Neuronal differentiation of embryonic stem cells

AbstractNeuronal differentiation from totipotent precursors in vitro, is thought to require two signals: first a biophysical state (cellular aggregation) followed by a biochemical signal (retinoic acid treatment). In investigating the properties of retinoic acid-differentiated embryonic stem cell lines. However, we noted that retinoic acid treatment without prior aggregation, is sufficient to induce expression of the neuronal markers GAP-43 and NF-165. In agreement, immunohistochemistry revealed the presence of GAP-43 positive cells in these embryonic stem cell monolayers after three days of retinoic acid (RA) treatment. Furthermore an NF-165 positive subpopulation of cells was clearly observed after 4–5 days of RA treatment. The expression of these neuronal markers coincided with the appearance of electrically excitable cells, as assayed with whole cell patch clamp recording. We conclude that for neuronal differentiation of totipotent embryonic stem cells in vitro, one biochemical signal, i.e. retinoic acid treatment, is sufficient

Sensitization of the histamine H1 receptor by increased ligand affinity.

Histamine regulates a variety of physiological processes including inflammation, gastric acid secretion, and neurotransmission. The cellular response to histamine is subject to dynamic control, and exaggerated histamine reactivity in response to cysteinyl leukotrienes and other stimuli is important in a variety of different pathological conditions. The molecular mechanisms controlling histamine responsiveness are still unresolved. In investigating histamine responses in embryonic stem (ES5) and F9 embryonic carcinoma cells, we encountered a novel mechanism controlling the cellular reaction to histamine. Unstimulated cells displayed neither

Regulation of receptor protein-tyrosine phosphatase α by oxidative stress

The presence of two protein-tyrosine phosphatase (PTP) domains is a striking feature in most transmembrane receptor PTPs (RPTPs). The function of the generally inactive membrane-distal PTP domain (RPTP-D2) is unknown. Here we report that an intramolecular interaction between the spacer region (Sp) and the C-terminus in RPTPα prohibited intermolecular interactions. Interestingly, stress factors such as H(2)O(2), UV and heat shock induced reversible, free radical-dependent, intermolecular interactions between RPTPα and RPTPα-SpD2, suggesting an inducible switch in conformation and binding. The catalytic site cysteine of RPTPα-SpD2, Cys723, was required for the H(2)O(2) effect on RPTPα. H(2)O(2) induced a rapid, reversible, Cys723-dependent conformational change in vivo, as detected by fluorescence resonance energy transfer, with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) flanking RPTPα-SpD2 in a single chimeric protein. Importantly, H(2)O(2) treatment stabilized RPTPα dimers, resulting in inactivation. We propose a model in which oxidative stress induces a conformational change in RPTPα-D2, leading to stabilization of RPTPα dimers, and thus to inhibition of RPTPα activity

Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure

One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes (hESC-CMs) with dexamethasone, a synthetic glucocorticoid, activated glucocorticoid signaling which in turn improved their calcium handling properties and contractility. L-type calcium current and action potential properties were not affected by dexamethasone but significantly faster calcium decay, increased forces of contraction and sarcomeric lengths, were observed in hESC-CMs after dexamethasone exposure. Activating the glucocorticoid pathway can thus contribute to mediating hPSC-CMs maturation

A cardiomyocyte show of force: A fluorescent alpha-actinin reporter line sheds light on human cardiomyocyte contractility versus substrate stiffness

Cardiovascular disease is often associated with cardiac remodeling, including cardiac fibrosis, which may lead to increased stiffness of the heart wall. This stiffness in turn may cause subsequent failure of cardiac myocytes, however the response of these cells to increased substrate stiffness is largely unknown. To investigate the contractile response of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) to increased substrate stiffness, we generated a stable transgenic human pluripotent stem cell line expressing a fusion protein of α-Actinin and fluorescent mRubyII in a previously characterized NKX2.5-GFP reporter line. Cardiomyocytes differentiated from this line were subjected to a substrate with stiffness ranging from 4 kPa to 101 kPa, while contraction of sarcomeres and bead displacement in the substrate were measured for each single cardiomyocyte. We found that sarcomere dynamics in hPSC-CMs on polyacrylamide gels of increasing stiffness are not affected above physiological levels (21 kPa), but that contractile force increases up to a stiffness of 90 kPa, at which cell shortening, deducted from bead displacement, is significantly reduced compared to physiological stiffness. We therefore hypothesize that this discrepancy may be the cause of intracellular stress that leads to hypertrophy and consequent heart failure in vivo

Kvalita veřejných služeb

V první kapitole se zabývám definicí veřejného sektoru, popisuji metodologické přístupy a metody hodnocení kvality veřejným sektorem.V druhé části popisuji město Turnov, jak v samosprávné činnosti, tak i ve výkonu státní správy.Hodnocení jsem provedla formou dotazníku

Uncoupling DNA damage from chromatin damage to detoxify doxorubicin

The anthracycline doxorubicin (Doxo) and its analogs daunorubicin (Daun), epirubicin (Epi), and idarubicin (Ida) have been cornerstones of anticancer therapy for nearly five decades. However, their clinical application is limited by severe side effects, especially dose-dependent irreversible cardiotoxicity. Other detrimental side effects of anthracyclines include therapy-related malignancies and infertility. It is unclear whether these side effects are coupled to the chemotherapeutic efficacy. Doxo, Daun, Epi, and Ida execute two cellular activities: DNA damage, causing double-strand breaks (DSBs) following poisoning of topoisomerase II (Topo II), and chromatin damage, mediated through histone eviction at selected sites in the genome. Here we report that anthracycline-induced cardiotoxicity requires the combination of both cellular activities. Topo II poisons with either one of the activities fail to induce cardiotoxicity in mice and human cardiac microtissues, as observed for aclarubicin (Acla) and etoposide (Etop). Further, we show that Doxo can be detoxified by chemically separating these two activities. Anthracycline variants that induce chromatin damage without causing DSBs maintain similar anticancer potency in cell lines, mice, and human acute myeloid leukemia patients, implying that chromatin damage constitutes a major cytotoxic mechanism of anthracyclines. With these anthracyclines abstained from cardiotoxicity and therapy-related tumors, we thus uncoupled the side effects from anticancer efficacy. These results suggest that anthracycline variants acting primarily via chromatin damage may allow prolonged treatment of cancer patients and will improve the quality of life of cancer survivors