Binding and catalytic properties of Xenopus (6-4) photolyase.

Xenopus (6-4) photolyase binds with high affinity to DNA bearing a (6-4) photoproduct and repairs it in a light-dependent reaction. To clarify its repair mechanism of (6-4) photolyase, we determined its binding and catalytic properties using synthetic DNA substrate which carries a photoproduct at a single location. The (6-4) photolyase binds to T[6-4]T in double-stranded DNA with high affinity (KD = 10(-9)) and to T[6-4]T in single-stranded DNA and T[Dewar]T in double- and single-stranded DNA although with slightly lower affinity (KD = approximately 2 x 10(-8)). Majority of the T[6-4]T-(6-4) photolyase complex dissociates very slowly (koff = 2.9 x 10(-5) s-1). Its absolute action spectrum without a second chromophore in the 350-600 nm region closely matches the absorption spectrum of the enzyme. The quantum yield (phi) of repair is approximately 0.11. The fully reduced form (E-FADH-) of (6-4) photolyase is catalytically active. Direct analysis of the photoreactivated product showed that (6-4) photolyase restores the original pyrimidines. These findings demonstrate that cis, syn-cyclobutane pyrimidine dimer photolyase and (6-4) photolyase are quite similar, but they are different with regard to the binding properties.

Identification of 11-dehydro-2,3-dinorthromboxane B3 in human urine based on the mass spectral properties of 11-dehydrothromboxane B3 and related compounds.

11-Dehydro-2,3-dinorthromboxane B3 (an enzymatic metabolite of TXB3) was identified. Urine from a healthy male adult who had received daily administration of 1.8 g (0.6 g three times) of eicosapentaenoic acid (EPA) ethyl ester was collected. A urine sample was extracted with an affinity column of anti-11-dehydro-TXB2 monoclonal antibody. Conversion of 11-dehydro-2,3-dinor-TXB3 in the urine extract into the methyl ester (ME)-11-n-propylamide (PA)-9, 12, 15-dimethylisopropylsilyl (DMIPS) ether derivative was followed by gas chromatography with selected-ion monitoring (GC/SIM) (resolution: 8000) and GC/mass spectrometry (MS). High-resolution SIM result revealed that a major component corresponded to the [M - C3H7]+ (m/z 668.4198) ion, which may be specific for the structural integrity by analogy with the corresponding 11-dehydro-TXB3 derivative. Conventional GC/MS analysis confirmed that the peak of the interest was the title compound. The mass spectrum of the 11-dehydro-2,3-dinor-TXB3 ME-PA-DMIPS ether derivative was dominated by the ions of [M - CH3]+ (m/z 696) and [M - C3H7]+ (m/z 668, base peak) and ions containing a protected 11-carboxylic acid moiety together with the unidentified additional ions which were considered to derive from urinary endogenous substances. However, the quality was sufficient for use of mass spectral identification of 11-dehydro-2,3-dinor-TXB3, the beta-oxidation product of 11-dehydro-TXB3. Characteristic fragment ions of [M - C5H9-(dimethylisopropylsilanol)n]+ (m/z 524 and 406) yielding information about the position of the incorporated double bond were prominent, the same as the corresponding ions found in the mass spectrum of the 11-dehydro-TXB3 derivative. Consequently, the fragmentation products were closely related to those of the 11-dehydro-TXB3 derivative, except for an obvious shift produced by the lack of C-2/C-3 hydrocarbon units. The results demonstrate the endogenous formation of TXA3 in humans after administration of EPA ethyl ester and excretion of 11-dehydro-2,3-dinor-TXB3 into urine as one of the enzymatic metabolites of TXB3, together with the previously reported 11-dehydro-TXB3.

Microdetermination of 11-dehydrothromboxane B3 in human urine by gas chromatography--selected-ion monitoring using (18O2) analogue as an internal standard.

The microdetermination of 11-dehydrothromboxane B3 (11-dehydro-TXB3) in human urine is described. We prepared [18O2]11-dehydrothromboxane B3 for use as an internal standard. Samples containing an [18O2] analogue were extracted chromatographically using Sep Pak tC18 and a silica gel column. Conversion of the extracted 11-dehydro-TXB3 to 1-methyl ester-11-n-propylamide-9,12,15-dimethylisopropylsilyl ether derivative was followed by gas chromatography/selected ion monitoring (GC/SIM). Interfering substances from the urine matrix were eliminated during GC/SIM analysis using an MP-65HT column. Human urine was monitored using the ions of m/z 696.4511 (11-dehydro-TXB3), m/z 700.4597 (18O2]1l-dehydro-TXB3), m/z 698.4668 (11-dehydro-TXB3) and m/z 702.4918 ([2H4]11-dehydro-TXB2). 11-Dehydro-TXB3 and [18O2]11-dehydro-TXB3 appeared at the same retention time and were eluted 15 s after 11-dehydro-TXB2 on this capillary column. This difference in retention time was due to the double bond in the omega3 position in 11-dehydro-TXB3. A good linear response over the range of 10 pg to 10 ng/tube was demonstrated. We detected 11-dehydro-TXB3 in the range from 1.29 to 7.64 pg/mg creatinine in the human urine. In the healthy volunteers, the 11-dehydro-TXB3 level was less than 1% of that of the 11-dehydro-TXB2, but 11-dehydro-TXB3 was increased by dietary supplementation of eicosapentaenoic acid. This method can be applied to the determination of 11-dehydro-TXB3 in human urine.

