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171.
Molecular enzymology of lipoxygenases 总被引:1,自引:0,他引:1
Igor Ivanov Katharina Hofheinz Valerie B. O’Donnell Matthias Walther 《Archives of biochemistry and biophysics》2010,503(2):161-174
Lipoxygenases (LOXs) are lipid peroxidizing enzymes, implicated in the pathogenesis of inflammatory and hyperproliferative diseases, which represent potential targets for pharmacological intervention. Although soybean LOX1 was discovered more than 60 years ago, the structural biology of these enzymes was not studied until the mid 1990s. In 1993 the first crystal structure for a plant LOX was solved and following this protein biochemistry and molecular enzymology became major fields in LOX research. This review focuses on recent developments in molecular enzymology of LOXs and summarizes our current understanding of the structural basis of LOX catalysis. Various hypotheses explaining the reaction specificity of different isoforms are critically reviewed and their pros and cons briefly discussed. Moreover, we summarize the current knowledge of LOX evolution by profiling the existence of LOX-related genomic sequences in the three kingdoms of life. Such sequences are found in eukaryotes and bacteria but not in archaea. Although the biological role of LOXs in lower organisms is far from clear, sequence data suggests that this enzyme family might have evolved shortly after the appearance of atmospheric oxygen on earth. 相似文献
172.
173.
Cytosolic phosphoglucomutase (cPGM) interconverts glucose-6-phosphate and glucose-1-phosphate and is a key enzyme of central metabolism. In this study, we show that Arabidopsis (Arabidopsis thaliana) has two cPGM genes (PGM2 and PGM3) encoding proteins with high sequence similarity and redundant functions. Whereas pgm2 and pgm3 single mutants were undistinguishable from the wild type, loss of both PGM2 and PGM3 severely impaired male and female gametophyte function. Double mutant pollen completed development but failed to germinate. Double mutant ovules also developed normally, but approximately half remained unfertilized 2 d after pollination. We attribute these phenotypes to an inability to effectively distribute carbohydrate from imported or stored substrates (e.g. sucrose) into the major biosynthetic (e.g. cell wall biosynthesis) and respiratory pathways (e.g. glycolysis and the oxidative pentose phosphate pathway). Disturbing these pathways is expected to have dramatic consequences for germinating pollen grains, which have high metabolic and biosynthetic activities. We propose that residual cPGM mRNA or protein derived from the diploid mother plant is sufficient to enable double mutant female gametophytes to attain maturity and for some to be fertilized. Mature plants possessing a single cPGM allele had a major reduction in cPGM activity. However, photosynthetic metabolism and growth were normal, suggesting that under standard laboratory conditions cPGM activity provided from one wild-type allele is sufficient to mediate the photosynthetic and respiratory fluxes in leaves. 相似文献
174.
First LightCycler real-time PCR assay for the quantitative detection of Mycoplasma suis in clinical samples 总被引:1,自引:0,他引:1
Hoelzle LE Helbling M Hoelzle K Ritzmann M Heinritzi K Wittenbrink MM 《Journal of microbiological methods》2007,70(2):346-354
Mycoplasma suis cannot be cultivated in vitro. Therefore, PCR-based methods are irreplaceable for the diagnosis of M. suis infections especially when clinical symptoms are not evident. Currently, no easy and reliable method allowing the quantitative detection of M. suis is available. This report describes the development of a quantitative LightCycler PCR assay based on the msg1 gene of M. suis (LC MSG1 PCR). No PCR signals were obtained with closely related haemotrophic and non-haemotrophic mycoplasmas, with other bacteria, and with M. suis-free blood and tissue arguing for a high analytical specificity. Test sensitivity was found to be 100%, and test specificity 96.7%. To test the diagnostic suitability of the LC MSG1 PCR, 25 pigs with clinical porcine eperythrozoonosis and 25 healthy pigs were investigated. All ill pigs revealed a positive real-time PCR result whereas only one healthy pig was detected to be M. suis-infected. M. suis was quantitatively detected in 19 blood specimens of 100 sows from Switzerland and in 17 of 160 post-weaning piglets from Germany. In conclusion, this new LC MSG1 PCR assay represents a powerful tool for the improvement of the current M. suis diagnosis and for prevalence and pathogenesis studies. 相似文献
175.
A short and efficient synthesis of pentadeuterated 2,2,3,4,4-d5-19-nor-5alpha-androsterone 7 starting from 19-norandrost-4-ene-3,17-dione 1 by a d1-L-Selectride mediated stereo- and regioselective reduction of the 3-keto group is presented. The use of compound 7 as internal standard for the detection of anabolic steroids via mass spectrometric techniques such as gas chromatography-mass spectrometry (GC-MS) is discussed. 相似文献
176.
