Kimie Fukuyama

Effects of ultraviolet radiation on the mitotic cycle and DNA, RNA and protein synthesis in mammalian epidermis in vivo.

Abstract— Acute effects of ultraviolet radiation on the mitotic cycle and macromolecular synthesis were investigated on hairless mouse epidermis in vivo. Colcemid was used to arrest mitoses in metaphase and thus allow more accurate mitotic counts. The radioactive tracers, TdR‐3H, cytidine‐3H, and the amino acids, histidine‐3H and methionine‐3H were used to examine DNA, RNA and protein synthesis, respectively. Using these techniques, we found that wavelengths shorter than 320 nm markedly inhibited mitosis, increased the basal cell turnover time and depressed DNA, RNA and protein synthesis within the first few hours post‐irradiation. By 24hr, recovery and acceleration of these functions were in progress, reaching a peak by 48–72 hr and persisting though to a lesser degree for 7 days. This stage of acceleration was associated with epidermal hyperplasia and most likely represented post‐injury cell renewal.

Defect in DNA synthesis in skin of patients with xeroderma pigmentosum demonstrated in vivo.

Exposure of normal human skin in vivo to ultraviolet irradiation at wavelengths shorter than 320 nanometers stimtulated an unscheduled DNA synthesis in all of the cell layers of the epidermis and in the upper dermnial fibrocytes. The skin of patients with xeroderma pigmentosum did not show this response. correlation of these findings with previous tissue culture studies suggests that the defect in repair of the damaged DNA in xeroderma cells occurs in vivo as well as in vitro.

EARLY EFFECTS OF ULTRAVIOLET LIGHT ON DNA SYNTHESIS IN HUMAN SKIN IN VIVO.

The early effects of ultraviolet light (UV) irradiation on deoxyribonucleic acid (DNA) synthesis in human skin were studied in vivo. Volunteers exposed to 3 minimal erythema doses (MED) (8.2-40.8 × 106ergs/sq cm) were injected intradermally with tritiated thymidine (TdR-H3), immediately, 15 minutes, 3, 5, and 24 hours afterward, and biopsies processed for light microscopy autoradiography. Comparison of the results with findings in unirradiated human skin indicated: (1) a depression in the number of germinative basal cells synthesizing DNA prior to division at three and five hours after UV, as seen in other systems; (2) a population of sparsely labeled cells not only in the basal layer but also in the malpighian and granular layers and not seen in unirradiated skin or after several other types of acute injury. This aberrant type of TdR-3H incorporation has been seen in other systems and is thought to represent dark reactivation repair of irradiated DNA as occurs in microorganisms.

Effects of 8-methoxypsoralen-induced phototoxic effects on mammalian epidermal macromolecule synthesis in vivo.

Abstract— The influence of 8‐methoxypsoralen (8‐MOP) and ultraviolet (UVA; 315–400 nm) radiation‐induced phototoxic responses on DNA, RNA and protein synthesis and the DNA repair phenomenon were investigated utilizing the hairless mouse epidermis in vivo. The radioactive tracers TdR‐3H, cytidine‐3H and histidine‐3H were used to examine changes in these macromolecules. Using these techniques, we found that the 8‐MOP‐UVA phototoxic injury inhibited premitotic semiconservative DNA synthesis in the germanitive layer of the epidermis within the first few hours post‐irradiation. Recovery occurred by 24 h, followed by a progressive acceleration of this function over the next 7 days. No depression in RNA or protein formation was noted through 36 h. By 48 h the cells in the upper 1/2 to 1/3 of the epidermis lost their normal appearance and discontinued synthesizing these macromolecules. At 72 h RNA and protein synthesis was again active throughout the epidermis and the apparently dead cells had desquamated. At this time the epidermis was notably acanthotic and the epidermal cells were markedly enlarged. Examination for the dark repair response revealed no evidence of unscheduled DNA synthesis following irradiation indicating that the excision repair process was not demonstrable within the first 15min after the phototoxic injury. These responses differ in a number of parameters from the phototoxic reactions induced by UV rays shorter than 320 nm.