Analysis of 11-dehydrothromboxane B2 in human urine improved by use of gas chromatography/high-resolution selected ion monitoring with 18O2-labelled analogue as an internal standard.

Gas chromatography/high-resolution selected ion monitoring (GC/HR-SIM) for the determination of 11-dehydrothromboxane B2 (11-dehydro-TXB2) in human urine is described. Combined use of the lactone ring opening reaction and dimethylisopropylsilylation has been adopted for the analysis of 11-dehydro-TXB2, to give a new chemically stable derivative. The feasibility of using this derivative for the analysis of 11-dehydro-TXB2 in human urine has been evaluated. Following the addition of 18O2-labelled analogue, for use as an internal standard, the samples were extracted using chromatographic separation with Bond Elut C18 and silica gel. Derivatization of the extracted 11-dehydro-TXB2 was followed by GC/SIM at resolution of 5000. This method enables selective detection of 11-dehydro-TXB2 in human urine without serious interference from other endogenous substances. In 11 volunteers, the mean urinary excretions of 11-dehydro-TXB2 was 547.6 +/- 217.6 ng per day.

Microdetermination of 2,3-dinor-6-ketoprostaglandin F1 alpha in human urine using gas chromatography-high-resolution selected-ion monitoring.

The microdetermination of 2,3-dinor-6-ketoprostaglandin F1 alpha (I) in human urine is described. Samples to which the [2H4]-analogue was added as an internal standard were extracted by chromatographic sample preparation using a Bond Elut C18 cartridge and a silica gel column. Conversion of the extracted I into the 1-methyl ester-6-methoxime-9,11,15-trisdimethylisopropylsilyl ether derivative was followed by gas chromatography-high-resolution selected-ion monitoring (GC-HR-SIM). Interfering substances from the urine matrix were eliminated during GC-HR-SIM analysis using a DB-1 column. A good linear response over the range 10 pg-100 ng per tube was demonstrated. Compound I could be detected in the range 26-375 pg/ml of human urine. The proposed method can be applied to the determination of I in human urine.

Reverse flow in the intracranial arteries--the possible significance of comparative flow in the anterior cerebral and the basilar arteries.

Four infants with reverse flow patterns of the intracranial arteries are reported. Two with severe brain damage, had a reverse flow pattern in the anterior cerebral artery, which was recorded during the recovery stage from cardiac arrest. The other two patients showed a reverse flow pattern in the basilar artery and had a good prognosis. Reverse flow in the anterior cerebral artery suggests severe brain damage, but that in the basilar artery does not.

[Two-dimensional and Doppler echocardiographic evaluation of intrauterine blood flow dynamics in the fetuses with a ventricular septal defect].

In nine fetuses with ventricular septal defect (VSD), intracardiac blood flow was observed with color Doppler ultrasound. Interventricular shunt velocity, maximum systolic velocity in the ascending aorta and pulmonary artery, trans-atrioventricular blood flow velocity, and the flow pattern in the inferior vena cava were also measured with pulsed Doppler ultrasound. The cardio-thoracic ratio and ejection fraction were measured by B- and M-mood ultrasound. In all four fetuses with isolated VSD a bidirectional shunt was demonstrated. In the three normal developed fetuses time analysis showed a right (RT)-to-left (LT) shunt during systole and a LT-to-RT during diastole with maximum systolic velocities of 61cm/s, 42cm/s and 68cm/s. In the five fetuses with complexed cardiac malformation, two fetuses had a unidirectional shunt and three fetuses had a bidirectional shunt, which was a LT-to-RT during systole and a RT-to-LT during diastole. These studies indicate that although the systolic pressure in the right ventricle is slightly higher than that in the left, the pressure is almost the same in both ventricles. The presence of unidirectional shunting or a different time relationship from that of normal fetuses in bidirectional shunting would necessitate a careful check for other cardiac malformations.