Angiotensin-converting enzyme inhibition curbs tyrosine nitration of mitochondrial proteins in the renal cortex during the early stage of diabetes mellitus in rats.

Experiments were performed to evaluate the hypothesis that ACE (angiotensin-converting enzyme) inhibition (enalapril) suppresses 3-NT (3-nitrotyrosine) production in the renal cortex during the early stage of Type 1 DM (diabetes mellitus) in the rat. Enalapril was administered chronically for 2 weeks to subsets of STZ (streptozotocin)-induced DM and vehicle-treated sham rats. O(2)(-) (superoxide anion) and NO(x) (nitrate+nitrite) levels were measured in the media bathing renal cortical slices after 90 min incubation in vitro. SOD (superoxide dismutase) activity and 3-NT content were measured in the renal cortex homogenate. Renal cortical nitrated protein was identified by proteomic analysis. Renal cortical production of O(2)(-) and 3-NT was increased in DM rats; however, enalapril suppressed these changes. DM rats also exhibited elevated renal cortical NO(x) production and SOD activity, and these changes were magnified by enalapril treatment. 2-DE (two-dimensional gel electrophoresis)-based Western blotting revealed more than 20 spots with positive 3-NT immunoreactivity in the renal cortex of DM rats. Enalapril treatment blunted the DM-induced increase in tyrosine nitration of three proteins ACO2, GDH1 and MMSDH (aconitase 2, glutamate dehydrogenase 1 and methylmalonate-semialdehyde dehydrogenase), each of which resides in mitochondria. These data are consistent with enalapril preventing DM-induced tyrosine nitration of mitochondrial proteins by a mechanism involving suppression of oxidant production and enhancement of antioxidant capacity, including SOD activation.

Proteomic Analysis of Anti-inflammatory Effects of a Kampo (Japanese Herbal) Medicine "Shoseiryuto (Xiao-Qing-Long-Tang)" on Airway Inflammation in a Mouse Model.

Effects of a Kampo (Japanese herbal) medicine "shoseiryuto (SST, xiao-qing-long-tang in Chinese)", which has been used for the treatment of allergic bronchial asthma clinically, were examined on ovalbumin (OVA)-sensitized allergic airway inflammation model (i.e., bronchial asthma) in a mouse. When SST was orally administered at 0.5 g kg(-1) day(-1) from day 1 to 6 after OVA inhalation, SST reduced the inflammation in lung tissue, the number of eosinophils and the OVA-specific immunoglobulin E (IgE) antibody titer in bronchoalveolar lavage (BAL) fluids at 7 days after the OVA inhalation. SST also reduced the airway hyperreactivity at 6 days after the OVA inhalation. Proteomic analysis with the agarose two-dimensional electrophoresis showed that the expression of spectrin α2 was reduced in the lung tissue of OVA-sensitized mice and SST recovered the expression. Western blot and immunohistochemical analyses of lung tissue also confirmed this result. When prednisolone was orally administered at 3 mg kg(-1) day(-1) from day 1 to 6 after OVA inhalation, the inflammation in lung tissue, the number of eosinophils in BAL fluids and airway hyperreactivity were reduced in the OVA-sensitized mice. However, prednisolone did not reduce the OVA-specific IgE antibody titer in BAL fluids and did not recover the expression of spectrin α2 in lung tissue. These results suggest that at least a part of action mechanism of SST against OVA-sensitized allergic airway inflammation in a mouse model is different from that of prednisolone.

Comparative proteomic analysis of Salmonella enterica serovar Typhimurium ppGpp-deficient mutant to identify a novel virulence protein required for intracellular survival in macrophages.

BACKGROUND: The global ppGpp-mediated stringent response in pathogenic bacteria plays an important role in the pathogenesis of bacterial infections. In Salmonella enterica serovar Typhimurium (S. Typhimurium), several genes, including virulence genes, are regulated by ppGpp when bacteria are under the stringent response. To understand the control of virulence genes by ppGpp in S. Typhimurium, agarose 2-dimensional electrophoresis (2-DE) combined with mass spectrometry was used and a comprehensive 2-DE reference map of amino acid-starved S. Typhimurium strain SH100, a derivative of ATCC 14028, was established. RESULTS: Of the 366 examined spots, 269 proteins were successfully identified. The comparative analysis of the wild-type and ppGpp0 mutant strains revealed 55 proteins, the expression patterns of which were affected by ppGpp. Using a mouse infection model, we further identified a novel virulence-associated factor, STM3169, from the ppGpp-regulated and Salmonella-specific proteins. In addition, Salmonella strains carrying mutations in the gene encoding STM3169 showed growth defects and impaired growth within macrophage-like RAW264.7 cells. Furthermore, we found that expression of stm3169 was controlled by ppGpp and SsrB, a response regulator of the two-component system located on Salmonella pathogenicity island 2. CONCLUSIONS: A proteomic approach using a 2-DE reference map can prove a powerful tool for analyzing virulence factors and the regulatory network involved in Salmonella pathogenesis. Our results also provide evidence of a global response mediated by ppGpp in S. enterica.

