Owl monkey astrocytoma cells in culture spontaneously produce infectious JC virus which demonstrates altered biological properties.

A tumor cell suspension of an explanted JC virus (JCV)-induced owl monkey glioblastoma was inoculated intracranially into four recipient juvenile owl monkeys. Twenty-eight months following inoculation one owl monkey developed a glioblastoma, which was explanted into tissue culture. DNA from both the tumor tissue and tumor cells in culture hybridized to a JCV DNA probe by Southern analysis, indicating that free, as well as integrated, viral DNA may be present. At the time of the second culture passage, viral JCV DNA was extracted from these cells and cloned into a plasmid vector. Nucleotide sequencing of the regulatory region of the cloned DNA demonstrated homology with the prototype Mad-1 strain of JCV and revealed a 19-base-pair deletion in the second 98-base-pair tandem repeat that eliminated a second TATA box. This deletion is characteristic of the Mad-4 strain of JCV, which is highly neurooncogenic. By the third culture passage, 100% of the cells were T-antigen positive. Approximately one-third of the cells in culture hybridized to a biotinylated JCV DNA probe when in situ hybridization was used, a technique that only detects high-copy-number of replicating viral sequences. By the culture passage 5 and continuing through culture passage 14, viable JC virions could be recovered. The T protein synthesized by this virus, now termed JCV-586, differed from both the Mad-1 and Mad-4 strains in that it formed a stable complex with the cellular p53 protein in the tumor cells. Also, the JCV-586 T protein reacted to several monoclonal antibodies made to the simian virus 40 T protein that were not recognized by either the Mad-1 or Mad-4 strains.     

Dynamic regulation of aquaglyceroporin expression in erythrocyte cultures from cold- and warm-acclimated Cope's gray treefrog, Hyla chrysoscelis

Cope's gray treefrog, Hyla chrysoscelis,is a freeze-tolerant anuran which accumulates and distributes glycerol as a cryoprotectant before freezing. We hypothesize that HC-3, an aquaglyceroporin member of the MIP family of water pores, may play an important role in the process of freeze tolerance by mediating transmembrane passage of glycerol and water during cold-acclimation. The objectives of this study were two-fold: to examine HC-3 protein abundance and cellular localization in erythrocytes from cold- and warm-acclimated frogs and to develop and characterize an erythrocyte cell culture system for examining HC-3 gene regulation. Compared with warm-acclimated frogs, erythrocytes from cold-acclimated frogs had higher HC-3 protein expression and enhanced plasma membrane localization. Furthermore, erythrocytes from cold- and warm-acclimated frogs maintained in culture at 4 and 20°C exhibited time- and temperature-dependent regulation of HC-3 expression and an increase in the abundance of high molecular weight immunoreactive species within 24?hr of culture at 20°C. Deglycosylation of erythrocyte proteins resulted in the disappearance of the high molecular weight species, indicating that HC-3 is post-translationally modified by N-linked glycosylation. Erythrocytes cultured in media containing glycerol also showed an increased abundance of the high molecular weight bands and enhanced plasma membrane localization of HC-3, suggesting a role for glycerol in regulating HC-3 subcellular trafficking. Thus, the development of this erythrocyte cell culture system from H. chrysoscelis opened an opportunity to study the properties of cells with changing expression of an aquaglyceroporin, HC-3, and to explore the factors regulating that expression.     

Inhibition by caffeine of post-replication repair in Chinese hamster cells treated with cis platinum (II) Diamminedichloride: the extent of platinum binding to template DNA in relation to the size of low molecular weight nascent DNA.

Treatment of Chinese hamster lung V79-379A cells with the anti-tumour agent cis platinum (II) diamminedichloride, (cis Pt(II)), resulted in an immediate recuction in the rate of DNA synthesis. Sedimentation of newly synthesised DNA through alkaline sucrose gradients revealed it to be approximately the same size as that obtained from untreated cells. In contrast, in the presence of 0.75 mM caffeine, the rate of DNA synthesis rapidly returned to control levels, although sedimentation analysis showed the DNA synthesised in cis Pt(II)-treated cells to be of lower molecular weight than in untreated cells. The reduction in molecular weight was directly proportional to the initial dose of the platinum compound. Furthermore, the results of separate binding studies suggested that at several levels of reaction the new DNA was synthesised up to a size approximately equal to the interplatinum distance in the template strand. This has been interpreted as being the result of the formation of a gap in the daughter DNA strand opposite every DNA-platinum product in the template strand. If caffeine was removed from the culture medium, there was a rapid increase in the molecular weight of the nascent DNA strands. However, if caffeine remained in the medium, the DNA remained of lower molecular weight than in untreated cells. It is proposed that this effect of caffeine is the result of the inhibition of a post-replicative DNA repair process which allows the eventual synthesis of a continuous DNA strand on a template containing unexcised lesions. It is further proposed that inhibition of this post-replicative DNA repair process provides a molecular basis for the previously observed potentiation by caffeine of cis Pt(II)-induced chromosomal aberrations and lethality.     

