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71.
72.
PTP-MEG2 is an intracellular protein tyrosine phosphatase with a putative lipid-binding domain at the N-terminus. The present study reports expression, purification, and characterization of the full-length form of the enzyme plus a truncated form containing the catalytic domain alone. Full-length PTP-MEG2 was expressed with an adenovirus system and purified from cytosolic extracts of human 293 cells infected with the recombinant adenovirus. The purification scheme included chromatographic separation of cytosolic extracts on fast flow Q-Sepharose, heparin-agarose, l-histidyldiazobenzylphosphonic acid agarose, and hydroxylapatite. The enrichment of PTP-MEG2 from the cytosol was about 120-fold. The truncated form of PTP-MEG2 was expressed in E. coli cells as a non-fusion protein and purified by using a chromatographic procedure similar to that used for the full-length enzyme. The purified full-length and truncated enzymes showed single polypeptide bands on SDS-polyacrylamide gel electrophoresis under reducing conditions and behaved as monomers on gel exclusion chromatography. With para-nitrophenylphosphate and phosphotyrosine as substrates, both forms of the enzyme exhibited classical Michaelis-Menten kinetics. Their responses to pH, ionic strength, metal ions, and protein phosphatase inhibitors are similar to those observed with other characterized tyrosine phosphatases. Compared with full-length PTP-MEG2, the truncated DeltaPTP-MEG2 displayed significantly higher V(max) and lower K(m) values, suggesting that the N-terminal putative lipid-binding domain may have an inhibitory role. The full-length and truncated forms of PTP-MEG2 were also expressed as GST fusion proteins in E. coli cells and purified to near homogeneity through affinity columns. However, the specific phosphatase activities of the GST fusion proteins were 10-25-fold below those obtained with the correspondent non-fusion proteins. 相似文献
73.
Xu X Shi YC Gao W Mao G Zhao G Agrawal S Chisolm GM Sui D Cui MZ 《The Journal of biological chemistry》2002,277(50):48913-48922
Recent studies have suggested a possible role for presenilin proteins in apoptotic cell death observed in Alzheimer's disease. The mechanism by which presenilin proteins regulate apoptotic cell death is not well understood. Using the yeast two-hybrid system, we previously isolated a novel protein, presenilin-associated protein (PSAP) that specifically interacts with the C terminus of presenilin 1 (PS1), but not presenilin 2 (PS2). Here we report that PSAP is a mitochondrial resident protein sharing homology with mitochondrial carrier protein. PSAP was detected in a mitochondria-enriched fraction, and PSAP immunofluorescence was present in a punctate pattern that colocalized with a mitochondrial marker. More interestingly, overexpression of PSAP caused apoptotic death. PSAP-induced apoptosis was documented using multiple independent approaches, including membrane blebbing, chromosome condensation and fragmentation, DNA laddering, cleavage of the death substrate poly(ADP-ribose) polymerase, and flow cytometry. PSAP-induced cell death was accompanied by cytochrome c release from mitochondria and caspase-3 activation. Moreover, the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, which blocked cell death, did not block the release of cytochrome c from mitochondria caused by overexpression of PSAP, indicating that PSAP-induced cytochrome c release was independent of caspase activity. The mitochondrial localization and proapoptotic activity of PSAP suggest that it is an important regulator of apoptosis. 相似文献
74.
The deltapi vs pi(i) curve from monolayer surface pressure detection is a powerful method to characterize the membrane insertion. The deltapi vs pi(i) curve is fixed by two parameters, the pi(c) and the slope. The intersected point (pi(c)) of deltapi vs pi(i) curve with abscissa is generally used as a quantitative measure of the membrane insertion ability of a protein. In the current work we demonstrate a correlation between the variation in the slope of the deltapi vs pi(i) curve with protein hydrophobic exposure by performing monolayer experiments on three different proteins, human apolipoprotein H, trichosanthin, and mutant trichosanthin. The value of /slope/ increases gradually following the degree of hydrophobic exposure. These findings suggest that the two parameters, the pi(c) and the slope, will complement each other to interpret the lipid/protein interaction involved in membrane insertion. 相似文献
75.
