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Publications and source records attributed to K Mawatari.
The liquid chromatography-mass spectrometry (LC-MS) following on from the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique was applied for the analysis of proteins in a renal stone found in a hyperuricemic patient. This technique was sensitive enough to detect small quantities of proteins even in a renal stone.
The goldfish optic nerve can regenerate after injury. To understand the molecular mechanism of optic nerve regrowth, we identified genes whose expression is specifically up-regulated during the early stage of optic nerve regeneration. A cDNA library constructed from goldfish retina 5 days after transection was screened by differential hybridization with cDNA probes derived from axotomized or normal retina. Of six cDNA clones isolated, one clone was identified as the Na,K-ATPase catalytic subunit alpha3 isoform by high- sequence homology. In northern hybridization, the expression level of the mRNA was significantly increased at 2 days and peaked at 5-10 days, and then gradually decreased and returned to control level by 45 days after optic nerve transection. Both in situ hybridization and immunohistochemical staining have revealed the location of this transient retinal change after optic nerve transection. The increased expression was observed only in the ganglion cell layer and optic nerve fiber layer at 5-20 days after optic nerve transection. In an explant culture system, neurite outgrowth from the retina 7 days after optic nerve transection was spontaneously promoted. A low concentration of ouabain (50-100 nm ) completely blocked the spontaneous neurite outgrowth from the lesioned retina. Together, these data indicate that up-regulation of the Na,K-ATPase alpha3 subunit is involved in the regrowth of ganglion cell axons after axotomy.
We recently showed that FliC of Salmonella enteritidis increased human beta-defensin-2 (hBD-2) expression, and now describe the signaling responsible pathway. FliC increased the intracellular Ca(2+) concentration ([Ca(2+)](in)) in Caco-2 cells. The [Ca(2+)](in) increase induced by FliC was prevented by U73122 and heparin, but not by chelating extracellular Ca(2+) or pertussis toxin. The FliC-induced increase in hBD-2 promoter activity via nuclear factor kappaB (NF-kappaB) was also inhibited by chelation of intracellular Ca(2+) or by U73122. We conclude that FliC increased [Ca(2+)](in) via inositol 1,4,5-trisphosphate, which was followed by up-regulating hBD-2 mRNA expression via an NF-kappaB-dependent pathway.
Mutations in the ATP-binding cassette transporter 1 (ABCA1) gene have been recently identified as the molecular defect in Tangier disease (TD) and familial high density lipoprotein deficiency (FHA). We here report novel mutations in the ABCA1 gene in two sisters from a Japanese family with TD who have been described previously (S. Ohtaki, H. Nakagawa, N. Kida, H. Nakamura, K. Tsuda, S. Yokoyama, T. Yamamura, S. Tajima, A. Yamamoto, Atherosclerosis 49 (1983)) and a family with FHA. Both probands of TD and FHA developed coronary heart disease. Sequence analysis of the ABCA1 gene from the patients with TD revealed a homozygous G to A transition at nucleotide 3805 of the cDNA resulting in the substitution of Asp 1229 with Asn in exon 27, and a C to T at nucleotide 6181 resulting in the substitution of Arg 2021 with Trp in exon 47. Sequence analysis of the ABCA1 gene from the FHA patient revealed a homozygous 4 bp CGCC deletion from nucleotide 3787 to 3790 resulting in premature termination by frameshift at codon 1224. These mutations were confirmed by restriction digestion analysis, and were not found in 141 control subjects. Our findings indicate that mutations in the ABCA1 gene are associated with TD as well as FHA.
