Polyoma Pseudovirions II. Influence of Host Cell on Pseudovirus Production

The type of host cell influenced the relative amounts of pseudovirions and polyoma virions produced. The infection of primary mouse embryo cells resulted in the production of particles that were predominantly pseudovirions. Infection of baby mouse kidney or 3T3D cells yielded mainly infectious polyoma virus. The length of time that infection was allowed to continue also affected the amount of pseudovirions relative to polyoma virions. The longer the viral infection was allowed to proceed, the greater the quantity of pseudovirions produced. Pseudovirion production could be correlated with the fragmentation of host cell DNA to a size of approximately 3 x 10(6) daltons. The fragmentation of host cell DNA was much more extensive in primary mouse embryo cells than in the other cell types.     

Interactions of Polyoma and Mouse DNAs I. Lytic Infection of Bromodeoxyuridine-Prelabeled Mouse Embryo Cells

On CsCl isopycnic centrifugation of the DNA extracted from secondary mouse embryo (ME) cultures grown in the presence of 5-bromodeoxyuridine (BUdR) and 5-fluorodeoxyuridine (FUdR) for 40 h, 10 to 25% of the DNA was found to be unsubstituted, 70 to 80% was bromouracil-hybrid DNA, and 5 to 10% was heavy DNA. These results together with cell number determinations, autoradiography, and Feulgen microspectrophotometry revealed three types of cells in these cultures: (i) 60 to 80% of the cells replicated their DNA once, divided, and then stopped mitotic activity, (ii) 5 to 10% were going through a second round of DNA replication; whereas (iii) 10 to 30% did not replicate DNA during the BUdR-FUdR exposure. After the transfer of these cultures to normal medium (without BUdR-FUdR), up to 20% of the cells resumed DNA synthesis asynchronously within 60 h, but no increase in cell number was observed. BUdR-FUdR-treated cultures, which were infected with polyoma virus in the absence of the thymidine analogues, supported a lytic infection to the same extent as did untreated ME cultures. This was concluded from the similar number of cells, which were induced to synthesize DNA, from the similar replication rate of the viral DNA, from the similar number of cells containing polyoma capsid proteins, and from the similar yields of progeny virus determined by hemagglutination and plaque formation. Thus, BUdR-prelabeled ME cultures are suitable for the investigation of interactions of the polyoma and mouse genomes during the lytic infection.     

Effect of Phleomycin on Polyoma Virus Synthesis in Mouse Embryo Cells

The addition of phleomycin (25 mug) to primary mouse embryo cells infected with polyoma virus was found to cause 96% inhibition of the synthesis of infectious virus. When ribonucleic acid and protein synthesis was investigated in these cells by use of isotope incorporation, it was found that neither was inhibited drastically. Immunofluorescent staining studies with the use of antibody directed to the viral structural proteins showed that proteins were synthesized in the presence of the antibiotic. However, when deoxyribonucleic acid (DNA) synthesis was investigated, it was found that DNA synthesis in uninfected cells was completely inhibited within the initial 10 hr of phleomycin addition, whereas DNA synthesis in infected cells proceeded at a reduced rate. Selective DNA extraction (Hirt method) of phleomycin-treated infected cells demonstrated that synthesized viral DNA was salt-extractable, similar to that in infected control cells lacking phleomycin. This extracted DNA was further fractionated by ethidium bromide-cesium chloride density gradient equilibrium centrifugation. The phleomycin-treated preparations revealed twice as much component II (circular nicked and linear) as component I (supercoiled) DNA, whereas the DNA from normally infected control cells showed the reverse picture. It was also demonstrated that viral particles synthesized in the presence of phleomycin did not contain component I DNA. This packaged DNA was found to consist of fragments of both the host and viral types. Cells that were prelabeled with (3)H-thymidine and then treated with phleomycin demonstrated host DNA degradation. However, fragments formed from prelabeled host DNA were not encapsidated into viral particles.     

Influence of Diethylaminoethyl-Dextran on Uptake and Degradation of Polyoma Virus Deoxyribonucleic Acid by Mouse Embryo Cells

