Bovine mammary explant versus primary cell cultures: Effect of bovine somatotropin and insulinlike growth factor-I on DNA content and protein synthesis

Summary Cellular DNA, milk protein content, and protein secretion by bovine mammary explants were compared to cultures of confluent and growing primary bovine mammary secretory cells over 4 d. Explants were obtained at slaughter from eight Holstein cows (120 ± 35 d lactation). Primary cells were grown to confluence, cryopreserved, thawed, and cultured through five passages. Explants and cells were cocultured with liver and adipose tissue in the presence of somatotropin, insulinlike growth factor-I, and somatotropin + insulinlike growth factor-I. Cellular DNA and milk proteins were assayed using fluorescent probes and flow cytometry. Media proteins were assayed by densitometer scanning of electrophoresis gel bands. DNA content of explant, confluent, and growing primary cells increased similarly through the 96 h incubation. DNA content in G₀G₁ phase was increased by: (a) insulinlike growth factor-I in explant cells; (b) somatotropin, insulinlike growth factor-I, and their combination in confluent primary cells; and (c) the combination of somatotropin and insulinlike growth factor in growing primary cells. Approximately 65% of explant and confluent primary cells were in the G₀G₁ or differentiated phase compared to 47% for the growing primary cells. Whey protein content and secretion were similar among cell types. Explant cells contained and secreted more β-casein than primary cells but secretion trends for β-casein and k-casein were similar after 48 h for both cell types. Results suggest that primary cell cultures are comparable to explant cultures when used to study mechanisms of DNA and milk protein synthesis and secretion.     

DNA content analysis of insect cell lines by flow cytometry

The DNA content of insect cell lines (6 lepidoptera, 1 coleoptera and 1 diptera) was determined by flow cytometry. The DNA profiles of the 8 cell lines tested were different. They were characterized by the presence of several peaks (2 to 7) corresponding to different ploidy levels, by differences in the fluorescence intensity of each peak and by the proportion of cells in each peak. Two cell lines (Cf124 and BmN) were constituted of 2 distinct populations of cells. The DNA profiles of the cell lines were stable among the passages and during the length of time culture. This technique was demonstrated to be useful for the detection of mixed cell lines and nucleopolyhedrovirus cell infection, using Autographa californica MNPV. The flow cytometry gives interesting results on the cell cycle and the ploidy level; it appears as a good tool for insect cell lines characterization. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nuclear DNA content of Solanum species grown in vitro, as determined by flow cytometry

The DNA relative content in nuclei from several Solanum species, which were used as partners for somatic hybridization, were determined using a flow cytometry method. The nuclei were isolated mechanically or via protoplasts from leaves of in vitro grown plants. In the case of S. nigrum as well as S. tuberosum cv. Bzura and dihaploid clone H8105, the nuclei were also obtained from suspension cultured cells by lysis of protoplasts. The source and the method of nuclei isolation affected the pattern of nuclear DNA in the genotypes studied. The mesophyll nuclei showed two distinct peaks on the DNA histograms, whereas the DNA peaks produced by cell suspension nuclei were broad and less distinct. The DNA content in the nuclei, calculated from the DNA histograms of the samples and a DNA standard historgam (Trout Red Blood Cells, having DNA content of 5.05 pg per nucleus), were much lower in mesophyll nuclei than in those obtained from the cell suspension for the same genotypes. The results are discussed in respect of the genetic instability of Solanum genotypes. The usefulness of a flow cytometry approach in somatic hybridization research is also discussed.     

