Endless forms most stupid,icky, and small: The preponderance of noncharismatic invertebrates as integral to a biologically sound view of life

Big, beautiful organisms are useful for biological education, increasing evolution literacy, and biodiversity conservation. But if educators gloss over the ubiquity of streamlined and miniaturized organisms, they unwittingly leave students and the public vulnerable to the idea that the primary evolutionary plot of every metazoan lineage is “progressive” and "favors" complexity. We show that simple, small, and intriguingly repulsive invertebrate animals provide a counterpoint to misconceptions about evolution. Our examples can be immediately deployed in biology courses and outreach. This context emphasizes that chordates are not the pinnacle of evolution. Rather, in the evolution of animals, miniaturization, trait loss, and lack of perfection are at least as frequent as their opposites. Teaching about invertebrate animals in a “tree thinking” context uproots evolution misconceptions (for students and the public alike), provides a mental scaffold for understanding all animals, and helps to cultivate future ambassadors and experts on these little‐known, weird, and fascinating taxa.     

Analysis of a transformed cell line using antisense c-fos RNA

Simian sarcoma virus (SSV)-infected NIH-3T3 cells (SSV-NIH-3T3), express a homologue of platelet-derived growth factor, (PDGF) a powerful inducer of the c-fos gene. We have used these cells to test the hypothesis that autocrine stimulation by PDGF-like molecules leads to c-fos expression which is functional in the transformed phenotype. We have transfected SSV-NIH-3T3 cells with a c-fos antisense-RNA expression vector, pSVsof, or control plasmids. pSVsof-transfected cells exhibit markedly decreased c-fos mRNA and protein levels, restored density-dependent growth arrest and reduced (three of five clones) tumorigenicity compared to control lines. The results confirm that c-fos cooperates in the transformed phenotype of SSV-NIH-3T3 cells.     

Genotoxicity of theobromine in a series of short-term assays

Theobromine (3,7-dimethylxanthine) was evaluated for genotoxic activity in a series of in vitro assays. Theobromine was not mutagenic in the Ames assay up to a maximum concentration of 5000 micrograms/plate either with or without S9 activation. The compound also failed to induce significant levels of chromosome aberrations in CHO cells (with and without S9 activation) or transformation in Balb/c-3T3 cells. At the maximum tolerated concentration theobromine increased the frequency of TK-/- mutants in mouse lymphoma L5178Y cells. Increased frequencies were observed both with and without S9 activation and they were reproducible in 2 independent experiments. Statistically significant increases in SCEs were obtained in human lymphocytes and in CHO cells under nonactivation test conditions. The spectrum of results in this battery of tests indicate that theobromine treatment results in the expression of genotoxic potential in some assays and the observed activity appears qualitatively and quantitatively similar to that of caffeine, a closely related methylxanthine.     

Large-T-antigen-p53 complex formation is not cold sensitive in a cold-sensitive transformant induced by simian virus 40 mutant tsA1499

F111 rat cells transformed by simian virus 40 mutant tsA1499 are cold sensitive for the expression of transformation. Yet, unlike F111 cells transformed by tsA58, they do not lose the ability to stabilize the transformation-associated host cell protein p53 at the temperature at which transformation is extinguished.     

Mutational analysis of simian virus 40 small-t antigen.

Several point mutations in the simian virus 40 (SV40) small-t antigen have been analyzed for their effects on protein stability, transformation, transactivation, and binding of two cellular proteins. All mutations which affected cysteine residues in two cysteine clusters produced highly unstable small-t antigens. Four point mutations outside these clusters and one in-frame deletion mutant, dl890, produced stable proteins but reduced transformation efficiency. These were able to transactivate the EII promoter and bind the cellular proteins, suggesting that these activities are not sufficient for small-t-mediated enhancement of transformation.     

Simian virus 40 small-t antigen stimulates viral DNA replication in permissive monkey cells.

