Quantitative studies of nucleic acid in the cell by micro-spectrophotometry I. The critique and improvement of the microspectrophotometry

Some critical experiments have been carried out on the microspectrophotometry using the lymphocytes of a mouse, stained with Feulgen reaction, revealing that most reliable value can be attained by illuminating the material with a small spot-light and integrating the area surrounded by the extinction curve drawn by tracing along the diameter of the smeared and fixed cell.</p

Studies on the relation between heme and nucleic acid syntheses in erythroid cell. II. Nucleic acid synthesis in erythroblast of anemic rat treated with aminopterin and bromouracil

With the bone marrow of anemic rats, which had received the repeated injections of phenylhydrazine once a day for three to four days, the effects of aminopterin and bromouracil on the nucleic acid metabolism of erythroblasts were observed in vivo experiment. The injection of aminopterin suppressed DNA synthesis with the lowered labeling index as observed by the incorporation of &#179;H-thymidine into DNA in vitro. But the grain count per cell showed the level similar to that of anemic control. RNA synthesis was not interfered by AP injections. These results indicate that AP mainly suppresses the thymidilate kinase. Bromouracil showed no such effect even on the administration of a large dose. On the basis of the data obtained from the experiment by using AP, a discussion was made on the correlation between DNA synthesis, nuclear function and the cell specialization.</p

The retina visual cycle is driven by cis retinol oxidation in the outer segments of cones

Vertebrate rod and cone photoreceptors require continuous supply of chromophore for regenerating their visual pigments after photoactivation. Cones, which mediate our daytime vision, demand a particularly rapid supply of 11-cis retinal chromophore in order to maintain their function in bright light. An important contribution to this process is thought to be the chromophore precursor 11-cis retinol, which is supplied to cones from Müller cells in the retina and subsequently oxidized to 11-cis retinal as part of the retina visual cycle. However, the molecular identity of the cis retinol oxidase in cones remains unclear. Here, as a first step in characterizing this enzymatic reaction, we sought to determine the subcellular localization of this activity in salamander red cones. We found that the onset of dark adaptation of isolated salamander red cones was substantially faster when exposing directly their outer vs. their inner segment to 9-cis retinol, an analogue of 11-cis retinol. In contrast, this difference was not observed when treating the outer vs. inner segment with 9-cis retinal, a chromophore analogue which can directly support pigment regeneration. These results suggest, surprisingly, that the cis-retinol oxidation occurs in the outer segments of cone photoreceptors. Confirming this notion, pigment regeneration with exogenously added 9-cis retinol was directly observed in the truncated outer segments of cones, but not in rods. We conclude that the enzymatic machinery required for the oxidation of recycled cis retinol as part of the retina visual cycle is present in the outer segments of cones

Quantitative studies of nucleic acid in the cell by microspectrophotometry III. Nucleic acid contents in the cancer cells

1. The DNA contents in mature lymphocytes of the mouse, rat and man are kept almost constant. 2. The variety in the DNA contents in tumor cells is attributed to the rapid DNA synthesis taking place at the interphase, though the degenerating cells and the cells in abnormal mitosis can not be discarded as the source of the variety in DNA content. 3. The RNA content in AH-130 (ascites hepatoma) is less than that in normal liver cells.</p

Absorption and Screening in Phycomyces

In vivo absorption measurements were made through the photosensitive zones of Phycomyces sporangiophores and absorption spectra are presented for various growth media and for wavelengths between 400 and 580 mµ. As in mycelia, ß-carotene was the major pigment ordinarily found. The addition of diphenylamine to the growth media caused a decrease in ß-carotene and an increase in certain other carotenoids. Growth in the dark substantially reduced the amount of ß-carotene in the photosensitive zone; however, growth on a lactate medium failed to suppress ß-carotene in the growing zone although the mycelia appeared almost colorless. Also when diphenylamine was added to the medium the absorption in the growing zone at 460 mµ was not diminished although the colored carotenoids in the bulk of the sporangiophore were drastically reduced. Absorption which is characteristic of the action spectra was not found. Sporangiophores immersed in fluids with a critical refractive index show neither positive nor negative tropism. Measurements were made of the critical refractive indices for light at 495 and 510 mµ. The critical indices differed only slightly. Assuming primary photoreceptors at the cell wall, the change in screening due to absorption appears too large to be counterbalanced solely by a simple effect of the focusing change. The possibility is therefore advanced that the receptors are internal to most of the cytoplasm; i.e., near the vacuole

