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Methamphetamine (MAP) is a commonly used, addictive drug, and a powerful stimulant that dramatically affects the central nervous system. In this study, we used the conditioned place preference (CPP) paradigm in order to study the reinforcing properties of MAP and the herewith associated changes in proteins within the mesolimbic dopamine system. A CPP was induced by MAP after three intermittent intraperitoneal injections (1 mg/kg) in rats and protein profiles in the nucleus accumbens, striatum, prefrontal cortex, cingulate cortex and hippocampus were compared with a saline-treated control group. In addition, a group of animals was run through extinction and protein profiles were compared with a non-extinguished group. Protein screening was conducted using two-dimensional electrophoresis analysis which identified 27 proteins in the group that showed MAP-induced CPP. Some of the proteins were confirmed by Western lot analysis. Identified proteins had functions related to the cytoskeleton, transport/endocytosis or exocytosis (e.g. profilin-2 and syntaxin-binding protein), and signal transduction, among others.

The U-box motif is a conserved domain found in the diverse isoforms of E3 ubiquitin ligase in eukaryotes. From water-stressed hot pepper (Capsicum annuum L. cv Pukang) plants, we isolated C. annuum putative U-box protein 1 (CaPUB1), which encodes a protein containing a single U-box motif in its N-terminal region. In vitro ubiquitination and site-directed mutagenesis assays revealed that CaPUB1 possessed E3 ubiquitin ligase activity and that the U-box motif was indeed essential for its enzyme activity. RNA gel-blot analysis showed that CaPUB1 mRNA was induced rapidly by a broad spectrum of abiotic stresses, including drought, high salinity, cold temperature, and mechanical wounding, but not in response to ethylene, abscisic acid, or a bacterial pathogen, suggesting its role in the early events in the abiotic-related defense response. Because transgenic work was extremely difficult in hot pepper, in this study we overexpressed CaPUB1 in Arabidopsis (Arabidopsis thaliana) to provide cellular information on the function of this gene in the development and plant responses to abiotic stresses. Transgenic Arabidopsis plants that constitutively expressed the CaPUB1 gene under the control of the cauliflower mosaic virus 35S promoter had markedly longer hypocotyls and roots and grew more rapidly than the wild type, leading to an early bolting phenotype. Microscopic analysis showed that 35S∷CaPUB1 roots had increased numbers of small-sized cells, resulting in disordered, highly populated cell layers in the cortex, endodermis, and stele. In addition, CaPUB1-overexpressing plants displayed increased sensitivity to water stress and mild salinity. These results indicate that CaPUB1 is functional in Arabidopsis cells, thereby effectively altering cell and tissue growth and also the response to abiotic stresses. Comparative proteomic analysis showed that the level of RPN6 protein, a non-ATPase subunit of the 26S proteasome complex, was significantly reduced in 35S∷CaPUB1 seedlings as compared to the wild type. Pull-down and ubiquitination assays demonstrated that RPN6 interacted physically with CaPUB1 and was ubiquitinated in a CaPUB1-dependent manner in vitro. Although the physiological function of CaPUB1 is not yet clear, there are several possibilities for its involvement in a subset of physiological responses to counteract dehydration and high-salinity stresses in transgenic Arabidopsis seedlings.

To elucidate an understanding into H-Ras protein network, we have established various oncogene H-Ras-expressing NIH/3T3 mouse embryonic fibroblast cell clones, which are expressing G12V H-Ras, G12R H-Ras, and G12V/T35S H-Ras proteins under the tight control of expression by an antibiotic doxycycline. Here we provide a catalog of proteome profiles in total cell lysate derived from the oncogenic and partial loss of function 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 both commonly in oncogenic and partial loss of function H-Ras expression system. Thus, we tried to dissect H-Ras signaling pathway, especially a downstream effector molecule, Raf in NIH/3T3 cells using proteomics tools. In this study, we centralized upon the proteome profile changes as common targets for oncogenic H-Ras and a partial loss of function H-Ras in the H-Ras-expressing cells. Thirteen protein spots were selected as what the staining intensities on the gels for 2-DE images from both kinds of cells were consistently changed in their protein expression level. Differentially regulated expression was further confirmed for some subsets of candidates by semiquantitative RT-PCR and Western blot analysis using specific antibodies. Taken together, our results obtained and present here show that the comparative analysis of proteome from oncogenic and partial loss of function H-Ras-expressing cells has yielded interpretable data to elucidate the protein network directly and/or indirectly.

