Comparison of nasal valve dysfunction treatment outcomes for temperature-controlled radiofrequency and functional rhinoplasty surgery: a systematic review and meta-analyses.

BACKGROUND: Nasal valve dysfunction (NVD) is a substantial contributor to nasal airway obstruction. Minimally-invasive temp-erature-controlled radiofrequency (TCRF) treatment of the nasal valve is available and comparison with surgical techniques is warranted. METHODOLOGY: Databases: Medline (PubMed), Embase, Cochrane Library. POPULATION: adults with preprocedural nasal obstruction symptom evaluation (NOSE) score >=45. Treatment effects were derived from a random effects model and reported as weighted mean difference in NOSE score between baseline; 3, 6, and 12 months postprocedure. RESULTS: Of 2529 initial articles, 5 studies describing TCRF treatment and 63 studies describing functional rhinoplasty were included. Pooled effect sizes for TCRF treatment and functional rhinoplasty were comparable in all analyses. CONCLUSIONS: TCRF treatment of the internal nasal valve for NVD was associated with sustained effects comparable to functional rhinoplasty addressing the nasal valve only, rhinoplasty without concomitant turbinate treatment, and all rhinoplasty.

IDH2-deficient mice develop spinal deformities with aging.

Spinal deformities such as scoliosis and kyphosis are incurable, and can lead to decreased physical function, pain, and reduced quality of life. Despite much effort, no clear therapies for the treatment of these conditions have been found. Therefore, the development of an animal model for spinal deformity would be extremely valuable to our understanding of vertebral diseases. In this study, we demonstrate that mice deficient in the mitochondrial enzyme isocitrate dehydrogenase 2 (IDH2) develop spinal deformities with aging. We use morphological analysis as well as radiographic and micro-CT imaging of IDH2-deficient mice to characterize these deformities. Histological analysis showed increased abnormalities in IDH2-deficient mice compared to wild type mice. Taken together, the results suggest that IDH2 plays a critical role in maintaining the spinal structure by affecting the homeostatic balance between osteoclasts and osteoblasts. This indicates that IDH2 might be a potent target for the development of therapies for spinal deformities. Our findings also provide a novel animal model for vertebral disease research.

3',4',7,8-Tetrahydroxyflavone inhibits RANKL-induced osteoclast formation and bone resorption.

Osteoclasts, which are specialized bone multinuclear cells, are responsible for bone lytic diseases such as osteoporosis. 3',4',7,8-tetrahydroxyflavone is a flavonoid from Acacia confusa. In the present study, we found that 3',4',7,8-tetrahydroxyflavone markedly inhibited receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastic differentiation from mouse bone marrow-derived macrophages (BMMs). 3',4',7,8-tetrahydroxyflavone also reduced the mRNA expression levels of osteoclastic marker genes including the calcitonin receptor (CTR) and cathepsin K. In addition, 3',4',7,8-tetrahydroxyflavone decreased the bone resorption activity of osteoclasts on dentin slices. We found that 3',4',7,8-tetrahydroxyflavone inhibited RANKL-induced expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), a key transcription factor of osteoclast differentiation. Furthermore, ectopic overexpression of a constitutively active form of NFATc1 completely rescued the anti-osteoclastogenic effect of 3',4',7,8-tetrahydroxyflavone, suggesting that the anti-osteoclastogenic effect was mainly attributed to the reduction in NFATc1 expression. Taken together, our data suggest that 3',4',7,8-tetrahydroxyflavone inhibits osteoclast differentiation and bone loss and may therefore be considered a promising drug candidate for treating or preventing bone-lytic diseases.

An integrated microfluidic device for influenza and other genetic analyses.

An integrated microfluidic device capable of performing a variety of genetic assays has been developed as a step towards building systems for widespread dissemination. The device integrates fluidic and thermal components such as heaters, temperature sensors, and addressable valves to control two nanoliter reactors in series followed by an electrophoretic separation. This combination of components is suitable for a variety of genetic analyses. As an example, we have successfully identified sequence-specific hemagglutinin A subtype for the A/LA/1/87 strain of influenza virus. The device uses a compact design and mass production technologies, making it an attractive platform for a variety of widely disseminated applications.