Identification of novel immunohistochemical tumor markers for primary hepatocellular carcinoma; clathrin heavy chain and formiminotransferase cyclodeaminase.

UNLABELLED: Early diagnosis of hepatocellular carcinoma (HCC) greatly improves its prognosis. However, the distinction between benign and malignant tumors is often difficult, and novel immunohistochemical markers are necessary. Using agarose two-dimensional fluorescence difference gel electrophoresis, we analyzed HCC tissues from 10 patients. The fluorescence volumes of 48 spots increased and 79 spots decreased in tumor tissues compared with adjacent nontumor tissue, and 83 proteins were identified by mass spectrometry. Immunoblot confirmed that the expression of clathrin heavy chain (CHC) and Ku86 significantly increased, whereas formiminotransferase cyclodeaminase (FTCD), rhodanese, and vinculin decreased in tumor. The protein expression in tumor and nontumor tissues was further evaluated by immunostaining. Interestingly, CHC and FTCD expression was strikingly different between tumor and nontumor tissues. The sensitivity and specificity of individual markers or a combination for the detection of HCC were 51.8% and 95.6% for CHC, 61.4% and 98.5% for FTCD, and 80.7% and 94.1% for CHC+FTCD, respectively. Strikingly, the sensitivity and specificity increased to 86.7% and 95.6% when glypican-3, another potential biomarker for HCC, was used with FTCD. Moreover, CHC and FTCD were useful to distinguish early HCC from benign tumors such as regenerative nodule or focal nodular hyperplasia, because the sensitivity and specificity of the markers are 41.2% and 77.8% for CHC, 44.4% and 80.0% for FTCD, which is comparable with those of glypican-3 (33.3% and 100%). The sensitivity significantly increased by combination of these markers, 72.2% for CHC+FTCD, and 61.1% for CHC+glypican-3 and FTCD+glypican-3, as 44.4% of glypican-3 negative early HCC were able to be detected by either CHC or FTCD staining. CONCLUSION: Immunostaining of CHC and FTCD could make substantial contributions to the early diagnosis of HCC.

Detection of biomarkers for alcoholism by two-dimensional differential gel electrophoresis.

BACKGROUND: Up to now, gamma-glutamyltransferase (gamma-GTP) and carbohydrate-deficient transferrin (CDT) have been used as markers for alcoholism most widely, but they are not satisfactory regarding sensitivity/specificity. Therefore, for novel markers need to be searched. METHODS: To detect new biomarkers for alcoholism, albumin and immunoglobulinG were first removed from serum. Then, protein profiles of 12 serum samples before and after 3 months of abstinence treatment were examined using agarose 2-dimensional differential gel electrophoresis (agarose2-D DIGE). Two-dimensional differential gel electrophoresis images were analyzed using Shimadzu 2-D Evolution Software. RESULTS: Eight spots whose expression were significantly altered after abstinence were detected. Of these, 2 proteins increased and 6 proteins decreased after treatment. CONCLUSIONS: Altered expressions of several serum proteins after abstinence therapy were detected. They are promising markers for clinical application of alcoholism.

Disease proteomics of high-molecular-mass proteins by two-dimensional gel electrophoresis with agarose gels in the first dimension (Agarose 2-DE).

