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H Baumann

Publications and source records attributed to H Baumann.

At least 145 records · Page 8Linked to original sources
PubMed1991

Characterization and functional expression of a cDNA encoding egasyn (esterase-22): the endoplasmic reticulum-targeting protein of beta-glucuronidase.

Egasyn (esterase-22), a member of the nonspecific carboxylesterase multigene family (E.C. 3.1.1.1), is the endoplasmic reticulum (ER)-targeting protein of beta-glucuronidase. We utilized the polymerase chain reaction (PCR) in the eventual isolation of murine egasyn cDNAs. PCR primers were based upon: (1) partial amino acid sequences derived from egasyn peptides and (2) a conserved active site region shared by carboxylesterases. The amino acid sequence deduced from the PCR product matched that obtained from egasyn protein. This product was utilized as a probe to screen a cDNA library. Two cDNAs whose composite sequence encoded an open reading frame of 562 amino acids were isolated. A message size of 1700-2000 bp was revealed by RNA blot hybridization analysis. S1 nuclease protection analyses detected mRNA in liver, kidney, lung, and submandibular gland, but not in spleen, brain, and testes. Genetic mapping confirmed the location of an egasyn cDNA fragment in cluster 1 of the esterase region on chromosome 8. Transfection of COS cells with the 2022-bp cDNA resulted in the expression of esterase activity, which comigrated on native gels with liver esterase-22. The features of the deduced amino acid sequence of the egasyn cDNA are compared with previously characterized carboxylesterases and with other lumenal ER proteins.

Amino Acid Sequence↗
PubMed1991

Acute phase mediated change in glycosylation of rat alpha 1-acid glycoprotein in transgenic mice.

Transgenic mouse lines carrying the gene for rat alpha 1-acid glycoprotein (AGP) express the protein in the plasma at concentrations equal to or exceeding that of acute phase rats. Owing to the high basal level, these transgenic mice represent a unique experimental system for defining the largely unknown function of AGP. Since the carbohydrate moiety of AGP has been found to be changed during acute phase and the oligosaccharide structure to be important for immunomodulating activity of the protein, the rat AGP in transgenic mice was characterized by lectin-affinity immuno-electrophoresis. Unlike in the rat, the AGP in the transgenic mouse plasma consisted primarily of strongly concanavalin A-reactive forms. Acute phase mediated a several-fold increase in the total plasma concentration of AGP concomitant with a shift toward moderately concanavalin A-reactive forms. A similar change in concanavalin A-reactive forms was observed for the endogenous acute phase plasma protein haptoglobin. To define the role of inflammatory factors in AGP production, primary cultures of hepatocytes were prepared. In contrast to in vivo, the AGP recovered from tissue culture medium represented primarily the concanavalin A-non-reactive form. Treatment of the cells with recombinant human interleukin-1, interleukin-6 and dexamethasone stimulated the production of concanavalin A-reactive AGP forms. The data indicate that the glycosylation pattern of plasma-resident AGP is modulated by acute phase, but that the profile of AGP forms does not coincide with that secreted by hepatocytes in tissue culture. This finding demands an assessment of which of the possible glycosylated forms of AGP is functionally significant in vivo.

Animals↗
PubMed1991

NF-AB, a liver-specific and cytokine-inducible nuclear factor that interacts with the interleukin-1 response element of the rat alpha 1-acid glycoprotein gene.

The 142-bp cytokine response element of the rat alpha 1-acid glycoprotein (AGP) gene is a complex of several additively contributing regulatory sequences. By using deletions and point mutations, a minimal interleukin-1 (IL-1) response element was localized to the region from positions 1 to 36 within the 5'-most AB fragment of the cytokine response element. Two distinct sequence motifs were contained within this element, both of which were required to achieve full IL-1 response in rat and human hepatoma cells. This element showed a minor response to phorbol ester treatment only in human hepatoma cells. Southwestern (DNA-protein) blot analysis of nuclear proteins of rat liver and hepatoma cells revealed the presence of a heat-labile nuclear factor (NF-AB). NF-AB migrated as a basic protein with an apparent molecular mass of 37 kDa and bound specifically to the DNA sequence at positions 10 to 37 of the AB fragment. The NF-AB binding activity was detected neither in the cytoplasmic fraction of rat hepatoma cells nor in nuclear extracts from control or acute-phase rat kidney. The binding activity of NF-AB correlated with the transcriptional activity of the endogenous AGP gene in rat liver and hepatoma cells. Nuclear extract from human HepG2 cells showed a similar binding activity with an apparent molecular mass of 34.5 kDa. The human NF-AB binding activity was detectable only after 13 h of cytokine treatment and was not induced by phorbol ester. Tissue distribution, DNA sequence binding specificity, and kinetics of cytokine induction of NF-AB do not coincide with the characteristics of any other described factors that have been associated with cytokine regulation. Therefore, NF-AB is considered a new candidate involved in IL-1 regulation of the rat AGP gene.

Animals↗
PubMed1991

Effects of interleukin-6 and leukemia inhibitory factor on the acute phase response and DNA synthesis in cultured rat hepatocytes.

Human recombinant interleukin-6 (IL-6) and human recombinant leukemia inhibitory factor (LIF) similarly stimulate synthesis of typical acute-phase proteins in the primary rat hepatocyte cultures. LIF is, however, less effective in increasing uptake of alpha-aminoisobutyric acid than IL-6. Antiserum to human IL-6 abolishes induced protein synthesis and amino acid uptake elicited by hrIL-6 but has no effect on the acute-phase response of rat liver cells stimulated by LIF. Both IL-6 and LIF inhibit basal and epidermal growth factor-induced DNA synthesis in rat hepatocytes.

Acute-Phase Proteins↗
PubMed1990

Interaction of cytokine- and glucocorticoid-response elements of acute-phase plasma protein genes. Importance of glucocorticoid receptor level and cell type for regulation of the elements from rat alpha 1-acid glycoprotein and beta-fibrinogen genes.

Dexamethasone increased the transscriptional activity of several acute-phase plasma protein genes in cytokine-treated HepG2 cells, suggesting the presence of functional glucocorticoid receptors (GR). The level of GR was, however, insufficient for stimulation of transiently transfected gene constructs containing glucocorticoid-response elements (GRE). By complementation of HepG2 cells with a GR expression vector, a cell system was generated that allowed analysis of the interaction between GRE and cytokine-response elements of the rat alpha 1-acid glycoprotein gene and identification of the principal regulatory elements of the rat beta-fibrinogen gene. Although the expression of plasmid-derived GR mRNA was reduced by dexamethasone treatment, the concentration of GR was sufficient for full, short-term stimulation of GRE-containing vectors. By comparing the pattern of regulation of the cloned GRES in HepG2 and mouse L-cells, an equivalent, cell-type independent dexamethasone response was monitored for the alpha 1-acid glycoprotein element but a response limited to HepG2 cells was found for the beta-fibrinogen element. The data indicate that, although substantial differences exist in the organization and composition of the regulatory elements of the two genes, the overall function is, nevertheless, remarkably similar.

Acute-Phase Proteins↗
PubMed1990

Molecular cloning, characterization and functional expression of the rat liver interleukin 6 receptor.

A series of cDNA clones coding for the rat liver interleukin 6 receptor (IL6-R) were isolated from an acute-phase library. The identity of the clones was established (a) by DNA sequence analysis and comparison with the known human leukocyte IL6-R and (b) by demonstrating that the clones generated specific IL6 ligand binding activity and IL6-dependent regulation of acute-phase gene control elements after transfection into appropriate recipient cells. Two types of cDNA clones were obtained corresponding to two mRNA species of different length, both encoding an identical protein of 462 amino acid residues. The two prototype clones, pRIL6RC.21 and pRIL6RC.6 contained 3'-untranslated regions of 550 and 3100 nucleotides, respectively. The sequence motifs TTATTTAT and ATTTA associated with the regulation of mRNA stability and translation efficiency were present only in the longer mRNA species. The deduced amino acid sequence of the rat liver IL6-R was 53% identical with the human leukocyte IL6-R. Both receptors contained conserved structural features in their extracellular domains, including the signal peptide, a C2 domain characteristic of the immunoglobulin superfamily, and two domains shared among members of a family of cytokine and growth factor receptors. The strongly conserved intracellular portion of the rat liver IL6-R lacked recognizable signal transduction domains. The cDNA clones were used to demonstrate that rat liver IL6-R mRNA concentrations were increased 4.2-fold at 12 h after the induction of an experimental acute-phase response. Clone pRIL6R.21ex, but not clone pRIL6RC.6ex, generated specific IL6 ligand binding activity after transfection into human Jurkat cells that lack the endogenous IL6-R. By contrast, only pRIL6RC.6ex reconstituted a response of human Hep3B-2 and HepG2 hepatoma cells to mouse IL6. These human hepatoma cells were highly responsive to human IL6 but did not respond to physiologic concentrations of murine IL6. After cotransfection with pRIL6RC.6ex and plasmids containing the chloramphenicol acetyltransferase reporter gene under the control of IL6 response elements of acute-phase plasma protein genes, these cells showed a strong stimulation of the reporter gene by recombinant mouse IL6. Thus, both cell surface ligand binding activity and the complete IL6 signal cascade terminating in the transcriptional induction of IL6-dependent promoters were successfully reconstituted. Therefore, both IL6-R mRNA species code for functionally active receptor, depending on the target cell, but only the longer mRNA species coded for significant receptor levels in human hepatoma cell lines.

Amino Acid Sequence↗
PubMed1990

Transcriptional control of the rat alpha 1-acid glycoprotein gene.

Dexamethasone strongly stimulates the production of alpha 1-acid glycoprotein (AGP) in rat liver. The regulated expression of AGP in cultured liver cells has been variably ascribed to the activation of AGP gene transcription, to change in the nuclear processing of AGP gene transcripts, or to both. Treatment of HTC cells with dexamethasone proportionally enhanced the rate of AGP gene transcription and the concentration of AGP mRNA. To assess whether hormone treatment is indeed capable of affecting posttranscriptionally the level of rat AGP mRNA, a hormone-independent expression vector containing the rat AGP gene was transiently introduced into HepG2 cells. None of the hormone combinations known to modulate expression of the endogenous AGP influenced the steady-state concentration of mature mRNA that was derived from the transgene. Hence, the existence of a hormone-sensitive posttranscriptional regulatory pathway for rat AGP gene expression could not be substantiated.

Animals↗
PubMed1990

Molecular characterization and acute phase expression of the multiple Mus caroli alpha 1-acid glycoprotein (AGP) genes. Differences in glucocorticoid stimulation and regulatory elements between the rat and mouse AGP genes.

In this study, we have analyzed alpha 1-acid glycoprotein (AGP) production in the wild mouse strain Mus caroli to assess whether the stimulation of AGP by inflammatory mediators has been conserved during rodent evolution and to determine what DNA elements manifested the hormonal induction in mouse AGP gene sequences. To this end, we isolated the M. caroli AGP genes and characterized their expression. Southern blot hybridization of M. caroli genomic DNA revealed the existence of approximately eight genes per haploid genome, and eight distinct genes were identified from a M. caroli lambda genomic DNA library. Two actively transcribed and acute phase-regulated genes (AGP genes 1 and 8) were identified by sequence correlation with the two different cDNAs isolated from an acute phase liver cDNA library. Two-dimensional gel analysis of in vitro transcription and translation products from these two cDNAs displayed a pattern of protein precursors identical with that shown by in vitro translation of the endogenous AGP mRNA. A glucocorticoid-responsive element (GRE) in M. caroli AGP gene 8 was localized to a unique sequence distal to the relative position of the rat AGP gene GRE. The mouse region analogous to the rat GRE did not show glucocorticoid-mediated induction of an indicator gene although greater than 90% sequence similarity is maintained. GRE function in this mouse region was improved by introducing a point mutation that restores the rat AGP GRE consensus sequence, although the relative induction obtained was less than the wild-type rat AGP GRE.

Amino Acid Sequence↗
PubMed1990

Inducible and tissue-specific expression of rat alpha-1-acid glycoprotein in transgenic mice.

alpha-1-Acid glycoprotein (AGP), which is produced in the mammalian liver and secreted into the blood-stream, is regulated by steroid hormones and by mediators of the acute phase response. In vitro transfection studies have shown that the response to glucocorticoids requires a cis-acting regulatory element, termed the glucocorticoid response element, that is located within 120 bp of the transcriptional start-site of the gene; induction by the acute phase mediators requires a different element, termed the distal regulatory element (DRE), that is located about 5 kb upstream of the start-site. To determine if these elements function in vivo, we have produced and characterized transgenic mice containing rat AGP gene constructs with and without the DRE. Five transgenic lines were produced from a 9.5-kb genomic AGP containing 4.7 kb of the 5' flanking region; this construct lacks the DRE. Another transgenic line was derived from a 10.7-kb clone that contains 5.3 kb of 5' flanking sequences including the DRE. All transgenic mice produced high levels of immunologically detectable rat AGP in the circulation, comparable to or in excess of that found in normal rats. There were correspondingly high concentrations of rat AGP transcripts in the liver. Transgene expression in all lines was induced in response to dexamethasone and during acute inflammation resulting from LPS treatment. The DRE-containing transgene underwent a greater induction in response to LPS than to dexamethasone; the transgene lacking the DRE responded similarly to both treatments. In cultured primary hepatocytes, the DRE-containing transgene was induced by the acute phase cytokines IL-1 and IL-6, and by dexamethasone, administered individually or in combination; the transgene lacking the DRE responded only to dexamethasone, and was not affected by the peptide hormones. Together, these results provide in vivo evidence supporting the notion that a minimum of two upstream sequences are responsible for the inflammatory induction of rat AGP. One element, which is located within the smaller 9.7-kb restriction fragment, is responsive to glucocorticoids and is likely to be the glucocorticoid response element located close to the transcriptional start site. The other element, the DRE, is located much further upstream and is responsible for imparting responsiveness to the acute phase cytokines.

Animals↗
PubMed1990

Fermentation of bovine endothelial cells for preparation of endothelial cell-surface heparan sulphate.

The polysaccharide chains of a proteoheparan sulphate located on the endothelial cell surface are responsible for athrombogenicity of blood vessel walls. Mass cultivation of endothelial cells is the only way to isolate adequate amounts of this proteoheparan sulphate. In order to establish a method for fermentation of bovine endothelial cells, colonization of microcarriers, growth phase and cultivation of confluent carriers were optimized. The colonization process was varied relative to the number of beads, number of cells, total volume and kind of vessel. Two basal media were tested at different serum contents by growth assays. The same basal media without serum were supplemented with mitogen, bovine lipoprotein, insulin and transferrin and tested by activity assays on confluent cultures. The best method yields more than 80% of the cells on microcarriers. During the fermentation glucose and lactate concentrations were measured at constant perfusion rate and glucose consumption and lactate production were determined. Under optimized conditions we achieved a final cell titre of 4 x 10(9) cells/l and a calculated cell density of 7-9 x 10(4) cells/cm2 offered substrate surface. The minimal doubling time of the cell culture was about 18 h under optimized fermentation conditions. Removal of the core-protein by enzymatic digestion or beta-elimination releases the endothelial cell surface heparan sulphate.

Animals↗
PubMed1990

[Iniencephaly: prenatal and postnatal findings].

A 33-year-old para-3 was admitted in the 33rd week of gestation because of a suspected foetal anomaly. Ultrasound examination showed polyhydramnios, exaggerated cervico-thoracic lordosis and significant shortening of the spine because of a reduced number of vertebrae. The facial profile was flat, the foetal movements were rare and slow, and the extremities normal. A biopsy of the placenta revealed a normal female karyotype. Based on ultrasound examination, the diagnosis of iniencephaly was made. Because of the fatal prognosis of this malformation, labour was induced at 35 weeks of gestation. The patient delivered spontaneously. The infant died after 90 minutes. The postmortem examination confirmed the diagnosis of iniencephaly. Iniencephaly is a very rare malformation comprising a bone defect at the occiput, malformation of the cervical and thoracic vertebrae, spina bifida, and retroflexion of the head. The aetiology is not clear. 90% of the probands are female. The malformation is incompatible with survival after birth.

Abnormalities, Multiple↗
PubMed1990

Distinct regulation of the interleukin-1 and interleukin-6 response elements of the rat haptoglobin gene in rat and human hepatoma cells.

The transcription rate of the haptoglobin (Hp) gene is stimulated by interleukin-1 (IL-1), IL-6, and dexamethasone in rat hepatoma (H-35) cells. To identify the cis-acting regulatory elements responsive to these hormones, various lengths of 5' Hp gene-flanking regions, including the promoter, were inserted into chloramphenicol acetyltransferase gene expression vectors and transiently introduced into H-35 cells. The first 4 kb of 5' region mediated a severalfold increase in expression after treatment with IL-6 and dexamethasone. No response to IL-1 was detectable. When, however, upstream sequences were deleted to position -165 relative to the transcription start site, a significant stimulation by IL-1 was gained without appreciably affecting the IL-6 response. With the apparent removal of an inhibitory sequence, the promoter-proximal 165-bp region also displayed a severalfold enhanced response to the combination of dexamethasone, IL-1, and IL-6. The sequence from -165 to -147, termed the A-element, was found to be crucial for all hormone regulatory functions. Two copies of the A-element linked to a heterologous promoter responded to the three hormones, but to a lesser degree than in the Hp gene promoter context. The regulatory elements of the rat Hp gene were similarly active in human hepatoma cells. Optimal regulation by IL-6 in HepG2 cells was, however, independent of the A-element. The A-element functioned in these cells exclusively as an IL-1 response sequence. The results suggest that genomic sequences upstream of the rat Hp gene suppress the regulation by specific cytokines more prominently in transient expression assays than in the normal chromosomal context. Moreover, the functional comparison indicated that specific regulatory regions of the rat Hp gene do not function identically in different hepatic cell types.

Animals↗
PubMed1990

Highly conserved upstream regions of the alpha 1-antitrypsin gene in two mouse species govern liver-specific expression by different mechanisms.

alpha 1-Antitrypsin (AT), the major elastase inhibitor in mammalian serum, is produced primarily in the liver. We have characterized AT gene structure and expression in the mouse species Mus caroli, which expresses high levels of AT in the kidneys as well as in the liver. Analysis of cDNA and genomic clones showed that the AT gene in M. caroli exhibits high sequence homology (greater than 90%) to the gene in laboratory mice (M. domesticus) throughout the coding and 5'-flanking regions. Despite this extensive sequence conservation, the functional organization of cis-acting regulatory elements governing liver-specific expression is strikingly different between these species. Transient-transfection assays showed that the proximal region of the M. caroli promoter (i.e., between -120 and -2 relative to the transcriptional start site) is 10-fold more active than the analogous region of M. domesticus in driving the expression of an indicator gene in cultured liver cells. The increased activity of the proximal region of the M. caroli AT promoter appears to be the result of one or both of the two base substitutions at positions -46 and -48. The weak proximal promoter in M. domesticus is compensated for by the presence of upstream, liver-specific enhancers between -199 and -520; the analogous region in M. caroli is inactive. Thus, during the course of evolution, the modest 7% sequence divergence that has occurred between the 5'-flanking regions of the AT genes in these two species has generated distinct, yet equally effective, modes of hepatocyte-specific expression.

Amino Acid Sequence↗
PubMed1990

Structure, hormonal regulation, and identification of the interleukin-6- and dexamethasone-responsive element of the rat haptoglobin gene.

Hepatic expression of the haptoglobin (Hp) gene in mammalian species is stimulated severalfold during an acute-phase reaction. To identify the molecular mechanism responsible for this regulation, the single-copy rat Hp gene has been isolated. The genomic sequences showed a high degree of homology with the primate Hp gene. Activity of the rat Hp gene was increased in cultured liver cells by interleukin-1 (IL-1), IL-6, and glucocorticoids. The genomic Hp gene sequence spanning from -6500 to +6500, when transiently introduced into human hepatoma (HepG2) cells, directed IL-6- and dexamethasone-stimulated expression of rat Hp mRNA and protein. No response to IL-1 was detected, suggesting that the corresponding regulatory element(s) might lie outside of the tested gene sequences. An IL-6- and dexamethasone-responsive element has been localized to the promoter proximal region -146 to -55. Although the nucleotide sequences of this rat Hp gene region showed substantial divergence from that of the human gene, analysis of sequential 5' and 3' deletion constructs indicated an arrangement of functional IL-6 response elements in the rat Hp promoter sequence comparable to that of the human homolog. The magnitude of IL-6 regulation through the rat Hp gene promoter was severalfold lower than that of the human Hp gene. The reduced activity could be ascribed to a single-base difference in an otherwise conserved sequence corresponding to an active element in the human gene. The IL-6 response of the rat Hp element was improved severalfold by substituting that base with the human nucleotide.

