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Biomedical subjects

G M Rosen

Publications and source records attributed to G M Rosen.

At least 19 recordsLinked to original sources
PubMed1992

Generation of superoxide by purified brain nitric oxide synthase.

Brain nitric oxide synthase (NOS), which utilizes NADPH and calcium/calmodulin as cofactors for metabolizing L-arginine to nitric oxide (NO) and L-citrulline, contains recognition sites for the flavins FAD and FMN. Using a spin-trapping technique combined with electron spin resonance spectroscopy, we report that brain NOS generates superoxide O2-. in a calcium/calmodulin-dependent manner. The "specific inhibitors" of NOS, NG-monomethyl L-arginine (L-NMMA), and NG-nitro-L-arginine methyl ester (L-NAME), have different effects on O2-. generation. For L-NMMA, O2-. production is unaffected, while for L-NAME, inhibition of this free radical is concentration-dependent.

Amino Acid Oxidoreductases↗
PubMed1992

Spin trapping evidence for myeloperoxidase-dependent hydroxyl radical formation by human neutrophils and monocytes.

Using the electron spin resonance/spin trapping system, 4-pyridyl 1-oxide N-tert-butylnitrone (4-POBN)/ethanol, hydroxyl radical was detected as the alpha-hydroxyethyl spin trapped adduct of 4-POBN, 4-POBN-CH(CH3)OH, from phorbol 12-myristate 13-acetate-stimulated human neutrophils and monocytes without the addition of supplemental iron. 4-POBN-CH(CH3)OH was stable in the presence of a neutrophil-derived superoxide flux. Hydroxyl radical formation was inhibited by treatment with superoxide dismutase, catalase, and azide. Treatment with a series of transition metal chelators did not appreciably alter 4-POBN-CH(CH3)OH, which suggested that hydroxyl radical generation was mediated by a mechanism independent of the transition metal-catalyzed Haber-Weiss reaction. Kinetic differences between transition metal-dependent and -independent mechanisms of hydroxyl radical generation by stimulated neutrophils were demonstrated by a greater rate of 4-POBN-CH(CH3)-OH accumulation in the presence of supplemental iron. Detection of hydroxyl radical from stimulated monocyte-derived macrophages, which lack myeloperoxidase, required the addition of supplemental iron. The addition of purified myeloperoxidase to an enzymatic superoxide generating system resulted in the detection of hydroxyl radical that was dependent upon the presence of chloride and was inhibited by superoxide dismutase, catalase, and azide. These findings implicated the reaction of hypochlorous acid and superoxide to produce hydroxyl radical. 4-POBN-CH(CH3)OH was not observed upon stimulation of myeloperoxidase-deficient neutrophils, whereas addition of myeloperoxidase to the reaction mixture resulted in the detection of hydroxyl radical. These results support the ability of human neutrophils and monocytes to generate hydroxyl radical through a myeloperoxidase-dependent mechanism.

Electron Spin Resonance Spectroscopy↗
PubMed1992

The role of a sleep disorder center in evaluating sleep violence.

To review the state-dependent nature of violence and present a clinically useful classification of sleep violence, this article reviews our experience with sleep-related violence, establishing a differential diagnosis, methods of evaluation, and treatment options. The study occurs in a full-service clinical sleep disorders center evaluating approximately 1000 patients annually with an active participation of 16 physicians representing seven specialties. The patients were self-, physician-, or court/social service-referred for evaluation of violent or injurious behaviors associated with the sleep period. Interventions were dependent on the final diagnosis following clinical and (usually) sleep laboratory evaluation. The main outcome measures were self-reported. During routine clinical evaluations at a multidisciplinary sleep disorder center, it has become apparent that violence is often state-dependent, occurring only during the sleep period, resulting from a number of both neurologic and psychiatric conditions (including malingering and Munchausen syndrome by proxy). In such cases, careful clinical and laboratory evaluation usually results in a specific diagnosis, with effective therapeutic recommendations. Violence may be state-dependent. It is clear that violent behaviors may arise from the sleep period, often without conscious awareness on the part of the subject. This has social, forensic, and clinical implications, and may help contribute to the understanding of violence in general.