We have investigated the effect of free radicals on the electrical gap junctions between horizontal cells in the carp retina. In our previous study, L-buthionine sulfoximine, an inhibitor of glutathione synthesis, caused uncoupling of horizontal cells four days after injection. In the present study, we have used paraquat, a generator of exogenous reactive oxygen species, to investigate whether it was the depletion of glutathione or an increase in the level of reactive oxygen species which resulted in horizontal cell uncoupling after L-buthionine sulfoximine injection. Intracellular recordings were made from L-type horizontal cells at various time-points after intravitreal injection of paraquat. Injection of 25nmol paraquat caused an increase in response amplitude to central spot light stimuli by two days after injection, which continued for a further two to three days and had almost disappeared by seven days after injection. There was also a sharp increase in reactive oxygen species production, peaking at four days and disappearing by seven days after injection, and an accompanying depletion and a restoration of glutathione levels with a similar time-course. Marking cells with Lucifer Yellow clearly illustrated uncoupling of horizontal cells after paraquat injection. If paraquat and L-buthionine sulfoximine were injected simultaneously, the increase in response to central spots was observed as early as one day after injection. This response amplitude was not more enhanced than that observed after L-buthionine sulfoximine injection alone, although a dramatic increase in the level of reactive oxygen species was observed. From these results, we suggest that reactive oxygen species are involved in uncoupling, while recovery from uncoupling is dependent on glutathione. Furthermore, we conclude that a balance between glutathione and reactive oxygen species levels is the most important factor controlling gap junctional intercellular communication of L-type horizontal cells in the carp retina.
For the fluorimetric determination of isatin in human urine and serum, HPLC-postcolumn photoirradiation using a mobile phase has been developed. Isatin in the urine or serum sample was separated on a Capcell Pak C1 column (250 x 4.6 mm id). The mobile phase consisted of 70 mmol l-1 phosphate buffer (pH 7.2)-tetrahydrofuran (85 + 15% v/v) containing 5 mmol l-1 hydrogen peroxide, which was irradiated with germicidal light to induce fluorescence (lambda ex 302 nm, lambda em 418 nm). The addition of tetrahydrofuran to the mobile phase led to the peaks showing good separation as well as increased sensitivity. The calibration graph for isatin was linear over the range of 0.16-10.7 ng. The pretreatment of the acidified urine or serum samples consisted of diluting steps or deproteinizing steps using perchloric acid, respectively. The mean recovery of isatin from urine and serum was greater than 94%.
This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Carol C. Cunningham and Victor R. Preedy. The presentations were (1) Ribosomal content, ribosomal localization and the levels of ribosomal protein mRNA and rRNA in rat skeletal muscle exposed to ethanol, by Alistair G. Paice, John E. Hesketh, Timothy J. Peters, and Victor R. Preedy; (2) Altered hepatic mitochondrial ribosome structure after chronic ethanol administration, by Vinood B. Patel and Carol C. Cunningham; (3) Clinical aspects of hepatic protein metabolism and alcohol, by Elena Volpi; and (4) Effects of oral intake of alanine plus glutamine on ethanol metabolism and ethanol-related depression in motor activity, by Kazunori Mawatari, H. Masaki, M. Mori, and Kunio Torii.
OBJECTIVES: The optimal therapeutic range for laboratory evaluation of oral anticoagulant therapy is now defined by the prothrombin time international normalized ratio (PT-INR). However, the thrombo test (TT), an alternative method to measure intensity of anticoagulation, is also currently used throughout Japan. The relationship between PT-INR and TT (%) has yet to be clarified. This study investigated the relationship between PT-INR and TT (%). METHODS: The PT-INR and TT (%) were simultaneously measured of 505 consecutive samples from patients treated with warfarin in our hospital. Fourteen functions were used for regression analyses: a fractional function (Y = a/X + b), a square root function (Y = aX0.5 + b), a natural logarithmic function (Y = a.lnX + b), a power series function (Y = aXb), a quotient function (Y = abX), and polynomial functions [Y = anXn + an - 1Xn - 1 +......+ a1X1 + b, (1 < or = n < or = 9)]. The results were confirmed by the same methods in 383 samples and 296 samples from another two laboratories. RESULTS: The power series function showed the most significant (p < 0.0001) and highest adjusted R2 (0.858) correlation, with a regression formula of TT (%) = e4.48 (PT-INR)-2.09 in our laboratory. Using the same analyses, the power series function also showed the most significant and highest adjusted R2 in samples from the other two laboratories. CONCLUSIONS: This study showed that a power series function is the most appropriate for expressing the relationship between PT-INR and TT (%) among the 14 functions. The function between PT-INR and TT (%) is mainly derived from the relationship between TT (%) and TT (sec). Both internal validity and external validity confirmed the relationship between PT-INR and TT (%).
