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111.
Sørensen JB Cha A Latorre R Rosenman E Bezanilla F 《The Journal of general physiology》2000,115(2):209-222
When attached outside the voltage-sensing S4 segment of the Shaker potassium channel, the fluorescent probe tetramethylrhodamine (TMRM) undergoes voltage-dependent fluorescence changes (DeltaF) due to differential interaction with a pH-titratable external protein-lined vestibule (Cha, A., and F. Bezanilla. 1998. J. Gen. Physiol. 112:391-408.). We attached TMRM at the same sites [corresponding to M356C and A359C in the wild-type (wt) channel] in a deletion mutant of Shaker where all but the five amino acids closest to S4 had been removed from the S3-S4 linker. In the deletion mutant, the maximal DeltaF/F seen was diminished 10-fold, and the DeltaF at M356C became pH independent, suggesting that the protein-lined vestibule is made up in large part by the S3-S4 linker. The residual DeltaF showed that the probe still interacted with two putative quenching groups near the S4 segment. One group was detected by M356C-TMRM (located outside of S3 in the deletion mutant) and reported on deactivation gating charge movement when applying hyperpolarizing voltage steps from a holding potential of 0 mV. During activating voltage steps from a holding potential of -90 mV, the fluorescence lagged considerably behind the movement of gating charge over a range of potentials. Another putative quenching group was seen by probes attached closer to the S4 and caused a DeltaF at extreme hyperpolarizations (more negative than -90 mV) only. A signal from the interaction with this group in the wt S3-S4 linker channel (at L361C) correlated with gating charge moving in the hyperpolarized part of the Q-V curve. Probe attached at A359C in the deletion mutant and at L361C in wt channel showed a biphasic DeltaF as the probe oscillated between the two groups, revealing that there is a transient state of the voltage sensor in between, where the probe has maximal fluorescence. We conclude that the voltage sensor undergoes two distinct conformational changes as seen from probes attached outside the S4 segment. 相似文献
112.
Monkey (Mk) CD9 antigen has been shown previously to increase the diphtheria toxin (DT) sensitivity of cells when co-expressed with Mk proHB-EGF (DT receptor). We have elucidated here the mechanism whereby Mk CD9 influences Mk proHB-EGF and present evidence that Mk CD9 is a coreceptor for DT. We observed that Mk CD9 not only increased the DT sensitivity but also increased the DT receptor affinity of cells. Furthermore, the higher the Mk CD9/Mk proHB-EGF ratio, the higher the affinity. In contrast, mouse (Ms) CD9 did not increase the toxin sensitivity or receptor affinity of cells when co-expressed with Mk proHB-EGF. Using Mk/Ms chimeric CD9 molecules, we determined that the second extracellular domain of Mk CD9 is responsible for both increased sensitivity and receptor affinity. This domain of Mk CD9 also interacts with Mk proHB-EGF in a yeast two-hybrid system. Our findings thus suggest that Mk CD9 has a direct physical interaction with Mk proHB-EGF to form a DT receptor complex and that this contact may change the conformation of the receptor to increase DT binding affinity and consequently increase toxin sensitivity. We thus propose that Mk CD9 is a coreceptor for DT. 相似文献
113.
Minocycline inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease 总被引:39,自引:0,他引:39
114.
115.
Ivanova EP Romanenko LA Gorshkova NM Chaĭkina EL 《Zhurnal mikrobiologii, epidemiologii, i immunobiologii》2000,(5):22-25
The results of the microbiological monitoring of potential causative agents of sapronoses in the water of the Bogatinskoye reservoir revealed that in the summer period of 1998 the mass accumulation of virulent Aeromonas sobria (up to 25% of the total number of heterotrophic bacteria) took place. The autumn period was characterized by a decrease in the number of A. sobria and the detection of bacteria of the genus Vibrio (up to 22%) with V. mimicus and V. metschnikovii identified among them in the water ecosystems of the southern regions of the Maritime Territory. 相似文献
116.
Human cytomegalovirus (HCMV)-specific monoclonal antibody, SCMVM34, recognizes the early antigen encoded by UL44 of HCMV. This antigen is confined to the nucleus of HCMV-infected cells. This study was performed to characterize the DNA-binding activity of the protein encoded by UL44 of HCMV. The nuclear and cytoskeletal fraction of HCMV-infected cells was subjected to 0.4 M NaCl extraction, DEAE-Sephacel ion exchange chromatography, DNA-cellulose chromatography and SDS-PAGE analysis with monitoring of the reactive protein using SCMVM34 monoclonal antibody. The molecular weights of the resultant proteins were found to be 34, 40 and 52 kDa. The internal peptide fragments were isolated by tryptic digestion and reverse-phase HPLC. The internal amino acid sequence analysis of the peptides from the HPLC profile revealed that the antigen recognized by SCMVM34 monoclonal antibody was ppUL44. The reactive antigen began to be eluted from 250 mM NaCl (Tris-HCl pH 7.4) in DNA cellulose. The 34 kDa protein seems to bind to DEAE more tightly than the 52 kDa protein. The surface charge of 34 kDa might be more basic. Conclusively, the antigen recognized by SCMVM34 was the protein encoded by HCMV UL44, which was localized in the nuclei after HCMV infection, and was the DNA-binding protein with the characteristic that the surface charge of the molecule was more basic, as the molecular weights of the protein were decreased. 相似文献
117.