Health Facilities↗
PubMed1992

Sleep-terror disorder in children: the role of self-hypnosis in management.

This paper describes four children, ages 8 to 12 years, with frequent, prolonged, or dangerous disorders of arousal. None had any significant psychological or behavioral problems. Each had a polysomnogram that showed sudden arousals out of slow-wave sleep associated with complex behavior. All responded to a short course of imipramine, 20 to 60 mg at bedtime, followed by and in conjunction with training in relaxation and mental imagery (self-hypnosis). Once the correct diagnosis was made, the treatment strategy was to (1) demystify the symptom complex through education, (2) establish prompt control of the symptoms with the use of imipramine, (3) train the children in self-regulation with self-hypnosis, and (4) discontinue the medication while maintaining control of the arousals. Over a 2-3 year follow-up all children remain asymptomatic. This is the first report of successful use of self-hypnosis for the treatment of polysomnogram-proven disorders of arousal in the pediatric population. Also reported are seven additional children who were treated equally successfully with hypnosis without the use of medication.

Adolescent↗
PubMed1992

Culture of bovine pulmonary artery endothelial cells on Gelfoam blocks.

Conventional methods of endothelial cell culture on monolayers and beads require enzymatic digestion, traumatic scraping, or centrifugation to transfer cells to other experimental systems. Gelfoam, a porous gelatin block, not only supports the growth of bovine pulmonary artery endothelial cells but also allows the rapid transfer of cell-laden blocks from one experimental system to another with minimal intervention. This property has been shown to be especially useful for the rapid fixation of endothelial cells for microscopy using standard histologic methods. Histology confirmed that the trabecular nature of the substrate allows endothelial cells to line the interstices of the sponge matrix and grow in a configuration that simulates the appearance of the endothelium in small vessels and capillaries. The inoculation of 1 x 10(5) endothelial cells on 7.5 mg Gelfoam (24 x 8 x 2 mm blocks) was enhanced by fibroblast growth factor and resulted in cell attachment by day 2 with a cell doubling time of 1.7 days. In addition, endothelial cells completely infiltrated 1, 5 and 7.5 mg Gelfoam blocks, as verified by histology. Assays to quantify cell number and protein were easily performed. To facilitate cell counting, the Gelfoam matrix was rapidly removed by the addition of 0.05 mg/ml collagenase, a concentration that interfered minimally with the assay for cellular protein concentration. The data demonstrate that Gelfoam is a suitable support growth matrix for the in vitro culture of bovine pulmonary artery endothelial cells.

Animals↗
PubMed1992

Light-dependent generation of superoxide from human erythrocytes.

Using the cytochrome c reduction method, we investigated light-dependent erythrocytic superoxide production. After 4 h light and dark exposure of erythrocytes from eight healthy human subjects, an average of 18.6% more superoxide was generated by erythrocytes exposed to light. Pretreatment of erythrocytes with the superoxide dismutase inhibitor N,N-diethyldithiocarbamate increased detection of superoxide while pretreatment with the anion channel blocker 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene decreased detection. These findings indicate that substantially more spontaneously generated superoxide is produced and escapes from normal erythrocytes at ambient oxygen tensions on exposure to light. This excess generation and escape of superoxide from erythrocytes exposed to light may result in tissue photosensitization, especially in the retina of the eye, where high oxygen tension, blood and chronic light exposure occur simultaneously.

Adult↗
PubMed1991

Light-dependent spin trapping of hydroxyl radical from human erythrocytes.

The generation of reactive oxygen species from human erythrocytes has previously been demonstrated. Furthermore, erythrocytic protoporphyrin IX has been shown to generate superoxide and singlet oxygen when exposed to light. These findings suggest that a component of erythrocytic reactive oxygen species production may be light-dependent. By inhibiting erythrocyte superoxide dismutase, catalase, and glutathione peroxidase with N,N-diethyldithiocarbamate or sodium cyanide, we demonstrate the light-dependent generation of hydroxyl radical in human erythrocytes using spin trapping/Electron Spin Resonance spectroscopy. This finding may be significant in tissues where blood is exposed to light, such as in the eye.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1991

N-hydroxylamine is not an intermediate in the conversion of L-arginine to an activator of soluble guanylate cyclase in neuroblastoma N1E-115 cells.

