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Biomedical subjects

M Gassmann

Publications and source records attributed to M Gassmann.

At least 19 recordsLinked to original sources
PubMed2006

Formation of Neu/ErbB2-induced mammary tumors is unaffected by loss of ErbB4.

The four members of the ErbB family of receptor tyrosine kinases are involved in development and tumorigenesis of the mammary gland. Whereas the epidermal growth factor receptor, ErbB2 and ErbB3 are positively associated with various cancers, clinical studies of ErbB4 in breast cancer are contradictory. Results from tissue culture analyses and some clinical studies suggested that ErbB4 is either a tumor suppressor or is a negative regulator of ErbB2-driven tumors. Neu-Cre-ErbB4(flox/null) mice in which ErbB4 was inactivated by Cre-lox-mediated recombination in the mammary gland developed MMTV-Neu-driven mammary tumors with a similar latency period to mice with one or two wild-type ErbB4 alleles. Moreover, there was no difference in the histologies of tumors that developed, nor in the propensity to form lung metastases. Taken together these results suggest that ErbB4 is not a potent, highly penetrant tumor suppressor, nor is it a factor in Neu-mediated tumorigenesis in this model.

Animals↗
PubMed2005

Na-K-ATPase in rat cerebellar granule cells is redox sensitive.

Redox-induced regulation of the Na-K-ATPase was studied in dispersed rat cerebellar granule cells. Intracellular thiol redox state was modulated using glutathione (GSH)-conjugating agents and membrane-permeable ethyl ester of GSH (et-GSH) and Na-K-ATPase transport and hydrolytic activity monitored as a function of intracellular reduced thiol concentration. Depletion of cytosolic and mitochondrial GSH pools caused an increase in free radical production in mitochondria and rapid ATP deprivation with a subsequent decrease in transport but not hydrolytic activity of the Na-K-ATPase. Selective conjugation of cytosolic GSH did not affect free radical production and Na-K-ATPase function. Unexpectedly, overloading of cerebellar granule cells with GSH triggered global free radical burst originating most probably from GSH autooxidation. The latter was not followed by ATP depletion but resulted in suppression of active K(+) influx and a modest increase in mortality. Suppression of transport activity of the Na-K-ATPase was observed in granule cells exposed to both permeable et-GSH and impermeable GSH, with inhibitory effects of external and cytosolic GSH being additive. The obtained data indicate that redox state is a potent regulator of the Na-K-ATPase function. Shifts from an "optimal redox potential range" to higher or lower levels cause suppression of the Na-K pump activity.

Adenosine Triphosphate↗
PubMed2005

Chronic excessive erythrocytosis induces endothelial activation and damage in mouse brain.

Excessive erythrocytosis results in severely increased blood viscosity, which may have significant detrimental effects on endothelial cells and, ultimately, function of the vascular endothelium. Because blood-brain barrier stability is crucial for normal physiological function, we used our previously characterized erythropoietin-overexpressing transgenic (tg6) mouse line (which has a hematocrit of 0.8-0.9) to investigate the effect of excessive erythrocytosis on vessel number, structure, and integrity in vivo. These mice have abnormally high levels of nitric oxide (NO), a potent proinflammatory molecule, suggesting altered vascular permeability and function. In this study, we observed that brain vessel density of tg6 mice was significantly reduced (16%) and vessel diameter was significantly increased (15%) compared with wild-type mice. Although no significant increases in vascular permeability under normoxic or acute hypoxic conditions (8% O2 for 4 h) were detected, electron-microscopic analysis revealed altered morphological characteristics of the tg6 endothelium. Tg6 brain vascular endothelial cells appeared to be activated, with increased luminal protrusions reminiscent of ongoing inflammatory processes. Consistent with this observation, we detected increased levels of intercellular adhesion molecule-1 and von Willebrand factor, markers of endothelial activation and damage, in brain tissue. We propose that chronic excessive erythrocytosis and sustained high hematocrit cause endothelial damage, which may, ultimately, increase susceptibility to vascular disease.

Animals↗
PubMed2005

Neuroprotection by hypoxic preconditioning: HIF-1 and erythropoietin protect from retinal degeneration.

Hypoxic exposure of cells or organisms induces expression of a number of hypoxia responsive genes through the activation of the hypoxia-inducible factor-1 (HIF-1). One of the most prominent HIF-1 targets is erythropoietin that has beneficial effects on ischemia-related injury in the brain. Exposure to low environmental oxygen concentrations can be used as a preconditioning paradigm to protect cells or tissues against a variety of harmful conditions. Here, we summarize recent work on neuroprotection of retinal photoreceptors and ganglion cells induced by hypoxic preconditioning or by systemically elevated levels of Epo in mouse plasma.