Disruption of the structure of the Golgi apparatus and the function of the secretory pathway by mutants G93A and G85R of Cu, Zn superoxide dismutase (SOD1) of familial amyotrophic lateral sclerosis.

The Golgi apparatus of motor neurons (GA) is fragmented in sporadic amyotrophic lateral sclerosis (ALS), in familial ALS with SOD1 mutations, and in mice that express SOD1G93A of familial ALS, in which it was detected months before paralysis. In paralyzed transgenic mice expressing SOD1G93A or SOD1G85R, mutant proteins aggregated not only in the cytoplasm of motor neurons, but also in astrocytes and oligodendrocytes. Furthermore, aggregation of the G85R protein damaged astrocytes and was associated with rapidly progressing disease. In order to gain insight into the functional state of the fragmented GA, we examined the effects of S0D1 mutants G93A and G85R in Chinese Hamster Ovary Cells (CHO). In contrast to cells expressing the wt and G93A, the G85R expressers had no SOD1 activity. However, cells expressing both mutants, and to a lesser degree the wt, showed decreased survival, fragmentation of the GA, and dysfunction of the secretory pathway, which was assessed by measuring the amount of cell surface co-expressed CD4, a glycoprotein processed through the GA. The G93A and wt proteins were partially recovered in detergent insoluble fractions; while the recovery of G85R was minimal. Both mutants showed equal reductions of cell survival and function of the secretory pathway, in comparison to the wt and cells expressing mutant alsin, a protein found in rare cases of fALS. These results are consistent with the conclusion that the two SOD1 mutants, by an unknown mechanism, promote the dispersion of the GA and the dysfunction of the secretory pathway. This and other in vitro models of mutant SOD1 toxicity may prove useful in the elucidation of pathogenetic mechanisms.

Dietary taurine intake and serum taurine levels of women on Jeju Island.

The purpose of this study was to investigate the dietary taurine intake and serum taurine levels of women on Jeju Island in Korea. Sixty six married women aged 43.5 +/- 7.1 volunteered for this study: 34 from the city area and 32 from two fishing-farming areas. Diet samples were collected from the participants; the samples included three meals (breakfast, lunch and supper), including snacks, drinks and whatever else the participants had eaten for 24 hours. Taurine levels in the diet and serum were determined as the dabsyl derivative by HPLC with a Rf-detector. The intake of taurine ranged from 8.4 to 767.6 mg/day and its mean value was 163.9 +/- 150.2 mg/day (mean +/- SD). There was a significant difference between the two groups: 114.9 +/- 78.7 for the women from the city area and 215.9 +/- 187.9 mg/day for the women from the fishing-farming areas (p<0.001). The taurine intake of the total diet, including all snacks and drinks, was 2300 +/- 584 g/day for the city area and 2342 +/- 528 g/day for the fishing-farming areas. The daily protein intake was 58.8 +/- 16.4 g for the women of the city area and 65.5 +/- 17.1 g for the women of the fishing-farming areas. There was a significant correlation between the intake of fish/shellfish and taurine (p=0.001) while there was no correlation between the intake of protein and taurine (p=0.057). The taurine levels in serum ranged from 68.6 to 261.6 micromol/L and the mean value was 169.7 +/- 41.5 micromol/L. There was no significant difference between the women from the city area and the women from the fishing-farming areas in serum taurine levels. The correlations of serum taurine levels with serum retinol levels (p=0.016) and alpha-tocopherol (p=0.014) levels were significant. These results suggest that taurine intake is dependent on the fish/shellfish intake and that taurine may play an important role in the retention of antioxidative nutrients.

Correlation of plasma homocysteine and mitochondrial DNA content in peripheral blood in healthy women.

