Hydrogen-Bonding Interactions at the DNA Terminus Promote Extension from Methylguanine Lesions by Human Extender DNA Polymerase ζ.

Chemically induced DNA lesions can become DNA replication substrates that are bypassed by low-fidelity DNA polymerases. Following nucleotide misinsertion opposite a DNA lesion, the extension step can contribute to preserving such errors and lead to genomic instability and cancer. DNA polymerase ζ, a B-family polymerase, is proficient as an extender polymerase that catalyzes elongation; however, the chemical factors that impact its DNA replication are not understood. This study addresses the question of how DNA polymerase ζ achieves extension by examining the ability of recombinant human DNA polymerase ζ to extend from a series of methylated guanine lesions. The influence of H-bonding was examined by placing structurally altered nucleoside analogues and canonical bases opposite G, O6-MeG, N1-MeG, and N2-MeG. We determined that terminal base pairs with the highest proclivity for H-bonding were most efficiently extended in both primer extension assays and steady-state kinetic analysis. In contrast, when no H-bonding was possible at the DNA terminus, the least efficient steady-state kinetics were observed. To evaluate H-bonding protein minor groove interactions that may underlie this phenomenon, we performed computational modeling with Escherichia coli DNA polymerase II, a homologue for DNA polymerase ζ. The modeling data together with the primer extension assays demonstrate the importance of having a carbonyl group on the primer strand that can interact with a lysine residue found to be conserved in many B-family polymerases, including human Pol ζ. These data provide a model whereby interbase H-bonding interactions at the DNA terminus promote lesion bypass and extension by human DNA polymerase ζ.

N'-3-(Trifluoromethyl)phenyl Derivatives of N-Aryl-N'-methylguanidines as Prospective PET Radioligands for the Open Channel of the N-Methyl-d-aspartate (NMDA) Receptor: Synthesis and Structure-Affinity Relationships.

N-Methyl-d-aspartate (NMDA) receptor dysfunction has been linked to several neuropsychiatric disorders, including Alzheimer's disease, epilepsy, drug addiction, and schizophrenia. A radioligand that could be used with PET to image and quantify human brain NMDA receptors in the activated "open channel" state would be useful for research on such disorders and for the development of novel therapies. To date, no radioligands have shown well-validated efficacy for imaging NMDA receptors in human subjects. In order to discover improved radioligands for PET imaging, we explored structure-affinity relationships in N'-3-(trifluoromethyl)phenyl derivatives of N-aryl-N'-methylguanidines, seeking high affinity and moderate lipophilicity, plus necessary amenability for labeling with a positron-emitter, either carbon-11 or fluorine-18. Among a diverse set of 80 prepared N'-3-(trifluoromethyl)phenyl derivatives, four of these compounds (13, 19, 20, and 36) displayed desirable low nanomolar affinity for inhibition of [(3)H](+)-MK801 at the PCP binding site and are of interest for candidate PET radioligand development.

Effects of uremic solutes on reactive oxygen species in vitro model systems as a possibility of support the renal function management.

BACKGROUND: In view of the prevalence of oxidative stress in chronic kidney disease (CKD) patients, the loss of low-molecular-weight biomolecules by hemodialysis and the antioxidant potential of some uremic solutes that accumulate in CKD, we used in vitro model systems to test the antioxidant potential of the following uremic solutes: uric acid, hippuric acid, p-cresol, phenol, methylguanidine, L-arginine, L-tyrosine, creatinine and urea. METHODS: The in vitro antioxidant efficiencies of the uremic solutes, isolated or in mixtures, were tested with the following assays: i) ABTS radical cation decolorization assay; ii) hypochlorous acid (HOCl/OCl(-)) scavenging activity; iii) superoxide anion radical (O2(•-)) scavenging activity; iv) crocin bleaching assay (capture of peroxyl radical, ROO(•)); v) hydrogen peroxide (H2O2) scavenging activity. RESULTS: Four of the tested uremic solutes (p-cresol, phenol, L-tyrosine, uric acid) were effective antioxidants and their IC50 were found in three model systems: ABTS(•+), HOCl/OCl(-) and crocin bleaching assay. In the 4-solutes mixtures, each one of the solute captured 12.5% for the IC50 of the mixture to ABTS(•+) or HOCl/OCl(-), exhibiting a virtually exact additive effect. In the 2-solutes mixtures, for ROO(•) capture, it was observed the need of more mass of uremic solutes to reach an IC50 value that was higher than the projected IC50, obtained from the IC50 of single solutes (25% of each, in the binary mixtures) in the same assay. In model systems for O2(•-) and H2O2, none of the uremic solutes showed scavenging activity. CONCLUSIONS: The use of the IC50 as an analytical tool to prepare and analyze mixtures allows the determination of their scavenging capacities and may be useful for the assessment of the antioxidant status of biological samples under conditions of altered levels of the endogenous antioxidant network and/or in the employment and monitoring of exogenous antioxidant therapy.

