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991.
Roessl U Wiesbauer J Leitgeb S Birner-Gruenberger R Nidetzky B 《Biotechnology journal》2012,7(8):1014-1024
Effective inhibition of protein aggregation is a major goal in biopharmaceutical production processes optimized for product quality. To examine the characteristics of process-stress-dependent aggregation of human granulocyte colony-stimulating factor (G-CSF), we applied controlled stirring and bubble aeration to a recombinant non-glycosylated preparation of the protein produced in Escherichia coli. We characterized the resulting denaturation in a time-resolved manner using probes for G-CSF conformation and size in both solution and the precipitate. G-CSF was precipitated rapidly from solutions that were aerated or stirred; only small amounts of soluble aggregates were found. Exposed hydrophobic surfaces were a characteristic of both soluble and insoluble G-CSF aggregates. Using confocal laser scanning microscopy, the aggregates presented mainly a circular shape. Their size varied according to incubation time and stress applied. The native intramolecular disulfide bonds in the insoluble G-CSF aggregates were largely disrupted as shown by mass spectrometry. New disulfide bonds formed during aggregation. All involved Cys(18) , which is the only free cysteine in G-CSF; one of them had an intermolecular Cys(18(A)) -Cys(18(B)) crosslink. Stabilization strategies can involve external addition of thiols and extensive reduction of surface exposition during processing. 相似文献
992.
993.
Background
There is some evidence that the association of fish and marine fatty acids with stroke risk differs between men and women. We investigated the gender-specific associations of habitual intake of the marine fatty acids eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) and fish on incident stroke in a population-based study in the Netherlands.Methods
We prospectively followed 20,069 men and women, aged 20–65 years, without cardiovascular diseases at baseline. Habitual diet was assessed with a validated 178-item food frequency questionnaire. Incidence of stroke was assessed through linkage with mortality and morbidity registers. Cox proportional hazards models were used to estimate multivariable-adjusted hazard ratios (HR) and 95% confidence intervals (95%CI).Results
During 8–13 years of follow-up, 221 strokes occurred. In women, an inverse dose-response relation (P-trend = 0.02) was observed between EPA-DHA intake and incident stroke, with an HR of 0.49 (95% CI: 0.27–0.91) in the top quartile of EPA-DHA (median 225 mg/d) as compared to the bottom quartile (median 36 mg/d). In men, the HR (95%CI) for the top quartile of EPA-DHA intake was 0.87 (0.51–1.48) (P-trend = 0.36). Similar results were observed for fish consumption and stroke incidence.Conclusion
A higher EPA-DHA and fish intake is related to a lower stroke risk in women, while for men an inverse association could not be demonstrated. 相似文献994.
AU-rich elements (AREs) are regulatory sequences located in the 3' untranslated region of many short-lived mRNAs. AREs are recognized by ARE-binding proteins and cause rapid mRNA degradation. Recent reports claimed that the function of AREs may be--at least in part--relayed through the miRNA pathway. We have revisited this hypothesis using dicer knock-out mouse embryonic fibroblasts and cultured Drosophila cells. In contrast to the published results, we find no evidence for a general requirement of the miRNA pathway in the function of AREs. Endogenous ier3 mRNA, which is known to contain a functional ARE, was degraded rapidly at indistinguishable rates in wild type and dicer knock-out mouse embryonic fibroblasts. In cultured Drosophila cells, both ARE-containing GFP reporter mRNAs and the endogenous cecA1 mRNA were resistant to depletion of the mi/siRNA factors dcr-1, dcr-2, ago1 and ago2. Furthermore, the Drosophila miRNA originally proposed to recognize AU-rich elements, miR-289, is not detectably expressed in flies or cultured S2 cells. Even our attempts to overexpress this miRNA from its genomic hairpin sequence failed. Thus, this sequence cannot serve as link between the miRNA and the AU-rich element mediated silencing pathways. Taken together, our studies in mammalian and Drosophila cells strongly argue that AREs can function independently of miRNAs. 相似文献
995.
996.
Stephan DA Howell GR Teslovich TM Coffey AJ Smith L Bailey-Wilson JE Malechek L Gildea D Smith JR Gillanders EM Schleutker J Hu P Steingruber HE Dhami P Robbins CM Makalowska I Carpten JD Sood R Mumm S Reinbold R Bonner TI Baffoe-Bonnie A Bubendorf L Heiskanen M Kallioneimi OP Baxevanis AD Joseph SS Zucchi I Burk RD Isaacs W Ross MT Trent JM 《Genomics》2002,79(1):41-50
997.
Respiratory energy losses related to cell weight and temperature in ciliated protozoa 总被引:2,自引:0,他引:2
Summary Respiratory energy losses in five species of ciliated protozoa, Tetrahymena pyriformis Ehrenberg, Vorticella microstoma Ehrenberg, Paramecium aurelia Ehrenberg, Spirostomum teres Claparède and Lachmann and Frontonia leucas Ehrenberg, were investigated at 8.5° C, 15° C and 20° C using Cartesian diver microrespirometry. Q
10 values of 1.15–2.24 were found for four of the species between 8.5–15° C, while in S. teres a Q
10 of 12.98 occurred between these temperatures. Between 15–20° C T. pyriformis and P. aurelia had Q
10 values of 3.73 and 1.56, respectively. Linear double log regressions of oxygen consumption vs. dry weight were derived at each temperature and regression coefficients (b) of 0.2723 (8.5° C), 0.4364 (15° C) and 0.4171 (20° C) were obtained. The results are explained and discussed in relation to previous work on the energetics of ciliated protozoa. 相似文献
998.