Hyperhomocysteinemia is an independent risk factor of cardiovascular disease and associated with insulin resistance, although their causal relationship remains unclear. A previous report has shown that high concentration of homocysteine damages mitochondrial gene expression, function and structure. As we found recently, the mitochondrial DNA (mtDNA) contents are inversely correlated with insulin resistance parameters. Thus there is possibility that plasma total homocysteine (tHcy) level is somewhat correlated with mtDNA content. Sixty healthy women (mean age 40.3+/-20.9 yr, range 18-78 yr) were recruited to investigate the correlation of plasma tHcy level and mtDNA content in peripheral blood. A significant negative correlation was found between plasma tHcy levels and mtDNA content (r=-0.507, P<0.01). Plasma tHcy and mtDNA content have an independent effect on each other and on insulin resistance (HOMA-insulin resistance (IR) score) respectively in multiple regression model. Plasma tHcy showed positive correlations with age (r=0.407), W/H ratio (r=0.370), total cholesterol (r=0.338), LDL-cholesterol (r=0.317) and insulin resistance (HOMA-IR score) (r=0.261); and a negative correlation with folate (r=-0.273). MtDNA content showed negative correlations with age (r=-0.407), BMI (r=-0.440), W/H ratio (r=-0.659), SBP (r=-0.350), total cholesterol (r=-0.340), triglyceride (r=-0.376), LDL-cholesterol (r=-0.349), fasting plasma insulin (r=-0.483), and insulin resistance (HOMA-IR score) (r=-0.423); and a positive correlation with folate (r=0.299). In this study, there was a significant inverse correlation between plasma tHcy level and mtDNA content. Further study will be warranted to elucidate the mechanism by which two factors are associated.

Mutated plant lectin library useful to identify different cells.

The 24 nucleotides comprising the carbohydrate-recognition domain of Maackia amurensis hemagglutinin (MAH) cDNA were randomly mutated. The mutant lectins were expressed as glutathione-S-transferase fusion proteins in Escherichia coli and 16 clones were randomly chosen. Although all of 16 recombinant lectins reacted strongly with anti-MAH polyclonal antibody, the carbohydrate-recognition domain of each was unique. As shown by agglutination studies, each mutant MAH lectin was able to bind to erythrocytes from one or more of five animal species in very distinct patterns. Thus, novel plant lectin libraries can be used to discriminate in a highly specific manner among a variety of cell types. This technology may prove to be very useful in a number of different applications requiring a high level of specificity in cell identification.

The formation of plasmid DNA loaded pharmaceutical powders using supercritical fluid technology.

The invention of novel drugs based on biological macromolecules requires the development of specialized formulation methods. Supercritical fluid technology offers the possibility to produce dry powder formulations suitable for inhalation or needle-free injection. In this article we describe the first application of a process involving supercritical carbon dioxide for the production of plasmid DNA-loaded particles. The technique of solution enhanced dispersion by supercritical fluids (SEDS) is used to coformulate the 6.9 kb plasmid pSV beta with mannitol as excipient. After initial experiments showed a high degradation of the plasmid during powder formation, a systematic investigation of the process revealed pH effects to be crucial for the recovery of intact DNA. The application of high-buffer concentration led to an increase of the recovered supercoiled proportion from 7% to 80%.

Protein glycation: creation of catalytic sites for free radical generation.

In a glycation reaction, alpha-dicarbonyl compounds such as deoxyglucosone, methylglyoxal, and glyoxal are more reactive than the parent sugars with respect to their ability to react with amino groups of proteins to form inter- and intramolecular cross-links of proteins, stable end products called advanced Maillard products or advanced end products (AGEs). The AGEs, which are irreversibly formed, accumulate with aging, atherosclerosis, and diabetes mellitus, and are especially associated with long-lived proteins such as collagens, lens crystallins, and nerve proteins. It was suggested that the formation of AGEs not only modifies protein properites but also induces biological damage in vivo. In this report, we summerize results obtained from our studies for (1) identifying the structure of the cross-linked radical species formed in the model system-the reaction between alpha-dicarbonyl methylglyoxal with amino acids, and (2) the reactivity of the radical center of the protein created by the similar reaction. These results indicate that glycation of protein generates active centers for catalyzing one-electron oxidation-reduction reactions. This active center, which exhibits enzyme-like character, is suggested to be the cross-linked Schiff-based radical cation of the protein. It mimics the characteristics of the metal-catalyzed oxidation system. These results together indicate that glycated proteins accumulated in vivo provide stable active sites for catalyzing the formation of free redicals.

