Microspectrophotometric Studies Research Articles

Microspectrophotometric studies of the effect of 60Co irradiation on DNA synthesis in germ cells of the face fly

Cytological and histochemical analyses of spermatogenesis in the face fly, Musca autumnalis DeGeer, were conducted following cobalt irradiation of pharate adults 96 hr after the onset of puparium formation. Three dose levels (600, 2000, and 4000 rad.) were employed and gonads analysed at four time intervals (1, 2, 5, and 10 days) post-irradiation using phase contrast microscopy, light microscopy after aceto-orcein staining, and cytophotometry following Feulgen staining. The 2000 and 4000 rad. doses resulted in a progressive decrease in the number of secondary spermatogonia and primary spermatocytes undergoing premitotic or premeiotic DNA synthesis, a decrease in the overall number of primary spermatocytes in the testes, and a near absence of dividing cells. Following the 600 rad. treatment, there was a slight increase in the number of cells exhibiting elevated Feulgen-DNA levels (i.e. undergoing DNA synthesis) 2 days post-irradiation, an increase in the number of primary spermatocytes 2 and 3 days post-irradiation, and an initial 1-day decrease in the number of dividing nuclei followed by a 2- to 3-day mitotic increase over control levels. The combined data indicate that at the higher dose levels (2000–4000 rad.) there is an inhibition of DNA synthesis resulting in an interruption of the spermatogenic process. After the 600 rad. exposure, there is no observable decrease in DNA synthesis, but there is an apparent retardation in the rate of spermatogenesis. These results support the concept that differential radiosensitivity of germ cells is influenced, in part, by differences in radiation induced inhibition of DNA synthesis.

Microspectrophotometric and electron microscopic studies of bone marrow in hereditary sideroblastic anaemia.

Ultrastructural studies in 3 patients with hereditary sideroblastic anaemia have shown iron-laden mitochondria in a proportion of the non-dividing, late polychromatic erythroblasts. In contrast to the situation in primary acquired sideroblastic anaemia, the mitochondria of the proliferating erythroblasts were rarely infiltrated with iron; and a combined quantitative cytochemical and autoradiographic study showed no significant arrest of cell proliferation. It is likely that impaired haemoglobin synthesis is more important than ineffective erythropoiesis in the pathogenesis of theanaemia in this disease

DNA synthesis in polyploid and binucleate hepatic cells in the regenerating rat liver of different ages.

In many vertebrate species, polyploid and binucleate cells are found in the adult hepatic parenchyma. The number of polyploid and binucleate hepatic cells increases with age. In this paper, the proliferative capacity of polyploid and binucleate cells were studied, in order to analyse a progressive reduction with age in the regenerative capacity of tissues. Three groups of rats, 4-week, 6-week and 15-week-old, were used in this experiment. The animals were partiall y hepatectomized, received an injection of triti ated-thymidine at 18 hours after the operation, and were sacrificed 30 minutes later. The livers were then subjected to the combined autorad iograph ic and microspectrophotometric studies. The labeling index was calculated for each hepatic cell type, which was classified according to nuclear polyploidy and binuclearit y. Spatial distribu tion of polyploid cells in the liver lobule and that of hepatic cells labeled with tritiated-th ymidine were also studied, and were taken into consideration to interpret values of the labeling index. The results of these studies indicate that value of the labelin g index of polyploid hepatic cells is genera lly smaller than that of diploid cells, and tha t value of the labeling index of binucleate cells is also smaller than that of mononucleat e cells. These results strongly suggest that the proliferative capacity of hepatic cells is reduced with polyploidization.

Spectral analysis of L-typeS-potentials and their relation to photopigment absorption in a fish ( Eugerres plumieri) retina

The spectral sensitivities of the exclusively hyperpolarizing ( L-type)S-potentials were studied in a fish ( Eugerres plumieri) retina. One sensitivity curve with a maximum at 498 nm was found under strictly scotopic conditions, and a second curve with a peak at 606 nm was also obtained in the dark adapted retina when stimuli of higher intensities were used. In the light adapied retina, three different sensitivity curves were found in different cells, with peaks at 476, 568 and 606 nm. Microspectrophotometric studies were performed on isolated cones from the same fish retina Three different types were found, with maximal absorptions at 471, 568 and 604 nm. The correspondence between photoreceptor absorptions and spectral sensitivities ofL-potentials show that the latter, which are recorded from horizontal cells, receive information from the different receptors independently. Mixing of signals from different receptors does not take place at or before the locus of recording theL-potentials.