This study evaluates the role of N-hydroxylamine (NH2OH) in activating soluble guanylate cyclase in the mouse neuroblastoma clone N1E-115. It has been proposed that NH2OH is a putative intermediate in the biochemical pathway for the generation of nitric oxide (NO)/endothelium-derived relaxing factor (EDRF) from L-arginine. NH2OH caused a time- and concentration-dependent increase in cyclic GMP formation in intact cells. This response was not dependent on Ca2+. In cytosol preparations the activation of guanylate cyclase by L-arginine was dose-dependent and required Ca2+ and NADPH. In contrast, NH2OH itself did not activate cytosolic guanylate cyclase but it inhibited the basal activity of this enzyme in a concentration-dependent manner. The formation of cyclic GMP in the cytosolic fractions in response to NH2OH required the addition of catalase and H2O2. On the other hand, catalase and/or H2O2 lead to a decrease in L-arginine-induced cyclic GMP formation. Furthermore, NH2OH inhibited L-arginine- and sodium nitroprusside-induced cyclic GMP formation in the cytosol. The inhibition of L-arginine-induced cyclic GMP formation in the cytosol by NH2OH was not reversed by the addition of superoxide dismutase. These data strongly suggest that NH2OH is not a putative intermediate in the metabolism of L-arginine to an activator of guanylate cyclase.

Animals↗
PubMed1991

Phagocyte-derived free radicals stimulated by ingestion of iron-rich Staphylococcus aureus: a spin-trapping study.

Phagocytic cells generate superoxide (O2-) and hydrogen peroxide (H2O2), creating the substrates for hydroxyl radical (HO.) in the presence of redox active metals. Previously it was shown that HO. is not a physiologic product of human neutrophils or monocytes but can be generated in the presence of high concentrations of iron. This study was undertaken to determine whether bacterial iron could be used for the generation of HO. The growth of Staphylococcus aureus under iron-rich conditions increased bacterial iron concentration and phagocytosis of iron-rich bacteria allowed neutrophils to accumulate threefold more iron than ingestion of iron-starved organisms. Neither neutrophils nor monocytes ingesting iron-rich S. aureus generated iron-catalyzed HO. at levels detectable by spin-trapping techniques. No differences in the killing of iron-rich organisms by neutrophils was noted. The results suggest that HO. does not play a role in the killing of S. aureus by human neutrophils, regardless of their ability to deliver iron to the cell.

Electron Spin Resonance Spectroscopy↗
PubMed1991

Application of spin trapping to human phagocytic cells: insight into conditions for formation and limitation of hydroxyl radical.

In recent years spin trapping techniques have been used extensively to better understand the free radical biology of phagocytic cells. These results demonstrate that spin trapping is of adequate sensitivity to detect superoxide and/or hydroxyl radical generated by these cells, and that spin trapping is capable of measuring phagosomal radicals as well. However, neither neutrophils, monocytes, nor monocyte derived macrophages generate hydroxyl radical in the absence of exogenous iron. Furthermore, neutrophil lactoferrin and myeloperoxidase limit the magnitude (and in the case of lactoferrin the duration) of hydroxyl radical formed by neutrophils in an iron catalyzed system. Since monocytic phagocytes possess no lactoferrin, and limited myeloperoxidase, hydroxyl radical may play an important role in the inflammatory behavior of mononuclear phagocytes.

Cyclic N-Oxides↗
PubMed1991

Use of a low-frequency ESR spectrometer: implications for spin-trapping free radicals, in situ.

We have adapted the low-frequency ESR spectrometer, designed and built by H.J. Halpern, to the physiologic needs of organ preparations operating at 250 MHz. Initial studies have allowed us to detect nitroxides in an isolated perfused heart. These in situ measurements were made with nitroxides specifically designed to mimic the lipophilic nature of 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxyl (DMPO-OH). These spin labels provided information about the influence of dynamic factors of the heart, such as flow rate, different cell populations and unequal distribution between compartments on our ability to conduct and interpret spin trapping experiments. They also clarified the sacrifice in sensitivity involved in operating at the lower frequencies. To deal with this later problem, we have increased the sensitivity of the spin trapping method by synthesizing a family of 15N- and deuterium-containing DMPO analogs and by determining their ability to spin trap free radicals generated by the model superoxide system of xanthine/xanthine oxidase. Finally, since activated neutrophils are one of the few cells known to generate free radicals as part of their physiologic function, we used these phagocytic cells, as a source of superoxide.

