Ubiquitination and degradation of neuronal nitric-oxide synthase in vitro: dimer stabilization protects the enzyme from proteolysis

It is established that neuronal NO synthase (nNOS) is ubiquitinated and proteasomally degraded. The metabolism-based inactivation of nNOS and the inhibition of heat shock protein 90 (hsp90)-based chaperones, which are known to regulate nNOS, both lead to enhanced proteasomal degradation of nNOS. The mechanism of this selective proteolytic degradation, or in essence how the nNOS becomes labilized and recognized for ubiquitination and subsequent degradation, has not been determined. In the current study, we used a crude preparation of reticulocyte proteins, which contains ubiquitin-conjugating enzymes and the proteasome, to determine how nNOS is labilized. We found that the inactive monomeric heme-deficient nNOS (apo-nNOS) is rapidly degraded in vitro, consistent with the finding that both metabolism-based inactivation and inhibition of hsp90-based chaperones cause the formation of apo-nNOS and enhance its degradation in vivo. In the current study, we discovered that destabilization of the dimeric nNOS, as determined by measuring the SDS-resistant dimer, is sufficient to trigger ubiquitin-proteasomal degradation. Treatment of nNOS with NG-nitro-L-arginine or 7-nitroindazole led to stabilization of the dimeric nNOS and decreased proteasomal degradation of the enzyme, consistent with that observed in cells. Thus, it seems that the dimeric structure is a major determinant of nNOS stability and proteolysis.     

TC-1734: an orally active neuronal nicotinic acetylcholine receptor modulator with antidepressant, neuroprotective and long-lasting cognitive effects

The development of selective ligands targeting neuronal nicotinic acetylcholine receptors to alleviate symptoms associated with neurodegenerative diseases presents the advantage of affecting multiple deficits that are the hallmarks of these pathologies. TC-1734 is an orally active novel neuronal nicotinic agonist with high selectivity for neuronal nicotinic receptors. Microdialysis studies indicate that TC-1734 enhances the release of acetylcholine from the cortex. TC-1734, by either acute or repeated administration, exhibits memory enhancing properties in rats and mice and is neuroprotective following excitotoxic insult in fetal rat brain in cultures and against alterations of synaptic transmission induced by deprivation of glucose and oxygen in hippocampal slices. At submaximal doses, TC-1734 produced additive cognitive effects when used in combination with tacrine or donepezil. Unlike (-)-nicotine, behavioral sensitization does not develop following repeated administration of TC-1734. Its pharmacokinetic (PK) profile (half-life of 2 h) contrasts with the long lasting improvement in working memory (18 h) demonstrating that cognitive improvement extends beyond the lifetime of the compound. The very low acute toxicity of TC-1734 and its receptor activity profile provides additional mechanistic basis for its suggested potential as a clinical candidate. TC-1734 was very well tolerated in acute and chronic oral toxicity studies in mice, rats and dogs. Phase I clinical trials demonstrated TC-1734's favorable pharmacokinetic and safety profile by acute oral administration at doses ranging from 2 to 320 mg. The bioavailability, pharmacological, pharmacokinetic, and safety profile of TC-1734 provides an example of a safe, potent and efficacious neuronal nicotinic modulator that holds promise for the management of the hallmark symptomatologies observed in dementia.     

The 677 C-T methylenetetrahydrofolate reductase mutation does not predict increased maternal homocysteine during pregnancy

OBJECTIVE: To test the hypothesis that, regardless of the presence of the 677 C-T methylenetetrahydrofolate reductase (MTHFR) mutation, maternal homocysteine concentrations will not be significantly different in women who are taking prenatal vitamins containing folic acid, and to test this relationship in preeclampsia because homocysteine concentrations are higher in preeclamptic pregnancies. METHODS: Fifty-seven pregnant white women (control and preeclamptic) with and without the 677 C-T MTHFR mutation were studied. Total plasma homocysteine and plasma folic acid were analyzed. RESULTS: Homocysteine concentrations were not different by MTHFR genotype (wild type 677 CC 8.7 +/- 5.6 microM versus mutant 677 TT 9.0 +/- 5.7 microM, P =.84) in preeclamptic or normal pregnancies. However, mean homocysteine concentrations were significantly increased in preeclamptic pregnancies compared with those in normal pregnancies (10.6 +/- 7.3 microM versus 7.2 +/- 3.0 microM, P <.03) as previously reported. CONCLUSION: The 677 C-T MTHFR polymorphism does not significantly affect maternal homocysteine concentrations in most women taking prenatal vitamins including women with preeclampsia. The increase in plasma folic acid likely affects maternal homocysteine more than the MTHFR genotype. If homocysteine is considered a thrombophilia risk factor, the concentration of the amino acid and not a particular genotype should be determined.     

