New aspects of the histology of the mandibular condyle in the rat

The function of the multipotential mesenchymal cells in the mandibular condyle was studied histochemically and histologically in 27 Long Evans/Turku rats. Sagittal sections from the temporomandibular joint were stained with haematoxylin and eosin, toluidine blue, or van Gieson's stain. A weakly orthochromatically stained fibrous layer was followed in the upper region by a weakly metachromatically stained mesenchymal cell layer. Deep within this was a strongly metachromatically stained layer of immature chondroblasts. The metachromasia of the matrix of these layers disappeared abruptly in an anterior direction and gradually in a posterior direction. The changes in the staining reactions are explained by the fact that mesenchymal cells can differentiate into chondrogenic or osteogenic cells depending on the environmental conditions. A new hypothesis is presented according to which regulation of the direction of condylar growth is achieved by choosing the cells for chondrogenesis more posteriorly or anteriorly from among the mesenchymal cells covering the whole condylar cartilage.     

Maxilon Blue Rl: A New Metachromatic Monoazo Dye

Maxilon blue RL, a basic monoazo dye developed by Geigy S. A., stains metachromatically acid mucopolysaccharide-containing elements in histological sections. This property is due to a chromatographically pure blue fraction which is the main component of the dye. The following routine has been developed for staining sections of formalin-calcium fixed paraffin-embedded tissues. Dewax in xylene and hydrate the sections through alcohol; stain in 0.05% aqueous Maxilon blue RL, from 30-60 sec; wash in distilled water; dehydrate either in tertiary butanol, 2 to 3 min, after removing excess water from the slide by blotting; or, rinse in 70% ethyl alcohol and dehydrate in 2 changes, 2 min each, of absolute alcohol; clear in xylene; mount in DPX or in Canada balsam. Acid mucopolysaccharides are colored red to violet; other basophilic elements, blue.     

Fate of Transforming Deoxyribonucleate in Bacillus subtilis

The majority of donor deoxyribonucleate (DNA) at early stages after uptake was found in a complex with a cell component which changes its buoyant behavior on equilibrium density gradients. Analysis of the recipient cell lysates, after treatment to dissociate the complex, showed about two-thirds of the donor molecules in denatured form and the rest associated with recipient DNA. Incubation of cells after DNA uptake leads to the disappearance of denatured donor DNA and to the increase of donor label associated with recipient DNA. Some characteristics of a component from intact cells or spheroplasts with affinity for denatured Bacillus subtilis DNA are described.     

Identification of apoptotic cells by formamide-induced dna denaturation in condensed chromatin.

In this article we describe a novel effect of formamide on DNA of apoptotic nuclei and present a method for specific detection of apoptotic cells based on this effect. Our observations show that formamide induces DNA denaturation in apoptotic nuclei but has no such effect on DNA of non-apoptotic cells. Formamide-induced DNA denaturation combined with detection of denatured DNA with a monoclonal antibody (MAb) against single-stranded DNA made it possible to specifically identify the apoptotic cells. This procedure produced intense staining of the condensed chromatin in the apoptotic nuclei. In contrast, necrotic cells from cultures treated with sodium azide, saponin, or hyperthermia did not bind this antibody, demonstrating the specificity of the formamide-MAb assay for the apoptotic cells. However, TUNEL stained 90-100% of necrotic cells in all three models of necrosis. Because the MAb did not stain cells with single- or double-stranded DNA breaks in the absence of apoptosis, we conclude that staining of the apoptotic nuclei is not influenced by DNA breaks and is induced by specific changes in condensed chromatin, such as damage to the DNA-histone interactions. Importantly, the formamide-MAb technique identified apoptotic cells in frozen sections and in histological sections of formalin-fixed, paraffin-embedded tissues.     