Effect of exercise on the mitochondrial DNA content of peripheral blood in healthy women.

Exercise decreases insulin resistance and increases maximal exercise capacity as estimated from maximal oxygen uptake (VO2max). Recent reports have demonstrated that the mitochondrial DNA (mtDNA) content of blood is correlated with VO2max in healthy subjects (mean age 31 years) and is inversely correlated with insulin resistance parameters. The aim of this study was to determine the effect of regular exercise on the mtDNA content in the peripheral blood of 16 healthy young women of mean age 24.8 (SD 6.2) years and 14 healthy older women of mean age 66.7 (SD 5.8) years. The exercise programme lasted for 10 weeks and consisted of three sessions a week, each of 1 h and aiming to attain 60%-80% of VO2max. The mtDNA content of peripheral blood was measured by competitive polymerase chain reaction. The VO2max had significantly increased following the exercise programme [from 33.1 (SD 3.4) to 35.2 (SD 3.4) ml x kg(-1) min(-1) in the young and from 24.3 (SD 5.3) to 30.3 (SD 7.3) ml x kg(-1) x min(-1) in the older women, both P < 0.05]. Exercise decreased systolic blood pressure, and concentrations of triglyceride, low density lipoprotein-cholesterol (LDL-C), glucose and insulin in the blood of the young and of total cholesterol, LDL-C and glucose in that of the older women. High density lipoprotein-cholesterol (HDL-C) in the young women was increased by exercise. The mtDNA content significantly increased following the exercise programme in both groups [from 27.1 (SD 17.9) to 52.7 (SD 44.6) amol x 5 ng(-1) genomic DNA in the young and from 15.3 (SD 10.2) to 32.1 (SD 30.0) amol x 5 ng(-1) genomic DNA in the older women, both P < 0.05]. There was a significant positive correlation between the change in mtDNA content and the change in VO2max (r = 0.74 in the young and r = 0.71 in the older women, both P < 0.01). In conclusion, 10 weeks of moderate intensity, regular exercise increased the mtDNA content in peripheral blood and decreased insulin resistance parameters. This data suggests that increase in the mtDNA content may be associated with increased insulin sensitivity.

Quantitation of supercoiled circular content in plasmid DNA solutions using a fluorescence-based method.

A method for quantifying the proportion of supercoiled circular (SC) forms in DNA solutions is described. The method (SCFluo) takes advantage of the reversible denaturation property of SC forms and the high specificity of the PicoGreen fluorochrome for double-stranded (ds)DNA. Fluorescence values of forms capable of reversible denaturation after a 5 min heating, 2 min cooling step are normalised to fluorescence values of total dsDNA present in the preparation. For samples with a SC content >20-30%, good regression fits were obtained when values derived from densitometric scanning of an agarose gel and those derived from the SCFluo method were compared. The method represents an attractive alternative to currently established methods because it is simple, rapid and quantitative. During large-scale processing and long-term storage, enzymatic, chemical and shear degradation may substantially decrease the SC content of plasmid DNA preparations. Regulations for pharmaceutical grade products for use in gene therapy and DNA vaccination may require >90% of the plasmid to be in the SC form. In the present study the SC content of 6.9, 13 and 20 kb plasmid preparations that had been subjected to chemical and shear degradation was successfully quantified using the new method.

Enzyme-like activity of glycated cross-linked proteins in free radical generation.

