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Karl-Heinz Krause

Publications and source records attributed to Karl-Heinz Krause.

At least 19 recordsLinked to original sources
PubMed2006

Expression and function of alpha-smooth muscle actin during embryonic-stem-cell-derived cardiomyocyte differentiation.

Three alpha-muscle actin isoforms are sequentially expressed during in vivo cardiac development. alpha-Smooth muscle actin is first and transiently expressed, followed by alpha-skeletal and finally alpha-cardiac actin. The significance of these transitions in actin gene expression during myogenesis remains to be determined. To understand whether actin isoforms have specific functions during cardiac development and cardiomyocyte contractility, we have hampered alpha-smooth muscle and alpha-skeletal actin expression and organization during embryonic stem cell differentiation towards cardiomyocyte. We show that the sequence of actin isoform expression displays similar pattern in the in vitro model and in mouse heart embryogenesis. Treatment with an interfering fusion peptide containing the N-terminal sequence of alpha-smooth muscle actin during a time window preceding spontaneous beating, prevents proper cardiac sarcomyogenesis, whereas alpha-skeletal actin-fusion peptide has no effect. Knockdown of alpha-smooth muscle actin in embryonic stem cells using RNA interference also affects cardiac differentiation. The application of both fusion peptides on beating embryoid bodies impairs frequency. These results suggest specific functional activities for actin isoforms in cardiogenesis and cardiomyocyte contractility.

Actins↗
PubMed2006

Aging: a revisited theory based on free radicals generated by NOX family NADPH oxidases.

Reactive oxygen species (ROS), often also referred to as free radicals, play an important role in aging. It is widely assumed that mitochondria are the predominant source of ROS relevant for the aging process. In this hypothesis article, we suggest that the role of ROS generated by NOX family NADPH oxidases has been largely overlooked in aging theories. NOX NADPH oxidases form a seven member gene family and are high level ROS-generating enzymes. As opposed to mitochondria, which generate ROS as a byproduct of their metabolism, NOX enzymes are professional ROS generators. From an experimental point of view, there is now abundant evidence for the involvement of NOX enzymes in age-associated diseases. The role of NOX enzymes in the aging process itself and their relative contribution as compared to mitochondria needs further investigations.

Aging↗
PubMed2006

Genetic engineering of embryonic stem cells.

We describe a novel generation of lentiviral vectors that are particularly well suited for work with embryonic stem cells. The possibility of selecting cell lines with antibiotics and the rapid insertion of any combination of promoters and genes of interest makes them a powerful tool in the generation of transgenic ES cell lines. This vector can also greatly facilitate studies aimed at the improvement of neuronal engineering from ES cells, by making it possible to monitor the emergence and differentiation of neurons.

Animals↗
PubMed2006

The NADPH oxidase NOX4 drives cardiac differentiation: Role in regulating cardiac transcription factors and MAP kinase activation.

Reactive oxygen species (ROS) generated by the NOX family of NADPH oxidases have been described to act as second messengers regulating cell growth and differentiation. However, such a function has hitherto not been convincingly demonstrated. We investigated the role of NOX-derived ROS in cardiac differentiation using mouse embryonic stem cells. ROS scavengers prevented the appearance of spontaneously beating cardiac cells within embryoid bodies. Down-regulation of NOX4, the major NOX isoform present during early stages of differentiation, suppressed cardiogenesis. This was rescued by a pulse of low concentrations of hydrogen peroxide 4 d before spontaneous beating appears. Mechanisms of ROS-dependent signaling included p38 mitogen-activated protein kinase (MAPK) activation and nuclear translocation of the cardiac transcription factor myocyte enhancer factor 2C (MEF2C). Our results provide first molecular evidence that the NOX family of NADPH oxidases regulate vertebrate developmental processes.

Animals↗
PubMed2006

Aberrant expression of BARD1 in breast and ovarian cancers with poor prognosis.

