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41.
Ventini DC Damiani R Sousa AP de Oliveira JE Peroni CN Ribela MT Bartolini P Tonso A Soares CR Pereira CA 《Applied biochemistry and biotechnology》2011,164(4):401-409
Since the recombinant thyroid-stimulating hormone (rhTSH) is secreted by stably transfected Chinese hamster ovary (CHO-hTSH)
cells, a bioprocess consisting of immobilizing the cells on a substrate allowing their multiplication is very suitable for
rhTSH recovering from supernatants at relative high degree of purity. In addition, such a system has also the advantage of
easily allowing delicate manipulations of culture medium replacement. In the present study, we show the development of a laboratory
scale bioprocess protocol of CHO-hTSH cell cultures on cytodex microcarriers (MCs) in a 1 L bioreactor, for the preparation
of rhTSH batches in view of structure/function studies. CHO-hTSH cells were cultivated on a fetal bovine serum supplemented
medium during cell growth phase. For rhTSH synthesis phase, 75% of supernatant was replaced by animal protein-free medium
every 24 h. Cell cultures were monitored for agitation (rpm), temperature (°C), dissolved oxygen (% DO), pH, cell concentration,
MCs coverage, glucose consumption, lactate production, and rhTSH expression. The results indicate that the amount of MCs in
the culture and the cell concentration at the beginning of rhTSH synthesis phase were crucial parameters for improving the
final rhTSH production. By cultivating the CHO-hTSH cells with an initial cell seeding of four cells/MC on 4 g/L of MCs with
a repeated fed batch mode of operation at 40 rpm, 37 °C, 20% DO, and pH 7.2 and starting the rhTSH synthesis phase with 3 × 106 cells/mL, we were able to supply the cultures with enough glucose, to maintain low levels of lactate, and to provide high
percent (∼80%) of fully covered MCs for a long period (5 days) and attain a high cell concentration (∼9 × 105 cells/mL). The novelty of the present study is represented by the establishment of cell culture conditions allowing us to
produce ∼1.6 mg/L of rhTSH in an already suitable degree of purity. Batches of produced rhTSH were purified and showed biological
activity. 相似文献
42.
Sándor Bátori Dorottya Csányi Daniella Takács Orsolya Egyed Zsuzsanna Riedl György Hajós 《Tetrahedron》2019,75(2):180-185
N-aminopyridyl ketone salts were reacted with formamide to yield heteroaromatic pyrido[2,1-f][1,2,4]triazinium salts. Upon storage of these products in the presence of water, formation of covalent hydrates have been observed. Reaction of the same starting compound with urethane yielded 3-chloropyrido[2,1-f][1,2,4]triazinium salt which readily reacted with secondary amines to afford 3-amino derivatives. An analogous ring closure reaction of 2-formylaminomethyl- and formaminobenzylpyridine allowed the synthesis of the partially reduced 3,4-dihydropyrido[2,1-f][1,2,4]triazinium compounds. The cyclization procedure was also applied for the synthesis of the related pyrimido[2,1-f][1,2,4]triazinium salt. 相似文献
43.
Dr. Arina Dalaloyan Dr. Andrea Martorana Dr. Yoav Barak Diana Gataulin Prof. Eitan Reuveny Dr. Andrew Howe Prof. Michael Elbaum Dr. Shira Albeck Dr. Tamar Unger Dr. Veronica Frydman Dr. Elwy H. Abdelkader Prof. Gottfried Otting Prof. Daniella Goldfarb 《Chemphyschem》2019,20(14):1860-1868
It is an open question whether the conformations of proteins sampled in dilute solutions are the same as in the cellular environment. Here we address this question by double electron-electron resonance (DEER) distance measurements with Gd(III) spin labels to probe the conformations of calmodulin (CaM) in vitro, in cell extract, and in human HeLa cells. Using the CaM mutants N53C/T110C and T34C/T117C labeled with maleimide-DOTA-Gd(III) in the N- and C-terminal domains, we observed broad and varied interdomain distance distributions. The in vitro distance distributions of apo-CaM and holo-CaM in the presence and absence of the IQ target peptide can be described by combinations of closed, open, and collapsed conformations. In cell extract, apo- and holo-CaM bind to target proteins in a similar way as apo- and holo-CaM bind to IQ peptide in vitro. In HeLa cells, however, in the presence or absence of elevated in-cell Ca2+ levels CaM unexpectedly produced more open conformations and very broad distance distributions indicative of many different interactions with in-cell components. These results show-case the importance of in-cell analyses of protein structures. 相似文献
44.
