Stochastic model for mast cell proliferation in culture of murine peritoneal cells

We recently identified two types of mast cell colonies derived from murine peritoneal cells: type 1 and type 2. Type 1 mast cell colonies consisted of berberine sulfate(+)- safranin(+) connective tissue-type mast cells (CTMC) and were derived from mature CTMC in the heaviest fraction obtained by Percoll density gradient centrifugation. In contrast, type 2 mast cell colonies consisted of alcian blue(+)- berberine sulfate(-)- safranin(-) mucosal mast cells (MMC) and were derived from immature progenitors in low density fractions. We replated a total of 60 type 1 and 60 type 2 mast cell colonies and examined their capability for producing secondary colonies. Although all of the primary colonies yielded secondary colonies, the replating efficiencies of individual colonies varied over a wide range. Cumulative distributions of secondary colonies from both type 1 and type 2 primary colonies could be fitted well by gamma distributions obtained by computer simulation. These findings are in agreement with the stochastic model for CTMC- and MMC proliferation. Cytological analyses of secondary colonies from primary type 1 colonies revealed heterogeneous distributions of alcian blue(+)- safranin(-)- berberine sulfate(-) mast cells, suggesting that transdifferentiation from mature CTMC to safranin(-)- berberine sulfate(-) mast cells is also governed by stochastic mechanisms.     

Brom Cresol Green and Brom Phenol Blue as Indicators of Respiration Deficiency in Yeast

Strains of Saccharomyces which contained cells of respirationally normal (wild-type) and of respiration-deficient (RD) mutants were grown on untrient agar plates containing 20-23 mg/liter of either brom cresol green (BCG) or brom phenol blue (BPB). Glucose content of the media was varied experimentally in the range of 1.5-6%. After 2-4 days of incubation, normal colonies were very palely stained whereas RD colonies were drak green with BCG and dark blue with BPB. Optimum glucose content in the medium was 2-3% for S. cerevisiae, S. carlbergensis and S. chevalieri, but Fleischmann's (baker's) yeast developed the best color contrast with 4-5% glucose.     

Blood Cell Ultrastructure of the Ascidian Botryllus schlosseri L. II. Pigment Cells

Colonies of Botryllus schlosseri L., bred in the laboratory and genetically selected as regards the blue and/or reddish pigments, were used. The following phenotypes were investigated under the electron microscope: (a) blue colonies without reddish pigment; (b) reddish colonies without blue pigment; (c) colonies with both blue and reddish pigments; (d) colonies with neither blue nor reddish pigments. In the pigmented colonies, a specialized blood pigment cell type was recognized that, in giant membrane-limited vacuoles, contained a great number of granules. In general, the granules were similar in size, not individually limited by a membrane and were made up with electrondense material often arranged in concentric rings. Although there could be some variability within the same cell, in each phenotype the granules displayed a characteristic pattern so that the differences in colour of the granules, as seen in vivo, were paralleled by differences in the ultrastructural architecture. In the unpigmented colonies also, granulated vacuolar cells, rare in number but morphologically comparable to the pigment cells, were seen. On the basis of these results, the hypothesis of the existence of a prospective pigment cell and of a common origin for all the pigment cells of B. schlosseri is discussed.     

High radiosensitivity of the MOLT-4 leukaemic cell line

Radiation survival of MOLT-4, a leukaemic T-lymphocyte cell line, was measured by counting colonies formed in 0.8 per cent methyl cellulose. The survival curve was a simple exponential and showed the cells to be radiation sensitive, with D0 = 0.49 +/- 0.02 Gy and extrapolation number n = 0.92 +/- 0.09. No increase in survival as measured by colony-forming ability or trypan blue dye exclusion was seen when the dose was split into two fractions, separated by a 5 h incubation period. Electron microscopy and trypan blue dye exclusion showed that 5 h after exposure to high doses, MOLT-4 cells began to die and displayed condensed, marginated chromatin and cellular vesiculation.     

Mechanism of the Selective Action of Eosin-Methylene-Blue Agar on the Enteric Group

The actual mechanism of the differentiation of lactose-fermenting and non-lactose-fermenting organisms on eosin-methylene-blue medium is not reported in the literature. The present study is an attempt to elucidate this problem.