火鸡疱疹病毒在鸡胚成纤维细胞上有效代次的测定及其免疫原性恢复的研究

本试验证明,火鸡疱疹病毒(HVT)FC126株在鸡胚成纤维细胞(CEF)传55代的病毒,仍有预防马立克氏病(MD)的效力,至第70代效力明显下降。若将HVT CEF第35代病毒复归火鸡连续传4代,能明显恢复其免疫原性,用C细胞传代则有效代次可达到75代。因而,此方法能有效地解决免疫原性下降的种毒的更新问题。本试验还为该种毒和疫苗的有效代次的使用范围提供了可靠依据。     

The selection of mosaic (MSC) phenotype after passage of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) through cell culture — a method to obtain plant mitochondrial mutants

Mosaic (MSC) mutants of cucumber (Cucumis sativus L.) appear after passage through cell cultures. The MSC phenotype shows paternal transmission and is associated with mitochondrial DNA rearrangements. This review describes the origins and phenotypes of independently produced MSC mutants of cucumber, including current knowledge on their mitochondrial DNA rearrangements, and similarities of MSC with other plant mitochondrial mutants. Finally we propose that passage of cucumber through cell culture can be used as a unique and efficient method to generate mitochondrial mutants of a higher plant in a highly homozygous nuclear background.     

Enhanced synthesis of a Mr = 55,000 dalton peptide by senescent human fibroblasts

The secretory protein profiles of early and late passage cultures of human fibroblasts were compared using polyacrylamide gel electrophoresis. In comparison with early passage cell cultures (40-50% lifespan completed), late passage (greater than 80% of lifespan completed) cell cultures exhibited enhanced production of several peptides in the Mr range 55-60,000. One of those peptides had an apparent molecular weight of Mr = 55,000 and was constitutively present in the late passage cell conditioned medium. Late passage cell cultures synthesized the Mr = 55,000 peptide in the presence or absence of fetal bovine serum. Serum did not enhance its production by early passage cells. Further, production of the peptide was not induced in early passage cell cultures whose proliferation was arrested either by serum starvation or by contact inhibition. Pulse chase studies demonstrated that the peptide appears in the culture medium within 60 min of labeling. There was no evidence that it is derived via degradation of other proteins present either in early passage or late passage cell conditioned media. Further, the production of the 55,000 dalton peptide did not appear to be regulated by factors present in conditioned media. The peptide was detected in the conditioned media produced by late passage cultures of several different cell strains.     

Proteins Specified by Herpes Simplex Virus XII. The Virion Polypeptides of Type 1 Strains

The polypeptides from purified virions of a herpes simplex 1 (human herpes-virus 1) strain, F1, which had been passaged a limited number of times in cell culture after isolation, formed 33 bands on electrophoretic separation in polyacrylamide gels cross-linked with N, N'-diallyltartardiamide in contrast to a maximum resolution of only 24 to 25 bands in gels cross-linked with N, N'-methylenebisacrylamide. This increase in the number of bands was due chiefly to an improved separation of glycosylated polypeptides from nonglycosylated polypeptides with which they co-electrophoresed on methylenebisacrylamide cross-linked gels. Purified virions of HSV-1 [F1] had a protein/DNA mass ratio of 10.7 +/- 0.96, and based on a DNA molecular mass of 85 x 10(6) to 100 x 10(6) the estimated weight of virion polypeptides ranges from 16.4 to 19.4 x 10(-16) g. The number of molecules of each polypeptide per virion ranged from less than 50 to 1,500. Comparison of the virion polypeptides of two HSV-1 strains with similar isolation and limited passage history with those of four HSV-1 strains with histories of numerous passages outside the human host showed a number of nonrandom variations in virion polypeptides. Thus, although the virion polypeptides of two strains with similar isolation and limited passage history could not be differentiated, strains with extended passage histories differed markedly from each other and from the limited passage strains in the number and electrophoretic mobility of noncapsid polypeptides and notably in those of the envelope.     

