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1.
Oxidation of methionine residues is involved in several biochemical processes and in degradation of therapeutic proteins. The relationship between conformational stability and methionine oxidation in recombinant human interleukin-1 receptor antagonist (rhIL-1ra) was investigated to document how thermodynamics of unfolding affect methionine oxidation in proteins. Conformational stability of rhIL-1ra was monitored by equilibrium urea denaturation, and thermodynamic parameters of unfolding (DeltaGH2O, m, and Cm) were estimated at different temperatures. Methionine oxidation induced by hydrogen peroxide at varying temperatures was monitored during "coincubation" of rhIL-1ra with peptides mimicking specific regions of the reactive methionine residues in the protein. The coincubation study allowed estimation of oxidation rates in protein and peptide at each temperature from which normalized oxidation rate constants and activation energies were calculated. The rate constants for buried Met-11 in the protein were lower than for methionine in the peptide with an associated increase in activation energy. The rate constants and activation energy of solvent exposed methionines in protein and peptide were similar. The results showed that conformational stability, monitored using the Cm value, has an effect on oxidation rates of buried methionines. The rate constant of buried Met-11 correlated well with the Cm value but not DeltaGH2O. No correlation was observed for the oxidation rates of solvent-exposed methionines with any thermodynamic parameters of unfolding. The findings presented have implications in protein engineering, in design of accelerated stability studies for protein formulation development, and in understanding disease conditions involving protein oxidation.  相似文献   
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The binding stoichiometry and affinities of the Shiga toxins, Stx1 and Stx2, for a series of uni- and oligovalent analogs of the Pk-trisaccharide were measured using the direct electrospray ionization mass spectrometry (ES-MS) assay. Importantly, it is shown that, for a given ligand, Stx1 and Stx2 exhibit similar affinities. The binding data suggest a high degree of similarity in the spatial arrangement and structural characteristics of the Pk binding sites in Stx1 and Stx2. The results confirm that both toxins recognize the alpha-D-Galp(1-->4)-beta-D-Galp(1-->4)-beta-D-Glcp carbohydrate motif of the cell surface glycolipid Gb3. This, taken together with the results of the chemical mapping study, suggests that the nature of the Pk binding interactions with Stx1 and Stx2 are similar. The affinities of Stx1-B(5) and Stx2 for the multivalent ligands reveals that site 2 of Stx2, which shares the same spatial arrangement as site 2 in Stx1, is the primary Pk binding site and that site 1 of Stx1 and of Stx2 can also participate in Pk binding.  相似文献   
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Histochemical, immunocytochemical and radioassay study was performed to detect the occurrence of NOS‐immunoreactive primary trigeminal sensory somata in the trigeminal ganglion, including their fiber components. Spinal trigeminal tract and sensory trigeminal nuclei were studied using the same methods. It was found that more than 30% of all somata in the trigeminal ganglion are NOS immunoreactive. Corresponding fibers were detected in the spinal trigeminal tract. NOS immunoreactive fibers of three different categories could be followed to terminate in the sensory trigeminal nuclei. Data presented here confirm that trigeminal sensory system is richly endowed with NOS and that NO is used to communicate between the first and second‐order trigeminal sensory neurons. Acknowledgements: Supported by VEGA Grant no. 2/3217/23PS9, STAA Grant no. 51‐013002 and by NIH grants NS 32794 and NS 40386 to M.M.  相似文献   
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Novel activities of bafilomycin A1, a macrolide antibiotic known as an inhibitor of V-ATPases, were discovered. Bafilomycin A1 induced uptake of potassium ions by energized mitochondria and caused mitochondrial swelling, loss of membrane potential, uncoupling of oxidative phosphorylation, inhibition of the maximal respiration rates, and induced pyridine nucleotide oxidation. The mitochondrial effects provoked by nanomolar concentrations of bafilomycin A1 were connected to its activity as a potent, K+-specific ionophore. The K+ ionophoric activity of bafilomycin A1 was observed also in black lipid membranes, indicating that it was an inherent property of the bafilomycin A1 molecule. It was found that bafilomycin A1 is a K+ carrier but not a channel former. Bafilomycin A1 is the first and currently unique macrolide antibiotic with K+ ionophoric properties. The novel properties of bafilomycin A1 may explain some of the biological effects of this plecomacrolide antibiotic, independent of V-ATPase inhibition.  相似文献   
