The effect of histochemical methylation on the phosphate groups of nucleic acids: Interpretation of the absence of nuclear basophilia

Summary The effect of hot methylation (hydrochloric acid-methanol) on nuclear stainability was investigated in order to determine whether the progressive loss of basophilia is due to methylation of the diester phosphate groups of nucleic acid.DNA spots on filter paper were unchanged in their stainability towards Toluidine Blue even after methylation for 4 days, while RNA, chondroitin sulphate and hyaluronic acid lost their affinity for this dye after 4 h methylation.In formalin-fixed sections, methylation for 4 h led to a loss of nuclear basophilia. There was no concomitant increase in nuclear relative to cytoplasmic stainability with Fast Green FCF at pH 9, as judged from the use of a comparison eyepiece, evaluation of colour transparencies or by microspectrophotometry. In contrast, extraction with trichloroacetic acid prior to or after methylation led to a much improved Fast Green staining of nuclei, comparable to the staining obtainable after treatment with trichloroacetic acid alone.In conclusion, there is no evidence that hot hydrochloric acid-methanol, as used in histochemistry, methylates the diester phosphate groups of nucleic acids. The loss of nuclear basophilia can be explained as a result of the excess positive charge on the chromatin following methylation of all the protein carboxyl groups. This effect is maximal after 3–4 h treatment with acid methanol at 60°C. Further methylation leads to depolymerization and extraction of DNA. RNA is depolymerized in less than 4h.     

Probing chromatin structure in the early phases of apoptosis

Abstract. A typical flow cytometric marker of apoptosis is the appearance of a hypodiploid peak. This phenomenon is related to the chromatin fragmentation and loss that occurs during the late stages of the process. We describe herein the changes occurring at the chromatin level in purified nuclei preparations obtained from human peripheral blood mononuclear cells in a time-course study, including the simultaneous evaluation of nuclear proteins and DNA stainability, light-scattering properties, and spectrophotometric determination of the protein content. An augmentation of fluoroscein isothiocyanate (FITC) stainability was noticed as early as 1 h after irradiation. As this phenomenon is not correlated to changes in actual protein content, one can conclude that modifications of basic protein accessibility occur from the early phases of the apoptotic process. Also DNA stainability augmented with time, generating the transient appearance of a hyperdiploid peak that preceded the appearance of the hypodiploid peak typical of the late stages of the process, and that shared with the latter the same light-scattering properties. Chromatin status was further explored by staining apoptotic nuclei using DNA probes with peculiar molecular weight. Propidium iodide (PI) and ethidium bromide (EB), but not the much bulkier 7-aminoactinomycin D (7-AAD), identified the nuclei with a transient increase in DNA stainability confirming that an increased dye accessibility to binding sites was responsible for the phenomenon. Remarkably, all dyes identified the same proportion of hypodiploid nuclei when an apoptotic nucleus shed its fragmented chromatin. Control experiments included differential interference contrast and fluorescence microscopy that showed the purity of nuclei preparations and the typical morphological apoptotic features. Finally, the simultaneous evaluation of DNA by PI and nuclear proteins by FITC in a time course study allowed a thorough assessment of changes occurring at the chromatin level in the diverse stages of apoptosis. It is suggested that proteolysis precedes endonucleolysis and probably renders it easier the final endonucleolytic step leading to DNA fragmentation and loss.     