Combined analysis of DNA methylation and cell cycle in cancer cells

DNA methylation is a chemical modification of DNA involved in the regulation of gene expression by controlling the access to the DNA sequence. It is the most stable epigenetic mark and is widely studied for its role in major biological processes. Aberrant DNA methylation is observed in various pathologies, such as cancer. Therefore, there is a great interest in analyzing subtle changes in DNA methylation induced by biological processes or upon drug treatments. Here, we developed an improved methodology based on flow cytometry to measure variations of DNA methylation level in melanoma and leukemia cells. The accuracy of DNA methylation quantification was validated with LC-ESI mass spectrometry analysis. The new protocol was used to detect small variations of cytosine methylation occurring in individual cells during their cell cycle and those induced by the demethylating agent 5-aza-2''-deoxycytidine (5AzadC). Kinetic experiments confirmed that inheritance of DNA methylation occurs efficiently in S phase and revealed a short delay between DNA replication and completion of cytosine methylation. In addition, this study suggests that the uncoupling of 5AzadC effects on DNA demethylation and cell proliferation might be related to the duration of the DNA replication phase.     

Quantitation of spermatogenesis by DNA flow cytometry: Comparative study among six species of mammals

Suspensions of testicular germ cells from six species of mammals were prepared and stained for the DNA content with a fluorochrome (ethidium bromide) adopting a common technique and subjected to DNA flow cytometry. While uniform staining of the germ cells of the mouse, hamster, rat and monkey could be obtained by treating with 0.5% pepsin for 60 min followed by staining with ethidium bromide for 30 min, that of the guinea pig and rabbit required for optimal staining pepsinization for 90 min and treatment with ethidium bromide for 60 min. The procedure adopted here provided a uniform recovery of over 80% of germ cells with each one of the species tested and the cell population distributed itself according to the DNA content (expressed as C values) into 5 major classes-spermatogonia (2C), cells in S-phase, primary spermatocytes (4C), round spermatids (1C), and elongating/elongated spermatids (HC). Comparison of the DNA distribution pattern of the germ cell populations between species revealed little variation in the relative quantities of cells with 2C (8–11%), S-phase (6–9%), and 4C (6–9%) amount of DNA. Though the spermatid cell populations exhibited variations (1C:31–46%, HCl:7–20% and and HC2:11–25%) they represented the bulk of germ cells (70–80%). The overall conversion of 2C to 1C (1C:2C ratio) and meiotic transformation of 4C cells to 1C (1C:4C ratio) kinetics were relatively constant between the species studied. The present study clearly demonstrates that DNA flow cytometry can be adopted with ease and assurance to quantify germ cell transformation and as such spermatogenesis by analysing a large number of samples with consistency both within and across the species barrier. Any variation from the norms in germ cell proportions observed following treatment, fore.g. hormonal stimulation or deprivation can then be ascribed due to a specific effect of the hormone/drug on single/multiple steps in germ cell transformation     

Improvement of flow cytometry analysis and sorting of bull spermatozoa by optical monitoring of cell orientation as evaluated by DNA specific probing.

Flow cytometry is a potential method for the separation of X and Y bearing spermatozoa, on the basis of their relative DNA content evaluated by the fluorescence emission intensity due to specific fluorochrome DNA staining. However, spermatozoa DNA is highly condensed and nuclei exhibit flat non spherical shape, which can produce artefacts impeding accurate analysis. In order to avoid these limitations, decondensation of DNA performed by enzymatic treatment and a modification of the flow cytometer that orients the spermatozoa relative to the laser beam are generally used. In this work, we describe alternative methods and materials for selection of 1) decondensed and thus dead spermatozoa without orientation, sorted on the basis of only the 10% spermatozoa containing the least DNA (expected Y) and the 10% spermatozoa containing the more DNA (expected X), or 2) native spermatozoa homogeneously oriented using a simultaneous measurement of Axial light loss (extinction) and Forward angle light scatter. For testing enrichment of each selected fraction we have worked out a molecular hybridization procedure using X and Y specific DNA probes. We analyse and sort bull spermatozoa on these basis: the purity obtained for these fractions is 80% without orientation after enzymatic treatment, and 70% on live spermatozoa "optically" oriented.     

