cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt).

We isolated mouse cDNA clones (Arnt2) that are highly similar to but distinct from the aryl hydrocarbon receptor (AhR) nuclear translocator (Arnt). The composite cDNA covered a 2,443-bp sequence consisting of a putative 2,136-bp open reading frame encoding a polypeptide of 712 amino acids. The predicted Arnt2 polypeptide carries a characteristic basic helix-loop-helix (bHLH)/PAS motif in its N-terminal region with close similarity (81% identity) to that of mouse Arnt and has an overall sequence identity of 57% with Arnt. Biochemical properties and interaction of Arnt2 with other bHLH/PAS proteins were investigated by coimmunoprecipitation assays, gel mobility shift assays, and the yeast two-hybrid system. Arnt2 interacted with AhR and mouse Sim as efficiently as Arnt, and the Arnt2-AhR complex recognized and bound specifically the xenobiotic responsive element (XRE) sequence. Expression of Arnt2 successfully rescued XRE-driven reporter gene activity in the Arnt-defective c4 mutant of Hepa-1 cells. RNA blot analysis revealed that expression of Arnt2 mRNA was restricted to the brains and kidneys of adult mice, while Arnt mRNA was expressed ubiquitously. In addition, whole-mount in situ hybridization of 9.5-day mouse embryos showed that Arnt2 mRNA was expressed in the dorsal neural tube and branchial arch 1, while Arnt transcripts were detected broadly in various tissues of mesodermal and endodermal origins. These results suggest that Arnt2 may play different roles from Arnt both in adult mice and in developing embryos. Finally, sequence comparison of the currently known bHLH/PAS proteins indicates a division into two phylogenetic groups: the Arnt group, containing Arnt, Arnt2, and Per, and the AhR group, consisting of AhR, Sim, and Hif-1alpha.

Dioxin suppresses the checkpoint protein, MAD2, by an aryl hydrocarbon receptor-independent pathway.

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown recently to be carcinogenic, but little is currently known about the molecular mechanism of TCDD affecting cell proliferation and carcinogenesis. In this report, we demonstrate that TCDD suppresses the expression of the checkpoint protein, Mad2. Suppression of Mad2 was also observed in aryl hydrocarbon receptor-deficient mouse embryonic fibroblasts, suggesting that TCDD suppresses Mad2 by a novel TCDD receptor signaling mechanism. In addition, HeLa cells treated with TCDD failed to arrest in mitosis after nocodazole treatment. The Mad2 protein plays a significant role in accurate chromosome segregation in mitotic cells. Our data suggest that TCDD may increase chromosomal instability through the suppression of Mad2 expression.

A complete structure of the mouse Ah receptor gene.

A complete sequence of the Ah receptor gene was cloned from a mouse genomic library by using the Ah receptor cDNA as a probe. The Ah receptor gene is 37.5 kb long and is split into 11 exons by 10 introns. Sequence analysis of the 5' flanking region of the Ah receptor gene reveals that there is neither a TATA box nor a CAAT box in the promoter region. Instead, this gene has a few GC boxes and other enhancer elements in the 5' upstream flanking region. Southern blot analysis indicated that the Ah receptor gene is a unique gene.

Polymorphic forms of the Ah receptor and induction of the CYP1A1 gene.

Induction of cytochrome P4501A1 (CYP1A1) by certain xenobiotics is mediated by the Ah receptor/Arnt complex. The present knowledge about the molecular process of induction is summarized with special attention to our recent work on characterization of the polymorphic forms of the Ah receptor.

Dioxin induces a novel nuclear factor, DIF-3, that is implicated in spermatogenesis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; dioxin), a member of a class of environmental pollutants represented by polychlorinated dibenzo-p-dioxins and dibenzofurans, is one of the most toxic artificial compounds ever developed. In this study, we identified a novel TCDD target gene, DIF-3 (dioxin inducible factor-3), by cDNA representational difference analysis. DIF-3 protein is a nuclear factor and possesses a zinc-finger motif at its N-terminus. High DIF-3 mRNA expression in the testes was demonstrated by Northern blot analysis and abundant DIF-3 protein was detected during spermatogenesis. Thus, these results suggest that DIF-3 may be a target gene mediating the reproductive toxicity induced by TCDD.

Effect of angiotensin-converting enzyme inhibitor on cardiopulmonary baroreflex sensitivity in patients with acute myocardial infarction.