Remnant-like particles, which have been recognized to be atherogenic derivatives of chylomicrons and very low density lipoproteins, can be measured using a new assay kit. The purpose of the present study was to investigate the association of remnant-like particles with the coagulation system that has an important role in the pathogenesis of myocardial infarction. We assayed blood levels of total cholesterol, triglyceride, HDL-cholesterol, apolipoproteins, remnant-like particles-cholesterol, remnant-like particles-triglyceride, fibrinogen, factor VII antigen, activated factor VII, and tissue factor in 111 patients with a history of myocardial infarction and 128 control subjects. In simple regression analysis, plasma levels of remnant-like particles-cholesterol and remnant-like particles-triglyceride showed a significant positive correlation with the levels of activated factor VII (r=0.319, p<0. 001, and r=0.286, p=0.002, respectively) and the activated factor VII/factor VII antigen ratio (r=0.241, p=0.011, and r=0.249, p=0.008, respectively) in patients with myocardial infarction. In contrast, there were no significant differences between remnant-like particles and activated factor VII in control subjects. In stepwise multivariate regression analysis, the significant determinants of activated factor VII were remnant-like particles-cholesterol (10.2%), apolipoproteins A-I (5.1%), and E (7.1%); for the activated factor VII/factor VII antigen ratio, remnant-like particles-triglyceride (6. 2%), age at blood sampling (5.1%), and apolipoprotein A-I (4.0%) in patients with myocardial infarction. However, the significant determinants of activated factor VII and the activated factor VII/factor VII antigen ratio were HDL-cholesterol (9.9% and 9.2%, respectively) in control subjects. It is concluded that remnant-like particles may be a risk factor for myocardial infarction by activating the extrinsic coagulation pathway.
PURPOSE: Growth factors and cytokines such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and transforming growth factor beta (TGF-beta) stimulate smooth muscle cell (SMC) proliferation and extracellular matrix (ECM) protein production by binding and activating their respective receptors. Recent investigations suggest that simultaneous activation of integrins, which are heterodimeric receptors for ECM, may also be required for growth factor and cytokine function. In this study, we tested the hypothesis that activation of two integrins, alpha v beta 3 and alpha 2 beta 1, both previously identified in vascular SMCs, is necessary for growth factor- and cytokine-induced vascular SMC dysfunction. METHODS: DNA synthesis was measured after stimulation of SMCs derived from human saphenous vein with the growth factors PDGF-BB, EGF, and bFGF. SMC fibronectin (Fn) production was measured (by means of Western blotting) in SMCs stimulated for 72 hours with TGF-beta1 or EGF. Both endpoints were measured in the presence and absence of antibodies that block the function of the alpha v beta 3 and alpha 2 beta 1 integrins as well as the alpha2 and beta1 subunits. RESULTS: The alpha v beta 3 integrin blocking antibody significantly inhibited PDGF-BB-, EGF-, and bFGF-induced SMC proliferation. The alpha v beta 3 integrin antibody also markedly inhibited TGF-1- and EGF-induced SMC Fn production. Neither the alpha 2 beta 1 integrin nor the alpha2 or the beta1 subunits inhibited either proliferation or matrix protein production in response to any of these agonists. CONCLUSION: The alpha v beta 3 integrin is required for growth factor- and cytokine-induced SMC proliferation and FN production, whereas alpha 2 beta 1 is not. Since activation of alpha v beta 3 is required for the activity of at least four distinct growth factors and cytokines, inhibition of this integrin might be used as a therapeutic tool for the prevention of intimal hyperplasia.
BACKGROUND: Vascular smooth muscle cell (SMC) migration, proliferation and extracellular matrix protein production are key steps in the formation of intimal hyperplasia, a process that leads to failure of vascular reconstructions. Protein kinase C (PKC) may be involved in all 3 cellular events. PKC consists of a family of 11 isotypes, 8 of which we have identified in human vascular SMCs. In this study we evaluate the role of PKCalpha as a second messenger for proliferation, migration and fibronectin production induced by human saphenous vein SMCs. METHODS: DNA synthesis was evaluated by using (3)H-thymidine incorporation. Mitogen-activated protein kinase (MAP-K) activation was quantified by Western blotting with an antibody to its phosphorylated substrate, Elk-1. Chemotaxis was evaluated by using a microchemotaxis chamber. SMC fibronectin was measured by Western blotting. For all experiments, PKCalpha was blocked with a selective inhibitor, Gö6976. RESULTS: Gö6976, at concentrations that allow selective inhibition of PKCalpha, inhibited platelet-derived growth factor-stimulated SMC proliferation and MAP-K activation by 30% to 40% and 30% to 60%, respectively. SMC chemotaxis was stimulated approximately 2-fold by the PKCalpha inhibitor. Neither basal nor transforming growth factor-betaI induced fibronectin production was affected by Gö6976. CONCLUSIONS: Our data suggest that PKCalpha is a positive mediator of SMC proliferation and MAP-K activity, a negative regulator of migration and has no effect on SMC fibronectin production. These data suggest that modulating activities of specific PKC isotypes might be useful in both the study and control of intimal hyperplasia.