Changes in accessibility of DNA to various fluorochromes during spermatogenesis

Accessibility of mouse testicular and vas deferens (vas) sperm cell DNA to acridine orange, propidium iodide, ellipticine, Hoechst 33342, mithramycin, chromomycin A3, 4'6-diamidino-2-phenylindole (DAPI), and 7-amino-actinomycin D (7-amino-AMD) was determined by flow cytometry. Permeabilized cells were either stained directly or after pretreatment with 0.06 N HCl. For histone-containing tetraploid, diploid, and round spermatid cells, HCl extraction of nuclear proteins caused an approximately sixfold increase of 7-amino-AMD stainability but had no significant effect on DAPI stainability. For these same cell types, the stainability with other intercalating (acridine orange, propidium iodide, ellipticine) and externally binding (Hoechst 33342, mithramycin, chromomycin A3) dyes was increased by 1.6- to 4.0-fold after HCl treatment. In sharp contrast, HCl treatment of vas sperm did not increase the staining level of 7-amino-AMD, DAPI, or propidium iodide but did increase the staining level for the other intercalating dyes (1.3- to 1.5-fold) and external dyes (1.3- to 1.9-fold). Elongated spermatids that contain a mixture of protein types including histones, transition proteins, and protamines demonstrated the greatest variability of staining with respect to type of stain and effect of acid extraction of proteins. In general, for nearly all dyes, the round spermatids had an increased level and tetraploid cells had a decreased level of stainability relative to the same unit DNA content of diploid cells. The observed differential staining is discussed in the context of chromatin alterations related to the unique events of meiosis and protein displacement and replacement during sperm differentiation.     

On the stainability of plant cell walls with alcian blue

This work is a continuation of a communication on the stainability of broad bean (Vicia faba L.) root tip cells with alcian blue, published some time ago. Following the standard method of staining with alcian blue, the cell walls are very strongly stained, the nuclei (except nucleolus) lightly, the nucleolus and cytoplasm are practically colourless. The weak dyeing of the nucleus is not equal throughout the whole section so that the comparison of stainability of cell walls and nuclei by itself cannot explain the staining with alcian blue. The results of this work on the staining of cell walls (if not including model experiments and experiments in vitro, which are not considered as decisive here) can be summarized as follows: the pH dependence of staining, the loss of stainability as a result of pectinase digestion, blocking of staining by methylation and regeneration of stainability by demethylation and, finally, the impossibility of staining in the presence of NaCl lead to the conclusion that the staining of the material studied in this work is primarily caused by the salt linkage of alcian blue with the free carboxyls of pectic substances. From the comparison of staining with alcian blue and with other basic dyes it follows that in the case of alcian blue some other factors may also take part and are the reason for the selectivity and firmness (fastness) of the staining of cell walls with this dye. Otherwise, the staining of plant cell walls with alcian blue corresponds quite well to the staining of carboxyls containing polysaccharides of animal tissues with this dye. By staining with alcian blue it was found impossible to distinguish between younger and older cell walls within the meristem. However, this staining is suitable for routine use when studying the meristematic tissue. It is often possible to use solutions of a higher pH than generally used.     

Feulgen DNA stainability of bone tumors after demineralization

Microspectrophotometric DNA analysis of archival bone tumor tissue is often impeded by previous acid demineralization, which destroys Feulgen DNA stainability. To find an alternative to acid for prospective DNA studies of bone tumors in tissue sections, Feulgen stainability of fresh osteosarcoma specimens after demineralization in neutral EDTA was investigated. The reliability of DNA analysis of weakly Feulgen-stained sections from archival tissue was also studied. Demineralization of four fresh specimens in EDTA slightly reduced Feulgen DNA stainability compared to nondemineralized preparations but did not affect the determination of ploidy level. Hydrolysis tests of one diploid and one hyperploid osteosarcoma showed that the staining relationship between control and tumor cells was not altered by EDTA pretreatment. For DNA studies of bone tumors requiring demineralization, EDTA offers a means of retaining nuclear Feulgen stainability. In 22 archival osteosarcoma specimens of varying Feulgen stainability, three different upper limits of light transmission (75, 85, and 95%) were applied to test the significance of background disturbances in relation to nuclear stain intensity. The relationship between the median total extinction of the control and tumor cell populations was not significantly affected by altering the upper transmission limit except in four poorly stained lesions. The control cells of these four specimens exhibited a median total extinction less than one-third of the maximum encountered. The results suggest that weakly stained archival specimens can be tested for selecting those appropriate for ploidy determination.     

HALE STAIN FOR SIALIC ACID-CONTAINING MUCINS : Adaptation to Electron Microscopy

The feasibility of using the Hale stain to identify cellular sialic acid-containing mucins by electron microscopy was investigated. Three kinds of mouse ascites tumor cells were fixed in neutral buffered formalin, exposed to fresh colloidal ferric oxide, treated with potassium ferrocyanide, imbedded in Selectron, and sectioned for electron microscopy. Additional staining with uranyl acetate and potassium permanganate was done after sectioning in order to increase contrast. Those cells known to be coated with sialomucin showed deposits of electron-opaque ferric ferrocyanide crystals in the areas where sialomucin concentrations were expected. When these cells were treated with neuraminidase beforehand, these deposits did not appear. It was concluded that, with the precautions and modifications described, the Hale stain can be successfully combined with electron microscopy to identify sialomucin.     

