Refine
Year of publication
- 2003 (17) (remove)
Document Type
- Article (16)
- Doctoral Thesis (1)
Language
- English (17) (remove)
Has Fulltext
- yes (17)
Is part of the Bibliography
- no (17)
Keywords
- Endothelium (1)
- Isoproterenol (1)
- Proteinkinase (1)
- VASP (1)
- analgesia based sedation (1)
- cAMP (1)
- cGMP (1)
- critical care (1)
- offset times (1)
- pharmacodynamics (1)
Institute
- Medizin (17) (remove)
Hepatitis E virus (HEV) is a positive-stranded RNA virus with a 7.2 kb genome that is capped and polyadenylated. The virus is currently unclassified : the organisation of the genome resembles that of the Caliciviridae but sequence analyses suggest that it is more closely related to the Togaviridae. HEV is an enterically transmitted virus that causes both epidemics and sporadic cases of acute hepatitis in many countries of Asia and Africa but only rarely causes disease in more industrialised countries. Initially the virus was believed to have a limited geographical distribution. However, serological studies suggest that that HEV may be endemic also in the United states and Europe even though it infrequently causes overt disease in these countries. Many different animal species worldwide recently have been shown to have antibodies to HEV suggesting that hepatitis E may be zoonotic. Although two related strains have been experimentally transmitted between species, direct transmission from animal to a human has not been documented. Our main objective in this study is to evaluate the suitability of current available HEV antibody assays for use in low-endemicity areas such as in Germany. Methods: We selected sera on the basis of at least borderline reactivity in the routinely used Abbot EIA. Most were tested as part of routine screening of long-term expatriates in endemic countries. The following assays (recombinant antigens : ORF2 and ORF3) were used: Abbot EIA, Genelabs ELISA, Mikrogen recomBlot and a 'Prototype' DSL-ELISA. We observed a wide range of sensitivity ( average of 56.8%) and specificity ( an average of 61.4%) in these used assays. These results implies that , these assays might be unreliable for detection of HEV infection in areas where hepatitis E is not endemic. However, most anti- HEV assays have not been correlated with the HEV RNA determined by reverse transcription. Many of these unexpected results and discrepancies can be alluded to the following reasons: I. The choice and the size of the HEV antigen. II. Duration of the antibody persistence III. A cross reactivity with different agent IV. Due to geographic species V. A low sensitivity of the available assays. VI. And infection with non-pathogenic HEV strain. (zoonotic strain?). We therefore suggest that, further studies will be required to improve the sensitivity and specificity of the available commercial assays on the market.
Introduction: This open label, multicentre study was conducted to assess the times to offset of the pharmacodynamic effects and the safety of remifentanil in patients with varying degrees of renal impairment requiring intensive care.
Methods: A total of 40 patients, who were aged 18 years or older and had normal/mildly impaired renal function (estimated creatinine clearance ≥ 50 ml/min; n = 10) or moderate/severe renal impairment (estimated creatinine clearance <50 ml/min; n = 30), were entered into the study. Remifentanil was infused for up to 72 hours (initial rate 6–9 μg/kg per hour), with propofol administered if required, to achieve a target Sedation–Agitation Scale score of 2–4, with no or mild pain.
Results: There was no evidence of increased offset time with increased duration of exposure to remifentanil in either group. The time to offset of the effects of remifentanil (at 8, 24, 48 and 72 hours during scheduled down-titrations of the infusion) were more variable and were statistically significantly longer in the moderate/severe group than in the normal/mild group at 24 hours and 72 hours. These observed differences were not clinically significant (the difference in mean offset at 72 hours was only 16.5 min). Propofol consumption was lower with the remifentanil based technique than with hypnotic based sedative techniques. There were no statistically significant differences between the renal function groups in the incidence of adverse events, and no deaths were attributable to remifentanil use.
Conclusion: Remifentanil was well tolerated, and the offset of pharmacodynamic effects was not prolonged either as a result of renal dysfunction or prolonged infusion up to 72 hours.
