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PubMed1984

Koilocytotic lesions of the cervix. The relationship of mitotic abnormalities to the presence of papillomavirus antigens and nuclear DNA content.

It has been reported that abnormal mitotic figures (AMFs) occur principally in aneuploid lesions and that aneuploidy is a diagnostic feature of non-endocrine-dependent epithelial cancer precursors and cancers. Recently, AMFs have been reported in cervical lesions interpreted as flat condylomata, and it has been suggested by several authors that AMFs may not be diagnostic or aneuploidy or neoplasia, particularly in human papillomavirus-(HPV)-induced lesions. Although it is conceivable that AMFs may be a regular feature of HPV infection, their association with cytologic atypia and their presence in higher grades of cervical intraepithelial neoplasia (CIN) suggests that AMFs may herald the presence of a different lesion than the pure flat condyloma. In the current study, koilocytotic cervical lesions thought to be HPV-induced were examined microscopically for the presence of AMFs, and the findings were correlated with the presence of HPV as determined by immunoperoxidase and nuclear DNA distribution patterns as measured by Feulgen microspectrophotometry. In unselected lesions originally diagnosed as flat cervical condylomata, AMFs were surprisingly common (22.6%), and did not correlate with the extent of koilocytosis. Immunoperoxidase (IMPO) stains were performed in 35 cases with AMFs, and were negative for HPV in 74.3% and positive in 22.8%. However, among the cases evaluated by IMPO, there was an inverse relationship between the presence of mitotic abnormalities and the expression of HPV antigen. Nine of 11 (81.8%) lesions containing AMFs were aneuploid, and 2 of 11 (18.2%) were polyploid. Abnormal mitotic figures have a range of morphology and frequency in koilocytotic cervical lesions. Although the biology of these lesions is not well-defined, the presence of AMFs may identify a subgroup of HPV-induced cervical atypias which represent a transition between flat cervical conylomata and CIN.

Animals↗
PubMed1979

Microdensitometry.

Microdensitometry, or microspectrophotometry, is the measurement of the concentration or mass of a chromophore in microscopically defined regions, and is governed by well-established laws of physics. Initially it proved of value in Feulgen cytophotometry of the relative amounts of DNA in individual nuclei of isolated cells. It has now achieved wide applicability to the measurement of cellular biochemical activity by means of stoichiometric chromogenic reactions. The validity of some of these measurements has been confirmed by comparative biochemical and microdensitometric assays. Thus microdensitometry, even of heterogeneously distributed chromophores, can be precise, provided that the technique is operated with due regard to its limitations within the laws of physics. The potential errors include: variation in thickness of tissue sections (path-length); scatter; glare; diffraction; occlusion of light by optically dense particles; and the inhomogeneity error. However, under correct conditions for the cytochemical reactions and for operating the microdensitometer, these potential errors become small or negligible. Thus the highly sensitive cytochemical bioassay of thyrotropin exemplifies the precision that can be achieved by controlled use of microdensitometry.

Animals↗
PubMed1990

Estimating anatomical-functional position coordinates in liver tissue.

Hepatocyte enzyme activity was demonstrated by examining adult C57BL/6 mouse liver cryostat sections under a succinate dehydrogenase (SDH) histochemical reaction, and quantified by microspectrophotometry and microdensitometry. The hepatocyte SDH activity gradient along the path between the portal veins (PV) and efferent terminal hepatic venules (THV) was analyzed by measuring the concentration of the chromophore precipitated in 10 consecutive hepatic parenchymal domains located along imaginary lines drawn across the entire PV-to-THV distance. The profiles of intensity or of normalized relative optical density obtained on a high number of lines were correlated with distance values along the PV-to-THV pathway, enabling us to establish a general mathematical function relating SDH activity (chromophore concentration) to position values on a scale of 0 to 10 corresponding to the theoretical PV-to-THV distance. The equation can be used to interpolate the SDH activity surrounding any intrahepatic object located between the PV and the THV, thus making it possible to calculate the object's anatomical-functional position coordinates in the liver acinus. To demonstrate how this method is used, we have calibrated the intrahepatic position of hemopoietic foci induced in the liver tissue of adult mice treated with phenylhydrazine (PHZ), and show that these foci are located on coordinate 3.31 (maximum range 1.25-4.86) of the sinusoidal domain-that is, on the borderline between Rappaport's acinar zones 1 and 2.