Detection of the HTLV-I gene on cytologic smear slides

OBJECTIVE: To apply the polymerase chain reaction (PCR) for detection of the HTLV-I gene from cytologic smear slides. STUDY DESIGN: Samples were from seven cases of serum anti-ATL antibody (ATLA)-positive T-cell lymphoma and three from ATLA-negative T-cell lymphoma. Six of the seven ATLA-positive cases were confirmed to be ATLL by Southern blotting. From the seventh case a fresh sample for blotting could not obtained. DNA was extracted from the cytologic smear slides of all 10 cases; they had been stained with Papanicolaou or May-Giemsa stain, digested with proteinase K and precipitated with phenol and ethanol. The target sequence in the pX region of the HTLV-I gene was amplified by PCR. RESULTS: All seven ATLA-positive cases, including one that had not yet been confirmed by Southern blotting, showed a single band, as predicted, while the three ATLA-negative cases showed no band. CONCLUSION: If cytologic smear slides are available but a fresh sample is not, the PCR method should provide evidence that the virus is present since in our study sufficient DNA templates were successfully extracted from the stained cytologic smear slides for detection of the virus.     

Polarographic reducibility of denatured DNA

The de polarographic behavior of native and denatured DNA at pH 7.0 was studied. Whereas native DNA was polarographically inactive under the given conditions, denatured DNA yielded a reduction polarographic step at the potential of about ?1.4 V. Native DNA produced a single desorption wave on ac polarograms, while denatured DNA yielded, in addition to this wave, another more negative wave approximately corresponding, as to its potential, to the dc polarographic step of denatured DNA. The behavior of apurinic acid was similar to that of denatured DNA. The course of DNA denaturation at elevated temperature was studied by means of the two above techniques and changes at temperatures below the melting temperature observed. This finding is in agreement with earlier results obtained by oscillopolarographic and the pulse-polarographic method.     

SpermBlue®: A new universal stain for human and animal sperm which is also amenable to automated sperm morphology analysis

Abstract

Our study was aimed at exploring a simple procedure to stain differentially the acrosome, head, midpiece, and flagellum of human and animal sperm. A further prerequisite was that sperm morphology of the stained samples could be analyzed using automated sperm morphology analysis (ASMA). We developed a new staining process using SpermBlue® fixative and SpermBlue® stain, which are iso-osmotic in relation to semen. The entire fixation and staining processes requires only 25 min. Three main steps are required. First, a routine sperm smear is made by either using semen or sperm in a diluting medium. The smear is allowed to air dry at room temperature. Second, the smear is fixed for 10 min by either placing the slide with the dried smear in a staining tray containing SpermBlue® fixative or by adding 1 ml SpermBlue® fixative to the slide. Third, the fixed smear is stained for 15 min by either immersing the slide in a staining tray containing SpermBlue® stain or adding four drops of SpermBlue® stain to the fixed smear. The stained slide is dipped gently in distilled water followed by air drying and mounting in DPX® or an equivalent medium. The method is simple and suitable for field conditions. Sperm of human, three monkey species, horse, boar, bull, ram, mouse, rat, domestic chicken, fish, and invertebrate species were stained successfully using the SpermBlue® staining process. SpermBlue® stains human and animal sperm different hues or intensities of blue. It is possible to distinguish clearly the acrosome, sperm head, midpiece, principal piece of the tail, and even the short end piece. The Sperm Class Analyzer® ASMA system was used successfully to quantify sperm head and midpiece measurements automatically at either 600 × or 1000 × magnification for most of the species studied.     

Studies on mitochondrial structure and function in Physarium polycephalum : I. Fine structure, cytochemistry, and H-uridine autoradiography of a central body in mitochondria

The fine structure, cytochemistry and autoradiography of the rod-shaped central body in the mitochondria of the slime mold, Physarum polycephalum, has been investigated. The central bodies are stained with Feulgen stain and, like the nucleoli, are stained metachromatically with azure B. At the ultrastructural level, they are composed of a semi-electron-dense axial region which is sensitive to treatment with DNase and an electron-dense peripheral region which surrounds the axial region and is sensitive to treatment with RNase. With electronmicroscopic autoradiography it has been shown that the central body and its peripheral region, after short exposure to ³H-uridine, incorporate ³H-uridine into a form, possibly RNA, which is insoluble in trichloroacetic acid and can be extracted with RNase though not with DNase. It is suggested that the central body is composed of an axial component which contains primarily DNA and a peripheral component which contains primarily RNA and that the RNA is synthesized in the central body.     

