Plaque Assay for Polyoma Virus on Primary Mouse Kidney Cell Cultures

A plaque assay for polyoma virus using primary baby mouse kidney cells is reported.     

Pluripotent Embryonal Stem Cell Lines Can Be Established from Disaggregated Mouse Morulae

Mouse pluripotent embryonal stem ( ES ) cell lines hitherto have been conventionally isolated from the 'inner cell mass' of mouse blastocysts. In this report, I describe a new and simplified method for establishing pluripotent cell lines from mouse morulae of the 16- to 20-cell stage, which were disaggregated by the use of EDTA. From 17 cell lines established in such a way, 7 were characterized with respect to their differentiation potential:
(i) When injected into syngeneic mice, the cells gave rise to solid, fully differentiated teratomas representing derivatives of all three germ layers. (ii) When cultured in suspension in vitro, the cells were able to differentiate into complex organized 'embryoid bodies' analogous to mouse early postimplantation embryos. These results strongly imply that embryonal stem cell lines isolated from mouse morulae are highly homologous to conventionally isolated ES cells.
In addition, my results indicate that murine pluripotent embryonal stem ( ES ) cell lines can be derived with more ease and higher efficiency from disaggregated morulae than from the 'inner cell mass' of blastocysts.     

A Novel Mesoderm-Specific cDNA Isolated from a Mouse Embryonal Carcinoma Cell Line

A novel mesoderm-specific cDNA clone has been isolated by differential screening of cDNA library from an embryonal carcinoma (EC) cell line MC12. The cDNA clone 121a is about 2.5 kb in length and apparently encodes a putative polypeptide of 335 amino acids which may be secreted or membrane anchored glycoprotein since it has a possible signal sequence and a potential N-linked glycosylation site. In situ hybridization using mouse embryos revealed that 121a expression was confined to mesoderm and its derivatives such as allantois, the mesodermal layer of amnion, chorion and yolk sac, somites, heart, etc. These findings suggest that 121 a may be essential for mesodermal differentiation or function, although nothing definite is known. Conservation of 121a homolog in mammals and even in Drosophila seems to support this presumption. Fluorescence in situ hybridization successfully localized 121a to B1 band of mouse chromosome 6.     

Effect of Ultraviolet Irradiation and Actinomycin D on Polyoma Virus Replication in Mouse Embryo Cell Cultures

Ultraviolet irradiation and actinomycin D impair the capacity of mouse embryo (ME) cells to support the replication of polyoma virus, but not of encephalomyocarditis (EMC) virus. The loss in capacity for polyoma virus synthesis was an “all-or-none” effect and followed closely upon the loss in cellular capacity for clone formation. Cells treated with either agent produced polyoma “T” antigen, but did not synthesize polyoma structural protein. Infection of untreated ME cells with polyoma virus produced marked stimulation of both deoxyribonucleic acid (DNA) synthesis and ribonucleic acid (RNA) synthesis. ME cell cultures irradiated with ultraviolet for 30 sec at 60 μw/cm² or treated with actinomycin D at 0.1 μg/ml for 6 hr prior to infection were incapable of synthesizing DNA or RNA, even after infection with polyoma virus. Irradiation of cells during infection produced cessation of synthesis of both RNA and DNA. Addition of actinomycin D during infection did not inhibit DNA synthesis but abolished RNA synthesis and reduced the yield of polyoma virus to 10% of that in untreated infected cultures. Both agents lost the ability to prevent replication of a full yield of polyoma virus when administered 30 hr after infection or later. The period after which neither agent inhibited polyoma replication corresponded with the period at which maximal RNA synthesis in untreated infected cultures had subsided. It can be concluded on the basis of the data presented that the functional integrity of the mouse embryo cell genome is required for the replication of polyoma virus, but not for EMC virus. Whereas the requirement for cellular DNA-dependent RNA synthesis for polyoma virus replication has been demonstrated, the exact nature of the host-cell function remains to be elucidated.     

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.     

Internalization of Embryonal Carcinoma Cells when Aggregated with Normal Mouse Embryos

In the present study, we examined in detail the process of forming chimeric blastocysts between B242g embryonal carcinoma (EC) cells and normal mouse embryos. Electron microscopic observations of the developing aggregates revealed that the embryonic cells spread over the surface of the EC cells, resulting in the internalization of EC cells in the aggregates. When a single blastomere of an 8-cell embryo was aggregated with EC cells, the blastomere spread over and engulfed the EC cells. These results strongly suggest that EC cells are segregated and become situated in the inside position as the development of an aggregate proceeds, and then they are incorporated into the ICM of a blastocyst.     

