Natural and Chernobyl-caused radioactivity in mushrooms,mosses and soil-samples of defined biotops in SW Bavaria

Summary Different mushrooms, mosses and corresponding soil samples have been collected mainly from two sites in the alpine region of southwestern Bavaria. At the end of the growthseason, September 1986, gamma spectroscopic analysis showed that the moss-, mould, and needle-layer contained considerably more ¹³⁴Cs and ¹³⁷Cs activity per unit fresh weight than eight different species of mushroom. These two isotopes were carried into the biotop mainly as a consequence of the Chernobyl accident. ¹³¹J could not be found any more in the samples ca. 5–6 months after the catastrophe. The activity of the cesium isotopes decreased with increasing soil depth. In the mushrooms the activity was relatively high in Xerocomus badius and surprisingly low in Boletus edulis; samples of the latter and of Cantharellus cibarius collected in September 1985 (before the accident) and kept deep frozen contained almost identical amounts of ¹³⁷Cs as those collected from August to October 1986. Mushrooms contained considerably more of the natural isotope ⁴⁰K than the needlelayers and the soil samples in the neighbourhood. In all mushrooms except Xerocomus badius the activity of ⁴⁰K was generally higher than the ¹³⁷Cs activity. The results indicate that except Xerocomus badius the analyzed mushrooms do not actively take up Cs from the soil, in contrast to K.     

Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.     

Dideoxynucleoside triphosphates inhibit a late stage of SV40 DNA replicationin vitro

Summary The role of DNA polymerases in the replication of SV40 DNA was studied using a T-antigen-dependent assay supplemented with a human KB cell extract. Inhibition of DNA polymerase α by addition of aphidicolin or monoclonal antibodies prevented DNA synthesis, confirming the requirement for this enzyme in replication. The replication process was unaffected by ddTTP at a concentration (5 μM) inhibitory to DNA polymerases β and γ, however, higher concentrations of ddTTP (200 μM) caused an apparent accumulation of relaxed circular plasmid with a concomitant decrease in DNA synthesis. An analysis of this replication intermediate indicated that it was formed during the replication reaction and that the replicative cycle was nearly complete. A kinetic study of ddTTP inhibition strongly suggested DNA polymerase ε (PCNA-independent DNA polymerase δ) was the target of the inhibitor and that this enzyme functions during the final stages of DNA replication.     

Idiotype network components are involved in the murine immune response to simian virus 40 large tumor antigen

Summary Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (SV40 T-Ag), a monoclonal antibody specific for SV40 T-Ag (Ab-1 preparation), and a monoclonal anti-idiotypic antibody (anti-Id), designated 58D, were used to analyze the humoral immune response of Balb/c mice either immunized with recombinant SV40 T-Ag or challenged with SV40-transformed cells. Inhibition assays indicated that antibodies from mice immunized with SV40 T-Ag and from those bearing SV40 tumor inhibited the SV40 T-Ag/Ab-1 reaction. These data suggested that the antibody response in immunized or tumorchallenged mice recognized similar epitope(s) on SV40 T-Ag to that detected by the monoclonal Ab-1. These anti-(SV40 T-Ag) response antibodies also inhibited the Ab-1/anti-Id reaction and recognized the anti-Id in direct binding assays. Together, these data indicate that murine anti-(SV40 T-Ag) responses shared an idiotope with a monoclonal anti-(SV40 T-Ag) Ab-1 preparation. This idiotope, which is recognized by the monoclonal anti-Id preparation, 58D, appears to be involved in the humoral immune response to SV40 T-Ag in both SV40-T-Ag-immunized and tumor-bearing mice. The monoclonal anti-Id preparation may represent a focal point for manipulating the humoral immune response to tumors induced by SV40-transformed cells.     

Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.     

Mammalian cell lines can be efficiently established in vitro upon expression of the SV40 large T antigen driven by a promoter sequence derived from the human vimentin gene.

The aim of this study was to investigate a new method to enhance the efficiency to create mammalian cell lines. Cell immortalization was achieved by intranuclear microinjection of a recombinant DNA construct composed of a constitutive promoter controlling the genes encoding immortalizing proteins; the sequences coding for the large T and small t antigens were fused downstream of regulatory elements from the vimentin gene, the activation of which characterizes the vast majority of cells growing in vitro. Data show that the efficiency of the immortalizing procedures using the SV40 early genes could be enhanced by the control elements derived from the human vimentin (HuVim) 5' sequences that contained nucleotides -878 to +93 from the CAP site. This HuVim 830-T/t recombinant was used to create cell lines from numerous primary cultures of different origins: rabbit, porcine and human endothelial cells, rabbit and bovine epithelial cells. A set of large T-expressing cells was derived, and these cells retained characteristics of differential cells: binding of Ulex europaeus lectin and synthesis of Factor VIII for human endothelial cells; network of cytokeratin for bovine oviductal cells and rabbit mammary cells.     

