新方法随机筛选血管内皮生长因子受体1激酶的抑制剂

目的:利用重组血管内皮细胞生长因子(VEGF)受体1激酶蛋白质建立大规模随机筛选分子模型.方法:重组人源VEGF受体1激酶蛋白质的催化活性通过酶联免疫法检测96孔板上的底物磷酸化程度得到.用大规模随机筛选寻找抑制剂,并在稳定表达VEGF受体1的细胞系上研究它们的性质.结果:在研究VEGF受体1激酶蛋白质大肠杆菌表达体系的基础上,建立了大规模筛选模型.通过对2800个有机化合物的筛选,找到了两个二取代呋喃类的VEGF受体1激酶抑制剂(A1和A5).其中化合物A1能抑制底物磷酸化,而化合物A5则对激酶的自磷酸化和底物磷酸化都能抑制.同时A1和A5都能影响转染细胞上的受体磷酸化作用.结论:利用重组受体激酶建立的分子模型为寻找抗肿瘤血管生成抑制剂提供了一个简单而有效的方法.     

Hydrogen peroxide overload increases adriamycin-induced apoptosis of SaOS(2)FM, a manganese superoxide dismutase-overexpressing human osteosarcoma cell line

We previously developed a new microscopic observation system that enables time-lapse quantitative analysis of apoptosis and necrosis. With this system we quantitatively analyzed adriamycin (ADR)-induced cell death using manganese superoxide dismutase (MnSOD)- and wild-type p53-gene transfectants on SaOS(2), a p53-deficient human osteosarcoma cell line. A highly MnSOD-overexpressing cell line, SaOS(2)FM(H), acquired ADR-tolerance compared to the parent cell line SaOS(2). The ADR-tolerance of SaOS(2)FM(H) diminished by L-buthionine-[S,R]-sulfoximine (BSO), which did not change ADR-sensitivity of SaOS(2), to the similar ADR-sensitivity of SaOS(2). A wild-type p53-expressing cell line, SaOS(2)wtp53, significantly increased in ADR-sensitivity compared to SaOS(2). This ADR-sensitivity of SaOS(2)wtp53 was enhanced by BSO. When isosorbide 5-mononitrate was combined with BSO, isosorbide 5-mononitrate increased ADR sensitivity of a moderately MnSOD-overexpressing cell line, SaOS(2)FM(L), decreased that of SaOS(2) FM(H), and did not change those of SaOS(2) and SaOS(2)wtp53 compared to BSO alone. Time-lapse microscopic observations during ADR treatment for 24 h indicated that the most cells of each cell line underwent apoptosis, and a few cells (less than 11%) died by necrosis. When cells were treated with iso-concentration of ADR, apoptosis of SaOS(2)FM(H) was less than that of SaOS(2). BSO, which did not change ADR-sensitivity of SaOS(2), increased appearance rate of ADR-induced apoptosis, but not necrosis of MnSOD-overexpressing cell lines. When iso-survival dose of ADR, which reduced surviving fraction to 0.01, was given for each cell line, no difference was observed in appearance of either apoptosis or necrosis between SaOS(2) and MnSOD-overexpressing cell lines. On the other hands, appearance of both apoptosis and the following secondary necrosis of SaOS(2) wtp53 was significantly accelerated compared to those of SaOS(2). These findings indicate that hydrogen peroxide overload on p53-independent pathway due to MnSOD overexpression plus BSO might increase the apoptosis frequency without acceleration of apoptotic process of each cell, resulting in negating ADR-tolerance of MnSOD-overexpressing cell lines.     

