Cellulose Acetate Membrane Electrophoresis Based Urinary Proteomics for the Identification of Characteristic Proteins.

BACKGROUND: Analysis of urinary proteins using cellulose acetate membrane electrophoresis (CAME) is a useful and challenging method for the recognition of damaged sites in the kidney. However, protein content of each CAME fraction is still not completely understood. METHODS: In this study, an effective method of protein extraction from each band fractionated by CAME was established, which enabled us to examine the extracted proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. RESULTS: Proteins were extracted from the gel and analyzed by mass spectrometry. In all, 31 proteins were identified, including 20 urinary proteins that were newly identified in the CAME-based analysis. CONCLUSION: This methodology was useful for identifying the proteins responsible for creating unique bands on CAME in a urine sample of a patient with drug-induced interstitial nephritis. These findings provide in-depth characterization of urinary protein contents in each CAME fraction.

An anilinoquinazoline derivative inhibits tumor growth through interaction with hCAP-G2, a subunit of condensin II.

We screened 46 novel anilinoquinazoline derivatives for activity to inhibit proliferation of a panel of human cancer cell lines. Among them, Q15 showed potent in vitro growth-inhibitory activity towards cancer cell lines derived from colorectal cancer, lung cancer and multiple myeloma. It also showed antitumor activity towards multiple myeloma KMS34 tumor xenografts in lcr/scid mice in vivo. Unlike the known anilinoquinazoline derivative gefitinib, Q15 did not inhibit cytokine-mediated intracellular tyrosine phosphorylation. Using our mRNA display technology, we identified hCAP-G2, a subunit of condensin II complex, which is regarded as a key player in mitotic chromosome condensation, as a Q15 binding partner. Immunofluorescence study indicated that Q15 compromises normal segregation of chromosomes, and therefore might induce apoptosis. Thus, our results indicate that hCAP-G2 is a novel therapeutic target for development of drugs active against currently intractable neoplasms.

A phthalimide derivative that inhibits centrosomal clustering is effective on multiple myeloma.

Despite the introduction of newly developed drugs such as lenalidomide and bortezomib, patients with multiple myeloma are still difficult to treat and have a poor prognosis. In order to find novel drugs that are effective for multiple myeloma, we tested the antitumor activity of 29 phthalimide derivatives against several multiple myeloma cell lines. Among these derivatives, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3- dione (TC11) was found to be a potent inhibitor of tumor cell proliferation and an inducer of apoptosis via activation of caspase-3, 8 and 9. This compound also showed in vivo activity against multiple myeloma cell line KMS34 tumor xenografts in ICR/SCID mice. By means of mRNA display selection on a microfluidic chip, the target protein of TC11 was identified as nucleophosmin 1 (NPM). Binding of TC11 and NPM monomer was confirmed by surface plasmon resonance. Immunofluorescence and NPM knockdown studies in HeLa cells suggested that TC11 inhibits centrosomal clustering by inhibiting the centrosomal-regulatory function of NPM, thereby inducing multipolar mitotic cells, which undergo apoptosis. NPM may become a novel target for development of antitumor drugs active against multiple myeloma.

Production of a reactive metabolite of troglitazone by electrochemical oxidation performed in nonaqueous medium.

In order to confirm the existence of reactive metabolites by LC-MS/MS analysis, they should be modified into stable compounds, because some reactive metabolites generated by biotransformation induce drug toxicity; however, they are unstable, with very short lives, and cannot be detected in their intact forms. To overcome these problems, electrochemical oxidation of troglitazone was performed in nonaqueous medium, since such reactive compounds are stable in the absence of water. Troglitazone, an antidiabetic agent, was withdrawn from the market because of serious hepatotoxicity in some patients. It has been considered that one or more reactive metabolites are involved in hepatotoxicity, although the mechanism of the adverse reaction is unclear. Using our method of electrochemical oxidation in nonaqueous medium, we obtained a product of troglitazone derivative that may be a clue to clarify the mechanism of toxicity. The product in the reaction mixture was separated by HPLC without chemical modification and detected using UV and ESI-MS. The mass spectrum of its molecular ion showed that it was an o-quinone methide derivative of troglitazone and identified as a reactive metabolite generated by liver microsome oxidation of the drug. The product was stable over 24 h at room temperature in anhydrous acetonitrile, but it reacted with N-(tert-butoxycarbonyl)-L-cystein methylester to produce an adduct that could be identified by its m/z value. Thus, the method of electrochemical oxidation in nonaqueous medium is considered to be useful to prepare and predict reactive metabolites of drugs that are unstable in aqueous medium or in vivo.