The color of colon forms on E.M.B. agar was found to depend on two factors: (1) the reaction of eosin with methylene blue to form a dye compound of either acidic or neutral nature, and (2) the production, by lactose-fermenting colonies, of a sufficiently low pH so that this dye compound is taken up by individual cells of the colony. Non-lactose-fermenting organisms are not colored because the compound is not taken up in alkaline reaction.

An explanation is offered to account for the occasional blue colonies found on E.M.B. medium. It is suggested that these colonies form a relatively high pH and thus cause slight dissociation of the compound. This dissociation would allow independent staining of the colonies by methylene blue.     

Direct selection for mutators in Escherichia coli

We have constructed strains that allow a direct selection for mutators of Escherichia coli on a single plate medium. The plate selection is based on using two different markers whose reversion is enhanced by a given mutator. Plates containing limiting amounts of each respective nutrient allow the growth of ghost colonies or microcolonies that give rise to full-size colonies only if a reversion event occurs. Because two successive mutational events are required, mutator cells are favored to generate full-size colonies. Reversion of a third marker allows direct visualization of the mutator phenotype by the large number of blue papillae in the full-size colonies. We also describe plate selections involving three successive nutrient markers followed by a fourth papillation step. Different frameshift or base substitution mutations are used to select for mismatch-repair-defective strains (mutHLS and uvrD). We can detect and monitor mutator cells arising spontaneously, at frequencies lower than 10(-5) in the population. Also, we can measure a mutator cascade, in which one type of mutator (mutT) generates a second mutator (mutHLS) that then allows stepwise frameshift mutations. We discuss the relevance of mutators arising on a single medium as a result of cells overcoming successive growth barriers to the development and progression of cancerous tumors, some of which are mutator cell lines.     

电穿孔导入的LacZ基因在人肺癌细胞中的表达

选用LacZ基因作为报告基因,用电穿孔法与pSV2neo质位共转化导入人肺巨细胞癌高转移细胞株,经G418及X－gal组化法双重筛检,获得LacZ基因表达阳性的细胞克隆。经X－gal组化染色,该克隆70％细胞蓝染,再经软琼脂培养后,形成的集落蓝染率约为65％,单个集落中的细胞蓝染率可达100％,在裸小鼠皮下或尾静脉接种这一LacZ基因表达阳性的克隆细胞后,在形成的肿瘤及肺内转移灶的冰冻切片中,X－gal染色可观察到蓝染的癌细胞。但由于细胞周期不同步、遗传稳定性以及表达调控因素的影响,则可能是本实验中部分细胞染色阴性的原因。我们的实验表明,LacZ基因有可能在肿瘤转移的研究中作为一种理想标志基因。     

TWO DISTINCT COLONIAL MORPHOTYPES OF AMPHORA COFFEAEFORMIS (BACILLARIOPHYCEAE) CULTURED ON SOLID MEDIA1

When cultured on different types of solid media, the marine-fouling diatom Amphora coffeaeformis (Ag.) Kütz. consistently formed two distinct colonial morphotypes named tight and fuzzy. Tight colonies were comprised mainly of small, morphologically distorted, nonmotile cells, whereas morphologically normal and highly motile cells formed the fuzzy colonies. Cells from tight colonies were less adherent to glass, grew more slowly in liquid media, and had a slightly decreased viability on plates with copper than cells from fuzzy colonies. Whereas the protein profiles of the two types of cells were nearly identical in polyacrylamide gels stained with Coomassie blue, cells from tight colonies produced a significantly lower amount of a protease-resistant, low M_r polysaccharide or glycoconjugate as detected in silver-stained gels. The frequency of appearance of the fuzzy and tight morphotypes was not influenced by the mode of nutrition or the type of substratum to which the algal cells adhered. However, certain formulations of solid medium and the presence of growth-inhibitory concentrations of copper in agar plates favored the formation of tight colonies. Due to their frequencies and patterns of appearance, it was clear that the two naturally formed morphotypes were not the consequence of spontaneous mutations, genetic rearrangement, or selection of stable natural variants, and we have hypothesized that they were linked to a normal physiological behavior. The tight colonial morphotype was used as a valuable marker to screen for true motility/adhesion mutants within an ultraviolet-mutagenized population of A. coffeaeformis. Seven mutants were isolated that were non-motile on agar plates, poorly adherent to glass, and distinguished from naturally formed cells from tight colonies by their inability to form fuzzy colonies upon subculture on solid media.     