Mutations in tumor-suppressor gene BARD1 have been found in inherited and spontaneous breast, ovarian and uterine cancers. BARD1 plays a critical role in DNA repair and ubiquitination as binding partner of BRCA1, with which it colocalizes to nuclear dots. Independently of BRCA1, BARD1 can induce p53-dependent apoptosis in response to genotoxic stress. Therefore, BARD1 or p53 might be defective in cancer cells spared from apoptosis. We investigated BARD1 and p53 expression in ovarian, breast and non-small-cell lung cancers. BARD1 expression was highly upregulated and cytoplasmic in most cancer cells, while weak nuclear staining was observed in the surrounding normal tissue. Maximal BARD1 expression was associated with the most malignant ovarian cancer, clear cell carcinoma. In breast cancer, BARD1 expression was correlated with poor differentiation and large tumor size, established factors of poor prognosis, as well as short disease-free survival. In contrast to breast and ovarian cancers, no correlation of BARD1 expression with either grade or stage could be determined for lung cancer. RT-PCR, performed on 10 ovarian cancers, revealed absence of the 5' portion of the BARD1 transcript in 7 tumors, and sequencing of the remaining 3 identified a missense mutation (A1291G) resulting in an amino acid change of glutamine 406 to arginine. These data suggest that genetic and epigenetic changes might lead to elevated cytoplasmic expression of BARD1 and that cytoplasmic BARD1 might be a poor prognostic factor for breast and ovarian cancers.

Adult↗
PubMed2006

Pax6-induced alteration of cell fate: shape changes, expression of neuronal alpha tubulin, postmitotic phenotype, and cell migration.

The transcription factor Pax6 plays an important role in the development of the central nervous system. To understand its mechanism of action, we transduced HeLa cells with a Pax6-expressing lentiviral vector. Upon transduction, HeLa cells markedly changed shape and formed neuritelike extensions. Pax6-transduced HeLa cells expressed high levels of neuronal alpha3 tubulin, demonstrating a partial transdifferentiation towards a neuronal phenotype. Neurons are postmitotic cells. Pax6-transduced HeLa cells became postmitotic through mechanisms involving up-regulation of p53 and cyclin-dependent kinase inhibitor p21. One of the most striking effects of Pax6 was observed by time-lapse videomicroscopy: cells started to dissociate from cell clusters and displayed intense migratory activity. Migration was accompanied by dynamic and reversible shape changes. Our results identified three elements of Pax6 action: (i) expression of neuron-specific genes; (ii) establishment of a postmitotic phenotype; and (iii) involvement in the regulation of cell shape and cell migration.

Animals↗
PubMed2005

Decreased blood pressure in NOX1-deficient mice.

To understand the role of the superoxide-generating NADPH oxidase NOX1 in the vascular system, we have generated NOX1-deficient mice. NOX1-deficient mice had a moderately decreased basal blood pressure. In response to angiotensin II they showed an almost complete loss of the sustained blood pressure response, while the initial increase was conserved. NOX1-deficient mice showed a marked reduction in aortic media hypertrophy. Angiotensin II-induced smooth muscle cell proliferation was conserved, but there was a marked decrease in extracellular matrix accumulation. Our results establish a role for NOX1 in blood pressure regulation and vascular angiotensin II response.

Angiotensin II↗
PubMed2005

Expression and activity of NOX5 in the circulating malignant B cells of hairy cell leukemia.

Hairy cells (HCs) are mature malignant B cells that contain a number of constitutively active signaling molecules including GTP-bound Rac1, protein kinase C, and Src family kinases. Because Rac1 is a component of the reactive oxidant species (ROS)-generating NADPH oxidase system, we investigated the role of this GTPase in ROS production in HCs. In this study, we show that ROS production in HCs involves a flavin-containing oxidase dependent on Ca2+, but not on GTP-Rac1 or protein kinase C. This suggests the involvement of the nonphagocytic NADPH oxidase NOX5, an enzyme found in lymphoid tissues, but not in circulating lymphocytes. By using RT-PCR and Southern and Western blotting and by measuring superoxide anion production in membrane fractions in the absence of cytosolic components, we demonstrate for the first time that HCs (but not circulating normal B cells or some other lymphoid cell types) express NOX5. We also demonstrate that inhibition of NADPH oxidase in HCs results in a selective increase in the activity of Src homology region 2 domain-containing phosphatase 1 (SHP-1). Furthermore, SHP-1 in HCs coimmunoprecipitates with tyrosine phosphorylated CD22 and localizes in the same cellular compartment as NOX5. This allows the inactivation of SHP-1 by NOX5-generated ROS and contributes to the maintenance of the constitutive activation of HCs.

