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.     

Menstruation disorders in insulin-dependent diabetes mellitus--epidemiology and causes

About 20% of all women with insulin-dependent diabetes mellitus (IDDM) have menstrual irregularities. Eight percent have amenorrhea. Fluctuations in blood glucose and insulin concentration are probably contributing factors, but the irregular menstrual cycles are mainly caused by disorders in the central ovulatory mechanisms. Hypothalamic GnRH release is regulated by several neuropeptides. Dopamine and opiates exert an inhibitory effect, and there is evidence for an abnormally high dopaminergic hypothalamic activity among women with IDDM. There might also be disorders of the opioid, serotonergic and GABA'ergic systems, but the consequences of there possibilities remain uncertain.     

HMPAO brain SPECT in acute carbon monoxide poisoning

Technetium-99m-hexamethylpropylene amine oxime (HMPAO) brain images with fanbeam SPECT, in combination with surface three-dimensional display, were used to detect basal ganglion and cerebral cortex anomalies in the acute phase of carbon monoxide (CO) poisoning. METHODS: Ten patients, aged 16-29 yr, with acute CO poisoning and no past history of neurologic disorders were enrolled in this study. After oxygen treatment, all 10 patients were investigated using 99mTc-HMPAO brain images with fanbeam SPECT and surface three-dimensional display. Meanwhile, 6 of 10 patients also received a brain CT scan. RESULTS: CT scan findings were negative in all 6 patients. Fanbeam SPECT demonstrated unilateral or bilateral hypoactivity of basal ganglia in 6 patients. Local hypoactivity anomalies were found in the brain cortex of 7 patients, using surface three-dimensional display of the brain. Only 2 of 10 patients had normal 99mTc-HMPAO brain images. CONCLUSION: This study suggests that, in comparison with traditional brain imaging techniques, 99mTc-HMPAO brain imaging with fanbeam SPECT in combination with surface three-dimensional display is a better tool for early detection of regional cerebral anomalies in acute CO poisoning.