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To clarify the role of phospholipase C (PLC) in arrythmias and cell injury during myocardial ischemia/reperfusion, we studied its effects on electrophysiology and [Ca2+]i in guinea pig hearts. After exposure to PLC (1 and 2 U/ml), the action potential durations of right ventricular papillary muscles were decreased. Delayed afterdepolarizations were observed in all of the preparations, and some developed into triggered activities. Developed tension decreased after an initial increase for the first 5 min, while resting tension increased consistently. The effects of PLC (0.02, 0.1, and 0.2 U/ml) on [Ca2+]i of ventricular myocytes were measured using fura-2 fluorescence. The ratio of rod-shaped cells to all cells decreased in a time- and a concentration-dependent manner. Perfusion with 0.1 U/ml PLC elevated [Ca2+]i from 56 +/- 5 nM to 245 +/- 47 nM before cell rounding, and to 1167 +/- 172 nM after cell rounding, suggesting that PLC causes Ca2+ overload. In conclusion, activation of PLC may play a role in arrhythmias and cell injury during ischemia/reperfusion. The increase in [Ca2+]i during ischemia/reperfusion may activate phospholipase, which would further increase [Ca2+]i to form a vicious cycle.

Study objective – To characterise cell injury during myocardial hypoxia and reoxygenation, [Ca2+]i was measured in guinea pig ventricular myocytes using digital images of fura-2 fluorescence. Design – [Ca2+]i and cell morphology were measured during and after the perfusion of 2 mmol·litre−1 sodium cyanide (NaCN). Experimental material – 28 ventricular myocytes isolated from guinea pig hearts. Measurements and main results – Eight out of 28 cells became rounded during the perfusion with NaCN, and [Ca2+]i increased from 48 (SEM 6) nmol·litre−1 to 163(24) (p

A new cationic dye, 1-(10-bromodecyl)-4-(4-aminonaphthylazo)-pyridinium bromide, was synthesized and evaluated as a new reagent for the determination of anionic surfactants. The reagent reacts with anionic surfactants, such as sodium dodecylsulphate and sodium dodecylbenzenesulphonate, to produce an ion associate in an aqueous medium. The colour change occurs simultaneously, and the colour development is very stable. This makes it possible to determine anionic surfactants directly by spectrophotometry without solvent extraction. The stoichiometric ratio of the ion associate was found to be 1:1 by the mole ratio method. The calibration graph was linear up to 2.5×10−6 mol/l. The apparent molar absorptivity of the ion associate was 5.3×104 l mol−1 cm−1 (at 595 nm). The relative standard deviation (n=10) for 1.2×10−6 mol/l sodium dodecylsulphate was 4.9%. The proposed method was applied to the determination of anionic surfactants in tap and river waters.

An 83-year-old woman was admitted to our hospital complaining of respiratory distress. She developed blepharoptosis and dysphagia two months previously. On admission, blood pressure was 150/84 mmHg. There were bilateral blepharoptosis and bulbar palsy. Tendon reflexes were brisk, but pathological reflexes were absent. Repetitive stimulation test demonstrated decrement. Tensilon test was positive. Thus, a diagnosis of myasthenic crisis was made. Vital capacity was 0.71, which necessitated intubation and artificial ventilation. During the first course of immunoadsorption plasmapheresis, blood pressure fell to 80/50 mmHg. ECG showed deeply inverted T waves in leads V2 through V6. Echocardiographic examination demonstrated extensive akinesis around the apex. CK was slightly increased. A diagnosis of "Takotsubo"-shaped cardiomyopathy was made. To maintain blood pressure, dopamine was continuously injected. Left ventricular function returned to normal within a week. No thymoma was seen on the chest CT scan. She was treated with pyridostigmine and tacrolimus. Ten weeks after admission, she was weaned off the ventilator and she had no dysphagia anymore. Anti-acetylcholine receptor antibody titers had decreased. In Japan, approximately 17% of patients with myasthenia gravis are in the seventh decade or older. Approximately 10% of the patients of this age group suffer from severe generalized form. Careful ECG monitoring is necessary while treating elderly patients with myasthenic crisis, because they are at potential risk of developing "Takotsubo"-shaped cardiomyopathy.

Analysis of reports about incidental and accidental events in nursing care were made using a reliability engineering method. Unnatural working hours, such as evening duty, night duty falling next to a holiday, two consecutive night-duty shifts, and two consecutive evening-duty shifts were major factors in the occurrence of errors. In a mixed-division ward (a ward containing patients belonging to different divisions), rule-based errors happened more frequently than in a single-division ward. Also, less experienced nursing staffs made errors more frequently than experienced nursing staffs.

