Characteristics of a thyroid hormone responsive reporter gene transduced into a Xenopus laevis cell line using lentivirus vector

We introduced a self-inactivation (SIN) lentivirus vector (LV) into Xenopus laevis cell lines and established a permanent cell line expressing a reporter gene in a 3,5,3'-l-triiodothyronine (T(3)) dependent manner. The SIN LV contained the luciferase gene downstream from the X. laevis T(3)-response elements (TREs) and the SV40 promoter, and the enhanced green fluorescent protein (EGFP) gene downstream from the cytomegalovirus (CMV) promoter. It was integrated into the genome of X. laevis XL58, XTC2, and KR cells. The SIN LV transduced the X. laevis cells as efficiently as mammalian cells; however, the expression of EGFP in the transgene decreased with increasing culture time. A cell clone exhibiting the highest TH-dependent luciferase gene expression (XL58-TRE-Luc clone) was isolated from the EGFP-positive XL58 cell pool and characterized. The minimum effective concentration of T(3) that significantly induced the luciferase gene expression was 10(-11)M in the XL58-TRE-Luc clone. The application of the luciferase gene assay using the permanent XL58-TRE-Luc clone for the screening of thyroid-disrupting chemicals revealed that tetrachlorobisphenol A, at 10(-6)M, had a weak T(3)-agonist activity, whereas trichlorobisphenol A, at 10(-8) - 10(-6)M had a weak T(3)-antagonist activity. Our results indicated that the permanent X. laevis cell line containing a T(3)-response transgene could be used as a bioassay, with small intra-assay variation, for the rapid screening, identification, and characterization of the thyroid-disrupting chemicals.     

Alcohol-Metabolizing Enzyme Polymorphisms and Alcoholism in Japan

The liver enzymes, alcohol dehydrogenase (ADH) and aldehyde de-hydrogenase (ALDH), which are responsible for the oxidative metabolism of ethanol, are polymorphic in humans. Cytochrome P450IIE1 , an ethanol-inducible isozyme of liver microsomal P450 , is also important in ethanol metabolism. Genetic polymorphisms in the 5'-flanking region of the human cytochrome P450IIE1 gene have recently been reported. We hypothesized that the polymorphisms of ADH , ALDH , and P450IIE1 modify the susceptibility to development of alcoholism. We determined the genotypes of the ADH2 , ALDH2 , and P450IIE1 loci of 96 Japanese alcoholics and 60 healthy male subjects, using leukocyte DNA by the restriction fragment-length polymorphism by polymerase chain reaction. The alcoholics had significantly higher frequencies of the ADH2 ¹ and ALDH2 ¹ alleles than did the healthy subjects. No significant difference in the frequency of the P45011E1 genotype was observed between the alcoholics and the healthy subjects. In conclusion, genetic polymorphisms of the ADH and ALDH genes, but not of the P45011E1 gene, influence the risk of developing alcoholism in Japanese.     

Therapeutic bronchoscopy for stenotic lesions of central airway]

We retrospectively studied 26 patients with stenotic lesions of the central airway who had undergone bronchoscopic treatment. Thirteen of the patients had malignant tumors, and 13 had benign lesions (1 with post-tracheostomy difficulty in tube retrieval, 2 with bronchial obstruction due to granulation after bronchoplasty, 1 with bronchial stenosis due to granulation after pulmonary lobectomy, 7 with endobronchial foreign bodies, and 2 with hemoptysis). The patients ranged from 5 to 88 years of age with an average of 59.7 years. As a rule, bronchoscopic treatment was performed with endotracheal intubation up to 1993, and with laryngeal mask placement in 1994 and thereafter. Of the 26 eligible patients, 24 (92%) experienced good or excellent outcomes. Emergency bronchoscopy may be useful in treating a variety of emergency airway conditions. Use of a laryngeal mask may benefit emergency patients, including infants and children requiring a secure airway without complications.     

Radioimmunoassay and immunological properties of eel calcitonin

The immunological properties of eel calcitonin (CT) were studied by checking the cross-reactivities of eel CT or antisera to eel CT to various CT or antisera to these CT. Antisera to eel CT, raised in guinea pig, did not crossreact with either synthetic salmon, porcine or human CT. The antisera to salmon CT weakly crossreacted with eel CT, whereas eel CT did not crossreact with either antisera to porcine or human CT.     

Engagement of the CrkL adaptor in interferon alpha signalling in BCR-ABL-expressing cells

Interferon alpha (IFNalpha) has significant clinical activity in the treatment of patients with chronic myelogenous leukaemia (CML), but the mechanisms of its selective efficacy in the treatment of the disease are unknown. The CrkL adaptor protein interacts directly with the BCR-ABL fusion protein that causes the malignant transformation and is constitutively phosphorylated in BCR-ABL-expressing cells. In the present study, we provide evidence that CrkL was engaged in IFNalpha-signalling in the CML-derived KT-1 cell line, which expresses BCR-ABL and is sensitive to the growth inhibitory effects of IFNalpha. CrkL is constitutively associated with BCR-ABL in these cells and treatment with IFNalpha had no effect on the BCR-ABL/CrkL interaction. After IFNalpha stimulation, CrkL associated with Stat5, which also underwent phosphorylation in an IFNalpha-dependent manner. The interaction of CrkL with Stat5 was facilitated by the function of both the SH2 and the N-terminus SH3 domains of CrkL. The resulting CrkL-Stat5 complex translocated to the nucleus and could be detected in gel shift assays using elements derived from either the beta-casein promoter or the promoter of the PML gene, an IFNalpha-inducible gene that mediates growth inhibitory responses. In addition to its interaction with Stat5, CrkL interacts with C3G in KT-1 cells and such an interaction regulates the downstream activation of the small GTPase Rap1, which also mediates inhibition of cell proliferation. Thus, despite its engagement by BCR-ABL in CML-derived cells, CrkL mediates activation of downstream signalling pathways in response to the activated type I IFN receptor and such signals may contribute to the generation of the anti-proliferative effects of IFNalpha in CML.     