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Bloom (BLM) and Werner (WRN) syndrome proteins are members of the RecQ family of SF2 DNA helicases. In this paper, we show that restricting the rotational DNA backbone flexibility, by introducing vinylphosphonate internucleotide linkages in the translocating DNA strand, inhibits efficient duplex unwinding by these enzymes. The human single-stranded DNA binding protein replication protein A (RPA) fully restores the unwinding activity of BLM and WRN on vinylphosphonate-containing substrates while the heterologous single-stranded DNA binding protein from Escherichia coli (SSB) restores the activity only partially. Both RPA and SSB fail to restore the unwinding activity of the SF1 PcrA helicase on modified substrates, implying specific interactions of RPA with the BLM and WRN helicases. Our data highlight subtle differences between SF1 and SF2 helicases and suggest that although RecQ helicases belong to the SF2 family, they are mechanistically more similar to the SF1 PcrA helicase than to other SF2 helicases that are not affected by vinylphosphonate modifications.  相似文献   
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Light and cytokinins are known to be the key players in the regulation of plant senescence. In detached leaves, the retarding effect of light on senescence is well described; however, it is not clear to what extent is this effect connected with changes in endogenous cytokinin levels. We have performed a detailed analysis of changes in endogenous content of 29 cytokinin forms in detached leaves of Arabidopsis thaliana (wild‐type and 3 cytokinin receptor double mutants). Leaves were kept under different light conditions, and changes in cytokinin content were correlated with changes in chlorophyll content, efficiency of photosystem II photochemistry, and lipid peroxidation. In leaves kept in darkness, we have observed decreased content of the most abundant cytokinin free bases and ribosides, but the content of cis‐zeatin increased, which indicates the role of this cytokinin in the maintenance of basal leaf viability. Our findings underscore the importance of light conditions on the content of specific cytokinins, especially N6‐(Δ2‐isopentenyl)adenine. On the basis of our results, we present a scheme summarizing the contribution of the main active forms of cytokinins, cytokinin receptors, and light to senescence regulation. We conclude that light can compensate the disrupted cytokinin signalling in detached leaves.  相似文献   
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Bladder Cancer Associated Protein (BLCAP, formerly Bc10), was identified by our laboratory as being down-regulated in bladder cancer with progression. BLCAP is ubiquitously expressed in different tissues, and several studies have found differential expression of BLCAP in various cancer types, such as cervical and renal cancer, as well as human tongue carcinoma and osteosarcoma. Here we report the first study of the expression patterns of BLCAP in breast tissue. We analyzed by immunohistochemistry tissue sections of normal and malignant specimens collected from 123 clinical high-risk breast cancer patients within the Danish Center for Translational Breast Cancer Research (DCTB) prospective study dataset. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. We observed weak immunoreactivity for BLCAP in mammary epithelial cells, almost exclusively localizing to the cytoplasm and found that levels of expression of BLCAP were generally higher in malignant cells as compared to normal cells. Quantitative IHC analysis of BLCAP expression in breast tissues confirmed this differential BLCAP expression in tumor cells, and we could establish, in a 62-patient sample matched cohort, that immunostaining intensity for BLCAP was increased in tumors relative to normal tissue, in more than 45% of the cases examined, indicating that BLCAP may be of value as a marker for breast cancer. We also analyzed BLCAP expression and prognostic value using a set of tissue microarrays comprising an independent cohort of 2,197 breast cancer patients for which we had follow-up clinical information.  相似文献   
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