؊4 M carbamylcholine for 16 h. However, the rate of down-regulation was lower compared with wild type receptors (t1 ⁄2 ‫؍‬ 9.9 versus 2.3 h). These results indicate that rapid internalization of hm2 receptors is facilitated by their phosphorylation with GRK2 and does not occur in the absence of the third intracellular loop, but downregulation of hm2 receptors may occur through both GRK2-facilitating pathway and third intracellular loopindependent pathways.Loss of the cell's response to agonist acting at G proteincoupled receptors can occur in three phases: uncoupling from G proteins, sequestration/internalization, and down-regulation of the receptors. Many G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases (GRKs) 1 in an agonist-dependent manner as a major mechanism in receptor regulation (for review, see Refs. 1 and 2). Muscarinic acetylcholine receptor m2 subtypes have also been shown to be phosphorylated by GRK2 (␤-adrenergic receptor kinase 1) (3, 4) and other GRKs (5, 6). In addition, phosphorylation of ␤ 2 -adrenergic receptors by GRK2 may be involved in the uncoupling of ␤ 2 -adrenergic receptors from G protein G s (1, 2). The phosphorylated ␤ 2 -adrenergic receptors were reported to be uncoupled from G proteins because of their interaction with ␤-arrestin (1, 2, 7). Indeed, phosphorylation by GRKs has been reported to correlate with uncoupling for several G protein-coupled receptors including muscarinic m2 (8), ␣ 1B -adrenergic (9), ␣ 2 -adrenergic (10), thrombin (11), dopamine D1A (12), and thyrotropin receptors (13). ␤-Arrestin and arrestin 3 (␤-arrestin 2) were shown to interact with m2 muscarinic as well as with ␤ 2 -adrenergic receptors (14,15). Sequestration/internalization of ␤ 2 -adrenergic receptors seemed to be independent of phosphorylation by GRK2 on the basis of results with ␤ 2 -adrenergic receptor mutants lacking phosphorylation sites or GRK-specific inhibitors (16 -20). On the other hand, the agonist-induced sequestration of hm2 receptors expressed in HEK293 cells is hampered by deletion of the third intracellular loop (I3-loop) which includes the GRK2 phosphorylation sites (21,22). Moreover, agonist-dependent phosphorylation and sequestration of m2 receptors expressed in COS-7 cells are facilitated by coexpression of GRK2 and attenuated by coexpression of a dominant-negative mutant of GRK2 (DN-GRK2) that lacks kinase activity (23). Recently, Ferguson et al. have reexamined the relationship between the phosphorylation by GRK2 and sequestration of ␤ 2 -adrenergic receptors, demonstrating that phosphorylation by GRK2 (24) or other GRKs (25) facilitates sequestration of ␤ 2 -adrenergic receptors. Phosphorylation facilitates ␤-arrestin binding to ␤ 2 -adrenergic receptors (26) and thereby appears to enhance sequestration, possibly interacting with clathrin (27), a major * This work was supported in part by grants from the Japan Society for the Promotion of Science, the Ministry of Education, Science, and Culture of Japan, and the Japan Health Science Founda...

This paper is available online at http://dmd.aspetjournals.org ABSTRACT:Genotype analysis of the aldehyde dehydrogenase (ALDH)-2 gene was performed using an improved simplified method, and effects of the genotype on the metabolism of a variety of aldehydes in different fractions of human liver cells were investigated. The effects of sex, aging, smoking, drinking alcohol, liver function, and various drugs on ALDH activity were also analyzed. Of the 39 subjects, eight were heterozygotes of the wild (ALDH2*1) and mutant (ALDH2*2) alleles, and the others were homozygotes of the wild allele. ALDH activity toward acetaldehyde in liver mitochondria from subjects with a mutant allele was less than 10% of that with two alleles of wild-type, and the activities toward formaldehyde, propionaldehyde, n-butyraldehyde, capronaldehyde, and heptaldehyde were also significantly lower in the ALDH2*1/*2 rather than ALDH2*1/*1 group. However, the metabolism of octylaldehyde, decylaldehyde, retinaldehyde, benzaldehyde, 3-hydroxybenzaldehyde, and 2,5-dihydroxybenzaldehyde was similar in the two genotypes. Changes in activity in the cytosolic fraction were similar to those in mitochondria. There was no significant difference in ALDH activity in microsomes between the two groups. Total activities of ALDH toward acetaldehyde and other shortchain aliphatic aldehydes in supernatant fractions of homogenized liver were affected in a manner similar to that in mitochondria. Our results suggest that the single nucleotide polymorphisms of the ALDH2 gene only alter the metabolism of aldehydes with a short aliphatic chain. Furthermore, sex, drinking alcohol, and smoking had little effect on ALDH activity, although the activity in elderly individuals tended to be lower albeit statistically insignificant.

[Structure: see text] A 1,4-bis(phenyl)-1,4-dihydro[60]fullerene resulting from an efficient nucleophilic substitution has been obtained by reaction of a fullerene epoxide, C60O, with nucleophilic aromatic compounds in the presence of boron trifluoride etherate as a Lewis acid.