Thrombosed varicose vein in the sternocleidomastoid muscle

A thrombosed varicose vein arising in the neck is quite rare. A 68-year-old Japanese male was referred to our hospital because of a mass in his left upper neck. The mass did not change in size in response to strain resulting from Valsalva's manouvre. Magnetic resonance imaging (MRI) showed iso-signal intensity of the mass on T1-weighted images and a target-like signal arrangement (concentric hyper-, hypo-, hyper-signal intensity from outside to inside) on T2-weighted images. Surgical excision revealed that the tumour arose from the intramuscular small vein in the sternocleidomastoid muscle. The pathological examination revealed the mass to be a thrombosed varicose vein with capillarization in the dilated vein wall. The de-oxygenation and degradation of haemoglobin were thought to be responsible for these characteristic MRI findings. The concentric signal distribution on MRI strongly suggested this pathology.     

In vivo characteristics of HPV-immortalized and carcinogen transformed oral keratinocytes

PURPOSE: To characterize in vivo features of HPV-immortalized and carcinogen transformed oral keratinocytes. METHODS: The growth and squamous differentiation of IHGK (immortalized human gingival keratinocyte with HPV), IHGKN [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, (NNK)]-carcinogen transformed keratinocytes, and two head and neck squamous cell carcinoma (HNSCC) cell lines, HN30 and HN12, were tested by injecting these cells into SCID mice. The growth, histological features, and expression of PCNA, Involucrin, and high molecular weight keratin of the tumors formed were compared among these cell lines. RESULTS: All cell lines formed a palpable lesion at 2 weeks; however, only HN30 continued to grow. IHGK and IHGKN cells formed palpable nodules within 2 weeks with no further growth after 4 to 5 weeks, and no regression of the nodule was noted at 12 weeks. HN12 cells did not form tumor nodules unless these cells were co-injected with immortalized fibroblasts. Both IHGK and IHGKN cells formed a well-circumscribed epithelial lesion with islands of differentiated squamous cells bound by a myxoid matrix. Nests of basal-horny squamous cells centrally differentiated into anucleate squamous cells. IHGK and IHGKN nodules had more squamous differentiation than HN12 and HN30 and further differentiated over time. IHGK and IHGKN cells expressed differentiation (involucrin and high molecular weight keratin) and proliferation (PCNA) markers that suggest that IHGK and IHGKN behave as well-differentiated squamous lesions when compared with malignant HN12 and HN30 nodules. IHGK and IHGKN cells showed an initial growth phase followed by terminal differentiation, and then a phase characterized by regression and host inflammatory stage. CONCLUSIONS: The growth, histology, and expression of differentiation and proliferation markers of IHGK and IHGKN lesions into SCID mice demonstrate that these cells are endowed with a limited malignant potential. Our in vivo model with these intermediate cell lines can provide a short-term analysis for studying the biology of HNSCC progression and the activity of chemoprevention agents.     

Transfusion transmitted virus infection in patients on maintenance haemodialysis and in hospital workers

A newly discovered DNA virus, transfusion transmitted virus (TTV), was isolated from a post-transfusional hepatitis patient in Japan. A high prevalence (32-46%) of TTV infections in patients receiving maintenance haemodialysis (HD) has been reported but the occupational risk of TTV on HD units has not yet been determined. We determined the prevalence of TTV in workers in the same HD unit and the risk factors for TTV infection in HD patients, using logistic regression analysis. The prevalence of TTV DNA was 59.6% in 198 HD patients, significantly higher than that in the HD unit (13 of 39, 33.3%;P= 0.002) and non-HD healthcare workers (20 of 75, 26.7%; P= 0.001). A logistic regression analysis showed that male gender and negative test results for hepatitis G virus RNA were risk factors for TTV infection, but prior blood transfusion and duration of HD were not. Stepwise selection of multiple regression analysis showed that the presence of hepatitis C virus RNA was the only significant predictor for high serum ALT activity, and that the presence of TTV DNA was not. These results indicate that TTV is one of the prevalent human viruses transmissible either parenterally or nonparenterally in HD patients, but the occupational risk of TTV infection in HD unit workers is as low as in other healthcare workers. The pathogenic effects of TTV on the liver appear to be limited.     