Suppression of erythropoiesis by actinomycin D. 3. The effect of actinomycin D on the hemoglobin level of bone marrow erythrocytes and erythroblasts

For the purpose of revealing whether or not hemoglobin synthesis is inhibited by the AMD, the author estimated the hemoglobin level of AMD treated anilmals by microspectrophotometer, and found that the hemoglobin levels of all the developmental stages of erythroid cells were not inhibited by the AMD. The data indicated that about one half of mRNA for hemoglobin is synthesized in the early stage of specialization with the supplementary synthesis at the later stages and all these mRNA is stable and insensitive to AMD.</p

Studies on the DNA metabolism of erythroid cell. I. DNA level of erythroblastic nuclei of rabbit bone marrow, observation of normal, blood depleted, and phenylhydrazine anemias, and their recovery by red cell transfusion

For the purpose to reveal the changes in the metabolism of erythroblast in varied specialization stages the author observed the Feulgen DNA level of rabbit erythroblasts by microspectrophotometry. Observations were made on normal and anemic animals, and those receiving a mass red cell transfusion at the recovery stage of anemia where the early denucleation is stimulated. Observations have revealed that in normal erythropoiesis the DNA contents are kept at n to 2 n level from the proerythroblast to late basophilic stage, but in later stages, polychromatic and orthochromatic, DNA level per cell decreases gradually with advance of the cell specialization reaching the minimum level, nearly haploid level, at orthochromatic stage where most cells are believed to be denucleated. In blood depleted animals nearly the same pattern of DNA level was observed in connection with erythroid specialization as that in normal animal, except a relatively high DNA level in the later specialization. In the cases of the hemolytic anemia a similar tendency has been observed but the minimum level of DNA remains at a higher level, hypo-diploid level, in poly- and orthochromatic stages. Twenty-four hours after the mass red cell transfusion by which severe anemia has been recovered to the original level within one hour, the pattern of the DNA level of the erythroblast returns to the normal one showing a very low DNA level at the polyand the orthochromatic stages. The data indicate that the DNA synthesis of erythroblast kept at n to 2 n levels until the late basophilic stage begins to decline at polychromatic stage and reaches nearly haploid level at orthochromatic stage, but in active hemopoiesis the DNA synthesis is stimulated and the DNA contents are kept at a high level even in the late specialization stages, showing no relation between the denucleation and the low DNA level.</p

Multiple Cone Visual Pigments and the Potential for Trichromatic Colour Vision in Two Species of Elasmobranch

Elasmobranchs (sharks, skates and rays) are the modern descendents of the first jawed vertebrates and, as apex predators, often occupy the highest trophic levels of aquatic (predominantly marine) ecosystems. However, despite their crucial role in the structure of marine communities, their importance both to commercial and to recreational fisheries, and the inherent interest in their role in vertebrate evolution, very little is known about their visual capabilities, especially with regard to whether or not they have the potential for colour vision. Using microspectrophotometry, we show that the retinae of the giant shovelnose ray (Rhinobatos typus) and the eastern shovelnose ray (Aptychotrema rostrata) contain three spectrally distinct cone visual pigments with wavelengths of maximum absorbance (lamda_max) at 477, 502 and 561 nm and at 459, 492 and 553 nm, respectively. The retinae of R. typus and A. rostrata also contain a single type of rod visual pigment with lamda_max at 504 and 498 nm, respectively. R. typus, living in the same estuarine waters as A. rostrata, were found to have identical visual pigments to R. typus inhabiting coral reef flats, despite a considerable difference in habitat spectral radiance. This is the first time that multiple cone visual pigments have been measured directly in an elasmobranch. The finding raises the possibility that some species are able to discriminate colour - a visual ability traditionally thought to be lacking in this vertebrate class - and it is evident that the visual ecology of elasmobranchs is far more complex than once thought