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951.
Katja Conrath Alice S. Pereira Carlos E. Martins Cristina G. Timóteo Pedro Tavares Silvia Spinelli Joerg Kinne Christophe Flaudrops Christian Cambillau Serge Muyldermans Isabel Moura Jose J. G. Moura Mariella Tegoni Aline Desmyter 《Protein science : a publication of the Protein Society》2009,18(3):619-628
Nitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N2O. NOR is composed of two subunits: the large one (NorB) is a bundle of 12 transmembrane helices (TMH). It contains a b type heme and a binuclear iron site, which is believed to be the catalytic site, comprising a heme b and a non-hemic iron. The small subunit (NorC) harbors a cytochrome c and is attached to the membrane through a unique TMH. With the aim to perform structural and functional studies of NOR, we have immunized dromedaries with NOR and produced several antibody fragments of the heavy chain (VHHs, also known as nanobodies™). These fragments have been used to develop a faster NOR purification procedure, to proceed to crystallization assays and to analyze the electron transfer of electron donors. BIAcore experiments have revealed that up to three VHHs can bind concomitantly to NOR with affinities in the nanomolar range. This is the first example of the use of VHHs with an integral membrane protein. Our results indicate that VHHs are able to recognize with high affinity distinct epitopes on this class of proteins, and can be used as versatile and valuable tool for purification, functional study and crystallization of integral membrane proteins. 相似文献
952.
Sónia Carneiro António L. Amaral Ana C. A. Veloso Teresa Dias António M. Peres Eugénio C. Ferreira Isabel Rocha 《Biotechnology progress》2009,25(3):882-891
The development of monitoring methods for assessing the physiological state of microorganisms during recombinant fermentation processes has been encouraged by the need to evaluate the influence of processing conditions in recombinant protein production. In this work, a technique based on microscopy and image analysis was developed that allows the simultaneous quantification of parameters associated with viability and fluorescent protein production in recombinant Escherichia coli fermentations. Images obtained from light microscopy with phase contrast are used to assess the total number of cells in a given sample and, from epifluorescence microscopy, both protein producing and injured cells are evaluated using two different fluorochromes: propidium iodide and enhanced yellow fluorescent protein. This technique revealed the existence of different cell populations in the recombinant E. coli fermentation broth that were evaluated along four batch fermentations, complementing information obtained with standard techniques to study the effects of the temperature and induction time in recombinant protein production processes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 相似文献
953.
954.
Kunjan R. Dave Richard Anthony DeFazio Ami P. Raval Oleksandr Dashkin Isabel Saul Kimberly E. Iceman† Miguel A. Perez-Pinzon‡ Kelly L. Drew† 《Journal of neurochemistry》2009,110(4):1170-1179
During the pre-hibernation season, arctic ground squirrels (AGS) can tolerate 8 min of asphyxial cardiac arrest (CA) without detectable brain pathology. Better understanding of the mechanisms regulating innate ischemia tolerance in AGS has the potential to facilitate the development of novel prophylactic agents to induce ischemic tolerance in patients at risk of stroke or CA. We hypothesized that neuroprotection in AGS involves robust maintenance of ion homeostasis similar to anoxia-tolerant turtles. Ion homeostasis was assessed by monitoring ischemic depolarization (ID) in cerebral cortex during CA in vivo and during oxygen glucose deprivation in vitro in acutely prepared hippocampal slices. In both models, the onset of ID was significantly delayed in AGS compared with rats. The epsilon protein kinase C (εPKC) is a key mediator of neuroprotection and inhibits both Na+ /K+ -ATPase and voltage-gated sodium channels, primary mediators of the collapse of ion homeostasis during ischemia. The selective peptide inhibitor of εPKC (εV1-2) shortened the time to ID in brain slices from AGS but not in rats despite evidence that εV1-2 decreased activation of εPKC in brain slices from both rats and AGS. These results support the hypothesis that εPKC activation delays the collapse of ion homeostasis during ischemia in AGS. 相似文献
955.
956.
John R. Collette Richard J. Chi Douglas R. Boettner Isabel M. Fernandez-Golbano Rachael Plemel Alex J. Merz Maria Isabel Geli Linton M. Traub Sandra K. Lemmon 《Molecular biology of the cell》2009,20(14):3401-3413
Clathrin is involved in vesicle formation in the trans-Golgi network (TGN)/endosomal system and during endocytosis. Clathrin recruitment to membranes is mediated by the clathrin heavy chain (HC) N-terminal domain (TD), which forms a seven-bladed β-propeller. TD binds membrane-associated adaptors, which have short peptide motifs, either the clathrin-box (CBM) and/or the W-box; however, the importance of the TD binding sites for these motifs has not been tested in vivo. We investigated the importance of the TD in clathrin function by generating 1) mutations in the yeast HC gene (CHC1) to disrupt the binding sites for the CBM and W-box (chc1-box), and 2) four TD-specific temperature-sensitive alleles of CHC1. We found that TD is important for the retention of resident TGN enzymes and endocytosis of α-factor; however, the known adaptor binding sites are not necessary, because chc1-box caused little to no effect on trafficking pathways involving clathrin. The Chc1-box TD was able to interact with the endocytic adaptor Ent2 in a CBM-dependent manner, and HCs encoded by chc1-box formed clathrin-coated vesicles. These data suggest that additional or alternative binding sites exist on the TD propeller to help facilitate the recruitment of clathrin to sites of vesicle formation. 相似文献
957.