Comparative analysis of DNA nuclear content by flow cytometry on strawberry plants propagated via runners and regenerated from meristem and callus cultures

ABSTRACT

DNA variation may occur in plant species grown either in vivo or in vitro. In this study flow cytometric analyses were undertaken on Fragaria x ananassa Duch. runner plants, and on plants regenerated from callus cultures of leaf explants and from meristem cultures. Our aims were to investigate DNA variation in runner plants of different cultivars, and to compare DNA content in plants of the same cultivar obtained by different propagation procedures (i.e. from meristems or callus cultures). Plants growing in vitro and in the greenhouse were also compared. A good regeneration ability was observed in all the cultivars, with different percentages of shoot formation. No significant differences were detected in multiplication rate and rooting percentage within cultivars. This work documents the occurrence of DNA variations in strawberry plants in vivo and in vitro. Flow cytometric measurements of DNA content showed the presence of 4C nuclei, besides 2C nuclei, in runner plants of cultivar Pajaro. DNA content variations (2C/4C nuclei) were observed in plants regenerated from callus cultures. These variations were lost after transfer of the plants to the greenhouse, except for cultivar Don. The extent of such DNA variations was influenced by genotype. Our study confirms earlier reports indicating that DNA variation induced by in vitro culture could be lost or retained after transfer of the plants to the greenhouse.     

Selection, establishment and characterization of cell lines derived from a chemically-induced rat mammary heterogeneous tumor, by flow cytometry, transmission electron microscopy, and immunohistochemistry

Summary In order to isolate, characterize, and establish culture cell lines with different diagnostic and prognostic significance, derived from multiclonal neoplasms, a ductal infiltrating mammary tumor was induced in rats by 7,12-dimethylbenz[a]anthracene. Clones with different DNA/protein content, being the DI of 1.16, 1.30, and 1.60, respectively, were observed in the primary tumor. Biparametric flow cytometry suggested that the clone at 1.30 is made up of two subpopulations with different protein and slightly different DNA contents. The culture, after a few passages, exhibited the presence of aneuploid cells and the absence of diploid components, demonstrating that only tumor cells survived. The limiting dilution method gave rise to four lines with DI of 1.16, 1.25, 1.30, and 1.50; a mean chromosome number of 45, 46, 47, and 88, respectively; and different morphological and ultrastructural features. These characteristics were stable during the experimental procedure, that is, for about 20 passages. Conversely, the detection of cytoskeletal proteins indicated that the tumor epithelial cells underwent early dedifferentiation into sarcoma-like cells showing markers of stromal cell type and thus exhibiting phenotypic instability in vitro, a feature reported in many advanced human breast cancers in vivo. In conclusion, this cellular model represents the in vivo situation and appears suitable for in vitro studies of tumor cell characteristics and might be used to predict clinical behavior.     

Nuclear DNA content of the Solanum spp. in the series Etuberosa as determined by laser flow cytometry

Nuclear DNA content was determined in three accessions of Solanum brevidens, three accessions of S. etuberosum, and one accession of S. fernandezianum, which are diploid (2n = 2×= 24), closely related, non tuber-bearing wild potato species belonging to the series Etuberosa (Solanaceae). The plants were grown in vitro at 18°C or at 25°/22°C (day/night). S. brevidens was also grown in soil in the glasshouse at 25°/19°C (day/night), and in growth chambers at 18°C or 32°C. Leaf nuclei were isolated using a chopping method and stained with propidium iodide. Chicken red blood cells (CRBC; 2.33 pg) were added to the samples of nuclei as internal standards. The fluorescence of plant nuclei relative to CRBC was measured with an EPICS PROFILE flow cytometer. The 2C values of in vitro-grown S. brevidens and S. etuberosum were similar (1.48–1.54 pg, depending on the accession), but they were smaller than the 2C value of S. fernandezianum (1.63 pg). The 2C values of S. brevidens and S. etuberosum were generally smaller than those of the diploid species S. berthaultii (1.60–1.61 pg) and the diploid clones of S. tuberosum (1.60–1.72 pg). A similar relative difference of nuclear DNA content was found also between tetraploid S. brevidens and tetraploid S. tuberosum (2C = 3.15–3.16 pg and 3.50–3.62 pg, respectively). High (32°C) and low (18°C) growth temperatures caused abnormal changes in morphology and reduced fertility in S. brevidens in the growth chamber. The 2C values of S. brevidens grown at 25°/19°C (day/night) or at 32°C were similar, whereas the 2C values were c. 10% lower at 18°C.     

