Masashi Okada

A sensitive fluorometric method for the determination of aliphatic diamines and polyamines in biological materials by high-speed liquid chromatography.

Abstract A high-speed liquid chromatographic method for the determination of fluores-camine-labeled diamines and polyamines was applied to rat liver, human serum, and urine. The samples, after addition of 1,6-hexanediamine and N-3-aminopropyl-1,6-hexanediamine as internal standards, were appropriately pretreated by perchloric acid precipitation and/or acid hydrolysis. The resultant filtrates were subjected to CM-cellulose column chromatography in pyridine-acetic acid buffer. Stepwise elution with 0.2 and 1 m buffer gave fairly pure diamine and polyamine fractions. The evaporated fractions were dissolved in borate buffer and reacted with fluorescamine by either Procedure I or II, depending on the amine content. Aliquots of the reaction mixture were subjected to high-speed liquid chromatography for measurement of the amines. It was possible by this method to assay biological samples containing less than 100 pmol of the amines with about 5% relative standard deviation.

Biochemical studies on glucuronic acid and glucaric acid: 1. Quantitative chemical determination of d-glucaric acid in urine

Abstract A quantitative chemical method for the determination of urinary d -glucaric acid is described. Following the separation of d -glucaric acid by ion-exchange column chromatography using Dowex 1 X-8 borate, a portion of the sample is treated under neutral conditions (pH ca. 6.8) with periodic acid; the glyoxylic acid obtained is condensed with phenylhydrazine. The glyoxylic acid phenylhydrazone thus formed undergoes mild oxidation with potassium ferricyanide in the presence of excess phenylhydrazine, resulting in the intensely colored 1,5-diphenylformazan, the absorbancy of which is determined at 520 mμ. This method has been applied to determine d -glucaric acid excretion in human, rat, and guinea-pig urine; the results are compared with those obtained by the enzymic method of Marsh.

Phospholipase Cδ1 associates with importin β1 and translocates into the nucleus in a Ca2+‐dependent manner

Phospholipase C (PLC)δ1 shuttles between the nucleus and the cytoplasm. Here, we demonstrate that treatment of MDCK cells and PC12 cells with ionomycin causes nuclear accumulation of ectopically expressed and endogenous PLCδ1, respectively, suggesting that signals that increase [Ca2+]i trigger nuclear translocation. To clarify the molecular mechanisms involved in this translocation, we have examined whether PLCδ1 binds with importins. PLCδ1 interacted with importin β1 in a Ca2+‐dependent manner in vitro even in the absence of importin α. A PLCδ1 mutant E341A, which lacks Ca2+‐binding to the catalytic core, did not show this interaction at any physiological Ca2+ concentration and did not translocate into the nucleus after ionomycin treatment when expressed in MDCK cells. These results suggested that the nuclear import of PLCδ1 is mediated by its Ca2+‐dependent interaction with importin β1.

Phosphatidylserine induces functional and structural alterations of the membrane-associated pleckstrin homology domain of phospholipase C-δ1

The membrane binding affinity of the pleckstrin homology (PH) domain of phospholipaseu2003C (PLC)‐δ1 was investigated using a vesicle coprecipitation assay and the structure of the membrane‐associated PH domain was probed using solid‐state 13C NMR spectroscopy. Twenty per cent phosphatidylserine (PS) in the membrane caused a moderate but significant reduction of the membrane binding affinity of the PH domain despite the predicted electrostatic attraction between the PH domain and the head groups of PS. Solid‐state NMR spectra of the PH domain bound to the phosphatidylcholine (PC)/PS/phosphatidylinositol 4,5‐bisphosphate (PIP2) (75u2003:u200320u2003:u20035) vesicle indicated loss of the interaction between the amphipathic α2‐helix of the PH domain and the interface region of the membrane which was previously reported for the PH domain bound to PC/PIP2 (95u2003:u20035) vesicles. Characteristic local conformations in the vicinity of Ala88 and Ala112 induced by the hydrophobic interaction between the α2‐helix and the membrane interface were lost in the structure of the PH domain at the surface of the PC/PS/PIP2 vesicle, and consequently the structure becomes identical to the solution structure of the PH domain bound to d‐myo‐inositol 1,4,5‐trisphosphate. These local structural changes reduce the membrane binding affinity of the PH domain. The effects of PS on the PH domain were reversed by NaCl and MgCl2, suggesting that the effects are caused by electrostatic interaction between the protein and PS. These results generally suggest that the structure and function relationships among PLCs and other peripheral membrane proteins that have similar PH domains would be affected by the local lipid composition of membranes.