Restriction endonuclease EcoO109 from Escherichia coli H709c with heptanucleotide recognition site 5'-PuG/GNCCPy

A new restriction endonuclease, EcoO109, has been isolated from Escherichia coli H709c by polyethyleneimine (PEI) precipitation, DEAE-cellulose chromatography and heparin agarose chromatography. The yield was high, more than 3000 units/g of wet cells. The EcoO109 endonuclease recognizes and cleaves a nucleotide sequence of (formula: see text), in the presence of 10 mM Mg2+. The enzyme will be useful for structural analysis and molecular cloning of DNA because of the stability, high yield and easy handling of the producer strain.     

Stable expression of antibiotic resistance genes using a promoter fragment of the U1 snRNA gene

As U1 snRNA is produced in all mammalian cell types, antibiotic resistance genes driven by this promoter would be ideally suited as genetic selection markers. However, although the U1 snRNA gene is transcribed by RNA polymerase II, its native product is not a messenger RNA, but a splicing cofactor. To test whether this promoter could nevertheless produce a functional mRNA, sensitive reporter genes expressing resistance to the antibiotics hygromycin-B and bleomycin were constructed with either the U1 snRNA promoter or the SV40 early promoter. Resistant cell lines could only be obtained with constructs equipped with a functional polyadenylation signal. With the U1 snRNA promoter about three times fewer colonies were obtained than with the SV40 early promoter. Another potential advantage of the U1 snRNA promoter is that, in contrast to the promoters commonly used to express genetic selection markers, the enhancer-like element contained in the U1 snRNA promoter had only a minimal stimulative effect, only detectable with the most sensitive methods, on an adjacent mRNA-producing gene. The U1 snRNA promoter was also capable of expressing bleomycin resistance in the context of a self-inactivating retrovirus vector, whereby it was discovered that the mouse 3T3 cells used in this experiment were 10 times more sensitive to bleomycin than human or hamster cell lines.Abbreviations ble bleomycin resistance gene - Hm hygromycin - hpt hygromycin phosphotransferase gene - neo neomycin (geneticin) phosphotransferase gene     

Comparison of the murine humoral immune response to recombinant simian virus 40 large tumor antigen: Epitope specificity and idiotype expression

Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (SV40 T-Ag) was used to immunize inbred strains of mice to compare the humoral immune responses. Specifically we examined the epitope specificities and idiotype (Id) expression on anti-(SV40 T-Ag) responses induced in BALB/c and C57BL/6 inbred strains of mice. The predominant SV40 T-Ag epitopes recognized by the anti-(SV40 T-Ag) responses appeared to differ between these two inbred strains, this being based on the ability of sera to inhibit the binding of several murine monoclonal antibodies specific for SV40 T-Ag. In addition, anti-(SV40 T-Ag) responses produced in C57BL/6 mice failed to express a previously described cross-reactive Id expressed in the anti-(SV40 T-Ag) response in BALB/c mice. This cross-reactive Id is detected by a mouse monoclonal anti-Id, designated 58D, which has been shown to represent a potential focal point for manipulating the humoral immune response to SV40-induced tumors in BALB/c mice. Together, these data indicate that the functional duality of the humoral immune response, as assessed by epitope recognition and Id expression, differs between these two inbred strains of mice when immunized with a recombinant SV40 T-Ag.     

利用参数模型提取诱发脑电（EP）及其在40Hz EEG和EP间关联研究中的应用

提出一种基于参数模型的信息处理方法,用于诱发脑电的提取,这种方法可以得到单次ＥＰ波形的无偏,最小方差估计,用刺激强度实验验证了该方法在实际情况下的有效,进一步将其应用于刺激前ＥＥＧ与ＥＰ的关联研究,得到了刺激前４０Ｈｚ的ＥＥＧ成分与晚潜伏期听觉诱发脑电幅度有极显著负相关的结果,表明刺激前及状态确定对ＥＰ有影响。