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Oulehle Filip Wright Richard F. Svoboda Miroslav Bače Radek Matějka Karel Kaňa Jiří Hruška Jakub Couture Raoul-Marie Kopáček Jiří 《Ecosystems》2019,22(4):725-741
Ecosystems - Forest ecosystems worldwide are subjected to human-induced stressors, including eutrophication and acidification, and to natural disturbances (for example, insect infestation,... 相似文献
33.
Erica Mura Joan Edwards Sandra Kittelmann Kerstin Kaerger Kerstin Voigt Jakub Mrázek Giuseppe Moniello Katerina Fliegerova 《Fungal biology》2019,123(3):240-246
Anaerobic fungi are potent fibre degrading microbes in the equine hindgut, yet our understanding of their diversity and community structure is limited to date. In this preliminary work, using a clone library approach we studied the diversity of anaerobic fungi along six segments of the horse hindgut: caecum, right ventral colon (RVC), left ventral colon (LVC), left dorsal colon (LDC), right dorsal colon (RDC) and rectum. Of the 647 ITS1 clones, 61.7 % were assigned to genus level groups that are so far without any cultured representatives, and 38.0 % were assigned to the cultivated genera Neocallimastix (35.1 %), Orpinomyces (2.3 %), and Anaeromyces (0.6 %). AL1 dominated the group of uncultured anaerobic fungi, particularly in the RVC (88 %) and LDC (97 %). Sequences from the LSU clone library analysis of the LDC, however, split into two distinct phylogenetic clusters with low sequence identity to Caecomyces sp. (94–96 %) and Liebetanzomyces sp. (92 %) respectively. Sequences belonging to cultured Neocallimastix spp. dominated in LVC (81 %) and rectum (75.5 %). Quantification of anaerobic fungi showed significantly higher concentrations in RVC and RDC compared to other segments, which influenced the interpretation of the changes in anaerobic fungal diversity along the horse hindgut. These preliminary findings require further investigation. 相似文献
34.
Jean-Marc Bielser Jakub Domaradzki Jonathan Souquet Hervé Broly Massimo Morbidelli 《Biotechnology progress》2019,35(3):e2790
Perfusion cell culture, confined traditionally to the production of fragile molecules, is currently gaining broader attention in the biomanufacturing of therapeutic proteins. The development of these processes is made difficult by the limited availability of appropriate scale-down models. This is due to the continuous operation that requires complex control and cell retention capacity. For example, the determination of an optimal perfusion and bleed rate for continuous cell culture is often performed in scale-down bioreactors and requires a substantial amount of time and effort. To increase the experimental throughput and decrease the required workload, a semi-continuous procedure, referred to as the VCDmax (viable cell density) approach, has been developed on the basis of shake tubes (ST) and deepwell plates (96-DWP). Its effectiveness has been demonstrated for 12 different CHO-K1-SV cell lines expressing an IgG1. Further, its reliability has been investigated through proper comparisons with perfusion runs in lab-scale bioreactors. It was found that the volumetric productivity and the CSPRmin (cell specific perfusion rate) determined using the ST and 96-DWP models were successfully (mostly within the experimental error) confirmed in lab-scale bioreactors, which then covered a significant scale-up from the half milliliter to the liter scale. These scale-down models are very useful to design and scale-up optimal bioreactor operating conditions as well as screening for different media and cell lines. 相似文献
35.
The interactions of two different porphyrins, without axial ligands-5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin-Cu(II) tetrachloride (Cu(II)TMPyP) and with bulky meso substituents-5,10,15,20-tetrakis(N,N,N-trimethylanilinium-4-yl)porphyrin tetrachloride (TMAP), with (dG-dC)10 and (dA-dT)10 were studied by combination of vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopy at different [oligonucleotide]/[porphyrin] ratios, where [oligonucleotide] and [porphyrin] are the concentrations of oligonucleotide per base-pair and porphyrin, respectively. The combination of VCD and ECD spectroscopy enables us to identify the types of interactions, and to specify the sites of interactions: The intercalative binding mode of Cu(II)TMPyP with (dG-dC)(10), which has been well described, was characterized by a new VCD "marker" and it was shown that the interaction of Cu(II)TMPyP with (dA-dT)10 via external binding to the phosphate backbone and major groove binding caused transition from the B to the non-B conformer. TMAP interacted with the major groove of (dG-dC)10, was semi-intercalated into (dA-dT)10, and caused significant variation in the structure of both oligonucleotides at the higher concentration of porphyrin. The spectroscopic techniques used in this study revealed that porphyrin binding with AT sequences caused substantial variation of the DNA structure. It was shown that VCD spectroscopy is an effective tool for the conformational studies of nucleic acid-porphyrin complexes in solution. 相似文献
36.