Conditions of transformation by DNA of Neurospora crassa.

The DNA uptake and transformation of inositol-requiring recipient Neurospora strains were investigated. Exponentially growing cultures can accumulate 5-10 fold quantities of donor DNA than older ones. The rate of DNA uptake depends on the physiological state of the recipient cell, and on the molecular weight of donor DNA. The exocellular DNase activity of the recipient culture may influence the DNA uptake and the transformation process. "Young" inositol-requiring Neurospora crassa cultures can be transformed by wild type DNA reproducibly, but with low efficiency.     

Loss of viral genomes from hamster tumor cells and nonrandom alterations in patterns of methylation of integrated adenovirus type 12 DNA.

The insertion stability and DNA methylation patterns of integrated adenovirus type 12 (Ad12) genomes were investigated in Ad12-induced tumors and in tumor cell lines established from them as a function of time of passage under culture conditions. Upon subcultivation of cells from some of the tumors, the viral genomes were eliminated, apparently in a stepwise process with segments of the left termini of Ad12 DNAs persisting the longest. Morphological variants of these tumor cells lost all viral DNA and yet retained the oncogenic phenotype. All 13 independently isolated clones from one revertant line were devoid of Ad12 DNA. It could not be ruled out that very short sequence elements of viral DNA, such as promoters or enhancing sequences, could have persisted in these variants. The extent of viral DNA methylation was minimal in Ad12-induced tumors, although the viral genome was not extensively expressed, if at all. Upon passage in culture, the levels of viral DNA methylation increased. It was interesting that establishment of the final methylation pattern of integrated Ad12 DNAs required many cell generations after the fixation of foreign DNA in the host genome. The shift in methylation was nonrandom. The late parts of the inserted viral genomes became methylated more extensively than did the early gene segments.     

A few-polyhedra mutant and wild-type nucleopolyhedrovirus remain as a stable polymorphism during serial coinfection in Trichoplusia ni

Few-polyhedra (FP) mutants of nucleopolyhedroviruses (NPVs) are a well-known phenomenon during serial passage of virus in cell culture. Under these circumstances such mutants produce low yields of occlusion bodies (OBs) and poorly occlude virions, but they are selected for through advantageous rates of budded virus replication. Spontaneous insertion of transposable elements originating from host cell DNA into the viral fp25 gene has been shown to be a common cause of the phenotype. A model of NPV population genetics predicts that mutants with these characteristics might persist within stable polymorphisms in viral populations during serial passage of virus in vivo. However, this hypothesis was previously untested, and FP mutants have not been recovered from field isolates of NPVs. We isolated and characterized an FP mutant that arose during routine passage of Autographa californica multinucleocapsid NPV (AcMNPV) in cell culture and identified a transposable element within the fp25 gene. We tracked the fates of coinfecting wild-type and FP mutant AcMNPV strains through serial passage in fifth-instar Trichoplusia ni larvae. The levels of both strains remained stable during successive rounds of infection. We applied the data obtained to a model of NPV population genetics in order to derive the frequency distribution of the multiplicity of cell infection in infected insects and estimated that 4.3 baculovirus genomes per OB-producing cell would account for this equilibrium.     

Nascent DNA Synthesis in Ultraviolet Light-Irradiated Mouse, Human, and Chinese Hamster Cells

The technique of alkaline sucrose gradient centrifugation was used to study newly synthesized DNA in control and ultraviolet light-irradiated mouse L, human HeLa, and Chinese hamster ovary cells. Nascent DNA molecular weight distributions did not appear to differ among the three cell lines for unirradiated cells. However, at short times after ultraviolet light irradiation, human HeLa cells appeared to synthesize more low molecular weight DNA than either mouse L or Chinese hamster ovary cells. Since this difference was not related to differences in either the rate of DNA synthesis or amount of ultraviolet damage in the irradiated cells it appeared to be a phenotypic characteristic of the cell lines tested. A parallel was noted for these three cell lines between an increase in the synthesis of low molecular weight DNA, detected on alkaline sucrose gradients, and cell killing as measured by the ability of irradiated cells to form colonies.     