Inactivating mutations in the adenomatous polyposis coli gene (APC), and activating mutations in RAS, occur in a majority of colorectal carcinomas. However, the relationship between these changes and tumorigenesis is poorly understood. RAS-induced activation of the ERK pathway was reduced by overexpressing APC in DLD-1 colorectal cancer cells. ERK activity was increased by Cre-virus-induced Apc knockout in primary Apc(flox/flox) mouse embryonic fibroblasts, indicating that APC inhibits ERK activity. ERK activity was increased by overexpression and decreased by knock down of beta-catenin. The activation of Raf1, MEK and ERK kinases by beta-catenin was reduced by co-expression of APC. These results indicate that APC inhibits the ERK pathway by an action on beta-catenin. RAS-induced activation of the ERK pathway was reduced by the dominant negative form of TCF4, indicating that the ERK pathway regulation by APC/beta-catenin signaling is, at least, partly caused by effects on beta-catenin/TCF4-mediated gene expression. The GTP loading and the protein level of mutated RAS were decreased in cells with reduced ERK activity as a result of APC overexpression, indicating that APC regulates RAS-induced ERK activation at least partly by reduction of the RAS protein level. APC regulates cellular proliferation and transformation induced by activation of both RAS and beta-catenin signaling.

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.

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.

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.

Although elevated expression and increased tyrosine phosphorylation of focal adhesion kinase (FAK) are crucial for tumor progression, the mechanism by which FAK promotes oncogenic transformation is unclear. We have therefore determined the role of FAK phosphorylation at tyrosine 861 in the oncogenic transformation of NIH3T3 fibroblasts. FAK phosphorylation at tyrosine 861 was increased in both constitutively H-Ras-transformed and H-Ras-inducible NIH3T3 cells, in parallel with cell transformation. However, H-Ras-inducible cells transfected with the nonphosphorylatable mutant FAK Y861F showed decreased migration/invasion, focus forming activity and anchorage-independent growth, compared with either wild-type or kinase-defective FAK. In contrast to unaltered FAK/Src activity, the association of FAK and p130(CAS) was decreased in FAK Y861F-transfected cells, and FAK phosphorylation at tyrosine 861 enhanced this association in vitro. Consistently, FAK Y861F-transfected cells were defective in activation of c-Jun NH(2)-terminal kinase and in expression of matrix metalloproteinase-9 during transformation. Taken together, these results strongly suggest that FAK phosphorylation at tyrosine 861 is crucial for H-Ras-induced transformation through regulation of the association of FAK with p130(CAS).