Animals↗
PubMed1990

Spin-trapping of superoxide by 5,5-dimethyl-1-pyrroline N-oxide: application to isolated perfused organs.

Of the available techniques used to identify free radicals, spin-trapping offers the unique opportunity to simultaneously measure and distinguish among a variety of important biologically generated free radicals. For superoxide and hydroxyl radical, the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) is most frequently used. However, this nitrone has several drawbacks. For example, its reaction with superoxide is slow, having a second-order rate constant around 10 M-1 s-1. Because of this, high concentrations of DMPO are essential in order to observe the corresponding spin-trapped adduct, 5,5-dimethyl-2-hydroperoxy-1-pyrrolidinyloxy. This may, in some cases, lead to cellular toxicity. In an attempt to circumvent this serious limitation, it has been proposed that an indirect approach be employed to detect and identify free radicals generated as a consequence of ischemia/reperfusion injury. In the direct (most frequently used) approach, the spin trap is first added to an isolated perfused organ under the appropriate experimental conditions. Then, the infusion buffer containing the spin-trap adduct(s) is placed into an quartz flat cell to be inserted into an ESR spectrometer. In the indirect method, the spin trap is added to the perfusate, which had previously exited the organ. Therefore, with this method one can prevent any spin-trap-mediated toxicities to the isolated perfused organ. However, because of the very rapid rate of free radical reactions catalyzed by either superoxide or hydroxyl radical, it is questionable whether ESR spectra recorded using this indirect method result from the actual spin-trapping of free radicals. In this report, we evaluated the indirect spin-trapping technique in light of the kinetic considerations discussed above.

Cyclic N-Oxides↗
PubMed1990

Hydroxyl radical is not a product of the reaction of xanthine oxidase and xanthine. The confounding problem of adventitious iron bound to xanthine oxidase.

The reaction of xanthine and xanthine oxidase generates superoxide and hydrogen peroxide. In contrast to earlier works, recent spin trapping data (Kuppusamy, P., and Zweier, J.L. (1989) J. Biol. Chem. 264, 9880-9884) suggested that hydroxyl radical may also be a product of this reaction. Determining if hydroxyl radical results directly from the xanthine/xanthine oxidase reaction is important for 1) interpreting experimental data in which this reaction is used as a model of oxidant stress, and 2) understanding the pathogenesis of ischemia/reperfusion injury. Consequently, we evaluated the conditions required for hydroxyl radical generation during the oxidation of xanthine by xanthine oxidase. Following the addition of some, but not all, commercial preparations of xanthine oxidase to a mixture of xanthine, deferoxamine, and either 5,5-dimethyl-1-pyrroline-N-oxide or a combination of alpha-phenyl-N-tert-butyl-nitrone and dimethyl sulfoxide, hydroxyl radical-derived spin adducts were detected. With other preparations, no evidence of hydroxyl radical formation was noted. Xanthine oxidase preparations that generated hydroxyl radical had greater iron associated with them, suggesting that adventitious iron was a possible contributing factor. Consistent with this hypothesis, addition of H2O2, in the absence of xanthine, to "high iron" xanthine oxidase preparations generated hydroxyl radical. Substitution of a different iron chelator, diethylenetriaminepentaacetic acid for deferoxamine, or preincubation of high iron xanthine oxidase preparations with chelating resin, or overnight dialysis of the enzyme against deferoxamine decreased or eliminated hydroxyl radical generation without altering the rate of superoxide production. Therefore, hydroxyl radical does not appear to be a product of the oxidation of xanthine by xanthine oxidase. However, commercial xanthine oxidase preparations may contain adventitious iron bound to the enzyme, which can catalyze hydroxyl radical formation from hydrogen peroxide.

Animals↗
PubMed1990

Receptor-mediated generation of an EDRF-like intermediate in a neuronal cell line detected by spin trapping techniques.