We evaluated the effect of angiotensin-converting enzyme inhibition (quinapril) on cardiopulmonary baroreflex sensitivity in 30 patients with uncomplicated myocardial infarction (quinapril group, 15 patients; placebo group, 15 patients) at 5 and 10 days after the onset of myocardial infarction. This study indicates that quinapril improved cardiopulmonary baroreflex and thus reduced sympathetic outflow in patients with acute myocardial infarction.

Parathyroid hormone (PTH) down-regulates PTH/PTH-related protein receptor gene expression in UMR-106 osteoblast-like cells via a 3',5'-cyclic adenosine monophosphate-dependent, protein kinase A-independent pathway.

Parathyroid hormone (PTH) regulates osteoblast function via a G protein-linked PTH/PTH-related protein (PTHrP) receptor. We have studied the mechanisms of PTH/PTHrP receptor gene repression by PTH in UMR-106 osteoblast-like cells. Inhibition of PTH/PTHrP receptor mRNA expression by rat (r) PTH(1-34) and Insulin-like growth factor-I (IGF-I) at 10(-7)M was significant at 1 h and 3 h, and maximal at 2 h and 6 h. A maximal decrease in receptor mRNA abundance by rPTH(1-34) and IGF-I was maintained for 24 h. Inhibition of receptor gene expression by rPTH(1-34) was mimicked in UMR-106 cells by the addition of forskolin (an adenylyl cyclase activator), or 8-(4-chlorophenylthio)-adenine 3',5'-cyclic monophosphate (8-pCPTcAMP; a cAMP analogue). Although H89, a selective protein kinase A (PKA) inhibitor, completely inhibited PKA activity stimulated by rPTH(1-34), forskolin or 8-pCPTcAMP, suppression of PTH/PTHrP receptor mRNA synthesis induced by these substances in UMR-106 cells was not affected by H89. In primary osteoblast cultures, rPTH(1-34) inhibited synthesis of PTH/PTHrP receptor mRNA irrespective of H89. The down-regulation effect of rPTH(1-34) was also unaltered by PD98059 (an extracellularly regulated kinase 1/2 mitogen-activated protein kinase pathway inhibitor). Pretreatment with cycloheximide, a protein synthesis inhibitor, did not alter the inhibition of PTH/PTHrP receptor mRNA expression by rPTH(1-34), indicating that receptor mRNA suppression does not require new protein synthesis. Transcriptional activation of PTH/PTHrP receptor gene promoter (U3P or U4P)-luciferase constructs was decreased by rPTH(1-34), forskolin and 8-pCPTcAMP irrespective of H89. Thus, PTH transcriptionally down-regulates PTH/PTHrP receptor gene expression in osteoblast-like cells via a cAMP-dependent, PKA-independent pathway.

Budd-Chiari syndrome and extrahepatic portal obstruction associated with congenital antithrombin III deficiency.

We report a patient with Budd-Chiari syndrome (BCS) and extrahepatic portal obstruction (EHO) associated with congenital antithrombin (AT) III deficiency. A 35-year-old man was admitted to Nishi Kobe Medical Center for evaluation of abnormal intrahepatic veins. By various imaging modalities, BCS and EHO were diagnosed. Laboratory data revealed parallel decreases in activity and antigen concentration of AT III despite normal liver function. Taken together, the etiology of both BCS and EHO was considered to be thrombosis, associated with congenital AT III deficiency. Two years after beginning warfarin therapy, the patient has no symptoms and his liver function remains normal. Anticoagulant therapy is considered useful for preventing progression of the disease.

A case of localized primary sclerosing cholangitis mimicking intrahepatic bile duct cancer.

A 75-year-old woman with chronic hepatitis was regularly followed-up in our hospital. A computed tomography (CT) scan revealed an obviously enlarged intrahepatic bile duct in the posterior branch of the left lateral segment. Percutaneous cholangiography revealed an enlarged posterior branch of the left lateral segment and a narrow stenotic region at the root of this branch. We diagnosed her as having intrahepatic bile duct cancer, and a left lateral segmentectomy of the liver was performed. However, microscopic examination of the resected specimens revealed peribiliary fibrosis in the stenotic bile duct and other areas of the intrahepatic bile duct with no malignant cells. Thus, the final diagnosis was made to be primary sclerosing cholangitis. We must consider primary sclerosing cholangitis in the differential diagnosis of localized stenosis of the intrahepatic bile duct.