DNA exit ramps are revealed in the binding landscapes obtained from simulations in helical coordinates.

DNA molecules are highly charged semi-flexible polymers that are involved in a wide variety of dynamical processes such as transcription and replication. Characterizing the binding landscapes around DNA molecules is essential to understanding the energetics and kinetics of various biological processes. We present a curvilinear coordinate system that fully takes into account the helical symmetry of a DNA segment. The latter naturally allows to characterize the spatial organization and motions of ligands tracking the minor or major grooves, in a motion reminiscent of sliding. Using this approach, we performed umbrella sampling (US) molecular dynamics (MD) simulations to calculate the three-dimensional potentials of mean force (3D-PMFs) for a Na+ cation and for methyl guanidinium, an arginine analog. The computed PMFs show that, even for small ligands, the free energy landscapes are complex. In general, energy barriers of up to ~5 kcal/mol were measured for removing the ligands from the minor groove, and of ~1.5 kcal/mol for sliding along the minor groove. We shed light on the way the minor groove geometry, defined mainly by the DNA sequence, shapes the binding landscape around DNA, providing heterogeneous environments for recognition by various ligands. For example, we identified the presence of dissociation points or "exit ramps" that naturally would terminate sliding. We discuss how our findings have important implications for understanding how proteins and ligands associate and slide along DNA.

New N-aryl-N'-(3-(substituted)phenyl)-N'-methylguanidines as leads to potential PET radioligands for imaging the open NMDA receptor.

An expansive set of N-aryl-N'-(3-(substituted)phenyl)-N'-methylguanidines was prepared in a search for new leads to prospective PET ligands for imaging of the open channel of the N-methyl-d-aspartate (NMDA) receptor in vivo. The N-aryl rings and their substituents were varied, whereas the N-methyl group was maintained as a site for potential labeling with the positron-emitter, carbon-11 (t1/2=20.4min). At micromolar concentration, over half of the prepared compounds strongly inhibited the binding of [(3)H]TCP to its binding site in the open NMDA receptor in vitro. Four ligands displayed affinities that are similar or superior to those of the promising SPECT radioligand ([(123)I]CNS1261). The 3'-dimethylamino (19; Ki 36.7nM), 3'-trifluoromethyl (20; Ki 18.3nM) and 3'-methylthio (2; Ki 39.8nM) derivatives of N-1-naphthyl-N'-(phenyl)-N'-methylguanidine were identified as especially attractive leads for PET radioligand development.

The nonenzymatic decomposition of guanidines and amidines.

To establish the rates and mechanisms of decomposition of guanidine and amidine derivatives in aqueous solution and the rate enhancements produced by the corresponding enzymes, we examined their rates of reaction at elevated temperatures and used the Arrhenius equation to extrapolate the results to room temperature. The similar reactivities of methylguanidine and 1,1,3,3-tetramethylguanidine and their negative entropies of activation imply that their decomposition proceeds by hydrolysis rather than elimination. The influence of changing pH on the rate of decomposition is consistent with attack by hydroxide ion on the methylguanidinium ion (k2 = 5 × 10(-6) M(-1) s(-1) at 25 °C) or with the kinetically equivalent attack by water on uncharged methylguanidine. At 25 °C and pH 7, N-methylguanidine is several orders of magnitude more stable than acetamidine, urea, or acetamide. Under the same conditions, the enzymes arginase and agmatinase accelerate substrate hydrolysis 4 × 10(14)-fold and 6 × 10(12)-fold, respectively, by mechanisms that appear to involve metal-mediated water attack. Arginine deiminase accelerates substrate hydrolysis 6 × 10(12)-fold by a mechanism that (in contrast to the mechanisms employed by arginase and agmatinase) is believed to involve attack by an active-site cysteine residue.

