[show abstract][hide abstract] ABSTRACT: NF-κB is a multifunctional transcription factor involved in diverse biological processes. It has been well documented that NF-κB can be activated in response to various stimuli. While signal-inducible NF-κB activation mechanisms have been extensively characterized, exogenous signal-independent intrinsic NF-κB activation processes remain poorly understood. Here we show that IκB kinase β (IKKβ) can be intrinsically activated in the nucleus by a homeobox protein termed Nkx3.2 in the absence of exogenous IKK-activating signals. We found that ubiquitin chain-dependent, but persistent, interactions between Nkx3.2 and NEMO (also known as IKKγ) can give rise to constitutive IKKβ activation in the nucleus. Once the Nkx3.2-NEMO-IKKβ complex is formed in the nucleus, IKKβ-induced Nkx3.2 phosphorylation at Ser148 and Ser168 allows βTrCP to be engaged to cause IκB-α ubiquitination independent of IκB-α phosphorylation at Ser32 and Ser36. Taken together, our results provide a novel molecular explanation as to how an intracellular factor such as Nkx3.2 can accomplish persistent nuclear IKK activation to enable intrinsic and constitutive degradation of IκB in the nucleus in the absence of exogenous NF-κB-activating signals, which, in turn, plays a role in chondrocyte viability maintenance.
Molecular and cellular biology 05/2011; 31(14):2802-16. · 6.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: During DNA polymerase switching, the Xenopus laevis Cip/Kip-type cyclin-dependent kinase inhibitor Xic1 associates with trimeric proliferating cell nuclear antigen (PCNA) and is recruited to chromatin, where it is ubiquitinated and degraded. In this study, we show that the predominant E3 for Xic1 in the egg is the Cul4-DDB1-XCdt2 (Xenopus Cdt2) (CRL4(Cdt2)) ubiquitin ligase. The addition of full-length XCdt2 to the Xenopus extract promotes Xic1 turnover, while the N-terminal domain of XCdt2 (residues 1 to 400) cannot promote Xic1 turnover, despite its ability to bind both Xic1 and DDB1. Further analysis demonstrated that XCdt2 binds directly to PCNA through its C-terminal domain (residues 401 to 710), indicating that this interaction is important for promoting Xic1 turnover. We also identify the cis-acting sequences required for Xic1 binding to Cdt2. Xic1 binds to Cdt2 through two domains (residues 161 to 170 and 179 to 190) directly flanking the Xic1 PCNA binding domain (PIP box) but does not require PIP box sequences (residues 171 to 178). Similarly, human p21 binds to human Cdt2 through residues 156 to 161, adjacent to the p21 PIP box. In addition, we identify five lysine residues (K180, K182, K183, K188, and K193) immediately downstream of the Xic1 PIP box and within the second Cdt2 binding domain as critical sites for Xic1 ubiquitination. Our studies suggest a model in which both the CRL4(Cdt2) E3- and PIP box-containing substrates, like Xic1, are recruited to chromatin through independent direct associations with PCNA.
Molecular and cellular biology 09/2010; 30(17):4120-33. · 6.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Peroxiredoxins (Prxs) are a group of peroxidases containing a cysteine thiol at their catalytic site. During peroxidase catalysis, the catalytic cysteine, referred to as the peroxidatic cysteine (C(P)), cycles between thiol (C(P)-SH) and disulfide (-S-S-) states via a sulfenic (C(P)-SOH) intermediate. Hyperoxidation of the C(P) thiol to its sulfinic (C(P)-SO(2)H) derivative has been shown to be reversible, but its sulfonic (C(P)-SO(3)H) derivative is irreversible. Our comparative study of hyperoxidation and regeneration of Prx I and Prx II in HeLa cells revealed that Prx II is more susceptible than Prx I to hyperoxidation and that the majority of the hyperoxidized Prx II formation is reversible. However, the hyperoxidized Prx I showed much less reversibility because of the formation of its irreversible sulfonic derivative, as verified with C(P)-SO(3)H-specific antiserum. In an attempt to identify the multiple hyperoxidized spots of the Prx I on two-dimensional PAGE analysis, an N-acetylated Prx I was identified as part of the total Prx I using anti-acetylated Lys antibody. Using peptidyl-Asp metalloendopeptidase (EC 126.96.36.199) peptide fingerprints, we found that N(alpha)-terminal acetylation (N(alpha)-Ac) occurred exclusively on Prx II after demethionylation. N(alpha)-Ac of Prx II blocks Prx II from irreversible hyperoxidation without altering its affinity for hydrogen peroxide. A comparative study of non-N(alpha)-acetylated and N(alpha)-terminal acetylated Prx II revealed that N(alpha)-Ac of Prx II induces a significant shift in the circular dichroism spectrum and elevation of T(m) from 59.6 to 70.9 degrees C. These findings suggest that the structural maintenance of Prx II by N(alpha)-Ac may be responsible for preventing its hyperoxidation to form C(P)-SO(3)H.
