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Background Temozolomide-resistant (TMZ-R) glioblastoma is very difficult to treat, and a novel approach to overcome resistance is needed. Materials and methods The efficacy of a combination treatment of STAT3 inhibitor, STX-0119, with rapamycin was investigated against our established TMZ-resistant U87 cell line. Results The growth-inhibitory effect of the combination treatment was significant against the TMZ-R U87 cell line (IC50: 78 μM for STX-0119, 30.5 μM for rapamycin and 11.3 μM for combination of the two). Western blotting analysis demonstrated that the inhibitory effect of STX-0119 on S6 and 4E-BP1 activation through regulation of YKL-40 expression occurred in addition to the inhibitory effect of rapamycin against the mTOR pathway. Conclusion These results suggest that the STAT3 pathway is associated with the mTOR downstream pathway mediated by YKL-40 protein, and the combination therapy of the STAT3 inhibitor and rapamycin could be worth developing as a novel therapeutic approach against TMZ-resistant relapsed gliomas.

Dioxins and their surrogates were continuously monitored during the startup of two municipal waste in cinerators (MWIs). The surrogates studied included low-volatility organohalogen compounds (LVOH) sampled by online systems, as well as chlorobenzenes (CBs) and chlorophenols (CPs). The changes in levels of LVOH, CBs, and CPs corresponded well with the trend of the toxicity equivalent quantity (TEQ). The correlation of LVOH with TEQ was fairly good, whereas that of CBs and CPs with TEQ was not consistent. The correlation of LVOH with TEQ involved a memory effect related to the delayed emission of less volatile compounds. The isomer analysis of the dioxins present under startup conditions showed evidence of the memory effect, i.e., highly chlorinated isomers were emitted slowly, whereas low-chlorinated isomers and LVOH decreased rapidly as the temperature rose. LVOH cannot act as a perfect dioxin surrogate, but it can give a versatile, quick, and comprehensive warning of the presence of organohalogen compounds because it is free from the memory effect and covers many kinds of organohalogen compounds, including dioxins.

Of all potential biological therapeutics, monoclonal antibody (mAb)-based therapies are becoming the dominant focus of clinical research. In particular, smaller recombinant antibody fragments such as single-chain variable fragments (scFv) have become the subject of intense focus. However, an efficient affinity ligand for antibody fragment purification has not been developed. In the present study, we designed a consensus sequence for the human antibody heavy or light chain-variable regions (Fv) based on the antibody sequences available in the ImMunoGeneTics information system (IMGT), and synthesized these consensus sequences as template Fv antibodies. We then screened peptide ligands that specifically bind to the repertoire-derived human Fv consensus antibody using a 12-mer-peptide library expressed-phage display method. Subsequently, 1 peptide for the VH template and 8 peptides for the VK template were selected as the candidate ligands after 4 rounds of panning the phage display. Using peptide-bead-based immunoprecipitation, the code-4 and code-13 peptides showed recovery rates of the VH and VK templates that were 20-30% and 40-50%, respectively. Both peptides exhibited better recovery rates for trastuzumab scFv (approximately 40%). If it were possible to identify the best combination of VH and VK-binding peptides among the ligand peptides suitable for the human mAb Fv sequence, the result could be a promising purification tool that might greatly improve the cost efficiencies of the purification process.

We investigated dioxin formation and removal in a commercial thermal waste treatment plant employing a gasification and melting process that has become widespread in the last decade in Japan. The aim was to clarify the possibility of dioxin formation in a process operation at high temperatures and the applicability of catalytic decomposition of dioxins. Also, the possible use of dioxin surrogate compounds for plant monitoring was further evaluated. The main test parameter was the influence of changes in the amount and type of municipal solid waste (MSW) supplied to the thermal waste treatment plant which from day to day operation is a relevant parameter also from commercial perspective. Here especially, the plastic content on dioxin release was assessed. The following conclusions were reached: (1) disturbance of combustion by adding plastic waste above the capability of the system resulted in a considerable increase in dioxin content of the flue gas at the inlet of the bag house and (2) bag filter equipment incorporating a catalytic filter effectively reduced the gaseous dioxin content below the standard of 0.1 ng toxic equivalency (TEQ)/m3 N, by decomposition and partly adsorption, as was revealed by total dioxin mass balance and an increased levels in the fly ash. Also, the possible use of organohalogen compounds as dioxin surrogate compounds for plant monitoring was further evaluated. The levels of these surrogates did not exceed values corresponding to 0.1 ng TEQ/m3 N dioxins established from former tests. This further substantiated that surrogate measurement therefore can well reflect dioxin levels.

