Ryohei Ogura

Changes of membrane fluidity and Na+, K+-ATPase activity during cellular differentiation in the guinea pig epidermis

SummaryThe dynamic properties of plasma membrane in epidermal cells were determined by means of electron spin resonance using two kinds of doxyl stearic acid spin labeling agents: 5-DSA and 12-DSA. 5-DSA and 12-DSA are stearic acid analogues with a nitroxide radical ring at the 5th and 12th carbon positions, and these motions reflect molecular motion of lipid bilayer surrounding the hydrophilic region and the hydrophobic region, respectively. Guinea pig epidermal cells were separated into three regions of keratinocytes by Percoll density gradient centrifugation; the upper, middle, and lower epidermal cells. The order parameter S values for 5-DSA and 12-DSA incorporated into the isolated keratinocytes increased, suggesting a decrease in the plasma membrane fluidity, as cells approached the upper epidermal cell layer. The Na+, K+-ATPase activity as a plasma membrane-bound enzyme was determined in each epidermal cell region, and was found to decrease gradually as the cells approached the upper layer. Accordingly, the differentiation of epidermal cells in the keratinization process was found to be assiciated with a decrease in plasma membrane fluidity and with a decline of Na+, K+-ATPase activity.

Effects of vitamin E, vitamin B2 and selenite on DNA single strand breaks induced by sodium chromate (VI)

The effects of vitamin E, vitamin B2 and selenite on DNA single strand breaks induced by Na2CrO4 were examined by alkaline elution. Incubation of Chinese hamster V-79 cells with alpha-tocopherol succinate (vitamin E) for 24 h prior to exposure to Na2CrO4 resulted in a decrease of DNA breaks produced by this compound. However, similar pretreatment with riboflavin (vitamin B2) or Na2SeO3 resulted in an enhanced formation of breaks induced by Na2CrO4. Pretreatment with Na2SeO3 resulted in increased levels of glutathione in these cells while levels of glutathione remained the same with vitamin E or vitamin B2. These results suggest that Na2CrO4 induced DNA breaks appear to be mediated by the formation of free radicals and/or cellular reductive metabolism.

Morphological studies of different mitochondrial populations in monkey myocardial cells

SummaryThe ultrastructure of mitochondria in monkey myocardial cells was investigated by scanning electron microscopy, thin sections and freeze-fracturing. Mitochondria with well-developed cristae were distributed around the nucleus, between the myofibrils and beneath the sarcolemma. Those clustered near the the poles of the nucleus were generally spherical in shape. Interfibrillar mitochondia were arranged in longitudinal rows between the myofibrils, were elongated and usually about the same length as a sarcomere. Subsarcolemmal mitochondria varied in size and shape, being rod-like, spherical, polygonal or horseshoe-like. There were usually two profiles of subsarcolemmal mitochondria in each section of sarcomere, although sometimes one or three occurred, and they were typically oriented perpendicularly to the myofibrils. These morphological differences among mitochondria could reflect functional and/or mechanical properties in the various cellular locations.

Vitamin B2-enhancement of sodium chromate (VI) — Induced DNA single strand breaks: ESR study of the action of vitamin B2

Incubation of Chinese hamster V-79 cells with Na2CrO4 plus vitamin B2 resulted in an increase of Na2CrO4-induced DNA single strand breaks. Electron spin resonance (ESR) studies showed that vitamin B2 enhanced the formation of both hydroxyl radical and tetraperoxochromate (V) during the reaction of Na2CrO4 with hydrogen peroxide. Furthermore, ESR studies demonstrated that a chromium (V) species with a g value of 1.977 was formed by the reaction of Na2CrO4 with vitamin B2. These results indicate that chromate reacts with vitamin B2 to form chromium (V) species and also suggest that the enhancement effect of vitamin B2 on chromate-induced DNA single strand breaks may result from an increase of chromium (V)-related hydroxyl radical formation.

Effect of vitamin E on cytotoxicity, DNA single strand breaks, chromosomal aberrations, and mutation in Chinese hamster V-79 cells exposed to ultraviolet-B light.