Animals↗
PubMed1993

Long-distance running causes site-dependent decrease of cartilage glycosaminoglycan content in the knee joints of beagle dogs.

OBJECTIVE: To study the effects of a long-term (1-year) program of running exercise (up to 40 km/day) on the thickness and glycosaminoglycan (GAG) content of articular cartilage in the knee and humeral head cartilage of young dogs. METHODS: Samples for histologic analysis were obtained from 12 different locations of the joints. We conducted a detailed, area-specific analysis, measuring the thickness of articular cartilage and analyzing the distribution of Safranin O stain that binds stoichiometrically to GAG as determined by quantitative microspectrophotometry. RESULTS: Running exercise decreased the GAG content of the uncalcified articular cartilage in the weight-bearing summits of the femoral condyles by 5-13% (P < 0.05), while at margins of these areas the GAG content was equivalent to control levels. In the lateral condyle of the femur, the reduction was most prominent in the superficial zone (up to 28% decrease; P < 0.05), and extended into the intermediate zone (11% decrease; P < 0.05). GAG content was also significantly reduced in the superficial zone at the lateral condyle of the tibia and the head of the humerus, by 35% (P < 0.01) and 15% (P < 0.05), respectively. Running did not alter GAG concentration in the patellofemoral region. CONCLUSION: The GAG depletion caused by 40-km/day running exercise is restricted to prominent weight-bearing areas of the joint and begins from the superficial cartilage without signs of degeneration. The different degree and type of joint loading can explain the site-dependent cartilage response to long-distance running. The loss of GAGs was possibly due to breakdown of proteoglycans, which could not be compensated for by neosynthesis of molecules. With time, this may affect the condition of articular cartilage, especially if the joint is exposed to loading for lengthy periods.

Animals↗
PubMed1990

Peculiar ultraviolet absorbing moieties of human osteoarthritic cartilage.

The ultraviolet absorbing components of human cartilage have been measured by microspectrophotometry. The characteristics of the chondrocytes appeared to be identical, irrespective of the pathology. However the matrix of osteoarthritis cartilage contained components that absorbed maximally in the region of 270 to 250 nm; such components were not found in the matrix of cartilage of non-arthritic joints. Substances that absorb maximally in this region of the ultraviolet could generate free radicals.

Aged↗
PubMed1978

The DNA content of Purkinje cells in mammals.

Nerve cells have generally been assumed to have a diploid DNA content, typical of non-dividing somatic cells. However several reports have suggested that certain nerve cells types, notably Purkinje cells of the cerebellum, are polyploid. Other studies have contradicted these findings, stating Purkinje cells to be diploid. In this paper we reinvestigate the DNA status of Purkinje cells, in a variety of mammalian species. Cell DNA content is measured on tissue smears by Feulgen microspectrophotometry. Results show that for all species examined by us, Purkinje cells have, without exception, a DNA content comparable to that of somatic cells. A critical appraisal of the techniques used in those studies claiming a tetraploid DNA content for Purkinje cells leads us to believe our findings to be correct.

Animals↗
PubMed1987

Flow cytometric analysis of isolated rat liver nuclei during growth.

The development of hepatocyte polyploidy in rats aged up to 4 months was analyzed by flow cytometry using both scatter and fluorescent parameters to distinguish DNA diploid and DNA tetraploid populations and to discriminate between parenchymal and non-parenchymal compartments. The precise origin of each class of nuclei was assessed in whole liver homogenate using purified hepatocytes, obtained by liver perfusion followed by separation on Percoll gradient, and identifying the peaks corresponding to parenchymal nuclei. The results indicate that preparative procedures involving homogenization of the rat liver tissue caused loss of the DNA octaploid population. Data on the relative proportion of the different DNA ploidy elements during rat liver development, which are in good agreement with those observed by cell analysis by means of microspectrophotometry, indicate the usefulness of flow cytometry as a choice method for the analysis of ploidy distribution.