A rapid and sensitive spectrophotometric method for the determination of sodium linear-dodecylbenzenesulfonate (DBS) with 1-pentyl-4-(4-aminonaphthylazo)-pyridinium bromide (PANP) in the presence of sodium dodecyl sulfate (SDS) is proposed. The method is based on the formation of the ion associate of DBS with PANP, and does not require any solvent-extraction procedure. PANP was found to react selectively with DBS to form an ion associate, which caused a new light-absorption near to 416nm. The apparent molar absorptivity of the DBS-ion associate with PANP was found to be 4.6×104 dm3 mol-1 cm-1 at 416nm. A calibration graph was linear over the ranges from 0.1 to 2.0×10-6mol dm-3 DBS. The relative standard deviation (n=10) for 1.2×10-6mol dm-3 DBS was 2.0%. The proposed method can be applied preferably to the determination of DBS in river-water samples.

A rapid and sensitive spectrophotometric method for the simultaneous determination of sodium dodecyl sulphate (SDS) and sodium linear-dodecylbenzenesulphonate (DBS) with 1-stearyl-4-(4-aminonaphthylazo)-pyridinium bromide (SAPB) is described using the difference at the maximum absorption wavelength of the SDS- and the DBS-ion associate. SDS and DBS have been determined independently by measuring their respective absorbances at the maximum absorption wavelength. The apparent molar absorptivities of the SDS- and the DBS-ion associate are 8.0×104 l mol−1 cm−1 at 445 nm and 4.5×104 l mol−1 cm−1 at 424 nm, respectively. The calibration graph for SDS is linear in the range from 0.1 to 1.0×10−6 mol/l in the presence of 1.2×10−6 mol/l DBS and for DBS from 0.8 to 2.0×10−6 mol/l in the presence of 8.0×10−7 mol/l SDS. The relative standard deviation (n=15) for 8.0×10−7 mol/l SDS is 3.4% and for 1.6×10−6 mol/l DBS 2.1%. The proposed method has been applied to the simulatenous determination of SDS and DBS in river water samples.

We measured [Ca2+]i of guinea pig and rat myocytes with Ca2+ waves, using fura-2 fluorescence image processing. In guinea pig myocytes, Ca2+ waves were absent during the control perfusion period, but could be induced by the addition of strophanthidin (100μM) or sodium cyanide (NaCN: 2mM) to the perfusate. The [Ca2+]i increased from the control values of 69±5nM and 46±2nM, to 263±9 (p

Eleven pyridinium azo dyes with straight-chain alkyl groups CnH2n+1−(n=6−18) and bromoalkyl groups BrCmH2m−(m=6−12) were synthesized with the intention of developing reagents for the determination of low levels of anionic surfactants in an aqueous medium. The effect of the alkyl chain length of these reagents on the reactivity with anionic surfactants such as sodium dodecylsulphate (SDS), sodium linear-dodecylbenzenesulphonate (DBS), sodium dodecylsulphonate (DS) and sodium laurate (SL) was studied. It was found that the alkyl chain length played an important role in the formation of ion associates and the composition of the ion associates formed. These reagents were classified into four groups with respect to the reactivity with anionic surfactants. The first group (n,m=6) reacts only with DBS. The second group (n,m=8) reacts with SDS, DBS and DS. The third group (n,m=10, 12) reacts with SDS, DBS and DS; however, the colour intensity of the DBS-ion associate was unstable. The fourth group (n=14, 16, 18) reacts with all anionic surfactants examined, and the composition of the ion associates with SDS and DS was 2:1 ([reagent]/[surfactant]) though that of the ion associates of the three reagent groups mentioned above was 1:1. The optimal conditions for the determination of anionic surfactants in river water with 1-octyl-4-(4-aminonaphthylazo)-pyridinium bromide was examined. The calibration graph was linear up to 3×10−6 mol/l, and the apparent molar absorptivity of the ion associate was 3.8×104 l mol−1 cm−1 (at 427 nm). The relative standard deviation for 2.4×10−6 mol/l SDS was 4.9%. Recoveries of 88–107% were found for 8.0×10−7 mol/l SDS in river water samples.