We have studied receptor-mediated generation of an activator of soluble guanylate cyclase in cultured mouse neuroblastoma cells (clone N1E-115) by ESR/spin trapping spectroscopy. A spin adduct was detected during the activation of muscarinic receptors by carbamylcholine in the presence of the spin trap 3,5-dibromo 4-nitrosobenzene sulphonate (DBNBS). The spin adduct does not correspond to that originating from the free radical nitric oxide or hydroxylamine. The same adduct was generated in cytosol preparations from N1E-115 cells incubated with L-arginine, NADPH, in the presence of calcium. The use of isotopically labelled guanidino-N15-L-arginine supported the generation of a DBNBS spin trapped adduct originating from the guanidino moiety of L-arginine. Superoxide dismutase (SOD) stabilized the precursor of the spin adduct as well as the activator of soluble guanylate cyclase derived from L-arginine. Our results provide direct evidence for the receptor-mediated formation of a diffusible precursor of NO. derived from L-arginine.

Animals↗
PubMed1990

Application of spin traps to biological systems.

Since 1971, when nitroxides were first reported to be bioreduced, several cellular enzymes, in addition to ascorbic acid, have been found to catalyze the reduction of nitroxides to their corresponding hydroxylamines. Numerous studies have demonstrated that cellular bioreduction of nitroxides are both dependent upon the structure of the nitroxide and cell type. For example, pyrrolidinyloxyls are considerably more resistant to bioreduction than their corresponding piperidinyloxyls. In addition, cellular levels of reductases present in freshly isolated rat hepatocytes are considerably greater than concentrations found in freshly isolated rat enterocytes. Thus, through the proper selection of a cell type and an appropriate nitroxide, one can study cellular-mediated free radical processes. With the discovery that alpha-hydrogen-containing nitroxides, including 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxyl (DMPO-OH) decompose rapidly in the presence of superoxide and thiols, the ability to determine if hydroxyl radical is generated during stimulation of human neutrophils, is in doubt. To explore the limits of spin trapping in this context, we have studied the effect of varying the rates of superoxide production, in the presence and absence of thiols, on the decomposition of DMPO-OH. In parallel studies, we have found that t-butyl alpha-methyl-4-pyridinyl-N-oxide nitroxide (4-POBN-CH3) will not degrade in the presence of superoxide and a thiol. From these studies, we have determined that if hydroxyl radicals were generated as an isolated event in the presence of a continual flow of superoxide, spin trapping might not be able to detect its formation. Otherwise, spin trapping should be able to measure hydroxyl radicals, if continually generated, during activation of human neutrophils.

Cells↗
PubMed1990

Hematogenous photosensitization. A mechanism for the development of age-related macular degeneration.

Age-related macular degeneration (ARMD) is one of the leading causes of severe visual loss in the United States. Numerous risk factors have been investigated, but the pathogenesis of ARMD has remained elusive. The authors propose that ARMD develops as a direct result of photosensitization of the vascular endothelium of the choriocapillaris, Bruch's membrane, and the retinal pigment epithelium (RPE) by superoxide anion and singlet oxygen generated by photoactive compounds in blood. Using electron-spin resonance spectrometry, the free-radical trap, 5,5-dimethyl-1-pyrroline-N-oxide, and the singlet-oxygen trap, 2-(9,10-dimethoxyanthracentyl)-t-butylhydroxylamine, the authors demonstrate that the photoactive compound, protoporphyrin IX (PP IX), a naturally occurring precursor molecule of hemoglobin found in erythrocytes and plasma, generates superoxide anion and singlet oxygen. The amount of reactive-oxygen species produced by this system is dependent on the concentration of PP IX and the intensity and wavelength of the light delivered. Furthermore, the production of these photooxidants is significantly reduced by filtering the excitatory wavelengths of PP IX. These photogenerated oxidants could damage the vascular endothelium of the choriocapillaris, Bruch's membrane, and the RPE, necessitating a reparative process. This could result in features characteristically seen in ARMD such as a thickened Bruch's membrane, RPE atrophy, and hyperplasia. Prevention of phototoxic damage by this mechanism could involve enhancing protective enzymes, increasing scavenger substances, or supplying appropriate filters to eliminate the exciting wavelengths of light.

Aging↗