嫌色性肾细胞癌17例临床病理学分析

目的探讨嫌色性肾细胞癌的临床病理特征、诊断与鉴别诊断要点。方法对17例嫌色性肾细胞癌进行组织形态学、免疫组化染色及Hale’s胶样铁染色观察,结合文献对其临床表现、病理形态特点及鉴别诊断进行探讨。结果嫌色性肾细胞癌17例,大体肿瘤直径3-10.5cm。镜下肿瘤由嫌色细胞和嗜酸细胞构成,呈片状、梁状和腺泡状分布。嫌色细胞体积较大,多角形,胞膜清晰,胞质半透明细网状,胞核皱缩,可见核沟及核异型,核仁不明显;而嗜酸细胞胞质嗜酸,可见明显的核周空晕。免疫组化:EMA 100%阳性,CD10 52.9%阳性,Vimentin阴性,CK7 88.2%阳性,P504S29.4%阳性,CD11794.1%阳性。Hale’s胶样铁染色100%阳性。17例中12例随访6个月到3年,仅1例在术后15个月发现肝脏转移,其余均未发现复发及转移。结论嫌色性肾细胞癌是一种少见的肾肿瘤,恶性程度相对较低,预后良好。掌握该肿瘤独特的病理学特征,对鉴别其他肾上皮性肿瘤有重要帮助。     

Different stainability of Purkinje cells.

Regarding the different stainability when using the Luxol fast blue methods, two kinds of Purkinje cells of the rat are described: Luxol-positive and Luxol-negative cells. Since, by this method, phospholipids are demonstrated, the author suggests the prospective varying functional conditions of these cells. Different tinction of Purkinje cells has been confirmed also by other methods (gallocyanin-chromalaun, thionine, toluidine blue, lithium-haematoxylin, chromalaun-haematoxylin-phloxine and acid phosphatase) in both animal and human material. After 96 h of immobilization the different stainability of Purkinje cells becomes more marked, which penomenon can be as well explained with regard to the functional point of view. Similar differences, though less marked, were found also in neurosecretory cells of the nucelus supra-opticus of the rat and in the nuclear region of the ganglion semilunare Gasseri cells in man. Finally, the author refers to the relations between the Luxol blue staining method and Baker's method employing acid haematoxylin for demonstration of phospholipids in certain kinds of nervous system cells, taking into consideration Kroon's findings.     

Feulgen-positive staining of the cell nuclei in fossilized leaf and fruit tissues of the Lower Eocene Myrtaceae

The Feulgen reaction and the staining of preparations with two DNA-specific fluorochromes, Hoechst 33258 and 4',6-diamidino-2-fenilindol (DAPI), were used to study the preservation of DNA in the fossilized leaf and fruit tissues of the Lower Eocene Myrtaceae, Paramyrtacicarpus plurilocularis and Paramyrtaciphyllum agapovii collected in Yakutia (Siberia, Russia). It was shown that chromatin structures of the fossilized plants form stable red-purple complexes with the Schiff's fuchsin sulphuric acid reagent in situ . This coloration is specific for DNA, in particular, for the deoxyribose residues. It means that the cell nuclei of these 53–55 Myr old plants preserve a part of the deoxyribose backbone of DNA molecules. On the other hand, there was no, or only a very weak, staining of the cell nucleus with fluorochromes DAPI or Hoechst 33258, which specifically bind to the double-stranded DNA and do not bind to either the single-stranded DNA or RNA molecules. The stainability of fossil plant cell preparations with alcian blue shows that there are also polysaccharides containing carboxyl groups in the cell walls of fossilized leaf and fruit tissues of the Lower Eocene Myrtaceae.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 150 , 315–321.     