Journal of Biological Chemistry 04/2009; 284(20):13455-65. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although diagnosis of Plasmodium vivax malaria has been difficult when it is present at a low parasite density, it was recently revealed that an antibody assay was a good method of screening for malaria in blood banks. However, the use of this method for the diagnosis of malaria is limited due to the persistence of specific immunoglobulin (Ig) G. Therefore, we evaluated specific IgM antibody responses against the C-terminal region of the merozoite surface protein 1 of P. vivax (PvMSP1c) in sera obtained from patients with vivax malaria using various assays. The IgM capture enzyme-linked immunosorbent assay showed good sensitivity (97.7%; 308/315) and specificity (99.1%, 446/450). In addition, the results of this assay were not related to parasite density, and a high reactivity was observed when there was a low level of parasitemia. Furthermore, we found that patients with cases of malaria that had relapsed still had the IgM titers against PvMSP1c. Therefore, the use of IgM ELISA for the detection of specific IgM that was not involved in memorial immune activity could be an alternative tool for the diagnosis of malaria and blood screening, even in areas in which malaria is endemic.
Parasitology Research 09/2008; 103(3):625-31. · 2.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: The thiol (-SH) of the active cysteine residue in peroxiredoxin (Prx) is known to be reversibly hyperoxidized to cysteine sulfinic acid (-SO(2)H), which can be reduced back to thiol by sulfiredoxin/sestrin. However, hyperoxidized Prx of an irreversible nature has not been reported yet. Using an antibody developed against the sulfonylated (-SO(3)H) yeast Prx (Tsa1p) active-site peptide (AFTFVCPTEI), we observed an increase in the immunoblot intensity in proportion to the H(2)O(2) concentrations administered to the yeast cells. We identified two species of hyperoxidized Tsa1p: one can be reduced back (reversible) with sulfiredoxin, and the other cannot (irreversible). Irreversibly hyperoxidized Tsa1p was identified as containing the active-site cysteine sulfonic acid (Tsa1p-SO(3)H) by mass spectrometry. Tsa1p-SO(3)H was not an autoxidation product of Tsa1p-SO(2)H and was maintained in yeast cells even after two doubling cycles. Tsa1p-SO(3)H self-assembled into a ring-shaped multimeric form was shown by electron microscopy. Although the Tsa1p-SO(3)H multimer lost its peroxidase activity, it gained approximately 4-fold higher chaperone activity compared with Tsa1p-SH. In this study, we identify an irreversibly hyperoxidized Prx, Tsa1p-SO(3)H, with enhanced molecular chaperone activity and suggest that Tsa1p-SO(3)H is a marker of cumulative oxidative stress in cells.