A comparative autoradiographic study of keratohyalin granules containing cystine and histidine

Ultrastructural localization of cystine containing protein in keratohyalin granules of newborn rats was studied and compared with that of histidine containing protein using radioisotope-labeled precursors and autoradiographic techniques. The frequency of radioactive labels appearing over keratohyalin granules after injection of ( 3 H)cystine was not as high as after injection of ( 3 H)histidine. However, analysis of grain counts indicated that a cystine containing protein concentrates in the dense homogenous deposits (DHD) of keratohyalin granules. The number of labels in the center of keratohyalin granules increased. The incidence of labels over the keratohyalin granulelike inclusions in the nuclei of granular cells also increased with time. Histidine containing protein in contrast concentrated elsewhere in keratohyalin granules than in DHD. These findings demonstrate that keratohyalin granules are made of heterogenous constituents of different chemical composition; at least one is a protein relatively rich in cystine and another contains high levels of histidine.

Immunochemical comparison of histidine-rich protein in keratohyalin granules and cornified cells

(1) Combination of techniques for extraction and purification of histidine rich protein established by several investigators were employed for comparison of histidine-rich protein in granular cells and cornified cells of newborn rats. (2) Histidine-rich protein extracted from the same cell fraction by two different techniques either in 1 M potassium phosphate buffer (Ugel) or in 4 M urea (Dale) showed identical elution profiles on CM 52 cellulose ion exchange chromatography and the same SDS polyacrylamide gel electrophoretic patterns. (3) Histidine-rich protein from granular cells contained polypeptides of larger molecular sizes than those in histidine-rich protein from cornfield cells, although amino acid composition of the two histidine-rich protein was non-distinguishable (histidine residue was more than 7%). (4) Antibodies raised in rabbits by injection of histidine rich protein from granular cells and that from cornfield cells immunologically cross-reacted. Furthermore, the antisera were found to be reactive over both keratohyalin granules and cornified cells, but not epidermal cells of the lower strata.

Nuclear changes during keratinization of normal human epidermis.

Previous workers have shown step-by-step changes in the cytoplasm of the granular cells during differentiation (keratinization) of human epidermis, including enlargement of the keratohyalin granules and the appearance and migration to the distal plasma membrane of the membrane coating granules. Our study of the granular cell nucleus shows changes correlated with the cytoplasmic differentiation: With accumulation of cytoplasmic keratohyalin, the nucleus becomes flattened and the nuclear membrane becomes indented. Nuclear bodies are infrequently seen as concentrically arranged fibrils. The nucleolus loses its initial netlike (nucleolonemal) pattern and becomes dense. At the same time round, homogeneous dense inclusions appear in the nucleus with special concentration around the nucleolus. These dense nuclear inclusions have been observed in epithelia of other species and may reflect the process of keratinization.

Ultrastructural studies of newborn rat epidermis after trypsinization.

Ultrastructural changes observed in newborn rat epidermal cells after trypsinization were studied. Trypsin separated epidermis from dermis by dissolving the electron dense basement lamina and caused the separation of the desmosomes at their intercellular junctions by dissolving the interdesmosomal structure. Separated epidermal cells rounded up and exhibited numerous cytoplasmic processes as a result of retraction of semidesmosomes toward the center of cells. Cytoplasmic organelles, such as mitochondria, microtubules, endoplasmic reticulum, and tonofilaments, were situated near the nucleus and the cytoplasm between the retracted tonofilaments and the plasma membrane contained mostly ribosomes. The altered morphology of epidermal cells resembles that found in pathological skin conditions such as pemphigus, malignancy, and wound healing. After trypsinization a fine periodicity 300 A became apparent in tonofilaments. This finding should be taken into account in physicochemical studies concerned with the characterization of tonofilaments.

Biological properties of human melanoma cells in culture

SummaryThree human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and the metastatic melanoma seems to display a higher degree of malignant transformation than the primary.

Effect of Ultraviolet Light on RNA and Protein Synthesis in Differentiated Epidermal Cells

STUDIES of the effects of microbeam ultraviolet light on growing tissue culture cells have shown that nucleolar RNA synthesis decreases rapidly but protein synthesis is either not affected1 or is decreased as a result of inhibition of nuclear RNA synthesis2. The influence of ultraviolet light energy on formation of macromolecules in more differentiated cells, however, has not been adequately studied. In the present study we examined the effects of heterochromatic ultraviolet light on RNA and protein synthesis in granular cells of human epidermis in vivo. These cells are differentiated epidermal cells which do not replicate, but do actively synthesize RNA and protein.