Effects of prolonged water washing of tissue samples fixed in formalin on histological staining

The effects of prolonged water washing after fixation for 48 h in 10% (v/v) phosphate-buffered neutral formalin on the quality of representative histological staining methods were evaluated using samples of liver, kidney, spleen and thymus collected from three male Crl:CD(SD)(IGS) rats and one male beagle dog. Because door-to-door courier services in Japan prohibit handling formalin, our goal was to confirm that formalin fixed wet tissue samples could be stored in tap water rather than formalin during transportation of the samples without decreasing the quality of their staining or immunohistochemistry. Each tissue sample was allocated randomly to one of three groups: 12 min, 3 days and 7 days of washing in running tap water; samples then were routinely embedded in paraffin and sectioned. The sections were stained with hematoxylin and eosin, periodic acid-Schiff, azan, and the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. Immunohistochemical staining for Factor VIII, ED-1 and CD3 also was assessed. Prolonged water washing for up to 7 days did not affect the morphology or stainability by standard histological methods, or the intensity and frequency of positive reactions using the TUNEL method. Only immunohistochemical staining of Factor VIII was altered in both the rat and dog sections after 7 days of water washing. The intensity of positive reactions of Factor VIII immunohistochemistry after 7 days water washing was still strong enough to detect microscopically. Therefore, prolonged water washing for up to 7 days after formalin fixation does not have seriously detrimental effects on the quality and characteristics of paraffin sections stained by various methods, including immunohistochemistry.     

Pollen development of Rheum nobile Hook.f. & Thomson (Polygonaceae), with reference to its sterility induced by bract removal

The Himalayan alpine herb, Rheum nobile , terminates in a stout conical compound raceme concealed by large translucent bracts. It bears many fruits even under hostile conditions such as low temperature or persistent cloudy weather. To clarify the role of the bracts, the structure and the development of the pollen grains were examined after removing the bracts to expose the flowers to the open air for 9 days. Half of the individuals with bracts removed showed 0 to 1 % of pollen stainability and the pollen grains were variable in shape and size. It was also observed that the bracts of Rheum nobile increased the temperature of inflorescence by about 10deg;C above ambient daytime temperatures. These results suggest that one of the causes for the inhibition of pollen development was low temperature. The remainder, however, indicated high stainability of 70–100%. It is suggested that the extreme difference of pollen stainability between two groups of Rheum nobile exposed to the surroundings may be related to the stage of microsporogenesis. Bracts of Rheum nobile might play an important role in normal reproduction under low temperature at high altitudes.     

A simple and high-throughput method to assess maturation status of bovine oocytes: Comparison of anti-lamin A/C-DAPI with an aceto-orcein staining technique

A precise, accurate, nonambiguous and high-throughput method is required to assess nuclear maturation of mammalian oocytes. The objectives of this study were to compare the efficiency and ease of use of a simplified fluorescence imaging (anti-lamin A/C and 4′,6-diamidino-2-phenylindole [DAPI]) technique to the existing technique (aceto-orcein staining) for the evaluation of nuclear maturation of bovine oocytes, and to determine the kinetics of bovine oocyte maturation using an anti-lamin A/C-DAPI technique. In Experiment 1, oocytes were matured in vitro and stained with aceto-orcein and anti-lamin A/C-DAPI staining techniques. The proportions of oocytes lost during procedures and those that could not be classified (because of ambiguous morphology) during evaluation were lower (P < 0.0001) in oocytes stained with anti-lamin A/C-DAPI (9% and 2%) than those stained with aceto-orcein (31% and 13%), respectively. Anti-lamin A/C-DAPI was a quick procedure which could be completed within 7 h after completion of the maturation (compared with > 24 h for the aceto-orcein method). Furthermore, > 200 oocytes could be stained in one batch with anti-lamin A/C-DAPI technique. In Experiment 2, nuclear maturation kinetics of bovine oocytes at various time intervals (0, 6, 12, and 22 h) during in vitro maturation (IVM) was evaluated using the anti-lamin A/C-DAPI technique. Germinal vesicle, germinal vesicle breakdown, metaphase I, and metaphase II oocytes were predominant at 0, 6, 12, and 22 h of IVM, respectively. We concluded that the anti-lamin A/C-DAPI was an efficient and simple technique for nonambiguous evaluation of nuclear maturation status of large numbers of oocytes in a short interval.     

