SUMOylation of nonstructural 5A protein regulates hepatitis C virus replication.

Viruses exploit cellular SUMOylation machinery to favour their own propagation. We show that NS5A is a target protein of small ubiquitin-like modifier (SUMO) and is SUMOylated at lysine residue 348. We demonstrated that SUMOylation increased protein stability of NS5A by inhibiting ubiquitylation, and SUMOylation was also required for protein interaction with NS5B. These data imply that SUMO modification may contribute to HCV replication. Indeed, silencing of UBC9 impaired HCV replication in Jc1-infected cells, and HCV replication level was also significantly reduced in SUMO-defective subgenomic replicon cells. Taken together, these data indicate that HCV replication is regulated by SUMO modification of NS5A protein. We provide evidence for the first time that HCV exploits host cellular SUMO modification system to favour its own replication.

Direct application of the PCR restriction analysis method for identifying NTM species in AFB smear-positive respiratory specimens.

The early differentiation of Mycobacterium tuberculosis from non-tuberculous mycobacteria (NTM) and the identification of NTM species are crucial for the proper management of patients with smear-positive sputum. We evaluated the usefulness of a polymerase chain reaction restriction analysis (PRA) method based on the rpoB gene for identifying NTM species in a study of 121 smear-positive respiratory specimens with presumed NTM. The PRA method amplified mycobacterial DNA in 72 specimens (60%) and differentiated NTM species correctly in 68 (94%). The PRA method could be a useful and rapid method for identifying NTM species in smear-positive respiratory specimens when urgent clinical decisions are required.

S-thiolation of creatine kinase and glycogen phosphorylase b initiated by partially reduced oxygen species.

S-thiolation of cardiac creatine kinase and skeletal muscle glycogen phosphorylase b was initiated by reduced oxygen species in reaction mixtures containing reduced glutathione. Both proteins were extensively modified at similar rates under conditions in which the oxidation of glutathione was inadequate to cause S-thiolation by thiol-disulfide exchange. Creatine kinase was both S-thiolated and non-reducibly oxidized at the same time at low glutathione concentration. The amount of each modification was decreased by adding additional reduced glutathione, and with adequate glutathione oxidation was prevented while S-thiolation was still very active. S-thiolation of glycogen phosphorylase b was not significantly affected by glutathione concentration and non-reducible oxidation of glycogen phosphorylase b was not observed. These experiments suggest that oxyradical or H2O2-initiated processes may be an important mechanism of protein S-thiolation during oxidative stress, and that the cellular concentration of glutathione may be an important factor in S-thiolation of different proteins. Both creatine kinase and glycogen phosphorylase b competed favorably with ferricytochrome c for superoxide anion in the standard xanthine oxidase system for the generation of oxyradicals and H2O2. These proteins were as effective as ascorbate and much more effective than reduced glutathione in this regard. Ascorbate was also an effective inhibitor of oxyradical-initiated S-thiolation of creatine kinase, suggesting a role of superoxide anion in protein S-thiolation. Other experiments showed that both catalase and superoxide dismutase could partially inhibit protein S-thiolation. Thus, reduced oxygen species may react with protein sulfhydryls resulting in S-thiolation by a mechanism that involves the reaction of an activated protein thiol with reduced glutathione.

Ibuprofen protects ischemia-induced neuronal injury via up-regulating interleukin-1 receptor antagonist expression.

The inflammatory response accompanies and exacerbates the developing injury after cerebral ischemia. Ibuprofen, a non-steroidal anti-inflammatory drug, has been shown to attenuate injuries in animal models of various neurological diseases. In the present study, we investigated ibuprofen's neuroprotective effects in rats exposed to transient forebrain ischemia and in cultures exposed to oxygen glucose deprivation (OGD). Rats treated with ibuprofen after transient forebrain ischemia displayed long-lasting protection of CA1 hippocampal neurons. There were selective increases in interleukin-1 receptor antagonist gene and protein expression in ibuprofen-treated OGD microglia. Furthermore, treatment with ibuprofen in neuron/microglia co-cultures increased the number of surviving HC2S2 neurons against OGD whereas IL-1ra neutralizing antibody reversed the ibuprofen-induced neuroprotection. The data indicate that ibuprofen-induced IL-1ra secretion is involved in neuroprotection against ischemic conditions.

