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171.
The present study characterizes the unfolding and folding processes of recombinant manganese peroxidase. This enzyme contains five disulfide bonds, two calcium ions, and one heme prosthetic group. Circular dichroism in the far UV was used to monitor global changes of the protein secondary structure, whereas UV-visible spectroscopy of the Soret band provided information about local changes in the heme cavity. The effects of reducing agents, oxidizing agents, and denaturants on this process were investigated. In addition to affecting the secondary structure content, these factors also affect the binding of the heme and the calcium ions, both of which have a significant effect on the folding process. Our results also show that denaturants induce irreversible changes, which are most likely due to the inability of the denatured protein to rebind either calcium or the heme. Breaking of disulfide bonds by 30 mM dithiothreitol causes complete unfolding of recombinant manganese peroxidase. The unfolding process was also studied at low and high pH, where the protein reaches the final unfolded state through two different intermediate states. The data also indicate that only the acidic folding-unfolding process is reversible. Our results indicate a complex synergistic relationship between the secondary structure content, the tertiary structure arrangement, and the binding of the heme and the calcium ions and disulfide bridge formation. 相似文献
172.
173.
Chromosomal distribution of heterochromatin protein 1 (HP1) in Drosophila: a cytological map of euchromatic HP1 binding sites 总被引:2,自引:0,他引:2
The Heterochromatin Protein 1 (HP1) is a conserved protein which is best known for its strong association with the heterochromatin of Drosophila melanogaster. We previously demonstrated that another important property of HP1 is its localization to the telomeres of Drosophila, a feature that reflects its critical function as a telomere capping protein. Here we report our analysis of the euchromatic sites to which HP1 localizes. Using an anti-HP1 antibody, we compared immunostaining patterns on polytene chromosomes of the Ore-R wild type laboratory strain and four different natural populations. HP1 was found to accumulate at specific euchromatic sites, with a subset of the sites conserved among strains. These sites do not appear to be defined by an enrichment of known repetitive DNAs. Comparisons of HP1 patterns among several Drosophila species revealed that association with specific euchromatic regions, heterochromatin and telomeres is a conserved characteristic of HP1. Based on these results, we argue that HP1 serves a broader function than typically postulated. In addition to its role in heterochromatin assembly and telomere stability, we propose that HP1 plays an important role in regulating the expression of many different euchromatic regions. 相似文献
174.
Oxidative stress induces protein phosphatase 2A-dependent dephosphorylation of the pocket proteins pRb,p107, and p130 总被引:7,自引:0,他引:7
Cicchillitti L Fasanaro P Biglioli P Capogrossi MC Martelli F 《The Journal of biological chemistry》2003,278(21):19509-19517
Oxidative stress induces cell death and growth arrest. In this study, the regulation and the functional role of the retinoblastoma family proteins pRb, p107, and p130 in the cellular response to oxidative stress were investigated. Treatment of endothelial cells with H2O2 induced rapid hypophosphorylation of the retinoblastoma family proteins. This event did not require p53 or p21Waf1/Cip1/Sdi1 and was not associated with cyclin/cyclin-dependent kinase down-modulation. Four lines of evidence indicate that H2O2-induced hypophosphorylation of pRb, p107, and p130 was because of the activity of protein phosphatase 2A (PP2A). First, cell treatment with two phosphatase inhibitors, okadaic acid and calyculin A, prevented the hypophosphorylation of the retinoblastoma family proteins, at concentrations that specifically inhibit PP2A. Second, SV40 small t, which binds and inhibits PP2A, when overexpressed prevented H2O2-induced dephosphorylation of the retinoblastoma family proteins, whereas a SV40 small t mutant unable to bind PP2A was totally inert. Third, PP2A core enzyme physically interacted with pRb and p107, both in H2O2-treated and untreated cells. Fourth, a PP2A phosphatase activity was co-immunoprecipitated with pRb, and the activity of pRb-associated PP2A was positively modulated by cell treatment with H2O2. Because DNA damaging agents inhibit DNA synthesis in a pRb-dependent manner, it was determined whether the PP2A-mediated dephosphorylation of the retinoblastoma family proteins played a role in this S-phase response. Indeed, it was found that inhibition of PP2A by SV40 small t over-expression prevented DNA synthesis inhibition induced by H2O2. 相似文献
175.