Comparison of methods based on annexin-V binding, DNA content or TUNEL for evaluating cell death in HL-60 and adherent MCF-7 cells

HL-60 and MCF-7 cells were treated with 0.15 μ M camptothecin (CPT) or with the solvent dimethylsulfoxide (DMSO) for the controls, for 2, 3 and 4 h or for 24, 48 and 72 h, respectively. The apoptotic index (AI) was then evaluated in parallel by the following flow cytometric methods: (1) double staining of unfixed cells with fluoresceinated annexin V and propidium iodide (PI), this after detachment by trypsinization in the case of MCF-7 cultures; (2) prefixation in 70% ethanol, extraction of degraded, low molecular weight DNA with 0.2 M phosphatecitrate buffer and analysis of the DNA content stained with PI; (3) TUNEL, i.e. labelling of DNA strand breaks with biotin-dUTP, followed by staining with streptavidin-fluorescein and counterstaining with PI. In HL-60 cells, the three methods gave similar results for the AI (3–4% in the controls and at 2 h of CPT treatment, and 35–43% at 3 and 4 h after CPT). This indicates that CPT-induced membrane alteration and DNA fragmentation occurred concomitantly in those cells. For MCF-7 cells, CPT-induced apoptosis developed more slowly, the AI, whether based on annexin V or on DNA content, remained unchanged at 24 h, then was increasing to 8% at 48 h and to 25% at 72 h of treatment. In these cells, the TUNEL index did not increase prior to 72 h, and the increase was minor (up to 9% vs. 2–3% in the controls) at 72 h of the treatment. This indicates that in MCF-7 cells DNA strand breaks cannot be effectively labelled, which may be due to inaccessibility of 3-OH ends in the breaks to exogenous terminal deoxynucleotidyl transferase. The mechanism of endonucleolytic DNA fragmentation thus may be different, depending on the cell type.     

Flow cytometric analysis of rodent epididymal spermatozoal chromatin condensation and loss of free sulfhydryl groups

Flow cytometric measurements were made on acridine orange (AO) and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methyl-coumarin (CPM)-stained epididymal- and vas deferens-derived spermatozoal nuclei to follow the course of chromatin condensation and oxidation of free sulfhydryl groups, respectively, during passage through mouse and rat posttesticular reproductive tracts. Alterations of mouse and rat spermatozoal chromatin during transition from a testicular elongated spermatids to epididymal caput spermatozoa resulted in a threefold loss of DNA stainability with AO. Passage of spermatozoa from the caput to corpus epididymis was accompanied by an approximate 15% loss of DNA stainability, which was maintained at that level throughout passage into the vas deferens. AO stainability of epididymal spermatozoal nuclei was generally independent of -SH group stainability. CPM stainability of rat spermatozoal nuclei free -SH groups was 83%, 18%, and 11% of caput spermatozoal values for corpus, cauda epididymis, and vas deferens, respectively. Comparable values for mice were 69%, 20%, and 18%. CPM stainability was relatively homogeneous for these mouse and rat reproductive tract regions, except mouse corpus epididymis spermatozoal nuclei stained very heterogeneously. Rat spermatozoa detained by ligature up to 7 days in the caput, corpus, and cauda epididymi had CPM staining values equal to or below those of normal vas spermatozoa, indicating that disulfide (S-S) bonding is intrinsic to the spermatozoa and is independent of the epididymal environment. These data suggest that chromatin condensation and loss of spermatozoal DNA stainability during passage from the testis to the vas deferens are independent of S-S bonding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the depth of burn injury by collagen stainability

Heat-denatured collagen in burned skin stains red instead of blue in Masson's trichrome stain. This change in stainability corresponds to the loss of birefringence in slides examined in polarized light. The depth of the abnormal staining of the skin slices was proportional to the time and temperature of the heat exposure. It is concluded that the change in collagen stainability from blue to red relates to the loss of crystallinity or parallel alignment of the collagen fibers. It is further proposed that change in the stainability of collagen in the burns could be used to delineate the depth of the thermal skin injury or the effectiveness of the surgical excision or debridement of the wound by dressing materials.     