Curcumin attenuates renal ischemia and reperfusion injury-induced restrictive respiratory insufficiency.

OBJECTIVE: Pulmonary failure, instead of kidney failure, is one of the leading causes of acute kidney injury (AKI)-related death. Volume overload was previously regarded as the primary cause of lung injury, presumably by impaired renal fluid clearance. Recent evidence suggested that proinflammatory cytokines, chemokines, and free radicals released during AKI are playing a crucial role in the lung injury. We aimed to examine the protective efficacy of lung function with curcumin pretreatment. METHODS: AKI was induced by 45 minutes of kidney ischemia (bilateral occlusion of renal pedicles) followed by 3 hours of reperfusion. Rats were divided into 3 groups: sham-operated, kidney ischemia and reperfusion (I/R), and a group with 2 days of oral pretreatment with curcumin (12.5 mg/kg/d) before I/R injury. The pulmonary function test (PFT) was conducted at baseline and after 3 hours of reperfusion, yielding parameters of lung volumes, chord compliance (Cchord), inspiratory resistance (RI), and forced expiratory volume at the first 200 millisecond (FEV200). We also examined levels of protein concentration (PC), methylguanidine (MG), tumor necrosis factor-α (TNF-α), and malondialdehyde (MDA) in the bronchoalveolar lavage (BAL). RESULTS: Ischemic AKI-induced restrictive lung disease was demonstrated by the decreased Cchord, total lung capacitance (TLC), and FEV200, in addition to the increased lavage PCBAL, MG, TNF-α, and MDA level. Curcumin pretreatment ameliorated lung function impairment and alveolar vascular protein leak and attenuated lung inflammation. CONCLUSIONS: The protective effect of curcumin pretreatment against restrictive lung disease is most likely associated with decreasing hydroxyl radical, lipid peroxidation, and inflammation in the lungs and improving alveolar vascular permeability.

Liver reperfusion-induced decrease in dynamic compliance and increase in airway resistance are ameliorated by preischemic treatment with melatonin through scavenging hydroxyl radicals in rat lungs.

OBJECTIVES: Acute lung injury is frequently observed in patients subsequent to liver ischemia and reperfusion (I/R) injury. However, the changes in pulmonary function, eg, lung dynamic compliance (C(dyn)) and airway resistance (RI), are not well understood. We sought to study the alternations in pulmonary function during liver I/R and the protective effects of preischemic treatment with melatonin. METHODS: Animals were divided into 3 groups: sham-operated, liver I/R, and intraperitoneal (i.p.) pretreatment with melatonin (15 mg/kg). Liver I/R was performed by clamping the hepatic artery and portal vein for 30 minutes followed by releasing for 2 hours. The C(dyn) and RI were studied at baseline and at 2 hours of reperfusion. We assessed the level of pulmonary hydroxyl radicals by methylguanidine (MG) content in the bronchoalveolar lavage fluid (BALF) as well as the liver damage using plasma levels of lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT). RESULTS: After 2 hours of liver reperfusion, C(dyn) was reduced by ∼25%, while RI increased by ∼16% (P < .05). The decreased C(dyn) and increased RI were markedly attenuated by melatonin pretreatment (P < .05). Melatonin pretreatment also protected the liver against I/R injury (P < .05), as seen by reduced LDH, GOT and GPT along with markedly reduced hydroxyl radicals (P < .05). CONCLUSIONS: Preischemic treatment with melatonin protected lung function against damage by liver I/R. The improvement in lung function was strongly associated with decreased hydroxyl radicals in the lungs.

Preischemic treatment with melatonin attenuates liver reperfusion-induced impairment of cardiac function.

