Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry.

Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC ) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC(50) values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.

Essential thiol requirement to restore pterin- or substrate-binding capability and to regenerate native enzyme-type high-spin heme spectra in the Escherichia coli-expressed tetrahydrobiopterin-free oxygenase domain of neuronal nitric oxide synthase.

Nitric oxide (NO) synthases (NOS) are thiolate-ligated heme-, tetrahydrobiopterin (BH(4))-, and flavin-containing monooxygenases which catalyze the NADPH-dependent conversion of L-arginine (L-Arg) to NO AND citrulline. NOS consists of two domains: an N-terminal oxygenase (heme- and BH(4)-bound) domain and a C-terminal reductase (FMN- and FAD-bound) domain. In this study, we have spectroscopically examined the binding of L-Agr and BH(4) to the dimeric, BH(4)-free ferric neuronal NOS (NNOS) oxygenase domain expressed in Escherichia coli separately from the reductase domain.

Low-temperature stabilization and spectroscopic characterization of the dioxygen complex of the ferrous neuronal nitric oxide synthase oxygenase domain.

Nitric oxide (NO), an intercellular messenger and an immuno-cytotoxic agent, is synthesized by the family of nitric oxide synthases (NOS), which are thiolate-ligated, heme-containing monooxygenases that convert L-Arg to L-citrulline and NO in a tetrahydrobiopterin (BH4)-dependent manner, using NADPH as the electron donor. The dioxygen complex of the ferrous enzyme has been proposed to be a key intermediate in the NOS catalytic cycle. In this study, we have generated a stable ferrous-O2 complex of the oxygenase domain of rat neuronal NOS (nNOS) by bubbling O2 through a solution of the dithionite-reduced enzyme at -30 degrees C in a cryogenic solvent containing 50% ethylene glycol.

Tumor growth inhibition and regression induced by photothermal vascular targeting and angiogenesis inhibitor retinoic acid.

The effect of photothermal vascular targeting, alone and in combination with antiangiogenic therapy, was evaluated using tumors produced in mice by transplantation of KB cells. Tumor growth inhibition and regression followed vascular damage produced by pulsed dye laser (PDL) radiation. Administration of the antiangiogenic agent all-trans-retinoic acid (RA) was associated with smaller average tumor volumes in the presence and absence of PDL irradiation, but this effect was not statistically significant.

A 585-nanometer pulsed dye laser treatment of laryngeal papillomas: preliminary report.

Objectives/hypothesis: Standard management of recurrent respiratory papillomatosis (RRP) currently consists of CO2 laser microsurgical ablation of papillomas. Because of the recurrent nature of this viral disease, patients are often faced with significant cumulative risk of soft tissue complications. As a minimally traumatic alternative to management of RRP, we have investigated the use of the 585-nm pulsed dye laser (PDL) to cause regression of papillomas by selective eradication of the tumor microvasculature.

Diagnosis of head and neck precancerous lesions in an animal model using fluorescence spectroscopy.

Laser-induced fluorescence (LIF) of tissues depends on their biochemical and histomorphologic characteristics. LIF spectroscopic properties of 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced precancerous and early cancerous lesions in a hamster buccal pouch mucosa model were studied. Fluorescence spectra from neoplastic lesions showed a characteristic fluorescence peak in the red region of the visible spectrum centered between 630 and 640 nm when excited with 410-nm light.

Escherichia coli associated cellulitis in broilers: correlation with systemic infection and microscopic visceral lesions, and evaluation for skin trimming.

In Alberta, cellulitis condemnations average 0.5% and are among the highest in Canada. Presently, all cellulitis-affected birds are condemned for fear of systemic infections and public health implications.

Substrate binding-induced changes in the EPR spectra of the ferrous nitric oxide complexes of neuronal nitric oxide synthase.