Localization of species conserved zona pellucida antigens in mammalian ovaries

The mammalian zona pellucida (ZP) consists of three glycoproteins (ZP1, ZP2 and ZP3), which are variably conserved among species at the genomic and amino acid levels. In order to evaluate the expression of ZP during ovarian development, a population of antibodies was selected that recognize species conserved antigenic domains of the three ZP proteins. Domain specific antibodies were selected from sera of rabbits immunized with all three native pig ZP proteins by elution of antibodies bound to each of the three human ZP recombinant proteins expressed from cDNAs, using the baculovirus expression system in insect cells. Immunoblot analysis was used to characterize the specificity of the antibodies and immunohistochemistry was used to evaluate the stage specific expression of ZP proteins during ovarian follicular development of the mouse, baboon and human. This study demonstrates that the conserved domains of all three ZP proteins are localized in the oocyte extracellular ZP matrix as well as in a subset of granulosa cells. However, this expression does vary among species with respect to the stage and cell type during early stages of ovarian follicular development. These antibodies should serve as excellent markers for evaluating early stages of human ovarian follicular development and in the development of contraceptive agents.     

Single-center comparison of purely laparoscopic,hand-assisted laparoscopic,and open radical nephrectomy in patients at high anesthetic risk

BACKGROUND AND PURPOSE: The laparoscopic approach for management of high-risk patients with renal-cell carcinoma (RCC) may reduce perioperative and postoperative morbidity. The aim of this study was to compare the outcome of purely laparoscopic radical nephrectomy (LRN), hand-assisted laparoscopic radical nephrectomy (HALRN), and open radical nephrectomy (ORN) for renal tumors in a population of patients at high risk for perioperative complications. PATIENTS AND METHODS: All patients undergoing radical nephrectomy for presumed RCC between August 1999 and August 2001 at Vanderbilt University Medical Center and having an American Society of Anesthesiologists (ASA) score of >/=3 were reviewed. Patients with known metastasis, local invasion, caval thrombi, or additional simultaneous surgical procedures were excluded from analysis. Thirteen patients underwent LRN, eight patients underwent HALRN, and 26 underwent ORN. The patient demographics were similar in the three groups. The groups were compared with regard to intraoperative and postoperative parameters. Statistical analysis was done using chi-square testing for categorical variables and analysis of variance (ANOVA) for continuous variables. Differences in outcomes were examined using ANOVA and Dunnett's T for pairwise comparisons. RESULTS: The ASA 4 patients had significantly longer hospital stays and total hospital costs than the ASA 3 patients. The mean operative time in the ASA 3 patients was similar in the three groups: 2.8 hours, 2.8 hours, and 2.5 hours for the LRN, HALRN, and ORN patients, respectively. Both the LRN patients (22.9 mg of morphine sulfate equivalent) and the HALRN patients (42.1 mg) required less pain medication than the open surgery patients (97.7 mg). When the total hospital costs were compared, LRN was less costly than HALRN ($6089 v $7678; P = 0.57) and open surgery ($6089 v $7694; P = 0.04). The complication rate in the LRN, HALRN, and ORN group was 0%, 25%, and 27%, respectively, although the differences were not statistically different (P = 0.12). CONCLUSIONS: Both LRN and HALRN can be performed safely in patients with significant comorbid conditions. Careful preoperative preparation, intraoperative monitoring, and awareness of laparoscopy-induced oliguria can preclude inadvertent overhydration, hemodilution, and congestive heart failure. Both LRN and HALRN result in less pain medication requirement and faster return to oral intake than ORN, and LRN results in fewer perioperative complications than HALRN or ORN in patients at high perioperative risk. The LRN technique has a 21% lower total cost than both HALRN and ORN.