Abstract— The effect of pretreatment with vitamin E on cytotoxicity, DNA single strand breaks, and chromosomal aberrations as well as on mutation induced by ultraviolet‐B light (UV‐B) was investigated in Chinese hamster V‐79 cells. Cellular pretreatment with non‐toxic levels of 25 μMα‐tocopherol succinate (vitamin E) for 24 h prior to exposure resulted in a 10‐fold increase in cellular levels of α‐tocopherol. Using a colony‐forming assay, this pretreatment decreased the cytotoxicity of UV‐B light. However, alkaline elution assays demonstrated that pretreatment with vitamin E did not affect the number of DNA single strand breaks caused by UV‐B light. In addition, UV‐B exposure produced a dose‐dependent induction of chromosomal aberrations and mutations at the HGPRT locus, and neither of these actions of UV‐B was influenced by pretreatment with the vitamin. These results suggest that vitamin E protects cells from UV‐B‐induced cytotoxicity, possibly through its ability to scavenge free radicals. The results also suggest that the extent of genotoxicity induced by UV‐B light may not correlate directly with the cytotoxic action of this wavelength region in sunlight.

Reduction of chromium(VI) in Chinese hamster V-79 cells.

The cellular reduction of chromate(VI) was studied by electron spin resonance spectrometry. Incubation of Chinese hamster V-79 cells with Na2CrO4 resulted in the formation of both chromium(V) and chromium(III) complex in a manner dependent on time (30 min-2 h) and concentration (50–500 μM). Following removal of extracellular chromate, the level of chromium(V) complex decreased quickly during the first hour but more slowly for the next hour, whereas the level of chromium(III) remained unchanged, indicating that chromium(III) is the ultimate ion of this metal in cells. Alkaline elution studies demonstrated that treatment of cells with Na2CrO4 induced DNA single-strand breaks that decreased quickly and DNA-protein crosslinks that persisted for 2 h after removal of this metal. These results suggest that the cellular levels of chromium(V) and chromium(III) may be associated with the formation of DNA damage induced by chromium (VI).

Biochemical diagnosis of reduced salivary gland function

The volume of the secretion of saliva is decreased in elderly people. The volume of salivary secretion varies directly with acid DNase activity. Neutral DNase activity, however, shows no significant variation.

NON‐DIMER DNA DAMAGE IN CHINESE HAMSTER V‐79 CELLS EXPOSED TO ULTRAVIOLET‐B LIGHT

To understand and characterize non‐dimer DNA damage and cytotoxicity induced by ultraviolet‐B light (UV‐B,290–320 nm), an alkaline elution technique for analysis of DNA damage was used on Chinese hamster V‐79 cells. Ultraviolet‐B exposure produced a dose‐dependent induction of DNA single strand breaks and DNA‐protein crosslinks; however, there was an absence of DNA‐DNA interstrand crosslinks. Neither of these types of DNA damage were repaired within a 24 h incubation of the cells following a single UV‐B exposure; rather the damage increased. Using a colony forming assay, we found that UV‐B exposure resulted in an increase of cytotoxicity in a dose‐dependent fashion. In addition, UV‐B exposure inhibited DNA and RNA synthesis. The role of non‐dimer DNA damage in the cytotoxicity induced by UV‐B is discussed.

Lipid peroxidation and radical formation in methyl linoleate following ultraviolet light exposure.

To gain information useful in understanding radical reactions of sunlight‐induced lipid peroxidation in the skin, methyl linoleate (ML) was used as a material for a study of electron spin resonance (ESR). The ESR spectrum of ML exposed to UV light was examined under aerobic and anaerobic conditions at low temperature. No ESR signal was detected from the unexposed ML, even at —150°C. However, exposure to UV resulted in the appearance of an ESR signal at —150°C. With the elevation of temperature from —150°C, the ESR signal became asymmetric in shape and the g value increased under aerobic conditions, but these alterations did not occur under anaerobic conditions. Lipid peroxidation through a radical reaction initiated by UV exposure was discussed.

Distribution of enzymes of nucleic acid metabolism in cow snout epidermis

Certain enzymes participating in the degradation of DNA have been measured in cow snout epidermis, liver, kidney and intestinal epithelium. High activities were found in epidermis compared to other tissues.