Goldfarb D Lipkin Y Potapov A Gorodetsky Y Epel B Raitsimring AM Radoul M Kaminker I 《Journal of magnetic resonance (San Diego, Calif. : 1997)》2008,194(1):8-15
The set-up of a new microwave bridge for a 95 GHz pulse EPR spectrometer is described. The virtues of the bridge are its simple and flexible design and its relatively high output power (0.7 W) that generates pi pulses of 25 ns and a microwave field, B(1)=0.71 mT. Such a high B(1) enhances considerably the sensitivity of high field double electron-electron resonance (DEER) measurements for distance determination, as we demonstrate on a nitroxide biradical with an interspin distance of 3.6 nm. Moreover, it allowed us to carry out HYSCORE (hyperfine sublevel-correlation) experiments at 95 GHz, observing nuclear modulation frequencies of 14N and 17O as high as 40 MHz. This opens a new window for the observation of relatively large hyperfine couplings, yet not resolved in the EPR spectrum, that are difficult to observe with HYSCORE carried out at conventional X-band frequencies. The correlations provided by the HYSCORE spectra are most important for signal assignment, and the improved resolution due to the two dimensional character of the experiment provides 14N quadrupolar splittings. 相似文献
45.
Augusti DV Augusti R Carazza F Cooks RG 《Chemical communications (Cambridge, England)》2002,(19):2242-2243
Rapid and simple chiral analysis of thalidomide solutions is demonstrated by using electrospray ionization tandem mass spectrometry and analysis of cluster ion dissociation by the kinetic method. Average deviations of 1% between the actual and experimental enantiomeric compositions are observed. 相似文献
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47.
The addition of dialkylphosphites to methylvinylketone giving dialkyl‐3‐oxobutylphos‐ phonates was studied applying different reagents, such as NaOR/ROH, NaOH/H2O under PTC, DBU, and R3Al (R = Me, Et) under different conditions to find the optimum choice regarding efficiency and selectivity. Possible extensions to a few other model compounds were also investigated. © 2007 Wiley Periodicals, Inc. Heteroatom Chem 18:226–229, 2007; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/hc.20266 相似文献
48.
Cibele B. Zamboni Sabrina Metairon Laura C. Oliveira Simone M. Simons A. M. Chudzinski-Tavassi Daniella G. L. Oliveira 《Journal of Radioanalytical and Nuclear Chemistry》2012,291(2):433-437
The determination of elemental concentrations for Br, Ca, Cl, Fe, K, Mg, Na, S, Sr, and Zn in blood samples from White New Zealand rabbits was performed applying the NAA technique. Twenty whole blood samples (12 male and 8 female) collected in research centers from Brazil (Aggeu Magalhães in Recife and Butantan Institute in São Paulo) were investigated, using the IEA-R1 nuclear reactor at IPEN/CNEN-SP-Brazil. These data can be used as references to perform biochemistry analyses in veterinary medicine using small quantities of whole blood (100–400 μL), simplifying the collection and the preparation of biological samples (it is not necessary to perform the serum separation nor to use specific reactants). Furthermore, the knowledge of the biochemical values in blood allows us to check the similarities with the blood estimations in human beings, which is an important condition for selecting laboratory animals. Finally, these data suggest a great similarity of the inorganic tissue profile of rabbits (White New Zealand) and humans. 相似文献
49.
Daniella?G.?L.?Oliveira Simone?M.?Simons Ana?M.?Chudzinski-Tavassi Cibele?B.?ZamboniEmail author 《Journal of Radioanalytical and Nuclear Chemistry》2012,291(2):385-388
The Amblyomma cajennense tick species is considered one of the most important and widespread species in Brazil. It salivary secretion has been a target of several studies in biocenology, as the vector of diseases and in investigations related to antihemostatic properties and antitumor. To complement this investigation, neutron activation analysis (NAA) was applied to determine concentrations of elements in saliva samples of this tick species. The saliva samples (50–554 μL) were collected at Butantan Institute (São Paulo city, Brazil) and they were investigated using the IEA-R1 nuclear reactor at IPEN/CNEN-SP-Brazil. These data were compared with the values established for Amblyomma americanum and Amblyomma variegatum species emphasizing agreement only for Cl, K and Na with the A. americanum species, suggesting similarities in the mechanisms that regulate the osmotic pressure in this hematophagous animal. The knowledge of these limits contributes for tick saliva characterization as well as for the understanding of the many physiological processes, especially those related to salivary secretion. 相似文献
50.
Klein EL Raitsimring AM Astashkin AV Rajapakshe A Johnson-Winters K Arnold AR Potapov A Goldfarb D Enemark JH 《Inorganic chemistry》2012,51(3):1408-1418
In our previous study of the fatal R160Q mutant of human sulfite oxidase (hSO) at low pH (Astashkin et al. J. Am. Chem. Soc.2008, 130, 8471-8480), a new Mo(V) species, denoted "species 1", was observed at low pH values. Species 1 was ascribed to a six-coordinate Mo(V) center with an exchangeable terminal oxo ligand and an equatorial sulfate group on the basis of pulsed EPR spectroscopy and (33)S and (17)O labeling. Here we report new results for species 1 of R160Q, based on substitution of the sulfur-containing ligand by a phosphate group, pulsed EPR spectroscopy in K(a)- and W-bands, and extensive density functional theory (DFT) calculations applied to large, more realistic molecular models of the enzyme active site. The combined results unambiguously show that species 1 has an equatorial sulfite as the only exchangeable ligand. The two types of (17)O signals that are observed arise from the coordinated and remote oxygen atoms of the sulfite ligand. A typical five-coordinate Mo(V) site is compatible with the observed and calculated EPR parameters. 相似文献