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Patterns of oxygen consumption during establishment of cephalocaudal polarity in the early chick embryo.

Oxygen uptake was studied during the establishment of cephalocaudal polarity in the very early chick embryo, i.e., 10 hr before (stage VI) and at laying (stage X). Oxygen fluxes in minute regions of the intact blastoderms were measured in vitro by scanning microspectrophotometry in the presence or absence of glucose. The oxygen consumption of the whole blastoderm remained constant (6 nmol O2 X hr-1) throughout the period studied, although the number of cells increased more than twofold. The regional oxygen fluxes varied from 0.41 to 1.13 nmol O2 X hr-1 X mm-2 at stage VI and from 0.42 to 0.70 nmol O2 X hr-1 X mm-2 at stage X. At stage VI, the oxygen flux in the center of the blastoderm was significantly higher than that in its periphery. This pattern remained evident when the values were corrected for cell number or for cytoplasmic volume. At stage X, there was a tendency for the oxygen fluxes to decrease from the posterior to the anterior regions of the area pellucida. Thus the pattern of oxidative metabolism in the late uterine embryos seems to change from radial to bilateral. This change of symmetry probably reflects the process of formation of the embryonic axis. In addition, the fact that the oxygen uptake was similar in the presence or absence of glucose suggests that early chick embryos metabolize essentially intracellular stores.

Analysis of Variance↗

Comparison between slide and flow cytophotometric DNA measurements in breast tumors.

Nuclear DNA analysis was performed in 37 human mammary adenocarcinomas in order to elucidate the difficulties and pitfalls connected with the interpretation of DNA histograms obtained using different methodologic approaches. For each tumor, DNA profiles were obtained by means of slide microspectrophotometry on a fine needle aspirate, slide cytophotometry on a 4-micron histologic section and flow cytometry on a suspension prepared from a cube of fresh tissue. When the DNA histograms were interpreted according to criteria usually applied to discriminate low-grade malignant tumors from high-grade malignant tumors, some tumors classified as euploid by one method were classified as aneuploid by another method. The main reasons for this weak correlation seem to be in specimen preparation and in tumor cell representation within the specimen between the methods. Another reason is that slide and flow techniques exhibit different sensitivities for malignancy-associated nuclear DNA changes: minor alterations of the DNA content of the tumor stemlines seem to be more exactly reported by means of the flow technique whereas structural alterations of the nuclear chromatin seem to be more sensitively recorded by means of the slide technique. It is suggested that thorough control of each step of the various DNA analysis procedures and the use of information obtainable by slide and flow techniques taken together may significantly improve the prognostic value of DNA measurements.

Adenocarcinoma↗

Frequency and distribution of estrogen receptor-positive cells in normal, nonlactating human breast tissue.

We have analyzed the frequency and distribution of cells expressing estrogen receptor (ER) in cryosections of normal human breast tissue using quantitative microspectrophotometry and the estrogen receptor immunocytochemical assay. We found that the human mammary gland contained a small but distinct population of ER-positive cells, comprising approximately 7% of the total epithelial cell population from all biopsies. Stromal cells were found to be ER negative. The ER-positive cells were distributed as scattered single cells, with the highest frequency and intensity of measured staining in the lobules as compared to the interlobular ducts. Moreover, on the average, 87% of the ER-positive cells were luminal epithelial cells or occupied an intermediate position in the duct wall. The intermediate cells were found not to express basal cell phenotype as determined by combined immunocytochemistry to ER and "common acute lymphoblastic leukemia antigen" selectively decorating myoepithelial cells (B.A. Gusterson et al., J. Natl. Cancer Inst., 77: 343-349, 1986).

Adolescent↗

Coronary adjustments to graded hypotension in rabbits.

The purpose of this study was to determine how the myocardium can compensate (change O2 supply/consumption parameters) regionally during acute hemorrhagic hypotension. Twenty-one open chest, anesthetized rabbits were subjected to one of three treatments: (1) control (nonhemorrhaged), (2) hemorrhage to 70 mm Hg, (3) hemorrhage to 50 mm Hg. Myocardial blood flows were determined by using radioactive microspheres before and during hemorrhage. At the end of the experiment the hearts were removed and stored in liquid N2 and analyzed for subepicardial and subendocardial arterial and venous O2 saturation by using microspectrophotometry, and from this, O2 consumption was determined. During hemorrhage, blood pressures were significantly depressed for all groups compared to the control group. Myocardial blood flow was depressed 20% in the 70 mm Hg group and coronary resistance fell 15% compared to control. In the 50 mm Hg group, flow was reduced 38% and resistance decreased 31% compared to the 70 mm Hg group. No regional (subepicardial-subendocardial) differences in flow or resistance occurred during hypotension. These decreases in resistance were observed despite a 32% drop in O2 consumption in the 70 mm Hg group and 44% in the 50 mm Hg group compared to the control. O2 extraction remained unchanged for all groups as did the O2 supply/consumption balance. Thus, despite decreases in O2 consumption during acute hemorrhage, rabbit hearts decreased coronary resistance so as to maintain the O2 regional supply/consumption balance constant. Since O2 extraction did not change, these hearts were not ischemic, even in the subendocardium.

