Binding mode of 4',6-diamidino-2-phenylindole and ethidium to poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex.

Optical spectroscopic properties of 4',6-diamidino-2-phenylindole (DAPI) and ethidium bromide complexed with poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex were compared in this study. When complexed with both duplex and triplex, ethidium is characterized by hypochromism and a red shift in the absorption spectrum, a complicate induced circular dichroism (CD) band in the polynucleotide absorption region, and a negative reduced linear dichroism signal in both polynucleotide and drug absorption regions. The spectral properties for both duplex- and triplex-bound ethidium are identical and both can be understood by the intercalation binding mode. In contrast, the absorption and CD spectra of DAPI complexed with triplex differ from those of the DAPI-duplex complex, although both complexes can be understood by the intercalation binding mode. Considering that the third strand runs along the major groove of the template duplex, we conclude that the DAPI molecule partially intercalates near the major groove of the duplex, where the third strand can affect its spectroscopic properties.

Structural effects of Ti underlayer on CoCrPt magnetic films.

The effects of long-range and short-range orders of Ti underlayer thickness on the magnetic properties of sputtered Co72 Cr21 Pt7 films were investigated using synchrotron X-ray scattering and X-ray absorption near-edge structure spectroscopy. The results were consistent with that of magnetic measurements and X-ray photoelectron spectroscopy. For thin Ti underlayers (10 nm), the oxidation of Ti and significant mixing of other elements within this underlayer did not promote texture development, further resulting in poor texturing of magnetic films and undesirable magnetic properties. Increased crystallinity and texture of metallic Ti in thicker underlayers enhanced the magnetic peak alignment and its properties.

Hepatic injury and lipid peroxidation during ischemia and reperfusion.

We determined the relationship between lipid peroxidation and alterations in hepatic secretory and microsomal function during various periods of hepatic ischemia/reperfusion. Rats were pretreated with alpha-tocopherol or vehicle and then subjected to 30, 60, and 90 min, no-flow hepatic ischemia in vivo with 1 or 5 h of reperfusion. Serum aminotransferase (ALT) level, wet-dry weight ratio, and lipid peroxidation were increased at 1 and 5 h of reperfusion, and these changes were significantly attenuated by alpha-tocopherol. Na+, K+-ATPase activity, and glucose-6-phosphatase activity were significantly decreased in 90-min ischemic rats, and these decreases were ameliorated by alpha-tocopherol. After 90 min of ischemia, bile flow, cholate output, and bilirubin output were markedly decreased by ischemia/reperfusion, and alpha-tocopherol restored the secretion. Cytochrome P450 content was decreased by ischemia/reperfusion and restored by alpha-tocopherol to the level of that found in the sham-operated group. Aminopyrine N-demethylase activity was decreased, and aniline p-hydroxylase was increased in 60-min ischemic rats. The changes in the activities of the two enzymes were prevented by alpha-tocopherol. Our findings suggest that ischemia/reperfusion diminishes hepatic secretory functions and microsomal drug metabolizing systems in proportion to the duration of ischemia and reperfusion in vivo, and this is associated with increased lipid peroxidation.

Effect of ursodeoxycholic acid on ischemia/reperfusion injury in isolated rat heart.

In this study, the effects of ursodeoxycholic acid (UDCA) on ischemia/reperfusion injury were investigated on isolated heart perfusion model. Hearts were perfused with oxygenated Krebs-Henseleit solution (pH 7.4, 37 degrees C) on a Langendorff apparatus. After equilibration, isolated hearts were treated with UDCA 20 to 160 microM or vehicle (0.04% DMSO) for 10 min before the onset of ischemia. After global ischemia (30 min), ischemic hearts were reperfused and allowed to recover for 30 min. The physiological (i.e. heart rate, left ventricular developed pressure, coronary flow, double product and time to contracture formation) and biochemical (lactate dehydrogenase; LDH) parameters were evaluated. In vehicle-treated group, time to contracture formation was 21.4 min during ischemia, LVDP was 18.5 mmHg at the endpoint of reperfusion and LDH activity in total reperfusion effluent was 54.0 U/L. Cardioprotective effects of UDCA against ischemia/reperfusion consisted of a reduced TTC (EC25=97.3 microM), reduced LDH release and enhanced recovery of cardiac contractile function during reperfusion. Especially, the treatments of UDCA 80 and 160 microM significantly increased LVDP and reduced LDH release. Our findings suggest that UDCA ameliorates ischemia/reperfusion-induced myocardial damage.

Protective effect of ethanol against acetaminophen-induced hepatotoxicity in mice: role of NADH:quinone reductase.