Characterization of a novel and specific inhibitor for the pro-apoptotic protease Omi/HtrA2 总被引:10,自引:0,他引:10
Cilenti L Lee Y Hess S Srinivasula S Park KM Junqueira D Davis H Bonventre JV Alnemri ES Zervos AS 《The Journal of biological chemistry》2003,278(13):11489-11494
Omi/HtrA2 is a mammalian serine protease with high homology to bacterial HtrA chaperones. Omi/HtrA2 is localized in mitochondria and is released to the cytoplasm in response to apoptotic stimuli. Omi/HtrA2 induces cell death in a caspase-dependent manner by interacting with the inhibitor of apoptosis protein as well as in a caspase-independent manner that relies on its protease activity. We describe the identification and characterization of a novel compound as a specific inhibitor of the proteolytic activity of Omi/HtrA2. This compound (ucf-101) was isolated in a high throughput screening of a combinatorial library using bacterially made Omi-(134-458) protease and fluorescein-casein as a generic substrate. ucf-101 showed specific activity against Omi/HtrA2 and very little activity against various other serine proteases. This compound has a natural fluorescence that was used to monitor its ability to enter mammalian cells. ucf-101, when tested in caspase-9 (-/-) null fibroblasts, was found to inhibit Omi/HtrA2-induced cell death. 相似文献
176.
Conspecific pilferage but not presence affects Merriam's kangaroo rat cache strategy 总被引:7,自引:1,他引:6
We investigated the effects of pilferage on caching behaviorin the Merriam's kangaroo rat by manipulating two factors associatedwith pilferage: the presence of a conspecific, and the opportunityfor pilferage. In one experiment we assessed animals in either"Stealer" or "Victim" roles and measured changes in caching,space use, and behavior after caches were pilfered. Victimsshifted from a majority scatter-hoarding to a majority larder-hoardingstrategy after their caches were pilfered by the Stealer. InExperiment 2, we measured changes after exposure to a conspecificwhen there was no pilferage, with or without prior exposureto pilferage from Experiment 1. Merriam's kangaroo rats werevigilant when a conspecific was present, but did not changecache strategy. Prior exposure did not have any major effecton caching or behavior. Food storage is an economic decisionthat is often made by a solitary forager. Our results suggestthat social competition nonetheless influences such economic
decisions, even in a nonsocial forager. 相似文献
177.
Cataldi A Miscia S Centurione L Rapino M Bosco D Grifone G Valerio VD Garaci F Rana R 《Journal of cellular biochemistry》2002,85(3):553-560
The suppressive role of endogenous regucalcin (RC), which is a regulatory protein of calcium signaling, in the enhancement of protein phosphatase activity (PPA) in the cytosol and nucleus of kidney cortex in calcium-administered rats was investigated. Calcium content in the kidney cortex was significantly increased at 0.5-5 h after a single intraperitoneal administration of calcium chloride solution (10 mg Ca/100 g body weight) to rats. The analysis with Western blotting of RC protein showed that RC levels in the cytosol and nucleus were significantly increased 0.5-5 h after the administration of calcium (10 mg/100 g). PPA toward phosphotyrosine, phosphoserine, and phosphothreonine was found in the cytosol and nucleus of kidney cortex. PPA toward three phosphoamino acids in the cytosol and nucleus was significantly increased by the administration of calcium (10 mg/100 g). The presence of anti-RC monoclonal antibody (25 ng/ml) in the enzyme reaction caused a significant increase in PPA toward phosphotyrosine, phosphoserine, and phosphothreonine in the cytosol and nucleus of kidney cortex in normal rats. The effect of anti-RC monoclonal antibody (25 ng/ml) in increasing PPA toward three phosphoamino acids in the cytosol and nucleus was significantly enhanced in calcium-administered rats. The effect of anti-RC monoclonal antibody (25 ng/ml) in increasing PPA in the cytosol and nucleus of normal rats and calcium-administered rats was completely abolished by the addition of RC (10(- 6) M) in the enzyme reaction mixture. The present study suggests that endogenous RC suppresses the enhancement of PPA in the cytosol and nucleus of kidney cortex in calcium-administered rats. 相似文献
178.
