Staining human lymphocytes and onion root cell nuclei with madder root

We performed staining experiments on cells using natural dyes and different mordants using techniques that are used for wool and silk dyeing. The natural dye sources were madder root, daisy, corn cockle and yellow weed. Ferrous sulfate, copper sulfate, potassium tartrate, urea, potassium aluminum sulfate and potassium dichromate were used as mordants. Distilled water, distilled water plus ethanol, heptane, and distilled water plus methanol were used as solvents. All dye-mordant-solvent combinations were studied at pH 2.4, 3.2 and 4.2. The generic staining procedure was to boil 5–10 onion roots or stimulated human lymphocyte (SHL) preparations in a dye bath on a hot plate. Cells were examined at every half hour. For multicolor staining, madder-dyed lymphocytes were decolorized, then stained with Giemsa. The AgNOR technique was performed following the decolorization of Giemsa stained lymphocytes. Good results were obtained for both onion root cells and lymphocytes that were boiled for 3 h in a dye bath that included 4 g madder root, 4 g ferrous sulfate as mordant in 50 ml of 1:1 (v/v) methanol:distilled water. The pH was adjusted to 4.2 with 6 ml acetic acid. We conclude that madder root has potential as an alternative dye for staining biological materials.     

RNA synthesis in nuclei of rat liver and of human lymphocytes.

Physico-chemical properties and RNA synthesis in the rat liver and human lymphocytes have been compared in a nuclear system in vitro. Human lymphocytes were isolated from blood of healthy donors and of chronic lymphocytic leukaemia patients. The isolated nuclei served as the source of polymerase and template DNA. 3H-CTP was incorporated into the acid insoluble fraction linearly for 60 min. The nuclei of lymphocytes contained small amounts of RNA and protein, and the isolation procedure was complicated. Rat liver nuclei seem to be less prone to clumping at high pH values and may incorporate much more 3H-CTP. The nuclear synthesis was compared with incorporation of 3H-rU and 32P-orthophosphate into nuclear RNA of intact lymphocytes. Normal cells easily incorporated 32-P, and in contrast leukaemic cells incorporated 3H-rU to a greater extent.     

The DNA-content (Feulgen) of nuclei during mitosis in a root tip of onion

Summary The microphotometric two-wavelength method is demonstrated to permit, even with only an improvised optical set-up, DNA-measurements which would not be possible, at any rate not with a comparable accuracy, with the conventional method.The data, in agreement with earlier results, show that the largest interphase nuclei in the root meristem have already completed their DNA-synthesis.The mean DNA-contents per set of chromatids at late interphase, prophase, and telophase, appeared to be virtually identical. A natural variation in DNA-content, if any, between individual nuclei at these stage would have a coefficient of variation of significantly less, and most likely much less, than 7.3%. The data from metaphase to middle anaphase are, for technical reasons, not yet conclusive. An indicated, moderate, excess of Feulgen-dye in these as compared with earlier and later stages may not be real.The results are fully compatible with a working hypothesis of strict constancy of the DNA-content per chromatid and of its precise doubling during DNA-synthesis (for necessary qualifications see the discussion). It is pointed out that apparently contradictory results by certain other authors do not, for methodological reasons, yet establish disproof of this hypothesis.Contribution from the Program in Cytology, Department of Botany, University of Wisconsin, Madison, supported in part by grants to Dr.C. Leonard Huskins from the Rockefeller Foundation, the American Cancer Society, and the Research Committee of the Graduate School with funds supplied by the Wisconsin Alumni Research Foundation.supported in part by the U. S. Public Health Service and the Wallace C. and Clara A. Abbott Memorial Fund.     

Staining and fluorescence-activated cell sorter analysis of human lymphocytes using antibodies to a short, chemically synthesized human IL-2 peptide

A rabbit antibody against a chemically synthesized peptide (p84), encompassing residues 59-72 of mature human interleukin 2 (IL-2), has been shown to react specifically with natural or recombinant IL-2. This antibody was used in immunoperoxidase and immunofluorescence techniques for identification of IL-2-containing cells in human peripheral blood or tonsils. Lymphocytes were stimulated with T-cell mitogens (PHA, PWM), fixed, and incubated with affinity-purified anti-p84 antibody, followed by appropriately conjugated secondary antibodies. FACS analysis demonstrated a low fluorescence intensity in 5 to 15% of unstimulated cells. In contrast, 40-60% of mitogen-stimulated cells were stained at a high fluorescence intensity. Staining was inhibited by preincubating the anti-p84 antibody with the homologous peptide or recombinant IL-2, but not by unrelated peptides. In immunoperoxidase staining, anti-p84 antibody reacted selectively with an enriched T-cell population which was 95% Leu 5+, 80% Leu 3+, and 60% Tac+. Thus, this antibody to a synthetic IL-2 peptide reacts selectively with activated T cells, and may serve, therefore, as a useful tool for visualization and enumeration of IL-2-containing cells in blood and tissues.     