Cardiac troponin T(cTnT) is one of the most myocardial-specific markers for the diagnosis of acute myocardial infarction(AMI). Recently, the rapid bedside cTnT assay(Trop T rapid assay sensitive version), which can provide qualitative determinations within 15 min, has been developed for the emergency clinical setting. To evaluate the usefulness of rapid bedside cTnT assay, we performed the Trop T test and measured serum levels of myoglobin(Mb), creatine kinase MB isoenzyme(CK-MB) and cTnT in 256 consecutive emergency patients with suspected AMI(65 found to have AMI and 191 without AMI). The diagnostic sensitivities for AMI of Trop T, Mb and CK-MB measurements were 66%, 92% and 52%, respectively, whereas the specificities were 80%, 18% and 74%, respectively. The diagnostic accuracy for AMI of Trop T(77%) was significantly higher than that of Mb(37%, p < 0.001) and CK-MB(69%, p < 0.05). The sensitivity for AMI of Mb(86%) was significantly(p < 0.001) higher than that of Trop T (31%) and CK-MB(31%) in patients admitted < or = 3 hr after the onset of AMI. In contrast, the sensitivities of Trop T(80% and 100%) in patients admitted at 3-6 hr and > 6 hr showed no significant differences from those of Mb(100% and 96%). Furthermore, Trop T in patients admitted > 6 hr had significantly(p < 0.01) higher sensitivity compared with CK-MB(69%). The mortality rate in the non-AMI group during hospitalization in patients with positive Trop T test(39%) was significantly(p < 0.001) higher than that in patients with negative test(9%). When the positive Trop T test was regarded as > or = 0.10 ng/ml of serum cTnT, Trop T test had the best concordance of 92% with a quantitative of cTnT assay.
BACKGROUND: It remains difficult for surgeons to choose between an in-flow and sequential arterial reconstruction in patients with multisegment arterial occlusive disease. In addition, the exact criterion for the proper revascularization procedures of these patients also remains obscure. METHODS: The profundapopliteal collateral index (PPCI) was determined in all patients with occlusions of both the aortoiliac and superficial femoral arteries prior to undergoing an arterial bypass. The PPCI in the inflow bypass (IB) was also compared with the sequential bypass (SB). RESULTS: The symptoms of all patients undergoing either IB or SB improved. Preoperatively, the average PPCI in IB patients was significantly lower than that in SB patients. In addition, no significant difference was observed in the increased average rate of the ankle brachial index (ABI) between IB and SB. CONCLUSIONS: The PPCI is an accurate predictor of the hemodynamic potential of the geniculate collaterals. In cases with a low PPCI, especially in patients with multisegment arterial occlusive disease, in-flow procedures alone may often be sufficient for the successful treatment of such patients. The PPCI is thus considered to be useful for selecting the optimal revascularization procedures.
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A method for the determination of N1-Methylnicotinamide (NMNA) was developed by the fluorescence formed by UV irradiation after separation by HPLC with a mobile phase containing hydrogen peroxide and 18-crown-6. This method was applied to human urine and serum.