B cell hyperactivity and its relation to distinct clinical features and the degree of disease activity in patients with systemic lupus erythematosus

Peripheral blood B cells that were actively proliferating, those actively secreting immunoglobulin, and those expressing an early activation marker, Ba antigens, on the surfaces were quantitated in 25 patients with systemic lupus erythematosus (SLE). B cell hyperactivity was found in almost all of the SLE patients, as demonstrated by any one of these measures of B cell activity. Moreover, we observed a strong positive correlation between the degree of disease activity and the amount of spontaneous incorporation of 3H-thymidine by B cells; the magnitude of the increase in frequency of spontaneous Ig-secreting cells in peripheral blood correlated strikingly with certain clinical features in these patients. Our findings suggest that there is heterogeneity of B cell hyperactivity in individual patients with SLE and, thus, that clinical subsets of SLE can be identified on the basis of B cell hyperactivity.     

Electrocardiographic characteristics of repetitive monomorphic right ventricular tachycardia originating near the His-bundle

INTRODUCTION: Most idiopathic nonreentrant ventricular tachycardia (VT) and ventricular premature contractions (VPCs) arise from the right or left ventricular outflow tract (OT). However, some right ventricular (RV) VT/VPCs originate near the His-bundle region. The aim of this study was to investigate ECG characteristics of VT/VPCs originating near the His-bundle in comparison with right ventricular outflow tract (RVOT)-VT/VPCs. METHODS AND RESULTS: Ninety RV-VT/VPC patients underwent catheter mapping and radiofrequency ablation. ECG variables were compared between VT/VPCs originating from the RVOT and near the His-bundle. Ten patients had foci near the His-bundle (HIS group), with the His-bundle local ventricular electrogram preceding the QRS onset by 15-35 msec (mean: 22 msec) and His-bundle pacing produced a nearly identical ECG to clinical VT/VPCs. The HIS group R wave amplitude in the inferior leads (lead III: 1.0 +/- 0.6 mV) was significantly lower than that of the RVOT group (1.7 +/- 0.4 mV, P < 0.05). An R wave in aVL was present in 6 of 10 HIS group patients, while almost all RVOT group patients had a QS pattern in aVL. Lead I in HIS group exhibited significantly taller R wave amplitudes than RVOT group. HIS group QRS duration in the inferior leads was shorter than that of the RVOT group. Eight of 10 HIS group patients exhibited a QS pattern in lead V1 compared to 14 of 81 RVOT group patients. HIS group had larger R wave amplitudes in leads V5 and V6 than RVOT group. CONCLUSION: VT/VPCs originating near the His-bundle have distinctive ECG characteristics. Knowledge of the characteristic QRS morphology may facilitate catheter mapping and successful ablation.     

Serum tenascin levels in chronic liver disease

To evaluate the diagnostic significance of tenascin, the extracellular matrix glycoprotein in chronic liver disease, serum tenascin levels were measured by a newly developed ELISA in 21 patients with chronic persistent hepatitis, in 55 with chronic active hepatitis, in 59 with liver cirrhosis, in 31 with hepatocellular carcinoma, in 26 with acute hepatitis and in 66 healthy subjects. The serum tenascin level was significantly elevated in the patients with chronic active hepatitis, liver cirrhosis, hepatocellular carcinoma, and acute hepatitis when compared with the healthy subjects (P<0.001). The serum tenascin level also increased with increasing severity of chronic liver diseases. A significant correlation was observed between the serum tenascin levels and serum levels of various extracellular matrix proteins such as type III procollagen N-aminoterminal peptide (PIIIP), laminin and the 7S domain of type IV collagen (P<0.001). A strong positive correlation was observed between the serum tenascin levels and histologic findings, particularly in the degree of hepatic fibrosis. This is the first report documenting serum tenascin level increases in patients with various chronic liver diseases. The measurement of the serum tenascin levels may provide additional information relevant to the study of connective tissue.     

Characterization of Mandibular Bones in Senile Osteoporotic Mice

At present, little is known about the age-related changes in jaw bones. The aim of this study was to characterize the mandibles of 6 month-old senile osteoporotic mice, SAMP6, and compare with those of age-matched controls, SAMR1. In comparison to SAMR1, SAMP6 showed thinner cortical bone, lower bone volume, and poorly organized collagen matrix. The collagen fibril diameter in SAMP6 was significantly smaller than that of SAMR1. In SAMP6 both collagen content and cross-links were lower than those of SAMR1, but the ratio of the major mature cross-link (pyridinoline) to its precursor reducible cross-link (dehydrodihydroxylysinonorleucine/its ketoamine) was higher in comparison to SAMR1. In addition, the extent of lysine hydroxylation of collagen was higher in SAMP6 than that of SAMR1. These results indicate that not only the quantity of collagen but also its quality are altered in SAMP6 and may result in the age-associated osteoporotic defects of mandibles.