Mode of action of 3-substituted propylamine cytotoxicity in culture cells.

Cytotoxic effects on MDBK cells of various 3-substituted propylamines, including spermine and spermidine, were tested in culture in the presence of calf serum, and the possible mode of action was studied from the viewpoint of oxidative deamination leading to acrolein formation. The compounds were roughly classified in two groups with IC50 values of 0.1 and 0.4 mM under the conditions used. Phenyl derivatives of 3-substituted propylamines, 3-benzylaminopropylamine, and polyamines were included in the first group with IC50 values of 0.1 mM, and acrolein was liberated from these compounds after incubation with bovine plasma amine oxidase. Alkyl derivatives of 3-substituted propylamines (with IC50 values of 0.4 mM), on the other hand, were unable to release acrolein after the oxidative deamination, which was further confirmed by a lack of liberation of acrolein from the authentic 3-butoxypropanal. These observations indicated that both the acrolein originating from the 3-substituted propanals, and the propanal as such, were possibly involved in manifestation of the cytotoxicity of the 3-substituted propylamines. Thus, spermine and spermidine may exert their cytotoxic effects on the cells in vitro by a combination of two mechanisms involving acrolein and oxidized polyamines.

Essential Role of Monocytes in the In Vitro Production of IL-4 and Nonspecific IgE Antibody by Peripheral Blood Lymphocytes from Mice Sensitized s.c. Once with Cedar Pollen

To explore which cytokine or cell is essential for the production of antibodies (Abs) of the IgE class in allergic diseases, we injected cedar pollen into wild-type, interferon-gamma(-/-) (IFN-gamma(/)), or interleukin-4(-/-) (IL-4(-/-)) BALB/c mice through four (i.n., i.p., s.c., and i.v.) different routes without adjuvant. Wild-type or IFN-gamma(-/-), but not IL-4(-/-), mice sensitized once or twice showed a significant increase in total IgE Ab in their serum, revealing the essential role of IL-4 in the production of total IgE Ab. We separated peripheral blood mononuclear cells (PBMCs) from untreated or sensitized mice into monocyte-rich, lymphocyte-rich, and granulocyterich populations by Percoll density-gradient centrifugation or into specific antigen cells by flow cytometry, cultured the cells in various combinations, and examined the levels of cytokines and IgE Ab released into the medium. The PBMCs from mice sensitized s.c. once, but not those from untreated animals, produced significant amounts of IL-4 and total IgE Ab, whereas the lymphocyte-rich population alone did not. Unexpectedly, IL-4 and IgE Ab production was restored by the addition of Mac-1(+) cells in the monocyte-rich fraction to the lymphocyte-rich fraction. These results indicate the essential role of monocytes in the production of IL-4 and total IgE Ab by lymphocytes during the initial stage of sensitization.

Influence of membrane curvature on the structure of the membrane-associated pleckstrin homology domain of phospholipase C-δ1

The effects of geometric properties of membranes on the structure of the phospholipase C-delta1 (PLC-delta1) pleckstrin homology (PH) domain were investigated using solid state (13)C NMR spectroscopy. Conformations of the PLC-delta1 PH domain at the surfaces of multilamellar vesicles (MLV), small unilamellar vesicles (SUV), and micelles were examined to evaluate the effects of membrane curvature on the PH domain. An increase in curvature of the water-hydrophobic layer interface hinders membrane-penetration of the amphipathic alpha2-helix of the PH domain that assists the membrane-association of the PH domain dominated by the phosphatidylinositol 4,5-bisphosphate (PIP(2)) specific lipid binding site. The solid state (13)C NMR signal of Ala88 located at the alpha2-helix indicates that the conformation of the alpha2-helix at the micelle surface is similar to the solution conformation and significantly different from those at the MLV and SUV surfaces which were characterized by membrane-penetration and re-orientation. The signal of Ala112 which flanks the C-terminus of the beta5/beta6 loop that includes the alpha2-helix, showed downfield displacement with decrease in the interface curvature of the micelles, SUV and MLV. This reveals that the conformation of the C-terminus of the beta5/beta6 loop connecting the beta-sandwich core containing the PIP(2) binding site and the amphipathic alpha2-helix is sensitive to alterations of the curvature of lipid bilayer surface. It is likely that these alterations in the conformation of the PLC-delta1 PH domain contribute to the regulatory mechanisms of the intracellular localization of PLC-delta1 in a manner dependent upon the structure of the molecular complex containing PIP(2).

