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931.
Ristriani T Nominé Y Laurent C Weiss E Travé G 《Protein expression and purification》2002,26(3):90-367
Interpretation of protein mutagenesis experiments requires the ability to distinguish functionally relevant mutations from mutations affecting the structure. When a protein is expressed soluble in bacteria, properly folded mutants are expected to remain soluble whereas misfolded mutants should form insoluble aggregates. However, this rule may fail for proteins fused to highly soluble carrier proteins. In a previous study, we analysed the biophysical status of HPV oncoprotein E6 fused to the C-terminus of maltose-binding protein (MBP) and found that misfolded E6 moieties fused to MBP formed soluble aggregates of high molecular weight. By contrast, preparations of properly folded E6 fused to MBP were monodisperse. Here, we have used this finding to evaluate the quality of 19 MBP-fused E6 site-directed mutants by using a light scattering assay performed in a fluorimeter. This assay guided us to rule out structurally defective mutants and to obtain functionally relevant E6 mutants selectively altered for two molecular activities: degradation of tumour suppressor p53 and DNA recognition. 相似文献
932.
933.
p53 is one of the most mutated genes in human cancer. We have performed the molecular characterization of p53 and have searched
for correlations with etiological factors and clinical parameters in primary and secondary liver tumors.
A systematic study was carried out, innovative in many respects, to determine the mutational pattern of all 11 exons of p53
and analysis was extended also to exons 1–4 and 9–11 and the exon/intron junctions.
Our analyses were performed on case histories of 114 patients from the European area and highlighted p53 mutation patterns
different from those reported in the literature for the same tumors. In our case history, different tumors of the same organ
showed a different frequency and distribution of mutations.
In analyzed tumor types, gene status was a prognostic indicator of survival because patients undergoing liver resection without
mutated p53 had a more favorable prognosis than mutated patients. This suggests p53 molecular diagnosis could become a further
criterion in the decision for surgery and possible therapies.
We describe the ideal conditions for polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP), and
direct sequencing, which we have set in order to optimize yields, sensitivity, and time of what might become a massive molecular
screening. 相似文献
934.
The ATP-binding cassette (ABC) transporters are a large family of proteins responsible for the translocation of a variety
of compounds across the membranes of both prokaryotes and eukaryotes. The inter-protein and intra-protein interactions in
these traffic ATPases are still only poorly understood. In the present study we describe, for the first time, an extensive
yeast two-hybrid (Y2H)-based analysis of the interactions of the cytoplasmic loops of the yeast pleiotropic drug resistance
(Pdr) protein, Pdr5p, an ABC transporter of Saccharomyces
cerevisiae. Four of the major cytosolic loops that have been predicted for this protein [including the two nucleotide-binding domain
(NBD)-containing loops and the cytosolic C-terminal region] were subjected to an extensive inter-domain interaction study
in addition to being used as baits to identify potential interacting proteins within the cell using the Y2H system. Results
of these studies have revealed that the first cytosolic loop (CL1) – containing the first NBD domain – and also the C-terminal
region of Pdr5p interact with several candidate proteins. The possibility of an interaction between the CL1 loops of two neighboring
Pdr5p molecules was also indicated, which could possibly have implications for dimerization of this protein.
Electronic Publication 相似文献
935.
Cocco T Cutecchia G Montedoro G Lorusso M 《Journal of bioenergetics and biomembranes》2002,34(4):251-258
A study is presented on the interaction of carvedilol with mitochondria isolated from several rat organs. It is shown that carvedilol causes a moderate uncoupling effect under non phosphorylating succinate supported respiration of intact mitochondria, as well as a marked inhibition of coupled respiration with NAD-dependent substrates. The inhibitory effect was also found in the bovine heart purified Complex I as well as in experiments with mitochondrial particles, where the individual redox segments of the respiratory chain were analysed. It is also shown that carvedilol, though exhibiting an intrinsic scavenger activity, caused reactive oxygen species to be produced as a consequence of its inhibitory effect on the steady-state respiration. Under these conditions the pro-oxidant activity of carvedilol appears to prevail over its scavenging activity, and a net generation of ROS is promoted. 相似文献
936.
