Masa-Oki Yamada

Accurate determination of DNA content in single cell nuclei stained with Hoechst 33258 fluorochrome at high salt concentration.

SummaryIn an attempt to achieve accurate quantification of DNA levels in cell nuclie, we studied the influence of salt concentration on the fluorescence of cell nuclei complexed with Hoechst-33258 (Hoe) fluorochrome. The fluorescence of cell nuclei was compared with that of extracted DNA as well as that of nucleosome core. Conformational changes in these complexes were examined by measuring both fluorescence anisotropy and fluorescence lifetime in the nanosecond region. The results showed that the fluorescence of DNA-Hoe was quenched by the nucleosomal structure, there being an associated increase in anisotropy and a decrease in the fluorescence lifetime; however, the fluorescence was restored to the orginal level by the addition of a high concentration of NaCl, CsCl, or LiCl. The reduction in fluorescence may have been due to loss of fluorescence energy caused by collision of the fluorophore with histones in the nucleosome. The addition of 1 M NaCl to the medium used for staining with Hoe greatly stabilized the fluorescence of DNA in cell nuclei. The DNA content of individual cell nuclei was determined by comparing the fluorescence of these nuclei with that of a standard DNA solution. For lymphocytes and liver ploidy cells, reasonably accurate values were obtained by applying the present method.

NaCl-aided Hoechst 33258 staining method for DNA quantification and its application

SummaryWe investigated the effect of salt on the fluorescence staining procedure for quantification of the amount of DNA in cell nuclei in situ. For this, NaCl was added at various concentrations to the Hoechst 33258 fluorochrome (Hoe) medium for staining DNA. The fluorescence intensity of free DNA-Hoe solution was not changed by the addition of NaCl, but that of the nuclei-Hoe complex in situ increased 4-fold on increasing the NaCl concentration up to 1 M. SDS polyacrylamide gel electrophoresis showed that histones H1, H2A, and H2B dissociated from cell nuclei in the presence of 1 M NaCl, resulting in increasing accessibility of DNA to the fluorochrome.The applicability of the NaCl-aided fluorescence staining method was evaluated by measuring the ploidy classes of various cells. The amount of DNA in spermatozoa is half that in 2n hepatocytes, but by the conventional Hoe staining procedure the fluorescence intensity of spermatozoa is higher than that of 2n hepatocytes, due to differences in accessibility of the dye to DNA. In contrast, by the NaCl-aided procedure, the fluorescence intensity of 2n hepatocytes was twice that of spermatozoa. The effectiveness of the NaCl-aided Hoe staining method was checked using cultivated human gingival cells and hepatocytes of LEC rats with hereditary hepatitis. In all cases, reasonable proportionality between the fluorescence intensity and the amount of DNA was observed.

Topographic estimations by component spectroanalysis of two formazans of nitroblue tetrazolium in tissue sections

SummaryA component-spectroanalysis technique was used to study the multicolor properties of histochemically stained tissue sections. We developed a method that makes it possible to obtain separately both the spectral patterns and spatial distributions of different color components in tissue sections. To illustrate the application of this technique, we examined the extinction spectrum of reduced nitroblue tetrazolium (NBT), which is used for the detection of dehydrogenase activity. Upon the reduction of NBT, mono-and diformazans are formed, and these exhibit overlapping extinction spectra. When succinate dehydrogenase (SDH) activity in rat liver lobules was examined using NBT, monoformazan was found to be present at higher concentrations than diformazan and to have a uniform distribution, whereas the concentration of diformazan increased with a steep gradient between the center and periphery of lobules. In rat skeletal muscle fibers, diformazan was present at higher concentrations than monoformazan. The level of SDH activity was topographically represented by the hydrogen concentration calculated from the concentrations of the two formazans. This method is effective for separating multiple components such as mono-and diformazans in histochemical reactions.