Preoperative triglycerides predict post-coronary artery bypass graft survival in diabetic patients: a sex analysis.

OBJECTIVE: Hypertriglyceridemia is commonly observed in association with diabetes. Despite cross-sectional studies and isolated longitudinal analyses in patients without coronary artery disease, the suggestion that triglyceride levels are relevant to subsequent cardiovascular events in the setting of diabetes remains controversial. This study evaluates the predictive value of serum triglyceride levels on mortality in post-coronary artery bypass graft (CABG) diabetic patients with subsequent analysis by sex. RESEARCH DESIGN AND METHODS: This longitudinal observational study involving a large metropolitan hospital consists of 1,172 diabetic post-CABG patients (792 men and 380 women) with lipid data collected between the years 1982 and 1992. Cox proportional hazards regression models were used to estimate the risk of mortality and cardiac events associated with triglyceride levels in the highest quartile (> 2.90 mmol/l for men and > 3.12 mmol/l for women). RESULTS: Elevated preoperative serum triglyceride values in post-CABG subjects with diabetes were correlated with increased overall mortality (hazard ratio [HR] 1.26, 95% CI 1.00-1.59). The greatest influence of triglyceride levels was observed on overall (1.89, 1.30-2.73) and event-free survival (1.49, 1.06-2.08) in women. High triglyceride values were also modestly related to risk of cardiac events in diabetic men (1.28, 0.99-1.66). CONCLUSIONS: These data suggest that increased preoperative triglyceride levels predict increased late mortality and cardiac event risk in diabetic post-CABG patients, more strongly in women than in men.

Early c-Fos induction after cerebral ischemia: a possible neuroprotective role.

The role of c-Fos in neurodegeneration or neuroprotection after cerebral ischemia is controversial. To investigate whether early c-Fos induction after ischemia is associated with neuroprotection, rats were subjected to 10 minutes of transient forebrain ischemia and c-Fos expression was examined. Resistant dentate granule cells and neurons in CA2-4 displayed more robust immunoreactivity than vulnerable neurons in the CA1 region of hippocampus during early hours of reperfusion. By 6 hours after reperfusion, c-Fos immunoreactivity was greatly diminished in all areas of the hippocampus. Administration of N-acetyl-O-methyldopamine (NAMDA), a compound previously shown to protect CA1 neurons against ischemia, increased c-Fos immunoreactivity in the CA1 vulnerable region at 6 hours after ischemia and protected SK-N-BE(2)C neurons from oxygen glucose deprivation. Further in vitro study showed that NAMDA potentiated phorbol-12 myristate-13 acetate (PMA)-induced c-Fos expression, AP1 binding activity, and late gene expression determined by chloramphenicol acetyltransferase (CAT) activity from AP1 containing tyrosine hydroxylase promoter-CAT fusion gene in SK-N-BE(2)C neurons. In vivo and in vitro results showed that a neuroprotectant, NAMDA, in concert with another stimulus (for example, ischemia or PMA) up-regulates c-Fos expression and suggested that the early rise of NAMDA-induced c-Fos expression in vulnerable CA1 neurons may account for neuroprotection by means of up-regulating late gene expression for survival.

Oxidative damage in tissues of rats exposed to cigarette smoke.