Animals↗
PubMed2005

Hypoxic responses of Na+/K+ ATPase in trout hepatocytes.

Reduction in oxygenation induces inhibition of Na+/K+ ATPase in a number of cells and tissues, including hepatocytes. When not reversed, decrease in Na+/K+ pump activity leads to a gradual Na+ accumulation, cell swelling and death. However, when accompanied by suppression of dissipative cation pathways, it has also been shown to be a beneficial adaptive strategy used by some hypoxia-tolerant species to reduce ATP consumption during prolonged periods of anoxia. This study aims to investigate acute hypoxic responses of the Na+/K+ ATPase in primary cultures of trout hepatocytes. Gradual decrease in oxygenation was followed by an instantaneous transient dose-dependent downregulation of the Na+/K+ ATPase transport activity, but was without an effect on hydrolytic function of the enzyme. Hypoxia-induced inhibition of active K+ influx was reversed spontaneously when hypoxic incubation time exceeded 20 min. The stimulating effect of prolonged hypoxic exposure on the Na+/K+ pump is most probably secondary to hypoxia-induced activation of the Na+/H+ exchanger with the following Na+ accumulation leading to Na+/K+ ATPase activation. Hypoxia-induced inhibition of the Na+/K+ pump was not caused by ATP depletion or global oxidative stress. However, local controlled production of reactive oxygen species seems to play an important role in hypoxia-induced regulation of the Na+/K+ ATPase. Treatment of cells with mercaptopropionyl glycine (MPG), a scavenger of OH*-, abolished hypoxia-induced inhibition of the Na+/K+ ATPase. Earlier on we have shown that activation of Na+/H+ exchanger under hypoxic conditions can be opposed by MPG treatment as well. Taken together our results suggest that regulation of both oxygen-sensitive transporters may be accomplished by local changes in free radical production.

Adenosine Triphosphate↗
PubMed2003

Pivotal role of reduced glutathione in oxygen-induced regulation of the Na(+)/K(+) pump in mouse erythrocyte membranes.

This study addresses the mechanisms of oxygen-induced regulation of ion transport pathways in mouse erythrocyte, specifically focusing on the role of cellular redox state and ATP levels. Mouse erythrocytes possess Na(+)/K(+) pump, K(+)-Cl(-) and Na(+)-K(+)-2Cl(-) cotransporters that have been shown to be potential targets of oxygen. The activity of neither cotransporter changed in response to hypoxia-reoxygenation. In contrast, the Na(+)/K(+) pump responded to hypoxic treatment with reversible inhibition. Hypoxia-induced inhibition was abolished in Na(+)-loaded cells, revealing no effect of O(2) on the maximal operation rate of the pump. Notably, the inhibitory effect of hypoxia was not followed by changes in cellular ATP levels. Hypoxic exposure did, however, lead to a rapid increase in cellular glutathione (GSH) levels. Decreasing GSH to normoxic levels under hypoxic conditions abolished hypoxia-induced inhibition of the pump. Furthermore, GSH added to the incubation medium was able to mimic hypoxia-induced inhibition. Taken together these data suggest a pivotal role of intracellular GSH in oxygen-induced modulation of the Na(+)/K(+) pump activity.

Adaptation, Physiological↗
PubMed2003

Oxygen-regulated expression of TGF-beta 3, a growth factor involved in trophoblast differentiation.

The transforming growth factor-beta 3 (TGF-beta 3) is involved in oxygen-dependent differentiation processes during placental development and pregnancy disorders. However, the importance of oxygen partial pressure for the regulation of TGF-beta 3 expression is presently unclear. We and others presented preliminary evidence that the hypoxia-inducible factor-1 (HIF-1) confers TGF-beta 3 transcription but it was unknown whether this occurred directly or indirectly. To analyze how HIF-1 regulates TGF-beta 3 gene transcription, we cloned and sequenced the mouse TGF-beta 3 promoter region. Multiple putative HIF-1 binding sites (HBSs) were identified, many of which co-localized with two G+C rich CpG islands 5' to the TGF-beta 3 transcription start site. A 6.8 kb fragment of the TGF-beta 3 promoter induced reporter gene expression under hypoxic conditions or when treated with an iron chelator known to stabilize and activate the HIF-1 alpha subunit. Deletion of a 2.4 kb fragment upstream of the distal CpG island abolished inducibility of reporter gene expression. Two HBSs (HBS1 and HBS6) that bound the HIF-1 protein could be identified within this 2.4 kb fragment. These results suggest that TGF-beta 3 gene expression is directly regulated by HIF-1.