OBJECTIVES: Cardiac functional impairment is frequently observed in patients with end-stage liver disease and after reperfusion of an ischemic liver. Excessive production of reactive oxygen species (ROS) through activation of Kupffer cells and leukocytes during reperfusion may play important roles. We evaluated the cardiac protective effects of preischemic treatment with melatonin. METHODS: Studies were performed on 3 groups of male Sprague-Dawley rats; shame-operated controls, liver ischemia and reperfusion (I/R), and melatonin pretreatment prior to I/R. Liver I/R was performed by clamping the hepatic artery and portal vein for 30 minutes, followed by releasing the clamps for 2 hours. The cardiac function was assessed using a high-fidelity dual pressure-volume catheter positioned in the left ventricle (LV). We also evaluated heart injury using plasma creatine kinase-MB (CKMB), and Troponin I (cTnI). The level of hydroxyl radical production was evaluated using plasma methylguanidine (MG). RESULTS: LV function was severely impaired after 2 hours of reperfusion; stroke volume and LV contractility were significantly reduced (P < .05). Markedly increased CKMB and cTnI indicated serious myocardial injury. Preischemic treatment with melatonin protected the heart as seen by the reduced plasma CKMB and cTnI (P < .05), and decreased systemic hydroxyl radical production. CONCLUSIONS: Liver I/R severely impaired cardiac functions by production of hydroxyl radicals. Melatonin pretreatment effectively scavenged oxidants and hydroxyl radicals, protecting cardiac function against liver I/R-induced injury.

Effects of the antioxidants lycium barbarum and ascorbic acid on reperfusion liver injury in rats.

OBJECTIVE: Ischemia/reperfusion (I/R) of the rat liver can induce liver injury through mechanisms involving oxidative and nitrosative stresses. In this study we examined the effects of antioxidants Lycium barbarum (LB) and ascorbic acid on I/R-induced liver injury in rats. METHODS: Liver ischemia was induced by clamping the common hepatic artery and portal vein of rats for 40 minutes. Thereafter, flow was restored with reperfusion for 90 minutes. Blood samples collected before ischemia and after reperfusion were analyzed for alanine transaminase (ALT), lactic dehydrogenase (LDH), hydroxyl radical, and nitric oxide (NO) levels. Pharmacologic interventions included administration of ascorbic acid (100 mg/kg, i.p., 1 hour before I/R) or LB, an extract of Gogi berries: 600 mg in 100 mL of drinking water for 2 weeks prior to experimentation. RESULTS: This protocol resulted in elevation of blood concentrations of NO, hydroxyl radical, ALT, and LDH (P < .001) in the I/R-induced liver injury group. Ascorbic acid significantly attenuated the reperfusion liver injury by attenuating hydroxyl radical (P < .01) and NO (P < .05) release. The LB aggravated I/R-induced liver injury by increasing hydroxyl radical release with no effect on NO release. DISCUSSION AND CONCLUSIONS: This I/R protocol resulted in oxidative and nitrosative stress and liver injury. Ascorbic acid showed significant protective effects on reperfusion liver injury by attenuating hydroxyl radical and NO release. In contrast, LB aggravated liver injury by increasing hydroxyl radical release.

Impact of increasing haemodialysis frequency versus haemodialysis duration on removal of urea and guanidino compounds: a kinetic analysis.

BACKGROUND: Patients with renal failure retain a large variety of uraemic solutes, characterized by different kinetic behaviour. It is not entirely clear what the impact is of increasing dialysis frequency and/or duration on removal efficiency, nor whether this impact is the same for all types of solutes. METHODS: This study was based on two-compartmental kinetic data obtained in stable haemodialysis patients (n = 7) for urea, creatinine (CREA), guanidinosuccinic acid (GSA) and methylguanidine (MG). For each individual patient, mathematical simulations were performed for different dialysis schedules, varying in frequency, duration and intensity. For each dialysis schedule, plasmatic and extraplasmatic weekly time-averaged concentrations (TAC) were calculated, as well as their %difference to weekly TAC of the reference dialysis schedule (three times weekly 4 h). RESULTS: Increasing dialysis duration was most beneficial for CREA and MG, which are distributed in a larger volume (54.0 +/- 5.9 L and 102.6 +/- 33.9 L) than urea (42.7 +/- 6.0 L) [plasmatic weekly TAC decrease of 31.5 +/- 3.2% and 31.8 +/- 3.8% for CREA and MG with Q(B) of 200 mL/min, compared to 25.7 +/- 3.2% for urea (P = 0.001 and P < 0.001)]. Increasing dialysis frequency resulted only in a limited increase in efficiency, most pronounced for solutes distributed in a small volume like GSA (30.6 +/- 4.2 L). Increasing both duration and frequency results in weekly TAC decreases of >65% for all solutes. Comparable results were found in the extraplasmatic compartment. CONCLUSION: Prolonged dialysis significantly reduces solute concentration levels, especially for those solutes that are distributed in a larger volume. Increasing both dialysis frequency and duration is the superior dialysis schedule.