Portal venous volume flow: in vivo measurement by time-domain color-velocity imaging.

The portal venous velocity and flow volume in 39 patients (16 with liver cirrhosis, 11 with chronic hepatitis, 12 without liver disease) were measured using both color velocity imaging quantification (CVI-Q) and conventional Doppler flowmetry. The average portal venous velocity and flow volume values obtained using the two methods were similar. The correlation coefficients for the paired measurements show positive correlations (velocity: 0.73, p < 0.0001; volume: 0.50, p = 0.001). However, the coefficients of variation between the two methods were not good (velocity: 14.9%, volume: 26.4%). In conventional Doppler flowmetry, the mean velocity to maximum velocity ratio (Vmean:Vmax) is assumed to be constant (Vmean:Vmax = 0.57 in this study). However, the Vmean:Vmax ratios calculated from the flow profile in CVI-Q were 0.67 +/- 0.13 in the patients with liver cirrhosis, 0.58 +/- 0.13 in the patients with chronic hepatitis, and 0.53 +/- 0.08 in the patients without liver disease. Therefore, a measurement method that takes the blood flow profile into account, such as CVI-Q, might be useful for the quantitative measurement of the portal venous velocity and volume.

cDNA cloning and predicted primary structure of scallop sarcoplasmic reticulum Ca(2+)-ATPase.

Sarcoplasmic reticulum (SR) Ca(2+)-ATPase of the scallop cross-striated adductor muscle was purified with deoxycholate and digested with lysyl endopeptidase for sequencing of the digested fragments. Overlapping cDNA clones of the ATPase were isolated by screening the cDNA library with an RT-PCR product as a hybridization probe, which encodes the partial amino acid sequence of the ATPase. The predicted amino acid sequence of the ATPase contained all the partial sequences determined with the proteolytic fragments and consisted of the 993 residues with approximately 70% overall sequence similarity to those of the SR ATPases from rabbit fast-twitch and slow-twitch muscles. An outline of the structure of the scallop ATPase molecule is predicted to mainly consist of ten transmembrane and five 'stalk' domains with two large cytoplasmic regions as observed with the rabbit ATPase molecules. The sequence relationship between scallop and other sarco/endoplasmic reticulum-type Ca(2+)-ATPases is discussed.

Endoscopic resection of small inflammatory fibroid polyp of the colon.

Inflammatory fibroid polyp (IFP) is a solitary intestinal lesion of unknown etiology. Although IFP is benign, laparotomy for the resection of colonic IFP is performed in most cases because the polyp is usually large. We report a successful endoscopic resection of cecal IFP. It is considered that colonic IFP should be resected endoscopically if the polyp is small and is located submucosally.

Colonic ulceration caused by administration of loxoprofen sodium.

A 54-year-old female with chronic headache was admitted to our hospital because of hematochezia. She had routinely taken loxoprofen sodium because of severe headache. Emergent colonoscopic examination revealed ulceration of the cecum. After administration of loxoprofen sodium was discontinued and administration of sulfasalazine was initiated, her intestinal bleeding subsided. Two months after discontinuation of loxoprofen sodium, the colonoscopic examination revealed scar formation at the site of cecal ulceration. In this case, it was conceivable that the administration of loxoprofen sodium might have induced colonic ulceration.

[Tumor hemodynamics in hepatic nodules associated with liver cirrhosis: relationship between cancer progression and tumor hemodynamic change].

Tumor hemodynamics including arterial vascularity (AV) and portal perfusion (PP) were evaluated in histologically confirmed 55 hepatic nodules associated with cirrhosis using ultrasonographic (US) angiography during intraarterial carbon dioxide microbubbles injection and CT during arterial portography. Tumor hemodynamic patterns were classified into 6 types as follows: Type I (n = 10): PP (+), AV (hypo); Type I' (n = 2): PP (+), AV (iso); Type II (n = 5): PP (-), AV (hypo); Type III (n = 8): PP (-), AV (iso); Type IV (n = 25): PP (-), AV (hyper), Type V (n = 5): PP (partially +), AV (vascular spot in hypovascular). Eight nodules of Type I were diagnosed as benign nodules histologically including adenomatous hyperplasia (AH) (n = 6) and regenerative nodule (n = 2). Hundred percent (5/5) of Type II and 88% (7/8) of Type III nodules were well-differentiated HCC, in contrast to 8% (2/25) of Type IV nodules, typical HCCs. Fatty metamorphosis was observed in 75% (6/8) of Type III nodules, in contrast to 16% (4/25) of typical (classical) HCC nodules (Type IV). We concluded that at the malignant transformation from AH to HCC, reduction of portal blood flow in the nodule precedes the initiation of the increase of the arterial tumor vessel. Moreover, early stage HCC could exhibit hypovascular (Type I, II), isovascular (Type III), or vascular spot in hypovascular pattern (Type V) compared with a typical HCC (Type IV). It was also suggested that the more mature as a neoplasms the HCC becomes, the more the arterial tumor vessel in the nodule increases and fatty metamorphosis of well-differentiated HCC is highly related with tumor hemodynamic condition, i.e., hypoperfusion state from both arterial and portal vessel.

