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981.
982.
983.
Isolation of Vibrio vulnificus during winter months is difficult due to the entrance of these cells into the viable but nonculturable (VBNC) state. While several studies have investigated in vitro gene expression upon entrance into and persistence within the VBNC state, to our knowledge, no in situ studies have been reported. We incubated clinical and environmental isolates of V. vulnificus in estuarine waters during winter months to monitor the expression of several genes during the VBNC state and compared these to results from in vitro studies. katG (periplasmic catalase) was down-regulated during the VBNC state in vitro and in situ compared to the constitutively expressed gene tufA. Our results indicate that the loss of catalase activity we previously reported is a direct result of katG repression, which likely accounts for the VBNC response of this pathogen. While expression of vvhA (hemolysin) was detectable in environmental strains during in situ incubation, it ceased in all cases by ca. 1 h. These results suggest that the natural role of hemolysin in V. vulnificus may be in osmoprotection and/or the cold shock response. Differences in expression of the capsular genes wza and wzb were observed in the two recently reported genotypes of this species. Expression of rpoS, encoding the stress sigma factor RpoS, was continuous upon entry into the VBNC state during both in situ and in vitro studies. We found the half-life of mRNA to be less than 60 minutes, confirming that mRNA detection in these VBNC cells is a result of de novo RNA synthesis. 相似文献
984.
Marchès O Batchelor M Shaw RK Patel A Cummings N Nagai T Sasakawa C Carlsson SR Lundmark R Cougoule C Caron E Knutton S Connerton I Frankel G 《Journal of bacteriology》2006,188(8):3110-3115
EspF of enteropathogenic Escherichia coli targets mitochondria and subverts a number of cellular functions. EspF consists of six putative Src homology 3 (SH3) domain binding motifs. In this study we identified sorting nexin 9 (SNX9) as a host cell EspF binding partner protein, which binds EspF via its amino-terminal SH3 region. Coimmunoprecipitation and confocal microscopy showed specific EspF-SNX9 interaction and non-mitochondrial protein colocalization in infected epithelial cells. 相似文献
985.
Schlenker O Hendricks A Sinning I Wild K 《The Journal of biological chemistry》2006,281(13):8898-8906
The eukaryotic signal recognition particle (SRP) and its receptor (SR) play a central role in co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum. The SR is a heterodimeric complex assembled by the two GTPases SRalpha and SRbeta, which is membrane-anchored. Here we present the 2.45-A structure of mammalian SRbeta in its Mg2+ GTP-bound state in complex with the minimal binding domain of SRalpha termed SRX. SRbeta is a member of the Ras-GTPase superfamily closely related to Arf and Sar1, while SRX belongs to the SNARE-like superfamily with a fold also known as longin domain. SRX binds to the P loop and the switch regions of SRbeta-GTP. The binding mode and structural similarity with other GTPase-effector complexes suggests a co-GAP (GTPase-activating protein) function for SRX. Comparison with the homologous yeast structure and other longin domains reveals a conserved adjustable hydrophobic surface within SRX which is of central importance for the SRbeta-GTP:SRX interface. A helix swap in SRX results in the formation of a dimer in the crystal structure. Based on structural conservation we present the SRbeta-GTP:SRX structure as a prototype for conserved interactions in a variety of GTPase regulated targeting events occurring at endomembranes. 相似文献
986.