On the nature of the metachromatic cells of pancreatic islets

Summary Islet cells with cytoplasmic granules metachromatically stained by toluidine blue are found in the pancreas of dog, cat, pig, rabbit, teleostian fish, monkey and man, but not in the rat, mouse and guinea pig; dubious results are obtained on duck and ox islets. These cells do not react with the staining methods for the demonstration of A- and B-cells; conversely, they are silver-impregnated by a modification of Davenport's method, and, at least in dog islets, can be readily identified with dark-field microscopy and stained blue with the Mallory-Heidenhain trichrome stain. No obvious changes of this cell type are found either in synthalintreated dogs or in diabetic men.The Authors suggest that the islet argyrophil-metachromatic cells (A₁-cells of Swedish Authors) are identical with D-cells and very likely represent a morphologically and functionally indipendent cell type.     

Denaturation and condensation of DNA in situ induced by acridine orange in relation to chromatin changes during growth and differentiation of Friend erythroleukemia cells

DNA in situ is progressively denatured when the cells or nuclei are treated with increasing concentration of acridine orange (AO). This transition can be monitored by flow cytometry as a decrease in green fluorescence. The complexes of denatured DNA and AO undergo immediate condensation and aggregation; this step is manifested by appearance of red luminescence and formation of precipitates that can be detected by electron microscopy. The precipitates form preferentially in heterochromatin as well as in ribosomes and polysomes. Their formation and further aggregation affects cellular light scatter properties in both the forward and right-angle direction. The AO-induced DNA denaturation and condensation was studied in nuclei of Friend erythroleukemia cells from exponentially growing, differentiated or quiescent cells. The DNA in nuclei of quiescent cells, from plateau-phase cultures, was the most sensitive to denaturation; it denatured (measured by changes in luminescence) at an AO concentration between 50 and 80 microM with the midpoint of the transition (Cd) at 70 microM. DNA in nuclei of differentiated cells (dimethyl-sulfoxide-induced erythroid differentiation) was more resistant (Cd = 77-83 microM), whereas DNA in exponentially growing cells was the most resistant (Cd = 86 microM). Extraction of proteins with 0.1 M HCl at 0 degree C abolished the differences between the cells and shifted the transition to a lower AO concentration (Cd = 46 microM). For comparison, the midpoint transitions representing condensation of free, nucleic acids measured as light scatter changes occurred at 13, 22, 31 and 53 microM of AO, for rRNA, tRNA, and denatured and native-calf thymus DNA, respectively. Denaturation and condensation of DNA, which can be induced by AO either in isolated nuclei or viable permeabilized or fixed cells provides a new approach to discriminate cell subpopulations with different chromatin structure by flow cytometry. The molecular mechanisms of this phenomenon are discussed.     

Denaturation of deoxyribonucleic acid in situ effect of formaldehyde.

In situ denaturation of nuclear deoxyribonucleic acid (DNA) is studied by use of acridine orange to differentially stain native versus denatured DNA, and a flow-through cytofluorometer for measurements of cell fluorescence. Thermal- or acid-induced DNA denaturation is markedly influenced by formaldehyde. Two mechanisms of the formaldehyde action are distinguished. If cells are exposed to the agent during heating, DNA denaturation is facilitated, most likely by the direct action of formaldehyde as a "passive" denaturing agent on DNA. If cells are pretreated with formaldehyde which is then removed, DNA resistance to denaturation increases, presumably due to chromatin cross-linking. It is believed that both effects occur simultaneously in conventional techniques employing formaldehyde to study DNA in situ, and that the extent of each varies with the temperature and cell type (chromatin condensation). Thus, profiles of DNA denaturation of cells heated with formaldehyde do not represent characteristics of DNA denaturation in situ; DNA denaturation under these conditions is modulated by the reactivity of chromatin components with formaldehyde rather than by DNA interactions with the macromolecules of nuclear mileu.     