Based on an anonymous questionnaire obtained from 102 Japanese in-patients at a rural Japanese hospital who underwent coronary angiography, the patients understanding of information about the procedure, their perception of consent and their attitude toward it were investigated. The patients were able to recall 63.8% of the contents of the information. There was a statistically significant correlation between the patients' educational status and the recall test score of the information. Older (65 years old and above) patients had a tendency to entrust decision making to their physicians. Only 19.6% of the patients made the decision to undergo a coronary angiography by themselves. After being informed of the risks 40.2% of the patients felt more anxious. In conclusion, our patients could not recall the contents of the information sufficiently. There still existed a tendency to entrust important decisions to the physician especially in older patients. The patients had a tendency not to face difficult problems solely by themselves but with the support of family members.

We examined the effect of ulinastatin in comparison with prednisolone (PSL) and salazosulfapyridine (SASP), well-known drugs for ulcerative colitis (UC), on two experimental UC models induced by dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNB) in rats. Ulinastatin at the doses of 3000 approximately 10,000 units/kg/day (i.v.) significantly ameliorated the formation of erosion and infiltration of inflammatory cells in colonic mucosa in DSS-induced rat UC models. Moreover, ulinastatin at the dose of 10,000 units/kg/day (i.v.) significantly suppressed inflammation with ulcer in the colonic mucosa in TNB-induced rat UC models. PSL at the dose of 1 mg/kg/day (p.o.) also was as effective as ulinastatin on the above two UC models, while SASP at the dose of 100 mg/kg/day (p.o.) was less effective than ulinastatin and PSL. In addition, ulinastatin inhibited the activities of elastase and cathepsin G from human leukocytes, and it suppressed TNF alpha, IL-8 and superoxide production by rat macrophages and rabbit leukocytes in vitro. These results suggest that the suppression of inflammatory mediators produced by leukocytes is involved in the mechanism of the anti-UC action of ulinastatin.

Background High-grade gliomas including glioblastoma multiforme (GBM) are among the most malignant and aggressive of tumors, and have a very poor prognosis despite a temozolomide-based intensive treatment. Therefore, a novel therapeutic approach to controlling recurrence is needed. In the present study, we investigated the effect of activated dendritic cell (DC) (α-type-1 polarized DC)-based immunotherapy on high-grade glioma patients with the HLA-A2 or A24 genotype. Methods Nine patients with recurrent high-grade gliomas including 7 with GBMs who fulfilled eligibility criteria were enrolled into a phase I study of monocyte-derived DC-based immunotherapy. HLA-genotyping revealed 1 case of HLA-A*0201 and 8 cases of A*2402. Enriched monocytes obtained using OptiPrepTM from leukapheresis products on day1, were incubated with GM-CSF and IL-4 in a closed serum-free system, and activated on day6 with TNF-α, IL-1β, IFN-α, IFN-γ, and poly I/C. After pulsing with a cocktail of 5 synthetic peptides (WT-1, HER2, MAGE-A3, and MAGE-A1 or gp100) restricted to HLA-A2 or A24 and KLH, cells were cryopreserved until used. Thawed DCs were injected intradermally in the posterior neck at a dose per cohort of 1.0, 2.0 and 5.0× 107/body. Results The frequency of CD14+ monocytes increased to 44.6% from 11.9% after gradient centrifugation. After a 7-day-incubation with cytokines, the mean percentage of DCs rated as lin-HLA-DR+ in patients was 56.2 ± 19.1%. Most DCs expressed high levels of maturation markers, co-stimulatory molecules and type-1 phenotype (CD11c+HLA-DR+) with a DC1/2 ratio of 35.6. The amount of IL-12 produced from activated DCs was 1025 ± 443 pg/ml per 105 cells. All 76 DC injections were well tolerated except for transient liver dysfunction with grade II. Six patients showed positive immunological responses to peptides in an ELISPOT assay, and positive skin tests to peptide-pulsed DC and KLH were recognized in 4 cases. The clinical response to DC injections was as follows :1 SD and 8 PD. Interestingly, the SD patient, given 24 DC injections, showed a long-term recurrence-free and immunological positive response period. Conclusions These results indicate peptide cocktail-treated activated α-type-1 DC-based immunotherapy to be a potential therapeutic tool against recurrent high-grade glioma with mainly HLA-A*2402. Trial registration Current non-randomized investigational trial UMIN-CTR UMIN ID: 000000914.