Tetrazolium staining by optical scanning overestimates colony size and number of colonies counted

We measured the effect that staining with 2-(P-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) had on the number and size distribution of tumor colonies counted using an optical image analyzer (FAS II). Staining increased the number of tumor colonies counted. By using opaque tumor cells or pigmented melanoma cells and measuring colony growth kinetics, we demonstrated that the use of INT staining to assist in counting tumor colonies artificially increased the size of viable tumor cell aggregates by adding a red precipitate to the outside surface of the cells. Laboratories that are using the INT method for drug screening are probably measuring colonies down to and below 42 microns in diameter. These small colonies could result from as few as one or two divisions. Thus, potentially useful drugs may be missed in the screen because of the presence of abortive colonies: i.e., lethally damaged cells completing only one or two divisions.     

The use of toluidine blue for in situ detection of micro-organisms in foods

The use of toluidine blue to stain cryosections of food samples to detect sites of microbial growth was examined. The method successfully detected colonies and single cells of both yeasts and bacteria at magnifications of x 400 or below in the majority of foods studied. The method demonstrates great potential for studying the micro-environments in which micro-organisms grow.     

Use of the oblique illumination method for characterizing N. meningitidis cultures]

The authors used the method of oblique illumination for detection of the state of the population of meningococcus cultures. In studying 48 strains there were revealed three types of fluorescence of the colonies: bright orange with a transition into greenish-light blue along the lower margin (the I type), bright light blue with a narrow orange or green upper margin (the II type) and greyish-light blue colonies (the III type). The type of fluorescence was not associated with the sero-group specificity. Populations of meningococcus cultures, depending on conditions and duration of growth on the nutrient medium could consist both of the colonies with the same type of fluorescence, and also represent a combination of colonies with a different type of fluorescence. The colonies of the I and II types of fluorescence had marked group specificity, but mutation occurred during passage and the III type of fluorescence formed with a loss of serological specificity. Continuous selection of the colonies of the I and the II types of fluorescence permitted to preserve the population with specific properties necessary for production of diagnostic and vaccine preparations for a long time.     

Staining method of poly(3-hydroxyalkanoic acids) producing bacteria by nile blue

Summary Using an ethanol solution of nile blue, we have developed an efficient method to detect the colonies of poly(3-hydroxyalkanoic acids) (PHA) producing bacteria on the agar plate. When the bacterial colonies with PHA granules were stained with nile blue, the stained colonies fluoresced bright orange on the irradiation of UV light. In the fluoresce emission spectra, fluorescence intensity increased with an increase in the PHA content of bacterial cells.Alcaligenes eutrophus andA.latus colonies with poly(3-hydroxybutyric acid) (PHB) homopolymer exhibited an emission maximum at 580nm on the excitation at 490nm. On the other hand,Pseudomonas oleovorans andP.putida with medium-chain-length (mcl-) PHA copolymers of C6, C8 and C10 units exhibited an emission maximum at 570nm.     

DNA topoisomerase inhibitors block erythropoiesis and delay hemoglobinization in vitro

To examine the importance of topological constraints on DNA during erythroid development, we measured the effects of camptothecin and teniposide, two tumoricidal agents which are also specific inhibitors of type I and type II topoisomerases respectively, on the formation of hematopoietic colonies by cultured human bone marrow cells. When added to bone marrow culture, each inhibitor alone impairs the formation of early BFU-E-derived colonies, late CFU-E-derived colonies and mixed hematopoietic (CFU-GEMM-derived) colonies by up to 100%. Inhibition of colony formation is directly related to the time of inhibitor addition and the inhibitor concentration tested. Although either inhibitor alone reduces colony formation by 90%, when added together at a submaximal concentration, camptothecin and teniposide exert a synergistic suppressive effect. Furthermore, addition of topoisomerase inhibitors to culture impairs hemoglobinization of colony erythroblasts in a time-dependent fashion. In contrast to the effects of topoisomerase inhibitors, the antiproliferative agent aphidicolin reduces erythroid colony number and size without altering hemoglobinization of colony erythroblasts. Since neither topoisomerase inhibitor alters the morphology of cultured cells, the capacity of cells to exclude trypan blue or the potential to form erythroid colonies through the interval required for the first progenitor cell division, it is unlikely that camptothecin or teniposide are cytotoxic to hematopoietic cells. Human mononuclear cells enriched in bone marrow lymphocytes and nucleated erythroblasts from both human and mouse sources release DNA into the detergent soluble fraction. Release requires functional topoisomerases and is altered by acute exposure to topoisomerase inhibitors. Our results suggest that topoisomerases are critical not only to proliferation but also to differentiation of human marrow erythroid progenitor cells and stem cells in culture.     