Quantitative interference microscopy of polytene chromosomes II Cytochemical studies via ultramicrospectrophotometry

Quantitative microspectrophotometric studies were performed on third instar salivary gland chromosomes and nucleoli of Drosophila melanogaster. These data correlated well with interference microscopy data. In general, the relative RNA concentration was several times greater in the nucleoli than in the bands, with no RNA detected in the interbands. DNA was present in both bands and interbands; the distribution ratio of the regions was approx. 20 to 1, respectively. RNA was present in variable degrees in certain bands. Protein staining methods revealed that arginine staining was confined mainly to the histone fraction in the bands, while SH staining was highest in the residual protein of the interbands. The high levels of non-histone proteins in the interband regions were probably not directly associated with the nucleohistone component. The distribution of DNA did not correlate proportionally with the protein distribution. It was evident, therefore, that simple coiling of the chromonemata could not entirely explain the distribution of nucleic acids and protein. A method of automated autoradiograph grain counting, characterized by its extreme accuracy and rapidity, was applied to in vivo 3H-thymidine labeled third instar glands. A lessening of the rate of incorporation with increasing ploidy was observed.

Visual Pigment Density in Single Primate Foveal Cones

The feasibility is demonstrated of microspectrophotometric studies on primate photoreceptors aligned at right angles to the test beam, rather than axially illuminated. Pigment densities, and hence absorption per unit thickness, are approximately equal in primate rods and foveal cones. These pigment densities are similar to those reported for frog rods and fish cones.

Variation of myocardial nucleolar abundance with heart weight.

SummaryThe number of nucleoli in myocardial nuclei was determined for human hearts in the weight range of 300-640 g. The variation of nucleolar abundance was significant, but slight, with larger hearts tending to show more nucleoli per nucleus. The maximum number of nucleoli per nucleus was six, regardless of the heart weight. It is concluded that even if the amount of DNA per nucleus increases during the development of myocardial hypertrophy, as suggested by microspectrophotometric studies, the number of potential nucleolar organizers remains fixed at six per nucleus.

THE RELATIONSHIP OF THE "SPHERE CHROMATOPHILE" TO THE FATE OF DISPLACED HISTONES FOLLOWING HISTONE TRANSITION IN RAT SPERMIOGENESIS

Cytochemical, radioautographic, and microspectrophotometric studies bearing on the relationship of histone transition to the origin and development of the protein and RNA components of the "sphère chromatophile" in the developing spermatogenic cells of the albino rat are presented. These studies show that the sphère chromatophile has many features in common with somatic nuclei: it contains histonelike basic proteins rich in lysine, with lesser amounts of arginine. No evidence is found for the presence of a protamine in this granule. The sphère chromatophile is rich in RNA, but contains no DNA. The failure of a positive reaction with basic protein stains, unless the RNA is first removed, indicates either a chemical bonding or a very close association between the RNA and basic protein. The basic protein and RNA components of the sphère chromatophile appear to have different origins in the cell. A sequence of stages in the development of the lysine-rich basic protein component of this structure commences with the appearance of tiny grains in those spermatid nuclei which are beginning to replace lysine-rich histones with arginine-rich histones. Subsequently, similar-staining cytoplasmic grains appear, which coalesce to form the sphère chromatophile in the cytoplasm. Labeling studies show that the basic protein component is synthesized at about the time of the last premeiotic DNA (and histone) synthesis. The results of the microspectrophotometric measurements support the idea that the basic protein lost from the spermatid nucleus is the source of the basic protein in the sphère chromatophile.

Cytochemical studies on cytoplasmic RNA-associated basic proteins in oocytes, somatic cells, and ribosomes.

Cytochemical studies on oocytes from representatives of several animal groups, of somatic tissues known to possess high levels of cytoplasmic RNA, and of isolated ribosomes indicated that basic proteins associated with the ribosomes may be stained by the methods currently in use for demonstrating histones. These proteins were resistent to weak acid extraction in fixed material and were labile to acetylation, but pretreatment with hot 5% TCA caused a measurable increase in staining with a modified Sakaguchi reaction. Such proteins were not confined to oocytes, but could be demonstrated with the TCA-alkaline fast green method in mouse pancreas and liver cells, and by the ammoniacal-silver method in all cells with high levels of cytoplasmic RNA. Microspectrophotometric studies onAscidia nigra andClavelina picta oocytes indicated that the highest concentration of cytoplasmic basic proteins was found in smaller oocytes, but the concentration of alkaline fast green stainable cytoplasmic RNA-associated basic proteins decreased in a pattern that differed significantly from that of stainable levels of cytoplasmic RNA. The implications of these findings to current concepts in developmental biology were discussed.