Cell division induced by mechanical stimulation in starved Tetrahymena thermophila: cell cycle without synthesis of macronuclear DNA

Starved Tetrahymena thermophila cells underwent synchronous cell division 2 h after a mechanical stimulation. The macronucleus showed no obvious increase in DNA content before the cell division in the starvation medium, and the DNA content was decreased after the cell division. On the other hand, when the starved cells were given nutrient-supplied medium immediately after the mechanical stimulation, cell division was delayed for 3 h. This period was almost the same as that for G1 cells in the stationary culture to first division after transfer to fresh nutrient medium. These results suggest that the mechanical stimulation induces an early division of starved cells, skipping the macronuclear S-phase with the starved cells probably becoming trapped in G1. Starved cells that had finished division soon formed mating pairs with cells of the opposite type. These observations lead us to propose that cell division in starvation conditions may be necessary to reduce macronuclear DNA content prior to the mating of T. thermophila.     

In vitro studies of the metabolism of [14C]- n-alkanes using ruminal fluid of sheep as substrate

Whether the rumen microbes are able to synthesize and/or degrade long-chain alkanes in anaerobic conditions remains a question to be answered before these hydrocarbons can be confidently used as duodenal flow or rumen transit markers. In this context, an experiment in vitro was carried out to establish whether within a rumen liquor fermentation system, n-alkanes can be derived from de-waxed structures of the plant or from non-alkane wax components (long-chain fatty alcohols, long-chain fatty acids and esters), or may be metabolized by bacteria to other components or to shorter-chain hydrocarbons. Ryegrass was labelled with 14C in growth chambers under controlled conditions in order to use it as a substrate. The labelled material obtained was separated in three fractions: labelled alkanes, labelled de-waxed plant and labelled wax components without the alkanes. These fractions were used for three different incubations in vitro, which objectives were as follows: 1. To check whether rumen bacteria can synthesize alkanes from carbon structures other than waxes (e.g. sugars). 2. To verify whether rumen bacteria can metabolize the n-alkanes to other compounds. 3. To check whether rumen bacteria can synthesize n-alkanes from other carbon compounds from waxes. The results showed that there was neither bacterial synthesis nor metabolism of the n-alkanes in in vitro conditions.     

In vitro analysis of proliferating epithelial cell populations from the mouse mammary gland: Fibroblast-free growth and serial passage

Summary Normal and neoplastic mouse mammary epithelial cells were cultured in nutrient medium containing D-valine substituted for L-valine. Fibroblast overgrowth was prevented and epithelial cell functions and morphology were retained in cultures maintained in, D-valine medium up to 2 months. A nonenzymatic technique was devised to dissociate epithelial cell monolayers. The combined use of this dissociation buffer and D-valine nutrient medium made it possible to passage serially normal and neoplastic mammary epithelial cells. Normal cells were derived from mammary glands of animals stimulated with exogenous hormones for various periods. The period of in vivo hormonal stimulation influenced the ability of normal mammary epithelial cells to attach and proliferate in primary and serially passaged cultures. A greater proportion of cells derived from glands following 2 to 4 weeks of hormonal stimulation were recovered after replating and showed higher labeling indices during serial passage than cells from unstimulated or 5- to 7-week stimulated groups. This investigation was supported by Grant No. CA 05388 from the National Cancer Institute and by Cancer Research Funds of the University of California.     