16S ribosomal DNA analysis of the faecal lactobacilli composition of human subjects consuming a probiotic strain Lactobacillus acidophilus NCFM 总被引:1,自引:0,他引:1
AIMS: The aims of this study were to evaluate the ability of exogenous Lactobacillus acidophilus strain NCFM to survive through the human gastro-intestinal (GI) tract, and to evaluate the selectivity of Rogosa SL medium for faecal lactobacilli. METHODS AND RESULTS: The composition of the faecal lactobacilli of 10 healthy subjects was monitored for two weeks prior to, two weeks during and two weeks after the administration of the Lact. acidophilus strain NCFM consumed with skim milk (daily dose 10(10) viable cells). Fresh faecal samples were collected, processed and cultured on Rogosa SL selective medium for lactobacilli enumeration. Colonies demonstrating various morphologies were identified and purified for 16S ribosomal DNA sequence analysis for speciation of colonial genotype. The species composition of cultivable faecal lactobacilli changed considerably during consumption of the strain NCFM. CONCLUSIONS: The probiotic Lact. acidophilus strain NCFM can survive through the human GI tract, but cannot colonize itself during the two-week consumption. Rogosa SL medium is selective for faecal lactobacilli. However, genetic analysis is required for colony speciation. SIGNIFICANCE AND IMPACT OF THE STUDY: It is demonstrated that continuous consumption is necessary to maintain a high population of the probiotic strain, and that the Rogosa SL medium is reliable. 相似文献
76.
Abeta is the core protein of extracellular plaque of Alzheimer's disease, and its neurotoxicity is relative to its conformation. In the current work, the effects of various factors, such as pH, ionic strength and lipid membranes, on the secondary structure of Abeta were studied by circular dichroism. In addition, we detected the exposure of hydrophobic sites of Abeta under different conditions using ANS fluorescence. The results showed that the hydrophobic exposure of the protein was correlated with the content of 3betasheet conformation in the phospholipid-containing environment. The beta-sheet content and hydrophobic exposure of Abeta both increased when reacted with pure PC vesicles, while no beta-sheet content and very low hydrophobic exposure were detected after reaction with 30% cholesterol containing PC vesicles. Since beta-sheet conformation is considered as the toxic conformation of Afbeta such correlation may be important for the pathology of AD. 相似文献
77.
The 100 kDa Type I isozyme of mammalian hexokinase has evolved by duplication and fusion of a gene encoding an ancestral 50 kDa hexokinase. Although the N- and C-terminal halves are similar in sequence, they differ in function, catalytic activity being associated only with the C-terminal half while the N-terminal half serves a regulatory role. The N- and C-terminal halves of rat Type I hexokinase have been coexpressed in M + R 42 cells. The halves associate noncovalently to produce a 100 kDa form that exhibits characteristics seen with the intact Type I isozyme but not with the isolated catalytic C-terminal half, i.e., characteristics that are influenced by interactions between the halves. These include a decreased K(m) for the substrate ATP and the ability of P(i) to antagonize inhibition by Glc-6-P or its analog, 1-5-anhydroglucitol-6-P. Thus, functional interactions between the N- and C-terminal halves do not require their covalent linkage. 相似文献
78.
79.
Building an efficient factory: where is pre-rRNA synthesized in the nucleolus? 总被引:9,自引:0,他引:9 下载免费PDF全文
Huang S 《The Journal of cell biology》2002,157(5):739-741
80.
Gene Expression Dynamics Inspector (GEDI): for integrative analysis of expression profiles 总被引:2,自引:0,他引:2
Genome-wide expression profiles contain global patterns that evade visual detection in current gene clustering analysis. Here, a Gene Expression Dynamics Inspector (GEDI) is described that uses self-organizing maps to translate high-dimensional expression profiles of time courses or sample classes into animated, coherent and robust mosaics images. GEDI facilitates identification of interesting patterns of molecular activity simultaneously across gene, time and sample space without prior assumption of any structure in the data, and then permits the user to retrieve genes of interest. Important changes in genome-wide activities may be quickly identified based on 'Gestalt' recognition and hence, GEDI may be especially useful for non-specialist end users, such as physicians. AVAILABILITY: GEDI v1.0 is written in Matlab, and binary Matlab.dll files which require Matlab to run can be downloaded for free by academic institutions at http://www.chip.org/~ge/gedihome.html Supplementary information: http://www.chip.org/~ge/gedihome.html 相似文献