Receptor tyrosine kinases of the epidermal growth factor (EGF) receptor family regulate essential cellular functions such as proliferation, survival, migration, and differentiation but also play central roles in the etiology and progression of tumors. We have identified short peptide sequences from a random peptide library integrated into the thioredoxin scaffold protein, which specifically bind to the intracellular domain of the EGF receptor (EGFR). These molecules have the potential to selectively inhibit specific aspects of EGF receptor signaling and might become valuable as anticancer agents. Intracellular expression of the aptamer encoding gene construct KDI1 or introduction of bacterially expressed KDI1 via a protein transduction domain into EGFR-expressing cells results in KDI1·EGF receptor complex formation, a slower proliferation, and reduced soft agar colony formation. Aptamer KDI1 did not summarily block the EGF receptor tyrosine kinase activity but selectively interfered with the EGF-induced phosphorylation of the tyrosine residues 845, 1068, and 1148 as well as the phosphorylation of tyrosine 317 of p46 Shc. EGF-induced phosphorylation of Stat3 at tyrosine 705 and Stat3-dependent transactivation were also impaired. Transduction of a short synthetic peptide aptamer sequence not embedded into the scaffold protein resulted in the same impairment of EGF-induced Stat3 activation.
The renin-angiotensin-aldosterone system plays a pivotal role in the regulation of salt and water homeostasis. Here, we demonstrate the expression and functional role of cGMP-dependent protein kinases (PKGs) in rat adrenal cortex. Expression of PKG II is restricted to adrenal zona glomerulosa (ZG) cells, whereas PKG I is localized to the adrenal capsule and blood vessels. Activation of the aldosterone system by a low sodium diet up-regulated the expression of PKG II, however, it did not change PKG I expression in adrenal cortex. Both, activation of PKG II in isolated ZG cell and adenoviral gene transfer of wild type PKG II into ZG cells enhanced aldosterone production. In contrast, inhibition of PKG II as well as infection with a PKG II catalytically inactive mutant had an inhibitory effect on aldosterone production. Steroidogenic acute regulatory (StAR) protein that regulates the rate-limiting step in steroidogenesis is a new substrate for PKG II and can be phosphorylated by PKG II in vitro at serine 55/56 and serine 99. Stimulation of aldosterone production by PKG II in contrast to stimulation by PKA did not activate StAR gene expression in ZG cells. The results presented indicate that PKG II activity in ZG cells is important for maintaining basal aldosterone production.
Mitochondrial proton-translocating NADH:ubiquinone oxidoreductase (complex I) couples the transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the mitochondrial inner membrane. Subunit PSST is the most likely carrier of iron-sulfur cluster N2, which has been proposed to play a crucial role in ubiquinone reduction and proton pumping. To explore the function of this subunit we have generated site-directed mutants of all eight highly conserved acidic residues in the Yarrowia lipolytica homologue, the NUKM protein. Mutants D99N and D115N had only 5 and 8% of the wild type catalytic activity, respectively. In both cases complex I was stably assembled but electron paramagnetic resonance spectra of the purified enzyme showed a reduced N2 signal (about 50%). In terms of complex I catalytic activity, almost identical results were obtained when the aspartates were individually changed to glutamates or to glycines. Mutations of other conserved acidic residues had less dramatic effects on catalytic activity and did not prevent assembly of iron-sulfur cluster N2. This excludes all conserved acidic residues in the PSST subunit as fourth ligands of this redox center. The results are discussed in the light of the structural similarities to the homologous small subunit of water-soluble [NiFe] hydrogenases.
Tetrahydrobiopterin, a critical factor in the production and role of nitric oxide in mast cells
(2003)
Mast cells (MC) are biologically potent, ubiquitously distributed immune cells with fundamental roles in host integrity and disease. MC diversity and function is regulated by exogenous nitric oxide; however, the production and function of endogenously produced NO in MC is enigmatic. We used rat peritoneal MC (PMC) as an in vivo model to examine intracellular NO production. Live cell confocal analysis of PMC using the NO-sensitive probe diaminofluorescein showed distinct patterns of intracellular NO formation with either antigen (Ag)/IgE (short term) or interferon-γ (IFN-γ) (long term). Ag/IgE-induced NO production is preceded by increased intracellular Ca2+, implying constitutive nitric-oxide synthase (NOS) activity. NO formation inhibits MC degranulation. NOS has obligate requirements for tetrahydrobiopterin (BH4), a product of GTP-cyclohydrolase I (CHI), IFN-γ-stimulated PMC increased CHI mRNA, protein, and enzymatic activity, while decreasing CHI feedback regulatory protein mRNA, causing sustained NO production. Treatment with the CHI inhibitor, 2,4-diamino-6-hydroxypyrimidine, inhibited NO in both IFN-γ and Ag/IgE systems, increasing MC degranulation. Reconstitution with the exogenous BH4 substrate, sepiapterin, restored NO formation and inhibited exocytosis. Thus, Ag/IgE and IFN-γ induced intracellular NO plays a key role in MC mediator release, and alterations in NOS activity via BH4 availability may be critical to the heterogeneous responsiveness of MC.
Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1β. We demonstrate here that the stable ATP analog adenosine 5′-O-(thiotriphosphate) (ATPγS) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5′-promoter region showed weak responses to ATPγS but confered a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3′-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATPγS potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3′-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATPγS. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATPγS, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1β-inducibility. By confocal microscopy we demonstrate that ATPγS increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.
Atovaquone is a substituted 2-hydroxynaphthoquinone that is used therapeutically to treat Plasmodium falciparum malaria, Pneumocystis carinii pneumonia, and Toxoplasma gondii toxoplasmosis. It is thought to act on these organisms by inhibiting the cytochrome bc1 complex. We have examined the interaction of atovaquone with the bc1 complex isolated from Saccharomyces cerevisiae, a surrogate, nonpathogenic fungus. Atovaquone inhibits the bc1 complex competitively with apparent Ki = 9 nm, raises the midpoint potential of the Rieske iron-sulfur protein from 285 to 385 mV, and shifts the g values in the EPR spectrum of the Rieske center. These results indicate that atovaquone binds to the ubiquinol oxidation pocket of the bc1 complex, where it interacts with the Rieske iron-sulfur protein. A computed energy-minimized structure for atovaquone liganded to the yeast bc1 complex suggests that a phenylalanine at position 275 of cytochrome b in the bovine bc1 complex, as opposed to leucine at the equivalent position in the yeast enzyme, is responsible for the decreased sensitivity of the bovine bc1 complex (Ki = 80 nm) to atovaquone. When a L275F mutation was introduced into the yeast cytochrome b, the sensitivity of the yeast enzyme to atovaquone decreased (Ki = 100 nm) with no loss in activity, confirming that the L275F exchange contributes to the differential sensitivity of these two species to atovaquone. These results provide the first molecular description of how atovaquone binds to the bc1 complex and explain the differential inhibition of the fungal versus mammalian enzymes.
There is evidence that endothelial nitric-oxide synthase (eNOS) is regulated by reciprocal dephosphorylation of Thr497 and phosphorylation of Ser1179. To examine the interrelationship between these sites, cells were transfected with wild-type (WT), T497A, T497D, S1179D, and T497A/S1179D eNOS and activity, NO release and eNOS localization were assessed. Although eNOS T497A, S1179D and T497A/S1179D eNOS had greater enzymatic activity than did WT eNOS in lysates, basal production of NO from cells was markedly reduced in cells transfected with T497A and T497A/S1179D eNOS but augmented in cells transfected with S1179D eNOS. Stimulating cells with ATP or ionophore normalized the loss of function seen with T497A and T497A/S1179D eNOS to levels observed with WT and S1179D eNOS, respectively. Despite these functional differences, the localization of eNOS mutants were similar to WT. Because both T497A and T497A/S1179D eNOS exhibited higher enzyme activity but reduced production of NO, we examined whether these mutations were “uncoupling” NO synthesis. T497A and T497A/S1179D eNOS generated 2-3 times more superoxide anion than WT eNOS, and both basal and stimulated interactions of T497A/S1179D eNOS with hsp90 were reduced in co-immunoprecipitation experiments. Thus, the phosphorylation/dephosphorylation of Thr497 may be an intrinsic switch mechanism that determines whether eNOS generates NO versus superoxide in cells.
Signal transducer and activator of transcription 5 (STAT5) is a transcription factor that activates prolactin (PRL)-dependent gene expression in the mammary gland. For the activation of its target genes, STAT5 recruits coactivators like p300 and the CREB-binding protein (CBP). In this study we analyzed the function of p300/CBP-associated members of the p160/SRC/NCoA-family in STAT5-mediated transactivation of β-casein expression. We found that only one of them, NCoA-1, acts as a coactivator for both STAT5a and STAT5b. The two coactivators p300/CBP and NCoA-1 cooperatively enhance STAT5a-mediated transactivation. For NCoA-1-dependent coactivation of STAT5, both the activation domain 1 and the amino-terminal bHLH/PAS domain are required. The amino-terminal region mediates the interaction with STAT5a in cells. A motif of three amino acids in an α-helical region of the STAT5a-transactivation domain is essential for the binding of NCoA-1 and for the transcriptional activity of STAT5a. Moreover we observed that NCoA-1 is involved in the synergistic action of the glucocorticoid receptor and STAT5a on the β-casein promoter. These findings support a model in which STAT5, in concert with the glucocorticoid receptor, recruits a multifunctional coactivator complex to initiate the PRL-dependent transcription.