The effect of UV irradiation on proliferation and life span of human diploid fibroblast-like cells

Summary The effect of low dose UV irradiation on the reinitiation of proliferative activity and on the life span of human diploid fibroblast-like cells is described. Cells were exposed to UV at confluence or after maintenance in an arrested state. Cell division was stimulated immediately after UV irradiation or after an additional post-UV incubation period. Arrested populations of all in vitro ages exhibited a greater sensitivity to UV and the reinitiation of proliferation was enhanced by post-UV incubation before stimulation. Ultraviolet light had no effect on life span regardless of in vitro cell age, culture state at the time of exposure, or the presence of a postirradiation period of arrest.     

Effects of feeder layers made of human,mouse, hamster,and rat cells on the cloning efficiency of transformed human cells

Summary The effect of feeder layers on cloning efficiency of transformed human cells was investigated. Embryonic human skin or lung fibroblasts; adult human skin fibroblasts; early passage cells from embryos of mouse, rat, and hamster; established mouse cell lines; 3T3 and 10T1/2 were used as feeder layers after they were lethally exposed to Co-60 gamma-rays at 3,000 rad. As test cells to study the effect of feeder layers on cloning efficiency, WI-38 CT-1 cells transformed in vitro by Co-60 gamma-rays and HGC cells cultured from a human gastric cancer were used. The effect of feeder layers on the cloning efficiency of the test cells was dependent on cell density of feeder layer cells, sources of the feeder layer cells, and kinds of test cells. An optimal density of feeder cels produced cloning efficiencies 3 to 15 times higher than in cultures without a feeder layer. Generally, high density of cells in feeder layers decreased the cloning efficiency of the test cells, presumably owing to contact inhibition of growth and depletion of essential nutrients by the feeder layer cells. Regarding the effect of the feeder layers made of human fibroblasts, there were no significant differences in population doubling levels; tissue origins of fibroblasts; or fibroblasts derived from normal individuals, patients with cancer, or with a genetically high familial incidence of cancer, hereditary adenomatosis of the colon and rectum. This study was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan.     

Characterization of a new human diploid cell line—IMR-91

Summary A human diploid fibroblast cell line has been established from the lung tissue of a male fetus. This has been characterized and frozen away in large quantity. A smaller quantity of fibroblastlike cells from skin has also been established, partially characterized, and placed in frozen storage from the same fetus. This project is in support of the National Institute on Aging research in general cell biology. The present lines designated IMR-91 lung and IMR-91 skin complement the previous human diploid fibroblast culture (IMR-90) established from a female fetus. The lack of random inactivation of one of the two X chromosomes in the present male line reduces the genetic heterogeneity inherent in the female line.     

Flow cytometry analysis of early DNA content changes in human and monkey cells following infection with simian virus 40

Simian virus 40 (SV₄₀) is capable of inducing cellular DNA synthesis in permissive and nonpermissive cells. Utilizing flow cytometry, we analyzed the DNA content changes in two diploid human cell strains and two monkey cell lines. The osteogenesis imperfects (OI) human skin fibroblasts were induced into DNA synthesis, and within one to two cell generations, a polyploid cell population was produced. With WI-38 phase II cells, a similar pattern of increased cycling of cells into DNA synthesis was observed; however, the majority (～60%) of the cells were blocked in the G₂ + M phase of the cell cycle. At later time intervals, an increase in the G₁ population was demonstrated. The two monkey cell lines responded to SV₄₀ virus with an accumulation of cells in the G₂ + M phase of the cell cycle. Thus, two diploid human cell strains exhibited different cell cycle kinetics early after infection with SV₄₀ virus. The one strain (WI-38) behaved similarly to the two monkey cell lines studied. The other strain (OI) responded similarly to nonpermissive (transformin) cells infected with SV₄₀ virus.     

A growth history comparison of the human diploid cells WI-38 and IMR-90: Proliferative capacity and cell sizing analysis

Summary Results of growth history studies on IMR-90 and WI-38 showed that the two cell strains were equivalent in population doublings achieved per life span. However, IMR-90 exhibited higher cell yields in phase II than did WI-38. In addition, entry of IMR-90 cells into phase III occurred more abruptly than in WI-38 cultures. Cell sizing analysis showed that phase II and phase III IMR-90 cell populations contained greater numbers of cells in the small volume categories. At senescence, both cell lines contained similar numbers of cells in all size categories. These data suggest that IMR-90 may not be equivalent in all respects to current stocks of WI-38.     

