Regulation of thyroid follicular cell function by intracellular redox-active copper.

Pyrrolidine dithiocarbamate (PDTC) is a metal-chelating compound that exerts prooxidant or antioxidant effects and is widely used to study redox regulation of cell function. In the present study, we investigated effects of PDTC on the function of rat thyroid follicular FRTL-5 cells. Treatment of the cells with PDTC resulted in a marked decrease in Pax-8 messenger RNA level and its DNA-binding activity. This decrease was associated with a significant reduction in thyroperoxidase (TPO) messenger RNA level. Expression of TTF-1 and thyroglobulin was not affected by PDTC. Treatment with PDTC also decreased DNA-binding activity of p53, a tumor suppressor protein, and increased cell proliferation rates. These changes were not observed by the treatment with another antioxidant, N-acetyl-L-cysteine, suggesting that the metal-chelating, prooxidant property of PDTC is responsible for its effects. Indeed, the intracellular level of copper was significantly increased by PDTC. Treatment with bathocuproinedisulfonic acid, a noncell-permeable chelator of Cu1+, abrogated the copper increase by PDTC and its effects on Pax-8 and TPO expression as well as on p53 binding. Taken together, these results indicate that the intracellular level of redox-active copper is crucial for Pax-8 and TPO expression and for proliferation of thyroid follicular cells.

Expression of E16/CD98LC/hLAT1 is responsive to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

We employed cDNA representational difference analysis to identify new genes that are upregulated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a human hepatoblastoma cell line HepG2. We isolated several TCDD-responsive cDNAs. Sequence analysis revealed that one of them encodes E16/CD98LC/hLAT1, an integral membrane protein involved in multiple cellular functions including cellular transport of L-type amino acids. Northern blot analysis confirmed the TCDD-dependent upregulation of the mRNA. Induction of E16/CD98LC/hLAT1 mRNA by TCDD did not require de novo protein synthesis as revealed by the experiment using cycloheximide. Consistent with the changes at mRNA level, the transport of 3H-leucine into HepG2 cells was significantly increased by TCDD treatment. These findings provide a novel aspect of biological effects of TCDD on human hepatocytes.

Highly diastereoselective dihydride formation by activation of methanol with IrCl((S)-binap)(PPh3).

Reaction of [IrCl((S)-binap)(PPh3)] ((S)-3) with methanol gave one of the diastereomers of the cis,mer-dihydride, cis,mer-OC-6-44-A-[IrCl(H2)((S)-binap)(PPh3)] ((S)-4a) stereoselectively, the structure of which was determined crystallographically, whereas the reaction of (S)-3 with H2 produced a 1:1 mixture of the diastereomers of the cis,mer-dihydride, (S)-4a and cis,mer-OC-6-44-C-[IrCl(H2)((S)-binap)(PPh3)] ((S)-4b).

[Isoflurane and sevoflurane impair left ventricular relaxation in dogs with fixed heart rate].

The effects of isoflurane (Iso) and sevoflurane (Sev) on left ventricular relaxation were evaluated in 22 open-chest dogs with fixed heart rate (130 beats.min-1) using atrial pacing. Fentanyl was injected intravenously to maintain anesthesia during the preliminary preparation. In both Iso and Sev groups (n = 11), left ventricular systolic pressure, mean aortic pressure and dp/dt max were significantly decreased at 0.5 MAC, but there was no significant change in left ventricular end-diastolic pressure. Left ventricular systolic function was depressed to the similar extent in both groups. In Sev group, -dp/dt max and time constant of isovolumic left ventricular pressure fall (T) increased significantly at 0.5 MAC but it increased at 1.5 MAC in Iso group. T at 0.5 MAC Sev was also significantly longer compared with T at equipotent Iso. These findings suggest that Sev may impair isovolumic left ventricular relaxation more strongly than Iso, and this may result from the difference of the effect of each agent on intracellular Ca2+ homeostasis in the myocardium.

[Left ventricular function during hypoxia--II: Effects of intramyocardial pH].