Marcus Clauss Tanja Froeschle Johanna Castell Jean-Michel Hatt Sylvia Ortmann W. Jürgen Streich Jürgen Hummel 《Acta theriologica》2005,50(3):367-376
The mean retention time (MRT) of ingesta in the gastrointestinal tract is one of the major determinants of herbivore digestive
physiology. We examined MRTs of fluids and particles in the gastrointestinal tract of six adult captive black rhinocerosesDiceros bicornis on conventional zoo diets. Fluid MRT ranged from 25–45 h and averaged 31 h. Particle MRT ranged from 28–59 h and averaged
38 h. In the six animals, both fluid and particle MRT declined as relative dry matter intake (g/kg metabolic body mass) increased.
Black rhinoceroses, which are large hindgut-fermenting browsers, retained ingesta for a shorter period relative to their body
size than grazing equids or grazing rhinoceros species. Our findings support the hypothesis that browsing hindgut fermenters
have relatively shorter MRTs than grazing hindgut fermenters. 相似文献
999.
Structural characteristics and chemical composition of birch (Betula pendula) leaves are modified by increasing CO2 and ozone 总被引:1,自引:0,他引:1
Elina Oksanen Johanna Riikonen†‡ Seija Kaakinen‡ Toini Holopainen† Elina Vapaavuori‡ 《Global Change Biology》2005,11(5):732-748
Impacts of ozone and CO2 enrichment, alone and in combination, on leaf anatomical and ultrastructural characteristics, nutrient status and cell wall chemistry in two European silver birch (Betula pendula Roth) clones were studied. The young soil‐growing trees were exposed in open‐top chambers over three growing seasons to 2 × ambient CO2 and/or ozone concentrations in central Finland. The trees were measured for changes in altogether 35 variables of leaf structure, nutrients and cell wall chemistry of green leaves, and 20 of the measured variables were affected by CO2 and/or O3. Elevated CO2 increased the size of chloroplasts and starch grains, number of mitochondria, P : N ratio, and contents of cell wall hemicellulose. Elevated CO2 decreased the total leaf thickness, specific leaf area, concentrations of N, K, Cu, S and Fe, and contents of cell wall α‐cellulose, uronic acids, acid‐soluble lignin and acetone‐soluble extractives. Elevated ozone led to thinner leaves, higher palisade to spongy ratio, increased number of peroxisomes and mitochondria, reduced content of Mn, Zn, Cu, hemicellulose and uronic acids, and lower Mn : N and Zn : N ratios. In the combined exposure, interactions were antagonistic. Ultrastructural changes became more evident towards the end of the exposure. Young leaves were tolerant against ozone‐caused oxidative stress, whereas oxidative H2O2 accumulation was found in older leaves. CO2 enrichment improved ozone tolerance not only through increased photosynthesis rates, but also through changes in cell wall chemistry (hemicellulose, in particular). However, nutrient imbalances due to ozone and/or CO2 may predispose the trees to other biotic and abiotic stresses. Down‐regulation and up‐regulation of photosynthesis under elevated CO2 through anatomical changes is discussed. 相似文献
1000.
Meshalkina LE Kochetov GA Brauer J Hübner G Tittmann K Golbik R 《Biochemical and biophysical research communications》2008,366(3):692-697
Transketolase from Saccharomyces cerevisiae exhibits a rarely reported activity with a methylated analogue of the native cofactor, 4′-methylamino-thiamin diphosphate. We demonstrated the kinetic stability of the dihydroxyethyl carbanion/enamine intermediate to be dependent on the functionality of the 4′-aminopyrimidine moiety of thiamin diphosphate [R. Golbik, L.E. Meshalkina, T. Sandalova, K. Tittmann, E. Fiedler, H. Neef, S. König, R. Kluger, G.A. Kochetov, G. Schneider, G. Hübner, Effect of coenzyme modification on the structural and catalytic properties of wild-type transketolase and of the variant E418A from Saccharomyces cerevisae, FEBS J. (2005) 272 1326-1342]. This paper extends these investigations of the function of the coenzyme’s aminopyrimidine in transketolase catalysis exemplified for the 4′-monomethylamino-thiamin diphosphate analogue. Here, we report near UV circular dichroism data and NMR-based analysis of reaction intermediates that give evidence for a strong destabilisation of the carbanion/enamine of DHE-4’-monomethylamino-thiamin diphosphate on the enzyme. A new negative band in near UV circular dichroism arising during turnover is attributed to the conjugate acid of the carbanion/enamine intermediate, an assignment additionally corroborated by 1H NMR-based intermediate analysis. As opposed to the kinetically stabilized carbanion/enamine intermediate in transketolase when reconstituted with the native cofactor, DHE-4′-monomethylamino-thiamin diphosphate is rapidly released from the active centers during turnover and accumulates in the medium on a preparative scale. 相似文献