Conditions for the invasion of male‐haploidy in diploid populations

Male‐haploidy has independently evolved several times in different phylogenetic groups and has led to various extant lineages in the insects, Arachnida and Rotifera. Although the stability of male‐haploidy as an evolutionary strategy is not well understood, various theories address the invasion of male‐haploidy in diploid populations. Here two of these theories: (i) the maternal transmission hypothesis (MTH) and (ii) the deleterious mutation hypothesis (DMH), are re‐investigated with an agent‐based model to understand the role of genetic drift as a mechanism facilitating the spread of male‐haploidy. These two hypotheses are analysed separately and comparatively, and the results suggest dominance of the MTH. In addition, comparison of the stochastic results to deterministic results using the same model structure shows how genetic drift can enhance the parameter space where male‐haploidy can be expected to invade.     

Establishment of the diploid gynogenetic hybrid clonal line of red crucian carp × common carp

This study investigated the gynogenetic cytobiological behavior of the third gynogenetic generation (G3), which was generated from the diploid eggs produced by the second gynogenetic generation (G2)of red crucian carp × common carp, and determined the chromosomal numbers of G3, G2×scatter scale carp and G2×allotetraploid hybrids of red crucian carp × common carp. The results showed that the diploid eggs of G2 with 100 chromosomes, activated by UV-irradiated sperm from scatter scale carp and without the treatment for doubling the chromosomes, could develop into G3 with 100 chromosomes.Similar to the first and second gynogenetic generations (G1 and G2), G3 was also diploid (2n=100) and presented the hybrid traits. The triploids (3n=150) and tetraploids (4n=200) were produced by crossing G2 with scatter scale carp, and crossing G2 with allotetraploids, respectively. The extrusion of the second polar body in the eggs of G2 ruled out the possibility that the retention of the second polar body led to the formation of the diploid eggs. In addition, we discussed the mechanism of the formation of the diploid eggs generated by G2. The establishment of the diploid gynogenesis clonal line (G1, G2 and G3) provided the evidence that the diploid eggs were able to develop into a new diploid hybrid clonal line by gynogenesis. By producing the diploid eggs as a unique reproductive way, the diploid gynogenetic progeny of allotetrapioid hybrids of red crucian carp × common carp had important significances in both biological evolution and production application.     

Studies of the inheritance of virulence in the entomopathogenic fungus Metarhizium anisopliae

A forced heterocaryon was established between two auxotrophic conidial color mutants of Metarhizium anisopliae. From the heterocaryon, a prototrophic somatic diploid was selected which, in turn, yielded somatic segregants. The virulence of the original mutants, the somatic diploid, and the somatic segregants was evaluated on three species of mosquitoes as well as on Ostrinia nubilalis larvae. The virulence of the somatic diploid was comparable to that of the wild-type parental strain while the auxotrophic somatic segregants exhibited virulence approximately equal to that of the auxotrophic components of the heterocaryon. Putative somatic diploids were obtained between morphological mutants of the two species varieties (M. anisopliae var. minor and var. major). The presumptive diploids were avirulent for the insect species to which the parental strains exhibited virulence.     

Stomatal length and frequency as a measure of ploidy level in black wattle, Acacia mearnsii (de Wild)

The length and frequency of stomata on leaf surfaces were examined as rapid techniques for future identification of ploidy level of Acacia mearnsii (de Wild). Diploid (2 n  = 2 x  = 26) and tetraploid (2 n  = 4 x  = 52) plants were germinated from chipped seed at 25°C and grown under nursery conditions. After one month, measurements showed that the mean stomatal length was 27.17 ± 0.474 µm for diploids and 40.24 ± 0.521 µm for tetraploids and these differed significantly from each other ( P  < 0.001). The frequency of stomata per leaf surface was shown to decrease significantly ( P  < 0.001) as the ploidy level increased, with a mean of 22.11 ± 0.495 for diploids and 10.26 ± 0.495 for tetraploids. It was concluded that stomatal length and stomatal frequency are rapid indirect methods to identify ploidy level in black wattle.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 141 , 177–181.     

高效建立129/ter、C57BL/6J小鼠胚胎干细胞系的方法学探讨

小鼠胚胎干细胞 (ＥＳ细胞 )是从小鼠囊胚内细胞团(ＩＣＭ)分离出来的、在体外培养过程中可维持未分化状态、正常二倍体核型及无限增殖能力 ,具有多能性或全能性的细胞系[1～ 3 ] 。ＥＳ细胞广泛应用于克隆动物制作、转基因动物生产、动物医学模型建立、真核细胞基因表达与调控的研究、细胞分化机制的探索、人及哺乳动物基因功能的研究以及细胞、组织、和器官的修复与移植研究。胚胎干细胞的研究和应用已成为生命科学研究的热点和前沿领域之一[4～ 8] 。自第一株 12 9小鼠ＥＳ细胞系建立以来 ,人们在生物学和医学等多个领域进行了广泛深入的…