NRP-152 is an androgen responsive, non-tumorigenic cell line, which shows basal epithelial cell characteristics under normal growth conditions. It has been noted that NRP-152 undergoes morphological and cytoskeletal changes toward its luminal counterpart NRP-154 when it is grown under growth restrictive conditions. We have extensively investigated the details of protein change of NRP-152 during transdifferentiation using proteomic techniques. NRP-152 cells were cultured under normal and growth restrictive media conditions for 3, 5 days. NRP-154 cells were normally cultured. Protein samples were submitted to 2D gel electrophoresis and silver stained. Protein patterns on the gels were comparatively analysed using Melanie III software. Protein spots exhibiting significant changes in NRP-152 cells during the time course were excised and subjected to in-gel tryptic digestion. After 6 days of growth restrictive conditions in NRP-152, the cells were morphologically changed resembling luminal phenotype. Of the 35 protein spots that were up-reglated, 20 proteins from 21 spots were identified by peptide mass fingerprinting and, of 21 proteins spots that were down-regulated, 10 proteins from 12 spots were identified as landmark proteins. Our study confirmed that basal NRP-152 cells were proportionally transdifferentiated into luminal featuring cells according to the duration of growth restrictive culture conditions. This suggests that human prostatic basal epithelial cells may be changed into luminal cells under certain conditions. Proteomic approach enabled us to identify 30 proteins involved in this differentiation with a single experiment. These proteins will be subjected to further functional studies to evaluate their possible roles related to cellular differentiation. These data strongly support that proteomics is a very powerful approach for studying physiologic and pathologic cellular changes such as differentiation and carcinogenesis.

Phosphoinositide-specific phospholipase C (PI-PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate and diacylglycerol, both of which act as secondary messengers in animal cells. In this report, we identified in Vigna radiata L. (mung bean) three distinct partial cDNAs (pVr-PLC1, pVr-PLC2, and pVr-PLC3), which encode forms of putative PI-PLC. All three Vr-PLC genes were transcriptionally active and displayed unique patterns of expression. The Vr-PLC1 and Vr-PLC2 transcripts were constitutively expressed to varying degrees in every tissue of mung bean plants examined. In contrast, the Vr-PLC3 mRNA level was very low under normal growth conditions and was rapidly induced in an abscisic acid-independent manner under environmental stress conditions (drought and high salinity). An isolated genomic clone, about 8.2 kb in length, showed that Vr-PLC1 and Vr-PLC3 are in tandem array in the mung bean genome. The predicted primary sequence of Vr-PLC3 (M(r)=67.4 kDa) is reminiscent of the delta-isoform of animal enzymes which contain core sequences found in typical PI-PLCs, such as the catalytic domain comprising X and Y motifs, a lipid-binding C2 domain, and the less conserved EF-hand domain. Results of in vivo targeting experiment using a green fluorescent protein (GFP) showed that the GFP-Vr-PLC3 fusion protein was localized primarily to the plasma membrane of the Arabidopsis protoplast. The C2 domain was essential for Vr-PLC3 to be targeted to the plasma membrane. The possible biological functions of stress-responsive Vr-PLC3 in mung bean plants are discussed.

The plant hormone ethylene has been shown to play an important role in root hair development in Arabidopsis. With the aid of proteomic analysis, we identified three distinct glutathione S-transferase (GST) isoforms, AtGSTF2, AtGSTF8, and AtGSTU19, expressed early in root epidermal establishment in Arabidopsis seedlings. The AtGSTF2 protein was specifically up-regulated by ethylene. A subsequent RNA expression study revealed that the AtGSTF2 gene was highly sensitive to ethylene, whereas the transcripts for AtGSTF8 and AtGSTU19 were constitutively present in new root tissue of 4-day-old seedlings. The steady-state level of AtGSTF2 mRNA was greatly reduced in the roots of ethylene-insensitive mutants, while mutation at the CTR1 locus, which confers an ectopic root hair phenotype, resulted in a markedly elevated level of AtGSTF2 transcript in young root tissue. Although the physiological function of ethylene-induced AtGSTF2 is not yet clear, there are several possibilities for its role during early root development.

The tumor suppressor, PTEN, located at 10q23, is one of the most frequently mutated tumor suppressors in a number of sporadic cancers and in two autosomal dominant harmatomas. It is considered one of the most important tumor suppressors in the post p53 era. To identify the molecules involved in the signal network regulated by PTEN using proteomic tools, a PTEN-inducible expression system was established in NIH 3T3 mouse embryonic fibroblast cells. We compared proteome images of PTEN-induced and non-induced cells by 2-dimensional electrophoresis. Twenty-nine differentially expressed protein spots were identified by MALDI-TOF MS and NSI MS/MS. We conclude that expression of PTEN by itself leads to protein profile changes, and those proteins affected are likely to be directly and/or indirectly involved in the function and physiology of the tumor suppressor.