We investigated the effects of intramyocardial acidosis on cardiac function during hypoxia in mongrel dogs (n = 50). Intramyocardial pH in subendocardium of left ventricle was measured continuously using a pH electrode. During hypoxia (PaO2 = 20 mmHg) caused by inhalation of low oxygen fraction, intramyocardial pH decreased significantly following initial enhancement. Intramyocardial pH correlated significantly with arterial pH, base excess, lactic acid (LA) levels, coronary venous PcO2, and coronary venous-arterial PcO2 difference. There were significant correlations between intramyocardial pH with maximum rate of rise of left ventricular pressure (LV dp/dt max), left ventricular end-diastolic pressure (LVEDP), LVEDP/LVP and the time constant of exponential isovolumic left ventricular pressure fall. High arterial LA levels tended to cease myocardial LA uptake and production, turning LA balance into zero. LV dp/dt max was low in such a condition. Myocardial LA production decreased intramyocardial pH, but LV dp/dt max was maintained at high levels of LA production. These observations suggest that myocardial LA production maintains left ventricular function with the increase of ATP by acceleration of anaerobic glycolysis. In conclusion, during hypoxia, intramyocardial acidosis was caused by the increase of arterial LA level, myocardial anaerobic glycolysis and ATP breakdown, and it worsened left ventricular contractile function and relaxation. Arterial LA levels can play a major part in intramyocardial acidosis, but the increase in myocardial LA production might be beneficial to left ventricular contractile function.

[Effects of sevoflurane on regional myocardial oxygen balance in the canine heart with coronary artery stenosis].

The effects of increasing inspired sevoflurane (Sev) concentrations (0.5, 1.0, 1.5, 2.0 MAC) on regional myocardial oxygen balance and metabolism were studied in 10 open-chest dogs with coronary artery stenosis. Regional myocardial oxygen balance was assessed by continuous recording of subendocardial and subepicardial oxygen tensions. After the basal measurement, the left circumflex coronary artery blood flow (CBFLCX) was reduced by 20% with a screw flow regulator. The institution of stenosis caused a slight but significant decline in subendocardial oxygen tension. Otherwise, there were no significant differences between basal values and those after the stenosis. During the subsequent inhalation of Sev at 0.5, 1.0 and 1.5 MAC, endocardial oxygen tension was maintained at the level of control value. Sev 2.0 MAC caused a severe reduction in systemic arterial pressure and resulted in a marked and significant decline of endocardial oxygen tension compared with control value. On the other hand, subepicardial oxygen tension remained unchanged during each Sev inhalation. The ratio of epicardial/endocardial oxygen tension did not show a significant decline with increasing Sev concentration. It seems that Sev may not possess sufficient potency to cause myocardial ischemia by redistribution of coronary blood flow.

Genetic and biochemical studies of the highly active esterases A' and B associated with organophosphate resistance in mosquitoes of the Culex pipiens complex.

The highly active esterases A' and B that cannot be dissociated from OP resistance in Culex pipiens from France and California are shown to have equivalent Km values (2.1 x 10(-6) M/min/mosquito) but different turnover rates (Vm = 2.13 and 0.57 x 10(-6) M/min/mosquito, respectively) and pH for maximum activity. Both enzymes have broad substrate specificities and at least one, esterase A', can hydrolyze OP insecticides. In addition, esterases A' and B are coded by two closely linked genes, Est-3 and Est-2, respectively (0.67 unit of crossing over), located on the same autosome as pl, a locus attributed to linkage group III. The estimated distance between Est-2 and pl was 9.4 units.

Pyrrolidine dithiocarbamate inhibits TNF-alpha-dependent activation of NF-kappaB by increasing intracellular copper level in human aortic smooth muscle cells.

Pyrrolidine dithiocarbamate (PDTC) is a metal-chelating compound that acts as antioxidant or pro-oxidant and is widely used to study redox regulation of cell function. In the present study, we investigated effects of PDTC and another antioxidant, N-acetyl-l-cysteine (NAC), on TNF-alpha-dependent activation of NF-kappaB in human aortic smooth muscle cells (HASMC). Treatment of the cells with TNF-alpha induced the activation of p65/p50 heterodimer NF-kappaB and increased the mRNA levels of monocyte chemoattractant protein (MCP)-1. Pretreatment with PDTC markedly suppressed the NF-kappaB activation and expression of MCP-1 by inhibiting IkappaB-alpha degradation. In contrast, NAC had no effect. PDTC concomitantly increased the intracellular levels of copper, and bathocuproinedisulfonic acid, a non-cell-permeable chelator of Cu(1+), inhibited the PDTC-induced increase in intracellular copper level and reversed the PDTC effects on IkappaB-alpha, NF-kappaB, and MCP-1. These results indicate that TNF-alpha-dependent expression of MCP-1 in HASMC is tightly regulated by NF-kappaB and that intracellular copper level is crucial for the TNF-alpha-dependent activation of NF-kappaB in HASMC.