Chitin synthesis in Spodoptera frugiperda wing imaginal discs: I. Chlorfluazuron,diflubenzuron, and teflubenzuron inhibit incorporation but not uptake of [14C]N-acetyl-D-glucosamine

The purpose of this study was to determine the effects of potent inhibitors of chitin synthesis on an organ culture test system as a basis for determining the mode of action of such compounds. Consequently, we investigated the action of chlorfluazuron (CFA), diflubenzuron (DFB), and teflubenzuron (TFB) on uptake and incorporation into chitin of [¹⁴C]N-acetyl-D-glucosamine ([¹⁴C]GlcNAc) in wing imaginal discs cultured in vitro. Spodoptera frugiperda wing imaginal discs provided a highly responsive test system for studying the inhibition of ecdysteroid-dependent chitin synthesis in a target tissue in vitro. All three inhibitors blocked ecdysteroid-dependent [¹⁴C]GlcNAc incorporation into chitin by the wing imaginal discs. The effectiveness of the inhibitors was not affected by the time of their application, i.e., exposures before, during, or after 20-hydroxyecdysone treatment were equally effective in inhibiting chitin synthesis. Thus, exposure of freshly dissected discs to CFA for periods as short as 15 min inhibited approximately 90% of the chitin synthesis measured 72 h later. In contrast to previous in vivo studies all three inhibitors were similar in their effectiveness in vitro. However, while all three compounds inhibited [¹⁴C]GlcNAc incorporation in a similar dose-dependent manner, only DFB and TFB reduced but did not block uptake of GlcNAc. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Utilization of [2-14C]tetrahydropteroylglutamic acid and 5-[G-3H]methyltetrahydropteroylglutamic acid as substrates for folate polyglutamate synthesis in fruit bats: Effect of vitamin B-12-deficiency

[2-¹⁴C]Tetrahydropteroylglutamic acid and 5-[G-³H]methyltetrahydropteroylglutamic acid were given intraperitoneally to fruit bats. Folate polyglutamates were formed in the liver from both substrates in different amounts and at different rates. The methylfolate pool appeared to remain separate from the tetrahydrofolate pool. More polyglutamate was formed from tetrahydropteroylglutamic acid than from 5-methyltetrahydropteroylglutamic acid. There was a fall in the folate content of the liver in the vitamin B-12-deficient bat and a more rapid incorporation of folates into polyglutamates but thereafter a more rapid loss of the labelled folate from liver.     

Design,synthesis, anticancer evaluation,molecular docking and cell cycle analysis of 3-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidine derivatives as potent histone lysine demethylases (KDM) inhibitors and apoptosis inducers

A novel series of pyrazolo[1,5-a]pyrimidines were synthesized and proved by their spectral and elemental analysis, some elected of the newly synthesized compounds were examined for their cytotoxic activity employing MTT assay on two cancer cell lines (Breast and Hela cancers). Compounds 5, 7e and 7i showed the higher cytotoxicity against two cancer cell lines with (IC₅₀ = 13.91 ± 1.4 and 22.37 ± 1.8 μM/L), (IC₅₀ = 6.56 ± 0.5 and 8.72 ± 0.9 μM/L) and (IC₅₀ = 4.17 ± 0.2 and 5.57 ± 0.4 μM/L) for two cancer cell lines breast and hela respectively, using doxorubicin as a reference drug. The most potent cytotoxic active compounds 5, 7e and 7i presented inhibitory activity against KDM (histone lysine demethylases) with IC₅₀ = 4.05, 1.91 and 2.31 μM, respectively. The most potent KDM inhibitor 7e (IC₅₀ = 1.91 μM) showed to cause cell cycle arrest at G2/M phase by 4 folds than control and induce total apoptotic effect by 10 folds more than control. In silico studies performed on the more potent cytotoxic active compounds 5, 7e and 7i included lipinisk's rule of five. Moreover, molecular docking study was utilized to explore the binding mode of the most active compounds to the target enzyme (PDB-ID: 5IVE). Also, some bioinformatics studies were carried out for compounds 7e and 7i using Swiss ADME (Swiss Institute of bioinformatics 2018).