A new protein architecture for processing alkylation damaged DNA: the crystal structure of DNA glycosylase AlkD.

DNA glycosylases safeguard the genome by locating and excising chemically modified bases from DNA. AlkD is a recently discovered bacterial DNA glycosylase that removes positively charged methylpurines from DNA, and was predicted to adopt a protein fold distinct from from those of other DNA repair proteins. The crystal structure of Bacillus cereus AlkD presented here shows that the protein is composed exclusively of helical HEAT-like repeats, which form a solenoid perfectly shaped to accommodate a DNA duplex on the concave surface. Structural analysis of the variant HEAT repeats in AlkD provides a rationale for how this protein scaffolding motif has been modified to bind DNA. We report 7mG excision and DNA binding activities of AlkD mutants, along with a comparison of alkylpurine DNA glycosylase structures. Together, these data provide important insight into the requirements for alkylation repair within DNA and suggest that AlkD utilizes a novel strategy to manipulate DNA in its search for alkylpurine bases.

Accumulation of methylguanidine and changes in guanidino compound levels in plasma, urine, and kidneys of furosemide-treated rats.

Antidiuresis and renal diseases alter the levels of guanidino compounds (GCs) in various tissues. Therefore, we hypothesized that diuresis could also disturb GC metabolism, storage, and elimination. In this study, rats were made diuretic to analyze GC levels in plasma, urine, and kidneys. Furosemide was chosen because of its wide use in various human pathologies. Rats were injected intraperitoneally 5 or 10 mg furosemide spread over a 24-hour cycle. Urine was collected over a period of 24 hours before and during furosemide treatment. Plasma was obtained from arterial blood. Renal zones were dissected. The GCs were determined by liquid chromatography. Five milligrams of furosemide provoked a significant increase in plasma and urine levels of GCs compared with those of the controls. The renal distribution and content of GCs were weakly modified by furosemide except for methylguanidine (MG). The level of MG was enhanced by 10 to 16 times in all renal zones. The MG level was 60% higher in renal zones of rats treated with 10 rather than 5 mg furosemide. The fractional excretion of MG was decreased by furosemide. Our data suggest that MG accumulation in kidney and plasma was caused by furosemide, which might induce MG synthesis, and that MG washout from tissue cells into urine by furosemide through the kidney may cause an increase in MG in the kidney.

Nicotinamide abrogates acute lung injury caused by ischaemia/reperfusion.

Poly (ADP-ribose) synthase or polymerase (PARS and PARP, respectively) is a cytotoxic enzyme which causes cellular damage. Nicotinamide, a compound of vitamin B complex, has been reported to exert an inhibitory effect on PARS or PARP. The present study tests the effects of nicotinamide on acute lung injury and associated alterations following ischaemia/reperfusion (I/R) of the isolated perfused rat's lung. I/R increased the lung weight (LW) to body weight ratio, LW gain, protein and dye tracer leakage, pulmonary arterial pressure and capillary permeability. The insult also increased nitrate/nitrite, methyl guanidine, tumour necrosis factor-alpha and interleukin-1beta in lung perfusate, while it decreased adenosine triphosphate content with an increase in PARP activity in lung tissue. Most of the I/R-induced changes were abrogated by post-treatment (30 min after I/R) with nicotinamide (100 mg.kg(-1) body weight). However, the increase in pulmonary arterial pressure was enhanced by nicotinamide post-treatment. Following I/R, the inducible nitric oxide synthase (iNOS) mRNA expression was enhanced. Nicotinamide reduced the iNOS expression. The results suggest that nicotinamide exerted a protective effect on the acute lung injury caused by ischaemia/reperfusion. The mechanisms may be mediated through the inhibition on the poly (adenosine diphosphate-ribose) polymerase activity, inducible nitric oxide synthase expression and the subsequent suppression of nitric oxide, free radicals and pro-inflammatory cytokines with restoration of adenosine triphosphate.