The structure and property of cross-linked amino acids and proteins produced by a three- carbon alpha-dicarbonyl methylglyoxal in glycation reaction were investigated. Our results showed that these reactions generated yellow fluorescent products and several free radical species. From the reaction with alanine, three types of free radicals were identified by EPR spectroscopy: 1) the cross-linked radical cation, methylglyoxal diaklylimine cation radical; 2) the methylglyoxal radical anion as the counterion; 3) the superoxide radical anion produced only in the presence of oxygen. Glycation of bovine serum albumin by methylglyoxal also generated the protein-bound, cross-linked free radical, probably the cation radical of the cross-linked Schiff base as observed with alanine. The glycated protein reduced ferricytochrome c to ferrocytochrome c in the absence of oxygen or added metal ions. This reduction of cytochrome c was accompanied by a large increase in the amplitude of the electron paramagnetic resonance signal originated from the protein-bound free radical. In addition, the glycated protein catalyzed the oxidation of ascorbate in the presence of oxygen while the protein-free radical signal disappeared. These results indicate that glycation of protein generates active centers for catalyzing one-electron oxidation-reduction reactions. This active center, which exhibits enzyme-like character, was suggested to be the cross-linked Schiff base/the cross-linked Schiff base radical cation of the protein. It mimics the characteristics of metal-catalyzed oxidation system. These results together indicate that glycated proteins accumulated in vivo provide stable active-sites for catalyzing the formation of free radicals.

A co-operative study: clinical characteristics of 334 Korean patients with moyamoya disease treated at neurosurgical institutes (1976-1994). The Korean Society for Cerebrovascular Disease.

A co-operative study was conducted to determine the clinical characteristics of patients with moyamoya disease who were diagnosed and treated at neurosurgical institutes in Korea before 1995. Twenty-six hospitals contributed 505 cases and among them, the clinical characteristics of 334 patients with definite moyamoya disease were evaluated. The number of patients began to increase from the late 1980s, and after that approximately 20 patients were treated each year. There were two age peaks: from six to 15 and from 31 to 40 years of age. Haemorrhagic manifestations occurred in approximately 43% of the patients. The major clinical manifestations were haemorrhage in adults (62.4%) and ischaemia in children (61.2%). Overall 54.5% of the patients experienced decreased consciousness levels, mainly due to intracranial haemorrhage or cerebral infarction. In the patients with ischemic manifestations, the adult patients were more likely to have cerebral infarction than the pediatric patients (80% vs. 39%) and the pediatric patients were more likely to have TIA (61% vs. 25%). Thirty eight percent of the patients underwent bypass surgery and 53% of these procedures were performed bilaterally. Treatment policies, including indications for bypass surgery and commonly used drugs, were somewhat different according to the institution. Overall favorable outcome was 73%, and the most significant factor affecting poor outcome was haemorrhagic manifestation. This article describes the characteristics of 334 patients with moyamoya disease, who were diagnosed and treated at neurosurgical institutes in Korea before 1995.

Transcriptional activation of the human manganese superoxide dismutase gene mediated by tetradecanoylphorbol acetate.

Transcriptional activation of human manganese superoxide dismutase (MnSOD) mRNA induced by a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined to identify the responsive transcriptional regulator. The effect of various deletions and mutations within the 5'-flanking region of the human MnSOD gene promoter was evaluated using the luciferase reporter system in A549 human lung carcinoma cells. Deletion of a region between -1292 and -1202 nucleotides upstream of the transcription start site abolished TPA-responsive induction, whereas deletion of the putative binding sequence for NF-kappaB or AP-1 did not. The region between -1292 and -1202 contains a cAMP-responsive element-like sequence, TGACGTCT, which we identified as the manganese superoxide dismutase TPA-responsive element, MSTRE. Site-specific mutation of the MSTRE abolished the TPA-responsive induction, validating the critical role of this sequence. We detected specific MSTRE activity from nuclear extracts and demonstrated by antibody supershift assay that this activity is closely related to CREB-1/ATF-1. TPA treatment rapidly induced phosphorylation of the CREB-1/ATF-1-like factor via the protein kinase C pathway. These results led us to conclude that the human MnSOD gene having the promoter construct used in this study is induced by TPA via activation of a CREB-1/ATF-1-like factor and not via either NF-kappaB or AP-1. In addition, we found that this induction was blocked by inhibitors of flavoproteins and NADPH oxidases, indicating involvement of enhanced generation of superoxide radical anion as an upstream signal.

