Cytofluorometric nuclear DNA determinations on the atrioventricular nodal cells in human hearts

In the human heart, it is well known that the polyploidization of working heart-muscle cells increases in proportion to increases in heart weight, but there has been no investigation of the process of polyploidization in the specialized heart-muscle cells of the cardiac conduction system which have a nerve-like function. In order to investigate the process of polyploidization in these cells, the nuclear DNA content of atrioventricular nodal cells was measured using cytofluorometry. Tissue samples taken from autopsied hearts without arrhythmias were embedded in paraffin blocks after Carnoy fixation. Blocks containing the atrioventricular conduction system were cut according to the serial sectioning method of Lev et al. The compact atrioventricular nodes were removed from thick paraffin sections (150 micron) under a stereomicroscope. The cells were then isolated by enzyme digestion and ultrasonic treatment. Smears of the isolated cells were double stained with azocarmin-G and acriflavine-Feulgen. Cytofluorometric DNA determinations of the DNA content of atrioventricular nodal cells were performed. Atrioventricular nodes were found to be composed of a large number of diploid cells and a small number of tetraploid cells. No octaploid cells were found. These findings reveal that the process of polyploidization in atrioventricular nodal cells is different from that found in working heart-muscle cells.     

Cytofluorometric nuclear DNA content analysis of breast tissues after frozen section diagnosis

OBJECTIVE: To establish a suitable method for measurement of nuclear DNA content in breast tissues from frozen storage after frozen section diagnosis. STUDY DESIGN: For fundamental research, rat liver samples preserved in a deep freezer were used. Four protocols were used (1. fixation with 70% ethanol followed by naked nuclei preparation; 2. fixation with 10% neutral buffered formalin followed by naked nuclei preparation; 3. preparation for naked nuclei prior to fixation with 70% ethanol; and 4. preparation for naked nuclei prior to fixation with 70% neutral buffered formalin). For clinical research, 13 separate fresh frozen breast tissue samples were analyzed after frozen section diagnosis. One contained a malignant phyllodes tumor (MPT) consisting of 2 components, benign epithelial cells and malignant stromal cells; 3 were benign tumors containing fibroadenoma; and 9 cases were carcinomas, consisting of 5 scirrhous, 3 papillotubular and 1 mucinous. RESULTS: Protocols 1, 2 and 3 were not suitable methods for our purpose because remaining cytoplasm or cohesive nuclei were observed. In protocol 4 the cytoplasm was completely undetectable, and nuclei were suitably separated for nuclear DNA content measurement. Benign epithelial cell component nuclei presented a diploid pattern, and the malignant stromal cell component nuclei indicated a euploid pattern in MPT. All 3 cases of benign constituents in fibroadenoma showed a diploid pattern, as did the 3 carcinoma cases (1 mucinous, 1 scirrhous and 1 papillary). Four scirrhous and 2 papillary carcinomas showed an aneuploid pattern. CONCLUSION: Our findings show that it is possible to measure nuclear DNA content of human frozen storage tissues after frozen section diagnosis.     

Feulgen slope determinations of urodele nuclear DNA amounts

Summary Measurements of the initial slope of the Feulgen hydrolysis curve allow the precise determination of specific nuclear DNA amounts. The low stain densities involved preclude density-dependent systematic error. Best estimates of the nuclear DNA amount for sixteen salamander species are presented.     

A mathematical model of human atrioventricular nodal function incorporating concealed conduction

This work develops a mathematical model for the atrioventricular (AV) node in the human heart, based on recordings of electrical activity in the atria (the upper chambers of the heart) and the ventricles (the lower chambers of the heart). Intracardiac recordings of the atrial and ventricular activities were recorded from one patient with atrial flutter and one with atrial fibrillation. During these arrhythmias, not all beats in the atria are conducted to the ventricles. Some are blocked (concealed). However, the blocked beats can affect the properties of the AV node. The activation times of the atrial events were regarded as inputs to a mathematical model of conduction in the AV node, including a representation of AV nodal concealment. The model output was compared to the recorded ventricular response to search for and identify the best possible parameter combinations of the model. Good agreement between the distribution of interbeat intervals in the model and data for durations of 5 min was achieved. A model of AV nodal behavior during atrial flutter and atrial fibrillation could potentially help to understand the relative roles of atrial input activity and intrinsic AV nodal properties in determining the ventricular response.     

