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Yeast System (yeast + system)
Selected AbstractsUncoupling proteins 2 and 3 interact with members of the 14.3.3 familyFEBS JOURNAL, Issue 9 2000Benoit Pierrat Uncoupling proteins (UCPs) are members of the superfamily of the mitochondrial anion carrier proteins (MATP). Localized in the inner membrane of the organelle, they are postulated to be regulators of mitochondrial uncoupling. UCP2 and 3 may play an important role in the regulation of thermogenesis and, thus, on the resting metabolic rate in humans. To identify interacting proteins that may be involved in the regulation of the activity of UCPs, the yeast two-hybrid system was applied. Segments of hUCP2 containing the hydrophilic loops facing the intermembrane space, or combinations of these, were used to screen an adipocyte activation domain (AD) fusion library. The 14.3.3 protein isoforms ,, ,, , were identified as possible interacting partners of hUCP2. Screening of a human skeletal muscle AD fusion library, on the other hand, yielded several clones all of them encoding the , isoform of the 14.3.3 family. Mapping experiments further revealed that all these 14.3.3 proteins interact specifically with the C-terminal intermembrane space domain of both hUCP2 and hUCP3 whereas no interactions could be detected with the C-terminal part of hUCP1. Direct interaction between UCP3 and 14.3.3 , could be demonstrated after in vitro translation by coimmunoprecipitation. When coexpressed in a heterologous yeast system, 14.3.3 proteins potentiated the inhibitory effect of UCP3 overexpression on cell growth. These findings suggest that 14.3.3 proteins could be involved in the targeting of UCPs to the mitochondria. [source] Analysis of the human APC mutation spectrum in a saccharomyces cerevisiae strain with a mismatch repair defectINTERNATIONAL JOURNAL OF CANCER, Issue 5 2003Kazunori Otsuka Abstract Somatic APC mutations in colorectal tumors with an RER phenotype reflect excessive frameshift mutations, especially in simple repetition tracts within the coding sequence. Because this type of mutation is characteristic of cells with a deficient DNA MMR system, the APC mutation signature of RER tumors may be attributable to a defect in the MMR system. However, there is little experimental evidence to prove that the spectrum of mutations and the APC gene distribution are directly influenced by MMR system defects. We therefore examined the mutation spectrum of the MCR of the APC gene after transfection into both MMR-proficient and MMR-deficient yeast strains and compared it with a previously reported human APC mutation database. Small insertions or deletions in mono- or dinucleotide repeats were more common in the MMR-deficient than in the MMR-proficient strain (91.2% vs. 38.1%, Fisher's exact test p < 0.0001). Furthermore, the 2 mutation hot spots, 4385,4394(AG)5 and 4661,4666(A)6, found in the yeast system corresponded with those in human tumors. Combining our data with those from human tumors, there appears to be hypermutable mutations in specific simple repetitive sequences within the MCR, which are more prevalent in MMR-deficient cells and RER tumors than in MMR-proficient cells and non-RER tumors. We therefore consider that the differences in the spectra of RER and non-RER tumors are attributable at least in part to the MMR system of the host cells. © 2002 Wiley-Liss, Inc. [source] Optimization of the expression of recombinant human activin A in the yeast Pichia pastorisBIOTECHNOLOGY PROGRESS, Issue 2 2010Dale Fredericks Abstract We report a new procedure to express recombinant human activin A using the methanolic yeast, Pichia pastoris. Optimization of culture procedures has involved comprehensive examination of the effects of culture vessel shape, volume of broth in the induction and expression cultures, methanol concentration, culturing temperature, and pH of the expression cultures. After this optimization, as well as modification of the native cleavage sites, a laboratory scale procedure has been established which routinely produced 2,10 mg/L amounts of this vital growth factor in the highly efficient, eukaryotic yeast system. This system avoids the need to produce this protein and similar TGF-, proteins in mammalian cell lines which, in addition to being costly, produce many native binding partners of these cystine knot proteins, a factor which can dramatically affect yields of the target protein. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Characterization of peach thaumatin-like proteins and their identification as major peach allergensCLINICAL & EXPERIMENTAL ALLERGY, Issue 9 2010A. Palacín Summary Background Peach is the most important fruit related to food allergy in the Mediterranean area. Pru p 3, its lipid transfer protein, has been described as the principal allergen responsible for cross-reactivities with other foods and pollen and the severity of clinical symptoms. However, the involvement of other allergenic families cannot be ruled out. Thaumatin-like proteins (TLPs) have been described as food allergen in several fruits, such as apple, cherry, kiwi and banana, and pollen. Objective To identify members of the TLP family in peach fruit and to characterize putative allergens. Methods Through two-dimensional (2D) electrophoresis of peach extract and immunodetections with a pool of peach-allergic patients, IgE-binding spots were identified and the corresponding proteins purified and characterized as allergens by in vitro and in vivo assays. Three isoforms, belonging to the TLP family, were purified by different chromatographic systems and characterized by N -terminal amino acid sequences, molecular weight determination (MALDI) and enzymatic activity analysis (,-1,3-gluconase test and inhibition growth of fungi). In the same way, their IgE-binding capacity and allergenic activity