Two patients with myocardial sarcoidosis are presented, both of whom underwent SPECT imaging with Tl-201 and Ga-67. The first had Ga-67 myocardial uptake with a Tl-201 defect, which disappeared with corticosteroid therapy. The second had multiple Tl-201 defects without Ga-67 uptake, which persisted despite corticosteroid therapy. Therefore, the combination of Tl-201 and Ga-67 imaging may be useful for recognizing myocardial sarcoidosis and for predicting the response to corticosteroid therapy.

The coextraction of scandium-yttrium-lumogallion [LMG; 4 - chloro - 6 - (2,4 - dihydroxyphenylazo) - 1 - hydroxybenzene - 2 - sulfonic acid] into diethyl ether has been studied. The acid dissociation constants of LMG, p K a2 and p K a3 were estimated to be 6.24 and 8.05 respectively. The composition of the complex extracted was determined by using the radioisotopes 46 Sc and 90 Y and by spectrophotometry of LMG and the ratio of the components was Sc:Y:LMG = 1:1:3. The coextraction scheme was discussed briefly.

Sulfate ion in river water is determined by flow injection analysis at a rate of 30 samples per hour; the sulfate contents are typically less than 30 ppm. The reagent solution contains dimethylsulfonazo-III, barium chloride, potassium nitrate and chloroacetate buffer in 70% ( v v ) ethanol, and is saturated with barium sulfate. The aqueous carrier stream is also saturated with barium sulfate. The sample is filtered and treated with Amberlite IR120-B cation-exchanger before injection into the carrier stream, and the decoloration of the barium—dimethylsulfonazo-III complex by sulfate is measured at 662 nm. The calibration graph is linear over the range 0–30 μg ml-1 for sulfate in water.

Effects of the urinary enzyme inhibitor ulinastatin on experimental arthritis were investigated. Ulinastatin at the dose of 30,000 units/kg/day, i.v., significantly restored the swelling of hind paw, inflammatory score and bone damage in adjuvant arthritic rats. Intraarticular administration of ulinastatin at 3000 units/site x 3, significantly suppressed the articular swelling and the elevated inflammatory parameters in the synovial fluid of carrageenin-induced arthritic rabbits. Moreover, ulinastatin at the dose of 50000 units/kg/day, i.v., significantly prevented the elevation of serum rheumatoid factor and articular lesions in MRL/l mice. In addition, ulinastatin significantly inhibited human granulocyte elastase and cathepsin G. These findings indicate that ulinastatin may be a useful therapeutic agent for arthritis.

Oxygen free radicals and Ca2+ overload have been implicated in the genesis of reperfusion induced arrhythmia and injury. Effects of hydrogen peroxide (H2O2) on action potentials and intracellular Ca2+ concentration ([Ca2+]i) were studied using guinea pig papillary muscles and ventricular myocytes. High concentration of H2O2 (10 mmol.litre-1) caused delayed afterdepolarisations in all six papillary muscles, and induced triggered activity in 3/6 preparations. Pretreatment with ryanodine (1 mumol.litre-1) abolished delayed afterdepolarisations and triggered activity induced by H2O2. [Ca2+]i and morphological changes in isolated ventricular myocytes of guinea pig were measured using fura-2. Quiescent and rod shaped myocytes became shortened and rounded (contracture) after the application of 0.1 and 1 mmol.litre-1 H2O2. [Ca2+]i increased from the control values of 53 (SEM 4) and 62(8) nmol.litre-1 to 110(29) and 105(24) nmol.litre-1 (p less than 0.05 v control) when cells were shortened during perfusion with 0.1 and 1 mmol.litre-1 H2O2, respectively. The values were 130(26) nmol.litre-1 (p less than 0.05 v control) and 100(18) nmol.litre-1 (p less than 0.05 v control) when the cells became rounded during perfusion with 0.1 and 1 mmol.litre-1 H2O2. We suggest that the arrhythmia caused by Ca2+ overload was induced by H2O2, possibly by lipid peroxidation of cell membrane. H2O2 was also shown to shorten cells and cause cell contracture (rounding). The mechanism of cell injury is not likely to be due to the Ca2+ overload, since the increase in [Ca2+]i during perfusion with H2O2 was not large.