Roles of superoxide radical anion in signal transduction mediated by reversible regulation of protein-tyrosine phosphatase 1B.

Growth factors induce intracellular production of reactive oxygen species in non-phagocytic cells and elevation of their phosphorylated protein tyrosine level. The latter can be achieved by activating protein-tyrosine kinases and/or inactivating protein-tyrosine phosphatases (PTPs). A highly abundant PTP, PTP-1B, is known to be inactivated by oxidation of its catalytic site Cys-215. We show that O-(2) is kinetically more efficient and chemically more specific oxidant than H(2)O(2) for inactivating PTP-1B. The second-order rate constant for the O-(2)- and H(2)O(2)-mediated inactivation is 334 +/- 45 M(-1) s(-1) and 42.8 +/- 3.8 M(-1) s(-1), respectively. PTP-1B oxidized by H(2)O(2) exhibits significantly more oxidized methionine residues and shows a lower degree of reversibility. The initial oxidative product, the Cys-215 sulfenic derivative, can easily be oxidized further to its irreversible sulfinic and sulfonic derivatives. This step is prevented by glutathionylation of the sulfenic derivative to form a S-glutathionylated PTP-1B, which can be reactivated by dithiothreitol or thioltransferase. Thus, a signal transduction mechanism mediated by the O-(2) and the participation of glutathione is proposed for the regulation of PTP-1B. This mechanism is supported by the in vivo demonstration that glutathionylated PTP-1B at Cys-215 is formed in A431 cells when they were treated with epidermal growth factor.

Regulation of PTP1B via glutathionylation of the active site cysteine 215.

The reversible regulation of protein tyrosine phosphatase is an important mechanism in processing signal transduction and regulating cell cycle. Recent reports have shown that the active site cysteine residue, Cys215, can be reversibly oxidized to a cysteine sulfenic derivative (Denu and Tanner, 1998; Lee et al., 1998). We propose an additional modification that has implications for the in vivo regulation of protein tyrosine phosphatase 1B (PTP1B, EC 3.1.3.48): the glutathionylation of Cys215 to a mixed protein disulfide. Treatment of PTP1B with diamide and reduced glutathione or with only glutathione disulfide (GSSG) results in a modification detected by mass spectrometry in which the cysteine residues are oxidized to mixed disulfides with glutathione. The activity is recovered by the addition of dithiothreitol, presumably by reducing the cysteine disulfides. In addition, inactivated PTP1B is reactivated enzymatically by the glutathione-specific dethiolase enzyme thioltransferase (glutaredoxin), indicating that the inactivated form of the phosphatase is a glutathionyl mixed disulfide. The cysteine sulfenic derivative can easily oxidize to its irreversible sulfinic and sulfonic forms and hinder the regulatory efficiency if it is not converted to a more stable and reversible end product such as a glutathionyl derivative. Glutathionylation of the cysteine sulfenic derivative will prevent the enzyme from further oxidation to its irreversible forms, and constitutes an efficient regulatory mechanism.

Enhanced free radical generation of FALS-associated Cu,Zn-SOD mutants.