Cytofluorometric measurements of nuclear DNA in adventitious buds and shoots of Picea abies regenerated in vitro

Nuclear DNA content of adventitious buds and shoots of Picea abies (L.) Karst. developed in vitro was compared to that of buds collected from field-grown trees. Protoplasts were isolated from the different tissues and after fixation and staining with ethidium bromide the DNA content of their interphase nuclei was determined cytofluorometrically. The DNA pattern of the different tissues was within the same range and had the same distribution with one main peak.     

Age-associated damage in mitochondrial DNA in human hearts

Damage to mitochondrial DNA seems to be involved in the etiology of age-associated degenerative diseases. The aim of this study is to elucidate effects of aging on human mitochondrial DNA. 8-Hydroxy-deoxyguanosine, a product of free radical damage to deoxyguanosine, is reported to cause random point mutations. In human mitochondrial DNA, 8-hydroxy-deoxyguanosine increased exponentially with age, and the population of mitochondrial DNA with deletion increased also exponentially with age. Furthermore, a clear correlation existed between the accumulation of 8-hydroxy-deoxyguanosine and that of mitochondrial DNA with deletion. We also determined the effects of aging on rat mitochondrial function together with 8-hydroxy-deoxyguanosine content in mitochondrial DNA. The activities of complexes I and IV of the mitochondrial electron transport chain decreased significantly in rats aged 100 weeks compared with those in rats aged 7 weeks. A concomitant increase in 8-hydroxy-deoxyguanosine was observed in mitochondrial DNA of rats aged 100 weeks. From our results, it is concluded that the age-associated accumulation of somatically acquired oxygen damage together with deletions in mitochondrial DNA might be important contributors to the deterioration of cardiac function associated with age.Abbreviations mtDNA mitochondrial DNA - 8-OH-dG 8-Hydroxy-Deoxyguanosine - dG Deoxyguanosine - HPLC/MS Micro-High Performance Liquid Chromatography/Mass Spectrometry     

Morphological study of sarcoplasmic reticulum in the atrioventricular node and bundle cells in guinea pig hearts

The osmium-ferrocyanide method for staining of the sarcoplasmic reticulum (SR) was used for a morphological investigation of the various components of the SR in the atrioventricular node and bundle (AVNB) cells of guinea pig hearts. On the basis of light microscopic observations, the AVNB tissue in guinea pig hearts can be divided into five regions: atrionodal junction, midnode, proximal bundle, distal bundle, and bundle branches. Electron microscopic observations revealed two types of junctional SR (j-SR) saccules in the cells from all the regions of AVNB tissue. One is similar to that seen in the working cardiac cells, i.e., flattened saccules with junctional granules. The second type is dilated and contains electron-dense granular material throughout its lumen. The flattened type is seen more often than the dilated type in atrionodal junctional cells and midnode cells, whereas the dilated type occurs more often in distal bundle cells and bundle branch cells. In most cells from the atrionodal junction and midnode regions, the j-SR saccules are apposed more often to sarcolemmal areas associated with nonspecialized regions of intercellular junctions than to other sarcolemmal areas. This distribution was not found in the distal bundle and bundle branch cells. Free SR tubules around the myofilament bundles are poorly developed in the midnode cells, generally in accord with the extent of development of myofibrils. Z-tubules are found in cells from all regions but are poorly developed in midnode cells. Corbular SR vesicles are found in cells from all the regions of AVNB tissues but are rare in midnode cells. Thus, each of the regions in the AVNB tissue has a different, characteristic distribution of SR components. Because of their possible relationship to the regulation of the intracellular concentrations of calcium, these differences in SR morphology may contribute to the diverse physiological properties of the different regions of the AV node and bundle.     