Bisphenol A (BPA), a xenoestrogen, has been reported to mimic the actions of estrogen or to affect the endocrine glands in vivo and in vitro. In this study, we examined whether in utero and lactational exposure to BPA altered the somatic growth and anogenital distance (AGD) of F 1 offspring (1, 3, and 9 weeks of age) in vivo in rats. Dams were orally administered with various doses of BPA (0, 4, or 40 mg/kg body weight (BW)/day) from gestation day (GD) 6 through postnatal day (PND) 20. There were no significant changes in body weight, liver weight, kidneys weight, testes weight, AGD, the ratio of AGD to BW, or the ratio of AGD to the cube root of BW in BPA exposed pups compared to the vehicle-exposed control. This suggests that prenatal and postnatal exposure (indirect exposure) to BPA (4-40 mg/kg/day, GD 6-PND 20) does not affect on somatic growth or AGD of F 1 generation of male and female rats.

The efficacy and safety of intrapleural LC9018 (Yakult Co. Ltd., Tokyo, Japan) with or without doxorubicin (Adriamycin; Adria Laboratories, Columbus, OH) were evaluated in a randomized, controlled trial performed in 95 patients with malignant pleural effusions secondary to lung cancer. Seventy‐six patients were eligible for the assessment of efficacy. The response rate for treatment with intrapleural doxorubicin plus LC9018 (38 patients) was 73.7%, which was significantly higher than the response rate of 39.5% for the control group treated with doxorubicin alone (38 patients) (P < 0.01). The LC9018 group also showed a significantly greater improvement in performance status (PS) and symptoms (chest pain, chest discomfort, and anorexia) than the control group (P < 0.05). A significant prolongation of survival was noticed in the LC9018 group (P < 0.05). The main side effects of LC9018 were fever and transient hepatic dysfunction, but there were no serious adverse reactions. These results suggest that the intrapleural instillation of LC9018 can be recommended for the treatment of malignant pleural effusions.

Long-term consumption of large amounts of alcohol is the main cause of chronic pancreatitis. All heavy drinkers, however, do not contract chronic pancreatitis. Although genetic predisposition to alcoholism and alcoholic liver disease has been reported, genetic susceptibility to alcoholic pancreatitis is still a matter of debate. To determine the relation between genotypes of alcohol-metabolizing enzymes and chronic alcoholic pancreatitis, we examined genotype patterns of aldehyde dehydrogenase 2 (ALDH 2), alcohol dehydrogenase 2 (ADH 2) and cytochrome P-4502E1 (CYP2E1) in 54 patients with chronic alcoholic pancreatitis who were diagnosed in general hospitals in all over Japan and compared with those in 30 patients with chronic nonalcoholic pancreatitis or in 46 alcoholics with normal pancreatic function. There were no significant differences in the distribution of genotypes of ALDH 2 and CYP2E1 among those three groups. As for the ADH 2 genotype, distribution of 2(1)/2(1), 2(1)/2(2), and 2(2)/2(2) was 35%, 30%, and 35% in alcoholics with normal pancreatic function; 4%, 39%, and 57% in the chronic alcoholic pancreatitis group; and 0%, 50%, and 50% in the chronic nonalcoholic pancreatitis group, respectively. The frequency of ADH 2(2) allele was significantly higher in the chronic alcoholic pancreatitis group, compared with alcoholics with normal pancreatic function; but, it was not significantly different from that in the chronic nonalcoholic pancreatitis group. We also examined the relation between pancreatic fibrosis or pancreatitis histologically diagnosed and genotypes of alcohol-metabolizing enzymes in alcoholic autopsy cases. Twenty of 31 cases showed moderate or severe pancreatic fibrosis and showed intralobular + interlobular fibrosis, which is characteristic in alcoholic pancreatitis or intralobular fibrosis. ADH 2(2) allele tended to show a high frequency in the intralobular + interlobular fibrosis group, compared with that in the intralobular fibrosis group (75.0% vs. 41.7%, p < 0.1). The chronic pancreatitis group had a significantly higher frequency of the ADH 2(2) allele than that in cases without such findings (87.5% vs. 58.7%, p < 0.05). However, the ALDH 2 and CYP2E1 genotypes showed no significant relation to the findings of pancreatic fibrosis or histological pancreatitis. These data suggest that the risk of chronic alcoholic pancreatitis diagnosed clinically and pathologically seems to be associated with the ADH 2(2) allele in the genotypes of alcohol-metabolizing enzymes.

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Exposure to polychlorobiphenyl (PCB) mix-tures at an early stage of development has been reported to affect endocrine glands; however, little is known about the precise toxicological properties of individual PCB. The present study was undertaken to determine whether prenatal exposure to 2,2Ј,4,4Ј,5,5Ј-hexachlorobiphenyl (PCB 153), a di-ortho-substituted non-coplanar congener, affects postnatal development in rat offspring. Pregnant Sprague-Dawley rats (Crj: CD (SD) IGS) were given PCB 153 (0, 16, or 64 mg/kg/ day) orally from gestational day (GD) 10 through GD 16, and developmental parameters in the male and female offspring were examined. We found no dose-dependent changes in body weight, body length (nose-anus length), tail length, or the weights of kidneys, testes, ovaries and uterus in offspring at 1 or 3 weeks of age. Liver weights were increased in the PCB 153-treated groups, although we observed a significant difference only in males. Anogenital distance was unaffected in the PCB 153-treated groups. We observed a significant dosedependent decrease in the plasma concentrations of thyroxine and tri-iodothyronine, whereas those of thyroid-stimulating hormone were not significantly changed. In addition, there were no dose-dependent changes in plasma concentrations of growth hormone and insulin-like growth factor-I in any dose group. These findings suggest that prenatal exposure to PCB 153 (GD 10-16, 16-64 mg/kg/day) may alter the thyroid status in rat offspring to some extent without affecting somatic growth or its related hormonal parameters.