The uptake of (32)P-labeled polyoma virus deoxyribonucleic acid (DNA) (I and II + III) by mouse embryo cells was increased from two- to fivefold in the presence of 500 mug of diethylaminoethyl-dextran (DEAE-D) per ml. This concentration of DEAE-D gives maximal enhancement of infectivity; however, the increase is many thousand-fold. As the DEAE-D concentration was increased from 0 mug/ml, uptake and infectivity increased to flat maxima and then decreased in a similar manner, except that at low DEAE-D concentrations uptake was relatively much greater than infectivity. Several other polycations also increased DNA uptake but did not enhance infectivity, and uptake of viral DNA was unaffected by the presence of mouse DNA, although infectivity was reduced. Thus, increased uptake is not the sole basis for the enhancement of infectivity produced by DEAE-D. The possibilities that DNA complexed with DEAE-D penetrates more rapidly or is stabilized against degradation do not completely account for enhancement since complexes formed in mixtures of DNA and DEAE-D, which sedimented heterogeneously from 40 to 60S, were infectious only for monolayers treated with DEAE-D. A more likely factor in enhancement is inhibition of the cellular nuclease activity detected, since virus DNA exposed to cells was much more degraded in the absence than in the presence of DEAE-D. The nuclease activity produced single-strand breaks in double-stranded DNA. Treatment of monolayers with deoxyribonuclease after adsorption of DNA in the presence of DEAE-D reduced cell-associated radioactivity by about 70%, although the number of plaques formed was not affected. In the absence of DEAE-D, 90 to 100% was removed by deoxyribonuclease. Thus, in both cases most of the DNA was adsorbed extracellularly. The greater deoxyribonuclease-resistant fraction in the presence of DEAE-D would be consistent with another possibility: that enhancement results from an increase in DNA penetration rate due to some action of DEAE-D on the cell.     

Deoxyribonucleotide Pools and Deoxyribonucleic Acid Synthesis in Mouse Embryo Cells Infected with Three Classes of Polyoma Virus Particles

Polyoma virus particles were purified by equilibrium centrifugation in CsCl. Particles from three regions of the density gradient were examined for infectivity, for their ability to induce expanded pools of deoxyribonucleic acid (DNA) precursors, and for their ability to stimulate the synthesis of DNA. The most infectious population of particles, the virions, having a buoyant density of 1.33 g/ml, gave the greatest stimulation of the DNA-synthesizing apparatus of mouse embryo cells. Empty particles at density 1.29 g/ml had no DNA stimulatory activity. A population of particles of intermediate density, referred to as pseudovirions, was also much less active than virions in stimulating DNA synthesis, and the limited stimulatory activity of the latter fraction may be accounted for by its measured contamination with infective particles.     

Interactions of Polyoma and Mouse DNAs III. Mechanism of Polyoma Pseudovirion Formation

In primary mouse kidney cell cultures infected with polyoma virus, the processes leading to virion and pseudovirion formation were studied. By photometric DNA quantitation, we followed the kinetics of mouse and polyoma DNA synthesis and the formation of low-molecular-weight fragmented mouse DNA (mouse f-DNA). Virus was harvested at different times and analyzed for its proportion of pseudovirions. The following correlations between the intracellular events and the production of virions and pseudovirions were found. (i) Syntheses of cellular and viral DNA were closely linked, both in time and in rates of synthesis. (ii) An increase of mouse f-DNA could only be detected several hours after the onset of mouse and polyoma DNA replication; its formation coincided in time with the appearance of progeny virus. (iii) The proportion of pseudovirions was not dependent on the amount of mouse f-DNA formed, but seemed to be inversely related to the amount of viral DNA synthesized. This was borne out by experiments in which DNA synthesis was partially inhibited by mitomycin C or after a synchronized onset of DNA replication. Under these conditions, virus preparations with a two- to threefold increased proportion of pseudovirions were obtained as compared with those from uninhibited cultures. Virus isolated from the remaining monolayer always had a higher proportion of pseudovirions than virus isolated at the same time from the supernatant medium only; also, the proportion of pseudovirions increased slightly with time after infection. Thus, according to the experimental conditions used, polyoma virus preparations with a low (10 to 20%) or a high (60 to 80%) proportion of pseudovirions can be obtained.     

激光辅助制作小鼠嵌合体胚胎的研究

目的：探索激光辅助体外制作小鼠嵌合体胚胎的方法。方法：激光辅助去除不同发育阶段体外受精胚胎的透明带,随机组合卵裂球体外培养,观察胚胎融合情况。结果：没有透明带的卵裂球体外能够“自发”融合,并且融合胚胎在体外培养环境中,能够发育至囊胚期,显微镜观察其形态基本与二倍体胚胎无差别。结论：激光辅助方法获得裸露的卵裂球能够在体外培养环境制作嵌合体胚胎。     

Immunochemical Procedure for Partial Purification of Newly Synthesized Proteins in Polyoma Virus-Infected Mouse Cells

A broad-spectrum antiserum has been obtained against nuclear proteins from normal mouse fibroblasts. Solid phase immunoadsorbants prepared from the serum specifically bind the corresponding antigens. The removal in this way of a significant fraction of host proteins leads to partial purification of viral and virus-induced polypeptides from polyoma virus-infected cell extracts. Four main peptides can be selected, with respective molecular weights of 90,000, 70,000, 46,000, and 41,000.     