Serial section method for cytofluorometric determinations of the DNA content of component cells of the human cochlea

We describe a method for measuring the DNA content of the component cells of the organ of Corti using serial sections of human cochleae obtained at autopsy. Cochleae were fixed in Carnoy's solution and embedded in Acrytron E, a water-miscible methacrylate resin. A procedure was developed to reduce the background fluorescence in methacrylate-embedded sections; the resin was pretreated with ion-exchange resin (Amberlite IRA-410). Experiments showed that pretreatment reduce the background fluorescence practically to zero. Seventy 3 microns-thick serial sections were prepared on fluorescence free glass slides and stained with azocarmin G and acriflavine-Feulgen. After postirradiation using blue excitation light, the amount of Feulgen-DNA present in the target nucleus in each section was determined using a microfluorometer. The amount of DNA in the entire nucleus was determined by adding together the DNA content of the segments of the nucleus. The characteristic appearance of the organ of Corti made it easy to detect these cells; under green excitation light the cells of this organ exhibited red cytoplasmic azocarmin-G fluorescence. Due to the relatively wide internuclear spaces, cytofluorometry fo individual nuclei could be performed without interference from the neighboring cells. Our technique using serial sections allowed us to measure the DNA content of individual cells and obtain histological information about particular cells and their neighboring cells. Several polyploid cells were found among the Hensen's cells in the cochlea, while all other component cells of the organ of Corti were diploid.     

Serial section method for cytofluorometric determinations of the DNA content of component cells of the human cochlea

Summary We describe a method for measuring the DNA content of the component cells of the organ of Corti using serial sections of human cochleae obtained at autopsy. Cochleae were fixed in Carnoy's solution and embedded in Acrytron E, a water-miscible methacrylate resin. A procedure was developed to reduce the background fluorescence in methacrylate-embedded sections; the resin was pretreated with ion-exchange resin (Amberlite IRA-410). Experiments showed that pretreatment reduce the background fluorescence practically to zero. Seventy 3 m-thick serial sections were prepared on fluorescence free glass slides and stained with azocarmin G and acriflavine-Feulgen. After postirradiation using blue excitation light, the amount of Feulgen-DNA present in the target nucleus in each section was determined using a microfluorometer. The amount of DNA in the entire nucleus was determined by adding together the DNA contnet of the segments of the nucleus. The characteristic appearance of the organ of Corti made it easy to detect these cells; under green excitation light the cells of this organ exhibited red cytoplasmic azocarmin-G fluorescence. Due to the relatively wide internuclear spaces, cytofluorometry of individual nuclei could be performed without interference from the neighboring cells. Our technique using serial sections allowed us to measure the DNA contnet of individual cells and obtain histological information about particular cells and their neighboring cells. Several polyploid cells were found among the Hensen's cells in the cochlea, while all other component cells of the organ of Corti were diploid.     

Mitoses and binucleated cells in perinatal human hearts

Cells from hardened formalin-fixed human hearts were isolated with potash lye. In perinatal hearts mitoses and evidence of irregular chromosomal movement such as chromatin bridges and micronuclei were observed in small numbers during normal development. A quantitative analysis of binucleated cells shows 8.8 +/- 5.3% in the left and 11.7 +/- 4.0% in the right ventricular wall around the time of birth. The number increases during the first year of life to 56.8 +/- 17.4% in the left and 42.0 +/- 18.1% in the right heart. This development precedes normal polyploidisation of the human heart by years.     

