Natural mutations of human XDH promote the nitrite (NO2-)-reductase capacity of xanthine oxidoreductase: A novel mechanism to promote redox health?

Several rare genetic variations of human XDH have been shown to alter xanthine oxidoreductase (XOR) activity leading to impaired purine catabolism. However, XOR is a multi-functional enzyme that depending upon the environmental conditions also expresses oxidase activity leading to both O2·- and H2O2 and nitrite (NO2-) reductase activity leading to nitric oxide (·NO). Since these products express important, and often diametrically opposite, biological activity, consideration of the impact of XOR mutations in the context of each aspect of the biochemical activity of the enzyme is needed to determine the potential full impact of these variants. Herein, we show that known naturally occurring hXDH mutations do not have a uniform impact upon the biochemical activity of the enzyme in terms of uric acid (UA), reactive oxygen species (ROS) and nitric oxide ·NO formation. We show that the His1221Arg mutant, in the presence of xanthine, increases UA, O2·- and NO generation compared to the WT, whilst the Ile703Val increases UA and ·NO formation, but not O2·-. We speculate that this change in the balance of activity of the enzyme is likely to endow those carrying these mutations with a harmful or protective influence over health that may explain the current equipoise underlying the perceived importance of XDH mutations. We also show that, in presence of inorganic NO2-, XOR-driven O2·- production is substantially reduced. We suggest that targeting enzyme activity to enhance the NO2--reductase profile in those carrying such mutations may provide novel therapeutic options, particularly in cardiovascular disease.

The immunodepletion of factor VII from human plasma using a monoclonal antibody.

A murine hybridoma cell line which secretes monoclonal antibody to factor VII has been prepared to facilitate the immunodepletion of this clotting factor from plasma. Specific monoclonal antibody was purified from mouse ascites tumours by protein A-Sepharose chromatography and shown to be of the IgG1 immunoglobulin subclass. On immunoblotting, this antibody reacted with a single protein band identical to purified factor VII. The purified monoclonal antibody was coupled to Sepharose 4B and was used to immuno-deplete factor VII from pooled normal human plasma. The prothrombin time of plasma immunodepleted in this way was 35 s compared to 12 s for the starting plasma. Specific factor assays of the immunodepleted plasma showed factor VII activity to be less than 1% while the levels of the other clotting factors were unchanged. The immunodepleted plasma was equivalent to severe congenital factor VII deficient plasma as a substrate for factor VII assays. Bound factor VII could be eluted from the immunoaffinity column with citrate buffer, pH 6.0, with good recovery.

Effects of heparin fractions of different affinities to antithrombin III and thrombin on the inactivation of thrombin and factor Xa by antithrombin III.

To investigate the relative contribution of heparin-binding thrombin and antithrombin III to the enhancement of the rate of inactivation of thrombin by antithrombin III, standard heparin was fractionated on matrix-linked thrombin and (or) antithrombin III. There was a good correlation between heparin affinity for antithrombin III and its ability to enhance the inactivation of thrombin and factor Xa. In addition, there was a good correlation between affinity of heparin for thrombin and its catalytic activity on the inactivation of thrombin by antithrombin III. Thus fractions with high affinity to thrombin had similar rate-enhancing activity for thrombin inactivation to that of fractions with high affinity to antithrombin III. Fractions with high affinity to both proteins were more potent than fractions with high affinity to either protein alone. No significant differences in mean molecular weight were observed among the various heparin fractions. A heparin fraction with very low affinity to thrombin and high affinity to antithrombin III was prepared by repeated fractionation of a low molecular weight heparin on the two affinity columns. This fraction had very weak rate-enhancing activity for the inactivation of thrombin by antithrombin III, but retained substantial activity for the inactivation of factor Xa. The results of these studies support the concept that, for both standard and low molecular weight heparin, the enhancement of the inactivation of thrombin by antithrombin III requires the interaction of the heparin with both thrombin and antithrombin III.

Effect of continuous infusion of gonadotrophin-releasing hormone in ewes at different times of the year.

Gn-RH (3 mug/hr for 8 hr) evoked a surge of LH in ewes during the anoestrous season which was of similar peak height to that found at oestrus but of shorter duration. When Gn-RH was given on 3 consecutive days, the response was considerably reduced on the 2nd and 3rd day. Follicles grew as a result of Gn-RH infusion but peripheral plasma oestrogen concentration did not increase. During the anoestrous season 9/18 ewes ovulated but only 1/6 did so at mid-anoestrus. Mature follicles or a CL were found in 15/18 ewes that had increased peripheral plasma progesterone concentrations for 1-5 days (11 ewes) or 9-14 days (4 ewes). The concentrations of progesterone found were always lower than those observed during the normal cycle. In the breeding season 10/11 ewes ovulated but there was no evidence that the induced CL were maintained.