-extracted rat liver polysomes was translated in a heterologous cell-free system derived from wheat germs. The labeled translation products were incubated with an antiserum against cytochrome c oxidase subunit V. After immunoprecipitation and affinity chromatography with protein-A-Sepharose, the isolated antigen-immunoglobulin complexes were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. Only one protein with an apparent molecular weight of 15 500 was visualized. In immunocompetition experiments with unlabeled individual cytochrome c oxidase subunits IV, V, VI or VII only subunit V could compete with the 15 500-Mr protein synthesized in vitro. Two-dimensional fingerprints of cytochrome c oxidase subunit V and the polypeptide synthesized in vitro showed a high degree of similarity. It is concluded that the cytochrome c oxidase subunit V is synthesized as a precursor with an amino-terminal extension of about 25 amino acids. It was possible to convert the precursor of cytochrome c oxidase subunit V synthesized in vitro to its mature form by intact mitochondria as well as by submitochondrial particles. A chain length of 830 +/- 70 nucleotides was estimated for the poly(A)-rich mRNA of the higher-molecular-weight precursor of rat liver cytochrome c oxidase subunit V. Assuming a molecular weight of 15 500 for the precursor a non-coding region of about 300 nucleotides must exist. In experiments on the site of synthesis it is shown that the poly(A)-rich RNA for the higher-molecular-weight precursor of cytochrome c oxidase subunit V is found in free, loosely and tightly membrane-bound polyribosomes.

Animals↗