The role of NAD(P)H:quinone reductase (QR; EC 1.6.99.2) in the alcohol-derived protective effect against hepatotoxicity caused by acetaminophen (APAP) was studied. In mice pretreated with dicoumarol (30 mg/kg), an inhibitor of QR, hepatic necrosis caused by APAP (400 mg/kg) was potentiated. Hepatocellular injuries induced by APAP, as assessed by liver histology, serum aminotransferase activities, hepatic glutathione (reduced and oxidized) contents, and liver microsomal aminopyrine N-demethylase activities, all were potentiated by pretreatment of mice with dicoumarol. Even in mice given APAP and ethanol (4 g/kg), in which APAP-inducible hepatic necrosis was abolished, the dicoumarol pretreatment again produced moderate hepatotoxicity and reversed the protective effect of ethanol. In mice pretreated with dicoumarol and ethanol, levels of APAP in blood and bile fluid between 90 and 240 min were higher than those in mice given ethanol. However, the biliary contents of sulfate and glucuronide conjugates of APAP were much lower than those in the ethanol group, particularly at early time points. In contrast, the biliary level of APAP-cysteine conjugate, which in the ethanol group was at its basal level, was increased maximally in the dicoumarol-pretreated mice. In the mice given dicoumarol and ethanol, the biliary APAP-cysteine conjugate level was increased moderately. These results suggest that ethanol inhibited not only the microsomal (CYP2E1 mediated) formation of a toxic quinone metabolite from APAP, but also accelerated the conversion of the toxic quinone metabolite produced back to APAP by stimulating cytoplasmic QR activity. In the presence of dicoumarol, however, QR activity was inhibited, and conversion of the toxic quinone metabolite back to APAP became inhibited and diminished the alcohol-dependent protective effect against APAP-induced hepatic injury.

Systemically administered capsazepine prevents the capsaicin-induced functional desensitization and loss of substance P-like immunoreactivity (SP-LI) in guinea-pig bronchi.

In this study, we investigated whether the systemically administered capsazepine can prevent the capsaicin-induced desensitization ex vivo in guinea-pig bronchi. Pretreatment with capsaicin (2.5, 5 and 10 mg/kg, s.c.) induced the functional desensitization and the loss of substance P-like immunoreactivity (SP-LI) with a similar potency (ED50: 3.31 +/- 0.57 and 4.81 +/- 0.89 mg/kg, respectively) in isolated guinea-pig bronchi. Capsazepine (30 mg/kg, s.c.) co-administered with capsaicin (5 mg/kg, s.c.) prevented the capsaicin (5 mg/kg, s.c.)-induced functional desensitization and loss of SP-LI. These results suggest that capsazepine can antagonize systemically the desensitizing action of capsaicin at the level of receptor, preventing the loss of SP-LI and the establishment of functional desensitization in guinea-pig bronchi.

Cardioprotective effects of KR-30450, a novel K+(ATP) opener, and its major metabolite KR-30818 on isolated rat hearts.

The cardiac effects of KR-30450 ((-)-(2R)-2-([1,3]-dioxolan-2-yl)-2-methyl-4-(2-oxopyrrolidin++ +-1-yl)-6-nitro-2H-1-benzopyran), a newly synthesized potassium channel activator, and its major metabolite KR-30818 ((-)-(2R)-2-hydroxymethyl-2-methyl-4-(2-oxopyrrolidin-1-yl)-6-nitr o-2H-1-benzopyran) were compared with those of lemakalim, a prototype of this class, in isolated globally ischemic rat hearts. KR-30450 and KR-30818 significantly improved reperfusion cardiac function (LVDP, left ventricular developed pressure; double product, LVDP x heart rate/1000), their potency being 5.2-fold and 0.7-fold greater than lemakalim (ED50 for recovering predrug double product: 0.10, 0.80 and 0.54 microM, respectively). KR-30450 and KR-30818 significantly attenuated reperfusion contracture and lactate dehydrogenase release with potency greater than and equal to lemakalim, respectively. They significantly increased time to contracture (TTC) during ischemia in a dose-dependent manner with a greater potency than lemakalim (EC25 for increasing TTC: 1.2, 2.1 and 3.2 microM, respectively). The protective effects of three compounds on the measured parameters were reversed by glyburide, a selective K+(ATP) blocker. In non-ischemic hearts, KR-30450 and lemakalim exerted weak negative inotropism at high concentrations and KR-30818 had no effects, whereas the three compounds significantly increased coronary flow at doses studied. Glyburide completely reversed preischemic cardiodepressant effects of these compounds but not their effects on coronary flow. In conclusion, KR-30450, a recently developed K+(ATP) opener, exerted more potent cardioprotective effects than lemakalim, and its major metabolite KR-30818 may play a significant role in its action in vivo.

