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61.
Birthelmer A Ehret A Riegert C Rothmaier AK Leemhuis J Jackisch R 《Journal of neurochemistry》2007,100(6):1613-1625
Electrically evoked release of serotonin (5-HT) and its modulation via 5-HT autoreceptors and alpha(2)-heteroreceptors was studied in primary cell cultures prepared from the embryonic (ED 15) rat mesencephalic brain region comprising the raphe nuclei. Cultures were grown for up to 3 weeks on circular glass coverslips. They developed a dense network of non-neuronal and neuronal cells, some of which were positive for tryptophan hydroxylase. To measure 5-HT release, the cultures were pre-incubated with [(3)H]5-HT (in the presence of the selective noradrenaline reuptake inhibitor oxaprotiline [1 micromol/L]), superfused with modified Krebs-Henseleit medium containing 6-nitroqipazine [1 micromol/L] and electrically stimulated using two conditions. Condition A: 360 pulses, 3 Hz, 0.5 ms, 90 mA, or condition B: 4 pulses 100 Hz, 0.5 ms, 90 mA (a condition which diminishes interactions with endogenously released transmitters during ongoing stimulation). After only 1 week in culture, the electrically evoked overflow of [(3)H] was Ca(2+) dependent and tetrodotoxin sensitive, suggesting an action-potential-induced exocytotic release of 5-HT. Using stimulation condition A in cultures grown for 2 weeks, both basal and evoked 5-HT release were strongly enhanced by methiotepine (1 micromol/L) but unaffected by the 5-HT(1B) autoreceptor agonist CP-93, 129 (1 micromol/L) and the alpha(2)-adrenoceptor agonist UK-14, 304 (1 micromol/L). Conversely, using stimulation condition B, not only CP-93, 129 (IC(50) 8.1 +/- 1.4 nmol/L) and UK-14, 304 (IC(50) 14.9 +/- 1.6 nmol/L) had inhibitory effects on cells grown for 2 weeks, but also the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino)tetralin. In conclusion, we describe for the first time electrically evoked release of 5-HT from primary cultures of fetal raphe cells and its modulation via 5-HT(1B) and 5-HT(1A) auto- and alpha(2)-heteroreceptors. Such cultured raphe cells may represent a suitable model to study expression and development of presynaptic receptors on serotonergic neurons in-vitro. 相似文献
62.
Polymorphisms in base-excision repair and nucleotide-excision repair genes in relation to lung cancer risk 总被引:7,自引:0,他引:7
De Ruyck K Szaumkessel M De Rudder I Dehoorne A Vral A Claes K Velghe A Van Meerbeeck J Thierens H 《Mutation research》2007,631(2):101-110
Polymorphisms in DNA repair genes may be associated with differences in DNA repair capacity, thereby influencing the individual susceptibility to smoking-related cancer. We investigated the association of 10 base-excision and nucleotide-excision repair gene polymorphisms (XRCC1 -77 T/C, Arg194Trp, Arg280His and Arg399Gln; APE1 Asp148Glu; OGG1 Ser326Cys; XPA -4 G/A; XPC PAT; XPD Asp312Asn and Lys751Gln) with lung cancer risk in Caucasians. Genotypes were determined by PCR-RFLP and PCR-single base extension assays in 110 lung cancer patients and 110 age- and sex-matched controls, and the results were analyzed using logistic regression adjusted for relevant covariates. A significant association between the APE1 Asp148Glu polymorphism and lung cancer risk was found, with adjusted odds ratios (OR) of 3.38 (p=0.001) for the Asp/Glu genotype and 2.39 (p=0.038) for the Glu/Glu genotype. Gene-smoking interaction analyses revealed a statistically significant interaction between cumulative cigarette smoking and the XRCC1 Arg399Gln and XPD Lys751Gln polymorphisms: these polymorphisms were significantly associated with lung cancer in nonsmokers and light smokers (<25 PY; OR=4.92, p=0.021 for XRCC1 399 Gln/Gln; OR=3.62, p=0.049 for XPD 751 Gln/Gln), but not in heavy smokers (> or =25 PY; OR=0.68, p=0.566 for XRCC1 399 Gln/Gln; OR=0.46, p=0.295 for XPD 751 Gln/Gln). Both the XRCC1 Arg194Trp and Arg280His as well as the OGG1 Ser326Cys heterozygous genotypes were associated with a significantly reduced risk for lung cancer (OR=0.32, p=0.024; OR=0.25, p=0.028; OR=0.51, p=0.033, respectively). No associations with lung cancer risk were found for the XRCC1 -77 T/C, the XPA -4 G/A and the XPC PAT polymorphisms. In conclusion, the APE1 Asp148Glu polymorphism is highly predictive for lung cancer, and cumulative cigarette smoking modifies the associations between the XRCC1 Arg399Gln and the XPD Lys751Gln polymorphisms and lung cancer risk. 相似文献
63.
