Effects of Fluorescent Light on Growth of Bovine Retinal Pigment Epithelial Cells In Vitro Incubated with Linoleic Acid or Linoleic Acid Hydroperoxide

Light-induced peroxidation of polyunsaturated fatty acids (PUFA) may generate lipid hydroperoxides, which may have toxic effects on retinal pigment epithelial (RPE) cells in vitro. We investigated the effects of cool-white fluorescent light on the RPE cells incubated with linoleic acids (LA) or linoleic acid hydroperoxides (LHP) and the influence of antioxidative enzymes. We measured the bovine RPE cell number after exposure to fluorescent light (610 and 1,200 lux) in the presence of LA or LHP. Furthermore, the effects of superoxide dismutase (SOD) and catalase on LA- or LHP-treated RPE cells were also examined. Both LA and LHP treatment increased RPE cell number under weak illumination (610 lux), but dose-dependently decreased the number of cells exposed to strong illumination (1,200 lux). With exposure to strong illumination, LA caused a greater reduction in RPE cell number than LHP. Multiple linear regression analysis showed that the number of RPE cells was significantly decreased in a manner dependent on the interactions of the illuminance of light and the concentrations of LA or LHP. The antioxidative enzymes significantly ameliorated the damage to RPE cells from LA or LHP and exposure to light. Therefore, the exposure to fluorescent light augmented the cytotoxic effects of LA and LHP on RPE cells, and this effect is likely to be mediated by reactive oxygen species.     

Development of Plasmodium berghei Ookinetes to Young Oocysts In Vitro

ABSTRACT. The mosquito stage of Plasmodium berghei was cultivated in vitro, with special attention to ookinete transformation into early oocyst. The ookinetes were obtained by in vitro culture of gametocytes taken from infected mice, purified by density gradient of metrizoic acid or a lymphocyte separation medium, and incubated either in acellular culture or in co-cultivations with mosquito cells. In acellular culture, the ookinetes were found to aggregate with each other and transformed from banana to round shapes. Their inner pellicular membranes and subpellicular microtubules partially disappeared, indicating that development to early oocyst had occurred. Co-cultivation with Aedes albopictus cells (C6/36 clone) revealed that ookinetes transformed into early oocyst in the medium, or invaded the cells and then transformed to early oocysts within the cell cytoplasm as well. However, all of these transformed cells failed to develop further, i.e. neither deposition of the oocyst capsule nor nuclear division was observed. Many ookinetes which failed to penetrate the Aedes cells were phagocytized within three days of culture. A significant difference between invaded and transformed oocysts and phagocytized ookinetes was seen in that the former lacked vacuole membrane. Co-cultivation with Toxorhynchites amboinensis cells (TRA-284-SFG clone) permitted transformation of ookinetes into early oocysts in the medium as in the acellular culture, but no ookinete invasion nor phagocytosis by the cell was observed.     

Establishment of a Murine Model for Metastasis of Cytokine-Producing Tumor to the Brain

The A375 cell line, derived from human malignant melanoma, has characteristics of interleukin-6 (IL-6) production. By using this cell line, we have investigated a murine metastasis model of IL-6-producing tumors to the brain by injecting A375 cells directly into the left cardiac ventricle. Nude mice were anesthetized with intraperitoneal injection of pentobarbital sodium. Next, A375 cells suspended in phosphate-buffered saline (PBS) were injected into the left cardiac ventricle of mice. An intracardiac injection of 10⁵ cells developed tumor colonies in the brain after 4 to 6 weeks. Metastatic cells were found in every lobe of the brain. An immunocytochemical study revealed IL-6 production by A375 cells at the metastatic sites in the brain. By the transfection of genes encoding proteins into A375 cells, a novel model of protein expression in the brain in vivo could be constructed. Our system does not require great skill. Our experimental model will facilitate future studies of the local effects of proteins in the brain.     

THE EFFECTS OF ACTINOMYCIN D ON MUSCLE CELLS OF ASCIDIAN EMBRYO*

The effect of actinomycin D on muscle cells development of the ascidian, Herdmania momus was studied ultrastructurally. No myofilament was formed when the drug was given at any stage before early tail-bud stage (stage 3). Some aggregates of myofilaments in various size were formed when the treatment was started at stage 4 (4.5 hr after fertilization at about 28°C). Above 60% of myofibrils of fully differentiated muscle cells were formed when the treatment was initiated at stage 5 (5 hr after fertilization). Muscle cells of the tadpoles treated from stage 7 (6 hr after fertilization), at which myofilaments were first detectable in normal development, differentiated almost normally. It is therefore suggested that most mRNAs for muscle proteins are synthesized preceding the onset of myofilament formation and are relatively stable. It is also suggested that mRNAs for myosin, actin and Z band materials are almost simultaneously synthesized. One of the characteristic features of the muscle cell development of the ascidian embryo is almost synchronous differentiation of relatively small numbers of cells (about 54–60 cells). The significance of these results is discussed in relation to mosaic natures of the muscle development.     

