Complete Nucleotide Sequence and Phylogenetic Analyses of Hepatitis B Virus Isolated from Two Pileated Gibbons

Virus-like particles (VLPs, named HmTV1-17), about 40nm in diameter were found in the violet root rot fungus Helicobasidium mompa Tanaka strain No. 17, which had been isolated from an apple tree. Purified preparations of HmTV1-17 contained two species of double-stranded RNA (dsRNA), designated 17L and 17S. cDNAs were constructed from HmTV1-17 genomic dsRNAs purified using CF-11 cellulose column chromatography. The sequences of 17L and 17S cDNA comprised 5207 and 2096bp, respectively. Although 17S has no large open reading flame (ORF) on either strand, 17L has two large overlapping ORFs. The 5 located ORF1 encodes the coat protein (CP, 788 amino acids), whereas the gene product of ORF2, which is in the –1 frame relative to ORF1, shows the typical features of a RNA dependent RNA polymerase (RDRP, 845 amino acids). Phylogenetic analysis based on RDRP showed that HmTV1-17 is closely related to Sphaeropsis sapinea SsRV1, a member of the genus Totivirus from filamentous fungus S. sapinea.     

Absence of Epstein-Barr virus (EBV) in intrahepatic cholangiocarcinoma confirmed by lack of EBV-coded nuclear RNA and latent membrane protein-1

AIMS: Studies are disclosing that Epstein-Barr virus (EBV) is involved in the aetiology of various neoplasms including undifferentiated carcinomas of the aerodigestive tract. The aetiology of intrahepatic cholangiocarcinoma (ICC), a malignant neoplasm arising from intrahepatic biliary epithelia, has yet to be fully evaluated. To date, two cases of EBV-related ICC have been reported, and they presented foci of lymphoepitheliomatous undifferentiated carcinoma components. METHODS AND RESULTS: To determine whether EBV is commonly involved in the developments of ICC, we performed in-situ hybridization and immunohistochemistry for EBV in 215 cases of ICC in Japan, using a probe against EBV-coded nuclear RNA (EBER) and a specific antibody against latent membrane protein-1 (LMP-1), respectively. We did not detect EBV-infected carcinoma cells in any of the ICC cases examined. No lymphoepitheliomatous undifferentiated carcinoma components were found either. CONCLUSION: The results suggest that EBV infection is unlikely to be involved in the pathogenesis of ICC.     

The AMeX method: a multipurpose tissue-processing and paraffin-embedding method. III. Extraction and purification of RNA and application to slot-blot hybridization analysis.

RNA was extracted from tissues processed by a new fixation and paraffin-embedding method (the AMeX method) and examined by Northern blot analysis and slot-blot analysis. The RNA extraction method for AMeX-processed tissue sections after the deparaffinization step was the same as that for fresh materials. The total amount of cellular RNA extracted from AMeX-processed mouse liver tissue was slightly less than that extracted from fresh tissue. In tissues of malignant lymphoma, the total amount of cellular RNA extracted from 25 sections each 20 microns thick was about 1.6-1.8 micrograms/mm2, regardless of the histological subtype and period of storage. The extracted RNA was moderately degraded, and usually could not be used for Northern blot hybridization analysis. The intensity of ethidium bromide staining and the hybridization signals of RNA extracted from AMeX-processed tissues were usually reduced in comparison with RNA from fresh material, but specific signals could be detected by slot-blot hybridization analysis. We have demonstrated previously that the AMeX method preserves high-molecular-weight DNA and various antigens. Since the present study showed that information on mRNA can be obtained from AMeX-processed tissue, the versatility and usefulness of this method were further proven.     

Topoanatomical measurements of protein-bound radioactivity in the rat brain after intraventricular application of [14C]leucine

The pattern of distribution of protein-bound radioactivity was studied in rats 4 h after stereotaxic injection of [¹⁴C]leucine into the lateral ventricle. For quantitative estimation of the distribution of radioactivity, the semiconductographic method was used. The patterns revealed in this way were in general agreement with patterns obtained by the autoradiographic technique. The semiconductographic measurements indicated a high labelling, especially of some thalamic and hypothalamic nuclei. With the exception of the base of the telencephalon, the cortical structures were very poorly labelled. A steep gradient from the ventricular system to the brain tissue was observed. Very high labelling of blood vessels and leptomeningeal cells, especially in the vicinity of the optic chiasma, was observed.This quantitative analysis of the distribution pattern indicated a marked difference in uptake of the radiolabelled amino acid from the cerebrospinal fluid and calls for caution in the interpretation of experiments in which the relation between brain function and macromolecular synthesis is studied by the intraventricular injection of a radiolabelled precursor.A method is also described which makes it possible to determine the density distribution within a non-radioactive tissue slice in a simple way.     

The effect of a series of fixatives on the AgNOR technique

With increasing interest being shown in nucleolar organizer regions (NORs) in pathology, it was considered of great importance to evaluate the effect of some of the more commonly used and more specialized fixatives on the demonstration of these moieties. NORs can be demonstrated in paraffin sections by a silver technique (AgNOR method) which was developed from a method used by cytogeneticists for the demonstration of NORs in chromosome spreads. The degree of staining is dependent on the fixation regime employed and results may vary greatly from one fixative to another. The fixative schedules and post-treatments used in this study were based on standard sequences from the literature. We have shown that, in general, alcohol-based fixatives give optimal results, Carnoy's fluid being especially recommended. Mercurial and dichromate-containing fixatives were found to have highly detrimental effects on NOR staining. 'Routine' 10 per cent formol saline fixation gave adequate results whereas 10 per cent neutral buffered formalin gave optimal staining, similar to alcohol-based fixation.     

卵巢恶性肿瘤VEGF mRNA和bFGF mRNA的表达及其意义

应用原位分子杂交技术研究卵巢恶性肿瘤组织中VEGFmRNA和bFGFmRNA的表达及其意义。结果发现 :浆液性腺癌和内胚窦癌两者表达阳性率高于粘液性腺癌和颗粒细胞瘤 ;临床分期Ⅰ期和未转移病例两者表达阳性率低于临床分期Ⅲ～Ⅳ期和转移病例。结果提示VEGFmRNA和bFGFmRNA表达与卵巢恶性肿瘤病理类型、临床分期和转移发生关系密切 ,阳性表达者可能预后较差。     

豚鼠内淋巴囊上皮细胞Na+,K+-ATP酶不同β亚基的mRNA表达

目的　进一步研究内淋巴囊上皮细胞Na ,K -ATP酶不同 β亚基的表达。方法　采用豚鼠内淋巴囊冰冻切片、原位杂交的方法检测Na ,K -ATP酶不同 β亚基mRNA在内淋巴囊上皮细胞中的表达。结果　在内淋巴囊上皮细胞胞浆内可见Na ,K -ATP酶不同 β亚基的阳性颗粒 ,β1亚基表达较弱 ,β2 亚基表达较强。结论　结合其他报道 ,内淋巴囊上皮细胞具有多种亚基异构体组成的Na ,K -ATP酶的表达 ,而结肠上皮、肾小管上皮的Na ,K -ATP酶几乎仅有α1β1型 ,提示其离子转运过程可能不同。