Journal of Biological Chemistry 09/2008; 283(43):28873-80. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Point mutations in three kinds of Ras protein (H-, K-, and N-Ras) that specifically occur in codons 12, 13, and 61 facilitate virtually all of the malignant phenotype of the cancer cells, including cellular proliferation, transformation, invasion, and metastasis. In order to elucidate an understanding into the oncogenic ras networks by H-, K-, and N-Ras/G12V, we have established various oncogenic ras expressing NIH/3T3 mouse embryonic fibroblast clones using the tetracycline-induction system, which are expressing Ras/G12V proteins under the tight control of expression by an antibiotics, doxycycline. Here we provide a catalog of proteome profiles in total cell lysates derived from three oncogenic ras expressing NIH/3T3 cells and a good in vitro model system for dissecting the protein networks due to these oncogenic Ras proteins. In this biological context, we compared total proteome changes by the combined methods of 2-DE, quantitative image analysis, and MALDI-TOF MS analysis using the unique Tet-on inducible expression system. There were a large number of common targets for oncogenic ras, which were identified in all three cell lines and consisted of 204 proteins (61 in the pH range of 4-7, 63 in 4.5-5.5, and 80 in 5.5-6.7). Differentially regulated expression was further confirmed for some subsets of candidates by Western blot analysis using specific antibodies. Taken together, we implemented a 2-DE-based proteomics approach to the systematical analysis of the dysregulations in the cellular proteome of NIH/3T3 cells transformed by three kinds of oncogenic ras. Our results obtained and presented here show that the comparative analysis of proteome from oncogenic ras expressing cells has yielded interpretable data to elucidate the differential protein expression directly and/or indirectly, and contributed to evaluate the possibilities for physiological, and therapeutic targets. Further studies are in progress to elucidate the implications of these findings in the regulation of Ras induced transformation.
[show abstract][hide abstract] ABSTRACT: Royal jelly (RJ) is an exclusive food for queen honey bee (Apis mellifera L.) that is synthesized and secreted by young worker bees. RJ is also widely used in medical products, cosmetics, and as health foods. However, little is known about RJ functionality and the total protein components, although recent research is attempting to unravel the RJ proteome. We have embarked on a detailed investigation of the RJ proteome, using a modified protein extraction protocol and two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with tandem mass spectrometry. Simultaneously, we examined total soluble protein from RJ collected at 24, 48, and 72 h after honey bee larvae deposition twice (in two flower blooming seasons), to check differences, if any, in RJ proteome therein. Both 1- and 2-D gels stained with silver nitrate revealed similar protein profiles among these three time points. However, we observed a clear difference in two bands (ca. MW of 55 and 75 kDa) on 1-D gel between the first and the second collection of RJ. A similar difference was also observed in the 2-D gel. Except for this difference, the protein profiles were similar at the 3 time points. As the RJ from 48 (or sometimes 72) is commercially used, we selected the RJ sample at 48 h for detailed analysis with the first collection. 1-DGE identified 90 and 15 proteins from the first and second selection, respectively; in total, 47 nonredundant proteins were identified. 2-DGE identified 105 proteins comprising 14 nonredundant proteins. In total, 52 nonredundant proteins were identified in this study, and other than the major royal jelly protein family and some other previously identified proteins, 42 novel proteins were identified. Furthermore, we also report potentially post-translationally modified (phosphorylation and glycosylation) RJ proteins based on the Pro-Q diamond/emerald phosphoprotein/glycoprotein gel stains; MRJP 2p and 7p were suggested as potential phosphoproteins. The 2-DGE data were integrated to develop a 2-D gel reference map, and all data are accessible through RJ proteomics portal (http://foodfunc.agr.ibaraki.ac.jp/RJP.html). Keywords: Apis mellifera; royal jelly; proteomics analysis; gel-based approach; mass spectrometry; proteins
Journal of Proteome Research 06/2008; 7(8). · 5.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mushroom can be defined as a macrofungus with a distinctive fruiting body. Mushrooms of class Basidiomycete are primarily wood degradation fungi, but serve as food and a part of traditional medicine used by humans. Although their life cycle is fairly well-established, the information on the molecular components, especially proteins are very limited. Here, we report proteomics analysis of two edible mushrooms (fruiting bodies) Sparassis crispa and Hericium erinaceum using one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) based complementary proteomics approaches. 1-DGE coupled with liquid chromatography and mass spectrometry identified 77 (60 nonredundant proteins) and 121 (88 nonredundant proteins) proteins from S. crispa and H. erinaceum, respectively. 2-DGE analysis revealed 480 and 570 protein spots stained with colloidal coomassie brilliant blue in S. crispa and H. erinaceum, respectively. Of the 71 and 115 selected protein spots from S. crispa and H. erinaceum 2D gel blots on polyvinyldifluoride (PVDF) membranes, respectively, 29 and 35 nonredundant proteins were identified by N-terminal amino acid sequencing. Identified nonredundant proteins from 1- or 2-DGE belonged to 19 functional categories. Twenty-one proteins were found common in both S. crispa and H. erinaceum proteomes, including 14-3-3 protein and septin. Together this study provides evidence for the presence of a large number of functionally diverse proteins, expressed in the fruiting body of two economically important mushrooms, S. crispa and H. erinaceum. Data obtained from 1-DGE and 2-DGE analyses is accessible through the mushroom proteomics portal http://foodfunc.agr.ibaraki.ac.jp/mushprot.html.