Four dicarboxylic acids (Reagents I, II, III and IV) containing ether linkages, HOOCCH2O-(CH2CH2O)nCH2COOH (n = 0–3), were synthesized. The acid dissociation constants and their chelate stability constants with alkaline earth metals were measured by pH titration method at an ionic strength μ = 0·10 and at 25·0 ± 0·1°C. As the total sum of the number of oxygen atoms of ether linkages and carbon atoms approaches infinity, the value of the ratio of the first to the second acid dissociation constant comes near to four. Therefore, it indicates that the molecules behave statistically, when the number of atoms between two carboxyl groups approaches infinity. The decreasing order of the stability constants of those chelates with alkaline earth metals is as follows: Reagents I and II (n = 0 and 1; Ca

o-Sulfobenzeneazochromotropic acid was prepared in the form of reddish brown needles of the monosodium salt, and the value of the first drssociation constant, pK/sub ai/, was estimated spectrophotometrically to be 9.7/sub 6/ (25 deg C, mu - 0.1). The reagent reacts with Th in the molar ratio 1: 1, displacing one H atom, and the approximate value of the apparent stabilrty constant of the complex, log K', was estimated to be 3.2 approximates 4.2 at pH values from 2.5 to 3.5 while the concentration of Th was about (4 approximates 8) x 10/sup -5/. (auth)

In order to clarify the effect of the hydroxyl group at the 5-position of chromotropic acid on the chelate stabilization, the following six reagents were synthesized by the coupling with 4-hydroxynaphthalene-2,7-disulfonic acid or 4,5-dihydroxynaphthalene-2,7-disulfonic acid(chromotropic acid): 2-(m-sulfophenylazo)-1-hydroxynaphthalene-3,6-disulfonic acid(I), 2-(o-methoxy-m-sulfophenylazo)-1-hydroxynaphthalene-3,6-disulfonic acid(II), 2-(o-hydroxy-m-sulfophenylazo)-1-hydroxynaphthalene-3,6-disulfonic acid(III), 2-(m-sulfophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid(IV), 2-(o-methoxy-m-sulfophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid(V), and 2-(o-hydroxy-m-sulfophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid(VI). The acid dissociation constants of these reagents and the stability constants of their metal chelates with magnesium and calcium were measured by the pH titration method at an ionic strength of 0.10 and at 25°C. From the results obtained, it has become apparent that ...

o-Carboxyphenyl-azo-chromotropic acid was synthesized as a colorimetric reagent for thorium. The acid dissociation constants of the reagent have been determined spectrophotometrically at 25°C and μ=0.1 (NaClO4). The pK1 (for the dissociation of the carboxyl group) and the pK2 (for the dissociation of the naphtholic hydroxyl groups) were 3.20 and 10.17 respectively. The reagent forms a violet 1 : 1 complex with thorium, showing an absorption maximum at 565 mμ in an acidic solution. The apparent stability constant of the thorium complex was measured spectrophotometrically.

o-, m-, and p-Nitrophenyl-azo-chromotropic acids were prepared as colorimetric reagents for thorium. The acid dissociation constants of the reagents have been determined spectrophotometrically, their pK1 values being 8.96, 8.60, and 8.77 for the and o-, m-, and p-compounds respectively at 25°C and when μ=0.1. The reagents form violet 1 : 1 complexes in an acidic solution. The apparent stability constants have been measured spectrophotometrically. The order of the apparent stability constants of the complexes agrees with that of the first acid dissociation constants of the reagents; m-nitro>p-nitro>o-nitro compounds.

The acid dissociation constants of phenylazochromotropic acid and its o-substituted derivatives with -CH3, -OCH3, -COCH3, -COOH, -CH2COOH, -COCOOH, -OCH2COOH, and -CH(OH)COOH in the ortho position to the azo group, and the stability constants of their 1 : 1 metal chelates with alkaline earth metals have been measured by potentiometric titation. The measurements have been carried out at 25°C and at μ=0.10 with KNO3. The effects of these substituents on the acid dissociation constants of the reagents and on the stability constants of these chelates were discussed. The pKa value for the naphtholic proton of the o-substituted reagents was larger than that of phenylazochromotropic acid, and the stability constants of these chelates with alkaline earth metals decreased as follows: Mg>Ca>Sr>Ba, while for the chelates of the o-OCH2COOH reagent the order was Ca>Mg>Sr>Ba, and for the o-CH(OH)COOH reagent it was Mg≈Ca>Sr>Ba. The plots of the stability constants, log K1 against the sum of the acid dissociation consta...

