Resazurin as an electron acceptor in glucose oxidase-catalyzed oxidation of glucose.

The behavior of resazurin (1) as an electron acceptor in glucose oxidase (GOD)-catalyzed oxidation of glucose under anaerobic conditions is described. When a mixture of 1, glucose, and GOD in phosphate buffer (pH 7.4, 0.1 M) was incubated at 25 degrees C, the resulting solution turned purple to fluorescent pink due to the deoxygenated product, resorufin (2). On incubation of 1 with GOD alone or with H2O2 under essentially the same conditions, no color change was seen, indicating that generation of 2 in the enzymatic reaction is brought about through reduction of 1 by the reduced form (GODred) of GOD, which was also supported by the voltammetric behavior of 1. However, it was found that the enzymatic transformation of 1 to 2 is of no practical use as an indicator reaction for glucose determination using only GOD due to a slow reaction of 1 with GODred. Based on a ping-pong type mechanism with a steady-state approximation, KM and kcat for 1 as an electron acceptor from GODred were estimated to be 15+/-1.3 microM and (5.0+/-0.5) x 10(-2) s(-1), respectively.

Hydrogen peroxide-induced deacetylation of acetyl resorufin as a novel indicator reaction for fluorometric detection of glucose using only glucose oxidase.

Hydrogen peroxide (H2O2)-induced deacetylation of non-fluorescent acetyl resorufin (1) to fluorescent resorufin (2) as a novel indicator reaction for fluorometric detection of glucose using only glucose oxidase (GOD) is described. When a 1:1:1 mixture of 1 (in CH3CN), glucose, and GOD (each in pH 7.4 phosphate buffer) was incubated at 25 degrees C under aerobic conditions, the resulting solution turned yellow to fluorescent pink due to 2. The formation of 2 was markedly retarded on incubation under anaerobic conditions. When a mixture of 1 and H2O2 was incubated under aerobic conditions, the formation of 2 was noted as in the case of the enzymatic reaction of 1. These results demonstrated that the observed color change is brought about through deacetylation of 1 to 2 induced by H2O2 generated in GOD-catalyzed oxidation of glucose. With regard to the fluorometric traces of the enzymatic reaction with 1 (0.2 mM), GOD (0.5 mg/ml), and glucose at 25 degrees C, fluorescence intensity exhibited a linear relationship against glucose concentration between 0.2 and 2.0 mm, with a correlation coefficient of 0.997. Neither ascorbic acid, uric acid, nor bilirubin significantly interfered with the transformation of 1 to 2 through GOD-catalyzed oxidation of glucose.

Resorufin as an electron acceptor in glucose oxidase-catalyzed oxidation of glucose.

Resorufin (1) has been found to act as an electron acceptor in glucose oxidase (GOD)-catalyzed oxidation of glucose. When a 1: 1: 1 mixture of solutions of 1 (5.0 microM), glucose, and GOD (4.0 mg/ml) in phosphate buffer (pH 7.4, 0.1 M) was incubated at 36 degrees C under aerobic conditions and the reaction was followed by a measurement of changes in fluorescence intensity due to 1, only two types of fluorometric traces were observed: (1) when a glucose solution of less than 0.7 mM was subjected to the enzymatic reaction, no consumption of 1 was observed; (2) the reaction with glucose at more than 1.0 mM always consumed 1, affording a regression fluorometric curve, and yet the obtained fluorometric traces could be almost superimposed on one another with no dependence on the glucose concentration. The reasons for the observed phenomena are discussed.

Demonstration of a ferric vibrioferrin-binding protein in the outer membrane of Vibrio parahaemolyticus.