Animals↗
PubMed2002

Expression of ErbB3, ErbB4, and neuregulin-1 mRNA during tooth development.

The receptor tyrosine kinases ErbB3 and ErbB4, which bind to various variants of neuregulin (NRG), play fundamental roles in neural development and in organs, which form through epithelial-mesenchymal interactions. Here, we demonstrate that NRG-1 and the receptors ErbB3 and ErbB4 are expressed locally during rodent tooth development. However, the mRNA expression patterns of ErbB3 and ErbB4 were distinctly different during odontogenesis. Examinations of teeth in genetically heart-rescued ErbB4-/- mice did not reveal any obvious deviation from the normal phenotype. The results suggest that ErbB3 and ErbB4 may participate in tooth morphogenesis. The specific interactions between NRG isoforms and ErbB receptors during this process remain to be determined.

Animals↗
PubMed2002

Generation and application of chicken egg-yolk antibodies.

Despite the fact that the use of chicken as immunization host brings many advantages to the production of polyclonal antibodies, the generation of egg yolk immunoglobulins (IgY) is rarely chosen. In this review, we report on the fast and efficient method for generation and affinity purification of IgY, in this case raised against the alpha-subunit of hypoxia-inducible factor-1 (HIF-1). The IgY antibody was successfully applied in a variety of methods and a number of different species for HIF-1alpha detection. In electrophoretic mobility shift assays, the IgY antibody recognized the native HIF-1 complex. The IgY antibody also detected HIF-1alpha protein on Western blots with extracts derived from human, monkey, pig, dog and mouse cell lines grown under hypoxic conditions. Immunofluorescence and immunoprecipitation experiments using the IgY antibody allowed detection and subcellular localization of HIF-1alpha in the nuclei of hypoxic cells. Chicken antibody production brings great benefit concerning the welfare of the immunized animals, due to non-invasive antibody harvesting with the added convenience of simple egg collection. An additional advantage is the fast and simple IgY isolation from egg yolk. IgY technology is a great improvement and should be considered as a good alternative to conventional polyclonal antibody production in mammals.

Animals↗
PubMed2001

Mammalian PASKIN, a PAS-serine/threonine kinase related to bacterial oxygen sensors.

The PAS domain is a versatile protein fold found in many archaeal, bacterial, and plant proteins capable of sensing environmental changes in light intensity, oxygen concentration, and redox potentials. The oxygen sensor FixL from Rhizobium species contains a heme-bearing PAS domain and a histidine kinase domain that couples sensing to signaling. We identified a novel mammalian PAS protein (PASKIN) containing a domain architecture resembling FixL. PASKIN is encoded by an evolutionarily conserved single-copy gene which is ubiquitously expressed. The human PASKIN and mouse Paskin genes show a conserved intron-exon structure and share their promoter regions with another ubiquitously expressed gene that encodes a regulator of protein phosphatase-1. The 144-kDa PASKIN protein contains a PAS region homologous to the FixL PAS domain and a serine/threonine kinase domain which might be involved in signaling. Thus, PASKIN is likely to function as a mammalian PAS sensor protein.

Amino Acid Sequence↗
PubMed2001

Erythropoietin expression in primary rat Sertoli and peritubular myoid cells.

Kidney and liver are the major organs of erythropoietin (Epo) synthesis. However, Epo messenger RNA (mRNA) has been detected in several organs, such as brain, lung, and testis. Furthermore, functional Epo receptors have been demonstrated on different cell types, including rat Leydig cells. The aim of the study was to identify testicular cells expressing Epo mRNA and to quantitate its levels by competitive reverse transcriptase-polymerase chain reaction (RT-PCR). Besides whole testis, Epo transcripts were found in Sertoli and peritubular myoid cells, while no signal was detected in Leydig cells. Exposure of Sertoli cells to CoCl(2) led to an increase of Epo mRNA level. Semiquantitative competitive RT-PCR presented an increase in the level of Epo mRNA in Sertoli cells stimulated by follicle-stimulating hormone, while exposure of peritubular myoid cells cultures to testosterone reduced Epo mRNA expression. Due to the blood-testis barrier, basal expression of Epo suggests a not yet defined function of this hormone in testis.