Visualization by chilling of protein bands in polyacrylamide gels containing 8 M urea: preparation and quantitation of foot-and-mouth disease virus capsid proteins

Protein bands become visible in polyacrylamide gels containing 8 m urea after chilling the gels in air for 5 to 10 min at ?70°C. Urea appears to crystallize preferentially as opaque bands in regions of the gel where protein reduces the amount of free water available as solvent for the urea molecules. Thus detected, the gel sections containing protein bands from foot-and-mouth disease virus can be immediately cut out, and their proteins obtained by electrophoretic elution or extraction procedures. Analysis of the proteins for purity and concentration is then carried out by electrophoresing measured aliquots on analytical gels, staining with Coomassie brilliant blue, scanning the gels for absorbance at 600 nm, and converting peak areas to micrograms of protein using Folin phenol standard curves determined for each purified capsid protein. The most basic capsid protein and its in virion proteolytic-cleavage products stain metachromatically.     

An improved immunocytochemical procedure for high-sensitivity detection of incorporated bromodeoxyuridine

This report describes an improved immunochemical procedure to stain cells in suspension for incorporated bromodeoxyuridine (BrdUrd) and total DNA content. The procedure consists of five steps: chromatin proteins are extracted by treating with 0.1 M HCl and 0.7% Triton X-100 to facilitate DNA denaturation and to minimize nonspecific staining; cellular DNA is denatured by heating to 100 degrees C in distilled water; BrdUrd in single-stranded DNA (ssDNA) is stained using an immunochemical procedure; autofluorescence is reduced using sodium borohydride (NaBH4); and DNA is stained with the fluorescent dye propidium iodide. With this procedure, the BrdUrd incorporated by CHO cells during periods as short as a few seconds can be detected using flow cytometry. In addition, the stoichiometry of the immunofluorescent staining procedure is high.     

Detection of S-Phase Cells in Smear Cytology Using in Vitro Bromodeoxyuridine Labeling

A technique Is described for rapid detection of S-pha?e cells of tumor tissues in smear specimens using bromodeoxyuridine (BrdU) immunostaining. Mouse NR-S1 tumors and human tumor specimens were prepared for smear cytology after incubation in RPMI 1640 culture medium containing 200 μM BrdU at 37 °C under 3 atm for 1 hr. Samples were fixed in 70% ethanol for 30 min and used immediately or air dried for 30 min. Samples were then denatured in either 4 N HC1 or 0.07 N NaOH to prepare partially single-stranded DMA. Fixation with air drying for 30 min followed by 30 min in 70% ethanol and 1 min denaturation with 0.07 N NaOH resulted in satisfactory staining quality. Cultured tumor specimens were processed for routine paraffin sections after smears were made for cytology. The labeling indices of the smear specimens and of the paraffin sections gave similar results. This technique should be useful in evaluating the cell proliferative potential of tumor tissue in smear cytology without processing paraffin sections.     

''Star'' activity and complete loss of specificity of CeqI endonuclease.

Restriction endonuclease CeqI, an isoschizomer of EcoRV, exhibits 'star' activity, a relaxation of specificity in the presence of Mn2+, dimethyl sulphoxide or glycerol. The enzyme cleaves a set of sequences that differ from the canonical GATATC by only one nucleotide in positions 2, 3, 4 or 5. Two of these sequences are not cleaved if modified by dam methylase. A further loss of specificity can be observed in circumstances less favourable for the enzyme, namely low-ionic-strength buffers of pH values below 6.0 or above 9.4. This activity seems to cleave DNA at any sequence, producing a smear instead of well-defined bands. Partial renaturation of the denatured enzyme gives rise to a similar non-specific nuclease activity.     