Proliferation kinetics of rabbit articular chondrocytes in primary culture and at the first passage

The in-vitro proliferation kinetics of young rabbit articular chondrocytes were compared in primary culture and at the first passage. The growth curves labelling and mitotic indices, percentage labelled mitosis (PLM) curves and DNA content distributions by flow-microfluorometric analysis during a 7-day growth period were determined in both cases. The length of the cell cycle and the doubling time calculated from the exponential part of the growth curve were quite similar: Tc = 19 hr and Td = 20 hr for the primary culture, Tc = 17 X 3 hr and Td = 20 hr for the first passage. However, the growth curve and the DNA distribution during the 7-day period showed some differences. The duration of the lag period studied by the growth curve was longer in the primary culture than at the first passage. This phenomenon was also observed using the FCM analysis. The growth fraction determination on the second day of culture was in accordance with the lower proliferation capacity of the cells in primary culture. These data suggest that it would be better to study growth kinetics and drug modifications in articular chondrocytes at the first passage than in primary culture.     

Reacquisition of a functional early region by a mouse transformant containing only defective simian virus 40 DNA.

Viral DNA in simian virus 40-transformed mouse cells is capable of rearranging with passage. In this report, we show that such rearrangement can include an alteration in viral protein expression. SVT2, a simian virus 40-transformed mouse BALB/c 3T3 cell line, synthesizes only a super T antigen of molecular weight 100,000 without synthesizing the lytic-size large T or small t antigens with molecular weights of 94,000 and 17,000, respectively. Analyses of the integrated viral DNA revealed an early region of 4.4 kilobases instead of the lytic-size 2.7 kilobases. However, upon subcloning in either plastic or agarose or after being in culture for several passages, the appearance of lytic-size large T and small t antigens was detected. Concurrently, an early region of 2.7 kilobases, in addition to one of 4.4 kilobases, was observed.     

Sequence of events leading to apoptosis in long term cultured HeLa cells

We have maintained HeLa cells in culture in the original medium for increasing times to induce growth arrest. Cell viability was evaluated by trypan blue dye exclusion assay. We observed that when cells are maintained in culture for several days, morphological hallmarks of apoptosis become evident. DNA synthesis rate, followed by (3)H-thymidine incorporation slowed down in long term cultured cells. This evidence was supported by the analysis of cell cycle progression determined by proliferating cell nuclear antigen (PCNA) immunostaining. Apoptotic cells have been characterized with respect to the sequential appearance of high molecular weight and internucleosomal DNA fragmentation. We have provided evidence that in this experimental model the first step in DNA degradation is represented by the formation of high molecular weight fragments, whereas nucleosomal DNA ladder is visible later on. The activation of the enzyme poly(ADP-ribose)polymerase, considered a marker of apoptotic death, has been observed. The results suggest that long term culture conditions activate the apoptotic programme.     

Effects of pH,conductivity, host cell protein,and DNA size distribution on DNA clearance in anion exchange chromatography media

Flowthrough anion exchange chromatography is commonly used as a polishing step in downstream processing of monoclonal antibodies and other therapeutic proteins to remove process‐related impurities and contaminants such as host cell DNA, host cell proteins, endotoxin, and viruses. DNA with a wide range of molecular weight distributions derived from Chinese Hamster Ovary cells was used to advance the understanding of DNA binding behavior in selected anion exchange media using the resin (Toyopearl SuperQ‐650M) and membranes (Mustang® Q and Sartobind® Q) through DNA spiking studies. The impacts of the process parameters pH (6–8), conductivity (2–15 mS/cm), and the potential binding competition between host cell proteins and host cell DNA were studied. Studies were conducted at the least and most favorable experimental conditions for DNA binding based on the anticipated electrostatic interactions between the host cell DNA and the resin ligand. The resin showed 50% higher DNA binding capacity compared to the membrane media. Spiking host cell proteins in the load material showed no impact on the DNA clearance capability of the anion exchange media. DNA size distributions were characterized based on a “size exclusion qPCR assay.” Results showed preferential binding of larger DNA fragments (>409 base pairs). © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:141–149, 2018     

Demonstration of Infectious Deoxyribonucleic Acid in Transformed Cells I. Recovery of Simian Virus 40 from Yielder and Nonyielder Transformed Cells