Helen Treichel Marcio Antonio Mazutti Francisco Maugeri Maria Isabel Rodrigues 《Journal of industrial microbiology & biotechnology》2009,36(7):895-900
A potential application of inulinase in the food industry is the production of fructoligosaccharides (FOS) by the transfructosilation
of sucrose. The FOSs present many interesting functional properties besides their ability to increase the shelf-life and flavor
of many products. The use of an industrial medium represents a good alternative to producing inulinase at low cost, since
the activity may improve, or at least remain the same, as that obtained using a synthetic medium. This work was an optimization
study of the inulinase production by Kluyveromyces marxianus NRRL Y-7571 using industrial pre-treated culture medium in a bioreactor employing a sequential strategy of experimental design.
Initially, a Plackett–Burman (Screening Design) design was used, where the studied variables were molasses, corn steep liquor,
yeast extract concentration, and agitation and aeration rates. After the analysis of the effects, a central composite rotational
design (CCRD) was carried out. The optimized condition for the inulinase production was: 250 g/l of molasses, 80 g/l of corn
steep liquor, 6 g/l of yeast extract, 300 rpm of agitation and 1.5 vvm aeration rate, which resulted in an enzymatic activity
of 1,317 ± 65 U/ml. 相似文献
958.
Willeke de Haan Jitske de Vries-van der Weij Isabel M. Mol Menno Hoekstra Johannes A. Romijn J. Wouter Jukema Louis M. Havekes Hans M.G. Princen Patrick C.N. Rensen 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2009,1791(3):191-197
Pregnane X receptor (PXR) agonism has been shown to affect multiple steps in both the synthesis and catabolism of HDL, but its integrated effect on HDL metabolism in vivo remains unclear. The aim of this study was to evaluate the net effect of PXR agonism on HDL metabolism in ApoE?3-Leiden (E3L) and E3L.CETP mice, well-established models for human-like lipoprotein metabolism. Female mice were fed a diet with increasing amounts of the potent PXR agonist 5-pregnen-3β-ol-20-one-16α-carbonitrile (PCN). In E3L and E3L.CETP mice, PCN increased liver lipids as well as plasma cholesterol and triglycerides. However, whereas PCN increased cholesterol contained in large HDL-1 particles in E3L mice, it dose-dependently decreased HDL-cholesterol in E3L.CETP mice, indicating that CETP expression dominates the effect of PCN on HDL metabolism. Analysis of the hepatic expression of genes involved in HDL metabolism showed that PCN decreased expression of genes involved in HDL synthesis (Abca1, Apoa1), maturation (Lcat, Pltp) and clearance (Sr-b1). The HDL-increasing effect of PCN, observed in E3L mice, is likely caused by a marked decrease in hepatic SR-BI protein expression, and completely reversed by CETP expression. We conclude that chronic PXR agonism dose-dependently reduces plasma HDL-cholesterol in the presence of CETP. 相似文献
959.
Morales Pedraza J Sánchez Noda EO Rodríguez Cardona RL Otero I 《Cell and tissue banking》2009,10(2):149-152
The first multi-tissue bank was founded at Havana in 1958. At that time, freeze-drying was used at the bank as a method of
preserving, as well as Cobalt 60 irradiation to sterilise bone tissue, heart valves and others. The impact of the IAEA program
in tissue banking activities in Cuba can be summarised as follows: (a) Increase in the production of sterilised tissues using
ionising radiation (bone, pig skin and amnion) for medical treatment in the tissue bank of the Hospital Frank Pais; (b) increase
of the quality of the productions of bone tissues, pig skin and amnion; (c) reduction in the import of tissues by increasing
the local production of tissues; (d) sustainability in the number of donors through the implementation of a public and professional
awareness campaign; (e) training of six persons in the Regional Training Centre of Buenos Aires; (f) qualification of one
person in the administration of a tissue bank and in the implementation of a Quality System. The amount of tissues produced
and sterilised using the ionising radiation techniques in the established banks was 25,510 units. The amount of patients treated
with sterilised tissues produced by the established banks was 2,448. 相似文献
960.
de Souza MI Salgueiro F Carnavale-Bottino M Félix DB Alves-Ferreira M Bittencourt JV Margis R 《Genetics and molecular biology》2009,32(3):546-556
Habitat fragmentation and a decrease in population size may lead to a loss in population genetic diversity. For the first time, the reduction in genetic diversity in the northernmost limit of natural occurence (southeastern Brazil) of Araucaria angustifolia in comparison with populations in the main area of the species continuous natural distribution (southern Brazil), was tested. The 673 AFLPs markers revealed a high level of genetic diversity for the species (Ht = 0.27), despite anthropogenic influence throughout the last century, and a decrease of H in isolated populations of southeastern Brazil (H = 0.16), thereby indicating the tendency for higher genetic diversity in remnant populations of continuous forests in southern Brazil, when compared to natural isolated populations in the southeastern region. A strong differentiation among southern and southeastern populations was detected (AMOVA variance ranged from 10%-15%). From Bayesian analysis, it is suggested that the nine populations tested form five "genetic clusters" (K = 5). Five of these populations, located in the northernmost limit of distribution of the species, represent three "genetic clusters". These results are in agreement with the pattern of geographic distribution of the studied populations. 相似文献