Anchorage-independent growth of normal human mesothelial cells: A sensitive bioassay for EGF which discloses the absence of this factor in fetal calf serum

Summary This laboratory recently reported that normal human mesothelial cells require epidermal growth factor (EGF) and hydrocortisone (HC), in addition to fetal calf serum and a complex defined medium component, in order to grow optimally in surface culture (9). We report here that this normal cell type also forms large colonies at high efficiency in semi-solid medium, but exhihits more stringent serum and EGF requirements for anchorage-independent than for surface growth. Mesothelial cells are unable to divide at all in semi-solid medium with added EGF or with less than 2% serum, whereas they grow slowly but progressively in surface culture under such conditions. In semi-solid medium containing 20% serum and HC, mesothelial cells are stimulated to divide by the addition of as little as 30 pg/ml purified EGF. Human urine or male mouse plasma could substitute for purified EGF, yielding growth commensurate with the levels of EGF in these biological fluids previously measured by others using radioreceptor and radioimmune assays. Thus growth of mesothelial cells in semi-solid medium can serve as a highly sensitive assay of EGF biological activity which is unaffected by the presence of serum proteins. In addition, our results demonstrate that fetal calf serum does not provide mitogenic levels of EGF to cultured cells, raising the question of the identity of plasma and serum mitogens. This work was supported by NIH grants RO1 AG02048 and RO1 CA26656 to James G. Rheinwald and by NIH postdoctoral fellowship F32 AG05303 to Paul J. La Rocca.     

泥鳅雄核发育纯合二倍体的产生

以机械方法挑去泥鳅(Misgurnus anguillicaudatus)×大鳞副泥鳅(Paramisgurnus dabryanus)(♀)属间杂交受精卵的雌核,得到泥鳅雄核发育单倍体胚胎。将这种单倍体胚胎的囊胚细胞核移植到大鳞副泥鳅去核卵中,获得了243个原肠胚胎,其染色体鉴定表明,29.6%的核移植体的染色体发生了加倍。在另一实验组中,从769个核移植卵得到了5尾2cm以上的个体。尾鳍染色体鉴定、肌肉LDH同工酶电泳和形态鉴别表明,这5尾核移植体为泥鳅雄核发育纯合二倍体。     

Isolation and characterization of diploid clones from adult and newborn rat liver cell lines

Summary A high frequency of diploid and near-diploid clones were developed from cell lines derived from adult and newborn rat liver using micropipettes. There were some differences in morphology, biochemical properties and growth rate between clones. Cloned cells had low levels of tyrosine transaminase activity, glucose-6-phosphatase activity and albumin content. A diploid clone and a pseudodiploid clone derived from adult rat liver cell line were positive for α-fetoprotein. This work was supported by a grant for cancer research from the Japanese Ministry of Education.     

A comparison of the DNA binding,cytotoxicity and repair synthesis induced in human fibroblasts by reactive derivatives of aromatic amide carcinogens

The cytotoxicity of three structurally-related direct-acting carcinogens, N-acetoxy-2-acetylaminofluorene, N-acetoxy-2-acetylaminophenanthrene and N-acetoxy-4-acetylaminobiphenyl, was compared in normal cells and in excision repair deficient xeroderma pigmentosum cells (XP12BE). All three proved significantly more cytotoxic to the XP cells than to the normal cells. At equicytoxic levels, substantially more residues were initially bound to the DNA of the normal cells than to the XP cells, suggesting that the former are able to remove a large percentage of the DNA bound residues before these can result in cell death. The ability of these cell strains to remove bound residues from DNA, to incorporate thymidine into parental strands of DNA during repair replication, and to recover from potentially lethal damage if held in the non-replicating, density-inhibited G_o state was compared as a function of dose and time. The XP12BE cells proved virtually incapable of excision repair of DNA damage induced by these carcinogens and of recovery. In contrast, normal cells recovered from the potentially lethal effects of these three compounds and did so at a rate comparable to their rate of removal of bound residues and of repair synthesis. In the excision-deficient XP12BE cells, DNA adducts induced by N-acetoxy-2-acetylaminophenanthrene proved 3- to 6-fold more cytotoxic than adducts induced by the other two carcinogens.     