桑蚕金属硫蛋白基因在大肠杆菌中的克隆和表达

用\%Bam\%HⅠ和\%Sac\%Ⅰ双酶切质粒pCM1\|1,获得酵母的MTI基因片段,用非放射性地高辛标记作为探针。提取桑蚕肥苏蚕卵的总DNA,分别用\%Eco\%RⅠ、\%Bam\%HⅠ和\%Hin\%dⅢ酶切,与MTI探针进行Southern杂交,出现较强的杂交信号。然后用\%Eco\%RⅠ完全酶切桑吞的总DNA,电洗脱法回收1～6kb的染色体片断,与\%Eco\%RⅠ酶切的M13-载体以3∶1比例连接,转化受体菌DH5α。筛选到4 000多个白色转化子,与探针MTI进行Southern杂交筛选阳性转化子,选择到有强杂交信号的三个转化子［编号为T1(pZHC\|1)\,T5(pZHC\|5)\,T7(pZHC\|7)\]。用12种限制性内切酶对pZHC\|5重组质粒进行酶切分析表明插入片段约12kb,在基因内有一个\%Hin\%dⅢ位点。抗性测定表明受体菌DH5α在含有50mmol/L CuSO\-4的培养基上生长,在含有52mmol/L CuSO\-4的培养基上不生长,而转化子确能在含有52mmol/L CuSO\-4以上的培养基上生长。上述研究结果表明12kb左右的插入片段含有桑吞的金属硫蛋白基因。     

Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma

From a transplantable mouse teratoma it has been possible to derive an established keratinizing cell line (XB) which grows well in cultures containing lethally irradiated 3T3 fibroblasts at the correct density. Single cells of the keratinizing line grow into colonies each consisting of a stratified squamous epithelium. The keratinizing nature of the colonies has been demonstrated by specific staining with Rhodanile blue, and by light and electron microscopy of sections through the colonies. A function of fibroblasts appears to be a strict requirement for keratinization and an important though less strict requirement for cell growth. The fibroblast function can be carried out by medium harvested from 3T3 cultures.It is possible to detect keratinizing colonies in primary cultures of disaggregated teratoma cells combined with 3T3 cells. Such colonies appeared in cultures of a transplantable teratoma with an overall frequency of 6 × 10⁻⁶ of the cells plated. Nonkeratinizing colonies of cells with otherwise very similar appearance were about 10 fold more abundant. Since both the keratinizing and the related nonkeratinizing colonies can be identified in the living state, it is possible to isolate them from the primary cultures.     

Clonal nature of murine hemopoietic blast cell colonies

Murine hemopoietic blast cell colonies obtained from spleen cells of 5-fluorouracil (5-FU)-treated mice give rise to many multilineage colonies including granulocyte - erythrocyte - macrophage - megakaryocyte (GEMM) colonies in secondary cultures. Progenitor cells for blast cell colonies are considered to be more primitive than colony forming units (CFU)-GEMM. To determine whether they are clonal, we examined the phosphoglycerate kinase-1 (PGK-1) isozyme type of colonies originally grown from spleen cells of 5-FU-treated mice which had PGK-1 isozyme mosaicism. PGK assays of whole secondary colonies derived from one blast cell colony showed that they were either of type A or type B but not both. These results suggest that murine hemopoietic blast cell colonies are clonal.     