B-Lymphocytes↗
PubMed2005

Rapid generation of stable transgenic embryonic stem cell lines using modular lentivectors.

Generation of stable transgenic embryonic stem (ES) cell lines by classic transfection is still a difficult task, requiring time-consuming clonal selection, and hampered by clonal artifacts and gene silencing. Here we describe a novel system that allows construction of lentivectors and generation of stable ES cell lines with > 99% transgene expression within a very short time frame. Rapid insertion of promoters and genes of interest is obtained through a modular recombinational cloning system. Vectors contain central polypurine tract from HIV-1 element and woodchuck hepatitis virus post-transcriptional regulatory element as well as antibiotic resistance to achieve optimal and homogenous transgene expression. We show that the system 1) is functional in mouse and human ES cells, 2) allows the generation of ES cells expressing genes of interest under the control of ubiquitous or tissue-specific promoters, and 3) allows ES cells expressing two constructs through selection with different antibiotics to be obtained. The technology described herein should become a useful tool in stem cell research.

Animals↗
PubMed2005

A key role for the microglial NADPH oxidase in APP-dependent killing of neurons.

Reactive oxygen species (ROS) and deposition of cleaved products of amyloid precursor protein (APP) are thought to contribute to neuronal loss observed in Alzheimer's disease (AD). The relationship between these factors was studied in a neuroblastoma and microglia co-culture system. Overexpression of wild-type APP (APP-wt) or APP with three mutations typical of familial AD (APP-3m) in SH-SY5Y neuroblastoma cells did not directly alter their morphology, growth rate, cell cycle or H(2)O(2) sensitivity. In a co-culture of APP-wt neuroblastoma cells with microglia, microglial cells generated ROS and neuronal cells died. The cell death was more pronounced in APP-3m-expressing neurons. Neuroblastoma cell death was attenuated by ROS-scavengers and was dose-dependently inhibited by the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). Macrophage cell lines behaved similarly to microglia in the co-culture model. However, a macrophage cell line deficient in the NADPH oxidase subunit, gp91phox, failed to kill neurons. These results suggest that APP-dependent microglia activation and subsequent ROS generation by the phagocyte NADPH oxidase play a crucial role in neuronal killing in a cellular model of AD.

Alzheimer Disease↗
PubMed2005

BARD1 induces apoptosis by catalysing phosphorylation of p53 by DNA-damage response kinase.

The BRCA1-associated RING domain protein BARD1 acts with BRCA1 in double-strand break repair and ubiquitination. BARD1 plays a role as mediator of apoptosis by binding to and stabilizing p53, and BARD1-repressed cells are resistant to apoptosis. We therefore investigated the mechanism by which BARD1 induces p53 stability and apoptosis. The apoptotic activity of p53 is regulated by phosphorylation. We demonstrate that BARD1 binds to unphosphorylated and serine-15 phosphorylated forms of p53 in several cell types and that the region required for binding comprises the region sufficient for apoptosis induction. In addition, BARD1 binds to Ku-70, the regulatory subunit of DNA-PK, suggesting that the mechanism of p53-induced apoptosis requires BARD1 for the phosphorylation of p53. Upregulation of BARD1 alone is sufficient for stabilization of p53 and phosphorylation on serine-15, as shown in nonmalignant epithelial cells and ovarian cancer cells, NuTu-19, which are defective in apoptosis induction and express aberrant splice variants of BARD1. Stabilization and phosphorylation of p53 in NuTu-19 cells, as well as apoptosis, can be induced by the exogenous expression of wild-type BARD1, suggesting that BARD1, by binding to the kinase and its substrate, catalyses p53 phosphorylation.