产黄青霉菌丝联合及病毒传递

通过营养要求、孢子颜色不同的带病毒和无病毒产黄青霉Penicillium chrysogenum菌株间的菌丝联合,在基本培养基(MM)上获得了营养互补的异核体。异核体菌落上出现亲本类型分离并产生深绿色角变或斑点。经孢子体积,DNA含量及营养类型测定证明角变和斑点为杂合二倍体。经电子显微镜观察,奥氏免疫双扩散,2.4％聚丙烯酰胺凝胶电泳及放射免疫测定检验了胞质融合或胞质和核融合后代病毒的传递,结果表明黄孢子后代带病毒而杂合二倍体及绿孢子后代均不带病毒。看来,这些绿孢子菌株(821和822)抗病毒感染。     

In vitro polyploidization of mature ostrich fern sporophytes through rejuvenation

An in vitro method is described for producing ostrich fern (Matteuccia struthiopteris (L.) Todaro) polyploids from mature sporophytes as a possible means of plant improvement in this economically important fern species. The procedure is based on rejuvenating adult sporophytes (2n) to enable the aposporous production of diploid (2n) gametophytes, and then mating the gametophytes to produce tetraploid (4n) sporophytes. The adult sporophytes were rejuvenated by culturing excised shoot tips for a minimum of three months in a liquid medium (Murashige and Skoog salts) under conditions of extreme carbohydrate deprivation (0.01% sucrose). Apospory was induced by culturing leaves excised from the rejuvenated shoots for two months on a semi-solid medium lacking sucrose, resulting in the production of diploid gametophytes. The gametophytes were transferred to fresh medium and grown to sexual maturity for one or two months, then floated on the surface of a liquid medium containing 0.01% sucrose for up to two months to promote opening of the sex organs. Subsequent self-fertilization resulted in the successful production of tetraploid sporophytes in 11 of the 14 diploid clones in which polyploidization was attempted. Tetraploids (4n=156) were confirmed by cytological examination. This method permits polyploidization of mature, fully characterized plants.     

Diploid males in the bumble beeBombus terrestris

The frequency of colonies that produce diploid males after brother-sister (50%) and nephew-niece (37.5%) matings proves that inB. terrestris the sex is determined by a single multi-allelic sex locus. The diploid males which develop normally into adults make up 50% of the diploid brood. In the laboratory the growth rate of colonies with diploid males is influenced only slightly. Of 41 colony founding queens caught out of a natural population, all produced a colony without any diploid males. Therefore, the number of sex alleles in this population is estimated to be at least 24. This means that in commercial rearing systems for bumble bees, involving several generations, the occurrence of diploid males can largely be prevented by a good scheme for crossings.     

Human diploid fibroblast growth on polystyrene microcarriers in aggregates

Polystyrene microcarriers were prepared in four size ranges (53–63 m, 90–125 m, 150–180 m and 300–355 m) and examined for ability to support attachment and growth of human diploid fibroblasts. Cells attached rapidly to the microcarriers and there was a direct relationship between cell attachment and microcarrier aggregation. Phasecontrast and scanning electron microscopic studies revealed that while aggregation was extensive, most of the aggregate consisted of void volume. Cell growth studies demonstrated that human diploid fibroblasts proliferated well in microcarrier aggregates, reaching densities of 2.5–3×10⁶ cells per 2 ml dish after 6 days from an inoculum of 0.5×10⁶ cells per dish. When cells were added to the microcarriers at higher density (up to 5×10⁶ cells per 2-ml culture), there was little net growth but the cells remained viable over a 7-day period. In contrast, cells died when plated under the same conditions in monolayer culture. When the microcarriers were used in suspension culture, rapid cell attachment and rapid microcarrier aggregation also occurred. In 100-ml suspension culture, a cell density of 0.7×10⁶ cells per ml was reached after 7 days from an inoculum of 0.1×10⁶ cells. Based on these data, we conclude that microcarrier aggregation is not detrimental to fibroblast growth. These data also indicate that small microcarriers (53–63 m) (previously thought to be too small to support the growth of diploid fibroblasts) can support fibroblast growth and this occurs primarily because microcarriers in this size range efficiently form aggregates with the cells.     

