Effects of Asp-369 and Arg-372 mutations on heme environment and function in human endothelial nitric-oxide synthase

Eight polar amino acid residues in the putative substrate-binding region from Thr-360 to Val-379 in human endothelial nitric-oxide synthase (eNOS) (Thr-360, Arg-365, Cys-368, Asp-369, Arg-372, Tyr-373, Glu-377, and Asp-378) were individually mutated. Only two of these residues, Asp-369 and Arg-372, were found to be essential for enzyme activity. A further series of mutants was generated by replacing these two residues with various amino acids and the mutant proteins were expressed in a baculovirus system. Mutant eNOS had a very low L-citrulline formation activity with the exception of D369E and R372K, which retained 27% and 44% of the wild-type enzyme activity, respectively. Unlike the wild-type enzyme, all mutants except D369E, R372K, and R372M had a low spin heme (Soret peak at 416 nm). All the Asp-369 mutants had higher Kd values for L-arginine (1-10 mM) than wild-type eNOS (0.4 microM) and an unstable heme-CO complex, and except for D369E, had a very low (6R)-5,6,7, 8-tetrahydro-L-biopterin (BH4) content. In contrast, each of Arg-372 mutants retained a considerable amount of BH4, had a moderate reduction in L-arginine affinity, and had a more stable heme-CO complex. 1-Phenylimidazole did not bind to wild-type eNOS heme, but bound to all Asp-369 and Arg-372 mutants (Kd ranged from 10 to 65 microM) except R372K. Heme spin-state changes caused by binding of 3, 5-lutidine appeared to depend on both charge and size of the side chains of residues 369 and 372. Furthermore, all Asp-369 and Arg-372 mutants were defective in dimer formation. These results suggest that residues Asp-369 and Arg-372 in eNOS play a critical role in oxygenase domain active-site structure and activity.     

Prolactin-releasing effect of buspirone in developing and adult male and female rats

The prolactin-releasing effects of buspirone, an azaspirodecanedione anxiolytic drug unrelated to the benzodiazepines in structure and pharmacologic properties, was examined in developing and adult male and female rats. The possibility that effects of this drug on hormone release could be modulated by neonatal brain sexual differentiation was also evaluated. A single injection of buspirone, 2 or 10 mg/kg body wt, increased serum prolactin (PRL) levels in both sexes; the increase was significant from Day 12 onward. The PRL-releasing effect increased with age. No significant sexual differences were observed in younger rats, but in peripubertal and adult animals, the hyperprolactinemic response was higher in the female. Neonatal androgenization of females or orchidectomy of males failed to modify the PRL-releasing action of buspirone. Serum titers of luteinizing hormone and follicle-stimulating hormone were not modified by buspirone at any age. The present results show for the first time the ontogeny of the PRL-releasing effect of buspirone in male and female rats, and provide evidence that the response is higher in the female and that the effect does not depend on brain sexual differentiation.     

Dinitrochlorobenzene skin test reactivity as a predictor of outcome in transplantation of juvenile onset diabetics

More than 50% of patients with chronic renal failure will be suppressed in their cell-mediated immune response to 2,4-dinitrochlorobenzene (DNCB). This applies in renal failure attributable to juvenile onset diabetes as well as other types of end stage renal disease. Significantly better kidney survival of both living related and cadaver grafts is seen in diabetic patients who are non-responsive to DNCB. Twelve-month kidney survival for DNCB-negative patients receiving living related allografts is 71% compared with 25% for DNCB-positive patients. Twelve-month kidney survival in cadaver recipients is 39% in DNCB-negative compared with 9% for DNCB-positive patients. Successful second grafts were done in DNCB-negative diabetic patients, however, all second grafts in DNCB-positive patients failed in less than 3 months. DNCB skin test reactivity as a measure of cell-mediated response is a valuable predictor of immunological outcome of transplantation in patients whose renal disease results from juvenile onset diabetes. Patient survival in DNCB nonresponders is nearly twice that of DNCB responders. Differences in outcome following transplantation could not be accounted for by HLA disparity, transfusion history, or other variables known to effect transplant outcome. Kidney and patient survival in DNCB-positive diabetic patients receiving either cadaver or living related allografts is sufficiently low as to identify them as a subpopulation of renal failure patients who should be treated by dialysis, or selected for special protocols which might provide immunological manipulation prior to transplantation to improve their treatment outcome.     

Brain abscess caused by Cladosporium trichoides

In 17 previously reported cases of cladosporiosis, no reliable therapy was described, and death occurred usually within one year of diagnosis. Pretreatment isolates from our two patients were inhibited by 6.2 mug/ml and 3.1mug/ml of flucytosine, respectively. Although both patients died, postmortem examination results showed that in one patient, the fungus had been eradicated. In the other patient, C trichoides that was isolated at postmortem examination was resistant to flucytosine treatment.     

Significance of renal visualization during bone scanning in Paget's disease

In 17 of 26 patients with extensive, symptomatic Paget's disease poor renal visualization was noted on bone scanning with 99mTc-diphosphonate. Renal function was normal in all patients. The intensity of the renal image proved to be inversely related to the extent and metabolic activity of the Pagetic process. This finding supports the hypothesis that in Paget's disease the balance between skeletal and renal extraction of circulating tracer amy be displaced in favor of the former.