Amino Acid Sequence↗
PubMed2005

Chemokine receptors in the central nervous system: role in brain inflammation and neurodegenerative diseases.

Chemokines were originally described as chemotactic cytokines involved in leukocyte trafficking. Research over the last decade, however, has shown that chemokine receptors are not restricted to leukocytes. In the brain, chemokine receptors are not only found in microglia (a brain macrophage), but also in astrocytes, oligodendrocytes and neurons. In this review, we describe the spatial and cellular distribution of chemokine receptors in the brain, distinguishing between constitutively and inducibly expressed receptors. We then discuss possible physiological functions, including neuronal migration, cell proliferation and synaptic activity. Evidence is emerging that chemokine receptors are also involved in neuronal death and hence neurodegenerative diseases. Chemokines may induce neuronal death either indirectly (e.g. through activation of microglia killing mechanisms) or directly through activation of neuronal chemokine receptors. Disease processes in which chemokines and their receptors are likely to be involved include multiple sclerosis (MS), Alzheimer's disease (AD), HIV-associated dementia (HAD) and cerebral ischemic disease. The study of chemokines and their receptors in the central nervous system (CNS) is not only relevant for the understanding of brain physiology and pathophysiology, but may also lead to the development of targeted treatments for neurodegenerative diseases.

Animals↗
PubMed2005

Expression of mRNA for ROS-generating NADPH oxidases in the aging stomach.

Oxidative damage is thought to play a key role in the aging of various organ systems. In this study, we have therefore analyzed mRNA expression of ROS-generating NADPH oxidases in the aging stomach. Gastric biopsies of hospitalized geriatric patients were analyzed for histology (Sidney classification), and real-time PCR was used to quantify mRNA expression of the superoxide-generating NADPH oxidases NOX1, NOX2, and NOX5. We found that stomach biopsies of elderly patients expressed NOX5 and NOX2 mRNA, but not NOX1. The mRNA expression of NOX5 (a lymphocyte NADPH oxidase) neither depended on age nor on the results of the stomach histology. In contrast, mRNA expression of NOX2 (phagocyte NADPH oxidase) was a function of two variables. Increased NOX2 mRNA levels were observed in biopsies with signs of chronic inflammation (p=0.01). Interestingly, however, there was also an age-dependent increase in NOX2 mRNA levels (p=0.01). We conclude that in elderly patients the gastric mRNA expression of the ROS-generating enzyme NOX2 increases as a function of age, possibly contributing to stomach aging and gastric vulnerability of the elderly.

Aged↗
PubMed2004

NOX3, a superoxide-generating NADPH oxidase of the inner ear.

Reactive oxygen species (ROS) play a major role in drug-, noise-, and age-dependent hearing loss, but the source of ROS in the inner ear remains largely unknown. Herein, we demonstrate that NADPH oxidase (NOX) 3, a member of the NOX/dual domain oxidase family of NADPH oxidases, is highly expressed in specific portions of the inner ear. As assessed by real-time PCR, NOX3 mRNA expression in the inner ear is at least 50-fold higher than in any other tissues where its expression has been observed (e.g. fetal kidney, brain, skull). Microdissection and in situ hybridization studies demonstrated that NOX3 is localized to the vestibular and cochlear sensory epithelia and to the spiral ganglions. Transfection of human embryonic kidney 293 cells with NOX3 revealed that it generates low levels of ROS on its own but produces high levels of ROS upon co-expression with cytoplasmic NOX subunits. NOX3-dependent superoxide production required a stimulus in the absence of subunits and upon co-expression with phagocyte NADPH oxidase subunits p47(phox) and p67(phox), but it was stimulus-independent upon co-expression with colon NADPH oxidase subunits NOX organizer 1 and NOX activator 1. Pre-incubation of NOX3-transfected human embryonic kidney 293 cells with the ototoxic drug cisplatin markedly enhanced superoxide production, in both the presence and the absence of subunits. Our data suggest that NOX3 is a relevant source of ROS generation in the cochlear and vestibular systems and that NOX3-dependent ROS generation might contribute to hearing loss and balance problems in response to ototoxic drugs.