Reperfusion liver injury induces down-regulation of eNOS and up-regulation of iNOS in lung tissues.

OBJECTIVES: Acute lung injury and inflammation can occur after hepatic ischemia/reperfusion (I/R). Little is known regarding the possible role of nitric oxide synthase expression in this complex type of lung injury. METHODS: Real-time polymerase chain reactions and immunohistochemistry were used to assess the mRNA and protein expression of eNOS and iNOS in lung tissue after I/R challenge to the liver. Ischemia was induced by clamping the hepatic artery and portal vein for 40 minutes. After flow was restored, the liver was reperfused for 300 minutes. Blood samples were collected to assay three inflammatory parameters: tumor necrosis factor (TNF)-alpha, hydroxyl radicals, and NO. Lung lavage samples were assayed for protein and myeloperoxidase. The expression of eNOS and iNOS in lung tissues (n = 3) was also evaluated after I/R challenge to the liver. The iNOS inhibitor aminoguanidine was also tested in this I/R model. RESULTS: Reperfusion of the liver produced increased blood concentrations of TNF, hydroxyl radicals, and NO (P < .001; n = 8). Bronchial lavage fluids showed higher levels of protein and myeloperoxidase in the I/R than in the sham-treated group (P < .01). eNOS expression was down-regulated and iNOS expression up-regulated in I/R lung tissues (n = 3). The iNOS inhibitor aminoguanidine (10 mg/kg) significantly attenuated the lung injury. CONCLUSIONS: I/R injury to the liver induced lung injury involving systemic inflammatory responses and iNOS expression. Administration of aminoguanidine significantly attenuated the injury, suggesting that iNOS expression may play a critical role in lung injury induced by I/R of the liver.

Guanidinium derivatives bind preferentially and trigger long-distance conformational changes in an engineered T4 lysozyme.

The binding of guanidinium ion has been shown to promote a large-scale translation of a tandemly duplicated helix in an engineered mutant of T4 lysozyme. The guanidinium ion acts as a surrogate for the guanidino group of an arginine side chain. Here we determine whether methyl- and ethylguanidinium provide better mimics. The results show that addition of the hydrophobic moieties to the ligand enhances the binding affinity concomitant with reduction in ligand solubility. Crystallographic analysis confirms that binding of the alternative ligands to the engineered site still drives the large-scale conformational change. Thermal analysis and NMR data show, in comparison to guanidinium, an increase in protein stability and in ligand affinity. This is presumably due to the successive increase in hydrophobicity in going from guanidinium to ethylguanidinium. A fluorescence-based optical method was developed to sense the ligand-triggered helix translation in solution. The results are a first step in the de novo design of a molecular switch that is not related to the normal function of the protein.

Activity of (-)-epigallocatechin 3-O-gallate against oxidative stress in rats with adenine-induced renal failure.

Methylguanidine (MG) is widely recognized as a strong uremic toxin. The hydroxyl radical (*OH) specifically plays an important role in the pathway of MG production from creatinine (Cr). In this study, we investigated whether oral administration of (-)-epigallocatechin 3-O-gallate (EGCg) suppresses MG production in rats with chronic renal failure after intraperitoneal Cr injection. MG production from Cr was significantly increased in rats with adenine-induced renal failure, which was more vulnerable to oxidative stress, compared with that in normal rats. However, oral administration of EGCg 30 min before and after Cr injection effectively inhibited MG production. Our findings suggest that EGCg, an excellent antioxidant from green tea, exerts protective activity in rats with chronic renal failure, resulting in suppression of Cr oxidation influenced by *OH.

Inhibition by heparin of protein kinase C activation and hydroxyl radical generation in puromycin aminonucleoside treated isolated rat hepatocytes.

