Oxidant damage to erythrocyte membrane in glucose-6-phosphate dehydrogenase deficiency: correlation with in vivo reduced glutathione concentration and membrane protein oxidation

Chronic nonspherocytic hemolytic anemia has been observed in a recently described glucose-6-phosphate dehydrogenase (G6PD) variant, G6PDWayne. The mechanical properties of these erythrocytes and other G6PD variants were examined. The deformability of G6PD-deficient erythrocytes was normal, as determined by osmotic scan ektacytometry, and was not significantly affected by hemolytic crisis. In the common varieties of G6PD deficiency, the mechanical stability of the red blood cell (RBC) membrane was greater than normal, but G6PDWayne membranes were abnormally susceptible to shear-induced fragmentation. There was no evidence for a concurrent genetic defect in spectrin, because self-association constants and tryptic digests were normal. The fragility of G6PDWayne membranes appeared to be a consequence of oxidative damage to membrane thiol groups associated with a low glutathione (GSH) level in these RBCs. Associations among GSH level, thiol oxidation, and membrane instability were also found when a larger group of G6PD-deficient RBCs were examined. In normal erythrocytes, 1-chloro-2,4-dinitrobenzene was used to reduce GSH levels by 50%. Membrane thiol oxidation and membrane fragility both increased when these cells were kept at 4 degrees C for 3 to 5 days. Our findings suggest that chronic depletion of GSH leads to the destabilization of membrane skeleton through oxidation of membrane protein thiols.     

News: White House panel creating database of endocrine disrupter-related research

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News: EPA tries "StarTrack" permits in New England

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News: EPA report targets 10 key research areas

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The effect of growth hormone replacement on cortisol metabolism and glucocorticoid sensitivity in hypopituitary adults

OBJECTIVE: Growth hormone (GH) replacement therapy in hypopituitary adults is associated with sodium and water retention. The underlying mechanisms are incompletely understood and a possible contribution of altered cortisol metabolism or action has not been evaluated. We have investigated the effect of GH replacement therapy on cortisol metabolism, cortisol binding globulin and in-vitro glucocorticoid sensitivity in a group of adult hypopituitary patients. DESIGN AND PATIENTS: We studied 19 adult hypopituitary patients (18 adult onset, M:F, 6:13), who were receiving conventional hydrocortisone (16 patients), thyroxine (14 patients), triiodothyronine (1 patient), sex steroid (9 patients), human chorionic gonadotrophin (1 patient) or desmopressin (6 patients) replacement during a 6-month, double blind controlled trial of GH therapy (active:placebo, 8:11) followed by a 6-month open phase of GH (mean dose: 0.2 IU/kg/week, range 0.051-0.27) and after a 6-week washout phase following discontinuation of GH therapy. MEASUREMENTS: Twenty-four-hour urine free cortisol, cortisol metabolites (CoM), ratio 11-hydroxy/11-oxo CoM (F/E) and ratio 5 alpha/beta tetrahydrocortisol were measured at 6 months, 12 months and after the 6 week washout phase. Serum cortisol binding globulin was measured basally, at 6 months, 12 months and after washout. Glucocorticoid sensitivity was determined in lymphocyte preparations from 8 patients, during GH therapy and after washout, using an in-vitro technique dependent on dexamethasone suppression of phytohaemagglutinin-stimulated thymidine incorporation into DNA. Plasma renin activity and aldosterone were measured after 6-12 months GH therapy and after washout. RESULTS: After 6 months of GH, in patients on hydrocortisone (n = 9), there were significant decreases in CoM (mean decrement 21%, P < 0.01), F/E (mean decreased from 1.27 to 1.0, P = 0.04; reference range 0.33-1.29) and 5 alpha/5 beta tetrahydrocortisol (mean decreased from 0.67 to 0.48, P = 0.01) and a subsequent increase after washout. Patients not on hydrocortisone (n = 2) demonstrated a normal basal F/E falling by 25% on GH therapy but no change in CoM. During 12 months of GH therapy, patients on hydrocortisone (n = 7) demonstrated a further trend to decrement in CoM (P = 0.09) which reversed after washout (P = 0.04). Urine free cortisol tended to fall during GH therapy and increased significantly following washout after 12 months treatment (P < 0.02). Serum cortisol binding globulin decreased by 20% (P < 0.05) during 12 months GH treatment but remained within the reference range. In-vitro studies demonstrated a trend to reduced glucocorticoid sensitivity on GH therapy; the maximum inhibition of phytohaemagglutinin by dexamethasone tended to be less on GH therapy (P = 0.052) and was also lower than in 29 normal volunteers (P < 0.05). There were no significant changes in plasma renin but there was a small increment in aldosterone in recumbent patients (P = 0.04) during the open phase of GH therapy in the placebo arm. CONCLUSIONS: GH therapy in hypopituitary adults is associated with an apparent reduction in availability of administered hydrocortisone as measured by urine cortisol metabolites and urine free cortisol. This effect is unlikely to be clinically significant except possibly in ACTH deficient subjects on suboptimal hydrocortisone replacement. The changes in F/E suggest that GH may directly or indirectly modulate the activity of 11 beta-hydroxysteroid dehydrogenase. The apparent decrease in glucocorticoid sensitivity during GH therapy, demonstrated in vitro, merits further investigation.     