Flow Cytoenzymology of the Early Differentiation of Mouse Embryonal Carcinoma Cells

Dual parameter flow cytoenzymology was used to detect biochemical differentiation of embryonal carcinoma cells, the undifferentiated, multipotent stem cells of teratocarcinomas. With the use of fluorogenic substrates, two enzyme systems, alkaline phosphatase (EC 3.1.3.1.) and carboxyl esterase (EC 3.1.1.), were studied. Embryonal carcinoma cells passaged in vitro for several years retained high alkaline phosphatase activities similar to those of embryonal carcinoma cells in embryoid bodies grown in vivo. Similar to the embryonal carcinoma cells in vivo, the in vitro embryonal carcinoma cells were capable of giving rise to progeny with greatly decreased levels of alkaline phosphatase. The embryonal carcinoma cell alkaline phosphatase was inhibited by l-p-bromotetramisole, suggesting a relationship between this enzyme and somatic, nonintestinal alkaline phosphatase isoenzymes. Determinations of esterase activities in viable teratocarcinoma cells showed that prior to any evidence of morphologic differentiation, the embryonal carcinoma cells are quite heterogeneous with regard to esterase activities.     

Collagen Synthesis in Mouse Embryonal Carcinoma Cells: Effect of Retinoic Acid

Abstract. In five lines of mouse embryonal carcinoma cells, PCC3/A1, PCC4, PCC4/Aza-R1, PCC7-S/Aza-R1, and F9, collagen synthesis was examined by immunofluorescence reaction using specific antibodies directed against collagen. All the embryonal carcinoma cell lines showed type IV collagen, and PCC7-S/Aza-R1 revealed the additional presence of type III collagen. When the F9 and PCC3/A1 EC cells were treated with retinoic acid and dibutyryl-cAMP, they differentiated into morphologically different cellular types. These cellular types showed new types of collagen. Thus, in treated F9 cells, type I, type III, and type V collagen were detected and in treated PCC3/A1 cells, type III and type V collagen were detected.
In two established cellular strains, PYS-2 corresponding to parietal endoderm and 3TDM-1 corresponding to trophoblastoma, collagen was identified by immunological reaction and electrophoretic mobility. The trophoblastoma cell line was characterized by the production of type I, type III, and type IV collagen, whereas endodermal PYS-2 revealed type IV collagen.     

Antigenic Heterogeneity in Cell Populations of Cloned Polyoma,Virus-induced Tumour Lines

WALLS and Negroni¹ isolated in tissue culture two cell clones from a polyoma virus-induced fibrosarcoma of H₃B/Bi mice. One (malignant cell line 7₁) produced transplantable tumours in adult syngeneic mice; the other (transformed non-malignant line 7₂) was not tumorigenic but induced resistance to the growth of the malignant cells. We have now confirmed these results and extended them to show that other malignant cells (tissue culture clones C₄ and C₅) from a polyoma virus-induced parotid tumour of syngeneic mice are also inhibited by pre-immunization with a non-malignant line (7₂). The number of cells required to induce tumours in 50% of the animals is ten times higher in the preimmunized mice than in the controls, for both fibrosarcoma and parotid malignant lines (Table 1).     

Relationships Among Virus Spread, Cytopathogenicity, and Virulence as Revealed by the Noncytopathic Mutants of Newcastle Disease Virus

We have studied protein synthesis in cultured cells infected with the six noncytopathic (nc) mutants of the Australia-Victoria strain (AV-WT) of Newcastle disease virus and their plaque-forming revertants. Virus-specific polypeptides accumulated at 30 to 63% of wild-type levels in nc mutant-infected cells and between 66 and 175% of wild-type levels in revertant-infected cells. An exception was the L polypeptide, which accumulated in nc mutant-infected cells at only 5 to 20% of the levels found in wild-type infection. The reduced accumulation of the L polypeptide did not appear to be due to increased degradation of that polypeptide. A new polypeptide (X) accumulated instead of polypeptide P in cells infected with mutants nc4 or nc16 and in virions released from them. Peptide mapping identified X as an altered form of P. A revertant of mutant nc4 (nc4S1), which forms larger hemadsorbing spots, but still does not form plaques, accumulated P instead of the X polypeptide. Thus, a lesion in P can affect virus spread without affecting cytopathogenicity. Virions of mutant nc7 and two naturally occurring avirulent strains of Newcastle disease virus (NJ LaSota and B1-Hitchner) contained polypeptides (F₇ and F_A, respectively) related to, but migrating more rapidly than, F₀ in sodium dodecyl sulfate-polyacrylamide gels. As previously reported for avirulent strains, a brief treatment of nc7 virions with trypsin converted F₇ to F and increased infectivity. Similarly, culturing nc7-infected cells in the presence of trypsin facilitated fusion from within and viral spread from cell to cell. A plaque-forming revertant of nc7 still accumulated F₇ in virions, indicating that the lesions responsible for the F₇ and noncytopathic phenotypes are genetically separable. The virulent parental strain, AV-WT, exhibited a mean embryo death time of 42 h. Both the larger-spot-forming revertant of nc4 (nc4S1) and the small-plaque-forming revertant of nc7 exhibited a decrease in mean embryo death time (increase in virulence) from 74 to 63 h. A second-step, plaque-forming revertant derived from nc4S1 (nc4S1R1) exhibited a further decrease in mean embryo death time from 63 to 44 h. The results suggest that the F_A-F₇ and X lesions affect the ability of virus to spread from cell to cell. In addition, these lesions appear to be genetically separable from those responsible for the noncytopathic phenotype. However, both types of lesions cause an extension of mean embryo death time and, thus, may be relevant to virulence in vivo.     