Animals↗
PubMed2006

DNA methylation in stingless bees with low and high heterochromatin contents as assessed by restriction enzyme digestion and image analysis.

BACKGROUND: The stingless bee genus Melipona has been divided into two groups, based on their heterochromatin content. Melipona quadrifasciata and Melipona rufiventris have low and high levels of heterochromatin, respectively. Since condensed chromatin may be rich in methylated DNA sequences, M. quadrifasciata and M. rufiventris nuclei may contain different amounts of methylated CpG. These differences could be assessed by comparing Feulgen-DNA values obtained by image analysis of cells treated with the restriction enzymes Msp I and Hpa II that distinguish between methylated and unmethylated DNA. Msp I and Hpa II cleave the sequence -CCGG-, but there is no cleavage by Hpa II if the cytosine of the central CG dinucleotide is methylated. METHODS: Malpighian tubules of M. quadrifasciata and M. rufiventris were treated with Msp I and Hpa II prior to the Feulgen reaction, and analyzed by automatic scanning microspectrophotometry. RESULTS: The Feulgen-DNA values for the heterochromatin of M. rufiventris and for the small heterochromatin and some euchromatin domains of M. quadrifasciata mostly decreased after treatment with Msp I, but were unchanged after treatment with Hpa II. CONCLUSION: CpG methylation, although detected in diverse chromatin compartments in different bee species, may induce silencing effects required for the same cell physiology.

Animals↗
PubMed2007

Chromatin supraorganization and extensibility in mouse hepatocytes with development and aging.

BACKGROUND: Chromatin supraorganization and extensibility, which lead to the formation of extended chromatin fibers (ECF), are affected by starvation and refeeding in adult mouse hepatocytes. It is expected that they could also change with mouse development and aging. METHODS: Methods used involved topochemistry, image analysis, microspectrophotometry, gravity action, and polarization microscopy. RESULTS: Increased nuclear areas and Feulgen-DNA amounts with advancing hepatocyte polyploidy were found with development and aging. A slightly less packed chromatin with more heterogeneously distributed condensation levels was detected in young and old mice. Con-A responsiveness was almost absent in young mice but very deep in aged mice. ECFs formed from nuclei of adult and aged mice but not from nuclei of young mice. The frequency of ECF formation with the long lysis protocol increased with aging. CONCLUSIONS: In young mice, a less packed chromatin state may be associated with more intense gene activity, thus increasing the DNA-nuclear matrix interactions, and inhibiting ECF formation. Reduced DNA-nuclear matrix interactions besides defects in heterochromatin formation may induce higher ECF formation and chromatin unpackaging in old mice. We suggest that differences in Con-A staining relate to different gene activity with advancing development and aging.

Aging↗
PubMedDate not supplied

Hepatocytes of Zones 1 and 3 conjugate sulfobromophthalein with glutathione.

The capacity of hepatocytes of Zones 1 and 3 of the liver acinus to conjugate sulfobromophthalein (BSP) with glutathione and to secrete the conjugate into bile was studied. BSP was infused into the in situ perfused rat liver in concentrations of 0.01 and 0.05 mM. Perfusions were performed either in the portal to hepatic vein direction (forward perfusion) or in the hepatic vein to portal vein direction (retrograde perfusion). Hepatocytes contributing to the uptake, metabolism and biliary secretion of BSP were directly assessed qualitatively by light microscopy, and also semiquantitatively by microspectrophotometry. BSP was taken up predominantly by hepatocytes of Zone 1 during forward perfusion and by those of Zone 3 during the retrograde perfusion of BSP. The biliary products of BSP metabolism by each acinar zone were subsequently assessed. Hepatocytes of both Zones 1 and 3 of the acinus secreted BSP into bile in the form of BSP-glutathione, and BSP-glutathione conjugate represented about 78% of the total BSP secreted into bile by each zone. The rate of BSP biliary secretion by both zones was similar at a concentration of 0.01 mM BSP, while a slight decrease (15%) in BSP secretory rate by hepatocytes of zone 3 was observed at concentrations of BSP near biliary Tm. Liver perfusion with exogenous BSP-glutathione provided results similar to those obtained with BSP. In contrast, the perfusion of 3,6- dibromosulphthalein , a compound which is not conjugated by hepatocytes, resulted in similar biliary secretory rates regardless of the direction of perfusion. These results indicate that: (a) the capacity for glutathione conjugation of BSP is distributed among hepatocytes of all acinar zones; (b) near biliary Tm, the biliary secretory rate of BSP by hepatocytes of Zone 3 is slower than that of hepatocytes of zone 1, and (c) in vivo, all hepatocytes likely contribute to the uptake, metabolism and biliary secretion of BSP.