Deoxyribonucleases in phytohemagglutinin-stimulated lymphocytes

The in situ assay of deoxyribonucleases in DNA-containing polyacrylamide gels following their separation by microdisc electrophoresis was used to determine the deoxyribonuclease pattern of human lymphocytes during stimulation with phytohemagglutinin (PHA). Two additional neutral deoxyribonuclease activities are detectable in stimulated cells, one only active with denatured DNA, the other active with native and denatured DNA as substrate, showing a maximum activity after 36 h and increasing in waves respectively. A group of acid deoxyribonuclease activities also shows a maximum after 36 h of stimulation. A neutral deoxyribonuclease active only with native DNA is missing in stimulated lymphocytes. It is suggested that the acid deoxyribonuclease activities and the neutral deoxyribonuclease active only with denatured DNA are involved in DNA synthesis, whereas the involvement of the neutral deoxyribonuclease active with native and denatured DNA in processing of DNA excreted in stimulated lymphocytes is discussed.     

Use of the initial portion of the melting curve for determining locally denatured sites in DNA radiation damage

A simple approach is proposed to determine locally denatured sites and stability characteristics of secondary DNA structure. The method is based on the analysis of the initial part of melting curve and the determination of changes in the optical density of DNA after heating up to a fixed temperature. The potentiality of the approach is illustrated by the experiments with DNA containing defects in the secondary structure caused by gamma-irradiation in vitro. The sensitivity of the method is less than 0.2 Gy.     

Staining Mitochondria in Fixed Blood Smears

Wet blood smears are placed immediately in Helly's fluid for 24 hr, transferred directly to a saturated solution of potassium dichromate for 48 hr and washed in running water for 2-4 hr. The slides are then treated with iodine and sodium thiosulf ate and washed several hours or overnight. Excess water is removed by blotting the slide around the smear, Altmann's aniline fuchsin is placed on the smear and the slide is heated over a spirit lamp until white fumes appear. After the slide cools the stain is poured off and the excess removed by washing with distilled water. Methyl green (1% aqueous) is dropped on the smear and left for approximately 30 sec. It is then passed rapidly through 2 changes of absolute ethanol and into xylene, from which it is mounted in Permount. This stains mitochondria, red blood corpuscles and specific granules of eosinophilic granulocytes red on a green background.     

Separation of genomic DNA from plasmid DNA by selective renaturation with immobilized metal affinity capture

In contrast to proteins, many nucleic acids can undergo reversible modification of their conformations, and this flexibility can be used to facilitate purification. Selective renaturation with capture is a novel method of removing contaminating genomic DNA from plasmid samples. Plasmid DNA quickly renatures after thermal denaturation and cooling (or alkaline denaturation followed by neutralization), whereas genomic DNA remains locally denatured after rapid cooling in mismatch-stabilizing high ionic strength buffer. Partially denatured genomic DNA can be selectively bound to a metal chelate affinity adsorbent through exposed purine bases, while double-stranded renatured plasmid DNA is not bound. Using this method we have readily achieved 1,000,000-fold clearance of 71 wt % contaminating E. coli genomic DNA from plasmid samples.     

Fluorescence characteristics of dynamic changes in the DNA structure of peripheral blood cells in irradiated rats

A simple approach is proposed to determine locally denatured sites and stability characteristics of secondary DNA structure. The method is based on the analysis of the initial part of melting curve and the determination of changes in the optical density of DNA after heating up to a fixed temperature. The potentiality of the approach is illustrated by the experiments with DNA containing defects in the secondary structure caused by gamma-irradiation in vitro. The sensitivity of the method is less than 0.2 Gy.