Structure of the principal colored product formed in the determination of glyoxylic acid

Abstract The structure of the principal colored product formed in the determination of glyoxylic acid which involves the oxidation of glyoxylic acid phenylhydrazone in the presence of excess phenylhydrazine is unequivocally identified with 1,5-diphenylformazan, as opposed to the structure, 1,5-diphenylformazan-3-carboxylic acid, previously proposed.

Blood supply--susceptible formation of melanin pigment in hair bulb melanocytes of mice.

Background: Allogeneic skin grafts onto C57BL/6 mice are rejected, and the rejected skin is replaced by surrounding skin with black hair. In contrast, syngeneic skin grafts are tolerated, and gray hair grows on the grafts. Methods: To explore the mechanism of gray hair growing on the tolerated skin grafts, we prepared full-thickness skin (2-cm square) autografts, 2 (2 cm + 2 cm) horizontal or vertical parallel incisions, and U-shaped (2 cm × 2 cm × 2 cm) flaps with or without pedicle vessels. The grafts, incisions, and flaps were fixed by suturing with string and protected by a transparent bandage. On day 14 after the operation, the bandages were removed to observe the color of the hair growing on the skin. Results: Skin autografts from wild-type or hepatocyte growth factor-transgenic (Tg) C57BL/6 mice survived with gray hair, whereas those from steel factor (Kitl)-Tg C57BL/6 mice survived with black hair. In addition, U-shaped flaps lacking both of the 2 main feeding vessels of wild-type mice had gray hair at the tip of the flaps. Light microscopy after staining with hematoxylin and eosin or dihydroxyphenylalanine showed that the formation of melanin pigment in the follicles, but not in the interadnexal skin, was susceptible to the blood supply. Conclusions: Melanin pigment formation in the hair bulb melanocytes appeared to be susceptible to the blood supply, and melanocytosis was promoted in the follicles and in the epidermis of Kitl-Tg C57BL/6 mice.

Bone Quality at the Sites of Loosened Screws in Mandibular Reconstruction Plates

1 Takashi Nuri, MD, PhD Koichi Ueda, MD, PhD Masashi Okada, MD, PhD Yuki Otsuki, MD Hiroyuki Iwanaga, MD Department of Plastic and Reconstructive Surgery Osaka Medical College Takatsuki, Osaka, Japan Sir, A lthough the osteocutaneous free flap is the best choice for mandibular reconstruction, it cannot be chosen sometimes because of the patient’s condition. Then, a metal reconstruction plate is chosen. However, some complications associated are reported.1–3 Plate exposure is the most common complication. Another complication is dislocation of the plate caused by loosening of the screws. The dislocation may cause plate exposure. We believe loosening of the screws is affected by the quality of mandibular bone. The purpose of this study is to investigate the quality of mandibular bone at the sites of loosened screws. Therefore, we retrospectively studied bone quality at the fixation sites using multidetector row computed tomography images. We studied a total of 46 fixation screw sites in 6 patients—1 woman and 5 men with a mean age of 70 years (range, 54–78 years)—with cancer. They had been treated with titanium reconstruction plates (Walter Lorenz, Jacksonville, Fla., 2.4 mm locking reconstructive plates) wrapped with rectus abdominis myocutaneous free flaps. They all had undergone multidetector row computed tomography imaging (Aquilion One, 16 and 32; Toshiba). There were a mean of 3.8 (range, 3–6) fixation screws at each location. The mean follow-up was 22.6 months (range, 6–42). Bone quality was assessed by measuring the thickness of the mandible, the thickness of the outer and inner tables of the cortical bone, and the cancellous bone density measured in average computed tomography values (Hounsfield units) in a region of interest that had been shown by pretreatment multidetector row computed tomography to be closely related to hydroxyapatite concentration4 (Fig. 1). The computed tomography values were accommodated between each unit. All analyses were performed using a software (INTAGE Realia). A total of 9 screws were loosened at both sides of the plate. Comparative studies were performed on these patients’ mandibular bones at sites where loosening did and did not occur. There were significant differences in the thickness of the lateral table of the cortical bone (P < 0.05) and in cancellous bone density (P < 0.05) at various sites (Table 1). However, no significant differences were recognized in the full thickness and inner table thickness of the mandible. These results indicate that cancellous bone density affects the stability of the fixation screws and also suggest that the thickness of the outer table of the mandibular bone affects plate stability. In patients with a mandibular reconstruction plate, biting force applied to the mandibular bone is transmitted to the contralateral mandible via the metal plate. When