Animals↗

[Microspectrophotometric research on the rhodopsin and carotenoid content of the retinal visual cells of hens maintained on a vitamin A-deficient diet].

Using microspectrophotometry, a study was made of the retina of adult hens, that had been kept on a diet for 6.5 months with deficiency of vitamin A. It has been shown that despite an expressed drop of quantity of vitamin A in the liver, in the external segments of the retina rods in A-avitaminosis hens, the concentration of rhodopsin remains normal.

Animals↗

[The validity of measuring methods for intranuclear DNA on thin sections. Theoretical and experimental study].

Computer modelling is used to simulate nuclear segments obtained by random sectioning through tissue. A few more computations lead to DNA measurement simulation. Simulation results in negatively skewed DNA frequency distributions. Both the right skewness and the coefficient of variation of measurements are increasing with the ploidy level because nuclear DNA content is assumed to be related to nuclear size in the chosen model. Observed mean values are biased underestimates of expected values but are strongly correlated to the degree of ploidy. Finally, the bias introduced by measuring nuclear segments instead of whole nuclei increases the variance of measurements but contributes to less than half the experimentally observed variance. Our conclusion is that microspectrophotometry on tissue sections is a valuable method for DNA content evaluation of small clusters of pathological cells as one may find in endoscopic biopsies.

Cell Nucleus↗

Nuclear deoxyribonucleic acid heterogeneity of ovarian borderline malignant serous tumors.

Sixteen borderline malignant serous ovarian tumors and seven well-differentiated invasive serous ovarian carcinomas were examined with the technique of Feulgen microspectrophotometry for the determination of nuclear deoxyribonucleic acid (DNA) ploidy patterns (diploid versus aneuploid) and ploidy levels of the stem cell lines. Of the nine stage I-II borderline malignant tumors, only one (11%) was aneuploid. In contrast, four of seven (57%) stage III borderline malignant neoplasms and all stage III carcinomas were aneuploid. The stem cell modal values in all borderline serous tumors were less than triploid (3N) while in five of seven carcinomas stem cell modal values were greater than triploidy. This contrast in ploidy patterns and ploidy levels may explain the differences in biologic behavior between borderline malignant serous tumors and invasive serous carcinomas of the ovary.

Adult↗

The retinal pigment epithelium of wild type (C57BL/6J +/+) and pearl mutant (C57BL/6J pe/pe) mice.

The retinal pigment epithelium (RPE) lying along the vertical meridian of the eyes of wild-type (+/+) and pearl mutant (pe/pe) mice was examined with electron microscopy and microspectrophotometry in order to compare differences in melanosome location, size, numerical density, volume density, and melanin concentration. In +/+ mice the fraction of melanosomes that lie in the apical processes was greater in the superior than in the inferior retina. Also, the numerical density and volume density of soma melanosomes were lower in the superior retina. The soma melanosomes were also located closer to the basal membrane and had a larger average diameter in the inferior retina. No significant differences in melanosome location were observed between light-adapted and dark-adapted retinas. In pe/pe mice the fraction of melanosomes in the apical processes was very low in both inferior and superior retinas. The numerical density and the volume density of soma melanosomes of pe/pe were significantly less than those of +/+. The soma melanosomes of pe/pe mice lie closer to the basal membrane than those of +/+, and they had a larger mean diameter than those of +/+. Electron micrographs depicted some melanosomes of pe/pe with irregular profiles and abnormal melanin deposition at their periphery. Microspectrophotometric measurements of individual melanosomes in semithin sections showed that maximal specific absorption and lambda max were nearly equal for both genotypes and both superior/inferior locations. These findings show that intramelanosomal melanin concentrations is normal for pe/pe. The basal membrane of the RPE of pe/pe was infolded over only 80% of the area covered by the basal surface, whereas the basal membrane in +/+ was infolded over more than 98% of the area covered. The boundary density of RPE basal infoldings was also lower in pe/pe than in +/+. Furthermore, some areas of the basal lamina of pe/pe RPE had a periodic, rather than an amorphous, structure. These findings suggest that the reduced retinal sensitivity found in intact pe/pe animals, but not in superfused, isolated pe/pe retinas, may result from impaired transport of a diffusible substance by the basal membrane of the RPE.

Animals↗

[Micro-quantitative determination of alpha1-antitrypsin by unidimensional capillary counter-immunodiffusion].