Journal of Proteome Research 06/2008; 7(5):1819-35. · 5.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Telomere maintenance is essential for continued cell proliferation and chromosome stability. Telomeres are maintained by telomerase and a collection of associated proteins. The telomeric protein telomeric repeat binding factor 1 (TRF1) negatively regulates telomere length by inhibiting access of telomerase at telomere termini. Here we report that TRF1 interacts with the beta subunit of casein kinase 2 (CK2) and serves as a substrate for CK2. CK2-mediated phosphorylation is required for the efficient telomere binding of TRF1 in vitro and in vivo. Inhibition of CK2 by the CK2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole decreased the ability of TRF1 to bind telomeric DNA. The resulting telomere-unbound form of TRF1 was then ubiquitinated and degraded by the proteasome. Partial knockdown of CK2 by small interfering RNA resulted in removal of TRF1 from telomeres and subsequent degradation of TRF1. Mapping of the CK2 target site identified threonine 122 as a substrate in TRF1. A threonine to alanine change at this position led to a diminished DNA binding due to reduced dimerization of TRF1. In addition, phosphorylation of threonine 122 seemed critical for TRF1-mediated telomere length control. Our findings suggest that CK2-mediated phosphorylation of TRF1 plays an important role in modulating telomere length homeostasis by determining the levels of TRF1 at telomeres.
Journal of Biological Chemistry 06/2008; 283(20):14144-52. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Manduca sexta is an excellent insect model for studying insect physiology, including hemolymph proteins. Larvae stages of this insect are highly damaging to tobacco leaves causing a drastic decrease in crop yield. Investigation on the larval biology should help in controlling its destructive potential, thus increasing crop yields. The hemolymph is the source of its immunity to disease and environmental factors, which invariably involves protein components. To better understand the physiology of M. sexta and the protein components expressed during its life cycle, two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with N-terminal amino acid sequencing and liquid chromatography-mass spectrometry, were employed to analyze the fifth instar larvae hemolymph proteins. These proteomics approaches together identified 123 proteins, which constituted a total of 58 nonredundant proteins and belonged to 10 functional categories. Defense (49%), transport and metabolism (15%), storage (9%), and metamorphosis (7%) categories were highly represented accounting for 80% of the identified proteins. Besides identification of previously reported proteins, 18 novel proteins were identified, which include the lipoprotein-releasing system transmembrane protein lolC, 50S ribosomal protein L24, inducible serine protease inhibitor 1, imaginal disk growth factor, protein disulfide-isomerase-like protein ERp57, etc. The 2-DGE data were integrated to develop a 2-D gel reference map. Data obtained from 1-DGE and 2-DGE analyses are accessible through the M. sexta proteomics portal ( http://foodfunc.agr.ibaraki.ac.jp/mansehemoprot.html). Together, this study provides evidence for the presence of a large number of functionally diverse protein families in the hemolymph of M. sexta. These proteins correlate well with the fifth instar stage, the transition from larvae to pupae.