Cigarette smoke is known to contain high concentrations of free radicals and oxidants. To examine the oxidative effect of cigarette smoking, we subjected rats to inhalation of cigarette smoke, and measured cellular free glutathione, the degree of protein S-thiolation, and 8-oxo-2'-deoxyguanosine (oxo8dG) in DNA. Inhalation of the cigarette smoke for 30 days, three times a day, resulted in a significant decrease of the total free glutathione contents in tissues, especially in the lung. Elevated levels of oxidized glutathione and protein S-thiolation were observed in the lung but not in other tissues. Increased contents of oxo8dG in DNA were found in all tissues analyzed. When rats were treated with buthionine sulfoximine (BSO, 80 mg/kg/day) to deplete glutathione, the oxidative effect of cigarette smoking was greatly potentiated. The effect of glutathione depletion was most evident in the lung. Cigarette smoking for only 7 days resulted in extreme depletion of the glutathione both in the lungs and in the liver of BSO-treated rats. Furthermore, oxo8dG in DNA increased markedly, especially in lung. The results verified that the lung is a primary target of cigarette smoke-induced oxidative damage, and cigarette smoke exerts its oxidative effects on the rest of the entire organs eventually. Our results indicate that glutathione plays crucial roles in protecting proteins and DNA from oxidation caused by cigarette smoking.

A neuroprotective role of extracellular signal-regulated kinase in N-acetyl-O-methyldopamine-treated hippocampal neurons after exposure to in vitro and in vivo ischemia.

In response to cerebral ischemia, neurons activate survival/repair pathways in addition to death cascades. Activation of cyclic AMP-response-element-binding protein (CREB) is linked to neuroprotection in experimental animal models of stroke. However, a role of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MAPK/ERK or MEK), an upstream kinase for CREB, and its relation to CREB phosphorylation in neuroprotection in cerebral ischemia has not been delineated. Previously, we reported that N-acetyl-O-methyldopamine (NAMDA) significantly protected CA1 neurons after transient forebrain ischemia [J Neurosci 19 (1999b) 87.8]. The current study is to investigate whether NAMDA-induced neuroprotection occurs via the activation of ERK and its downstream effector, CREB. NAMDA induced ERK1/2 and CREB phosphorylation with increased survival of HC2S2 hippocampal neurons subjected to oxygen-glucose deprivation. These effects were reversed by U0126, a MEK kinase inhibitor. Similarly, animals treated with NAMDA following ischemia showed increased ERK and CREB phosphorylation in the CA1 subregion of the hippocampus during early reperfusion period with increased number of surviving neurons examined 7 days following ischemia. The NAMDA-induced neuroprotection was abolished by U0126 administered shortly after reperfusion. The results showed that the ERK-CREB signaling pathway might be involved in NAMDA-induced neuroprotection following transient global ischemia and imply that the activation of the pathway in neurons may be an effective therapeutic strategy to treat stroke or other neurological syndromes.

Purification and molecular cloning of calobin, a thrombin-like enzyme from Agkistrodon caliginosus (Korean viper).

A thrombin-like enzyme, calobin, has been purified to homogeneity from the venom of Agkistrodon caliginosus by a procedure involving Bio-Gel P-100, Mono S, and Pro-RPC. The enzyme was identified as a monomer with a molecular weight of 34,000 on SDS-PAGE, and its isoelectric point was 6.2. Calobin acted on fibrinogen to form fibrin with a specific activity of 226 NIH equivalent units, and also exhibited arginine esterase activity. The enzyme predominantly cleaved the alpha-chain of fibrinogen with little degradation of the beta-chain. It contained abundant asparagine/aspartic acid residues, but very few tyrosine or methionine residues. The proteolytic activity of the enzyme with TAME as a substrate was higher than that of thrombin. However, it showed neither lysine esterase nor caseinolytic activity. The enzyme activity was strongly inhibited by PMSF, and moderately by benzamidine and soybean trypsin inhibitor, indicating it is a serine protease. On the other hand, the enzyme activity was not inhibited by hirudin or aprotinin. cDNA (1.6 kb) for calobin has been cloned from an A. caliginosus cDNA library. The cDNA sequence indicates that calobin is synthesized as a pre-zymogen of 262 amino acids, including a putative secretory signal peptide of 18 amino acids and a proposed zymogen peptide of 6 amino acid residues. The cDNA sequence encodes a 238-amino acid residue molecule exhibiting strong amino acid sequence homology to those of ancrod, batroxobin, and flavoxobin isolated from other snake venoms. Calobin contains 12 cysteine residues. As judged on alignment of the amino acid sequences of other thrombin-like enzymes (batroxobin, ancrod, and flavoxobin), calobin constitute the formation of six disulfide bridges. Amino acid residues, His43, Asp88, and Ser182, which are thought to be the catalytic active site are highly conserved. As calobin is a glycoprotein, its possible glycosylation site, Asn-X-Thr, is located at amino acid residues 81-83.