Heparin has been reported to have many actions similar to calcium-dependent protein kinase (PKC) inhibitors. We have found that puromycin aminonucleoside (PAN) increases hydroxyl radical generation and this was prevented by H-7, a PKC inhibitor in isolated rat hepatocytes. In this study, we investigate the effect of heparin on the increased hydroxyl radical generation as well as PKC activation by PAN in isolated rat hepatocytes. To estimate the amount of hydroxyl radical generation, we measured methylguanidine (MG) and creatol which are the products from the reaction of creatinine and hydroxyl radical. Synthetic rate of MG plus creatol in isolated rat hepatocytes incubated in Krebs-Henseleit bicarbonate buffer containing creatinine and tested reagents were recorded. This rate with or without PAN was 231 +/- 11 or 112 +/- 5.6 nmol/g wet cells/4 h (mean +/- S.E., n = 5), respectively. Heparin concentrations of 3.3, 6.6 and 10 U/ml inhibited MG plus creatol synthesis in the presence of PAN by 30, 38 and 39%, and without PAN by 8.4, 27 and 34%, respectively. Statistical significance was observed except for 3.3 U/ml without PAN. The ratio of PKC in membrane/cytoplasmic fraction, an indicator of PKC activation, increased 2.8- and 3-fold that of the 0 time after 60 and 120 min incubation with PAN while heparin at 10 U/ml almost completely suppressed this increase in the ratio of PKC. The PKC ratio of the membrane/cytoplasmic fraction obtained from hepatocytes with heparin alone or without PAN and heparin was almost unchanged during the tested period. Variation of PKC levels in membrane fraction is similar to that of PKC ratio of the membrane/cytoplasmic fraction. Increased creatol synthesis by PAN and its inhibition by heparin were observed in the same samples as those used for the PKC study. These results indicate that heparin inhibits the increase in hydroxyl radical generation induced by PAN through inhibition of PKC activation in isolated rat hepatocytes.

Site of methylguanidine production and factors that influence production levels.

The site of methylguanidine (MG) production in the kidney was investigated using animal models of renal disease and cultured renal epithelial cells. In rats with proximal tubular injury induced by adenine, the blood and urinary levels of MG increased as the severity of injury increased. In contrast, in cases of glomerular injury, there were no such changes in MG levels. Thus, it was apparent that proximal tubular injury served to promote MG production. In addition, a marked increase was observed in the intensities of bands attributable to 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OH in the electron spin resonance spectrum of the kidney in the rats given adenine. In these rats, the activity of the radical-scavenging enzymes superoxide dismutase, catalase, and glutathione peroxidase was decreased. This suggests that the formation of excessive radicals and deterioration of defense mechanisms that contribute to the development of oxidative stress underlie the enhanced MG production. The experiments using cultured cells revealed that an oxide of adenine, 2,8-dihydroxyadenine (DHOA), directly induced renal tubular injury. These findings indicate that the accumulation of creatinine due to DHOA, combined with oxidative stress, resulted in increased MG production.

The role of protein kinase C in the increased generation in isolated rat hepatocytes of the hydroxyl radical by puromycin aminonucleoside.

Puromycin aminonucleoside (PAN) has been known to induce proteinuria. The increased generation of reactive oxygen species (ROS) has been implicated in this toxicity of PAN. We have reported that PAN increases the synthesis of methylguanidine (MG) and creatol which are the products of the reaction of creatinine and the hydroxyl radical in isolated rat hepatocytes. However, the mechanism for the increased ROS induced by PAN is still unclear. In this paper, we investigate the role of protein kinase C (PKC) on the PAN induced reactive oxygen generation in isolated rat hepatocytes. Isolated hepatocytes were incubated in Krebs-Henseleit bicarbonate buffer containing 3% BSA, 16.6 mM creatinine and tested reagents. MG and creatol were determined by high-performance liquid chromatography using 9,10-phenanthrenequinone for the post-labeling. PAN increased MG and creatol synthesis in isolated rat hepatocytes by 60%. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a PKC inhibitor, at 10 and 100 microM significantly inhibited MG and creatol synthesis with or without PAN. The inhibition rate is dose dependent from 10 to 100 microM. H1004, a reagent used as control for H-7, did not affect (at 10 microM) or increased little (at 100 microM) the synthesis of MG and creatol. Ro31-8425, a potent PKC inhibitor, significantly inhibited (at 10 microM) MG synthesis in the presence of PAN. PKC in the membrane fraction, a marker of PKC activation, increased over the initial concentration by a factor of 1.65-fold at 60 min incubation and 2.16-fold at 120 min with PAN, while it changed little without PAN. These results indicate that PAN activates PKC resulting in increased hydroxyl radical generation in isolated rat hepatocytes.