Animals↗
PubMed2001

Dissecting hypoxia-dependent and hypoxia-independent steps in the HIF-1alpha activation cascade: implications for HIF-1alpha gene therapy.

The heterodimeric hypoxia-inducible factor (HIF)-1 is a master transcriptional regulator of oxygen homeostasis and a possible target for gene therapy of ischemic disease. Although the role of oxygen concentration in HIF-1a protein stabilization is well established, it is less clear whether and how oxygen-regulated mechanisms contribute to HIF-1a protein modifications, nuclear translocation, heterodimerization with the b-subunit, recruitment of cofactors, and gene trans-activation. Because the HIF-1a protein is proteolytically degraded under normoxic conditions, we established two HeLa Tet-Off cell lines (HT42 and HT43), which inducibly overexpress high levels of HIF-1a under normoxic conditions, allowing to distinguish hypoxia-dependent from hypoxia-independent activation mechanisms. Using these cells, we found that normoxically induced HIF-1a is localized to the nucleus, binds DNA, and trans-activates reporter and endogenous target genes. The levels of p53 expression remained unaffected. The MAP kinase inhibitor PD98059 attenuated HIF-1a protein modifications and trans-activation ability but not protein stabilization and DNA-binding activity. Because overexpressed HIF-1a is fully localized to the nucleus but displays only partial DNA-binding and trans-activation activity, mitogen-activated protein kinase-dependent phosphorylation might be required for full HIF-1 activation. HIF-1a protein was also overexpressed in vivo, following the transplantation of HT42 cells into nude mice, demonstrating the feasibility of HIF-1a gene transfer.

Animals↗
PubMed2001

Characterization of a hypoxia-inducible factor (HIF-1alpha ) from rainbow trout. Accumulation of protein occurs at normal venous oxygen tension.

The mammalian hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that controls the induction of several genes involved in glycolysis, erythropoiesis, and angiogenesis when cells are exposed to hypoxic conditions. Until now, the expression and function of HIF-1alpha have not been studied in fish, which experience wide fluctuations of oxygen tensions in their natural environment. Using electrophoretic mobility shift assay, we have ascertained that a hypoxia-inducible factor is present in rainbow trout cells. We have also cloned the full-length cDNA (3605 base pairs) of the HIF-1alpha from rainbow trout with a predicted protein sequence of 766 amino acids that showed a 61% similarity to human and mouse HIF-1alpha. Polyclonal antibodies against the N-terminal part (amino acids 12-363) and the C-terminal part (amino acids 330-730) of rainbow trout HIF-1alpha protein recognized rainbow trout and chinook salmon HIF-1alpha protein in Western blot analysis. Also, the human and mouse HIF-1alpha proteins were recognized by the N-terminal rainbow trout anti-HIF-1alpha antibody but not by the C-terminal HIF-1alpha antibody. The accumulation of HIF-1alpha was studied by incubating rainbow trout and chinook salmon cells at different oxygen concentrations from 20 to 0.2% O(2) for 1 h. The greatest accumulation of HIF-1alpha protein occurred at 5% O(2) (38 torr), a typical oxygen tension of venous blood in normoxic animals. The protein stability experiments in the absence or presence of a proteasome inhibitor, MG-132, demonstrated that the inhibitor is able to stabilize the protein, which normally is degraded via the proteasome pathway both in normoxia and hypoxia. Notably, the hypoxia response element of oxygen-dependent degradation domain is identical in mammalian, Xenopus, and rainbow trout HIF-1alpha proteins, suggesting a high degree of evolutionary conservation in degradation of HIF-1alpha protein.

Amino Acid Sequence↗
PubMed2001

Chronic inborn erythrocytosis leads to cardiac dysfunction and premature death in mice overexpressing erythropoietin.

The most common cause of an increase of the hematocrit is secondary to elevated erythropoietin levels. Erythrocytosis is assumed to cause higher blood viscosity that could put the cardiovascular system at hemodynamic and rheological risks. Secondary erythrocytosis results from tissue hypoxia, and one can hardly define what cardiovascular consequences are caused by chronic erythrocytosis or hypoxia. Herein, a novel transgenic (tg) mouse line is characterized that is erythrocytotic because of chronic overexpression of the human erythropoietin gene. These mice grow up well, reaching a hematocrit of about 0.80 in adulthood. Blood volume of adult tg mice was markedly increased by 75%. Unexpectedly, blood pressure was not elevated and cardiac output was not decreased. Still, the adult tg mice showed features of cardiac dysfunction with increased heart weight. In vivo, high-frequency echocardiography revealed marked ventricular dilatation that was confirmed by histologic examination. Furthermore, by transmission electron microscopy, a prominent intracellular edema of the cardiomyocytes was seen. Exercise performance of the tg mice was dramatically reduced, unmasking the severity of their compromised cardiovascular function. In addition, life expectancy of the tg mice was significantly reduced to 7.4 months. Our findings suggest that severe erythrocytosis per se results in cardiac dysfunction and markedly reduced life span.