Effect of exercise on the metabolism of vitamin B6 and some PLP-dependent enzymes in young rats fed a restricted vitamin B6 diet

The effect of exercise on vitamin B6 metabolism and PLP-dependent enzymes was studied in rats fed a diet with or without vitamin B6. Metabolism of some amino acids (citrulline, arginine, ornithine and threonine) inhibited in the B6-deficient rats was normalized during exercise. Exercise was also effective in storing vitamin B6 in the body by lowering excretion of vitamin B6, when intake of vitamin B6 was restricted. Aspartatae aminotransferase activity was higher in the red portion of the gastrocnemius muscle than that of the white one, whereas glycogen phosphorylase activity was vice versa and furthermore glycogen content in the white portion was very low in the vitamin B6-deficient rat. From the data obtained, it has been suggested that the red and white portions of the gastrocnemius muscle seemed to be more important in metabolizing amino acids and hydrolyze glycogen, respectively.     

Tc-99m DTPA renography in patients with collagen disease

PURPOSE: This study evaluated the use of Tc-99m DTPA renography in patients with collagen disease. MATERIALS AND METHODS: Tc-99m DTPA renal scintigraphy was performed in 28 patients with clinically diagnosed collagen disease. Twenty-two other patients who underwent renal scintigraphy and were subsequently shown to have no kidney abnormalities served as the control group. RESULTS: One quarter of the patients with collagen disease had abnormal findings of renography, despite the absence of abnormal laboratory data. Renograms were 100% sensitive for the detection of renal disease in patients with collagen disease, and their specificity was 53% when serum blood urea nitrogen, serum creatinine concentration, and proteinuria were the only measures of renal impairment considered. The glomerular filtration rate was not significantly different between the groups, but the bilateral time to peak was significantly greater in the patients with collagen disease. Hydronephrosis was present in 7% of patients with collagen disease. CONCLUSION: Renography is useful for detecting early changes of renal involvement when the clinical state of collagen disease is evaluated.     

Evidence for association of the myo-inositol monophosphatase 2 (IMPA2) gene with schizophrenia in Japanese samples

In our search for candidate genes for affective disorder on the short arm of chromosome 18, we cloned IMPA2, a previously unreported myo-inositol monophosphatase gene, that maps to 18p11.2. We determined its genomic structure and detected three new single nucleotide polymorphisms (SNPs). In the present study, we screened the gene further to search for additional polymorphisms in Japanese samples and identified seven other SNPs, including a novel missense mutation. These polymorphisms clustered into three regions of the gene. Three relatively informative SNPs, 58G>A, IVS1--15G>A and 800C>T from clusters 1, 2 and 3, respectively, were selected for association tests using a case-control design. The Japanese cohort included 302 schizophrenics, 205 patients with affective disorder and 308 controls. Genotyping was done either by melting curve analysis on the LightCycler or by sequencing. All three SNPs showed significant genotypic association (nominal P = 0.031--0.0001) with schizophrenia, but not with affective disorder. These findings increase the relevance of 18p11.2 to schizophrenia susceptibility because GNAL, which has been shown previously to be implicated in schizophrenia in an independent study, is in close physical proximity to IMPA2. Our findings suggest that IMPA2 or a gene nearby may contribute to the overall genetic risk for schizophrenia among Japanese.     

Cloning and characterization of a cDNA encoding beta-amyrin synthase involved in glycyrrhizin and soyasaponin biosyntheses in licorice

An oxidosqualene cyclase cDNA, termed GgbAS1, was isolated from cultured cells of licorice (Glycyrrhiza glabra) by heterologous hybridization with cDNA of Arabidopsis thaliana LUP1 lupeol synthase. The yeast transformed with an expression vector containing the open reading frame of GgbAS1 produced beta-amyrin, indicating that GgbAS1 encodes beta-amyrin synthase involved in the glycyrrhizin and soyasaponin biosyntheses in licorice. Northern blot analysis showed that the level of beta-amyrin synthase mRNA was drastically changed in the cultured licorice cells, whereas the mRNA level of cycloartenol synthase was relatively constant.