Nanoparticles in Catalysis

In this article, we report studies of two new forms of highly active supported catalysts. First, those derived from supported carbonylate clusters—nanocatalysts and second, those produced from the heterogenization of known chiral homogeneous systems. The utilization of established cluster compounds of precisely known composition and structure have proved invaluable in the preparation of mixed metal nanoparticles of well-defined composition. The attachment of these nanoparticles to the inner walls of mesoporous silica has led to the development of highly active and effective catalysts for a series of hydrogenation reactions, emphasizing the enhanced reactivity of these metal systems as a consequence of their size and of the low coordination numbers of the metal atoms involved. These attributes combined with the relative ease of characterization of both the active sites and their location has led to a detailed examination of the role of these nanosystems in a new approach to clean technology. In an alternative strategy, the use of heterogenized homogeneous chiral catalysts based on the ferrocenyl moiety and diamino ligands and linked to the inner surface of mesoporous materials either by a direct chemical bond or by an ionic interaction has also been explored. These catalysts have been shown to be highly effective in the enantioselective synthesis of organic compounds. Significantly, we have found that the mesopore (usually MCM-41) imposes spatial restrictions arising from the concavity of the inner surface and leads to greatly enhanced enantioselective (ee) performance.     

A new technique for laparoscopic splenectomy with massively enlarged spleens

Splenectomy is indicated in several hematological disorders and it can be particularly challenging in children with sickle cell disease, splenomegaly, and recurrent sequestration. Over the last 6 months, we have developed a new technique for laparoscopic splenectomy (LS) for hypersplenism and splenomegaly in five children with sickle cell disease. The average age of our patients was 6 years (range, 2-11), and the average weight was 18.7 kg (range, 13.2-30.1). On preoperative ultrasound, spleen size index ranged from 0.42 to 0.76. For the LS, four trochars were placed. One patient, who also underwent a laparoscopic cholecystectomy, had six trochars placed, two of which were used for both cholecystectomy and splenectomy. After laparoscopic mobilization of the spleen and hilar vascular stapling, a Steiner electromechanical morcellator was inserted through the 12-mm port to extract cores of splenic tissue until complete splenectomy was achieved. No patient required conversion to an open procedure or creation of a larger incision to remove the massively enlarged spleen. Operative time averaged 190 minutes; the combined LS and cholecystectomy took 245 minutes. Postoperative length of stay was <2 days for all patients. There were no complications, and no patient required postoperative transfusion. Based on these early findings, we conclude that intracorporeal coring of splenic tissue allows for safe and complete laparoscopic removal of very large spleens in small children. It provides expedient recovery and minimal postoperative pain and scarring. This new technique should enable surgeons to perform LS even in patients with massive splenomegaly, eliminating the need for large and cumbersome intracorporeal bags or the creation of additional incisions to remove the spleen.     

Fluorescence in situ hybridization (FISH): DNA probe production and hybridization criteria

We describe methods for the production of fluorescence in situ hybridization (FISH) probes and the utilization of these probes for the detection of complementary DNA sequences with accuracy and sensitivity for application in both basic research and clinical diagnosis. Due to the frequent use of FISH in many laboratories, it is important to apply the most convenient and reproducible approach. This review describes some of the most recent techniques, and includes versatile, effective and simple methods of probe production and fluorescence in situ hybridization. We also describe methods for the production of region-specific and chromosome-specific DNA probes and hybridization techniques for the visualization of these probes.