Under iron-restricted conditions, Vibrio parahaemolyticus produces a siderophore, vibrioferrin, accompanying expression of two major outer membrane proteins of 78 and 83 kDa. Autoradiographic analysis of nondenaturing polyacrylamide gel electrophoregrams of outer membrane preparations previously incubated with [35Fe]ferric vibrioferrin revealed a single radiolabeled band, in which the 78-kDa protein was detected predominantly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antiserum against the purified 78-kDa protein partially inhibited Fe-VF binding to isolated OMPs. The 78-kDa protein was cleaved by the treatment of whole cells with proteinase K, indicating that a portion of this protein is exposed on the surface of the outer membrane. The treated cells lost most of their iron uptake activity mediated by vibrioferrin. These results suggest that the ferric vibrioferrin-binding protein of 78 kDa may function as the receptor for ferric vibrioferrin involved in the initial step of vibrioferrin-mediated iron uptake. Immunoblot analysis using the antiserum against the 78-kDa protein demonstrated that the molecular mass and antigenic properties of the protein were highly conserved among V. parahaemolyticus strains examined. The antiserum also recognized an iron-repressible outer membrane protein of 78 kDa from iron-restricted V. alginolyticus strains, some of which appeared to produce vibrioferrin.

Patterns of failure and survival in locally advanced carcinoma of the uterine cervix treated with high dose-rate intracavitary irradiation system.

We retrospectively analyzed 71 patients with locally advanced carcinoma of the uterine cervix treated by irradiation using high dose-rate intracavitary brachytherapy between 1978 and 1985. Seven patients were Stage IIIa, 46 Stage IIIb, and 18 Stage IVa. Five-year survivals for Stage IIIa, IIIb, and IVa were 71.4, 60.9, and 16.7%, respectively. An analysis of patterns of failure demonstrated that loco-regional recurrences were observed in 1 (14.3%) for Stage IIIa, 6 (13.0%) for Stage IIIb, and 9 (50.0%) for Stage IVa. The incidence of recurrence outside the pelvis observed in Stage IIIb patients (7 para-aortic nodes, 5 distant metastases) was much higher than that of local recurrence. Five patients (7.0% of the total: 1 with Stage IIIa, 3 with Stage IIIb, 1 with Stage IVa) required surgery to manage the complications. These data suggest that a high dose-rate intracavitary irradiation system is an effective tool for the treatment of cervical cancer. Further efforts to control metastatic lesions outside the pelvis are required for patients with Stage IIIb. To increase a loco-regional control rate for patients with Stage IVa disease, it is important to give additional treatment such as chemotherapy in conjunction with radiation therapy.

Results of the combination of external-beam and high-dose-rate intracavitary irradiation for patients with cervical carcinoma.

We retrospectively analyzed 220 patients with squamous cell carcinoma of the uterine cervix treated by the combination of external-beam and high-dose-rate intracavitary brachytherapy between 1978 and 1986. Five-year survivals for Stage Ib (n = 15), IIa (n = 18), IIb (n = 107), IIIb (n = 50), and IVa (n = 18) were 72.3, 88.9, 68.9, 64.0, and 16.7%, respectively. The incidences of recurrence outside the pelvis in Stage IIb and IIIb patients (15.0 and 24.0%) were higher than those of local recurrences (10.3 and 12.0%). Eighteen patients (8.2% of the total) had serious intestinal complications. These serious complications were closely correlated with the external-beam dosages. External-beam therapy was performed at 1.8-2.0 Gy/day, and high-dose-rate brachytherapy using a remotely controlled afterloading system (RALS) was performed once a week with 6.0-7.5 Gy at points A/fraction. Treatment dosages to the whole pelvis (WP), with central shieldings (CS), and with RALS according to FIGO stage were as follows: Stages Ib, IIa, 30-40 Gy (WP) plus 24-30 Gy (RALS); Stages IIb, IIIb, 40 Gy (WP) plus 10-20 Gy (CS) plus 24-30 Gy (RALS); Stage IVa, 40-50 Gy (WP) plus 10-20 Gy (CS) plus 20-30 Gy (RALS). From these data, the combination of external-beam and high-dose-rate intracavitary irradiation is an effective therapy for cervical cancer. It is also suggested that an additional combined chemotherapy is necessary to control metastic lesions outside the pelvis for Stages IIb and IIIb.