We have tracked the evolutionary history of chromosomes homologous to HSA10 (PHYL-10) in primates using appropriate panels of PCP, YAC, and BAC probes. This approach allowed us to delineate more precisely the PHYL-10 constitution in the ancestor of catarrhine, platyrrhine, and prosimians. The results suggest that (i) in the ancestor of prosimians PHYL-10 was organized in two separate PHYL-10p and PHYL-10q chromosomes; (ii) in the progenitor of New World monkeys PHYL-10p was a separate chromosome, while PHYL-10q was associated with a chromosome homologous to HSA16; (iii) in the ancestor of Old World monkeys PHYL-10 was a unique chromosome with a marker order corresponding to the orang form. We have also analyzed the cat, chosen as an outgroup for its very conserved karyotype. In agreement with published data our experiments show that the PHYL-10 in cat is structured in two blocks, PHYL-10p and PHYL-10q, both as part of larger chromosomes. The overall data indicate that, contrary to common opinion, PHYL-10p and PHYL-10q were distinct chromosomes in the primate ancestor. Analysis of the Saimiri sciureus (SSC) PHYL-10q marker order showed that it was isosequential with the Callithrix jacchus PHYL-10q, as well as with the PHYL-10q platyrrhine ancestral form. The SSC centromere, nevertheless, was located in a different chromosomal region, therefore suggesting that a centromeric repositioning event occurred in this species. 相似文献
179.
Grechkin AN Chechetkin IR Mukhtarova LS Hamberg M 《Chemistry and physics of lipids》2002,120(1-2):87-99
Incubations of allene oxide synthases of flax or maize with the E,E-isomers of the 13- and 9-hydroperoxides of linoleic acid (E,E-13- and E,E-9-HPOD, respectively) at pH 7.5 afforded substantial yields of trans-disubstituted cyclopentenones. Under the conditions used, (Z,E)-HPODs were converted mainly into -ketols and afforded only trace amount of cyclopentenones. These findings indicated that changing the double bond geometry from Z to E dramatically increased the rate of formation of the pericyclic pentadienyl cation intermediate necessary for electrocyclization of 18:2-allene oxides and thus the yield of cyclopentenones. The well-known cyclization of the homoallylic allene oxide (12,13-EOT) derived from -linolenic acid 13-hydroperoxide (E,Z-13-HPOT) into cis-12-oxo-10,15-phytodienoic acid was suppressed at pH below neutral and was not observable at pH 4.5. In contrast, cyclization of the allene oxide ((9E)-12,13-EOD) derived from (E,E)-13-HPOD was slightly favoured at low pH. The finding that the cyclizations of 12,13-EOT and (9E)-12,13-EOD were differently affected by changes in pH suggested that the mechanisms of cyclization of these allene oxides are distinct. 相似文献
180.
Todeschini AR Girard MF Wieruszeski JM Nunes MP DosReis GA Mendonca-Previato L Previato JO 《The Journal of biological chemistry》2002,277(48):45962-45968
Trypanosoma cruzi, the protozoan parasite responsible for Chagas' disease, expresses on its surface an uncommon membrane-bound sialidase, known as trans-sialidase. trans-Sialidase is the product of a multigene family encoding both active and inactive proteins. We report here that an inactive mutant of trans-sialidase physically interacts with CD4(+) T cells. Using a combination of flow cytometry and immunoprecipitation techniques, we identified the sialomucin CD43 as a counterreceptor for trans-sialidase on CD4(+) T cells. Using biochemical, immunological, and spectroscopic approaches, we demonstrated that the inactive trans-sialidase is a sialic acid-binding protein displaying the same specificity required by active trans-sialidase. Taken together, these results suggest that inactive members of the trans-sialidase family can physically interact with sialic acid-containing molecules on host cells and could play a role in host cell/T. cruzi interaction. 相似文献