Changes of dictyosome ultrastructure during the cell cycle in onion root meristematic cells. A morphometric and stereological study

Summary Dictyosome ultrastructure changes during the cell cycle in onion root meristematic cells. Changes were mainly related to cisternae and intercisternal spaces morphology. Taking each dictyosome to be composed of three different regions (CIS, medial, and TRANS), several quantitative changes were detected in some of the compartments. Many of the planimetric parameters evaluated showed higher values in medical cisternae, while CIS and TRANS remained nearly constant. We have also found an increased activity of dictyosomes, as indicated by increase in the volume fraction of vesicular attached structures. This reaches maximum values at ana-telophase in coincidence with the onset and progression of cytokinesis.Abbreviations A anaphase - Ac mean area occupied by cisternae per stack section - C CIS - CCS cell cycle stage - D_A mean total dictyosome area - I_SA mean area occupied by intercisternal spaces per stack section - M metaphase - N mean number of cisternae per stack - P prophase - S.E. standard error - T telophase - T TRANS - V_v volume density     

Cytohistological and phytochemical study of madder root extracts obtained by ultrasonic and classical extractions

Introduction – Madder (Rubia tinctorum) has been used since ancient times as a source of pigments for dyeing and painting. Madder dyes are localised in roots and the native chemical population is composed of glycosiled and aglycone compounds. The aim of this study is to elaborate an efficient extraction process without any chemical denaturation of dyes. Objective – To compare an optimised ultrasonic process, using for madder dye extraction, with two conventional procedures and to determine the efficiency of ultrasound on these vegetable matrix. Methodology – Madder roots were extract in a methanol–water mixture in 37 : 63 (v/v) for ultrasound and 80 : 20 (v/v) for reflux and agitation. HPLC‐PAD analyses showed the anthraquinone proportion for each extraction process and their denaturing effects. Finally, cytohistological observations were made to show the consequence of each process on the cell organisation in madder roots. Results – The results showed that the amount of extracted dyes was higher with UAE than with agitation and reflux. HPLC‐PAD analysis revealed that the anthraquinone composition differed according to the extraction procedure. The UAE extracts presented an important richness in terms of anthraquinonic compounds that suggests a preserving effect. Cytohistological observations showed that the main alterations concerned the cell walls of phloem. After UAE the walls exhibited numerous pitted areas reflecting an ultrasound‐induced cavitation that enhances the extraction effectiveness of this method. Conclusion – The study has shown the improvement of madder roots extraction both quantitatively and qualitatively using the efficiency of ultrasound‐assisted extraction in comparison with magnetic agitation and reflux techniques. Copyright © 2009 John Wiley & Sons, Ltd.     

Growth kinetics of the Golgi apparatus during the cell cycle in onion root meristems

In onion root meristems, the number of dictyosomes per cell shows a kinetics of growth strongly related to the cell cycle. During the interphase of steady-state proliferative cells, the volume density and numerical density of the Golgi apparatus decrease to reach minimum values in late-interphase cells, characterized by their greatest length. This pattern is also found in the total volume occupied by Golgi apparatus. Once in mitosis, the above-mentioned parameters begin to increase reaching maximum mean values in telophase. After the experimental uncoupling of chromosome and growth cycles by presynchronization with hydroxyurea, we found a similar behaviour pattern in the Golgi apparatus: decreasing values during interphase and a triggering of Golgi-apparatus growth in prophase independently of the bigger cell sizes reached in mitosis as an effect of pretreatment with hydroxyurea. These results indicate a cyclic kinetics of this subcellular component in higher-plant meristems, coupled with early mitotic events.     