Izaurralde E 《European journal of cell biology》2002,81(11):577-584
Fully processed mRNAs are exported to the cytoplasm where they direct protein synthesis. A general feature of mRNA export is that it is an active, receptor-mediated process. The mRNA export receptors are thought to recognize and bind to the mRNA-export cargoes either directly or indirectly (via adaptor proteins) and facilitate their translocation across the central channel of the nuclear pore complex (NPC). On the cytoplasmic side of the NPC, the exported mRNA is released and the receptor returns to the nucleoplasm, without the cargo, to initiate additional rounds of export. Recent, studies in yeast and in higher eukaryotes have led to the elucidation of an evolutionarily conserved pathway for the export of bulk mRNA to the cytoplasm. 相似文献
937.
Calf lens A-crystallin isolated by reversed-phase HPLC demonstrates a slightly more hydrophobic profile than B-crystallin. Fluorescent probes in addition to bis-ANS, like cis-parinaric acid (PA) and pyrene, show higher quantum yields or Ham ratios when bound to A-crystallin than to B-crystallin at room temperature. Bis-ANS binding to both A- and B-crystallin decreases with increase in temperature. At room temperature, the chaperone-like activity of A-crystallin is lower than that of B-crystallin whereas at higher temperatures, A-crystallin shows significantly higher protection against aggregation of substrate proteins compared to B-crystallin. Therefore, calf lens A-crystallin is more hydrophobic than B-crystallin and chaperone-like activity of -crystallin subunits is not quantitatively related to their hydrophobicity. 相似文献
938.
Chie L Friedman FK Kung HF Lin MC Chung D Pincus MR 《Journal of Protein Chemistry》2002,21(5):367-370
We have previously found that a ras switch 1 domain peptide (PNC-7, residues 35–47) selectively blocks oocyte maturation induced by oncogenic (Val 12–containing) ras-p21 protein and also blocks c-raf–induced oocyte maturation. We now find that oncogenic ras-p21 does not inhibit oocyte maturation of a constitutively activated raf protein (raf BXB) that is lacking most of the first 301 amino terminal amino acids, including the major ras binding domain and accessory ras-binding regions. We also find that a dominant negative raf that completely blocks c-raf–induced maturation likewise does not block raf-BXB–induced maturation. We conclude that PNC-7 blocks ras by binding to the amino-terminal domain of raf and that raf BXB must initiate signal transduction in the cytosol. 相似文献
939.
Banerjee T Valacchi G Ziboh VA van der Vliet A 《Molecular and cellular biochemistry》2002,238(1-2):105-110
The present study attempts to establish the isoforms of hyaluronidase enzyme and their possible role in the spreading of toxins during envenomation. Screening of venoms of 15 snakes belonging to three different families revealed varied hyaluronidase activity in ELISA-like assay, but with relatively similar pH and temperature optima. The zymograms of individual venoms showed varied activity banding patterns and indicated the presence of at least two molecular forms of the enzyme. During envenomation, activity of hyaluronidase is considered crucial for the spreading of toxins and is presumed to distort the integrity of extracellular matrix through the degradation of hyaluronic acid in it. This property has been addressed through localization of hyaluronic acid in human skin and muscle tissue sections using the probe, biotinylated hyaluronic acid binding protein. Faint and discontinuous staining pattern of hyaluronidase treated tissue sections over intense staining of untreated tissue sections confirm the selective degradation of hyaluronic acid in extracellular matrix and thus provide an evidence for the spreading property of the enzyme. 相似文献
940.
Higashimoto K Soejima H Yatsuki H Joh K Uchiyama M Obata Y Ono R Wang Y Xin Z Zhu X Masuko S Ishino F Hatada I Jinno Y Iwasaka T Katsuki T Mukai T 《Genomics》2002,80(6):575-584
Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5' CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5' CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5' CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question. 相似文献