Staining with pyronin Y detects changes in conformation of RNA during mitosis and hyperthermia of CHO cells

Cellular RNA in Chinese hamster ovary (CHO) cells synchronized in mitosis (M) or G2 phase, as well as in interphase cells subjected to hyperthermia (42 degrees C, 10 min), was stained with acridine orange (AO), ethidium bromide (EB), or pyronin Y (PY) and the resultant fluorescence was measured by flow cytometry. Total RNA content detected after staining with AO increased in M as compared to G2-phase cells, consistent with continued RNA synthesis during G2 phase. The content of double-stranded RNA, stained with EB (after DNase treatment), was also somewhat higher in M cells. In contrast, the stainability of RNA with PY decreased by 27% in M- compared to G2-phase cells. Furthermore, a decrease in stainability of RNA with PY was observed in G2 cells compared to cells in G1 phase. In separate experiments, RNA stainability with AO or EB was generally unaffected when interphase CHO cells were exposed to 42 degrees C for 10 min, though this same treatment resulted in a 26% decrease in RNA stainability with PY. The decreased PY stainability of cellular RNA in M or heat-treated cells was observed at a relatively narrow range of dye concentration (1.0-2.0 micrograms/ml). The observed hypochromicity of RNA coincides with dissociation of polyribosomes into single ribosomes known to occur during mitosis and following exposure to hyperthermia. It is presumed that the phenomenon involves selective denaturation and condensation of ribosomal (r) RNA by PY in single ribosomes which does not occur in polyribosomes. While the molecular mechanisms responsible for stabilization of rRNA in polyribosomes preventing its denaturation and condensation by PY are unknown, PY appears to be a sensitive probe that can be used to detect and study these changes in rRNA confirmation in situ.     

Expression of myosin heavy chain isoforms in developing rat muscle spindles

The development of muscle spindles, with respect to the expression of myosin heavy chain isoforms was studied in rat hind limbs from 17 days of gestation up to seven days after birth. Serial cross-sections were labelled with antibodies against slow tonic, slow twitch and neonatal isomyosins, myomesin, laminin and neurofilament protein. At 17-18 days of gestation, a small population of primary myotubes expressing slow tonic myosin were identified as the earliest spindle primordia. These myotubes also expressed slow twitch and, to a lesser extent, neonatal myosin. At 19-20 days of gestation a second myotube became apparent; this staining strongly with anti-neonatal myosin. A day later this secondary myotube acquired reactivity to anti-slow tonic and anti-slow twitch myosins. By birth, a third myotube was present; this staining strongly with anti-neonatal myosin but otherwise unreactive with the other antibodies against myosin heavy chains. Three days after birth a fourth myotube, with identical reactivity to the third one, became apparent. Regional variation in the expression of isomyosins, which was present since birth in the two nuclear bag fibers was further enhanced: the nuclear bag staining strongly with anti-slow tonic and antineonatal in the equatorial region and with decreasing intensity towards the poles, whilst with anti-slow twitch the stainability was low in the equatorial and high in the polar region. The nuclear bag fiber showed a homogeneous staining: high with anti-slow tonic, moderate with anti-neonatal, and displayed stainability to anti-slow twitch myosin in the polar regions only. No regional variation was found along the chain fiber/myotube.(ABSTRACT TRUNCATED AT 250 WORDS)     

Appressorium morphogenesis and cell cycle progression are linked in the grass powdery mildew fungus Blumeria graminis

