Shiro Iijima

Changes of α1‐acid glycoprotein microheterogeneity in acute inflammation stages analyzed by isoelectric focusing using serum obtained postoperatively

The relationship between variations of α1‐acid glycoprotein (orosomucoid, AGP) microheterogeneity detected from isoelectric focusing (IEF) patterns and clinical stage of acute inflammation based on serum C‐reactive protein (CRP) levels and interleukin‐6 (IL‐6) levels was investigated. Serum samples were obtained from healthy subjects, and from patients with esophageal or stomach carcinoma before and after operation. Samples without neuraminidase treatment were used for AGP microheterogeneity analysis, and samples with neuraminidase treatment for AGP heterogeneity analysis. In AGP microheterogeneity, nine bands were detected in the range of pI 3.18—3.57 in sera obtained from healthy subjects. In patients, AGP microheterogeneity changed the first day after operation; the percentage of bands surrounding pI 3.5 increased, and the highest value appeared in sera taken the first or second day after operation and then decreased quickly. These bands showed reactivity for concanavalin A (Con A). The increase in Con A‐reactive AGP occurred later than the increase in IL‐6, and occurred earlier than the increase in CRP. On the seventh day after operation, the percentage of bands around pI 3.2 increased. These bands showed the reactivity for Datura stramonium agglutinin. On the other hand, in samples with neuraminidase treatment, little change of AGP heterogeneity was observed in most samples, which did not reflect the stage of inflammation. These findings suggested that AGP microheterogeneity detection was a useful marker for the clinical stage of inflammation.

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.

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.

New detection method after cellulose acetate membrane isoelectric focusing: Activity staining for lactate dehydrogenase, detection for sugar chains using lectin and simple Western blotting

To extend the clinical applications for cellulose acetate (CA) membrane isoelectric focusing, we designed two detection methods and two blotting methods that improve the characteristics of the CA membrane; that is, detection of the lactate dehydrogenase (LD) isozyme on the CA membrane, detection of the sugar chain in glycoprotein on the CA membrane, and simple and rapid methods for western blotting. In the detection of the LD isozyme, when 50% saturated ammonium sulfate solution was used as a fixing solution, five main LD bands and seven sub LD bands were detected clearly. By treating the CA membrane with 5% sulfosalicylic acid, glycoprotein was fixed on the CA prior to detection of the sugar chain of glycoprotein using Lens Culinaris (LCA), Phasseolus Vulgaris–L4 (PHA‐L4), Maackia Amurensis (MAM), and Sambucus Siedoldiana (SSA) lectins. Using this procedure, clear electrophoretic patterns were obtained. As simple and rapid methods for blotting proteins on polyvinylidene difluoride membranes, we designed natural contact blotting and suction blotting techniques. The natural contact blotting method did not need special apparatus. The suction blotting method completed protein blotting for 10 minutes. By both methods, clear electrophoretic patterns were obtained. J. Clin. Lab. Anal. 13:234–240, 1999.

Simultaneous analysis of microheterogeneity of immunoglobulins and serum protein fraction using high-voltage isoelectric focusing on six cellulose acetate membranes

A systematic detection method for the single performance of cellulose acetate (CA) membrane isoelectric focusing to detect six different types of information on protein abnormalities was developed. High‐voltage isoelectric focusing was carried out on six layers of CA membrane using a thermoelectric cooling apparatus. After electrophoresis, the proteins on the top, the third, the fourth, the fifth, and the bottom CA membrane were transferred to a polyvinylidene difluoride (PVDF) membrane by a simple contact printing procedure to detect IgM, κ‐chain, λ‐chain, IgA, and IgG, respectively. Each PVDF membrane revealed the microheterogeneity of these immunoglobulins using specified anti‐serum and enzyme immunostaining. The second CA membrane was stained with Coomassie brilliant blue G250 to detect serum protein patterns. All stained membranes showed clear electrophoretic patterns of immunoglobulin microheterogeneity. By our method, immunoglobulin abnormalities in serum could be screened out using six different types of information obtained simultaneously. J. Clin. Lab. Anal. 11:220–224, 1997.

Simultaneous analysis of serum immunoglobulins in patients with M protein using cellulose acetate membrane isoelectric focusing.

We developed a method for the simultaneous analysis of microheterogeneity of human serum IgG, IgA, IgM, IgD, and IgE, and serum protein pattern using cellulose acetate membrane isoelectric focusing, and analyzed in 11 healthy subjects and 67 patients with M protein (17 cases of multiple myeloma [MM] and 50 cases of monoclonal gammopathy of undetermined significance [MGUS]). Using this method, bands indicating the microheterogeneity of each immunoglobulin could clearly be detected. Among healthy subjects, the detected IgG, IgA, and IgM bands did not vary, but the detected IgE and IgD bands did vary. Therefore, IgA, IgM, and IgG were selected for comparison of serum immunoglobulins in MM and in MGUS. In the IgA‐type M protein group, normal IgM and IgG bands were decreased in MM patients compared to MGUS patients, while the M band and other bands were increased in MM patients compared to MGUS patients, but the differences between the two groups were not significant. In the IgG‐type M protein group, normal IgM, IgA, and IgG were significantly decreased in MM patients compared to MGUS patients. We examined the changes in electrophoretic pattern in six MM patients and eight MGUS patients with IgA‐type M protein after neuraminidase treatment. The width of the M band in MM patients with IgA‐type M protein decreased with neuraminidase treatment. On the other hand, the width of the M band in MGUS patients with IgA‐type M protein increased with neuraminidase treatment. We concluded that the decrease of the normal immunoglobulins in MM patients with IgG type M protein could be detected by this method, and IgA type of M protein binding sugar chain were different between MM and MGUS patients. J. Clin. Lab. Anal. 13:145–150, 1999.

Interprofessional education at the Keio University Faculty of Pharmacy

As part of a trial course on interprofessional education (IPE) at Keio University our Faculty of Pharmacy initiated a joint seminar with the university’s medical and nursing departments in 2008. We had two joint seminars: one in June and another in October. In the seminars, students and participants actively discussed several issues with regard to the national and private health care systems and medical malpractice. They also listened to feedback lectures held by experts from the medical and social system. At Kyoritsu University of Pharmacy, we had already held similar joint seminars three times since 2006. The participants were not only from our school but from various universities around Japan, given that our school was then only a small college for pharmaceutical students. Our considerable experience in the field of small group learning (SGL) helped make these seminars a success. By making full use of the SGL method we have successfully lead the discussion sessions at the IPE seminars attended by students from various medical fields. After the merging of our small pharmaceutical college with Keio University, we continued holding joint seminars for the three medical faculties at Keio University. It has not been without tribulations, however. The newly created interprofessional seminars faced several problems. The seeming lack of interest shown by the medical school decreased the level of participation of medical students, while too many facilitators are required for such seminars. To resolve these issues, we have made some changes to future activities in this area. To start with, we plan to change the course from an elective to a required subject for the students of the three medical faculties in Keio University.