Search

Research has been conducted on 7-methyl-6,8-bis(methyldisulfanyl)pyrrolo[1,2-a]pyrazine and methanethiosulfonic acid S-((6-methanesulfonylsulfanyl)-7-methyl)pyrrolo[1,2-a]pyrazin-8-yl) ester. The synthesis of these compounds has been carried out to investigate whether they can serve as alternative precursors to the major metabolites of the cancer chemopreventive oltipraz and whether their biological activities are similar.

Results demonstrate that heat shock protein 90 directly inhibits superoxide generation from neuronal nitric oxide synthase. Data further indicate that this inhibition may be responsible for the enhancement of heat shock protein's effect on the synthesis of nitric oxide from neuronal nitric oxide synthase.

Author(s): Y. Amy Lam [1]; T. Glen Lawson [2]; Murugesan Velayutham [3]; Jay L. Zweier [3]; Cecile M. Pickart (corresponding author) [1] The 26S proteasome is the chief site of [...]

Quantitation of superoxide generation and substrate utilization by vascular NAD(P)H oxidase. Am J Physiol Heart Circ Physiol 282: H466-H474, 2002; 10.1152/ajpheart.00482.2001.--In vascular tissues, an NAD(P)H oxidase is the main source of superoxide; however, there has been much uncertainty regarding its activity and the levels of superoxide it generates. This problem has limited overall progress in this field. Therefore, studies were performed and techniques developed to quantitatively assess the function of the vascular NAD(P)H oxidase, measuring its rate of superoxide production and substrate consumption in rat aortic homogenates and intact segments. NADPH/NADH oxidation was measured spectrophotometrically, and oxygen consumption was measured by electrochemical probe. Superoxide was detected and quantitated by electron paramagnetic resonance spin trapping. Under basal conditions, superoxide generation and oxygen consumption were negligible. After addition of NADPH or NADH (0.1 mM), superoxide was generated at rates of 0.41 [+ or -] 0.03 or 0.36 [+ or -] 0.04 nmol * mg [protein.sup.-1] * [min.sup.-1], respectively. Oxygen was consumed with a similar time course at rates of 1.5 [+ or -] 0.2 or 1.3 [+ or -] 0.3 nmol * mg [protein.sup.-1] * [min.sup.-1], and NADPH or NADH were oxidized at rates of 1.8 [+ or -] 0.4 and 1.5 [+ or -] 0.3 nmol * mg [protein.sup.-1] * [min.sup.-1], respectively. In intact aortic rings, superoxide was generated with rates of 4.0 [+ or -] 0.7 or 3.7 [+ or -] 0.7 pmol * mg [tissue.sup.-1] * [min.sup.-1], whereas oxygen was consumed at rates of 22.1 [+ or -] 5.0 or 14.5 [+ or -] 3.3 pmol * mg [tissue.sup.-1] * [min.sup.-1], for NADPH or NADH, respectively. These values are lower than those previously measured using lucigenin, which uncouples flavoenzymes, triggering additional superoxide generation. This quantitative approach for characterization of the vascular NAD(P)H oxidase activity should facilitate the further identification and cellular characterization of this enzyme(s) and its functional and signaling roles. vascular oxidase; free radicals; electron paramagnetic resonance; spin trap; 5,5'-dimethyl-pyrroline-N-oxide; lucigenin

Song, Yao, Jay L. Zweier, and Yong Xia. Determination of the enhancing action of HSP90 on neuronal nitric oxide synthase by EPR spectroscopy. Am J Physiol Cell Physiol 281: C1819-C1824, 2001.--Recent studies showed that heat shock protein 90 (HSP90) enhances nitric oxide (NO) synthesis from endothelial and neuronal NO synthase (eNOS and nNOS, respectively). However, these findings were based on indirect NO measurements. Moreover, although our previous studies showed that the action of HSP90 involves increased [Ca.sub.2+] /calmodulin ([Ca.sub.2+] /CaM) binding, quantitative measurements of the effect of HSP90 on CaM binding to nNOS have been lacking. With electron paramagnetic resonance spectroscopy, we directly measured NO signals from purified nNOS. HSP90 augmented NO formation from nNOS in a dose-dependent manner. Tryptophan fiuo-rescence-quenching quenching measurements revealed that HSP90 markedly reduced the Kd of CaM to nNOS (0.5 [+or -] 0.1 nM vs. 9.4 [+ or -] 1.8 nM in the presence and absence of HSP90, P < 0.01). [Ca.sup.2+] ionophore triggered strong NO production from nNOS-transfected cells, and this was significantly reduced by the HSP90 inhibitor geldanamycin. Thus these studies provide direct evidence demonstrating that HSP90 enhances nNOS catalytic function in vitro and in intact cells. The effect of HSP90 is mediated by the enhancement of CaM binding to nNOS. heat shock protein 90 modulation; nitric oxide synthase regulation; calmodulin binding affinity; tryptophan fluorescence; electron paramagnetic resonance spin trapping Received 25 June 2001; accepted in final form 7 August 2001

An investigation of preparation, characterization, and use of lithium phthalocyanine (LiPc) microcrystalline powder as a probe for electron paramagnetic resonance (EPR) oximetry applications is presented. The report discusses the application of this material to measure oxygen concentration in a biochemical, cellular (human) and in vivo (mice) biological systems.