Erdmann S Ricken A Merkwitz C Struman I Castino R Hummitzsch K Gaunitz F Isidoro C Martial J Spanel-Borowski K 《American journal of physiology. Endocrinology and metabolism》2007,293(5):E1365-E1377
In the corpus luteum (CL), blood vessels develop, stabilize, and regress. This process depends on the ratio of pro- and antiangiogenic factors, which change during the ovarian cycle. The present study focuses on the possible roles of 23,000 (23K) prolactin (PRL) in the bovine CL and its antiangiogenic NH(2)-terminal fragments after extracellular cleavage by cathepsin D (Cath D). PRL RNA and protein were demonstrated in the CL tissue, in luteal endothelial cells, and in steroidogenic cells. Cath D was detected in CL tissue, cell extracts, and corresponding cell supernatants. In the intact CL, 23K PRL levels decreased gradually, whereas Cath D levels concomitantly increased between early and late luteal stages. In vitro, PRL cleavage occurred in the presence of acidified homogenates of CL tissue, cells, and corresponding cell supernatants. Similar fragments were obtained with purified Cath D, and their appearance was inhibited by pepstatin A. The aspartic protease specific substrate MOCAc-GKPILF~FRLK(Dnp)-D-R-NH(2) was cleaved by CL cell supernatants, providing further evidence for Cath D activity. The 16,000 PRL inhibited proliferation of luteal endothelial cells accompanied by an increase in cleaved caspase-3. In conclusion, 1) the bovine CL is able to produce PRL and to process it into antiangiogenic fragments by Cath D activity and 2) PRL cleavage might mediate angioregression during luteolysis. 相似文献
177.
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
178.
Ehret A Birthelmer A Rutz S Riegert C Rothmaier AK Jackisch R 《Journal of neurochemistry》2007,102(4):1071-1082
Presynaptic receptors modulating the release of acetylcholine (ACh) were studied in fetal septal neurons cultured in a growth medium to which various drugs were added from day 3 in vitro (DIV 3) to DIV 14. The influence of these drugs on the function of the presynaptic muscarinic (M-) autoreceptor was determined at DIV 14 by measuring the inhibitory effect of the M-agonist oxotremorine on the electrically-evoked release of [(3)H]ACh from cultures pre-incubated with [(3)H]choline. The presence of the M-agonists oxotremorine (100 micromol/L) or carbachol (100 micromol/L) from DIV 3 to DIV 14, or from DIV 13 to DIV 14, abolished M-autoreceptor function at DIV 14, whereas the presence of the M-antagonist atropine (10 micromol/L from DIV 3 to DIV 14) during growth left M-autoreceptor function unaltered. Inhibition of ACh esterase by donepezil (1 micromol/L from DIV 3 to DIV 14) weakly decreased M-autoreceptor function at DIV 14; inhibition of neuronal firing by 0.1 tetrodotoxin (0.1 micromol/L from DIV 3 to DIV 14) did not tend to affect M-autoreceptor function at DIV 14. Co-cultivation of fetal septal and raphe neurons for 2 weeks yielded cell cultures containing both vesicular ACh transporter- and tryptophan hydroxylase-immunopositive cells. From these cultures, the release of both [(3)H]ACh and [(3)H]5-HT could be induced by electrical field stimulation. In co-cultured neurons versus septal-only ones the inhibitory effect of oxotremorine on the evoked release of [(3)H]ACh appeared almost normal, whereas that of the selective 5-HT(1B) agonist 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrollo[3,2-b]pyrid-5-one (CP-93,129) was completely abolished. The effects of CP-93,129 were also absent on DIV 14 in septal mono-cultures grown in the presence of CP-93,129 (10 micromol/L) from DIV 3 to DIV 14. It is therefore concluded that the regulation of presynaptic receptor function strongly depends on the concentrations of endogenous transmitters in the neuronal environment. 相似文献
179.
Hansen K Wagner B Hamel W Schweizer M Haag F Westphal M Lamszus K 《Journal of neurochemistry》2007,103(1):259-275
The neurotrophin receptor tropomyosin-related kinase A (TrkA) and its ligand nerve growth factor (NGF) are expressed in astrocytomas, and an inverse association of TrkA expression with malignancy grade was described. We hypothesized that TrkA expression might confer a growth disadvantage to glioblastoma cells. To analyze TrkA function and signaling, we transfected human TrkA cDNA into the human glioblastoma cell line G55. We obtained three stable clones, all of which responded with striking cytoplasmic vacuolation and subsequent cell death to NGF. Analyzing the mechanism of cell death, we could exclude apoptosis and cellular senescence. Instead, we identified several indications of autophagy: electron microscopy showed typical autophagic vacuoles; acridine orange staining revealed acidic vesicular organelles; acidification of acidic vesicular organelles was prevented using bafilomycin A1; cells displayed arrest in G2/M; increased processing of LC3 occurred; vacuolation was prevented by the autophagy inhibitor 3-methyladenine; no caspase activation was detected. We further found that both activation of ERK and c-Jun N-terminal kinase but not p38 were involved in autophagic vacuolation. To conclude, we identified autophagy as a novel mechanism of NGF-induced cell death. Our findings suggest that TrkA activation in human glioblastomas might be beneficial therapeutically, especially as several of the currently used chemotherapeutics also induce autophagic cell death. 相似文献
180.