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.

Malonyl-CoA decarboxylase (E.C.4.1.1.9) catalyzes the conversion of malonyl-CoA to acetyl-CoA. Although the metabolic role of this enzyme has not been fully defined, it has been reported that its deficiency is associated with mild mental retardation, seizures, hypotonia, cadiomyopathy, developmental delay, vomiting, hypoglycemia, metabolic acidosis, and malonic aciduria. Here, we isolated a cDNA clone for malonyl CoA decarboxylase from a rat brain cDNA library, expressed it in E. coli, and characterized its biochemical properties. The full-length cDNA contained a single open-reading frame that encoded 491 amino acid residues with a calculated molecular weight of 54, 762 Da. Its deduced amino acid sequence revealed a 65.6% identity to that from the goose uropigial gland. The sequence of the first 38 amino acids represents a putative mitochondrial targeting sequence, and the last 3 amino acid sequences (SKL) represent peroxisomal targeting ones. The expression of malonyl CoA decarboxylase was observed over a wide range of tissues as a single transcript of 2.0 kb in size. The recombinant protein that was expressed in E. coli was used to characterize the biochemical properties, which showed a typical Michaelis-Menten substrate saturation pattern. The Km and Vmax were calculated to be 68 microM and 42.6 micromol/min/mg, respectively.

Theiler’s murine encephalomyelitis virus (TMEV) induces immune-mediated demyelination after intracerebral inoculation of the virus into susceptible mouse strains. We isolated from a TMEV BeAn 8386 viral stock, a low-pathogenic variant which requires greater than a 10,000-fold increase in viral inoculation for the manifestation of detectable clinical signs. Intracerebral inoculation of this variant virus induced a strong, long-lasting, protective immunity from the demyelinating disease caused by pathogenic TMEV. The levels of antibodies to the whole virus as well as to the major linear epitopes were similar in mice infected with either the variant or wild-type virus. However, persistence of the variant virus in the central nervous system (CNS) of mice was significantly lower than that of the pathogenic virus. In addition, the T-cell response to the predominant VP1 (VP1 233–250 ) epitope in mice infected with the variant virus was significantly weaker than that in mice infected with the parent virus, while similar T-cell responses were induced against another predominant epitope (VP2 74–86 ). Further analyses indicated that a change of lysine to arginine at position 244 of VP1, which is the only amino acid difference in the P1 region, is responsible for such differential T-cell recognition. Thus, the difference in the T-cell reactivity to this VP1 region as well as the low level of viral persistence in the CNS may account for the low pathogenicity of this spontaneous variant virus.

Phospholipase C-beta1 (PLC-beta1), one of the PLC-beta isozymes, exists as two immunologically distinguishable polypeptides of 150 (PLC-beta1a) and 140 kDa (PLC-beta1b) which are encoded in two distinct transcripts and generated by alternative splicing of a single gene. In this study, the subcellular localization of the two phospholipases C-beta1 proteins was examined in rat C6Bu-1 glioma cells using immunological techniques. Immunoblot analysis revealed that the two forms of PLC-beta1 were detectable in both cytosolic and nuclear fractions. PLC-beta1a appeared to be located preferentially in the cytosol, whereas PLC-beta1b was found predominantly in the nuclei of C6Bu-1 cells. Immunocytochemical experiments confirmed the differential localization of the two PLC-beta1 species in C6Bu-1 cells. These results suggest that the two PLC-beta1 proteins may have different physiological roles in the cell.