Adaptor Proteins, Signal Transducing↗
PubMed2004

BARD1 expression during spermatogenesis is associated with apoptosis and hormonally regulated.

The BRCA1-binding RING-finger domain protein BARD1 may act conjointly with BRCA1 in DNA repair and in ubiquitination, but it may also induce apoptosis in a BRCA1-independent manner. In this study, we have investigated BARD1 expression during spermatogenesis. In contrast with BRCA1, which is expressed only in meiotic spermatocytes and early round spermatids, BARD1 is expressed during all stages of spermatogenesis. However, while spermatogonia expressed full-length BARD1 mRNA, later stages of spermatocyte precursors express predominantly a novel, shorter splice form BARD1beta. BARD1beta lacks the BRCA1-interacting RING finger but maintains its proapoptotic activity. Consistently, BRCA1 can counteract the proapoptotic activity of full-length BARD1 but not of BARD1beta. Several lines of evidence suggest that BARD1 is involved in proapoptotic signaling in testis: i) both BARD1 isoforms are mostly found in cells that stain positive for TUNEL, Bax, and activated caspase 3; ii) BARD1beta, capable of inducing apoptosis even in the presence of BRCA1, is specifically expressed in BRCA1-positive later stages of spermatogenesis; iii) antiapoptotic hormonal stimulation leads to BARD1 downregulation; and iv) BARD1 expression is associated with human pathologies causing sterility due to increased germ cell death. Our data suggest that full-length BARD1 might be involved in apoptotic control in spermatogonia and primary spermatocytes, while a switch to the BRCA1-independent BARD1beta might be necessary to induce apoptosis in BRCA1-expressing meiotic spermatocytes and early round spermatids.

Amino Acid Sequence↗
PubMed2004

Aging and infectious diseases in the developing world.

Although demographic aging does not remain restricted to industrialized countries, the medical challenge arising from the aging population will be distinct in the developing world. This is particularly true with respect to infectious diseases, which have a distinct spectrum in the elderly population, as well as a greater overall relevance in the developing world. Tropical diseases have a specific presentation and epidemiology in elderly patients. Infectious diseases with a worldwide distribution impact elderly patients in the developing world in a specific manner, which is most obvious with respect to human immunodeficiency virus and tuberculosis but is also true with respect to "trivial" manifestations of infection, such as diarrhea and pneumonia. Malnutrition contributes in a major way to the immunodeficiency of elderly patients in the developing world. Poorly controlled use of antimicrobial drugs leads to multidrug-resistant microorganisms, which, together with the limited resources available for drug treatment, makes appropriate treatment of infections in elderly patients in developing countries very difficult. Infections in elderly patients will have an increasing impact on the public health and economy of developing countries.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Nuclear-cytoplasmic translocation of BARD1 is linked to its apoptotic activity.

The tumor suppressor protein BARD1 plays a dual role in response to genotoxic stress: DNA repair as a BARD1-BRCA1 heterodimer and induction of apoptosis in a BRCA1-independent manner. We have constructed a series of BARD1 deletion mutants and analysed their cellular distribution and capacity to induce apoptosis. As opposed to previous studies suggesting an exclusively nuclear localization of BARD1, we found, both in tissues and cell cultures, nuclear and cytoplasmic localization of BARD1. Enhanced cytoplasmic localization of BARD1, as well as appearance of a 67 kDa C-terminal proteolytic cleavage product, coincided with apoptosis. BARD1 translocates to the nucleus independently of BRCA1. For recruitment to nuclear dots, however, the BRCA1-interacting RING finger domain is required but not sufficient. Protein levels of N-terminal RING finger deletion mutants were much higher than those of full-length BARD1, despite comparable mRNA levels, suggesting that the N-terminal region comprising the RING finger is important for BARD1 degradation. Sequences required for apoptosis induction were mapped between the ankyrin repeats and the BRCT domains coinciding with two known cancer-associated missense mutations. We suggest that nuclear and cytoplasmic localization of BARD1 reflect its dual function and that the increased cytoplasmic localization of BARD1 is associated with apoptosis.

Animals↗