Capsaicin-induced desensitization is prevented by capsazepine but not by ruthenium red in guinea pig bronchi.

In isolated guinea pig bronchi, the influence of ruthenium red, capsazepine and extracellular Ca2+ on capsaicin-induced desensitization was examined to investigate whether this desensitization was mediated via a specific receptor coupled with an ion channel. Pre-exposure of tissues to capsaicin (1, 3 or 10 microM) caused a dose-dependent desensitization to the second application of capsaicin. However, the contractile responses to exogenous tachykinins were not changed after pre-exposure of tissues to capsaicin. This capsaicin-induced desensitization was prevented by capsazepine (30 microM), but not by ruthenium red added to tissues 20 min before pretreatment with capsaicin (3 microM). While the excitatory contractile response to capsaicin was markedly reduced in the absence of extracellular Ca2+, the desensitization induced by capsaicin was not changed by the removal of extracellular Ca2+. In summary, the results from the present study suggest that in vitro functional desensitization induced by capsaicin in guinea pig bronchi may involve changes in the vanilloid receptor and occur through a ruthenium red-insensitive pathway.

Binding mode of porphyrins to poly[d(A-T)(2)] and poly[d(G-C)(2)].

We examined the binding geometry of Co-meso-tetrakis (N-methyl pyridinium-4-yl)porphyrin, Co-meso-tetrakis (N-n-butyl pyridinium-4-yl)porphyrin and their metal-free ligands to poly[d(A-T)(2)] and poly[d(G-C)(2)] by optical spectroscopic methods including absorption, circular and linear dichroism spectroscopy, and fluorescence energy transfer technique. Signs of an induced CD spectrum in the Soret band depend only on the nature of the DNA sequence; all porphyrins exhibit negative CD when bound to poly[d(G-C)(2)] and positive when bound to poly[d(A-T)(2)]. Close analysis of the linear dichroism result reveals that all porphyrins exhibit outside binding when complexed with poly[d(A-T)(2)], regardless of the existence of a central metal and side chain. However, in the case of poly[d(G-C)(2)], we observed intercalative binding mode for two nonmetalloporphyrins and an outside binding mode for metalloporphyrins. The nature of the outside binding modes of the porphyrins, when complexed with poly[d(A-T)(2)] and poly[d(G-C)(2)], are quite different. We also demonstrate that an energy transfer from the excited nucleo-bases to porphyrins can occur for metalloporphyrins.

Effect of Trolox C on hypoxia/reoxygenation-induced injury in isolated perfused rat liver.

Livers isolated from 18 hours fasted rats were subjected to N(2) hypoxia (for 45 min) followed by reoxygenation (for 45 min). The perfusion medium used was Krebs-Henseleit bicarbonate buffer (KHBB, pH 7.4). Lactate and alanine were added as gluconeogenic and ureagenic substrates and Trolox C was also added to perfusate. Oxygen consumption, lactate dehydrogenase (LDH), alanine transaminase (ALT), total glutathione, oxidized glutathione, bile flow, glucose and urea were measured. After hypoxia oxygen consumption significantly dropped but Trolox C had no influence on this decrease. ALT and LDH were significantly increased by hypoxia/reoxygenation. This increase was markedly attenuated in the presence of Trolox C. The total glutathione and oxidized glutathione efflux increased following hypoxia, which were prevented by the treatment of Trolox C. Bile flow rate decreased following hypoxia/reoxygenation but did not continue to decrease in the reoxygenation phase by Trolox C. Following hypoxia/reoxygenation glucose and urea releases decreased. Trolox C had no influence on inhibition of glucose and urea production. These results suggest that Trolox C protected the liver cells against hypoxia/reoxygenation injury, yielding further evidence for a causative role of oxidative stress in this model.

Organotropic formation and disappearance of 8-hydroxydeoxyguanosine in the kidney of Sprague-Dawley rats exposed to adriamycin and KBrO3.

A form of oxidative DNA damage, 8-hydroxydeoxyguanosine (8-OHdG), was comparatively determined for 48 h in the kidney and liver isolated from Sprague-Dawley rats i.p. treated with Adriamycin; potassium bromate (KBrO3), hydroquinone and vitamin A. HPLC-ECD analysis system showed that Adriamycin and KBrO3, renal carcinogens, induced higher levels of 8-OHdG in the target organ of kidney (12-13.8 residues/10(4) dG(deoxyguanosine)) compared to those in the liver (3.4-3.8 residues/10(4) dG) and showed highly persistent levels (8 residues, 10(4) dG) in the kidney. The data suggest that the organotropic persistence of 8-OHdG may provide a useful marker for identifying target organ systems in oxidative chemical carcinogenesis and screening free radical-generating carcinogens.