A versatile diatomic physiological messenger, nitric oxide (NO), is biosynthesized by a group of flavo-heme enzymes, the nitric oxide synthases. We have examined the active site of the neuronal isoform by EPR spectroscopy of the ferrous nitric oxide complex. The nitric oxide complex of the substrate-free enzyme exhibits a cytochrome P450-type EPR spectrum typical of a hexacoordinate NO-heme complex with a non-nitrogenous proximal axial heme ligand.

Effect of dynamic cooling on 585-nm pulsed dye laser treatment of port-wine stain birthmarks.

Background And Objective: The objective of this study was to determine the effectiveness of a dynamic cooling device (DCD), spraying the skin with a brief spurt of cryogen prior to the laser pulse, in reducing transient pain associated with 585-nm pulsed dye laser (PDL) treatment of port-wine stains (PWS), and reducing epidermal damage (hypo/hyperpigmentation) caused by this laser during PWS treatment.

Materials And Methods: Matched treatment sites were compared with and without the use of the cryogen spray in 47 patients at two investigational sites. Pain ratings, clearance of the PWS, and pigmentation changes were assessed.

The influence of chaotropic reagents on neuronal nitric oxide synthase and its flavoprotein module. Urea and guanidine hydrochloride stimulate NADPH-cytochrome c reductase activity of both proteins.

Changes in flavin and protein fluorescence of neuronal nitric oxide synthase (nNOS) and its flavoprotein module were studied in the presence of urea and compared with those previously reported for cytochrome P450 reductase (CPR) [R. Narayanasami, P. M.

Early diagnosis of upper aerodigestive tract cancer by autofluorescence.

Objective: To explore the potential of autofluorescence spectroscopy as a tool for early detection of upper aerodigestive tract cancer.

Design: Autofluorescence spectral characteristics of 19 untreated oral and oropharyngeal lesions in 13 patients were studied with excitation wavelengths of 370 and 410 nm generated by a nitrogen pumped dye laser. Ten healthy volunteers were recruited to characterize the fluorescence spectra of normal mucosa at different oral sites and to study individual variations.

Binding of intermediate, product, and substrate analogs to neuronal nitric oxide synthase: ferriheme is sensitive to ligand-specific effects in the L-arginine binding site.

The electron paramagnetic resonance spectra of purified neuronal nitric oxide synthase indicates that the binding of ligands to the arginine site perturbs the environment of the high-spin ferriheme in a highly ligand-specific manner. Four categories of high-spin complex can be distinguished; all are five-coordinate, and all retain the axial thiolate ligand, but they differ in their ligation geometries. These spectroscopic species reveal distinct local conformations which can be stabilized individually by the binding of L-arginine, N omega-hydroxy-L-arginine, N omega-methyl-L-arginine, and N omega-nitro-L-arginine.

Neuronal nitric oxide synthase and dystrophin-deficient muscular dystrophy.

Neuronal nitric oxide synthase (nNOS) in fast-twitch skeletal muscle fibers is primarily particulate in contrast to its greater solubility in brain. Immunohistochemistry shows nNOS localized to the sarcolemma, with enrichment at force transmitting sites, the myotendinous junctions, and costameres. Because this distribution is similar to dystrophin, we determined if nNOS expression was affected by the loss of dystrophin.

Neuronal nitric oxide synthase, a modular enzyme formed by convergent evolution: structure studies of a cysteine thiolate-liganded heme protein that hydroxylates L-arginine to produce NO. as a cellular signal.

The nitric oxide synthases (NOS-I, neuronal, NOS-II, inducible, and NOS-III, endothelial) are the most recent additions to the large number of heme proteins that contain cysteine thiolate-liganded protoporphyrin IX heme prosthetic groups. This group of oxygenating enzymes also includes one of the largest gene families, that of the cytochromes P450, which have been demonstrated to be involved in the hydroxylation of a variety of substrates, including endogenous compounds (steroids, fatty acids, and prostaglandins) and exogenous compounds (therapeutic drugs, environmental toxicants, and carcinogens). The substrates for cytochromes P450 are universally hydrophobic while the physiological substrate for the nitric oxide synthases is the amino acid L-arginine, a hydrophilic compound.