Pollinator limitaton in a deceptive orchid, Pogonia japonica, on a floating peat mat

Deceptive orchid species that offer no floral rewards to pollinators often experience pollinator limitation because they. The breeding system and fruit set were investigated in order to examine pollinator limitation for a population of a deceptive orchid, Pogonia japonica , on a floating peat mat in Mizorogaike Pond, western Japan. Fruit sets for outcross-pollinated, self-pollinated and pollinator-excluded flowers were 75%, 80%, and 0%, respectively. Thus, this species was self-compatible but neither autogamous nor apogamous. The fruit set for open-pollinated flowers was 22.9% and 17.4% in 1994 and 1995, suggesting that pollinator limitation did occur in the field. Larger flowers were more likely to develop into fruit than smaller ones in open pollination (36% vs 8%). Thus, flower size seems to affect fruiting of this species. No effective visits by major pollinators such as bees, which could carry pollinia, were observed during 16.5 h of observation during daytime. Instead, thrips were often found moving on gynostemium or pollinia and circumstantial evidence that suggests thrips may partly transfer pollen in pieces out of a granular pollinium of P. japonica was obtained. Pogonia japonica should come to use thrips as a pollinator to supplement low fruit set under limited flower visitation by major pollinators.     

MAGNESIUM REQUIREMENT IN FERTILIZATION PROCESS OF SEA URCHINS

The Mg²⁺ requirement in fertilization was investigated in sea urchins. It was found that when sea urchin eggs were inseminated in sea water free of Mg²⁺, little fertilization took place. Even when spermatozoa pre-treated with dissolved egg-jelly to induce the acrosome reaction, which needs Ca²⁺, were used, the fertilization rate remained quite low in the absence of Mg²⁺. In Strongylo-centrotus intermedius , the lowest concentration of Mg²⁺ required for 50% fertilization was 0.05 mM in the presence of 10 mM Ca²⁺, whereas that of calcium was 3 mM in the presence of 49 mM Mg²⁺. These critical concentrations increased when the concentration of the other ion decreased. Removal of Mg²⁺ or Ca²⁺ or both from the suspending medium had little adverse effect on sperm motility. The elevation of the fertilization membrane was also induced by butyric acid independent of the presence or absence of Mg²⁺ and/or Ca²⁺. These results indicate that Mg²⁺ are required at least in some process(es) between acrosome reaction and fertilization membrane elevation, such as sperm penetration or membrane fusion.     

Development and characterization of microsatellite markers in a clonal plant,Dioscorea japonica Thunb.

We developed 10 microsatellite loci from genomic DNA of a dioecious clonal plant, Dioscorea japonica. Out of 384 clones, 148 contained microsatellite repeats. Polymerase chain reaction primer pairs were designed for 95 of these clones from their sequence data, of which, 10 pairs produced successful amplification. Thirty‐eight individuals were genotyped for allelic diversity. We detected three to nine alleles per locus, and the expected heterozygosity ranged from 0.461 to 0.851.     

Abscisic Acid as a Stimulator for Rice Mesocotyl Growth

WHEN abscisic acid (ABA) is applied to a growing plant, it usually inhibits the growth of stem, leaf sheath and other plant parts^1–3. Here I report the possibility that ABA stimulates the growth of rice mesocotyl in darkness.     

Effects of dietary inositol with sucrose stimulation on chewing and swallowing motor patterns in larvae of the silkworm Bombyx mori

The effects of dietary inositol with sucrose stimulation on chewing and swallowing motor patterns in the larvae of Bombyx mori L. are investigated. Feeding activities of the larvae are significantly enhanced by a test diet containing an inositol–sucrose mixture compared with a test diet of sucrose only. Motor patterns of the mandibular closer muscle are accelerated with shorter burst durations and shorter inter‐burst intervals with the test diet of inositol–sucrose compared with sucrose. In terms of swallowing behaviours, inositol–sucrose shortens the duration of drinking. Motor patterns of the cibarial compressor muscle are accelerated with shorter burst durations and shorter inter‐burst intervals with the inositol–sucrose mixture. Peripheral interactions between inositol‐ and sucrose‐sensitive cells in the maxilla are not detected. Thus, such interactions cannot explain the positive effects of inositol on chewing and swallowing. Responses of inositol‐sensitive cells in the epipharyngeal sensillum are not affected by sucrose. These results suggest that dietary inositol can modify chewing and swallowing motor patterns when coupled with sucrose stimuli. These modifications may occur in the central neural networks involved in chewing and swallowing motor patterns but not in peripheral sensory interactions.     

Influence of Temperature on Lactate Utilization by Round Spermatids from Rat Testes

Rotenone-sensitive ¹⁴CO₂ formation from [¹⁴C]lactate and oxygen consumption by round spermatids were found to be greater at elevated temperatures than at 34°C. More than 96% of the total radioactivity of the metabolized [¹⁴C]lactate was recovered in the released CO₂ and the acid soluble fraction of the cells. There was practically no incorporation of [¹⁴C]latctate into the lipid, nucleic acid, and protein fractions. Intracellular level of ATP in spermatids was enhanced in the presence of lactate (20 mM) at 34°C (scrotal temperature), whereas it was decrease at 37°C (body temperature). However, this was reversible when the cells were transferred from the elevated temperature to 34°C. It was also found that oxygen consumption and CO₂ production were increased at 34°C by 2, 4-dinitrophenol (DNP), but decreased by oligomycin. On the other hand, oligomycin and DNP had no effect on oxygen consumption and ¹⁴CO₂ formation at the elevated temperature.
These findings provide evidence that lactate utilization by spermatids is coupled with oxidative phosphorylation at scrotal temperature, but becomes uncoupled at elevated temperature, although more lactate is consumed.