Familial amyotrophic lateral sclerosis (FALS) is an inherited disorder of motor neurons, which is associated with missense mutations in the Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene. Mice from the G93A transgenic line were reported to develop a syndrome of FALS. The fact that the symptoms occurred against a background of normal mouse Cu,Zn-SOD activity suggests that dominant, gain-of-function mutations in SOD play a role in the pathogenesis of FALS. We investigated the nature of this gain-of-function of FALS mutants. We have previously reported that Cu,Zn-SOD has the free radical-generating function in addition to normal dismutation activity. These two enzymic activities were compared by using mutants (G93A and A4V) and the wild-type Cu,Zn-SOD prepared by recombinant method. Our results showed that the wild-type, G93A, and A4V enzymes have identical dismutation activity. However, the free radical-generating function of the G93A and A4V mutants, as measured by the spin trapping and EPR method, is enhanced relative to that of the wild-type enzyme (wild type < G93A < A4V), particularly at lower H(2)O(2) concentrations. This is due to the decrease in the K(m) value for H(2)O(2), wild-type > G93A > A4V. The catalytic activity to generate free radicals is correlated to the clinical severity of the disorder induced by these mutant enzymes. Furthermore, we found that intact FALS mutants failed to enhance tyrosine nitration. Together our results indicate that the amyotrophic lateral sclerosis symptoms are not caused by the reduction of Cu,Zn-SOD dismutation activity with the mutant enzymes; rather, it is induced in part by enhancement of the free radical-generating function.

Reactivity of the flavin semiquinone of nitric oxide synthase in the oxygenation of arginine to NG-hydroxyarginine, the first step of nitric oxide synthesis.

Nitric oxide synthase (NOS) is a heme protein that catalyzes the oxygenation of L-arginine in the presence of NADPH to form nitric oxide, L-citrulline and NADP+, and proceeds via two partial reactions: 1) L-Arginine --> NG-hydroxy-L-arginine 2) NG-Hydroxy-L-arginine --> L-citrulline + nitric oxide Calmodulin, FAD, FMN and tetrahydrobiopterin are required for both reactions. Reactions 1 and 2 require the input of 2 and 1 electron equivalents, respectively. Under normal multiple turnover conditions, these electrons are ultimately derived from NADPH. We previously reported that NOS contains an endogenous reductant that, in the absence of NADPH, can support the single-turnover oxygenation of L-arginine to NG-hydroxy-L-arginine and a relatively small amount of L-citrulline [Campos, K. L., Giovanelli, J., and Kaufman, S. (1995) J. Biol. Chem. 270, 1721-1728]. This reductant has now been identified as the stable flavin semiquinone free radical (FSQ). Its oxidation appears to be coupled to the formation of NG-hydroxy-L-arginine and L-citrulline. The rate of FSQ oxidation is two orders of magnitude slower than the flux of electrons from NADPH through NOS during normal turnover of the enzyme, indicating that FSQ is not the proximal electron donor for heme under these conditions.

Oxidation-reduction properties of methylglyoxal-modified protein in relation to free radical generation.

Oxidation-reduction properties of methylglyoxal-modified protein in relation to free radical generation were investigated. Glycation of bovine serum albumin by methylglyoxal generated the protein-bound free radical, probably the cation radical of the cross-linked Schiff base, as observed in the reaction of methylglyoxal with L-alanine (Yim, H.-S., Kang, S.-O., Hah, Y. C., Chock, P. B., and Yim, M. B. (1995) J. Biol. Chem. 270, 28228-28233) or with Nalpha-acetyl-L-lysine. The glycated bovine serum albumin showed increased electrophoretic mobility suggesting that the basic residues, such as lysine, were modified by methylglyoxal. The glycated protein reduced ferricytochrome c to ferrocytochrome c in the absence of oxygen or added metal ions. This reduction of cytochrome c was accompanied by a large increase in the amplitude of the electron paramagnetic resonance signal originated from the protein-bound free radical. In addition, the glycated protein catalyzed the oxidation of ascorbate in the presence of oxygen, whereas the protein free radical signal disappeared. These results indicate that glycation of protein generates active centers for catalyzing one-electron oxidation-reduction reactions. This active center, which exhibits enzyme-like characteristic, was suggested to be the cross-linked Schiff base/the cross-linked Schiff base radical cation of the protein. It mimics the characteristics of the metal-catalyzed oxidation system. The glycated bovine serum albumin cross-linked further to the cytochrome c in the absence of methylglyoxal. The cross-linked cytochrome c maintains its oxidation-reduction properties. These results together indicate that glycated proteins accumulated in vivo provide stable active sites for catalyzing the formation of free radicals.