Animals↗
PubMed1975

Expression of SV40 T antigen during the cell cycle of sv40-transformed cells.

The expression of the nuclear SV40-induced T antigen was measured by microfluorimetry on individual, asynchronously growing SV40-transformed cells which had been stained with hamster T-antiserum by the indirect immunofluorescence method. The same individual cells were first measured for T antigen and then for DNA by Feulgen microspectrophotometry. A linear correlation was observed between the two parameters. T antigen expression was also measured in cell populations arrested at different phases of the cell cycle. Results of both types of experiments show that the expression of the gene (s) for T antigen in transformed cells increases during DNA replication and reaches its highest level in G2 nuclei. During mitosis T antigen is found in the cytoplasm.

Animals↗
PubMed1976

Tissue response to implanted polymers: the significance of sample shape.

Studies were designed to demonstrate the need for standardization of shape of samples used as implants to evaluate histotoxicity of polymer materials. Six medical-grade polymers (polypropylene, polyethylene, polyurethane, silicone rubber, poly(vinyl chloride), and Teflon) were extruded as rods with circular-, triangular-, and pentagonal-shaped cross sections, and were implanted in rat gluteal muscles for 14 days. Evaluation of the tissue response was assessed by quantitating cellular lysosomal acid phosphatase enzyme activity by using microspectrophotometry. All triangular-shaped implants showed the highest enzyme activity and cellular response; pentagon shapes showed less, and circular rods showed the lowest activity. The results demonstrate the need for standard sample shape for valid comparative studies of tissue response to implanted polymers.

Acid Phosphatase↗
PubMed1977

Dibutyryl cyclic AMP arrests the growth of cultivated Cloudman melanoma cells in the late S and G2 phases of the cell cycle.

DBcAMP reversibly arrests cultivated Cloudman melanoma cells in the late S and G2 phases of the cell cycle. This is supported by the measurement of DNA synthesis by autoradiography and measurement of cellular DNA by two methods--the diphenylamine reaction and microspectrophotometry of Feulgen stained cells. We also present evidence that (1) cell division is prevented if DBcAMP is added as late in the cycle as early S phase. (2) The inhibition of cell division does not appear to be caused by products of tyrosine oxidation. (3) The increase in cell size that occurs in the presence of DBcAMP reflects continued synthesis of protein in the absence of cell division.

Adenosine Monophosphate↗
PubMed1990

Bradykinin induces superoxide anion release from human endothelial cells.

The time-dependent release of superoxide anion (O2-) from bradykinin (Bk)-stimulated human umbilical vein endothelial cells (EC) was measured as the superoxide dismutase-inhibitable reduction of ferricytochrome C employing a novel application of microspectrophotometry. In the absence of Bk, O2- release by EC was not detectable. EC exposure to Bk (10(-6) to 10(-5) M) resulted in a rapid release of O2-. The release of O2- occurred within 5 minutes of exposure. O2- release was partially inhibited by indomethacin (63 +/- 6%), thus suggesting that arachidonic acid metabolism, through cyclooxygenase, contributes to EC O2- production. EC O2- release may be an important component in the pathophysiologic actions of Bk on vascular function.

Bradykinin↗
PubMed1993

Core proteins of soluble chondroitin sulfate proteoglycans purified from the rat brain block the cell cycle of PC12D cells.