On-line liquid chromatography and circular dichroism detection of stereo-isomers of alpha-tocopherol derivatives generated by an electrochemical reaction.

The electrochemical oxidation of (+/-)-alpha-tocopherol on a porous graphite electrode was performed in the presence of methanol, and successive separation and detection of the products were performed by an on-line liquid chromatography/mass spectrometry system. Three products were identified, one of which was determined to be alpha-tocopheryl quinone, because its m/z was 469 [M+Na](+). The other two products showed identical mass and UV spectra, and were suspected to be diastereomers of 9-methoxy-alpha-tocopheron, because their molecular weights were m/z 483 [M+Na](+), and also because it is known that the chemical oxidation of alpha-tocopherol by benzoyl peroxide or N-bromosuccinimide in the presence of methanol should provide 9-methoxy-alpha-tocopheron. To confirm that these two compounds were diastereomers, a circular dichroism detector was used. The signs of both peaks detected by the circular dichroism detector at 230 nm were opposite. In addition to observations of identical mass and ultraviolet spectra, these results indicated that the two products were diastereomers of 9-methoxy-alpha-tocopheron, whose stereochemistry is different at the newly generated chiral center of the 9-position. The on-line use of a circular dichroism detector with an electrochemical cell/liquid chromatography system may expand the utility of the system to study the metabolism of a chiral drug.

Successful preparation of metabolite of troglitazone by in-flow electrochemical reaction on coulometric electrode.

A simple, rapid and efficient system utilizing a coulometric electrode was developed for the preparation of drug metabolites. Trace amounts of reactants are usually generated in electrochemical reactions, which are not suitable for the sufficient preparation of products to obtain NMR and other spectral data for chemical structure confirmation or to obtain data from pharmacological activity screening tests of products. In the developed system, called the "in-flow electrochemical reaction system," a drug, troglitazone, was dissolved in a volatile flow solvent, and pumped into a coulometric electrode under optimized conditions, and the effluent was evaporated. Without any further purification, milligram amounts of a pure oxidation product of troglitazone could be obtained within several hours. The amount obtained was enough for (1)H- and (13)C-NMR analysis by which the structure could be confirmed and was found to be identical to one of the metabolites of troglitazone detected in human plasma. This system will be useful to prepare standard compounds of the required amount for pharmacokinetic study and for toxicokinetic study.

Urinary protein analysis in pre- and postoperative cancer patients.

Urinary proteins from six patients with esophageal cancer and two with stomach cancer were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analyses were performed on days-1 to 3, 5, 7, 10, 14, and 21 (or 22) after surgery. The protein patterns were scanned by densitometry and divided into nine fractions. The main proteins in the fractions (Fr.) were identified as follows: immunoglobulin G in Fr. A, Tamm-Horsfall glycoprotein (THP) in Fr. B, transferrin in Fr. C, albumin in Fr. D, alpha(1)-acid glycoprotein in Fr. E, alpha(1)-microglobulin in Fr. F, retinol binding protein in Fr. G, and beta(2)-microglobulin in Fr. I. The protein in Fr. H was not identified. The percentage of each fraction was calculated from the densitometry pattern of each lane. The percentage values were averaged among all the patients, and pre- and postoperative data were compared. The percentage of Frs. E, F, and G increased on days 1-7, and the changes in these three proteins were similar to changes in serum C-reactive protein (CRP). In particular, the percentage of Fr. G peaked within 1 day of operation, which was faster than for CRP. Conversely, other fractions decreased. These results suggest that urinary protein analysis is useful for monitoring the response to surgical stress.