Genetic analysis of anther and leaf disc culture in two clones of Solanum chacoense Bitt. and their reciprocal hybrids

Two diploid clones of self-incompatible Solanum chacoense Bitt. with androgenetic ability were tested for anther and leaf disc culture response together with eight of their reciprocal F1 hybrids. Large differences were found among genotypes in frequency of anther induction as well as in the phase of plant regeneration. Anthers harvested in June showed a significantly higher percentage of response (17.5%) at the induction phase than those collected in July (13.8%) or August (12.7%). The lowest induction frequency was observed in May (7.3%). By contrast, plant regeneration from induced anthers did not vary during this time. Genotypic differences were also observed in leaf disc response. The two parental clones and two of their hybrids failed to produced any shoots. Among the remaining genotypes, two had only sporadic occurrence of shoot formation, two gave an intermediate response (15% and 24% of their discs carried shoots), whereas the discs of the two remaining genotypes responded well to culture (68% and 77%). The genetic analysis performed on the reciprocal hybrids revealed that a positive significant correlation existed between anther induction and leaf disc response (Spearman's r=0.82; p=0.01). This suggests that, under our conditions, these two aspects of tissue culture might share a common system of genetic control. Estimates of broad sense heritabilities, for leaf disc culture, 83% were obtained and the number of effective factors involved in the control of tissue culture response, indicated a relatively simple genetic control. Finally, considering the potentialities opened by the use of RFLP analysis, it might be possible to find probes that are linked with genes involved in tissue culture competence.     

Effect of O6-alkylguanine-DNA alkyltransferase on the frequency and spectrum of mutations induced by N-methyl-N''-nitro-N-nitrosoguanidine in the HPRT gene of diploid human fibroblasts

N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) reacts with 12 nucleophilic sites in DNA to induce a variety of lesions, but O⁶-methylguanine (O⁶-MeG) and O⁴-methylthymine are the most effective premutagenic lesions produced, mispairing with thymine and guanine, respectively. O⁶-MeG is repaired by O⁶-alkylguanine-DNA alkyltransferase (AGT), which removes the methyl group from the O⁶ position and transfers it to itself, rendering the transferase inactive. When diploid human fibroblasts were exposed to 25 μM, O⁶-benzylguanine (O⁶-BzG) in the medium for 3 h, their level of AGT activity was dramatically reduced, to a level of at most 1.6% of the control. Populations of cells pretreated with this level of O⁶-BzG for 2 h or not pretreated, were exposed to MNNG at a concentration of 2, 4 or 6 μM in the presence or absence of O⁶-BzG and assayed for survival of colony-forming ability and the frequency of 6-thioguanine-resistant cells (mutations induced in the HPRT gene). O⁶-BzG (25 μM) was also present in the appropriate half of the cells during the 24 h immediately follwing exposure to MNNG. This 27-h exposure to O⁶-BzG alone had no cytotoxic or mutagenic effect on the cells but significantly increased the cytotoxicity and mutagenecity of MNNG, increasing the mutant frequency to that found previously in human cells constitutively devoid of AGT activity. At doses of 2 μM and 4 μM MNNG, the mutant frequency observed with the AGT-depleted cells was 120 × 10⁻⁶ and 240 × 10⁻⁶, respectively; in the cells with abundant AGT activity, these values were 10 × 10⁻⁶ and 20 × 10⁻⁶, respectively. DNA-sequence analysis of the coding region of the HPRT gene in 36 independent mutants obtained from MNNG-treated AGT-depleted populations and 36 from the control populations showed that even though AGT repair lowered the frequency of mutants by more than 90%, it did not affect the kinds of mutations induced by MNNG nor the strand distribution of the premutagenic guanine lesions. In mutants from the AGT-depleted cells, there were 26 base substitutions and 13 putative splice site mutations; in the control, there were 25 base substitutions and 11 splice site mutations. All but two substitutions involved G · C with 92% being G · C → A · T. In both sets, of the premutagenic lesions were located in the nontranscribed strand. Many ‘hot spots’ were seen, and there was evidence that AGT repaired more lesions from the 5′ half of the gene than from the 3′ half.