2-(1, 8-Dihydroxy-3, 6-disulfo-2-naphthylazo)-mandelic acid forms a stable violet complex with thorium ion at 1:1 molar ratio in an acidic solution. The acid dissociation constants of the reagent and the apparent stability constant of its thorium complex were estimated spectrophotometrically at 25°C and μ=0.1. It was found that the thorium complex formed by the present reagent was more stable than those of the other phenyl-azo-chromotropic acid derivatives reported in the preceding papers.

A new chelating agent, o-phosphonophenylazo-2-hydroxynaphthalene-3,6-disulfonic acid, has been synthesized. The acid dissociation constants of this ligand and the chelate stability constants with alkaline earth metals have been measured by the pH titration method at an ionic strengh of 0.10 and at 25.0±0.1°C. This ligand forms the acido complex, MHL, in the pH 5–7 region. The stability constants of the acido complex decrease in this order: log KSrHL≈logKCaHL>log KMgHL; whereas the decreasing order of the stability constants, logKML, is as follows: log KMgL>log KCaL>log KSrL.

The acid dissociation constants of o-phenol-, m-chloro-o-phenol- and o-anisolazochromotropic acids have been measured by potentiometric titration. The measurements have been carried out at 25.0±0.1°C and μ=0.10 (by KNO3). From these constants, the dissociation of the hydroxyl groups in the reagents has been discussed. The phenolic hydroxyl group was first dissociated, and then one of the naphtholic hydroxyl groups; the other naphtholic group was not dissociated under ordinary experimental conditions. The pKa1 and pKa2 values were 7.60 and 10.60 for o-phenolazochromotropic acid, and 7.56 and 10.35 for m-chloro-o-phenolazochromotropic acid. o-Phenol- and m-chloro-o-phenolazochromotropic acids formed 1 : 1 chelates with calcium and magnesium. The formation constants were measured potentiometrically; the logK’s for calcium were 5.01 and 5.22, and for magnesium, 6.15 and 6.22.

The acid dissociation constants of the three isomeric phosphonophenylazochromotropic acids and their chelate stability constants with alkaline earth metals have been measured by the pH titration method at an ionic strength of 0.10 and at 25.0±0.1°C. The stability order with respect to the ligands is as follows: ortho->meta->para-, and the stability constant of each ligand decreases in the order: Mg>Ca>Sr>Ba; this order is parallel to the reciprocal of the ionic radii of the metal ions. From a comparison between the chelate stability of o-phosphonophenylazochromotropic acid and that of Neo-Thorin, it has been concluded that the magnesium ion forms a more stable chelate bond with the phosphonic group than with the arsonic group.

Isolated rat heart myocytes were loaded with both the Ca2+ sensitive fluorescent probe fura-2/AM and the fluorescent pH indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM). Changes in [Ca2+]i and pHi were measured simultaneously using digitized video fluorescence microscopy. In measurement of [Ca2+]i and pHi, the ratios of dual-loaded cells were not different from single-loaded cells. Using this method, [Ca2+]i and pHi in myocytes were 48 +/- 7 nM and 7.17 +/- 0.05. It is concluded that [Ca2+]i and pHi could be measured simultaneously in isolated myocyte using dual-loading of fura-2 and BCECF.

To study the role of cytosolic calcium concentration [( Ca2+]i) in the pathophysiology of Ca2+ overload, we used a digital image of calcium indicator signals (fura-2 fluorescence) from guinea pig myocytes. [Ca2+]i increased from 69(SEM 5) nmol.litre-1 (n = 15) to 204(45) nmol.litre-1 (n = 14) after 10 min perfusion with 100 mumol.litre-1 strophanthidin. The effects of strophanthidin on cellular morphology were associated with variable increases in [Ca2+]i of myocytes. [Ca2+]i was 263(9) nmol.litre-1 in the spontaneous contracting cells with "Ca2+ wave", and 784(103) nmol.litre-1 in rounded cells. There was a significant relationship (r = 0.77, p less than 0.001) between elevation of [Ca2+]i and shortening of longitudinal length of the cells. After 20 min pretreatment with 10 mumol.litre-1 ryanodine, strophanthidin (100 mumol.litre-1) did not cause spontaneous contractile activity, and induced a slower increase in [Ca2+]i than found previously with strophanthidin alone. We suggest that [Ca2+]i played a primary role in mediating irreversible injury in this study, and that the absolute value of [Ca2+]i was correlated with morphological changes. We also suggest that the Ca2+ release from the sarcoplasmic reticulum was related to the increase in [Ca2+]i and the spontaneous contracting activity.