Microspectrophotometric Studies of Individual Erythrocytes from Thalassemia Patients.: ,I. A Case of Double Heterozygosity for, β,-Thalassemia and Panglobinopenia ?,

Abstract Microspectrophotometric hemoglobin determinations on individual red cells from the peripheral circulation of a patient clinically characterized as a case of thalassemia major show the presence of two main cell populations, one normochromic and one hypochromic, with a ratio of total hemoglobin per cell of 2 to 1, a subnormal β-chain production in all cells, at mean levels that are in a ratio of 4 to 1 in the two cell populations, and a strongly stimulated γ-chain production in both cell populations with no bimodality of distribution. The mean cellular content of δ-chain is augmented. The ratios of 2 to 1 for total hemoglobin production is explained on the basis of the hypothesis that the case is one of heterozygosity for a β-thalassemia gene and a panglobinopenia gene with a mosaic condition realized at least in the case of the panglobinopenia gene. "Panglobinopenia" is described as a condition leading to a general inhibition of hemoglobin synthesis, perhaps through an inhibition of heme synthesis. To account for the observed cellular mosaic, it is proposed that panglobinopenia and perhaps thalassemia are examples of the well known phenomenon of variegation. This interpretation is now to be tested by applying the same methods to the study of further cases of thalassemia patients.

Quantitative cytochemical studies on Paramecium, : V. Autoradiographic studies of nucleic acid syntheses

1. 1. Tritiated thymidine is incorporated into the macronuclear DNA of Paramecium aurelia throughout the second part of the interdivision interval but there is little if any incorporation during the first part. These observations are in good agreement with the previous studies with Feulgen microspectrophotometry. 2. 2. Tritiated cytidine and uridine are incorporated most rapidly into macronuclear RNA and only later appear in cytoplasmic RNA. On incubation in culture medium, macronuclear label decreases while cytoplasmic label increases. 3. 3. Both cytidine and uridine are incorporated into macronuclear RNA at nearly the same rate per unit volume throughout the interdivision interval. This contrasts with the earlier microspectrophotometric studies that showed no net increase in macronuclear RNA during the first part of the interval. 4. 4. These findings suggest that RNA is formed in the nucleus and moves into the cytoplasm as rapidly as it is formed. 5. 5. The relations suggest that the rate of synthesis of RNA may be proportional to the amount of DNA. The possible relations between the rates of synthesis of the nucleic acids and the rate of over-all growth of the cell are considered.

Effect of X-irradiation on the DNA content of individual nuclei of rabbit bone marrow

1. 1. Feulgen microspectrophotometric studies of DNA were conducted on individual nuclei of non-irradiated and irradiated femoral marrows of rabbits exposed to 800 r hemi-body X-irradiation. 2. 2. The major portion of the measured marrow nuclei from the non-irradiated marrow cells are in the range between 6 and 12 × 10 −9 mg of DNA per nucleus. These cells have been designated as synthesizing cells. 3. 3. The main consequence of X-irradiation to the exposed femurs has been to reduce the number of synthesizing cells. Studies conducted at 24 and 48 hours post-irradiation reveal a consistent decrease in the number of synthesizing cells. At intervals of X-irradiation less than 24 hours, the results were variable although indicative of a decrease in synthesizing cells. 4. 4. Studies on comparable nuclear sizes of X-rayed and non-X-rayed femoral marrow reveal a reduction of synthesizing cells in the larger nuclei (2.7–3.5 + arbitrary units). In the case of nuclear size ranges below 2.7 and erythroblasts (nuclear size range 2.0–2.7 arbitrary units) there are no significant effects of X-irradiation.

Autoradiographic and microspectrophotometric studies of DNA synthesis in excised tobacco pith tissue.

Pith tissue was cultured on modified White’s nutrient medium supplemented, except for controls, with 2 mg/l of IAA and/or 0,5 mg/l of kinetin. For autoradiographs sections were used from tissue grown on medium containing tritiated thymidine.

The desoxyribosenucleic acid (DNA) content in spermatozoa of fertile and infertile human males.

Microspectrophotometric studies were made on the amounts of DNA in individual spermatozoa of 21 human males with proven fertility and on 18 human males who are partners in sterile couples. The results were as follows: