Biomedical subjects

M Gassmann

Publications and source records attributed to M Gassmann.

At least 19 recordsLinked to original sources

PubMed1999

Erythropoietin modulates intracellular calcium in a human neuroblastoma cell line.

1. Recent investigations have shown that the glycoprotein erythropoietin (Epo) and its specific receptor (EpoR) are present in the mammalian brain including human, monkey and mouse. These findings suggest a local action of Epo in the nervous system. The aim of this study was to elucidate a possible functional interaction of Epo with neuronal cells. 2. To examine the influence of externally applied Epo on Ca2+ homeostasis the human neuroblastoma cell line SK-N-MC was chosen as a suitable in vitro model for undifferentiated neuronal cells. 3. Expression of the EpoR in SK-N-MC cells was detected by reverse transcription-PCR, Western blot and immunofluorescence analysis. 4. Patch-clamp studies of SK-N-MC cells confirmed the expression of T-type Ca2+ channels, whose peak macroscopic current was increased by the addition of recombinant human Epo (rhEpo) to the bathing medium. 5. Confocal laser scanning microscopy analysis of SK-N-MC cells confirmed a transient increase in intracellular free [Ca2+] in response to externally applied rhEpo. 6. The transient response to Epo was dependent on external Ca2+ and remained even after depletion of internal Ca2+ stores by caffeine or thapsigargin. However, after depletion the response to Epo was absent when cells were superfused with the T-type Ca2+ channel blocker flunarizine. 7. This study demonstrates that Epo can interact with neuronal cells by affecting Ca2+ homeostasis through an increase in Ca2+ influx via plasma membrane T-type voltage-dependent Ca2+ channels.

Blotting, Western↗

PubMed1999

General applicability of chicken egg yolk antibodies: the performance of IgY immunoglobulins raised against the hypoxia-inducible factor 1alpha.

Avian embryos and neonates acquire passive immunity by transferring maternal immunoglobulins from serum to egg yolk. Despite being a convenient source of antibodies, egg yolk immunoglobulins (IgY) from immunized hens have so far received scant attention in research. Here we report the generation and rapid isolation of IgY from the egg yolk of hens immunized against the alpha subunit of the human hypoxia-inducible factor 1 (HIF-1alpha). Anti-HIF-1alpha IgY antibodies were affinity purified and tested for their performance in various applications. Abundant HIF-1alpha protein was detected by Western blot analysis in nuclear extracts derived from hypoxic cells of human, mouse, monkey, swine, and dog origin whereas in hypoxic quail and frog cells, the HIF-1alpha signal was weak or absent, respectively. In electrophoretic mobility shift assays, affinity-purified IgY antibody was shown to recognize the native HIF-1 (but not the related HIF-2) complex that specifically binds an oligonucleotide containing the HIF-1 DNA binding site. Furthermore, IgY antibody immunoprecipitated HIF-1alpha from hypoxic cell extracts. Immunofluorescence experiments using IgY antibody allowed the detection of HIF-1alpha in the nucleus of hypoxic COS-7 cells. For comparison, the application of a mouse monoclonal antibody raised against the identical HIF-1alpha fragment was more restricted. Because chicken housing is inexpensive, egg collection is noninvasive, isolation and affinity purification of IgY antibodies are fast and simple, and the applicability of IgY is widespread, immunization of hens represents an excellent alternative for the generation of polyclonal antibodies.

Animals↗

PubMed1999

Oxygen tension modulates beta-globin switching in embryoid bodies.

Little is known about the factors influencing the hemoglobin switch in vertebrates during development. Inasmuch as the mammalian conceptus is exposed to changing oxygen tensions in utero, we examined the effect of different oxygen concentrations on beta-globin switching. We used an in vitro model of mouse embryogenesis based on the differentiation of blastocyst-derived embryonic stem cells to embryoid bodies (EBs). Cultivation of EBs at increasing oxygen concentrations (starting at 1% O2) did not influence the temporal expression pattern of embryonic (betaH1) globin compared to the normoxic controls (20% O2). In contrast, when compared to normoxically grown EBs, expression of fetal/adult (betamaj) globin in EBs cultured at varying oxygen concentrations was delayed by about 2 days and persisted throughout differentiation. Quantitation of hemoglobin in EBs using a 2,7-diaminofluorene-based colorimetric assay revealed the appearence of hemoglobin in two waves, an early and a late one. This observation was verified by spectrophotometric analysis of hemoglobin within single EBs. These two waves might reflect the switch of erythropoiesis from yolk sac to fetal liver. Reduced oxygenation is known to activate the hypoxia-inducible factor-1 (HIF-1), which in turn specifically induces expression of a variety of genes among them erythropoietin (EPO). Although EBs increased EPO expression upon hypoxic exposure, the altered beta-globin appearance was not related to EPO levels as determined in EBs overexpressing EPO. Since mRNA from both mouse HIF-1alpha isoforms was detected in all EBs tested at different differentiation stages, we propose that HIF-1 modulates beta-globin expression during development.

Aerobiosis↗

PubMed1999

Induction and nuclear translocation of hypoxia-inducible factor-1 (HIF-1): heterodimerization with ARNT is not necessary for nuclear accumulation of HIF-1alpha.

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of mammalian oxygen homeostasis. HIF-1 consists of two subunits, HIF-1alpha and the aryl hydrocarbon receptor nuclear translocator (ARNT). Whereas hypoxia prevents proteasomal degradation of HIF-1alpha, ARNT expression is thought to be oxygen-independent. We and others previously showed that ARNT is indispensable for HIF-1 DNA-binding and transactivation function. Here, we have used ARNT-mutant mouse hepatoma and embryonic stem cells to examine the requirement of ARNT for accumulation and nuclear translocation of HIF-1alpha in hypoxia. As shown by immunofluorescence, HIF-1alpha accumulation in the nucleus of hypoxic cells was independent of the presence of ARNT, suggesting that nuclear translocation is intrinsic to HIF-1alpha. Co-immunoprecipitation of HIF-1alpha together with ARNT could be performed in nuclear extracts but not in cytosolic fractions, implying that formation of the HIF-1 complex occurs in the nucleus. A proteasome inhibitor and a thiol-reducing agent could mimic hypoxia by inducing HIF-1alpha in the nucleus, indicating that escape from proteolytic degradation is sufficient for accumulation and nuclear translocation of HIF-1alpha. During biochemical separation, both HIF-1alpha and ARNT tend to leak from the nuclei in the absence of either subunit, suggesting that heterodimerization is required for stable association within the nuclear compartment. Nuclear stabilization of the heterodimer might also explain the hypoxically increased total cellular ARNT levels observed in some of the cell lines examined.

Animals↗

PubMed1998

Up-regulation of hypoxia-inducible factor-1alpha is not sufficient for hypoxic/anoxic p53 induction.

Oxygen-deprived regions of a solid tumor can induce tumor suppressor p53 expression and hence select for p53-mutant tumor cells with diminished apoptotic potential. It has been proposed that the hypoxia-inducible factor-1 (HIF-1) alpha subunit binds to p53 and protects it from proteasomal degradation. However, we found that hypoxic conditions that strongly induce HIF-1-dependent endogenous gene expression as well as HIF-1alpha protein neither induce p53-dependent gene expression nor p53 protein. The iron chelator deferoxamine induced both HIF-1alpha and p53, but p53 up-regulation could still be detected in HIF-1alpha-deficient cells, suggesting that mechanisms other than HIF-1alpha activation contribute to oxygen-regulated p53 induction.

Animals↗

PubMed1998

Oxygen-regulated erythropoietin gene expression is dependent on a CpG methylation-free hypoxia-inducible factor-1 DNA-binding site.

The hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator involved in the expression of oxygen-regulated genes such as that for erythropoietin. Following exposure to low oxygen partial pressure (hypoxia), HIF-1 binds to an hypoxia-response element located 3' to the erythropoietin gene and confers activation of erythropoietin expression. The conserved core HIF-1 binding site (HBS) of the erythropoietin 3' enhancer (CGTG) contains a CpG dinucleotide known to be a potential target of cytosine methylation. We found that methylation of the HBS abolishes HIF-1 DNA binding as well as hypoxic reporter gene activation, suggesting that a methylation-free HBS is mandatory for HIF-1 function. The in vivo methylation pattern of the erythropoietin 3' HBS in various human cell lines and mouse organs was assessed by genomic Southern blotting using a methylation-sensitive restriction enzyme. Whereas this site was essentially methylation-free in the erythropoietin-producing cell line Hep3B, a direct correlation between erythropoietin protein expression and the degree of erythropoietin 3' HBS methylation was found in different HepG2 sublines. However, the finding that this site is partially methylation-free in human cell lines and mouse tissues that do not express erythropoietin suggests that there might be a general selective pressure to keep this site methylation-free, independent of erythropoietin expression.

Animals↗

PubMed1998

Mouse hypoxia-inducible factor-1alpha is encoded by two different mRNA isoforms: expression from a tissue-specific and a housekeeping-type promoter.

Hypoxic induction of erythropoietin (Epo) and other oxygen-dependent genes is mediated by the hypoxia-inducible factor-1 (HIF-1), a heterodimeric transactivator consisting of an alpha and a beta subunit. We previously found that the mouse gene encoding HIF-1alpha harbors two alternative first exons (I.1 and I.2), giving rise to two different HIF-1alpha mRNA isoforms. Here, we show by RNase protection analysis that the exon I.1-derived mRNA isoform is differentially expressed in mouse tissues, being highest in kidney, tongue, stomach, and testis, but undetectable in liver, whereas the exon I.2 mRNA isoform is ubiquitously expressed. Sequence and methylation analysis showed that, in contrast to exon I.1, exon I.2 resides within a region showing typical features of a CpG island, known to be associated with the 5' end of housekeeping genes. We identified a 232-bp minimal exon I.2 promoter that strongly induced reporter gene expression in mouse L929 fibroblasts and Hepa1 hepatoma cells. In contrast to L929 cells, the exon I.1 promoter was inactive in Hepa1 cells and hypoxic exposure (1% O2) markedly reduced exon I.2 promoter activity in Hepa1 cells. Prolonged exposure of mice to hypoxia (7.5% O2 for up to 72 hours) also caused a decrease in liver HIF-1alpha mRNA, whereas aldolase mRNA levels increased. These findings might be related to the relatively low Epo levels in the adult liver.

Alternative Splicing↗

PubMed1998

Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha.

Hypoxia is an essential developmental and physiological stimulus that plays a key role in the pathophysiology of cancer, heart attack, stroke, and other major causes of mortality. Hypoxia-inducible factor 1 (HIF-1) is the only known mammalian transcription factor expressed uniquely in response to physiologically relevant levels of hypoxia. We now report that in Hif1a-/- embryonic stem cells that did not express the O2-regulated HIF-1alpha subunit, levels of mRNAs encoding glucose transporters and glycolytic enzymes were reduced, and cellular proliferation was impaired. Vascular endothelial growth factor mRNA expression was also markedly decreased in hypoxic Hif1a-/- embryonic stem cells and cystic embryoid bodies. Complete deficiency of HIF-1alpha resulted in developmental arrest and lethality by E11 of Hif1a-/- embryos that manifested neural tube defects, cardiovascular malformations, and marked cell death within the cephalic mesenchyme. In Hif1a+/+ embryos, HIF-1alpha expression increased between E8.5 and E9.5, coincident with the onset of developmental defects and cell death in Hif1a-/- embryos. These results demonstrate that HIF-1alpha is a master regulator of cellular and developmental O2 homeostasis.

Animals↗

PubMed1998

Fetuses from preeclamptic mothers show reduced hepatic erythropoiesis.

The fetal liver is the main hematopoietic organ during intrauterine life. Morphometrical studies were performed on liver sections to detect changes occurring with intrauterine growth retardation and preeclampsia. Compared with the controls (n = 10), fetuses from preeclamptic mothers showed a severe reduction of erythroid cells by 60% on average (n = 18). Closer examination revealed that the erythroid cells at early stages of differentiation were more affected (80% reduction) than at later stages (55%). Seven out of 18 fetuses from preeclamptic mothers did not show growth retardation but exhibited severely reduced hepatic erythropoiesis. We suggest that the prime factor for impaired red blood cell production is preeclampsia itself rather than intrauterine growth retardation. Regulation of erythropoiesis in utero might depend on the interaction of many hematopoietic growth factors, and preeclampsia might alter the balance. To test this notion, we quantitated erythropoietin in fetal blood and various cytokines in the amniotic fluid. An elevation of erythropoietin and interleukin (IL)-3 levels was seen in babies born under the conditions of preeclampsia, whereas the concentrations of granulocyte/macrophage-colony-stimulating factor (CSF), granulocyte-CSF, and IL-1 beta were reduced, and the levels of IL-6 and IL-8 remained constant. With preeclampsia, a discrepancy between elevation of erythrocyte numbers in peripheral blood and depression of hematopoiesis at the main production site, the fetal liver, is seen. Concomitantly, there is elevation of some but reduction of other hematopoietic cytokines. We envision that during the course of preeclampsia quantitation of hematopoietic growth factors might allow to predict the deterioration of in utero life conditions.

Amniotic Fluid↗

PubMed1998

Optimal erythropoietin expression in human hepatoma cell lines requires activation of multiple signalling pathways.

Hypoxia is thought to be a common precursor of coronary artery disease and malignant tumors, both diseases representing the leading causes of death in industrial nations. So far, investigations of oxygen-regulated erythropoietin (EPO) gene expression in the human hepatoma cell lines Hep3B and HepG2 allowed many important insights into the mechanisms of oxygen-sensing, signalling and regulation of an increasing number of oxygen-responsive genes. To differentiate the various signalling pathways involved in EPO production by these two cell lines, we examined several factors that positively influenced EPO expression. The results demonstrate a keen differential effect of cell density and oxygen concentration on EPO induction in Hep3B compared to HepG2 cells. Using optimized cell culture conditions, EPO production rates as high as 1 U EPO per 10(6) Hep3B cells in 24 h could be achieved. We also found a moderate but reproducible positive effect of CoCl2 on hypoxia-induced EPO expression in Hep3B but a negative CoCl2 effect on hypoxic induction in HepG2 cells. CoCl2 inhibited cell growth in a concentration-dependent manner. Interleukin-6 was synergistic with hypoxia on EPO induction in Hep3B as well as HepG2 cells, and dexamethasone enhanced this effect in Hep3B but not in HepG2 cells. The moderate CoCl2-dependent increase of EPO production observed in hypoxic Hep3B cells might indicate that CoCl2 and hypoxia do not necessarily act via, identical signalling pathways.

Carcinoma, Hepatocellular↗

PubMed1998

[Transgenic mice in basic research].

What are transgenic mice and what do we learn from them? In this review, we focus on the generation of "classical" transgenic and "knock-out" mice. The establishment of transgenic and gene-targeted mice provides an unique tool to study the function(s) of a given gene in the context of a whole organism. Based on selected examples, we demonstrate the potential of this transgenic technology to understand the interactions between cells, organs and organ systems in genetically engineered mice.

Alzheimer Disease↗

PubMed1997

Oxygen-regulated transferrin expression is mediated by hypoxia-inducible factor-1.

Transferrin (Tf) is a liver-derived iron transport protein whose plasma concentration increases following exposure to hypoxia. Here, we present a cell culture model capable of expressing Tf mRNA in an oxygen-dependent manner. A 4-kilobase pair Tf promoter/enhancer fragment as well as the 300-base pair liver-specific Tf enhancer alone conveyed hypoxia responsiveness to a heterologous reporter gene construct in hepatoma but not HeLa cells. Within this enhancer, a 32-base pair hypoxia-responsive element was identified, which contained two hypoxia-inducible factor-1 (HIF-1) binding sites (HBSs). Mutation analysis showed that both HBSs function as oxygen-regulated enhancers in Tf-expressing as well as in non-Tf-expressing cell lines. Mutation of both HBSs was necessary to completely abolish hypoxic reporter gene activation. Transient co-expression of the two HIF-1 subunits HIF-1alpha and aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF-1beta resulted in enhanced reporter gene expression even under normoxic conditions. Overexpression of a dominant-negative ARNT/HIF-1beta mutant reduced hypoxic activation. DNA binding studies using nuclear extracts from the mouse hepatoma cell line Hepa1 and the ARNT/HIF-1beta-deficient subline Hepa1C4, as well as antibodies raised against HIF-1alpha and ARNT/HIF-1beta confirmed that HIF-1 binds the Tf HBSs. Mutation analysis and competition experiments suggested that the 5' HBS was more efficient in binding HIF-1 than the 3' HBS. Finally, hypoxic induction of endogenous Tf mRNA was abrogated in Hepa1C4 cells, confirming that HIF-1 confers oxygen regulation of Tf gene expression by binding to the two HBSs present in the Tf enhancer.

Animals↗

PubMed1997

Neuregulin-2, a new ligand of ErbB3/ErbB4-receptor tyrosine kinases.

The neuregulins (NRGs) are a family of multipotent epidermal-growth-factor-like (EGF-like) factors that arise from splice variants of a single gene. They influence the growth, differentiation, survival and fate of several cell types. We have now discovered a set of new neuregulin-like growth factors, which we call neuregulin-2 (NRG-2): these are encoded by their own gene and exhibit a distinct expression pattern in adult brain and developing heart. Like NRG-1, the EGF-like domain of the new ligands binds to both the ErbB3- and ErbB4-receptor tyrosine kinases. However, NRG-2 stimulates different ErbB-receptor tyrosine-phosphorylation profiles from NRG-1. Our results indicate that NRG-1 and NRG-2 mediate distinct biological processes by acting at different sites in tissues and eliciting different biochemical responses in cells.

Amino Acid Sequence↗

PubMed1997

The mouse gene for hypoxia-inducible factor-1alpha--genomic organization, expression and characterization of an alternative first exon and 5' flanking sequence.

The ubiquitously expressed hypoxia-inducible factor-1 (HIF-1) is involved in expression of a large number of oxygen-regulated genes. HIF-1 is a heterodimer consisting of an alpha and a beta subunit, both belonging to the basic-helix-loop-helix Per-aryl hydrocarbon receptor nuclear translocator-Sim (PAS) family of transcription factors. Whereas HIF-1alpha is a novel member of this family, HIF-1beta is identical to the aryl hydrocarbon receptor nuclear translocator, previously recognized to be involved in xenobiotic metabolism. cDNA cloning revealed that mouse HIF-1alpha can be expressed as two mRNA isoforms containing alternative 5' untranslated regions and two different predicted translational start sites. We cloned and characterized 20.5 kb of the mouse HIF-1alpha gene (Hif1a) containing exon II-XV. The two alternative first exons, I.1 and I.2, are separated from exon II by approximately 24 kb and 17 kb, respectively. We also sequenced Hif1a exon I.1 and flanking regions, and mapped a single exon I.1 transcription initiation site. Reverse transcription PCR analysis of total RNA derived from normoxic and hypoxic mouse hepatoma and fibroblast cell lines suggested that the two alternative mRNA isoforms are constitutively coexpressed in these cells, and that two different promoters drive transcription of HIF-1alpha. A minimal exon I.1 promoter was identified which moderately activated heterologous gene expression, indicating that additional cis-elements are required for efficient HIF-1alpha transcription in vivo.

Animals↗

PubMed1997

Adjuvant therapy of pancreatic cancer using monoclonal antibodies and immune response modifiers.

Pancreatic cancer is a devastating disease with poor survival. At present, no effective adjuvant or palliative therapies are available. Unresponsiveness to chemotherapy, radiotherapy, and antihormonal treatment is one of the reasons that pancreatic cancer patients have an overall median survival time of 4-6 mo. This article summarizes clinical trials on immunotherapy of pancreatic cancer using the murine monoclonal antibodies (MAbs) 17-1A and BW 494. In addition, the use of MAb treatment in combination with immune response modifiers is discussed. In four clinical trials, MAb 17-1A was given by iv infusion to 100 patients with pancreatic cancer. In 30 of the these patents, antibody treatment was accompanied by gamma-interferon, also given intravenously. Complete response, partial response, and stable disease were reported in 1,5, and 23 patients, respectively. Passive immunotherapy using the MAb BW 494 was carried out in 148 pancreatic cancer patients in two phase I and two phase II trials. In 1 out of 75 patients a partial response and in 25 out of 74 patients stable disease were reported. However, in a controlled randomized trial enrolling 61 patients following Whipple resection, comparable survival times in patients with and without MAb BW 494 treatment led to the termination of further clinical trials with this antibody. New clinical studies using humanized MAbs in combination with immune response modifiers should be initiated to further evaluate immunotherapy as a treatment option in pancreatic cancer.

Antibodies, Monoclonal↗

PubMed1997

Neuregulins and neuregulin receptors in neural development.

The neuregulins are a family of closely related proteins that play important roles in neural and cardiac development, as well as in mammary carcinogenesis. The pleiotropic activities of these molecules are transduced by a set of receptor protein tyrosine kinases that exhibit structural similarity to the receptor for epidermal growth factor. Recent results have demonstrated essential roles for the neuregulins and their receptors in regulating cell number, determining cell fate, and establishing pattern in the developing central and peripheral nervous systems.

Animals↗

PubMed1997

Detection of erythropoietin in human liquor: intrinsic erythropoietin production in the brain.

Until now, erythropoietin (EPO) was thought to be produced exclusively in fetal liver and adult kidney and to regulate mammalian erythropoiesis. However, we recently showed that steady state levels of EPO mRNA could be induced up to 100-fold in primary mouse astrocytes cultured under hypoxic conditions, and also reported the presence of mRNA for EPO and its receptor in the brain of mouse, monkey and human. In extending these studies on humans we now show that immunoreactive EPO is present in ventricular cerebrospinal fluid (CSF) of 5 patients with traumatic brain injuries: EPO was found in 15 out of 15 CSF samples. There was no correlation between the serum EPO concentration and the concentration in the CSF. However, EPO concentrations in CSF correlated with the degree of blood-brain-barrier dysfunction. This suggests that EPO does not cross the intact blood-brain-barrier, implying that EPO is produced in the brain itself, most probably by astrocytes in an oxygen-dependent manner. In view that neuronal cells carry the EPO receptor, we propose that EPO acts in a paracrine fashion in the central nervous system and might function as a protective factor against hypoxia-induced damage of neurons.

Adult↗

PubMed1997

Hypoxia-inducible factor-1 alpha is regulated at the post-mRNA level.

The hypoxia-inducible factor-1 (HIF-1) is involved in the induction of oxygen regulated genes such as erythropoietin and vascular endothelial growth factor (VEGF). HIF-1 is a heterodimeric transcription factor consisting of an alpha and a beta subunit. The question of how HIF-1 itself is regulated remains to be elucidated. Studies performed in human Hep3B hepatoma cells suggested that the prevalent mode of HIF-1 action is an increase in HIF-1 alpha steady-state mRNA and protein levels after hypoxic exposure. In contrast to the reported very low basal HIF-1 alpha mRNA levels, however, we detected HIF-1 alpha mRNA in several cell lines cultured under normoxic conditions, although no HIF-1 DNA binding activity was observed. Following hypoxic induction, VEGF mRNA levels and HIF-1 DNA binding activity increased, but HIF-1 alpha mRNA levels remained largely unchanged. One possible explanation for this discrepancy might be that HIF-1 DNA binding activity does not follow HIF-1 alpha mRNA kinetics. We therefore incubated HeLaS3 cells in tonometers for 7.5 minutes up to four hours at either 20% O2 or 0.5% O2. Although there was some variation in HIF-1 alpha mRNA levels, we did not find significant changes over this time frame, suggesting that HIF-1 alpha is not transcriptionally regulated.

Animals↗