64.
Iron regulatory protein 2 (IRP2) is a critical switch for cellular and systemic iron homeostasis. In iron-deficient or hypoxic cells, IRP2 binds to mRNAs containing iron responsive elements (IREs) and regulates their expression. Iron promotes proteasomal degradation of IRP2 via the F-box protein FBXL5. Here, we explored the effects of oxygen and cellular redox status on IRP2 stability. We show that iron-dependent decay of tetracycline-inducible IRP2 proceeds efficiently under mild hypoxic conditions (3% oxygen) but is compromised in severe hypoxia (0.1% oxygen). A treatment of cells with exogenous H(2)O(2) protects IRP2 against iron and increases its IRE-binding activity. IRP2 is also stabilized during menadione-induced oxidative stress. These data demonstrate that the degradation of IRP2 in iron-replete cells is not only oxygen-dependent but also sensitive to redox perturbations. 相似文献
65.
S. Niemann Tanja Dammann-Kalinowski Anja Nagel Alfred Pühler W. Selbitschka 《Archives of microbiology》1999,172(1):22-30
The enterobacterial repetitive intergenic consensus (ERIC)-PCR method was employed to generate genomic amplification products
of Sinorhizobium meliloti strain 2011. Eleven distinctive PCR fragments obtained in PCR reactions by using the ERIC2 primer were cloned and their partial
or complete nucleotide sequences established. DNA sequences that extended past the ERIC2 primer region were not conserved
among the 11 PCR fragments and showed no sequence similarity to the enterobacterial ERIC consensus sequence. Thus, repetitive
ERIC or ERIC-like sequences seem not to be an integral part of the S. meliloti genome. An amplification product of S. meliloti 2011 was identified which was present in S. meliloti strains but absent in other rhizobial species. Based on the nucleotide sequence information, a pair of PCR primers was designed
and used for PCR amplification of sequences of S. meliloti laboratory strains 2011, L5–30, AK631 and 102F34. Nucleotide sequence analysis of the amplification products revealed a 100%
DNA sequence conservation. Database searches showed that the DNA fragment putatively encodes the C-terminal part of a protein
displaying similarity to 2-hydroxyacid dehydrogenases of various organisms. The newly designed PCR primers should be useful
for the rapid identification of S. meliloti isolates.
Received: 17 February 1999 / Accepted: 9 April 1999 相似文献
66.
The presence of the Z mutation (Glu342Lys) is responsible for more than 95% of α1-antitrypsin (α1AT) deficiency cases. It leads to increased polymerization of the serpin α1AT during its synthesis and in circulation. It has been proposed that the Z mutation results in a conformational change within the folded state of antitrypsin that enhances its polymerization. In order to localize the conformational change, we have created two single tryptophan mutants of Z α1AT and analyzed their fluorescence properties. α1AT contains two tryptophan residues that are located in distinct regions of the molecule: Trp194 at the top of β-sheet A and Trp238 on β-sheet B. We have replaced each tryptophan residue individually with a phenylalanine in order to study the local environment of the remaining tryptophan residue in both M and Z α1AT. A detailed fluorescence spectroscopic analysis of each mutant was carried out, and we detected differences in the emission spectrum, the Stern-Volmer constant for potassium iodide quenching and the anisotropy of only Trp194 in Z α1AT compared to M α1AT. Our data reveal that the Z mutation results in a conformational change at the top of β-sheet A but does not affect the structural integrity of β-sheet B. 相似文献
67.
Markus M. Heimesaat Ildiko R. Dunay Silvia Schulze André Fischer Ursula Grundmann Marie Alutis Anja A. Kühl Andrea Tamas Gabor Toth Miklos P. Dunay Ulf B. G?bel Dora Reglodi Stefan Bereswill 《PloS one》2014,9(9)
Background
The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) plays pivotal roles in immunity and inflammation. So far, potential immune-modulatory properties of PACAP have not been investigated in experimental ileitis.Methodology/Principal Findings
Mice were perorally infected with Toxoplasma (T.) gondii to induce acute ileitis (day 0) and treated daily with synthetic PACAP38 from day 1 to 6 post infection (p.i.; prophylaxis) or from day 4 to 6 p.i. (therapy). Whereas placebo-treated control mice suffered from acute ileitis at day 7 p.i. and succumbed to infection, intestinal immunopathology was ameliorated following PACAP prophylaxis. PACAP-treated mice exhibited increased abundance of small intestinal FOXP3+ cells, but lower numbers of ileal T lymphocytes, neutrophils, monocytes and macrophages, which was accompanied by less ileal expression of pro-inflammatory cytokines such as IL-23p19, IL-22, IFN-γ, and MCP-1. Furthermore, PACAP-treated mice displayed higher anti-inflammatory IL-4 concentrations in mesenteric lymph nodes and liver and higher systemic anti-inflammatory IL-10 levels in spleen and serum as compared to control animals at day 7 p.i. Remarkably, PACAP-mediated anti-inflammatory effects could also be observed in extra-intestinal compartments as indicated by reduced pro-inflammatory mediator levels in spleen (TNF-α, nitric oxide) and liver (TNF-α, IFN-γ, MCP-1, IL-6) and less severe histopathological sequelae in lungs and kidneys following prophylactic PACAP treatment. Strikingly, PACAP prolonged survival of T. gondii infected mice in a time-of-treatment dependent manner.Conclusion/Significance
Synthetic PACAP ameliorates acute small intestinal inflammation and extra-intestinal sequelae by down-regulating Th1-type immunopathology, reducing oxidative stress and up-regulating anti-inflammatory cytokine responses. These findings provide novel potential treatment options of inflammatory bowel diseases. 相似文献68.