The immunoprecipitation reaction for the determination of microamounts of alpha1-antitrypsin was conducted in the capillaries filled with a microvolume of a 1% agarose gel (the gel length was 7--8 mm, and the diameter--0.6 mm). Microamounts of the antigen and antiserum were applied to the surface of the opposite ends of the gel. Precision control of these manipulations was carried out by means of an ocular micrometer of a binocular lens. The amount of the formed immunoprecipitate in gel was calculated by the summated optic density after staining with amido black recorded by the method of double-wave microspectrophotometry. Sensitivity of the method was 0.3--1.0 ng of alpha1-antitrypsin. The linear plot between the amount of alpha1-antitrypsin and the amount of the precipitate formed was found within the range of 1--25 ng of protein. The error was about 10%.

Immunodiffusion↗

Partial trisomy for the distal long arm of chromosome 5 (region q34 leads to qter). A new clinically recognizable syndrome.

This report describes a family in which eight individuals in three generations had mental retardation in association with a characteristic pattern of clinical problems and physical abnormalities including short stature, eczema, hernias, delayed puberty, dysmorphic facies and digital anomalies. The family history was consistent with a chromosomal rearrangement with transmission through balanced carriers. Routine ASG banding studies showed extra chromosomal material on a chromosome 16 but failed to demonstrate any differences between the affected individuals and the presumed carriers. However, subsequent studies utilizing trypsin banding and microspectrophotometry of individual chromosomes demonstrated that the affected individuals were partially trisomic for the distal band of the long arm of chromosome 5 and that 0.273 units of a chromosome 5 were translocated to chromosome 16. This definitive cytogenetic diagnosis permitted accurate prenatal diagnosis to be carried out on the fetus of a balanced carrier female. The application of these techniques to previously obscure familial dysmorphic syndromes is recommended.

Abnormalities, Multiple↗

Feulgen-DNA-cytophotometry on mammary tumor cells from aspiration biopsy smears.

The nuclear DNA content of cells from 20 cases of mammary aspirates was estimated by microspectrophotometry. Smears stained according to Papanicolaou were destained and Feulgen-reacted. Among the smears there were 14 with doubtless tumor cells, four with a benign cell picture, and two of a doubtful nature. The mammary lesions were in all cases verfied by histology. Measurements showed that smears of benign tumors had a diploid or diploid to tetraploid distribution of DNA. The two doubtful smears also showed the same DNA pattern, although in one case the cells were from a papillary carcinoma and in the second case from a fibroadenoma. Malignomas with a cell picture, monomorphous-atypical in Papanicolaou staining exhibited a DNA histogram with a peak in the diploid to hyperdiploid ranges. Carcinomas showing a polymorphous-atypical cell picture were characterized either by polyploid values, by a large spread, or by aneuploidy. Hence, it is impossible to safely distinguish benign cases from carcinomas by the DNA pattern, since DNA distribution is fairly identical in part of the cases in either group.

Adenofibroma↗

[Comparative evaluation of dysplasia and early cancer of the stomach based on analysis of DNA content].

DNA content in the tumour cell nuclei was studied in 16 cases of early stomach carcinoma and in the adjacent areas of the foveolar epithelium dysplasia and metaplastic intestinal epithelium. One wave microspectrophotometry was used; the slides were stained with gallocyanin-chrome alum. The number of DNA synthesizing diploid cells was increasing with the increase of the dysplasia degree. The number of tetraploid cells was higher and the signs of aneuploidy appeared in the areas of noninvasive carcinoma. The stem line in early invasive carcinoma was represented by the diploid cells. The increase of cell atypia in tubular and papillary adenocarcinoma is followed by increase of the tetraploid cell number and by the appearance of polyploidy and aneuploidy. Both undifferentiated and goblet-cell carcinomas have similar parameters of the DNA histogram. DNA content allows an objective judgement on the degree of an early carcinoma cell atypia and differentiation of the advanced dysplasia and noninvasive carcinoma.

Adenocarcinoma↗

Presynaptic calcium diffusion and the time courses of transmitter release and synaptic facilitation at the squid giant synapse.

At the squid giant synapse, a presynaptic action potential is accompanied by an influx of calcium ions that continue to be detectable with arsenazo III microspectrophotometry for several seconds. Nevertheless, transmitter release occurs phasically, lasting only about 2 msec. If a second action potential follows within about 100 msec after the first, it releases more transmitter. In this paper, we present a mathematical model of intracellular calcium diffusion with binding to fixed cytoplasmic sites, active extrusion at the surface, and influx during an action potential, to predict the distribution of intracellular calcium following an action potential. With a square law relation between submembrane calcium and transmitter release, the model predicts the phasic release of transmitter and the magnitude and time course of synaptic facilitation following an action potential, as well as the relatively long persistence of free intracellular calcium.

