F-box/WD-repeat proteins pop1p and Sud1p/Pop2p form complexes that bind and direct the proteolysis of cdc18p.
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Ubiquitin-dependent proteolysis plays an important role in cell-cycle control [1] [2]. In budding yeast, the protein Skp1p, the cullin-family member Cdc53p, and the F-box/WD-repeat protein Cdc4p form the SCFCdc4p ubiquitin ligase complex, which targets the cyclin-dependent kinase (Cdk) inhibitor Sic1p for proteolysis [3] [4] [5] [6] [7] [8]. Sic1p is recruited to the SCFCdc4p complex by binding to the WD-repeat region of Cdc4p [5] [6], while Skp1p binds to the F-box of Cdc4p [9]. In fission yeast, two distinct Cdc4p-related proteins, Pop1p/Ste16p [10] [11] and the recently identified Sud1p/Pop2p [12], regulate the stability of the replication initiator Cdc18p and the Cdk inhibitor Rum1p. We show here that, despite their structural and functional similarities, the pop1 and pop2 genes fail to complement each other's deletion phenotypes, indicating that they perform non-redundant, but potentially interdependent, functions in proteolysis. Consistent with this hypothesis, Pop1p and Pop2p formed heterooligomeric complexes when overexpressed, and binding of Cdc18p to Pop2p was dependent on Pop1p. The Pop1p-Pop2p interaction was mediated by the amino-terminal domain of Pop2p which, when fused to full-length Pop1p, rescued the phenotype of a Deltapop1Deltapop2 double mutant. Thus, close physical proximity of two distinct F-box/WD-repeat proteins directs proteolysis mediated by the SCFPop ubiquitin ligase complex. (+info)
Computational studies on mutant protein stability: The correlation between surface thermal expansion and protein stability.
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Thermal stability of mutant proteins has been investigated using temperature dependent molecular dynamics (MD) simulations in vacuo. The numerical modeling was aimed at mimicking protein expansion upon heating. After the conditions for an expanding protein accessible surface area were established for T4 lysozyme and barnase wild-type proteins, MD simulations were carried out under the same conditions using the crystal structures of several mutant proteins. The computed thermal expansion of the accessible surface area of mutant proteins was found to be strongly correlated with their experimentally measured stabilities. A similar, albeit weaker, correlation was observed for model mutant proteins. This opens the possibility of obtaining stability information directly from protein structure. (+info)
Molecular dissection of mitogillin reveals that the fungal ribotoxins are a family of natural genetically engineered ribonucleases.
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Mitogillin and the related fungal ribotoxins are highly specific ribonucleases which inactivate the ribosome enzymatically by cleaving the 23-28 S RNA of the large ribosomal subunit at a single phosphodiester bond. The site of cleavage occurs between G4325 and A4326 (rat ribosome numbering) which are present in one of the most conserved sequences (the alpha-sarcin loop) among the large subunit ribosomal RNAs of all living species. Amino acid sequence comparison of ribotoxins and guanyl/purine ribonucleases have identified domains or residues likely involved in ribonucleolytic activity or cleavage specificity. Fifteen deletion mutants (each 4 to 8 amino acid deletions) in motifs of mitogillin showing little amino acid sequence homology with guanyl/purine ribonucleases were constructed by site-directed mutagenesis. Analyses of the purified mutant proteins identified those regions in fungal ribotoxins contributing to ribosome targeting and modulating the catalytic activity of the toxin; some of the identified motifs are homologous to sequences in ribosomal proteins and elongation factors. This mutational study of mitogillin together with the recently published x-ray structure of restrictocin (a close relative of mitogillin) supports the hypothesis that the specific cleavage properties of ribotoxins are the result of natural genetic engineering in which the ribosomal targeting elements of ribosome-associated proteins were inserted into nonessential regions of T1-like ribonucleases. (+info)
Detection of viable Listeria monocytogenes with a 5' nuclease PCR assay.
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A 5' nuclease assay has been developed to detect viable Listeria monocytogenes. The assay targets the hemolysin A (hlyA) transcript, which is found only in L. monocytogenes. The single-tube, reverse transcriptase (RT), fluorogenic probe-based assay was formatted by using Tth DNA polymerase whose activity was modulated by using the manganese-chelating morpholinepropanesulfonic acid (MOPS) buffer. This assay was quantitative over a 3-log-unit range of template concentrations when tested with an in vitro-transcribed hlyA mRNA. The viability of L. monocytogenes was reduced by heating at various temperatures and times up to a maximum of a 9-D inactivation. The location of the primer had a pronounced effect on the utility of the assay, and primers located in the most distal regions of the hlyA transcript appeared to correlate with the number of CFU while primers located more internal on the amplicon overestimated the cell viability. The assay with primers that included the 3' end of the transcript was an accurate indicator of viability as measured by CFU determination or staining with 5-sulfofluorescein diacetate. (+info)
Adhesive explant culture of allergic nasal mucosa: effect of emedastine difumarate, an anti-allergic drug.