Antioxidant activity of human serum albumin (HSA) increased steeply as the reaction mixture was shifted from neutral to alkaline pH. The antioxidant activity was also remarkably increased by Ca(2+) or a cationic detergent (cetyltrimethylammonium chloride). Carboxyl group modification of HSA resulted in about 40-fold increase of the antioxidant activity. The chemical modification study indicated that in addition to functional cysteine(s), cationic amino acid residues such as histidine, arginine and lysine appeared to involve in the antioxidant reaction. HSA also exhibited alkaline-pH dependent peroxidase activity to remove fatty acid hydroperoxide. At neutral pH, only two thiols of Cys-289 and free Cys-34 of HSA were modified by a thiol-specific modification reagent, 5-((((2-iodoacetyl)amino)ethy)amino)naphthalene-1-sulfonic acid (I14), regardless of the presence or absence of dithiothreitol (DTT), and the resultant antioxidant activity was not decreased, suggesting that Cys-289 and Cys-34 did not participate in the antioxidant reaction. At alkaline pH, I14 modified several additional HSA thiols in the presence, but did not in the absence of DTT. The antioxidant activity of the modified HSA was remarkably decreased to as much as 30% of the antioxidant activity given by the unmodified HSA in the absence of DTT. The HPLC pattern for tryptic peptides containing modified cysteine(s) derived from the I14-treated c-HSA (carboxyl group-modified HSA) at pH 7.0 with DTT was very similar to that of the I14-modified HSA at pH 8.0 with DTT. Taken together, these results suggest that activation of thiol-dependent antioxidant activity of HSA at alkaline pH is due to the conformational change favorable for the functional cysteine(s)-mediated catalysis. 相似文献
118.
Manoharan K Chae HS Cha JM Cho SH Shin SH Cho BH Lee WS 《Plant & cell physiology》2000,41(10):1143-1148
When carrot suspension cells were cultured on medium containing no carbon source (starvation), the levels of phosphatidylserine (PS) increased transiently 3-4 d after the initiation of starvation while levels of most other phospholipid (PL) species decreased. We previously reported that fatty acids of these PLs served as an alternative carbon source during starvation. The present study showed that cells possess two different biosynthetic pathways involving phosphatidylcholine (PC)/phosphatidylethanolamine (PE) exchange enzymes and PS synthase to synthesize PS. These activities peaked similarly 4 d after the initiation of starvation and coincided with the peak of PS level. The synthesis of serine was also significantly activated during starvation. The activity of phosphoserine aminotransferase (PSAT) which is involved in serine synthesis increased with a time course similar to that of the increase in the PS level. These observations suggest that the increase in PS level plays an important role in membranes which are degraded during starvation. 相似文献
119.
Open reading frame S/L of varicella-zoster virus encodes a cytoplasmic protein expressed in infected cells 下载免费PDF全文
Kemble GW Annunziato P Lungu O Winter RE Cha TA Silverstein SJ Spaete RR 《Journal of virology》2000,74(23):11311-11321
We report the discovery of a novel gene in the varicella-zoster virus (VZV) genome, designated open reading frame (ORF) S/L. This gene, located at the left end of the prototype VZV genome isomer, expresses a polyadenylated mRNA containing a splice within the 3' untranslated region in virus-infected cells. Sequence analysis reveals significant differences between the ORF S/Ls of wild-type and attenuated strains of VZV. Antisera raised to a bacterially expressed portion of ORF S/L reacted specifically with a 21-kDa protein synthesized in cells infected with a VZV clinical isolate and with the original vaccine strain of VZV (Oka-ATCC). Cells infected with other VZV strains, including a wild-type strain that has been extensively passaged in tissue culture and commercially produced vaccine strains of Oka, synthesize a family of proteins ranging in size from 21 to 30 kDa that react with the anti-ORF S/L antiserum. MeWO cells infected with recombinant VZV harboring mutations in the C-terminal region of the ORF S/L gene lost adherence to the stratum and adjacent cells, resulting in an altered plaque morphology. Immunohistochemical analysis of VZV-infected cells demonstrated that ORF S/L protein localizes to the cytoplasm. ORF S/L protein was present in skin lesions of individuals with primary or reactivated infection and in the neurons of a dorsal root ganglion during virus reactivation. 相似文献
120.
The Modified β-Ketoadipate Pathway in Rhodococcus rhodochrous N75: Enzymology of 3-Methylmuconolactone Metabolism 下载免费PDF全文
Rhodococcus rhodochrous N75 is able to metabolize 4-methylcatechol via a modified β-ketoadipate pathway. This organism has been shown to activate 3-methylmuconolactone by the addition of coenzyme A (CoA) prior to hydrolysis of the butenolide ring. A lactone-CoA synthetase is induced by growth of R. rhodochrous N75 on p-toluate as a sole source of carbon. The enzyme has been purified 221-fold by ammonium sulfate fractionation, hydrophobic chromatography, gel filtration, and anion-exchange chromatography. The enzyme, termed 3-methylmuconolactone-CoA synthetase, has a pH optimum of 8.0, a native Mr of 128,000, and a subunit Mr of 62,000, suggesting that the enzyme is homodimeric. The enzyme is very specific for its 3-methylmuconolactone substrate and displays little or no activity with other monoene and diene lactone analogues. Equimolar amounts of these lactone analogues brought about less than 30% (most brought about less than 15%) inhibition of the CoA synthetase reaction with its natural substrate. 相似文献