The effects of soluble chondroitin sulfate proteoglycans (CSPGs) purified from the rat brain on proliferation of and neurite outgrowth from PC12D cells (Katoh-Semba et al., J Neurosci Res 17:36, 1987) were investigated. When PC12D cells are cultured under standard conditions, they proliferate with a doubling time of about 2 days, irrespective of the presence or absence of NGF. However, the addition of a mixture of several types of purified soluble brain CSPG (50 nmol uronic acid/ml) to the culture medium prevented the increase in the number of PC12D cells as well as the nerve growth factor (NGF)-induced neurite extension. The dose for 50% inhibition (ID50) was 1.6 nmol/ml for cell proliferation and 2.7 nmol/ml for neurite elongation. The increase in cell number seemed to stop around 6 h after exposure to culture medium supplemented with brain-derived CSPGs, and even substratum-attached CSPGs were able to exert such inhibitory effects. Only brain-type CSPGs, not a cartilage-derived CSPG (PGH) or a hyaluronate-binding PGH, had such inhibitory effects. Furthermore, these inhibitory activities were associated only with the core proteins of brain-derived CSPGs, and not with polysaccharide chains from brain-derived CSPGs. Incorporation of [3H]thymidine into DNA did not decrease for at least the first 12 h. Consequently, the amount of DNA per cell after 48 h of culture was about twofold higher in cells treated with brain CSPGs than in nontreated cells after exposure to the medium with CSPGs. Microspectrophotometry revealed that the population of cells with a high DNA content was greater in the culture treated with brain-derived CSPGs than in the control culture. These findings indicate that purified soluble brain CSPGs block the cell cycle of PC12D cells at the G2 phase with resultant cessation of cell proliferation and the inhibition of neurite outgrowth.

Aggrecans↗
PubMed1976

Cytophotometric analysis of basic nuclear proteins during gametogenesis in the free-living nematode Panagrellus silusiae.

The cytochemical staining characteristics of basic nuclear proteins during gametogenesis in both sexes of the free-living nematode Panagrellus silusiae were monitored by absorption microspectrophotometry. During spermatogenesis, the levels of both of the nuclear-bound Sakaguchi and 1-fluoro-2, 4-dinitrobenzene (FDNB) reagents per Feulgen-DNA content increase prior to meiosis and subsequently drop off during spermiogenesis. During oogenesis, the amount of Sakaguchi-protein to DNA ratio rises slightly whereas the FDNB-protein to DNA ratio undergoes a meagre decline. Since the oocyte of Panagrellus accumulates DNA in excess of a 4C DNA equivalent, the relative constancy of the ratio of basic nuclear protein content to DNA amount during oogenesis signifies that these proteins are being continously produced during oocyte maturation. After treatment with alkaline fast green (pH 8.1) the cytoplasmic and nuclear regions of the gonocytes of both sexes yield such an intense reaction that microspectrophotometric measurements could not be taken. The basis of the cytoplasmic staining reaction to the fast green (pH 8.1) dye was not determined. The rapid, highly deterministic programme of embryogenesis in Panagrellus may require a substantial stockpiling of basic nuclear proteins during oogenesis.

Animals↗
PubMed1990

Adaptations of visual pigments to the photic environment of the deep sea.

This is a summary of studies that bear on the problems of the adaptation of visual pigments to the photic environment of the deep sea. The results suggest that the spectral absorption of these retinal pigments is shifted toward the blue in order to match the dim, blue-green downwelling light and/or the bioluminescence of organisms that are critical to the life of the species. Through such a spectral match, greater visual sensitivity is achieved for life in the special photic condition of their habitat. This adaptation has been found for chimaerid fishes, for elasmobranchs, for teleosts, for mammals, and for certain crustaceans and cephalopods. The most convincing evidence for such an adaptive match has been found in teleosts that have red-emitting photophores. In these fishes a photopigment with absorbance shifted toward the red has been found by extraction and microspectrophotometry. A few exceptions to this idea of an adaptive match have appeared in the literature, the cone pigments, especially, being examples of such offset pigments. The malacosteid fishes have been shown to have a red-shifted retinal pigment with 11-cis-3-dehydroretinal as the chromophore and some invertebrates have also adopted this molecule to adjust the spectral absorption to the photic environment or to the bioluminescence. These studies are beginning to reveal that visual biochemistry is basically the same in vertebrates and invertebrates and that the visual pigment protein arose early in phylogeny and has been retained, with appropraite modifications, to the present.

Animals↗