[Clinical utility of receptor imaging in the assessment of liver function].

Technetium-99m diethylene triamine pentaacetic acid-galactosyl human serum albumin (TcGSA) is a newly developed receptor-binding radiopharmaceutical, specific for the asialoglycoprotein receptor, which resides exclusively on the plasma membrane of hepatocytes. Clinical utility of TcGSA was evaluated in 3 control subjects with normal livers and in 54 patients with various liver diseases. The parameter, Receptor Index, was derived from liver and heart time-activity data and is the ratio of radioactivity of the liver over the radioactivity of the liver plus heart at 15 min after the intravenous injection of 3 mg of TcGSA. Receptor concentration ([R]o) was obtained by kinetic analysis of liver and heart time-activity data using pharmacokinetic nonlinear modeling. Values for the Receptor Index and [R]o was statistically different in the control subjects and in patients with mild, moderate, and severe liver diseases. Good correlations were obtained between the Receptor Index, [R]o and conventional liver function tests, such as Child-Turcotte criteria score, prothrombin time, and indocyanine green test. Receptor Index and [R]o were properly estimated even in patients with obstructive jaundice or remarkable portocaval shunt. These data suggest that the receptor imaging as well as its parameters, Receptor Index and [R]o, is a potentially practical and reliable diagnostic method for estimating the functioning hepatocyte mass and for assessing liver function.

[Hepatic receptor imaging with Tc-99m GSA: estimates of liver function in acute liver disease].

Technetium-99m galactosyl human serum albumin (TcGSA) is a synthesized radiolabeled analog ligand to asialoglycoprotein receptor, which resides only at a mammalian hepatocyte. TcGSA studies were performed on 16 patients with various acute liver disease and 3 controls with normal livers. Dynamic data were obtained by a gamma camera during 35 minutes after an intravenous injection of 3 mg (185 MBq) of TcGSA. The parameters of TcGSA time activity curves were obtained by dividing radioactivity of the liver by that of the liver plus heart at 15 min (Receptor Index), and by dividing radioactivity of the liver at 15 min by that at 3 min post injection (Clearance Index). The two parameters correlated well with prothrombin time, clinically estimated staging, and severity of acute liver disease. We have concluded that liver function study by the newly developed receptor imaging with TcGSA can be a sensitive and promising tool in estimating the severity and prognosis of acute liver disease.

[Small nodular lesions of the pancreas: differential diagnosis with ultrasound angiography].

Ultrasound angiography (USAG), sonographic imaging of the blood flow in an organ or tissue obtained by carbon dioxide infusion into the supplying artery, was performed on 28 pancreatic nodular lesions less than 3 cm in diameter. The hemodynamics of tumors observed with USAG were divided into three groups: hypovascular, isovascular, and hypervascular, compared with the adjacent pancreatic tissue. Most of hypovascular nodules were duct cell carcinoma (sensitivity 94.1%, specificity 90.4%), while isovascular lesion was the characteristic of inflammatory masses (sensitivity 100%, specificity 95.8%). Hypervascular cases included all of the mucin producing tumors and islet cell tumors but only one case of duct cell carcinoma. So you can almost exclude duct cell carcinoma as an diagnosis in vascular rich tumors (negative predictive value 83.6%). These results were compared with those on conventional x-ray angiograms and incremental CT scans. Ultrasound angiography enabled us to detect more slight differences of tumor vascularity than the other modalities. Thus we conclude that USAG can be a useful diagnostic aid in small mass lesions of the pancreas.