Cytokinins and nuclear RNA levels in onion root tips

Summary Onion root tips were grown in water, kinetin or 6-benzyladenine and levels of RNA in the chromatin region of nuclei were analyzed using visible light microscopy with basic-dye staining, and ultraviolet microscopy of unstained material. No evidence was found for a significant increase in nuclear RNA in response to cytokinin treatment.     

Ultrastructural changes in human leukemic cell nuclei

The ultrastructural changes of human leukemic cell nuclei have been investigated. Particular attention is paid to the alteration of the nuclear envelope and its constituents, i.e., the pores and the Zonula Nucleum Limitans which appear constantly involved in these pathologic processes. An alteration of the relationships between the components of the nuclear envelope and the chromatin itself may be responsible for the appearance of the most nuclear changes.     

Parental genomes mix in mule and human cell nuclei

Whether chromosome sets inherited from father and mother occupy separate spaces in the cell nucleus is a question first asked over 110 years ago. Recently, the nuclear organization of the genome has come increasingly into focus as an important level of epigenetic regulation. In this context, it is indispensable to know whether or not parental genomes are spatially separated. Genome separation had been demonstrated for plant hybrids and for the early mammalian embryo. Conclusive studies for somatic mammalian cell nuclei are lacking because homologous chromosomes from the two parents cannot be distinguished within a species. We circumvented this problem by investigating the three-dimensional distribution of chromosomes in mule lymphocytes and fibroblasts. Genomic DNA of horse and donkey was used as probes in fluorescence in situ hybridization under conditions where only tandem repetitive sequences were detected. We thus could determine the distribution of maternal and paternal chromosome sets in structurally preserved interphase nuclei for the first time. In addition, we investigated the distribution of several pairs of chromosomes in human bilobed granulocytes. Qualitative and quantitative image evaluation did not reveal any evidence for the separation of parental genomes. On the contrary, we observed mixing of maternal and paternal chromosome sets.     

The association of peroxisomes with the developing cell plate in dividing onion root cells depends on actin microfilaments and myosin

We have investigated changes in the distribution of peroxisomes through the cell cycle in onion (Allium cepa L.) root meristem cells with immunofluorescence and electron microscopy, and in leek (Allium porrum L.) epidermal cells with immunofluorescence and peroxisomal-targeted green fluorescent protein. During interphase and mitosis, peroxisomes distribute randomly throughout the cytoplasm, but beginning late in anaphase, they accumulate at the division plane. Initially, peroxisomes occur within the microtubule phragmoplast in two zones on either side of the developing cell plate. However, as the phragmoplast expands outwards to form an annulus, peroxisomes redistribute into a ring immediately inside the location of the microtubules. Peroxisome aggregation depends on actin microfilaments and myosin. Peroxisomes first accumulate in the division plane prior to the formation of the microtubule phragmoplast, and throughout cytokinesis, always co-localise with microfilaments. Microfilament-disrupting drugs (cytochalasin and latrunculin), and a putative inhibitor of myosin (2,3-butanedione monoxime), inhibit aggregation. We propose that aggregated peroxisomes function in the formation of the cell plate, either by regulating hydrogen peroxide production within the developing cell plate, or by their involvement in recycling of excess membranes from secretory vesicles via the -oxidation pathway. Differences in aggregation, a phenomenon which occurs in onion, some other monocots and to a lesser extent in tobacco BY-2 suspension cells, but which is not obvious in the roots of Arabidopsis thaliana (L.) Heynh., may reflect differences within the primary cell walls of these plants.Abbreviations BDM 2,3-butanedione monoxime - DAPI 4,6-diamidino-2-phenylindole - ER endoplasmic reticulum - GFP green fluorescent protein     

Staining of chromosomes in root tips of cloverin toto with aceto-gallocyanine

Приводится описание по личному опыту авторов вполне для краски хромосом кончиков корней клевера подходящего сквеч-метода, основанного на применении раствора галлоцианинового хромового лака в разбавленной уксусной кислоте.     

The onset of cell proliferation is stimulated by ascorbate free radical in onion root primordia

Summary— Ascorbate free radical (AFR) accelerates the quiescency-proliferation shift in onion root primordia. The acceleration was detected by the increase of [³H]thymidine incorporation and by the anticipated kinetics of the increase in the labeling index. The shortening of the onset of cell proliferation is attributted to the role of AFR in the energization of the plasma membrane.