Reduced glutathione oxidation ratio and 8 ohdG accumulation by mild ischemic pretreatment.

A critical role of oxidative stress has been implicated in ischemic brain damage. Mild ischemic pretreatment and/or synthesis of heat shock proteins (HSPs) has been suggested to protect against oxidative brain damage. However, experimental support of this suggestion have proven to be difficult partly because sensitive indices to assess oxidative consequences of ischemic brain damage were few. In this study, we have attempted to establish biochemical assay systems to quantitate oxidative brain damage following ischemia. We produced experimental brain ischemia in the Mongolian gerbil (Meriones unguiculatus) and examined the hippocampus for ischemic brain damage. The results obtained from ischemic gerbil hippocampus demonstrated that oxidative brain damage can be quantitated by determining glutathione oxidation ratio together with the accumulation of the oxidative DNA damage product, 8-hydroxy-2'-deoxyguanosine (8 ohdG). Our results also demonstrated a role for mild ischemic pretreatment and synthesis of HSPs against oxidative brain damage. We showed that mild 2-min ischemic pretreatment reduced the degree of both glutathione oxidation ratio and 8 ohdG accumulation in gerbil hippocampus subsequent to 10 min ischemic challenge. We also showed that the accumulation of HSP70 was closely associated with the reduction of oxidative brain damage. To our knowledge, this is the first report to investigate glutathione redox states and oxidative DNA damage levels to evaluate a protective role of mild ischemic pretreatment and HSP synthesis following brain ischemia. Our data validate the previous suggestions and provide new additional data that argue for the protective role of mild ischemic pretreatment and HSP70 synthesis against oxidative brain damage.

Measurement of glutathione oxidation and 8-hydroxy-2'-deoxyguanosine accumulation in the gerbil hippocampus following global ischemia.

Involvement of oxidative stress in ischemia/reperfusion-induced brain damage has been suggested. However, experimental support of this suggestion was limited partly because sensitive indices to assess oxidative consequences of ischemic brain damage were few. We have established biochemical assay systems to assess oxidative brain damage following ischemia. Mongolian gerbil brains were subjected to global ischemia/reperfusion, and the hippocampi were analyzed for oxidative damage by measuring temporal changes in glutathione and 8-ohdG following ischemia. Under oxidative stress, glutathione is known to be oxidized and subsequently depleted from cells. Therefore, glutathione content and its redox status can serve as sensitive indicators of oxidative damage. The accumulation of 8-ohdG has also been recognized as an excellent marker for oxidative DNA damage. The reduced and oxidized glutathione were measured by HPLC method following derivatization with 2,4-dinitrofluorobenzene. The 8-ohdG in DNA hydrolyzate was measured by HPLC with electrochemical detection. While total glutathione content decreased, glutathione oxidation ratio and 8-ohdG accumulation increased over a period of 30 min of reperfusion following ischemia. The results demonstrated that glutathione content, its oxidation ratio, and the accumulated 8-ohdG could be utilized as sensitive indices for the assessment of oxidative brain damage.

Cerebral activation and distribution of inducible hsp110 and hsp70 mRNAs following focal ischemia in rat.