Action Potentials↗

Peroxisomes (microbodies) in human liver: cytochemical and quantitative studies of 85 biopsies.

The number, intracellular distribution, and staining characteristics of human hepatocellular peroxisomes that had been made visible by cytochemical staining for catalase were evaluated in biopsies from 75 patients with hepatic, inflammatory, or malignant disease and ten normal individuals. Intensity of staining was found to be proportional to enzymatic activity by microspectrophotometry. Scanning transmission electron microscopy (STEM) image analysis demonstrated an inverse relationship between peroxisomal size and contrast. Peroxisomes were more abundant, and often concentrated in a perinuclear configuration in cholestatic and cirrhotic livers. Decreased peroxisomal staining was common in cholestasis, cirrhosis, hepatitis, and in almost all patients with malignancies, both with and without hepatic metastases.

Biopsy↗

Complex formation and intermolecular electron transfer between flavocytochrome b2 in the crystal and cytochrome c.

The present study addresses the question whether tetrameric flavocytochrome b2 in the crystal is catalytically competent and, if so, whether it is possible to prepare a functional complex of the crystalline enzyme with its physiological electron acceptor cytochrome c. By single crystal microspectrophotometry we show that the native reduced enzyme can be oxidized by oxygen or ferricyanide and that the oxidized enzyme can be reduced by the electron donor L-lactate. Reduced cytochrome c appears to diffuse through the liquid channels of flavocytochrome b2 crystals and, at low ionic strength, to accumulate in amounts stoichiometrically equivalent to the enzyme protomers. Both cytochromes can be oxidized by ferricyanide. In the presence of L-lactate, both cytochromes become reduced. Since reduction of cytochrome c by L-lactate requires the catalytic action of flavocytochrome b2, it is concluded that the structure of the crystalline enzyme not only allows for electron transfer from L-lactate to flavin and intramolecular electron transfer from flavin to heme, but also for the formation of a productive complex with cytochrome c.

Crystallization↗

Human papillomavirus infection and cervical intraepithelial neoplasia: histopathology and DNA content.

To evaluate the reliability of diagnostic criteria for separating intraepithelial squamous lesions into low- and high-risk categories, 25 lesions of the cervix were diagnosed as flat condyloma, atypical immature metaplasia, or cervical intra-epithelial neoplasia with koilocytosis based on well-defined histologic criteria. The presumption was that flat condyloma and atypical immature metaplasia would be diploid/polyploid as compared to low-grade cervical intraepithelial neoplasia, which should be aneuploid. Using the major histologic parameter of the presence or absence of abnormal mitoses to distinguish the low- and high-risk lesions, it was found that all of five typical flat condylomas were diploid/polyploid and seven of eight atypical immature metaplastic lesions were diploid/polyploid; 11 of 13 cervical intraepithelial neoplasms with koilocytosis, however, were aneuploid. An additional histologic parameter of anisocytosis (variation in nuclear size) appeared much less reliable for segregating these lesions than the nature of the mitoses. Lesions for which ploidy values were particularly difficult to predict were extremely well-differentiated koilocytotic lesions with occasional abnormal mitoses. Whether these are true polyploid lesions in which the abnormal mitoses are a response to the virus, or whether they are very early aneuploid lesions that cannot be confirmed by microspectrophotometry, remains to be determined.

Aneuploidy↗

[Cytologic study of glandular tumors in maxillofacial regions-- diagnostic application of nuclear DNA content].

We studied whether the DNA contents may be useful in the differential diagnosis of glandular tumors in the maxillofacial regions. In 25 of these tumors, the DNA contents were measured by microspectrophotometry, using 4 normal salivary glands as controls. We found that: (1) DNA histgram patterns were useful in differential diagnosis only in special cases. (2) The criteria were based on our decision and these tumors were classified into 3 types (a) Benign, (b) Low-grade malignant, (c) High-grade malignant. Our classification seemed to offer an objective means for differentiating between Benign & Malignant tumors of these types.

Adenocarcinoma↗

Fixation, decalcification, and tissue processing effects on articular cartilage proteoglycans.

Neutral buffered 4% formaldehyde fixation for 48 h preserved well the proteoglycan content of bovine articular cartilage. Neither subsequent demineralization in 10% EDTA, nor light microscopic tissue processing, reduced the hexosamine or uronic acid content of the tissue. Fixation in alcoholic solutions increased Safranin O binding as well as periodic acid Schiff reaction of the cartilage matrix as measured by microspectrophotometry. It is suggested that the enhanced staining of cartilage was due to better preservation of the glycoprotein oligosaccharides. Quaternary ammonium salts in the fixative suppressed the staining of cartilage matrix with Safranin O.

Animals↗