The simian virus 40 (SV40) large-T antigen is essential for SV40 DNA replication and for late viral gene expression, but the role of the SV40 small-t antigen in these processes is still unclear. We have previously demonstrated that small t inhibits SV40 DNA replication in vitro. In this study, we investigated the effect of small t on SV40 replication in cultured cells. CV1 monkey cell infection experiments indicated that mutant viruses that lack small t replicate less efficiently than the wild-type virus. We next microinjected CV1 cells with SV40 DNA with and without purified small-t protein and analyzed viral DNA replication efficiency by Southern blotting. Replication of either wild-type SV40 or small-t deletion mutant DNA was increased three- to fivefold in cells coinjected with purified small t. Thus, in contrast to our in vitro observation, small t stimulated viral DNA replication in vivo. This result suggests that small t has cellular effects that are not detectable in a reconstituted in vitro replication system. We also found that small t stimulated progression of permissive monkey cells--but not of nonpermissive rodent cells--from G0-G1 to the S phase of the cell cycle, possibly leading to an optimal intracellular environment for viral replication.     

Mutations which affect the inhibition of protein phosphatase 2A by simian virus 40 small-t antigen in vitro decrease viral transformation.

Three independent point mutations within residues 97 to 103 of the simian virus 40-small-t antigen (small-t) greatly reduced the ability of purified small-t to inhibit protein phosphatase 2A in vitro. These mutations affected the interaction of small-t antigen with the protein phosphatase 2A A subunit translated in vitro, and a peptide from the region identified by these mutations released the A subunit from immune complexes. When introduced into virus, the mutations eliminated the ability of small-t to enhance viral transformation of growth-arrested rat F111 cells. In contrast, the mutant small-t antigens were unimpaired in the transactivation of the adenovirus E2 promoter, an activity which was reduced by a double mutation in small-t residues 43 and 45.     

Presence in growth-arrested cells of cellular proteins that interact with simian virus 40 small-t antigen.

Two cellular proteins, 56K and 32K, found in association with simian virus 40 small-t antigen were not induced by viral infection. In addition, the proteins were expressed by cells in the growth arrest period, a time in which small-t function is of importance in infection and transformation.     

长爪沙鼠肝细胞体外分离培养体系的建立

目的建立长爪沙鼠原代肝细胞分离培养体系。方法以雄性长爪沙鼠为供体,采用组织消化法和Seglen两步灌流法分离肝细胞,以台盼蓝染色检测细胞得率和活率,过碘酸-希夫氏反应(PAS)鉴定肝细胞,倒置显微镜观察肝细胞形态变化,并使用含有多种细胞因子的培养基维持培养。结果组织消化法和Seglen两步灌流法平均每只长爪沙鼠可分别获得肝细胞(1.33±0.34)×107个、(3.97±1.15)×107个,细胞活率分别为(29.4±6.05)%、(80.3±4.56)%,这两种方法在细胞得率及活率方面存在显著差异。肝细胞内因有大量的糖原颗粒,经PAS染色后被染成红色。结果表明肝细胞在贴壁后72 h内,肝细胞形态发生显著变化。结论采用胶原酶经肝门静脉灌流分离肝细胞是一种高效获得肝细胞的方法。各种细胞因子有利于维持肝细胞在体外的生长分化,长爪沙鼠原代肝细胞分离培养体系的建立将为肝脏相关疾病研究和防治药物的开发提供技术支持。     

Physical properties of cytomegalorvirus immune complexes prepared with IgG neutralizing antibody, anti-IgG, and complement

Human cytomegalovirus (CMV) strain AD 169 was reacted with IgG antibody (ab) from a CMV-infected renal transplant patient. A portion of he virus was neutralized, but infectious CMV-ab complexes that could be neutralized by adding rabbit anti-human IgG (A-IgG) or complement (C) were also generated. The immune complexes were examined, and the following observations were made: 1) CMV ab sufficient to cause 94% neutralization did not induce measurable changes in virion size or density. 2) The CMV-ab complexes increased slightly in size after reaction with A-IgG. 3) C increased the size and density of CMV-ab complexes to a greater degree than A-IgG. Virus aggregation did not occur with ab alone or with ab + A-IgG. However, clumping may have occurred in the presence of ab + C. 4) C also damaged virus envelopes, rendering viral DNA sensitive to DNase. 5) CMV-ab complexes absorbed to host cells as efficiently as native CMV. A-IgG or C partially inhibited complex attachment to the cells and increased the rate of release of cell-attached CMV. These findings suggest that virus neutralization may occur in a multistage process by more than one mechanism depending on the immune reagents employed. The physical changes in virus particles caused by A-IgG or C may be contributing factors in the neutralization process.