Anja von Nahmen Andreas Post Hans-Joachim Galla M. Sieber 《European biophysics journal : EBJ》1997,26(5):359-369
Three compounds of the pulmonary surfactant – dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG),
and the surfactant associated protein C (SP-C) – were spread at the air-water interface of a Langmuir trough as a model system
to mimic the properties of natural surfactant. Fluorescence microscopical images of the film formed at the interface were
obtained during compression using a fluorescence dye bound covalently either to phosphatidylcholine or to SP-C. The images
were quantified using statistical methods in respect to relative areas and relative fluorescence intensities of the domains
found. In the early stage of compression, film pressure rose slightly and was accompanied by a phase separation which could
be recognized in the images by the formation of bright and dark domains. On further compression, after a steep increase of
film pressure, a plateau region of constant film pressure started abruptly. During compression in the plateau region, fluorescence
intensity of the bright domain formed in the early stage of compression increased. The increasing fluorescence intensity,
the non-Gaussian intensity distribution of the bright domain, and the small mean molecular area of the film in the plateau
region gave rise to the assumption that multilayer structures were formed in the late stage of compression. The formation
of the multilayer structures was fully reversible in repeated compression-expansion cycles including the plateau region of
the phase diagram. The ability of lipid/SP-C mixtures to form reversible multilayer structures during compression may be relevant
to stability in lungs during expiration and inhalation.
Received: 13 February 1997 / Accepted: 22 May 1997 相似文献
69.
Stability and function of eukaryotic genomes are closely linked to chromatin structure and organization. During cell division the entire genome must be accurately replicated and the chromatin landscape reproduced on new DNA. Chromatin and nuclear structure influence where and when DNA replication initiates, whereas the replication process itself disrupts chromatin and challenges established patterns of genome regulation. Specialized replication-coupled mechanisms assemble new DNA into chromatin, but epigenome maintenance is a continuous process taking place throughout the cell cycle. If DNA synthesis is perturbed, cells can suffer loss of both genome and epigenome integrity with severe consequences for the organism. 相似文献
70.
Anja Troeger Hee-Don Chae Mumine Senturk Jenna Wood David A. Williams 《The Journal of biological chemistry》2013,288(51):36451-36462
RhoH is a hematopoietic-specific, GTPase-deficient member of the Rho GTPase family that was first identified as a hypermutable gene in human B lineage lymphomas. RhoH remains in a constitutively active state and thus its effects are regulated by expression levels or post-translational modifications. Similar to other small GTPases, intracellular localization of RhoH is dependent upon the conserved “CAAX” box and surrounding sequences within the carboxyl (C) terminus. However, RhoH also contains a unique C-terminal “insert” domain of yet undetermined function. RhoH serves as adaptor molecule in T cell receptor signaling and RhoH expression correlates with the unfavorable prognostic marker ZAP70 in human chronic lymphocytic leukemia. Disease progression is attenuated in a Rhoh−/− mouse model of chronic lymphocytic leukemia and treatment of primary human chronic lymphocytic leukemia cells with Lenalidomide results in reduced RhoH protein levels. Thus, RhoH is a potential therapeutic target in B cell malignancies. In the current studies, we demonstrate that deletion of the insert domain (LFSINE) results in significant cytoplasmic protein accumulation. Using inhibitors of degradation pathways, we show that LFSINE regulates lysosomal RhoH uptake and degradation via chaperone-mediated autophagy. Whereas the C-terminal prenylation site is critical for ZAP70 interaction, subcellular localization and rescue of the Rhoh−/− T cell defect in vivo, the insert domain appears dispensable for these functions. Taken together, our findings suggest that the insert domain regulates protein stability and activity without otherwise affecting RhoH function. 相似文献