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201.
Bhattacharya AK Stolz F Kurzeck J Rüger W Schmidt RR 《Bioorganic & medicinal chemistry》2002,10(4):1129-1136
This paper describes SAR directed design and synthesis of novel beta(1-4)-glucosyltransferase (BGT) inhibitors. The designed inhibitors 1-5 provide conformational mimicry of the transition-state in glucosyltransfer reactions. The compounds were tested for in vitro inhibitory activity against (BGT) and the inhibition kinetics were examined. Three of the designed molecules were found to be potential inhibitors of BGT having IC50 values in micromolar (microM) range. Useful structure-activity relationships were established, which provide guidelines for the design of future generations of inhibitors of BGT. 相似文献
202.
Cruz HJ Conradt HS Dunker R Peixoto CM Cunha AE Thomaz M Burger C Dias EM Clemente J Moreira JL Rieke E Carrondo MJ 《Journal of biotechnology》2002,96(2):169-183
The production, purification and stability of quality (in terms of integrity and glycosylation) of an antibody/interleukin-2 fusion protein with potential application in tumour-targeted therapy expressed in BHK21 cells are described. Consistency of the product throughout time was determined by analysis of glycosylation of the fusion protein using MALDI-TOF mass spectroscopy and HPAEC-PAD combined with product integrity studies by SDS-PAGE and Western blotting. These investigations showed consistent expression in terms of integrity and of three major oligosaccharide structures of the fusion protein after 62 generations. The data obtained at this stage indicated the suitability of the cell line for production purposes. Different approaches for the production of this protein were subsequently carried out. The relative productivity of the recombinant fusion protein and general performance of the cells in two different protein-free medium (PFM) culture systems, continuous chemostat and continuous perfusion using a Centritech centrifuge as a cell retention device, were studied. The results indicate that the chemostat culture resulted in more stable and controllable nutrient environment, which could indicate better product consistency, in accordance with what has been observed under serum-containing conditions, in relation to the perfusion culture. Finally, product obtained from the chemostat culture was analysed and purified. The purification process was optimised with an increase in the overall yield from 38 to 70% being obtained, a significant improvement with important consequences for the implementation of an industrial-scale culture system. In conclusion, it was possible to produce and purify the recombinant antibody/interleukin-2 fusion protein assuring the quality and stability of the product in terms of integrity and glycosylation. Therefore, a candidate production process was established. 相似文献
203.
Masson P Schopfer LM Bartels CF Froment MT Ribes F Nachon F Lockridge O 《Biochimica et biophysica acta》2002,1594(2):313-324
Substrate inhibition is considered a defining property of acetylcholinesterase (AChE), whereas substrate activation is characteristic of butyrylcholinesterase (BuChE). To understand the mechanism of substrate inhibition, the pH dependence of acetylthiocholine hydrolysis by AChE was studied between pH 5 and 8. Wild-type human AChE and its mutants Y337G and Y337W, as well as wild-type Bungarus fasciatus AChE and its mutants Y333G, Y333A and Y333W were studied. The pH profile results were unexpected. Instead of substrate inhibition, wild-type AChE and all mutants showed substrate activation at low pH. At high pH, there was substrate inhibition for wild-type AChE and for the mutant with tryptophan in the pi-cation subsite, but substrate activation for mutants containing small residues, glycine or alanine. This is particularly apparent in the B. fasciatus AChE. Thus a single amino acid substitution in the pi-cation site, from the aromatic tyrosine of B. fasciatus AChE to the alanine of BuChE, caused AChE to behave like BuChE. Excess substrate binds to the peripheral anionic site (PAS) of AChE. The finding that AChE is activated by excess substrate supports the idea that binding of a second substrate molecule to the PAS induces a conformational change that reorganizes the active site. 相似文献
204.
Neglia G Gasparrini B Di Palo R De Rosa C Zicarelli L Campanile G 《Theriogenology》2003,60(1):125-133
The aim in this study was to compare two estrus synchronization protocols in buffaloes. Animals were divided into two groups: Group A (n=111) received 100 microg GnRH on Day 0, 375 microg PGF(2alpha) on Day 7 and 100 microg GnRH on Day 9 (Ovsynch); Group B (n=117) received an intravaginal drug release device (PRID) containing 1.55 g progesterone and a capsule with 10mg estradiol benzoate for 10 days and were treated with a luteolytic dose of PGF(2alpha) and 1000 IU PMSG at the time of PRID withdrawal. Animals were inseminated twice 18 and 42 h after the second injection of GnRH (Group A) and 60 and 84 h after PGF(2alpha) and PMSG injections (Group B). Progesterone (P(4)) concentrations in milk samples collected 12 and 2 days before treatments were used to determine cyclic and non-cyclic buffaloes, and milk P(4) concentrations 10 days after Artificial insemination (AI) were used as an index of a functional corpus luteum. Cows were palpated per rectum at 40 and 90 days after AI to determine pregnancies. All previously non-cyclic animals in Group B had elevated P(4) (>120 pg/ml milk whey) on Day 10 after AI. Accordingly, a greater (P<0.01) relative percentage of animals with elevated P(4) 10 days after AI were observed in Group B (93.2%) than in Group A (81.1%). However, there was no difference in overall pregnancy rates between the two estrus synchronization protocols (Group A, 36.0%; Group B 28.2%). When only animals with elevated P(4) on Day 10 after AI were considered, pregnancy rate was higher (P<0.05) for animals in Group A (44.4%) than Group B (30.3%). The findings indicated that treatment with PRID can induce ovulation in non-cyclic buffalo cows. However, synchronization of estrus with Ovsynch resulted in a higher pregnancy rate compared with synchronization with PRID, particularly in cyclic buffalo. 相似文献
205.