Further Studies on the Effect of 5-Iododeoxyuridine on Polyoma Virus Multiplication in Mouse Cells

5-Iododeoxyuridine (IUDR) inhibited production of infectious polyoma virus in mouse embryo cells and mouse kidney cells in culture. Deoxythymidine reversed its effect. IUDR did not inactivate infectivity of free virus particles. IUDR did not prevent adsorption and penetration of polyoma virus to cells. The events sensitive to IUDR treatment occurred at around 20 hours after infection. The cytopathic effects of polyoma virus, including emergence of DNA containing-inclusions in the nucleus, were observable in infected cells in which viral replication was completely arrested by IUDR. It was shown by fluorescent antibody technique in infected mouse embryo cells and by complement fixation test in infected mouse kidney cells that IUDR inhibited completely the synthesis of viral antigen. No virus-like particles were demonstrated in the IUDR-treated infected-mouse kidney cells by electron microscope examinations.     

Interactions of Polyoma and Mouse DNAs II. Polyoma-Induced Mouse DNA Replication and Pseudovirion Formation

Secondary mouse embryo (ME) cultures which had been grown prior to infection in the presence of 5-bromodeoxyuridine (BUdR) and 5-fluorodeoxy-uridine were found to be permissive for polyoma virus (16). The DNA extracted from the progeny virus yielded two bands on CsCl isopycnic centrifugation. The light band (LL) contained supercoiled circular (polyoma DNA I), open circular (polyoma DNA II), and linear (polyoma DNA III) molecules, as was seen by electron microscopy. The hybrid band (HL) contained exclusively linear molecules. This DNA was pure, density - labeled, pseudovirion DNA, i.e., fragmented HL mouse DNA. The quantitative comparison of HL and LL polyoma DNA III from six different virus preparations always revealed an excess of HL DNA, the ratio of HL/LL being between 1.2 and 2.2. These results led to the conclusion that in BUdR-prelabeled, polyoma-infected ME cells pseudovirion DNA is excised both from unreplicated and newly replicated regions of mouse DNA.     

Interferon Production and Resistance to Viral Infection Induced by Poly I · Poly C in Chick Embryo Cells

Two different growth media, one based on Eagle's minimum essential medium (MEM) and the other on Earle's balanced salt solution–lactalbumin hydrolysate–yeast extract (YLE), were used for growing primary chick embryo cells (CEC), and resistance to viral infection and interferon production induced by polyinosinic-polycytidylic acid (poly I·poly C) were compared. In CEC grown in Eagle's MEM, treatment with poly I·poly C at a concentration as low as 1.0 ng/ml was sufficient to induce a detectable resistance to infection with vesicular stomatitis virus (VSV), while more than 300-fold concentrated poly I·poly C was required to induce a similar resistance when the cells were grown in YLE. The cells grown in YLE did not produce an appreciable amount of interferon, whereas a significantly higher level of interferon was produced by the cells grown in Eagle's MEM. A similar phenomenon was observed in the interferon production of chick embryo cells treated with ultraviolet light (UV)-irradiated Newcastle disease virus (NDV) and in the induction of resistance to vaccinia virus in cells treated with poly I·poly C. It was found that the response of cells, bathing in one growth medium, to poly I·poly C was not affected by replacing it with the other at the same time with the addition of poly I·poly C, and that the response of CEC was strongly dependent upon the medium used for cultivation. These facts suggested that the observed difference in the response of cells to poly I·poly C was not due to a direct interaction between the inducer and medium components but to the physiological state of CEC established during their growth. Which component of YLE was responsible for such a lowered response of cells to poly I·poly C was also examined, and the marked reduction of PDD₅₀ by the replacement of lactalbumin hydrolysate of YLE with amino acids and the increase of PDD₅₀ by addition of lactalbumin hydrolysate to MEM suggested that lactalbumin hydrolysate might play an important role in this phenomenon.     

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 10⁶ acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.     

小鼠Kupffer细胞分离及细胞培养

目的 采用在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离Kupffer细胞（Kupffer cells,KCs）,探讨其在分离小鼠KCs的应用及其对KCs生物活性的影响.方法 根据原位灌注和梯度离心方法不同随机分为4组：无胶原酶原位灌注＋3层梯度离心组（A）、无胶原酶原位灌注＋双层梯度离心组（B）、胶原酶原位灌注＋3层梯度离心组（C）和胶原酶原位灌注＋双层梯度离心组（D）.采用F4/80（BM8）免疫染色及吞墨实验判断细胞纯度和功能、台盼蓝拒染实验判断细胞的活力,探讨不同方法KCs分离的效果及细胞活性.结果 刚分离的KCs细胞近似圆形,接种l h后收获细胞纯度较高,但细胞得率相对较低.培养4 h后KCs得率相对较高,培养28 d仍能存活.免疫荧光可显示分离的为KCs,台盼蓝染色显示各组细胞的活力均在90 %左右,在体胶原酶灌注和双层梯度离心可以增加KCs的得率,双层梯度离心法可以增加分离KCs的纯度.结论 在体胶原酶灌注对提高KCs得率较为重要,在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离小鼠KCs的的方法简便、高效、稳定,培养的KCs具有良好的细胞生物学性状.     