Amino Acid Sequence↗
PubMed1990

The cytokine response element of the rat alpha 1-acid glycoprotein gene is a complex of several interacting regulatory sequences.

Expression of the rat alpha 1-acid glycoprotein gene is stimulated by interleukin-1 (IL-1) and interleukin-6 (IL-6) and is synergistically enhanced by the combination of the two. The distal regulatory element (DRE), a 142-base-pair (bp) sequence located 5 kilobase pairs upstream of the transcriptional start site, appears to be crucial for this cytokine response. The cytokine-specific regulatory sequences within the DRE have been identified by inserting individual DRE subregions, selected combinations of these, or a few linker mutated fragments into a plasmid containing an enhancerless simian virus 40 promoter linked to the chloramphenicol acetyltransferase gene. The regulatory activity was determined in transiently transfected human and rat hepatoma cells. The IL-1 response region was confined to the 5'-most 62 bp of the DRE, and its function seemed to depend on at least two separate components. The same region was also responsive to phorbol ester treatment. The IL-6 regulatory function was dependent on a 54-bp sequence located within the 3' half of the DRE. When the IL-1 response region was recombined with the IL-6 regulatory region of the DRE or with IL-6 response elements of other plasma protein genes, a strong cooperative action by IL-1 and IL-6 was achieved. The functional DRE sequences were recognized by nuclear proteins extracted from rat liver and hepatoma cells. However, no cytokine-inducible binding activity was detectable, which suggests that transcriptional regulation through the DRE might be controlled by posttranslational modification of constitutively bound trans-acting factors.

Animals↗
PubMed1990

Intravenous thrombolytic therapy with a combination of single-chain urokinase-type plasminogen activator and recombinant tissue-type plasminogen activator in acute myocardial infarction.

The effects of simultaneous intravenous infusions of 12 mg recombinant tissue-type plasminogen activator (rt-PA) over 30 minutes and 48 mg single-chain urokinase-type plasminogen activator (scuPA) over 40 minutes were studied in 38 patients with acute myocardial infarction. Coronary arterial patency was assessed angiographically 60 minutes and 90 minutes after initiation of treatment. Patency was achieved in 19 of 31 patients (61.3%) (95% confidence limits, 42-78%) at 60 minutes and in 27 of 33 patients (81.8%) (95% confidence limits, 65-93%) at 90 minutes. Nonspecific plasminogen activation was monitored by measuring relevant plasma parameters. At 60 minutes and 120 minutes, the fibrinogen concentration decreased slightly to 82.8 +/- 24.3% and 91.2 +/- 17.4% of the preinfusion level, and the plasminogen concentration to 66.3 +/- 15.2% and 65.3 +/- 13.4%, respectively. A greater consumption of alpha 2-antiplasmin was observed, which decreased to 30.7 +/- 22.8% and 32.2 +/- 21.2% of the preinfusion level at 60 and 120 minutes, respectively. No bleeding necessitating transfusion was observed. Two patients (5.3%) died during hospitalization. The findings suggest that the combined intravenous infusion of rt-PA and scuPA at appropriate doses induces highly effective coronary thrombolysis equal to the best results obtained with either rt-PA or scuPA alone. This efficacy is coupled with high specificity. Thus, the data support the potential use of combinations of rt-PA and scuPA in place of monotherapy.

Drug Therapy, Combination↗
PubMed1990

Uteroplacental blood flow velocity waveforms as a predictor of adverse fetal outcome and pregnancy-induced hypertension.

The resistance index (RI) of uteroplacental blood flow velocity waveforms (VWF) was investigated by continuous Doppler ultrasound in 286 nonselected pregnant women with singleton pregnancies at 18-21 weeks (n = 129) and at 31-33 weeks (n = 157) of gestation to evaluate the validity of this method as a screening procedure for abnormal pregnancy outcome (intrauterine growth retardation, pregnancy-induced hypertension and fetal asphyxia). The sensitivity of predicting pregnancy outcome was 26.6% at 18-21 weeks and 17.6% at 31-33 weeks of gestation. It was concluded that this method is too insensitive as a screening tool for the prediction of intrauterine growth retardation and pregnancy-induced hypertension in a low-risk nonselected pregnant population.

Blood Flow Velocity↗