Journal of Proteome Research 04/2008; 7(3):938-59. · 5.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the present study, we profiled proteins in ssa1/2 mutant and wild-type using one-dimensional gel electrophoresis coupled with liquid chromatography and mass spectrometry to reveal a total of 322 proteins. Sixty and 84 nonredundant proteins were detected in ssa1/2 and wild-type, respectively, whereas 178 were common. A quantitative profiling proteomic approach using a modified N-terminal isotope tagging method was undertaken to determine quantitative changes in proteins between mutant and wild-type. Out of 210 identified proteins selected for quantification, 103 propionylated proteins were obtained. Eight only D0-propionylated protein (wild-type) and 4 only D5-propionylated proteins (ssa1/2) were detected; 90 proteins were overlapped in the ssa1/2 mutant and wild-type. In the ssa1/2 mutant, 28 proteins were up-regulated and 26 were down-regulated. The expression levels of the rest of 49 proteins were not changed compared with the wild-type. Furthermore, non-correlation between mRNA and protein expressions was found. Among up-regulated proteins, 19 proteins involved in protein synthesis, chromatin condensation, and silencing showed unchanged mRNA expression levels. Among down-regulated proteins, 21 proteins consisting mainly of transcription factors showed unchanged mRNA expressions. Surprisingly, several proteins involved in protein synthesis were also found among the down-regulated proteins. These results suggested that the proteins showing changed protein expressions and unchanged mRNA expressions were affected by the deletion of SSA1 and SSA2 genes at translational efficiency, mRNA degradation, or protein degradation. Moreover, we found the proteins related to chromosomal control were up-regulated in ssa1/2 mutant, a novel finding of this study, suggesting that the Ssa1/2p might contribute to chromosomal control.
Journal of Proteome Research 10/2007; 6(9):3465-74. · 5.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Post-translational addition of O-linked N-acetylglucosamine (O-GlcNAc) to p53 is known to occur, but the site of O-GlcNAcylation and its effects on p53 are not understood. Here, we show that Ser 149 of p53 is O-GlcNAcylated and that this modification is associated with decreased phosphorylation of p53 at Thr 155, which is a site that is targeted by the COP9 signalosome, resulting in decreased p53 ubiquitination. Accordingly, O-GlcNAcylation at Ser 149 stabilizes p53 by blocking ubiquitin-dependent proteolysis. Our results indicate that the dynamic interplay between O-GlcNAc and O-phosphate modifications coordinately regulate p53 stability and activity.
[show abstract][hide abstract] ABSTRACT: Proteomic technology provides useful tools to detect protein modification sites in vivo and in vitro. In this work, we applied proteomics to identify an essential amino acid residue involved in Malonyl-CoA Decarboxylase (MCD) catalysis. A reaction with acetic anhydride and MCD, under mild conditions without acetyl CoA as a substrate, resulted in the acetylation of six lysyl residues, K210, K58, K167, K316, K388, and K444. When acetyl CoA was added to the reaction, K210 was protected from acetylation, indicating a potential role for this residue in catalysis. In addition, K210 was the only lysyl residue, out of six, that was not endogenously acetylated. Because K210, K308, and K388 are conserved across species, they were site-specifically mutated to methionine which is size-wise similar to lysine but not protonated. The K308M and K388M MCD mutants retained 60% of their enzyme activities, whereas the K210M mutant was completely inactive. These results strongly suggest that K210 is an essential residue in rat MCD catalysis and is a likely proton donor to the alpha carbon of malonyl-CoA. Therapeutic inhibition of MCD may be a viable approach to treating various clinical pathologies associated with defective fatty acid metabolism.