Age Factors↗
PubMed2001

Attenuation of HIF-1 DNA-binding activity limits hypoxia-inducible endothelin-1 expression.

Hypoxia-inducible factors (HIFs) locate to HIF-binding sites (HBSs) within the hypoxia-response elements (HREs) of oxygen-regulated genes. Whereas HIF-1alpha is expressed ubiquitously, HIF-2alpha is found primarily in the endothelium, similar to endothelin-1 (ET-1) and fms-like tyrosine kinase 1 (Flt-1), the expression of which is controlled by HREs. We identified an unique sequence alteration in both ET-1 and Flt-1 HBSs not found in other HIF-1 target genes, implying that these HBSs might cause binding of HIF-2 rather than HIF-1. However, electrophoretic mobility shift assays showed HIF-1 and HIF-2 DNA complex formation with the unique ET-1 HBS to be about equal. Both DNA-binding and hypoxic activation of reporter genes using the ET-1 HBS was decreased compared with transferrin and erythropoietin HBSs. The Flt-1 HBS was non-functional when assayed in isolation, suggesting that additional factors are required for hypoxic up-regulation via the reported Flt-1 HRE. Interestingly, HIF-1 activity could be restored fully by point-mutating the ET-1 (but not the Flt-1) HBS, suggesting that the wild-type ET-1 HBS attenuated the full hypoxic response known from other oxygen-regulated genes. Such a mechanism might serve to limit the expression of this potent vasoconstrictor in hypoxia.

Base Sequence↗
PubMed2001

Mammalian gene expression in hypoxic conditions.

Hypoxia induces gene expression of specific genes such as erythropoietin (Epo) and vascular endothelial growth factor (VEGF) that allow physiological adaptation to the environmental conditions at the cellular, local, and systemic levels. Reduced oxygenation is also a common precursor of many pathological processes, including coronary artery defects, ischemia, and malignant tumour formation. The hypoxia-inducible transcription factor HIF-1, a heterodimer consisting of the oxygen-regulated alpha-subunit and the constitutively expressed beta or ARNT-subunit, serves as a master regulator of oxygen-dependent gene expression. We observed that upon hypoxic exposure of HeLa cells in tonometer, accumulation of HIF-1alpha occurred within two minutes, while reoxygenation strongly reduced HIF-1alpha levels within four to eight minutes. Thus, hypoxia leads to a rapid cellular adaptation. In another line of investigation, we analysed the impact of hypoxia-independent overexpression of Epo in transgenic mice. Despite a hematocrit of about 80% the transgenic mice did not develop hypertension or thromboembolic complications.

Journal Article↗
PubMed2001

Hypoxia affects expression of circadian genes PER1 and CLOCK in mouse brain.

The key elements of circadian clockwork and oxygen homeostasis are the PAS protein family members PER and CLOCK and hypoxia-inducible factor 1alpha (HIF-1alpha). The PAS domain serves as an interface for protein-protein interactions. We asked whether a cross-talk exists between the PAS components of hypoxic and circadian pathways. We found several isoforms of PER1 protein that exhibit tissue-specific size differences. In the mouse brain, a predominantly nuclear 48 kDa isoform that followed a daily rhythm was observed. The 48 kDa form was found in the nuclear fractions derived from mouse liver, Swiss3T3 fibroblasts, and N2A neuroblastoma cells. In mouse kidney and human 293 kidney cells, a 55 kDa PER1 form was detected. CLOCK was observed as a predicted 100 kDa protein in rat-1 cells and in all analyzed mouse tissues including brain, liver, kidney, and spleen. In contrast to PER1, CLOCK protein expression was not rhythmic. Exposure to hypoxia led to increased PER1 and CLOCK protein levels in mice. Based on coimmunoprecipitation experiments that showed protein-protein interaction between PER1 and the alpha subunit of HIF-1, we suggest that these hypoxic effects may be modulated by HIF-1alpha.-Chilov, D., Hofer, T., Bauer, C., Wenger, R. H., Gassmann, M. Hypoxia affects expression of circadian genes PER1 and CLOCK in mouse brain.

3T3 Cells↗