A potential function for inducible heat shock protein 70 (hsp70i) expression in the pathophysiology of ischemic brain has been well documented. The recently cloned hsp70 superfamily member, hsp110, was shown to be highly expressed in the brain and suggested to have a similar functional property as members of the hsp70 family. In this study, as an initial step to probe for its physiological significance in the ischemic brain, cerebral activation and distribution of hsp110 mRNA was comparatively evaluated with that of hsp70i mRNA by in situ hybridization. A rat focal cerebral ischemia model was employed to examine the distribution and localization of hsp110 and hsp70i mRNAs in both affected (ipsilateral) and unaffected (contralateral) hemispheres of the same animal. Our results demonstrated a significant accumulation of hsp110 as well as hsp70i mRNAs following ischemia; although the magnitude and kinetics of induction differ slightly, spatial expression profiles of hsp110 and hsp70i mRNAs were highly correlated in the affected region. In control brain, limited hybridization signal was observed with 3'-untranslated region (UTR) containing hsp110 probe, suggesting a possible existence of inducible hsp110 and a selective recognition of our 3'-UTR containing probe for the inducible hsp110 mRNA species. Subsequent 2D western analysis with Hsp110 specific Ab was consistent with our view, which resolved constitutive and inducible immunostained spots in rat ischemic brain. Considering a regulatory similarity as well as previously documented structural and functional similarities between hsp110 and hsp70i, we propose that coordinated cerebral activation of hsp110 and hsp70i is likely to be of significant relevance in the context of pathophysiology of ischemic brain. Further study is required to characterize the genetic and biochemical nature of rat inducible hsp110 identified in the current study.

Activation of estrogen receptor ß reduces blood-brain barrier breakdown following ischemic injury.

Estrogen receptors (ERs) play important roles in estrogen-mediated neuroprotection. However, their effects on blood-brain barrier (BBB) disruption with vasogenic edema after ischemic stroke have not been determined. We evaluated a role for ERß in the brain without effects in the peripheral reproductive organs for the amelioration of vasogenic edema following ischemic stroke. Transient focal ischemic stroke was induced in ovariectomized female C57BL/6 mice (age 10-11weeks) that were treated with the ERß-selective agonist diarylpropionitrile (DPN). BBB breakdown as determined by the extravasation of endogenous immunoglobulin G (IgG), vasogenic edema, and the infarct volume was significantly reduced by DPN compared to vehicle. Protein expressions of endothelial tight junction proteins (occludin and claudin-5) and the water channel protein aquaporin 4 in the ischemic cortex were not changed by DPN. However, protein levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1α (HIF-1α), a transcription factor that increases VEGF expression, were significantly decreased in the ischemic cortex by DPN. These results suggest that ERß contributes to the reduction of vasogenic edema caused by BBB breakdown via the inhibition of HIF-1α and VEGF following ischemic stroke.

Nanosized titanium dioxide enhanced inflammatory responses in the septic brain of mouse.

Nanosized titanium dioxide (TiO(2)) is used widely in various everyday products and can be applied to the medical field for diagnostic or therapeutic tools. However, its neurobiological responses have not been defined completely in the brain. To evaluate the acute inflammatory response to TiO(2) particles of two different sizes in normal and septic brains, male C57BL/6 mice were given intraperitoneal injections of fine (<1 microm) or ultrafine (21 nm) TiO(2), 30 min after vehicle or lipopolysaccaride (LPS). In the normal brain, neither fine nor ultrafine TiO(2) induced inflammation. However, in the brains of LPS-exposed mice, ultrafine TiO(2) significantly elevated proinflammatory cytokine interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) mRNAs, and IL-1beta protein levels. Also ultrafine TiO(2) increased the levels of reactive oxygen species and activated microglia 24 h after LPS challenge. In BV2 microglial cells stimulated with LPS, ultrafine TiO(2) enhanced TNF-alpha production and augmented nuclear factor-kB binding activity. These findings suggest that nanosized TiO(2) promotes an exaggerated neuroinflammatory responses by enhancing microglial activation in the pre-inflamed brain, in part.

The mechanisms of reduction of protein mixed disulfides (dethiolation) in cardiac tissue.