To investigate the role of Ca2+ metabolism and pH in diabetic cardiomyopathy, intracellular Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) of isolated myocytes were measured simultaneously using fura-2 and BCECF. We used diabetic (D.M.) rats at 8 weeks after the injection of streptozotocin (45 mg/kg, i.v.). (1) [Ca2+]i of D.M. myocytes was lower than that of controls (53 +/- 3 and 75 +/- 5 nM, mean +/- S.E., P less than 0.01). There was no difference in pHi (7.06 +/- 0.02 in D. M., 7.07 +/- 0.02 in control). There was no difference in the percentage of non-rounded cells at 30 min after the perfusion of glucose-free solution which contained 2 mM sodium cyanide (NaCN) between D.M. and controls (53% and 52%). When cells were rounded, the value of [Ca2+]i was significantly lower in D.M. myocytes than that in controls (172 +/- 21 and 421 +/- 106 nM, P less than 0.05). (2) When the cells were shortened or rounded in the high [Ca2+]o solution (24.5 mM), [Ca2+]i of D.M. rats was significantly lower than that of control rats. (3) The percentage of non-rounded cells at 30 min after the perfusion of NaCN increased in controls by 50 mM glucose (95%, P less than 0.01), but not in D.M. (47%). Insulin (25 mU/ml) and glucose (15 mM) increased the percentage of non-rounded cells in D.M. after 30 min perfusion with NaCN (88%, P less than 0.01 v.s. 53% without glucose nor insulin). It is suggested that there are disturbances of Ca2+ metabolism in D.M. myocytes, and that there is a close relation between cell injury and glucose utilization during metabolic inhibition.

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.

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.

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.

A new reagent, 1-(N-methylpyridinium-4-yl-azo)-4-(4-diethylaminophenylazo)naphthalene iodide, can react with sodium 4-dodecylbenzenesulfonate to show a color change in an aqueous phase in the presence of iodide ion. The calibration graph is straight line up to 1.2×10−5 M (1 M=1 mol dm−3); e=3.25×104 1 mol−1 cm−1 at 580 nm.

Higher carboxylic acid reacts with Bis[2-(5-chloro-2-pyridylazo)-5-diethylaminophenol] cobalt(III) chloride, Co-5-Cl-PADAP, to form a 1:1 ion-pair, which is extracted into chlorobenzene in a single extraction at pH 11. The molar absorptivities of the ion-pairs of C12, C14, C16 and C18 acids are 3.5, 6.6, 7.6 and 7.6×104 1 mol−1 cm−1 at 563 nm, respectively.

A new reagent, 1-(4-N-methylpyridiniumazo)-4-(4-aminophenylazo) naphthalene iodide, can react with sodium alkylbenzenesulfonate to show a color change in an aqueous medium. The calibration graph is straight line over the range from 8×10−6 to 3.0×10−5 M of sodium dodecylbenzenesulfonate. The practical molar absorptivity is 1.8×104 1 mol−1 cm−1 at 594 nm.

The synthesis of o-phosphonobenzeneazochromotropic acid from o- bromobenzenediazonium fluoborate is described. The absorption spectra of the reagent and of a 1: 2 mixture of the reagent and thorium were measured at various pH values. The reagent has a maximum in the region of 510 m , while that of the thorium complex falls at 555 m . The complex was found to have a reagent: thorium ratio of 1:1. (D.L.C.)

Copper(II)-chelate with chromotropic acid was investigated by potentiometric titration. The measurement have been carried out at 25.0±0.1°C and μ=0.10 (with KNO3). The acid dissociation constant of chromotropic acid, the formation constant of the 1 : 1 chelate (the molar ratio of ligand to copper(II) ion) and th hydrolysis constant of this chelate were determined.