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.     

Detection of Mouse Mammary Tumor Virus RNA in BALB/c Tumor Cell Lines of Nonviral Etiologies

A complementary DNA (cDNA) probe to mouse mammary tumor virus (MMTV) RNA was synthesized using calf thymus DNA oligonucleotides as a random primer. This probe was then used to study the expression of MMTV RNA in cell lines from BALB/c tumors induced in vivo either spontaneously or in response to viral, chemical, or hormonal stimuli. The cDNA had a length of approximately 400 to 500 nucleotides and specifically hybridized to MMTV RNA and BALB/c lactating mammary gland RNA, but not to Moloney leukemia virus RNA. Calf thymus DNA-primed cDNA could protect 50% of iodinated MMTV RNA from S1 nuclease digestion at cDNA-RNA ratios of 1:1 and 90% of labeled viral RNA at ratios of 10:1. Thermal denaturation of MMTV RNA-cDNA hybrids yielded a T(m) of 88.5 degrees C, indicative of a well-base-paired duplex. Screening of mouse mammary tumor cells for MMTV sequences revealed that three out of five lines of BALB/c origin had undetectable levels of viral RNA (相似文献   

Structural Roles of Polyoma Virus Proteins

The superhelical, closed circular form of polyoma deoxyribonucleic acid (DNA) (Co 1) is bound in a 25S DNA-protein complex to the viral histone-like proteins after alkaline disruption of the virion. Nicked viral DNA or linear DNA are largely free of protein. Most of the viral protein disruption is in the form of capsomeres, sedimenting principally at 10S and 7S. Despite the relatively constant ratio of 10S to 7S material in many preparations, (1:5.5 to 1:6.0, respectively), the two classes of capsomeres are indistinguishable by electron microscopy and contain only P(2), P(3), and P(4) in molar ratios of approximately 5:1:1 or 6:1:1, respectively. Material with sedimentation rates of approximately 1 to 3S is enriched for P(5) and contains small amounts of P(2), P(3), and P(4). During the in vitro reassembly of DNA-free, shell-like particles from disrupted virus, proteins P(1), P(2), P(3), P(4), and P(7) are reincorporated efficiently, whereas P(5) and P(6) are not. The presence in empty reassembled particles of histone-like protein, expecially P(7), implies that at least this one of the minor protein components of the virion may participate in protein-protein interactions with other components of the capsid.     

小鼠肝癌与肝正常细胞系ST3Gal和ST6Gal家族mRNA表达差异研究

探讨肝癌细胞系Hepa1-6与肝正常细胞系BNL CL.2唾液酸糖基转移酶ST3Gal和ST6Gal家族mRNA表达的差异以及与细胞膜唾液酸含量的关系,采用RT-PCR方法检测ST3Gal唾液酸转移酶家族6个成员以及ST6Gal唾液酸转移酶家族2个成员mRNA表达差异,用凝集素芯片检测细胞膜表面唾液酸表达情况,结果显示:与正常细胞系BNL CL.2相比,hepa1-6细胞内唾液酸转移酶ST3GalⅠ、ST3GalⅣ、ST3GalⅥ呈现高表达,ST3GalⅤ低表达,ST3GalⅡ、ST3GalⅢ表达无显著性差异,两细胞系内均为检测出ST6GalⅠ表达,ST6GalⅡ表达无显著差异;hepa1-6细胞膜α2-3和α2-6连接唾液酸含量均显著增加;提示ST3GalⅠ、ST3GalⅣ、ST3GalⅤ、ST3GalⅥ可能与肝癌发生过程相关,ST3GalⅠ、ST3GalⅣ、ST3GalⅥ可能与肝癌细胞膜α2-3唾液酸含量增加相关,ST6Gal家族对细胞膜α2-6连接唾液酸含量增加无贡献.     

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.