To investigate a physiological role of glutathione in the horizontal cells of carp retina, the gap junctional intercellular communication between horizontal cells was studied using the techniques of intracellular recording of light-induced responses and coupling of the fluorescence dye Lucifer Yellow. Intravitreal injection of 2.5 micromol L-buthionine sulfoximine, an inhibitor of glutathione synthesis, induced a dramatic reduction (20% of control) of retinal glutathione level two days after treatment. The low level of glutathione continued for a further four to five days, and thereafter gradually recovered to about 40% (20 days after injection) and 70% (50 days after injection) of the control level. The spatial properties of the photopic L-type horizontal cell response were examined by enlarging the diameter of the central spot and peripheral annulus over the recording point. In normal retinas, the response amplitude of horizontal cells was monotonically enhanced as the diameter of the spot increased (0.5-4.0 mm) and correspondingly the dye diffusion area was wide, as the injected Lucifer Yellow normally diffused to several neighboring cells. Treatment with L-buthionine sulfoximine significantly altered the spatial properties of horizontal cells by increasing the response amplitude to central spots and slightly decreasing that to peripheral annuli, which were observed by four days after injection. It also restricted intracellular Lucifer Yellow to one or two cells. Accompanying the recovery of the cellular level of glutathione, the spatial properties and dye coupling of horizontal cells were restored to normal. A time lag (two days) of initiation in retinal glutathione depletion and alteration of spatial or dye coupling properties of horizontal cells is discussed, together with reactive oxygen species accumulation.
OBJECTIVE: To determine if carcinoembryonic antigen (CEA) concentration doubling time (DT) can predict the course of disease and characterize tumor biology. DESIGN: Retrospective case series of patients who exhibited a distinct increase in circulating CEA during disease recurrence in the routine postoperative follow-up. SETTING: A tertiary general hospital. PATIENTS: Twenty patients with recurrent gastric carcinoma and 17 patients with recurrent colorectal carcinoma. INTERVENTION: The CEA DT was determined from semilogarithmic plots of CEA concentration time courses. MAIN OUTCOME MEASUREMENTS: The quantitative analyses of the CEA DT as related to various host and tumor characteristics, modes of recurrence, and life expectancy. RESULTS: The mean CEA DTs for gastric and colorectal carcinomas were 229 days and 86 days, respectively. Although there were no significant differences with regard to patient age, tumor size, gross appearance, and histological differentiation, women had shorter CEA DT than did men. Flow cytometric analysis showed that tumors with a higher proportion of cells in S phase (> or = 15%) had significantly shorter CEA DT than those with a lower S-phase fraction (< 15%). There was a significant correlation between the CEA DT and the length of survival after the initial CEA concentration increase in patients with recurrent gastric and colorectal carcinomas. In the multiple regression analysis of patients who had been treated with curative surgery and chemotherapy, CEA DT proved to be a major independent prognostic factor. CONCLUSIONS: Carcinoembryonic antigen DT predicts life expectancy in patients with adenocarcinoma of the gastrointestinal tract. Differences in survival time are closely associated with variations in the biological aggressiveness of individual tumors.
Late graft failure is still a significant problem particularly in vessels with poor runoff. In the present study, we examined whether poor runoff conditions contributed to either functional or morphological changes in experimental canine autogenous arterial grafts. Four weeks after grafting, isometric tension studies were performed on rings obtained from femoral arterial grafts with poor runoff and the findings were compared to rings from grafts with normal runoff. The flow rate and tau variation (wall shear stress variation) in the limbs with poor runoff (18.5 +/- 4.2 ml/min, 52.9 +/- 7.0 dyne/cm2) were significantly lower than those of the control limb (66.4 +/- 9.2 ml/min, 201.1 +/- 9.6 dyne/cm2). Acetylcholine (ACh), adenosine 5'-diphosphate (ADP), and A23187 (calcium ionophore) caused comparable endothelium-dependent relaxations in the two groups. Smooth muscle relaxation in response to sodium nitroprusside was also comparable in the two groups. No apparent intimal thickening of the arterial grafts was observed in the canine arterial grafts with normal or poor runoff. Scanning electron microscopy demonstrated mild endothelial cell damage in implanted autogenous arterial grafts. At 7 to 14 days after grafting, the endothelial cell layer in both groups of grafts appeared to be normal. These results are markedly different from our previous findings with vein grafts in which pronounced intimal thickening was associated with an impairment of the endothelium-dependent responses under conditions of poor runoff. Based on these findings, the intact endothelial function and the absence of intimal thickening under conditions of poor runoff may thus explain the improved patency of autogenous arterial grafts compared to the vein grafts in aortocoronary revascularization.