Agarose gel is the preferred electrophoretic medium currently used for separating high molecular mass (HMM) proteins (MW>100 kDa). Agarose gels are widely used for both SDS-agarose gel electrophoresis and agarose isoelectric focusing (IEF). A two-dimensional gel electrophoresis method employing agarose gels in the first dimension (agarose 2-DE) that is sufficiently good at separating up to 1.5mg of HMM proteins with molecular masses as large as 500 kDa has been used to separate proteins from various diseased tissues and cells. Although resolution of the agarose 2-DE pattern always depends on the tissue being analyzed, sample preparation procedures including (i) protein extraction with an SDS sample buffer; (ii) ultracentrifugation of a tissue homogenate; and (iii) 1% SDS in both stacking and separation gels of the second-dimension SDS-PAGE gel, are generally effective for HMM protein detection. In a comprehensive prostate cancer proteome study using agarose 2-DE, the HMM region of the gel was rich in proteins of particular gene/protein expression groups (39.1% of the HMM proteins but only 28.4% of the LMM ones were classified as transcription/translation-related proteins). Examples include transcription factors, DNA or RNA binding proteins, and ribosomal proteins. To understand oxidative stress-induced cellular damage at the protein level, a novel proteomic method, in which protein carbonyls were derivatized with biotin hydrazide followed by agarose 2-DE, was useful for detecting HMM protein carbonyls in tissues of both a diabetes model Ostuka Long-Evans Tokushima Fatty (OLETF) rat and a control Long-Evans Tokushima Otsuka (LETO) rat. In this paper, we review the use of agarose gels for separation of HMM proteins and disease proteomics of HMM proteins in general, with particular attention paid to our proteome analyzes based on the use of agarose 2-DE for protein separation followed by the use of mass spectrometry for protein identification.

Haplotype analysis reveals founder effects of thyroglobulin gene mutations C1058R and C1977S in Japan.

CONTEXT: Thyroglobulin (Tg) mutations were previously believed to be rare, resulting in congenital goitrous hypothyroidism. However, an increasing number of patients with Tg mutations, who are euthyroid to mildly hypothyroid, have been identified in Japan. OBJECTIVES: The purpose of this study was to investigate whether the three frequently found Tg mutations, namely C1058R, C1245R, and C1977S, were caused by a founder effect. RESULTS: We found 26 different mutations within the Tg gene in 52 patients from 41 families. Thirty-five patients were homozygous for the mutations, whereas the others were compound heterozygous. The occurrence of Tg mutation within the general Japanese population is one in 67,000. Patients with the C1245R mutation were found throughout Japan, whereas those with the C1058R mutation were confined to a small village on a southern island, and those with the C1977S mutation were restricted to a city. The eight patients with the C1058R mutation and the seven patients with the C1977S mutation all showed the same combinations of 18 single-nucleotide polymorphisms in the coding region of the Tg gene, which would appear in one in 810 million and one in 37 billion, respectively, control subjects. CONCLUSIONS: The frequently found mutations, C1058R and C1977S, were caused by founder effects. This result suggests that Tg mutations may provide a genetic basis for the cause of familial euthyroid goiter.

Identification of altered protein expression and post-translational modifications in primary colorectal cancer by using agarose two-dimensional gel electrophoresis.

PURPOSE: Although numerous proteome studies have been performed recently to identify cancer-related changes in protein expression, only a limited display of relatively abundant proteins has been identified. The aim of this study is to identify novel proteins as potential tumor markers in primary colorectal cancer tissues using a high-resolution two-dimensional gel electrophoresis (2-DE). EXPERIMENTAL DESIGN: 2-DE using an agarose gel for isoelectric focusing was used to compare protein profiling of 10 colorectal cancer tissues and adjacent normal mucosa. Altered expression and post-translational modification of several proteins were examined using Western blot analysis and immunohistochemistry. RESULTS: Ninety-seven proteins of 107 spots (90.7%) that were differentially expressed between matched normal and tumor tissues were identified by mass spectrometry. Among them, 42 unique proteins (49 spots) significantly increased or decreased in the tumors. They include eukaryotic translation initiation factor 4H, inorganic pyrophosphatase, anterior gradient 2 homologue, aldolase A, and chloride intracellular channel 1, whose elevated expression in tumor tissues was confirmed by Western blot analysis and immunohistochemistry. Interestingly, only isoform 1 of two transcript variants of eukaryotic translation initiation factor 4H was greatly up-regulated in most of the tumor tissues. Moreover, post-translational modifications of the prolyl-4-hydroxylase beta subunit and annexin A2 also were identified. CONCLUSIONS: We identified several novel proteins with altered expression in primary colorectal cancer using agarose 2-DE. This method is a powerful technique with which to search for not only quantitative but also qualitative changes in a biological process of interest and may contribute to the deeper understanding of underlying mechanisms of human cancer.

Proteomic method detects oxidatively induced protein carbonyls in muscles of a diabetes model Otsuka Long-Evans Tokushima Fatty (OLETF) rat.