Kupzig S Deaconescu D Bouyoucef D Walker SA Liu Q Polte CL Daumke O Ishizaki T Lockyer PJ Wittinghofer A Cullen PJ 《The Journal of biological chemistry》2006,281(15):9891-9900
GAP1(IP4BP) is a member of the GAP1 family of Ras GTPase-activating proteins (Ras GAPs) that includes GAP1(m), CAPRI, and RASAL. Composed of a central Ras GAP domain, surrounded by amino-terminal C(2) domains and a carboxyl-terminal pleckstrin homology/Bruton's tyrosine kinase domain, GAP1(IP4BP) has previously been shown to possess an unexpected GAP activity on the Ras-related protein Rap, besides the predicted Ras GAP activity (Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P., and Irvine, R. F. (1995) Nature 376, 527-530). Here we have shown that GAP1(IP4BP) is indeed an efficient Ras/Rap GAP, having K(m)s of 213 and 42 microm and estimated k(cat)s of 48 and 16 s(-1) for Ras and Rap, respectively. For this dual activity, regions outside the Ras GAP domain are required, as the isolated domain (residues 291-569) retains a pronounced Ras GAP activity yet has very low activity toward Rap. Interestingly, mutagenesis of the Ras GAP arginine finger, and surrounding residues important in Ras binding, inhibit both Ras and Rap GAP activity of GAP1(IP4BP). Although the precise details by which GAP1(IP4BP) can function as a Rap GAP remain to be determined, these data are consistent with Rap associating with GAP1(IP4BP) through the Ras-binding site within the Ras GAP domain. Finally, we have established that such dual Ras/Rap GAP activity is not restricted to GAP1(IP4BP). Although GAP1(m) appears to constitute a specific Ras GAP, CAPRI and RASAL display dual activity. For CAPRI, its Rap GAP activity is modulated upon its Ca(2+)-induced association with the plasma membrane. 相似文献
987.
A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change 总被引:7,自引:0,他引:7 下载免费PDF全文
Genetically encoded calcium biosensors have become valuable tools in cell biology and neuroscience, but some aspects such as signal strength and response kinetics still need improvement. Here we report the generation of a FRET-based calcium biosensor employing troponin C as calcium-binding moiety that is fast, is stable in imaging experiments, and shows a significantly enhanced fluorescence change. These improvements were achieved by engineering magnesium and calcium-binding properties within the C-terminal lobe of troponin C and by the incorporation of circularly permuted variants of the green fluorescent protein. This sensor named TN-XL shows a maximum fractional fluorescence change of 400% in its emission ratio and linear response properties over an expanded calcium regime. When imaged in vivo at presynaptic motoneuron terminals of transgenic fruit flies, TN-XL exhibits highly reproducible fluorescence signals with the fastest rise and decay times of all calcium biosensors known so far. 相似文献
988.
989.
With the completion of the major genome projects, one focus in biomedical research has shifted from the analysis of the rather static genome to the highly dynamic proteome. The sequencing of whole genomes did not lead to much anticipated insights into disease mechanisms; however, it paved the way for proteomics by providing the databases for protein identification by peptide mass fingerprints. The relative protein distribution within a cell or tissue is subject to change upon external and internal stimuli. Signal transduction events extend beyond a simple change in protein levels; rather they are governed by posttranslational modifications (PTMs), which provide a quick and efficient way to modulate cellular signals. Because most PTMs change the mass of a protein, they are amenable to analysis by mass spectrometry. Their investigation adds a level of functionality to proteomics, which can be expected to greatly aid in the understanding of the complex cellular machinery involved in signal transduction, metabolism, differentiation or in disease. This review provides an overview on posttranslational modifications exemplified on the model system cAMP-dependent protein kinase. Strategies for detection of selected PTMs are described and discussed in the context of protein kinase function. 相似文献
990.
Hermes S Bremm O Garczarek F Derrien V Liebisch P Loja P Sebban P Gerwert K Haumann M 《Biochemistry》2006,45(2):353-359
Previous time-resolved FTIR measurements suggested the involvement of an intermediary component in the electron transfer step Q(A)- --> Q(B) in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides [Remy and Gerwert (2003) Nat. Struct. Biol. 10, 637]. By a kinetic X-ray absorption experiment at the Fe K-edge we investigated whether oxidation occurs at the ferric non-heme iron located between the two quinones. In isolated reaction centers with a high content of functional Q(B), at a time resolution of 30 micros and at room temperature, no evidence for transient oxidation of Fe was obtained. However, small X-ray transients occurred, in a similar micro- to millisecond time range as in the IR experiments, which may point to changes in the Fe ligand environment due to the charges on Q(A)- and Q(B)-. In addition, VIS measurements agree with the IR data and do not exclude an intermediate in the Q(A)- --> Q(B) transition. 相似文献