Grybko MJ Bartnik JP Wurth GA Pores-Fernando AT Zweifach A 《The Journal of biological chemistry》2007,282(25):18009-18017
We have tested the idea that calcineurin, a calcium-dependent phosphatase that is critical for activating cytokine gene expression in helper T cells, plays a role in lytic granule exocytosis in cytotoxic T lymphocytes (CTLs). We used TALL-104 human leukemic CTLs as a model. Our results confirm an earlier report (Dutz, J. P., Fruman, D. A., Burakoff, S. J., and Bierer, B. E. (1993) J. Immunol. 150, 2591-2598) that immunosuppressive drugs inhibit exocytosis in CTLs stimulated either via the T cell receptor (TCR) or via TCR-independent soluble agents. Of the two recently reported alternate targets of immunosuppressive drugs (Matsuda, S., Shibasaki, F., Takehana, K., Mori, H., Nishida, E., and Koyasu, S. (2000) EMBO Rep. 1, 428-434 and Matsuda, S., and Koyasu, S. (2000) Immunopharmacology 47, 119-125), JNK is not required for lytic granule exocytosis, but we were not able to exclude a role for P38. Exocytosis could be inhibited by expressing GFP fused to a C-terminal fragment of CAIN (cabin 1), but not by expressing VIVIT-GFP. Finally, expressing either full-length or truncated constitutively active mutant calcineurin A enhanced lytic granule exocytosis. However, the mutant calcineurin was unable to support exocytosis when cells were stimulated in the absence of Ca2+ influx. Taken together, our results support the idea that activation of calcineurin is required for lytic granule exocytosis but suggest that it is not the sole Ca2+-dependent step. 相似文献
37.
Growth of the young is an important part of the life history in birds. However, modelling methods have paid little attention to the choice of regression model used to describe its pattern. The aim of this study was to evaluate whether a single sigmoid model with an upper asymptote could describe avian growth adequately. We compared unified versions of five growth models of the Richards family (the four‐parameter U‐Richards and the three‐parameter U‐logistic, U‐Gompertz, U‐Bertalanffy and U4‐models) for three traits (body mass, tarsus‐length and wing‐length) for 50 passerine species, including species with varied morphologies and life histories. The U‐family models exhibit a unified set of parameters for all models. The four‐parameter U‐Richards model proved a good choice for fitting growth curves to various traits – its extra d‐parameter allows for a flexible placement of the inflection point. Which of the three‐parameter U‐models was the best performing varied greatly between species and between traits, as each three‐parameter model had a different fixed relative inflection value (fraction of the upper asymptote), implying a different growth pattern. Fixing the asymptotes to averages for adult trait value generally shifted the model preference towards one with lower relative inflection values. Our results illustrate an overlooked difficulty in the analysis of organismal growth, namely, that a single traditional three‐parameter model does not suit all growth data. This is mostly due to differences in inflection placement. Moreover, some biometric traits require more attention when estimating growth rates and other growth‐curve characteristics. We recommend fitting either several three‐parameter models from the U‐family, where the parameters are comparable between models, or only the U‐Richards model. 相似文献
38.
39.
Swiss 3T3 fibroblasts were transfected with the provided plasmids of LacSwitch Inducible Mammalian Expression System (Stratagene). Stable transfectants were selected, expanded and characterised. At first, the production of CAT in these cell lines could be induced by IPTG treatment, but the inducibility was lost after a few months in culture in a reproducible manner. Further analysis revealed that the transfectants did not lose the cat gene nor the lac repressor protein. As a result, we conclude that LacSwitch Inducible Mammalian Expression System needs further modification for use in Swiss 3T3 fibroblasts. 相似文献
40.
Włodarczyk J Stoychev Galitonov G Kierdaszuk B 《European biophysics journal : EBJ》2004,33(5):377-385
Fluorescence and phosphorescence emission spectroscopy were employed to study the interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with its specific inhibitor, formycin A (FA), a close structural analogue of adenosine (natural substrate), in the absence and presence of phosphate (Pi, substrate). Formation of enzyme–FA complexes led to marked quenching of enzyme tyrosine intrinsic fluorescence and phosphorescence, with concomitant increases in fluorescence and phosphorescence of FA. Fluorescence resonance energy transfer from the protein Tyr160 residue to the FA base moiety was identified as a major mechanism of protein fluorescence quenching, increased by addition of Pi. The effects of enzyme–FA interactions on the nucleoside excitation and emission spectra for fluorescence and phosphorescence revealed shifts in the tautomeric equilibrium of the bound FA, i.e. from the N(1)–H tautomer (predominant in solution) to the N(2)–H form, enhanced by the presence of Pi. The latter was confirmed by enzyme–ligand dissociation constant (K
d) values of 5.9±0.4 and 2.1±0.3 M in the absence and presence of Pi, respectively. Addition of glycerol (80%, v/v) led to a lower enzyme affinity (K
d70 M), without changes in binding stoichiometry. Enzyme–FA complex formation led to a higher increase of the fluorescence than the phosphorescence band of the ligand, consistent with the fact that the N(2)–H tautomer is characterized by a weaker phosphorescence than the N(1)–H tautomeric form. These results show, for the first time, the application of phosphorescence spectroscopy to the identification of the tautomeric form of the inhibitor bound by the enzyme.Abbreviations Ado
adenosine
- FA
formycin A [3-(-d-ribofuranosyl)-7-aminopyrazolo[4,3-d]pyrimidine]
- FB
formycin B
- FRET
fluorescence resonance energy transfer
- Guo
guanosine
- Hepes
N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid)
- Ino
inosine
- m1FA
N(1)-methylformycin A
- m2FA
N(2)-methylformycin A
- m4FA
N(4)-methylformycin A
- m6FA
N(6)-methylformycin A
- m7Guo
N(7)-methylguanosine
- Pi
orthophosphate
- PNP
purine nucleoside phosphorylase
- Xao
xanthosine 相似文献