Oxidative stress is implicated in a broad variety of chronic and acute diseases, including such age-related diseases as diabetes. To understand at the protein level cellular damage caused by the stress, we developed a proteomic method, in which protein carbonyls were derivatized with biotin hydrazide followed by two-dimensional gel electrophoresis. The method, being capable of analyzing high-molecular-mass proteins as large as myosin heavy chains (molecular mass approximately 200 kDa), was applied to detecting protein carbonyls in muscles of a diabetes model Otsuka Long-Evans Tokushima Fatty (OLETF) rat and a control Long-Evans Tokushima Otsuka (LETO) rat. A number of proteins, including mitochondrial ATP synthase beta-chain, desmin, actin, and myosin, were found carbonylated. Our method would provide a means toward clarifying a comprehensive view of oxidative modifications of proteins during a long progression of age-related diseases and understanding the mechanism of the onset, progression, and complication of the diseases.

Changes in Electrophoretic Patterns of Oocyte Proteins during Oocyte Maturation in Oryzias latipes.

Changes in the two-dimensional SDS-electrophoretic patterns of extracts of maturing denuded oocytes of the medaka (Oryzias latipes) were surveyed. In oocytes without follicular constituents several proteins became detectable in the area between the acidic and slightly basic proteins on the two-dimensional electrophoretograms, while a few of the protein spots disappeared during the process of oocyte maturation. The former proteins were detected also in oocytes that were induced to mature in vivo without breakdown of the germinal vesicle. Several proteins newly observed in extracts of post-vitellogenic oocytes during maturation after breakdown of the germinal vesicle were also identified by two-dimensional electrophoresis. Of several proteins that exhibited noticeable changes in maturing oocytes, only one spot incorporated 14 C-labeled amino acid during maturation, suggesting that post-translational modification of many proteins occurred during oocyte maturation.

Separation techniques for high-molecular-mass proteins.

Many high-molecular-mass (HMM) proteins (MW>100 kDa) are known to be involved in cytoskeleton, defence and immunity, transcription and translation in higher eukaryotic organisms. Even in the post-genomic era, purification of HMM protein is the first important step to analyze protein composition in a tissue or a cell (proteomics), to determine protein tertiary structure (structural biology), and to investigate protein function (functional genomics). To separate a HMM protein from a protein mixture, ions, chaotropes (urea and thiourea), detergents and protease inhibitors in extraction media and buffer solutions either for liquid chromatography or for gel electrophoresis should be carefully chosen, since HMM proteins tend to be aggregates under denatured condition and their long polypeptide chains are easily attacked by intrinsic proteases during separation procedure. Among many liquid chromatography techniques, affinity chromatography either with sequence-specific DNA for transcription factor, or with monoclonal antibody specific for myosin heavy chain has been used for preparative isolation of the respective HMM proteins. Though SDS-PAGE could analyze the size and the quantity of megadalton proteins, the resolution of HMM proteins is relatively poor. A newly developed pulse SDS-PAGE would be able to raise the resolution of HMM proteins compared with the conventional SDS-PAGE. The 2-DE method is not particularly suitable in analyzing HMM proteins larger than 200 kDa. However, a 2-DE method that uses an agarose IEF gel in the first dimension (agarose 2-DE) has been shown to produce significant improvements in 2-DE separation of HMM proteins larger than 150 kDa and up to 500 kDa.

Disease proteomics of endocrine disorders revealed by two-dimensional gel electrophoresis and mass spectrometry.

Endocrine disorders such as dwarfism and diabetes show abnormalities in many different organs even if a certain hormone is the primary cause of the disease. One of the aims of proteomics is to elucidate an abnormal hormone network underlying dysfunction in the disease through quantitative and qualitative proteome analyses of various organs. In a comprehensive study of the rdw rat with hereditary dwarfism, we found the accumulation of ER proteins in the rdw thyroid. Contrary to the initial notion that the dwarfism of the rat was caused by genetic mutations related to pituitary hormones, the primary cause is a missense mutation in the thyroglobulin gene. To understand at the protein level cellular damage caused by oxidative stress, we developed a proteomic method and applied to detecting protein carbonyls in various organs of a diabetes model OLETF rat. The method would provide a means toward clarifying a comprehensive view of oxidative modifications of proteins in diabetes. We review 2-DE-based disease proteomics of endocrine disorders in general, with particular attention paid to our proteome projects by a 2-DE method with an agarose IEF gel in the first dimension (agarose 2-DE) and LC-MS/MS.