The effect of sub-lethal ALA-PDT on the cytoskeleton and adhesion of cultured human cancer cells

5-Aminolevulinic acid (ALA), a precursor of the endogenous photosensitizer protoporphyrin IX, is used in the photodynamic therapy (PDT) of cancer. Sub-lethal ALA-PDT (1-min irradiation with 370-450 nm blue light, 0.6 mW/cm(2) after 2-h incubation with 1 mM ALA) has been earlier shown to change cell morphology and to inhibit both trypsin-induced detachment of cultured cancer cells from the plastic substrata and cell attachment to the bottom of the plastic well plates. In the present study, we found that such treatment of human adenocarcinoma WiDr cells grown in dense colonies stimulated the formation of actin cortex between cells in the colonies and increased the number of actin stress fibres in some, but not in all, cells. However, ALA-PDT did not change the microtubular cytoskeleton in these cells. A similar treatment of glioblastoma D54Mg cells, which grow separately and communicate by protrusions, caused loss of fibrillar actin structures in growth cones, retraction of protrusions, and surface blebbing in some cells. The application of the cytoskeleton inhibitors cytochalasin D, colchicine or taxol showed that the inhibition of trypsin-induced detachment of photosensitized WiDr cells was related to ALA-PDT-induced changes in actin and microtubular cytoskeleton. Some signal transduction processes are suggested to be involved in ALA-PDT-induced changes in cytoskeleton, cell shape, and adhesion.     

Colony formation in agar by multipotential hemopoietic cells.

Agar cultures of CBA fetal liver, peripheral blood, yolk sac and adult marrow cells were stimulated by pokeweed mitogen-stimulated spleen conditioned medium. Two to ten percent of the colonies developing were mixed colonies, documented by light or electron microscopy to contain erythroid, neutrophil, macrophage, eosinophil and megakaryocytic cells. No lymphoid cells were detected. Mean size for 7-day mixed colonies was 1,800-7,300 cells. When 7-day mixed colonies were recloned in agar, low levels of colony-forming cells were detected in 10% of the colonies but most daughter colonies formed were small neutrophil and/or macrophage colonies. Injection of pooled 7-day mixed colony cells to irradiated CBA mice produced low numbers of spleen colonies, mainly erythroid in composition. Karyotypic analysis using the T6T6 marker chromosome showed that some of these colonies were of donor origin. With an assumed f factor of 0.2, the mean content of spleen colony-forming cells per 7-day mixed colony was calculated to vary from 0.09 to 0.76 according to the type of mixed colony assayed. The fetal and adult multipotential hemopoietic cells forming mixed colonies in agar may be hemopoietic stem cells perhaps of a special or fetal type.     

In vitro studies to prove the effect of sera of eosinophilia in colony formation

Serum samples from four patients with reactive eosinophilia and two patients with eosinophilic leukaemia were compared with normal sera with respect to formation of eosinophil colonies after addition of the sera to mononuclear cells from peripheral blood of healthy subjects. Supernatants from ConA stimulated guinea-pig spleen cells and human lymphocytes were tested in a similar way. Grown colonies were placed on glass slides and after staining with luxol fast blue the percentage of eosinophils was counted. The serum samples of the patients with reactive eosinophilia produced the greatest number of eosinophil colonies while supernatants of spleen and lymphocytes produced the greatest number of eosinophilic granulocytes. Our findings suggest the existence of a factor stimulating eosinophil colonies in the tested serum fractions. Beyond that an indication is given for a substance in the supernatants of spleen and lymphocyte suspensions which stimulates more intensively the maturing into eosinophilic granulocytes than the formation of colonies.     

Presence of cells in B-cell lineage in mixed (GEMM) colonies from murine marrow cells

Recent progress in clonal cell culture techniques makes it possible to detect pluripotent hemopoietic precursors from murine marrow cells. The precursors can proliferate, differentiate and form mixed colonies containing erythroblasts, granulocytes, macrophages and often megakaryocytes in viscid culture medium. In the present investigation, the presence of cells of B-cell lineage in mixed colonies was investigated. Experiments on colonies containing cIgM, cIgG, sIgM and sIgG bearing cells using goat IgG fluorescein-conjugated anti-mouse IgM, goat F(ab')2 fraction fluorescein-conjugated anti-mouse IgG and immunobeads revealed the presence of cytoplasmic IgM bearing cells in 47% of the colonies and surface IgM bearing cells in 74-84% of the colonies. Mixed colonies, however, did not contain either cIgG bearing cells or sIgG bearing cells. The results may indicate that some CFU-MIX proliferate and differentiate along B-cell lineage to sIgM or cIgM bearing cells in vitro.