Polarization Fluorometry to Determine Cell Density with Fluorochrome for DNA

There is a need for a rapid and sensitive technique to estimate a small number of cells. The total number of both living and dead cells suspended in a solution can be measured directly with the use of the Coulter counter or a flow-cytometer, but both of these measurements are inaccurate for small bacteria, such as an Escherichia coli cell (0.6 μm diameter × 2 μm length). The bacterial density is determined by the measurement of the turbidity of a cell suspension with a nepherometer or, more conveniently, with a spectrophotometer. However, foreign particles (e.g., dust, protein, or lipid) often increase turbidity and result in an error in the assay. Fluorometric assay for DNA content provides a sensitive estimation of the cell density free from turbidity changes. For example, Hill and Whatley used an antibiotic drug (mithramycin) as a labeling fluorochrome to measure cell DNA density with a lower limit of 0.5 μg. Recently, more sensitive quantification of DNA has been achieved with a bibenzimidazole dye, Hoechst 33258 (H33258). This dye reacts with the adenine-thymine-rich region of DNA, resulting in a high quantum efficiency for the determination. Downs and Wilfinger applied H33258 to quantify DNA measurement, and they achieved detection limits as low as 4 ng for extracted DNA from pituitary cells.

Fluorescence decay measurements for determining the relative content of ethidium bromide to DNA in situ in cell nuclei

SummaryA fluorometric method for the determination of the amount of ethidium bromide (EB) bound to DNA in situ in cell nuclei is discussed. Even when the EB content was very small, the molar ratio of DNA-phosphorus (DNA-p) to dye (P/D ratio) could be estimated by measuring the lifetime of the transient fluorescence of the EB-DNA complex as a function of the P/D ratio. To examine the relationship between the fluorescence intensity, lifetime, and P/D ratio, polyacrylamide gel film containing 4.7 mM DNA-P was used as a model DNA tissue, and its fluorescence was measured using a nanosecond microfluorometer. The fluorescence intensity showed a maximum at P/D=6. The fluorescence lifetime increased with the P/D ratio, and this was accompanied by a proportional increase in the quantum efficiency. Thus, the lifetime value was an effective parameter for the determination of the P/D ratio in situ in tissue. When this approach was applied to tissue sections of mouse liver treated with solutions of EB at concentrations of 10 and 50 μg/ml, the fluorescence lifetimes on cell nuclei were 18.9 and 17.4 ns with P/D ratios of 20 and 12, respectively, as based on the model-tissue experiments. When the P/D ratio was 20, the concentration of EB in the nucleus was approximately 1.5 mM, i.e., 60 times higher than that in the staining solution.

Association of neutral α-glucosidase with cytoplasmic granules of peritoneal macrophages☆

Abstract A neutral α-glucosidase (EC 3.2.1.20) activity was shown to be associated with granules which are sedimentable at 10 000 g after differential centrifugation of mouse peritoneal macrophage homogenates. When the post-nuclear supernatant was centrifuged in a sucrose density gradient, high activities for neutral α-glucosidase and β-glucuronidase (EC 3.2.1.31) were detected in the bottom fractions because of aggregation of the granules. Neutral α-glucosidase-containing granules were completely disaggregated by the addition of 20 units/ml of heparin and 10 mM Tris-HCl (pH 7.2), which caused only a partial disaggregation of β-glucuronidase-containing granules. The addition of a high concentration of heparin, Tris buffer, or KCl to the gradient gave the same patterns of disaggregation of the granules. Under the condition in which about 50% of the total β-glucuronidase activity was released into the medium, depending on phagocytosis, very little α-glucosidase was released. These observations suggested that neutral α-glucosidase may localize in non-lysosomal granules.

Quantitative topochemistry of rat liver enzymes during postnatal development in relation to activity-rest cycle.