Dethiolation of proteins (reduction of protein mixed disulfides) by NADPH-dependent and glutathione (GSH)-dependent enzymes, and by nonenzymatic reaction with GSH, was studied by electrofocusing methodology with glycogen phosphorylase b and creatine kinase as substrates. Phosphorylase b was not rapidly dethiolated by reduced glutathione alone, but a cardiac extract catalyzed rapid dethiolation by both an NADPH-dependent and a GSH-dependent process. In contrast, creatine kinase was actively dethiolated by GSH. This GSH-dependent dethiolation was not enhanced by a soluble extract of bovine heart. Creatine kinase was also not dethiolated by an NADPH-dependent process. Partial purification of the phosphorylase dethiolases showed that the NADPH-dependent dethiolase had both a high-molecular-weight and a low-molecular-weight component The properties of these components were similar to those of thioredoxin and thioredoxin reductase. These two components were sensitive to inhibition by phenylarsine oxide and inhibition was reversed by addition of a dithiol. In contrast, GSH-dependent dethiolation required a single component of low molecular weight. This process was less sensitive to phenylarsine oxide inhibition. These studies show that two cytosolic proteins, phosphorylase b and creatine kinase, were dethiolated by different mechanisms. Phosphorylase b was dethiolated by both NADPH-dependent and GSH-dependent enzymes found in a soluble extract of bovine heart. In contrast, creatine kinase was rapidly dethiolated nonenzymatically by GSH alone.

Reduction of protein mixed disulfides (dethiolation) by Escherichia coli thioredoxin: a study with glycogen phosphorylase b and creatine kinase.

The role of thioredoxin in the reduction of protein mixed disulfides (dethiolation) was studied by electrofocusing methodology with glycogen phosphorylase b and creatine kinase as substrates for the reaction. Glycogen phosphorylase b was effectively dethiolated by Escherichia coli thioredoxin with dithiothreitol as the reductant, while creatine kinase could not be dethiolated by this mechanism. The rate of dethiolation of phosphorylase b was dependent on the concentration of thioredoxin up to a maximum at 20 microM when the concentration of phosphorylase b was 4 microM in monomer. Rat heart contained a thioredoxin reductase activity that could use added E. coli thioredoxin to dethiolate phosphorylase b and the same concentration of thioredoxin as above was required. This activity was not expressed with creatine kinase as the substrate. Cardiac tissue was shown to have a similar endogenous dethiolating activity. These results suggest that thioredoxin may play an important role in dethiolating specific proteins that might become S-thiolated during oxidative stress of cardiac tissue.

Reduction (dethiolation) of protein mixed-disulfides; distribution and specificity of dethiolating enzymes and N,N'-bis(2-chlorethyl)-N-nitrosourea inhibition of an NADPH-dependent cardiac dethiolase.

The S-thiolated proteins phosphorylase b (Phb) and carbonic anhydrase III (CAIII) were prepared with [3H]glutathione in a reaction initiated with diamide. These substrates were used to measure the rate of reduction (dethiolation) of protein mixed-disulfides by enzymes with properties similar to those of thioredoxin and glutaredoxin. This enzyme activity is termed a dethiolase since the identities of the enzymes are still unknown. The dethiolation of either S-[3H]glutathiolated Phb or S-[3H]glutathiolated CAIII was employed in tissue assays and for study of two partially purified dethiolases from cardiac tissue. NADPH-dependent dethiolase activity was most abundant except in rat liver and muscle. Total dethiolase activity was approximately 10-fold higher in neutrophils, 3T3-L1 cells, and Escherichia coli than in other sources. Rat skeletal muscle had 3- to 4-fold higher dethiolase activity than rat heart or liver. These data indicate that protein dethiolase activity is ubiquitous and that normal expression of the two dethiolase activities varies considerably. A partially purified cardiac NADPH-dependent dethiolase acted on Phb approximately 1.5 times faster than CAIII, and a glutathione (GSH)-dependent dethiolase acted on Phb 3 times faster than CAIII. The Km for glutathione for the GSH-dependent dethiolase was 15 microM with Phb as substrate and 10 microM with CAIII. Thus, the GSH-dependent dethiolase is probably not affected by normal changes in the cardiac glutathione content (normally approximately 3 mM). Partially purified cardiac NADPH-dependent dethiolase was inactivated by BCNU (N,N'-bis(2-chloroethyl)-N-nitrosourea) and the GSH-dependent dethiolase was unaffected under similar conditions. In a soluble extract from bovine heart, 200 microM BCNU inhibited NADPH-dependent dethiolase by more than 60% but did not affect GSH-dependent activity. These results demonstrate that BCNU is a selective inhibitor of the NADPH-dependent dethiolase.