Journal of Proteome Research 07/2006; 5(6):1398-406. · 5.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: To elucidate the oncogenic H-Ras network, we have established various stable and inducible oncogenic H-Ras-expressing NIH/3T3 mouse embryonic fibroblast cell clones, which express G12V H-Ras and G12R H-Ras proteins under the influence of a strong cytomegalovirus promoter and under the tight control of expression by an antibiotic, doxycycline, respectively. Here we provide a catalogue of proteome profiles in total cell lysates derived from oncogenic H-Ras-expressing NIH/3T3 cells. In this biological context, we compared total proteome changes by the combined methods of 2-DE, quantitative image analysis and MALDI-TOF MS analysis using both a stable expression system as well as an inducible expression system. There were a large number of common targets for oncogenic H-Ras, which were identified in both cell lines and consisted of 64 proteins (36 up-regulated and 28 down-regulated). Differentially regulated expression was further confirmed for some subsets of candidates by Western blot analysis using specific antibodies. Taken together, the results presented here show that comparative analysis of the proteome from the oncogenic H-Ras-expressing cells yielded interpretable data to elucidate protein networks directly and/or indirectly.
[show abstract][hide abstract] ABSTRACT: The phosphatase and tensin homolog tumor suppressor (PTEN) belongs to a class of "gatekeeper" tumor suppressors together with p53, retinoblastoma and adenomatous polyposis. It is considered one of the most important tumor suppressors in the post p53 era. Previously to identify the molecules involved in the signaling network regulated by PTEN using proteomic tools, we reported global proteome profiles at different time points using the PTEN inducible NIH3T3 cells (Kim, S.-y., Kim, Y. S., Bahk, Y. Y., Mol. Cells 2003, 15, 396-405). However, the system had a critical limitation that NIH3T3 cell has endogenous wild-type PTEN and, thus to be exact, the induced PTEN could not give the answer about the real physiological roles of this tumor suppressor. Here, to find out PTEN-related protein network we have established various PTEN (wild-type, an activity inert C124G, and a lipid phosphatase deficient G129E)-expressing cell clones in U-87 MG human glioblastoma cells lacking detectable PTEN as a result of genetic lesions. In this biological context, we compared their morphological and expression patterns, and proteome images of each PTEN-expressing cell clone by 2-DE followed by identification with MALDI-TOF MS. We obtained some pieces of evidence that morphological change by PTEN expression is mediated by its protein phosphatase activity and their growth rate by the lipid phosphatase activity. The proteomic approaches showed that 30 proteins possibly correlated with PTEN's protein phosphatase activity (13 down-regulated and 17 up-regulated) and 20 with the lipid phosphatase activity (14 down-regulated and 6 up-regulated) were identified. Taken together, we conclude that the comparative analysis of proteome from various PTEN-expressing cells has yielded interpretable data to elucidate the protein network directly and/or indirectly caused by individual phosphatase activities of PTEN in vivo.
[show abstract][hide abstract] ABSTRACT: N-Terminal isotope tagging (NIT) is an important proteomic tool for quantifying proteins in complex mixtures. Here we describe a modified version of the isotope-coded propionylation procedure of Zhang et al. [Zhang et al., Rapid Commun. Mass Spectom. 16 (2002) 2325], which uses 'light' D0 and 'heavy' D10-propionic anhydride. The method has been extensively modified to improve both the kinetics and overall yield of propionylation. Using albumin as a model protein, the overall variation in quantification yields, calculated using several tryptic peptides, was within +/-10% (S.D. +/-0.2) error. The efficacy of the method is demonstrated by the quantitative differences obtained for vimentin in cell lysates of C2C12 myoblasts upon their myogensis to myotubules.