were tested by ELISA assays, basophil activation tests and skin prick tests (SPT). Results Two peach-TLPs, Pru p 2.0101 and Pru p 2.0201, were identified as IgE-binding spots by 2D electrophoresis. Another peach-TLP, Pru p 2.0301, was cloned and produced as recombinant protein in a yeast system. The three isoforms were purified and characterized as TLPs by immunoblotting with anti-chestnut TLP antibodies and anti-plant N -asparagine complex glycan (anti-cross-reactive carbohydrate determinant). All of them showed ,-1,3-glucanase activity and inhibition of fungal growth. The three TLPs were recognized by around 50% of the sera from 31 patients analysed in ELISA experiments. All three gave a positive response to an SPT and/or in basophil activation experiments. Conclusion Three isoforms, belonging to the TLP family, were identified in peach as principal allergens. Their prevalence, observed in in vitro, ex vivo and in vivo analyses, suggests that they are important allergens and should therefore be included in the routine diagnosis of peach allergy, at least in the Mediterranean area. Cite this as: A. Palacín, L. Tordesillas, P. Gamboa, R. Sanchez-Monge, J. Cuesta-Herranz, M. L. Sanz, D. Barber, G. Salcedo and A. Díaz-Perales, Clinical & Experimental Allergy, 2010 (40) 1422,1430. [source] Plant profilin isovariants are distinctly regulated in vegetative and reproductive tissuesCYTOSKELETON, Issue 1 2002Muthugapatti K. Kandasamy Abstract Profilin is a low-molecular weight, actin monomer-binding protein that regulates the organization of actin cytoskeleton in eukaryotes, including higher plants. Unlike the simple human or yeast systems, the model plant Arabidopsis has an ancient and highly divergent multi-gene family encoding five distinct profilin isovariants. Here we compare and characterize the regulation of these profilins in different organs and during microspore development using isovariant-specific monoclonal antibodies. We show that PRF1, PRF2, and PRF3 are constitutive, being strongly expressed in all vegetative tissues at various stages of development. These profilin isovariants are also predominant in ovules and microspores at the early stages of microsporogenesis. In contrast, PRF4 and PRF5 are late pollen-specific and are not detectable in other cell types of the plant body including microspores and root hairs. Immunocytochemical studies at the subcellular level reveal that both the constitutive and pollen-specific profilins are abundant in the cytoplasm. In vegetative cell types, such as root apical cells, profilins showed localization to nuclei in addition to the cytoplasmic staining. The functional diversity of profilin isovariants is discussed in light of their spatio-temporal regulation during vegetative development, pollen maturation, and pollen tube growth. Cell Motil. Cytoskeleton 52:22,32, 2002. © 2002 Wiley-Liss, Inc. [source] Devil inside: does plant programmed cell death involve the endomembrane system?PLANT CELL & ENVIRONMENT, Issue 9 2010JEAN-LUC CACAS ABSTRACT Eukaryotic cells have to constantly cope with environmental cues and integrate developmental signals. Cell survival or death is the only possible outcome. In the field of animal biology, tremendous efforts have been put into the understanding of mechanisms underlying cell fate decision. Distinct organelles have been proven to sense a broad range of stimuli and, if necessary, engage cell death signalling pathway(s). Over the years, forward and reverse genetic screens have uncovered numerous regulators of programmed cell death (PCD) in plants. However, to date, molecular networks are far from being deciphered and, apart from the autophagic compartment, no organelles have been assigned a clear role in the regulation of cellular suicide. The endomembrane system (ES) seems, nevertheless, to harbour a significant number of cell death mediators. In this review, the involvement of this system in the control of plant PCD is discussed in-depth, as well as compared and contrasted with what is known in animal and yeast systems. [source] Dynamic organization of COPII coat proteins at endoplasmic reticulum export sites in plant cellsTHE PLANT JOURNAL, Issue 6 2009Sally L. Hanton Summary Protein export from the endoplasmic reticulum (ER) is mediated by the accumulation of COPII proteins such as Sar1, Sec23/24 and Sec13/31 at specialized ER export sites (ERES). Although the distribution of COPII components in mammalian and yeast systems is established, a unified model of ERES dynamics has yet to be presented in plants. To investigate this, we have followed the dynamics of fluorescent fusions to inner and outer components of the coat, AtSec24 and AtSec13, in three different plant model systems: tobacco and Arabidopsis leaf epidermis, as well as tobacco BY-2 suspension cells. In leaves, AtSec24 accumulated at Golgi-associated ERES, whereas AtSec13 showed higher levels of cytosolic staining compared with AtSec24. However, in BY-2 cells, both AtSec13 and AtSec24 labelled Golgi-associated ERES, along with AtSec24. To correlate the distribution of the COPII coat with the dynamics of organelle movement, quantitative live-cell imaging analyses demonstrated that AtSec24 and AtSec13 maintained a constant association with Golgi-associated ERES, irrespective of their velocity. However, recruitment of AtSec24 and AtSec13 to ERES, as well as the number of ERES marked by these proteins, was influenced by export of membrane cargo proteins from the ER to the Golgi. Additionally, the increased availability of AtSec24 affected the distribution of AtSec13, inducing recruitment of this outer COPII coat component to ERES. These results provide a model that, in plants, protein export from the ER occurs via sequential recruitment of inner and outer COPII components to form transport intermediates at mobile, Golgi-associated ERES. [source] | |||||||||||||