Immunoaffinity isolation of urinary 8-hydroxy-2'-deoxyguanosine and 8-hydroxyguanine and quantitation of 8-hydroxy-2'-deoxyguanosine in DNA by polyclonal antibodies.

An immunoaffinity column is described that facilitates the analysis of oxidative DNA damage. DNA adducts excised from DNA are excreted in urine and can be assayed as a measure of DNA damage in individuals. Polyclonal antibodies that recognize 8-hydroxy-2'-deoxyguanosine (oh8dG), a biomarker of oxidative damage to DNA, have been produced and their binding properties characterized. The antibodies, raised in rabbits following immunization with protein carrier-hapten conjugates prepared by covalently linking periodate-treated 8-hydroxyguanosine (oh8G) to bovine serum albumin (BSA) or casein, bind oh8dG with high affinity and selectivity, as measured by a competitive radioimmunoassay (RIA). Antibodies obtained from the rabbits immunized with the casein conjugate exhibited a binding affinity for oh8dG of 6.9 x 10(8) M-1. Studies on the relative binding affinities of these polyclonal antibodies for oh8dG, unmodified nucleosides, or derivatives of guanine indicate that the antibodies are suitable for the preparation of immunoaffinity columns that permit us to rapidly isolate oh8dG and 8-hydroxyguanine (oh8Gua) from urine. The high selectivity of the antibodies for oh8dG and oh8G reduces the amount of urinary contaminants previously observed in samples prepared by solid phase extraction, thus greatly facilitating the isolation of these damage products from urine. The relative binding affinity of these antibodies for oh8Gua and 2'-deoxyguanosine were approximately 7.6 x 10(3) and 7.4 x 10(4) fold lower respectively, than the binding affinity for oh8dG. The antibody can be used to quantitate oh8dG in enzymatic hydrolyzates of DNA with values comparable to those obtained by HPLC with electrochemical detection (HPLC-EC).

Phosphorylase and creatine kinase modification by thiol-disulfide exchange and by xanthine oxidase-initiated S-thiolation.

The reaction of glycogen phosphorylase b and creatine kinase with glutathione disulfide, cystine, and cystamine was compared by direct analysis on electrofocusing gels. This method was useful for individual proteins or for mixtures of the proteins. Millimolar concentrations of glutathione disulfide were required for both proteins and the rate of modification of each protein was similar. The reaction of glutathione disulfide with creatine kinase was inhibited by reduced glutathione (GSH), but the effect on the reaction with phosphorylase was minimal. Cystine and cystamine were required in micromolar amounts to effectively form the disulfide adducts. Both proteins were modified by cystine but cystamine reacted only with phosphorylase. Cystamine (10 microM) was an effective inhibitor of the reaction of phosphorylase b with 2 mM glutathione disulfide. S-thiolation of creatine kinase inactivated the enzyme and a direct assay of the enzyme activity could be used to quantitate S-thiolation of this protein by each of the disulfides. The effect of each disulfide on enzyme activity confirmed the results obtained by gel electrofocusing. Glutathione disulfide and cystine both inactivated the enzyme while cystamine had no effect on the activity. S-thiolation of phosphorylase had no observable effect on any activity parameter, but it effectively prevented binding of phosphorylase to high-molecular-weight glycogen, probably at the glycogen storage site of phosphorylase. The rate of S-thiolation of a mixture of phosphorylase and creatine kinase by thiol-disulfide exchange with glutathione disulfide was compared to the rate of S-thiolation of these proteins by a xanthine oxidase-initiated process (presumably due to protein sulfhydryl activation by reactive oxygen species). The xanthine oxidase-initiated mechanism was somewhat faster than thiol-disulfide exchange with both proteins. It was shown that GSH inhibited S-thiolation of creatine kinase by this mechanism as well as by thiol-disulfide exchange. It is suggested that both mechanisms may play a role in protein S-thiolation in vivo. For proteins that are typified by creatine kinase, the concentration of GSH in the cells may determine whether the S-thiolated form of the protein accumulates. For proteins typified by phosphorylase b, the accumulation of S-thiolated forms may be more independent of GSH.