Conidial germination and differentiation – the so-called prepenetration processes – of the barley powdery mildew fungus (Blumeria graminis f. sp. hordei) are essential prerequisites for facilitating penetration of the host cuticle. Although the cell cycle is known to be pivotal to cellular differentiation in several phytopathogenic fungi there is as yet no information available concerning the relationship between cell cycle and infection structure development in the obligate biotroph B. graminis. The timing of specific developmental events with respect to nuclear division and morphogenesis was followed on artificial and host leaf surfaces by 4′,6-diamidino-2-phenylindole (DAPI) staining in combination with a pharmacological approach applying specific cell cycle inhibitors. It was found that the uninucleate conidia germinated and then underwent a single round of mitosis 5–6 h after inoculation. During primary germ tube formation the nucleus frequently migrated close to the site of primary germ tube emergence. This nuclear repositioning was distinctly promoted by very-long-chain aldehydes that are common host cuticular wax constituents known to induce conidial differentiation. The subsequent morphogenesis of the appressorial germ tube preceded mitosis that was spatially uncoupled from subsequent cytokinesis. Blocking of S-phase with hydroxyurea did not inhibit formation of the appressorial germ tube but prevented cytokinesis and appressorium maturation. Benomyl treatment that arrests the cell cycle in mitosis inhibited nuclear separation, cytokinesis, and formation of mature appressoria. Thus, we conclude that a completed mitosis is not a prerequisite for the formation and swelling of the appressorial germ tube, which normally provides the destination for one of the daughter nuclei, while appressorium maturation depends on mitosis.     

Bulk separation and long-term culture of oligodendrocytes from adult pig brain. I. Morphological studies

Abstract: A method is described by which oligodendrocytes from adult pig brains can be isolated. It results in a cellular preparation suitable for long-term culture. The entire procedure can be accomplished within 2–3 h. The purity of oligodendrocytes ranges between 80 and 95% depending on the Percoll gradient used and on the time in vitro . Yields between 2.5 and 4 × 10⁷ cells per brain and plating efficiencies on the order of 60% make the system very useful for biochemical investigations. It was shown by immunocytochemical studies that oligodendrocytes produce extensive networks of processes, some of them having elaborate membranous expansions. Anti-galactocerebroside (GC) antibodies as well as anti-myelin basic protein (MBP), anti-Wolfgram protein (WP), antiglial fibrillary acidic protein (GFAP), and monoclonal antibodies O1 and O4 are used to identify the cell types and to characterize the cellular composition of the cultures. Anti-GC and O1 are suitable markers for these oligodendrocytes. Both antibodies label similar cells, and the staining intensities are equally strong. In the case of O4, variable staining intensities are observed, and a few additional cells are labeled that are anti-GC⁻. After 3¹/2 weeks in culture, about 60% of the cells can be labeled by anti-MBP. Here too differences in staining intensities are observed. The anti-WP stain is too weak to be defined as positive. The percentage of GFAP⁺ cells lies in the range 15–20% at maximum. Cells were also mixed into collagen gels. This method appears to be more useful for outgrowth and branching of fibers than are monolayer systems. Drawbacks, however, include limited access for the antibodies and poor recovery of undamaged cells with their fibers.     

Nuclear chromatin texture and sensitivity to DNase I in human leukaemic CEM cells incubated with nanomolar okadaic acid

It is now known that the analysis of chromatin texture can be used in oncology as a sensitive detection method, either to define diagnostic classifications or to locate a lesion along a defined trend curve. However, the functional significance of these variations in textural features remains sometimes unclear. Several drugs have been shown to be able to modulate chromatin structure. Among them, the phosphatase inhibitor okadaic acid at low concentration can increase accessibility to DNA in chromatin of carcinoma cells. This paper demonstrates that short exposures (0–3h) to a 10-nM dose of okadaic acid induced an increased sensitivity to DNase I digestion in human CEM leukaemic cell nuclei and that this sensitization was associated to variations of nuclear texture characteristics, as evaluated by image cytometry. CEM cells treated with okadaic acid for 0–3h displayed changes in chromatin supraorganization with a more homogeneous and fine chromatin texture, as compared to control cells. This suggests that the appearance of an open configuration of chromatin structure as evaluated by biochemical methods corresponds to a more decondensed texture of nuclei measured by image cytometry. Longer exposures (6–24h) of CEM cells to 10nM okadaic acid lead to apoptosis. As reported previously for camptothecin-treated HL60 cells, okadaic acid-treated CEM cells display biphasic nuclear chromatin texture changes, i.e. a decondensation phase followed by the appearance of typical apoptotic cells with a smaller nuclear area and a highly condensed chromatin. Finally, using the multidrug-resistant CEM-VLB cell line, it was confirmed that these multidrug-resistant cells also display cross-resistance to okadaic acid, as this compound was unable to induce either increased DNase I sensitivity, apoptosis, or altered nuclear texture in this particular cell line.