Proteome analysis of focal adhesion kinase (FAK)-overexpressing cells.

We established several focal adhesion kinase (FAK) cDNA-transfected cells and found that FAK-transfected HL-60 (HL-60/FAK) cells are resistant to apoptosis induced with hydrogen peroxide, etoposide and radiation compared with the parental HL-60 or the vector-transfected (HL-60/Vect) cells. We carried out proteome analysis to study the mechanism of resistance to apoptosis in HL-60/FAK cells. Among 300 spots resolved in two-dimensional gels, ca. 10% of them were significantly increased in HL-60/FAK cells compared with HL-60/Vect cells, whereas ca. 2% of them were decreased or disappeared. These proteins were performed for further analysis by Western blots or N-terminal sequencing or mass spectrometry. Increased proteins included stress proteins such as hsp90, ribosomal proteins, and antioxidant enzymes such as peroxyredoxin 2. Some of these proteins are assumed to contribute to the antiapoptotic action of FAK.

Change in relative mobility of M protein following neuraminidase treatment in patients with multiple myeloma and monoclonal gammopathy of undetermined significance.

The aim of this study was to clarify the relationship between the relative mobility of M protein in various disease states using cellulose acetate membrane electrophoresis. To examine the carbohydrate chain of the M protein, sera from patients with multiple myeloma (MM), various cancers, and benign disease were treated with neuraminidase. The relative mobility in benign disease and MM patient sera following neuraminidase treatment varied among individuals, while that of IgG M protein in sera from cancer patients was from 0.2 to 0.3. Thus, the relative mobility in cancer patients was narrower than in those with MM or benign disease.However, after neuraminidase treatment, there was no significant difference between relative mobility in cancer patient's sera and those in other disease patients.

Reinvestigation of clinical value of Rivalta reaction of puncture fluid.

Rivalta reaction is still used as a puncture fluid test for differentiation of exudate and transudate. However, the test method of Rivalta reaction has not been standardized, or positive precipitates for the reaction have not been investigated. Thus, we clarified the measurement method, and investigated Rivalta reaction-positive proteins. Rivalta reaction-positive punctuates converted to negative when pH increased to 4.6 or higher, showing the necessity of pH adjustment of acetic acid solution to 3.6-4.2 in Rivalta reaction. Using pH 4.0 acetic acid solution, 8 types of proteins were identified in Rivalta reaction-positive turbid precipitates: C-reactive protein (CRP), alpha1-antitrypsin (alpha1-AT), alpha1-acid glycoprotein (alpha1-AG), haptoglobin (Hp), transferrin (Tf), ceruloplasmin (Cp), fibrinogen (Fg), and hemopexin (Hpx). Since these are acute reactive proteins, or proteins increased in malignant tumors and infections, positivity for Rivalta reaction at the specified pH may suggest pathological inflammation.

Development of a new method for measuring total urinary protein using acid violet 6B pigment.

We have developed a new method for measuring total urinary protein using acid violet 6B (AV6B) pigment. Nine purified components of human urinary proteins and urine samples collected randomly from 123 diabetic outpatients were used. There were 62, 36, and 25 cases of prenephropathy, early nephropathy, and overt nephropathy, respectively. All samples were measured by Coomassie brilliant blue G 250 (CBB), pyrogallol red-molybdate (PRM), and AV6B methods using an optical photometer. In healthy subjects, the major components of urinary proteins, such as gamma-globulins, IgG, IgA Tamm-Horsfall protein, and transferrin, the reactivity values of the AV6B and PRM methods were similar. The CBB method was the least sensitive of the three methods. In the urine samples from diabetic patients, the urinary protein values measured by the AV6B method were higher than those measured by the CBB method in the prenephropathy stage. The values obtained by the AV6B method (y) correlated well with those from the CBB method (x) (y=1.243x+3.61, r=0.904). When the values from the AV6B method (y) were compared to those from the PRM method (x), correlation was low (y=1.406x-29.15, r=0.786). In conclusion, the AV6B method was more useful than the CBB and PRM methods for low levels of urinary protein.