Characterization of tissue ablation with a continuous wave holmium laser.

Background And Objective: The pulsed holmium laser is a promising tool for tissue ablation but possesses some limitations. For example, it is capable of producing significant mechanical damage in certain tissues in the form of fissures and fractures. Because longer pulse durations should reduce mechanical damage, this study examined the tissue effects produced by a prototype continuous wave holmium laser.

Inhibition of nitric oxide synthase activity by Zn2+ ion.

We have found neural nitric oxide synthase (nNOS) activity to be completely and reversibly inhibited by Zn2+ ion with an apparent Ki of 30 microM. Zn2+ blocks NADPH-dependent reduction of heme iron in nNOS and also blocks the calmodulin-dependent superoxide-mediated cytochrome c reductase activity exhibited by nNOS. However, Zn2+ ion has no apparent effect on the calmodulin-independent direct reduction of cytochrome c by nNOS.

Characterization by electron paramagnetic resonance of the interactions of L-arginine and L-thiocitrulline with the heme cofactor region of nitric oxide synthase.

Nitric oxide synthase (NOS) catalyzes sequential NADPH- and O2-dependent mono-oxygenase reactions converting L-arginine to N omega-hydroxy-L-arginine and N omega-hydroxy-L-arginine to citrulline and nitric oxide. The homodimeric enzyme contains one heme/monomer, and that cofactor is thought to mediate both partial reactions. Here we show by electron paramagnetic resonance spectroscopy that binding of substrate L-arginine to neuronal NOS perturbs the heme cofactor binding pocket without directly interacting as a sixth axial heme ligand; heme iron is exclusively high spin.

Long-range effects on the retinal chromophore of bacteriorhodopsin caused by surface carboxyl group modification.

Carboxyl groups of bacteriorhodopsin (bR) that are modified by 1-ethyl-3-[3-(trimethylamino)-propyl]carbodiimide (ETC) have been identified. Reaction of deionized purple membrane with a 400-fold molar excess of ETC or [14C]ETC for 1 h at 0 degree C incorporates about 3.5 mol of ETC/mol of bR.

Modular structure of neuronal nitric oxide synthase: localization of the arginine binding site and modulation by pterin.

A putative dihydrofolate reductase (DHFR) module has been identified in neuronal nitric oxide synthase, consisting of amino acids 558-721, and is proposed to be the site of tetrahydrobiopterin (BH4) binding. This polypeptide has been expressed in E. coli as a fusion protein with glutathione S-transferase (GST), using the plasmid pGEX-4T1.

S-alkyl-L-thiocitrullines. Potent stereoselective inhibitors of nitric oxide synthase with strong pressor activity in vivo.

Nitric oxide synthase catalyzes the oxidation of a guanidino nitrogen of L-arginine to nitric oxide with concomitant formation of citrulline. Enzyme activity is inhibited by a variety of N omega-monosubstituted L-arginine analogs including N omega-alkyl-, N omega-amino-, and N omega-nitro-L-arginine derivatives. We report here that both constitutive and inducible isoforms of nitric oxide synthase are strongly inhibited by S-alkyl-L-thiocitrullines (N delta-(S-alkyl)isothioureido-L-ornithines) with n-alkyl groups of one to three carbons.

Pharmacokinetics, dosimetry and toxicity of holmium-166-DOTMP for bone marrow ablation in multiple myeloma.

Unlabelled: In this Phase I clinical trial, six multiple myeloma patients who had not responded to conventional therapy and were scheduled for bone marrow transplantation received a bone-seeking radiopharmaceutical for bone marrow ablation. The pharmacokinetics, dosimetry, and toxicity of this radiopharmaceutical were studied.

Methods: Patients received from 519 mCi to 2.