A quantitative histochemical method (Trident) has been adapted to measure the activities of 4 enzymes, succinate dehydrogenase (SD), isocitrate dehydrogenase (ICD), glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6-PGD), within the liver acini of the rat during the postnatal developmental period. Quantitative changes of these enzymes in livers of rats of 25 g and 50 g body weight were studied, with particular emphasis on the activity-rest cycle. The results indicate a time-dependent heterogeneous distribution of enzymes along the acinar zones and the pattern of localization is age-dependent. When the mean enzyme activity from each group in relation to the time of the day are compared, a mirror image of each other could be seen. In general, a high enzyme activity has been observed during the resting phase in 25-g rats and low in 50-g rats. During the developmental period, the mean ICD activity is diminished, whereas G6PD and 6-PGD are augmented, and SD activity remains unchanged.

Topogenesis of glycogen distribution pattern in post-natal rat liver in reference to the activity-rest cycle

The present study, on immature rats, revealed that in the liver with exclusively diploid cell population (MD), the glycogen content was half of the adult level and the 24 h distribution pattern was reverse, i.e. highest in the evening and lowest in the morning. In these animals, in general, the protein content was high and did not show any circadian rhythm but incidentally showed a time dependent zonal distribution pattern. The low glucose-6-phosphatase activity reciprocates with low glycogen content. The relative number of cells per unit area showed a time dependent distribution pattern. The liver with equal distribution of diploid and tetraploid classes (MD:MT), attained the classical circadian rhythm of glycogen with high morning and low evening oscillation pattern. The TRIDENT measurements manifest the zonal distribution pattern with high values at the perilobular region (PL) and low at the centrolobular region (CL). The relative cell number study along the acinus demonstrated more number of cells around the PL region than that at CL region, indicating a variation in the cell size. Eventually, the protein content showed a circadian rhythm in this group, with high amount in the morning. The zonal distribution pattern always revealed the high content near the perilobular region. This could be due to more number of cells per unit area in this region. The glucose-6-phosphatase showed a circadian rhythm. The typical high glucose-6-phosphatase in the perilobular region could be further subzonated into small groups of high activity surrounded by lighter zones, thus establishing the heterogeneous function of liver parenchymal cells.

In situ microfluorometry of kinetoplast and nuclear DNAs in Trypanosoma gambiense unusual repairment of DNA after treatment with bleomycin

The blood stream form of Trypanosoma gambiense was smeared on a nonfluorescent slide glass with 1 microgram/ml of Hoechst 33258 in 1 mM Tris-HCl buffer (pH 7.2) containing 1% 2-mercaptoethanol and subjected to the in situ microfluorometry. Effects of bleomycin (BL) on the kinetoplast (K)-DNA and nuclear (N)-DNA of T. gambiense were examined in the time course to 6 h after injection of BL into the infected mice. An enhancement of fluorescence occurred 30 min after the injection and then slowed down. This enhancement was due to DNA synthesis both in the K-DNA and N-DNA. This suggests that the strong repairment occurs in both DNAs after treatment with BL.

A Cyst-forming Eimeriina Found in the Kangaroo Imported from Australia

One pair of grey kangaroos was imported from Australia in July 1974. One month after arrival they successively died of serious intestinal bleeding. On biopsy, the epithelium was destroyed and replaced with necrotic degeneration with numerous dot-like flecks of bleeding from the stomach till the jejunum. On histopathological examinations, the epithelium was composed of swollen glandular cells which form giant cells with eccentrically displaced nuclei. The cell forms a cyst which develops solitarily and gathers together to form a racemous shape. These cysts are bound to the thick wall and the parasites are found in the vesicular space. Several sequences of the parasite growth are demonstrated. The parasites migrated initially into the cytoplasm and then to the nucleus of the glandular cell in which they grow, then the cyst is formed and finally the parasites flow out to penetrate again into normal cells. The parasite cell changes to crescent- or spindle-shape as a tachyzoite. The relative nucleic acid content of the parasite in the host cell or its nucleus is measured by spectrophotometry and fluorometry. The data suggest that the parasites are reproduced in intestinal glandular cells of the kangaroo.