Deoxyribonucleic acid (DNA) was extracted from virus-free simian virus 40 (SV40)-transformed hamster, mouse, and monkey cells and was inoculated into simian cells in the presence of diethylaminoethyl (DEAE)-dextran; infectious SV40 was recovered by using DNA from cell lines which fail to yield virus by the fusion technique as well as from cell lines which readily yield virus by fusion. The rescued virus was identified as SV40 by three methods: (i) neutralization of plaque formation by specific antiserum; (ii) induction of synthesis of viral-specific antigens detected by immunofluorescence; and (iii) presence of papovavirus particles seen by the electron microscope. Treatment of the transformed cell DNA with deoxyribonuclease or omission of the DEAE-dextran prevented the rescue of virus. Large amounts of transformed cell DNA were required (>10 mug/culture of 10(6) cells) to effect rescue of SV40 by passage through monkey cells. A linear response was obtained between the input of DNA with inocula between 10 and 45 mug of DNA/culture and the yield of SV40 recovered. Biological activity was demonstrable irregularly when the transformed cell DNA was assayed directly in the presence of DEAE-dextran. The DNA induced plaque formation in about 50% of the trials as well as the synthesis of SV40 tumor and viral antigens in rare simian cells. The infectious DNA appeared to be associated with cellular DNA. The infectivity was found in the pellet of precipitated DNA obtained by the Hirt technique and was inactivated by boiling for 15 min. These properties are characteristic of linear cellular DNA and not of free, circular SV40 DNA.     

旋毛虫肌幼虫细胞传代培养及超微结构观察

消化、分离观察旋毛虫(Trichinella spiralis)肌幼虫,获得肌幼虫细胞,用含10%胎牛血清的RPMI-1640培养液培养原代细胞,胰酶(含0.02?TA)消化法进行传代,透射电镜观察培养细胞超微结构,用多重PCR鉴定培养细胞。结果表明,在培养24～72h原代细胞开始贴壁,7～8d形成单层细胞,细胞间融合现象不明显,10～12d传一代。透射电镜显示旋毛虫细胞核为椭圆形,核膜、核仁清晰,核内染色质较丰富,胞浆含丰富的线粒体。细胞主要有两种类型:椭圆形和多角形,以椭圆形为主。多重PCR扩增培养细胞DNA,可见1条与旋毛虫肌幼虫DNA扩增产物相同的条带(173bp)。结果表明,旋毛虫肌幼虫细胞可在含10%胎牛血清的RPMI-1640培养液中传代培养。     

Mechanisms restricting DNA replication to once per cell cycle: MCMS, pre-replicative complexes and kinases

An important aspect of cell behaviour is that DNA replication happens only once per cell cycle. Replicated DNA is unable to re-replicate until cell division has occurred. Unreplicated DNA is in a replication-competent or 'licensed' state. The ability to replicate is lost in S phase and regained following passage through mitosis. Recent evidence has implicated an MCM (minichromosome maintenance) protein complex and the Cdc6 protein in determining replication competence. Regeneration of replication competence upon passage through mitosis entails changes in protein kinase activity, of which the MCMs are a likely target. Features of the mechanism that restricts DNA replication to once per cell cycle appear to be conserved throughout eukaryotes.     

p27Kip1 and Cyclin Dependent Kinase 2 Regulate Passage Through the Restriction Point

When quiescent cells are stimulated to re-enter the cell cycle, growth factors are required only until the restriction point in G1 phase. After this point the cell no longer requires growth factors, proliferative signaling molecules, or even protein synthesis in order to initiate DNA synthesis, which starts several hours later. Consequently, understanding the molecular nature of the restriction point constitutes one of the major goals in studies of growth regulation. We recently demonstrated that p27Kip1 (p27) regulates passage through G1 phase in actively proliferating cultures, and initiated these studies to determine if it is also involved in passage through the restriction point following stimulation of quiescent cells. In support of this suggestion, we found that passage through the restriction point requires mitogen-dependent suppression of the high p27 levels normally present in quiescent cells. Moreover, as the culture progresses to mid-G1 phase, the proportion of cells that pass the restriction point is increased by artificial suppression of p27 levels, while this proportion is reduced by elevation of p27 levels. p27 performs this critical function by regulating the subsequent activating phosphorylation of cyclin dependent kinase (CDK)2, which we also show is necessary for and closely associated with the initiation of DNA synthesis. We conclude that the p27 expression level at mid-G1 phase determines when a cell passes through the restriction point, and does so by regulating subsequent CDK2 activation.