206.
Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis 总被引:20,自引:0,他引:20
Schaible UE Winau F Sieling PA Fischer K Collins HL Hagens K Modlin RL Brinkmann V Kaufmann SH 《Nature medicine》2003,9(8):1039-1046
Protective immunity against Mycobacterium tuberculosis involves major histocompatibility complex class I (MHC-I)- and CD1-restricted CD8 T cells, but the mechanisms underlying antigen delivery to antigen-presenting molecules remain enigmatic. Macrophages, the primary host cells for mycobacteria, are CD1-negative. Here we show that M. tuberculosis phagosomes are secluded from the cytosolic MHC-I processing pathway and that mycobacteria-infected cells lose their antigen-presenting capacity. We also show that mycobacteria induce apoptosis in macrophages, causing the release of apoptotic vesicles that carry mycobacterial antigens to uninfected antigen-presenting cells (APCs). Inhibition of apoptosis reduced transfer of antigens to bystander cells and activation of CD8 T cells. Uninfected dendritic cells, which engulfed extracellular vesicles, were indispensable for subsequent cross-presentation of antigens, through MHC-I and CD1b, to T cells from mycobacteria-sensitized donors. This new 'detour' pathway for presentation of antigens from a phagosome-contained pathogen shows the functional significance of infection-induced apoptosis in the activation of CD8 T cells specific for both protein and glycolipid antigens in tuberculosis. 相似文献
207.
We demonstrate broad-field, non-scanning, two-photon excitation fluorescence (2PEF) close to a glass/cell interface by total internal reflection of a femtosecond-pulsed infrared laser beam. We exploit the quadratic intensity dependence of 2PEF to provide non-linear evanescent wave (EW) excitation in a well-defined sample volume and to eliminate scattered background excitation. A simple model is shown to describe the resulting 2PEF intensity and to predict the effective excitation volume in terms of easily measurable beam, objective and interface properties. We demonstrate non-linear evanescent wave excitation at 860 nm of acridine orange-labelled secretory granules in live chromaffin cells, and excitation at 900 nm of TRITC-phalloidin-actin/GPI-GFP double-labelled fibroblasts. The confined excitation volume and the possibility of simultaneous multi-colour excitation of several fluorophores make EW 2PEF particularly advantageous for quantitative microscopy, imaging biochemistry inside live cells, or biosensing and screening applications in miniature high-density multi-well plates.Abbreviations 1PEF one-photon excited fluorescence - 2PEF two-photon excited fluorescence - APD avalanche photo diode - CHO Chinese hamster ovary - DMEM Dulbecco's modified Eagle's medium - EGFP enhanced green fluorescent protein - EW evanescent wave - FCS fetal calf serum - GPI glycosylphosphatidylinositol - TIR total internal reflectionThis paper is dedicated to the memory of Prof. Horst Harreis (1940–2002) 相似文献
208.
209.
210.
Silvestre MD Lagarda MJ Farré R Martínez-Costa C Brines J Molina A Clemente G 《Biological trace element research》2000,76(3):217-227
The aim of this study was to establish the possible effects of the sampling protocol (between-breast, within-feed, and diurnal
differences) and the mother’s personal factors (age, parity, iron supple-mentation, smoking habits, and lactation period)
on the copper, iron, and zinc contents in human milk.
One hundred thirty-six human milk samples identified by their origin and sampling conditions were analyzed. The samples were
obtained from the 2nd to 15th d postpartum from 62 women. The data on the individuals required for the study were available.
Mineral determinations were analyzed by flame atomic absorption spectrometry following a standarized protocol.
The results showed that iron contents were higher in hind-milk samples and at the nighttime feeding and depended on the breast
from which the sample was taken. The copper and zinc concentrations showed no significant variations. There was no significant
relationship among the mothers’ age, parity, smoking habits, iron supplementation, and copper content. Milk from older women
had lower zinc contents than that of younger women. Increased amounts of iron were found in multiparous women. Between colostrum
and transitional milk, a sharp decrease in zinc content was observed, whereas copper and iron contents remained constant.
All of these results make it clear that standardized sampling protocols are needed in order to obtain comparable values. 相似文献