Journal of Chromatography B 12/2005; 826(1-2):91-107. · 2.49 Impact Factor
[show abstract][hide abstract] ABSTRACT: Tuberculosis caused by mycobacteria, mainly Mycobacterium tuberculosis, is a major infectious disease of the respiratory system. An early diagnosis followed by chemotherapy is the major control strategy. In an effort to identify the antigens suitable for immunodiagnosis and vaccines, the proteins secreted in a culture medium from the M. tuberculosis K-strain, which is the most prevalent among the clinical isolates in Korea and belongs to the Beijing family, were analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and compared with those from the M. tuberculosis H37Rv and CDC1551 strains. Eight proteins, Rv0652, Rv1636, Rv2818c, Rv3369, Rv3865, Rv0566c, MT3304, and Rv3160, were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) or liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and found to be relatively abundant in the culture medium from the M. tuberculosis K-strain but less so from the CDC1551 or H37Rv strains. In addition, Rv3874 (CFP-10), Rv-0560c and Rv3648c, which were expressed increasingly in the K and CDC1551 strains, were also identified using the same proteomics technology. All proteins were prepared by molecular cloning, expression in Escherichia coli followed by affinity purification. Among them, three proteins, rRv3369, rRv0566c, and rRv3874, were selected by prescreening and examined for their potential as serodiagnostic antigens using an enzyme-linked immunosorbent assay. When 100 sera from tuberculosis patients and 100 sera from the healthy controls were analyzed, rRV3369, rRv3874, and rRv0566c showed a sensitivity of 60%, 74%, and 43%, and a specificity of 96%, 97%, and 84%, respectively. These results suggest that the rRv3369 and rRv3874 proteins, which were expressed more abundantly in the more recently obtained clinical isolates of M. tuberculosis than in the laboratory-adapted H37Rv strain, are promising for use in the serodiagnosis of tuberculosis.
[show abstract][hide abstract] ABSTRACT: MCD (malonyl-CoA decarboxylase), which catalyses decarboxylation of malonyl-CoA, is known to play an important role in the regulation of malonyl-CoA concentration. Recently, it has been observed that the expression of MCD is significantly decreased in the hearts of the PPARalpha (peroxisome-proliferator-activated receptor alpha) (-/-) mice, where the rate of fatty-acid oxidation is decreased by the increased malonyl-CoA level [Campbell, Kozak, Wagner, Altarejos, Dyck, Belke, Severson, Kelly and Lopaschuk (2002) J. Biol. Chem. 277, 4098-4103]. This suggests that MCD may be transcriptionally regulated by PPARalpha. To investigate whether PPARalpha is truly responsible for transcriptional regulation of the rat MCD gene, transient reporter assay was performed in CV-1 cells. The promoter activity was increased by 17-fold in CV-1 cells co-transfected with PPARalpha/retinoid X receptor alpha expression plasmid. In sequence analysis of the promoter region, three putative PPREs (PPAR response elements) were identified, and promoter deletion analysis showed that PPRE2 and PPRE3 were functional. Electrophoretic mobility-shift assays revealed that PPARalpha/retinoid X receptor alpha heterodimer indeed bound to the two PPREs, and the binding specificity of PPARalpha on PPRE was also confirmed by experiments with mutated oligonucleotides. These results indicate that the elements behaved as a responsive site to PPARalpha activation. MCD mRNA levels in WY14643-treated rat hepatoma cells as well as in the liver of fenofibrate-fed Otsuka Long-Evans Tokushima fatty rats were also found to be increased, suggesting that PPARalpha can activate the rat hepatic MCD transcription by binding to the PPREs in the promoter. We propose that MCD performs an important role in understanding the regulatory mechanism between activated PPARalpha and fatty-acid oxidation by altering the malonyl-CoA concentration.
[show abstract][hide abstract] ABSTRACT: Most people infected with Plasmodium vivax malaria developed antibodies against the C-terminal region of P. vivax merozoite surface protein (PvMSP1c) and the antibodies are sustained for a period up to 10 months after anti-malarial treatment. The longer-term stability of the specific humoral response was evaluated indirectly by determining the antibody titers in the sera from healthy individuals who lived an area from which malaria had been eradicated (450 persons) and an area in which it had recurred (1,524 persons). There were considerable residual antibody responses to PvMSP1c in over 15% of sera from healthy individuals, but only those who had lived in the era when malaria was prevalent. This means that antibodies against PvMSP1c may persist for more than 30 years, the malaria-free duration. This long-term memory of humoral immunity supports the C-terminal region of merozoite surface protein 1 as an effective malaria vaccine, in addition to the neutralizing activity reported previously.
Parasitology Research 04/2004; 92(5):384-9. · 2.85 Impact Factor