DNA and nuclear protein measurement in columnar epithelial cells of human endometrium

Propidium iodide DNA flow cytometry, Feulgen-DNA, and nuclear light green protein scanning cytometry were performed in columnar epithelial cells of normal, nonmalignant human endometrium and endometrial adenocarcinomas. Columnar cells were identified by immunohistochemical staining for cytokeratin 18, an intermediate filament protein specifically present in columnar cell epithelium. DNA measurements derived from flow and scanning cytometry showed comparable results. The DNA content of the G0/G1 fraction of the adenocarcinomas had a considerable overlap with that of normal endometrium, with that of the carcinomas shifted toward higher values. For the carcinomas, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas. Most of the clinical stage I tumors showed a DNA content in the normal diploid region. Three of the four carcinomas of clinical stage II and higher had an increased DNA content. For the carcinomas, the percentage of cells in the proliferative fraction, as determined from scanning cytometric derived DNA histograms, was comparable to that of normal endometrium, or higher. No correlation was found with the histological grade. Tumors of clinical stage II and higher had intermediate values compared to carcinomas of lower stages. The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. Within the tumor population, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas, and clinical stage. Based on the aforementioned parameters, no discrimination could be obtained between normal and malignant endometrium. However, when the DNA content of the G0/G1 fraction was combined with the coefficient of variation of the nuclear protein/DNA ratio, a clear discrimination could be obtained with only two false-positive cases.     

Mechanism of polyuria and natriuresis in atrioventricular nodal tachycardia.

A woman with tachycardia associated with polyuria was investigated. Electrophysiological analysis showed that the tachycardia was an atrioventricular nodal re-entrant tachycardia. Programmed stimulation was then used to provoke and sustain the tachycardia for 40 minutes. Polyuria, with an appreciable increase in free water clearance, was observed. This was associated with reduction in plasma and urinary arginine vasopressin concentrations. Appreciable natriuresis also developed. These results support the hypothesis that the polyuria with increased free water clearance and the natriuresis occurring during sustained tachycardia in man are due to inhibition of secretion of vasopressin and the release of natriuretic factor.     

Cytofluorometric DNA base determination in vertebrate species with different genome sizes

The base composition of DNA was studied in 15 amphibian species and 28 reptile species by means of DAPI, a fluorochrome specific for adenine-thymine rich DNA (AT-rich DNA). The results obtained in reptiles and anuran amphibians coincided with biochemical data available for some species. In urodeles, on the contrary, the findings contrasted with biochemical data and suggest that DAPI is unable to stain all the AT-rich DNA in the erythrocytes of these organisms. It is concluded that the method is suitable for studying species with a small genome size, such as reptiles and anuran amphibians, but is not suitable for nuclei with a large genome size and a highly compact chromatin, such as urodele erythrocytes.     

Cytofluorometric determination of protein-bound thiols and DNA in cell nuclei

The aim of the present study was to establish a cytofluorometric method for the simultaneous determination of protein-bound sulfhydryl-groups (PSH) and DNA in isolated cell nuclei. DNA was stained with ethidiumbromide and PSH with N-iodoacetyl-N(5-sulfo-1-naphthyl) ethylendiamine (AEDANS). Disulfide groups of nuclear proteins were determined by the same method after reduction with sodium borohydride or thioglycollic acid. The method was established by using nuclei of human lymphocytes, which then served as a biological standard for further investigations of the nuclei of different mammalian cell types: nuclei from mouse liver cells and nuclei from the cells of two human melanoma cell lines. For non-proliferating lymphocytes distinct DNA- and PSH-values could be measured. The PSH-values detected in the nuclei of the other cell types were higher by comparison and varied within the cell cycle; i.e., PSH increased during the S-phase and was almost doubled during the cell generation cycle from G1- to G2-phase. Cell line and cell cycle-dependent variations of nuclear disulfides could also be detected. These results are discussed with respect to their radiobiological implications. In conclusion, thiol groups may represent one factor determining the radiosensitivity of cells, but they are not the only decisive one.