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Allergic reaction of the nose comprises of an immediate and a late reaction. To evaluate nasal allergic reactions, many experiments have been performed by investigators. In this study, we performed a new tissue culture technique (adhesive explant culture) to analyze the migration of cells into the culture medium from the cultured allergic nasal mucosa in response to an allergen. Basophilic cells (mast cells and basophils) and eosinophils, which were released into the culture medium after the allergen challenge, were evaluated by the analysis of histamine and eosinophil cationic protein (ECP) content in the culture medium. Histamine and basophilic cells in the culture medium were more abundant in the immediate phase (within 30 min) after challenge than in the late phase (from 30 min to 10 hr). On the other hand, ECP and eosinophils in the culture medium were more abundant in the late phase than in the immediate phase. The increase of histamine content in both phases were not inhibited by pre-treatment of emedastine difumarate (EME), an anti-allergic drug. However, the increase of ECP in the late phase was inhibited by pre-treatment with EME. Moreover, the number of EG2-positive cells was also decreased by pre-treatment with EME. These results suggest that EME might lower the activation of eosinophils in the late phase of the allergic reaction. The present study also indicates that this adhesive explant culture system is useful model for studying the cellular allergic responses to drugs ex vivo. (+info)
Expression in vitro of alternatively spliced variants of the messenger RNA for human apolipoprotein E receptor-2 identified in human tissues by ribonuclease protection assays.
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The apolipoprotein E receptor-2 (apoER2), also called LR7/8B, is a member of the low-density lipoprotein (LDL)-receptor family that is expressed in brain. We have identified mRNA splicing variants in human tissues by ribonuclease protection assays and found that some variants are preferentially amplified by reverse transcription-polymerase chain reaction (RT-PCR). Transcripts were found that lacked sequences encoding three repeats in the putative ligand-binding domain, the O-linked sugar domain or a novel region in the cytoplasmic domain. When mammalian expression vectors for eight potential protein isoforms were transfected into LDL-receptor-deficient Chinese hamster ovary cells, the proteins were all expressed on the cell surface, as detected by immunoblotting of cell extracts with a specific antipeptide antiserum to apoER2 before and after treatment of intact cells with pronase. Although cells expressing all the variants bound very low-density lipoprotein of beta mobility (beta-VLDL), it was with lower affinity and capacity than binding by the LDL-receptor and none was able to degrade beta-VLDL. Ligand blotting of cell extracts showed that all variants bound recombinant histidine6-tagged receptor-associated protein (His6-RAP) with high affinity, although variants lacking exon 5 bound less strongly. The presence of vestiges of the novel insert in the cytoplasmic domain of apoER2 in the LDL- or VLDL-receptor genes was investigated, but nucleotide sequencing showed that no sequences homologous to it could be detected in the final intron of these genes. (+info)
Diagnosing occupational asthma: use of induced sputum.
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The diagnosis of occupational asthma (OA) needs to be made with as much objective evidence as possible. If there is airway inflammation, measurement of this should be an asset. The objective of this study was to investigate whether there is an increase in induced sputum and blood eosinophils and eosinophil cationic protein (ECP) in OA after work exposure. Patients were assessed after a 2-4 week period at work and away from work with cell counts and ECP assays performed blind to the clinical data. They were considered to have OA if symptoms were worse at work and there was a fall in forced expiratory volume in one second (FEV1) > or =20% or in the provocative concentration of methacholine causing a 20% fall in FEV1 (PC20) of four-fold or more compared with away from work. Patients whose symptoms were worse at work but had a change in FEV1 of <20% and in methacholine PC20 of less than four-fold were considered as controls. Sixteen patients were studied. Ten had OA and six were controls. Patients with OA had a significant increase in median (interquartile range) sputum eosinophils and ECP when at work compared with the periods out of work, 10.0 (17.05) versus 0.8 (1.6)% (p=0.007) and 3,840 (6,076) versus 116 (180) microg x L(-1) (p=0.01). They also had a higher blood eosinophil count, 0.3 (0.5) x 10(9) versus 0.2 (0.1) x 10(9) x L(-1) (p=0.013), and a trend towards higher serum ECP levels, 44.0 (20.0) versus 32.0 (18.5) microg x L(-1) (p=0.07). In conclusion, the proportion of eosinophils and levels of eosinophil cationic protein in sputum are particularly high at work in patients with occupational asthma, suggesting that the measurement of these factors can supplement other physiological outcomes in establishing the diagnosis of occupational asthma. (+info)
Alpha2-macroglobulin and eosinophil cationic protein in the allergic airway mucosa in seasonal allergic rhinitis.
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As previously demonstrated in seasonal allergic rhinitis, increased microvascular permeability and eosinophil activation are key features of allergic airway inflammation. In the present study, the hypothesis that exudation of alpha2-macroglobulin may cause the appearance of eosinophil cationic protein (ECP) in the airway lumen was explored. Nasal lavages were carried out using the nasal pool device before and during the pollen season both at baseline and after histamine challenge in 10 children with allergic rhinitis. Nasal lavage fluid levels of alpha2-macroglobulin and ECP were determined. All patients experienced nasal symptoms of allergic rhinitis during the pollen season (p<0.01-0.05). Baseline nasal lavage fluid levels of alpha2-macroglobulin and ECP were increased during the season (p<0.01-0.05) and were found to be well correlated (p<0.0001). Histamine produced concentration-dependent plasma exudation before and during the pollen season, but it was only during the pollen season that this caused an increase in the lavage fluid levels of ECP (p<0.05). These data suggest that exudation of plasma and increased tissue levels and output of eosinophil cationic protein characterize nasal mucosal inflammation in children with seasonal allergic rhinitis. The plasma exudation process in part may account for lumenal entry of eosinophil cationic protein molecules that have been released in mucosal tissue compartments. A combination of induced exudation and nasal lavage may improve the yield of important markers of inflammation in studies of nasal diseases. (+info)