Proteomic analysis of primary esophageal squamous cell carcinoma reveals downregulation of a cell adhesion protein, periplakin.

Recent advances in two-dimensional electrophoresis (2-DE) such as fluorescent 2-D differential gel electrophoresis (2-D DIGE) has made it possible to detect and quantitate the critical changes involved in disease pathogenesis. We have previously identified novel proteins with altered expression in primary colorectal cancer using agarose 2-DE that has a higher loading capacity than immobilized pH gradient gel. The aim of this study is to identify novel proteins with altered expression in primary esophageal cancer using the powerful method of agarose 2-DE and agarose 2-D DIGE. Excised tissues from 12 patients of primary esophageal cancer were obtained. Proteins with altered expression between cancer and adjacent non-cancer tissues were analyzed by agarose 2-D DIGE and identified by mass spectrometry. Thirty-three proteins out of 74 spots with altered expression in tumors were identified. Among them, a 195-kDa protein, periplakin, was significantly downregulated in esophageal cancer, which was confirmed by immunoblotting. Immunohistochemistry showed that periplakin was mainly localized at cell-cell boundaries in normal epithelium and dysplastic lesions, while it disappeared from cell boundaries, shifted to cytoplasm, in early cancers and scarcely expressed in advanced cancers. These results suggest that periplakin could be a useful marker for detection of early esophageal cancer and evaluation of tumor progression.

Effect of thyroxine on abnormal pancreatic proteomes of the hypothyroid rdw rat.

A mutation in the thyroglobulin (Tg) gene is the primary cause of hereditary dwarfism and hypothyroidism in the rdw rat. Despite the Tg mutation that causes a Tg shortage, rdw rats survive. The present study examines the influences of this condition on the pancreatic proteome. Normal control (group 1; n = 19) and rdw rats that did not receive L-thyroxine (T4) (group 2; n = 27) were sacrificed from 4 to 56 weeks after birth. The rdw rats were supplemented either with daily intraperitoneal injections of T4 from 3 to 28 days after birth (group 3; n = 4) or with normal thyroid tissues grafted at 4 weeks of age (group 4; n = 3). Groups 3 and 4 were sacrificed 12 weeks after birth. Pancreatic proteomes analyzed by two-dimensional gel electrophoresis showed that levels of at least four pancreatic proteins were higher in group 2 than in group 1, and that those of four were lower. Cluster decomposition and principal component analysis of the eight protein contents showed that groups 1 and 2 were separated into two clusters and that pancreatic proteomes of group 4 were better normalized than those of group 3. Injecting T4 into group 3 was temporarily effective, whereas the thyroid graft to group 4 provided a continuous positive effect, which concurred with the increased body weight of the other two groups of rdw rats that received grafts of normal thyroids.

High molecular mass proteome of androgen-independent prostate cancer.

We made a comprehensive study of protein expression of androgen-independent (AI) prostate cancer by means of agarose two-dimensional gel electrophoresis (agarose 2-DE) followed by liquid chromatography-tandem mass spectrometric analysis. The agarose 2-DE method being good at separating high molecular mass (HMM) proteins and alkaline ones, so we were able to successfully reveal differences between the proteomes of androgen-dependent (AD) tumors and those of AI tumors. During the creation of agarose 2-DE protein maps, we successfully identified 295 proteins (91.0%) out of 324 spots excised in total. Excluding redundant and mouse serum proteins, we considered the remaining 225 proteins to be related to the cancer. We divided the 225 cancer-related proteins into HMM and low molecular mass (LMM) groups by their molecular mass being above or below 80 kDa. Functional classification of the proteins in these two groups showed clear differences between the two: more than half (54.8%) of the HMM proteins, but less than one-third (29.1%) of the LMM ones were classified among transcription- or translation-related proteins. Eighteen proteins were regulated when the tumor progressed from an AD to an AI state. Five of these proteins, including antioxidant protein 2, superoxide dismutase 1, thioredoxin peroxidase, GTP-binding protein beta chain homolog, and the ha1225 gene product, had a function to protect cells against oxidant stress-induced apoptosis. We suggest that prevention against oxidative stress is one of the key points for prostate cancer to obtain androgen independency. We also found proteins that had not been previously reported in prostate cancer by use of genomic or conventional 2-DE based proteomic approaches. The proteomic approach, using agarose 2-DE focused on HMM proteins, has the capability to find novel biomarkers of prostate cancer.