Phospholipase C-beta 4(PLC-beta 4), a new member of phospholipase C isozyme, was purified from bovine cerebellum. The cDNA encoding rat PLC-beta 4 has been cloned from a cDNA library prepared from rat brain. The predicted open reading frame encodes a protein of 1,176 amino acids with a calculated molecular weight of 134,552. The deduced amino acid sequence exhibits 39, 36, and 36% identity with the sequences of rat PLC-beta 1, human PLC-beta 2, and rat PLC-beta 3, respectively. The amino acid sequence of PLC-beta 4, especially, shows higher identity (50%) with norpA PLC sequence from Drosophila melanogaster than those of other PLC-beta subtypes, suggesting that the PLC-beta 4 might be a mammalian PLC equivalent of norpA PLC implicated in photosignal transduction in Drosophila.

Advancements in the field of proteomics have provided great opportunities for the development of diagnostic and therapeutic tools against human diseases. In this study, we analyzed haptoglobin and amyloid A protein levels of vivax malaria patients with combinations of depletion of the abundant plasma proteins, 2-dimensional gel electrophoresis (2-DE), image analysis, and mass spectrometry in the plasma between normal healthy donors and vivax malaria patients. The results showed that the expression level of haptoglobin had become significantly lower or undetectable in the plasma of vivax malaria patients due to proteolytic cleavage when compared to healthy donors on 2-DE gels. Meanwhile, serum amyloid A protein was significantly increased in vivax malaria patient's plasma with high statistical values. These 2 proteins are common acute phase reactants and further large scale evaluation with a larger number of patient's will be necessary to establish the possible clinical meaning of the existential changes of these proteins in vivax malaria patients. However, our proteomic analysis suggests the feasible values of some plasma proteins, such as haptoglobin and serum amyloid A, as associating factor candidates for vivax malaria.

Triple A syndrome is a rare genetic disorder caused by mutations in the achalasia-addisonianism-alacrima syndrome (AAAS) gene which encodes a tryptophan aspartic acid (WD) repeat-containing protein named alacrima-achalasia-adrenal insufficiency neurologic disorder (ALADIN). Northern blot analysis shows that the 2.1 kb AAAS mRNA is expressed in various tissues with stronger expression in testis and pancreas. We show that human ALADIN is a protein with an apparent molecular weight of 60 kDa, and expressed in the adrenal gland, pituitary gland and pancreas. Furthermore, biochemical analysis using anti-ALADIN antibody supports the previous finding of the localization of ALADIN in the nuclear membrane. The mutations S544G and S544X show that alteration of S544 residue affects correct targeting of ALADIN to the nuclear membrane.

Psoriasis is a common inflammatory skin disease that is thought to be mediated by activated T cells. In this study, the complementarity-determining region 3 (CDR3) in T cell receptors was examined for a common sequence motif among the T cells infiltrated in psoriatic lesional skin. A common specific CDR3 motif (Vbeta13-DWTSGV-Jbeta2.7) in lesions from psoriasis patients was identified by polymerase-chain-reaction-based spectratyping analysis and DNA sequencing. In addition, VDJ rearrangement with highly homologous amino acid composition in the CDR3 was observed in Vbeta15 of T cell receptors in lesions derived from psoriatic patients. Remarkably, T cell receptors containing the Vbeta13-DWTSGV-Jbeta2.7 were also found in the clinically normal skin from the psoriasis patients, which might seem to be responsible for the artificial production of psoriatic lesions. The identified CDR3 motif was highly expressed in cutaneous lymphocyte antigen (CLA+) cells of peripheral blood mononuclear cells from psoriasis patients compared with the expression in healthy individuals. This result showed that the infiltrated CLA+ T cells with the Vbeta13-DWTSGV-Jbeta2.7 motif in peripheral blood mononuclear cells from psoriasis patients might be involved in the development of psoriatic lesions. In addition, the results in this study suggest that the infiltrated T cells with the Vbeta13-DWTSGV-Jbeta2.7 motif in psoriatic lesions may be involved in the pathogenesis of psoriasis.