Evidence for a Stable Intermediate in Leukemia Virus Activation in AKR Mouse Embryo Cells

Analysis of the requirement for serum in the activation of the endogenous leukemia virus expression in AKR mouse embryo cells by 5-iododeoxyuridine shows that activation can be dissociated into two discrete serum-dependent events. The first involves incorporation of 5-iododeoxyuridine into DNA and results in the formation of a stable “activation intermediate” resembling the provirus formed during infection of stationary mouse embryo cells with exogenous leukemia virus. The second event, resulting in expression of the activation intermediate as synthesis of virus proteins, requires DNA replication but not 5-iododeoxyuridine.     

Mutant of Polyoma Virus with Impaired Adsorption to BHK Cells

A mutant of polyoma virus PY235 has an impaired adsorption to guinea pig red blood cells and BHK-21 hamster cells. Adsorption to 3T3 mouse cells is much less inhibited. These altered adsorption properties are responsible for the apparent inability of PY235 to cause cell transformation or hemagglutination.     

乙醛磺酸对小鼠胚胎细胞遗传和胚胎发育影响的研究

本文报道乙醛磺酸（ＳＡＤ）不仅会降低小鼠胚胎细胞的ｒＲＮＡ基因转录活性,而且还明显提高了胚胎细胞姐妹染色单体互换（ＳＣＥ）频率,导致胚胎发育停滞和胎儿死亡,最终使幼鼠出生率明显下降。当ＳＡＤ浓度为１０－５ｍｏｌ／Ｌ时,小鼠胚胎细胞ｒＲＮＡ基因转录活性降低为正常值的１５％,并引起ＳＣＥ频率成倍增加,幼鼠出生率仅为正常值的５０％;当ＳＡＤ浓度增至１０－３ｍｏｌ／Ｌ时,此时ｒＲＮＡ基因转录活性完全被抑制,而且ＳＣＥ频率增加为正常值的４．８倍,幼鼠出生率从正常值１００％降为４％。ＳＡＤ对小鼠胚胎细胞这种细胞遗传学效应和对胚胎发育影响的程度与该药浓度成正相关。此外,我们还对ｒＲＮＡ基因转录活性和ＳＣＥ的改变与哺乳类胚胎发育之间的关系进行了初步的讨论。     

Primary Cilia Are Not Required for Normal Canonical Wnt Signaling in the Mouse Embryo

Background

Sonic hedgehog (Shh) signaling in the mouse requires the microtubule-based organelle, the primary cilium. The primary cilium is assembled and maintained through the process of intraflagellar transport (IFT) and the response to Shh is blocked in mouse mutants that lack proteins required for IFT. Although the phenotypes of mouse IFT mutants do not overlap with phenotypes of known Wnt pathway mutants, recent studies report data suggesting that the primary cilium modulates responses to Wnt signals.

Methodology/Principal Findings

We therefore carried out a systematic analysis of canonical Wnt signaling in mutant embryos and cells that lack primary cilia because of loss of the anterograde IFT kinesin-II motor (Kif3a) or IFT complex B proteins (Ift172 or Ift88). We also analyzed mutant embryos with abnormal primary cilia due to defects in retrograde IFT (Dync2h1). The mouse IFT mutants express the canonical Wnt target Axin2 and activate a transgenic canonical Wnt reporter, BAT-gal, in the normal spatial pattern and to the same quantitative level as wild type littermates. Similarly, mouse embryonic fibroblasts (MEFs) derived from IFT mutants respond normally to added Wnt3a. The switch from canonical to non-canonical Wnt also appears normal in IFT mutant MEFs, as both wild-type and mutant cells do not activate the canonical Wnt reporter in the presence of both Wnt3a and Wnt5a.

Conclusions

We conclude that loss of primary cilia or defects in retrograde IFT do not affect the response of the midgestation embryo or embryo-derived fibroblasts to Wnt ligands.     

小鼠心脏神经嵴细胞的体外培养及其生物学特性

目的体外培养和鉴定心脏神经嵴细胞,探讨其生物学特性。方法取8·5d小鼠胚胎枕中部至第3体节神经管,组织块法无血清条件培养获心脏神经嵴细胞,采用转录激活因子2α(AP-2α)作为其生物学标记物,观察其迁移、分化等生物学特性。结果从胎鼠神经管中分离培养的细胞AP-2α表达阳性,具有迁移特性,传代后以含血清培养基培养后能自然分化成神经元和神经胶质细胞。结论体外培养可成功获得心脏神经嵴细胞,且具有迁移特性和多向潜能分化能力。