Institute for Advancement in Higher Education

This study was undertaken in order to investigate the effect of zinc (Zn) administration on induction of Znbinding metallothionein in rat liver with thioacetamide-induced cirrhosis, and the localization of metallothionein in the liver. Normal and cirrhotic rats received intraperitoneal injections with or without Zn. Subsequently, metal analyses, purification of metallothionein by gel filtration and immunohistochemical assessments of metallothionein were carried out. Although in Zn-injected cirrhotic rats, the Zn contents in the liver and plasma increased significantly depending upon the dose of Zn, the Zn contents in the liver and plasma of the cirrhotic rats were lower than those of normal rats after the same dose of Zn. The results of gel filtration also showed that the levels of Zn-metallothionein in the cirrhotic liver were reduced in comparison with those of the normal liver. By the immunohistochemical method, the presence of metallothionein in the parenchymal areas but not in the fibrotic areas of the cirrhotic liver was confirmed. These results suggested that the induced metallothionein was only located in the parenchymal areas. The metallothionein induced in the parenchymal areas was considered to play a role in protecting the parenchymal cells against the progression of fibrosis, because metallothionein has been thought to be involved in the cellular defense against oxidative stress.

In this study, to examine the mechanism of diethyl phthalate toxicity to cells, the effects of diethyl phthalate on apoptosis in a PC12 cell system were investigated by assaying apoptotic factors such as caspase-3, Bax, cytochrome c and DNA damage. Diethyl phthalate was shown to enhance the apoptosis induced by serum deprivation according to the results of DNA electrophoresis and TUNEL signal assays, although it could not induce apoptosis itself in the cells. This enhancement was thought to be because of an increase in caspase-3-like activity. In addition, the expression of bax and contents of cytochrome c in the cytosol showed a tendency to increase the cells exposed to diethyl phthalate. These results indicated that diethyl phthalate, a potential endocrine disrupter, affects the apoptotic system in PC12 cells. Diethyl phthalate may enhance oxidative stress such as that induced by reactive oxygen species in PC12 cells.

We examined the link of hippocampal Zn to the functional impairments with aging using senescence-accelerated mouse prone 10 (SAMP10) with deficits in learning and memory. Zn in hippocampal mossy fiber pathway was less distributed in aged SAMP10 than that in the age-matched control. Furthermore, expression of Zn transporter 3, ZnT3, which plays to accumulate Zn in synaptic vesicles in the mossy fiber pathway, was markedly reduced in the hippocampal region even in young SAMP10. Moreover, excessive presynaptic release of glutamate as well as glycine and expression of glial fibrillary acidic protein, a marker of neuronal cell injury, were observed in the hippocampus of aged SAMP10 compared to the control. The present results suggest that age-dependent deficiencies of Zn in synaptic vesicles of the mossy fiber pathway induced by low expression of ZnT3 cause glutamatergic excitotoxicity in the hippocampal neurons and the deterioration of learning and memory in SAMP10.

It has not been fully elucidated how endocrine-disrupting chemicals disrupt hormone functions or how strong their effects are compared with natural hormones. There is little information concerning the effects of tributyltin (TBT), one of the endocrine disrupters on living organisms. Although TBT at high cocentration induced apoptosis in PC12 cells, TBT at low concentration inhibited the DNA fragmentation in the

An endocrine disrupter, bisphenol-A is widely used in t he product ion of plast ics and coating s. Recent ly, it was reported that bisphenol-A affected neurotransmitters in the mammalian brain. On t he basis of these reports, it was considered that bisphenol-A affected neuronal different iat ion. In t his study, the morphological changes in nerve growth factor (NFG)-induced different iat ion caused by bisphenol-A were confir med using a PC12 cell system. When a low concentrat ion of bisphenol-A was added to medium containing NGF, it inhibit ed neurit e extension. In addit ion, to clarify whether bisphenol-A affects the early and late stages of the NGF-signaling pathway in cell different iat ion, changes of phosphorylat ion of MAP kinases and cAMP-response element binding protein (CREB) in PC12 cells treated with and wit hout BPA in medium containing NGF were invest igated using western blot analysis. As results, bisphenol-A significant ly inhibit ed phosphorylat ion of CREB and ERK1/2 MAPK.

In Java and Kalimantan in Indonesia, river water plays important roles in human life; for example, for transportation, and economic activities of the inhabitants. However, industrial, agricultural and domestic water is discarded into rivers directly in many developing countries, including Indonesia, since drainage systems have not been completely constructed. In this study, to evaluate the water quality and to compare those levels of environmental contaminants in developing and developed countries, water quality and contents of endocrine disrupters were measured in a total of 64 water samples (Indonesia; 28 samples and Japan; 36 samples) from 53 sites. The results indicated that, rivers in both capital cities, Jakarta and Tokyo, were contaminated. Water in rivers in Indonesia was not so heavily polluted as in Japan. Pollution in the river water in Indonesia appeared to be caused by the lack of sewerage systems. In addition, the findings on endocrine disrupters indicated that the concentration of alkylphenol in water samples was large enough to affect living organisms.

Alkyl esters of p-hydroxybenzoic acids (parabens), an endocrine disrupter, are used as preservatives in cosmetics and foods. In this study, to understand the relationship between parabens and differentiation in infants, the effects of parabens on apoptosis induced by serum deprivation in PC12 cells were investigated. In addition, apoptosis-related factors were assayed. As results, a tendency toward enhancement of apoptosis was observed in the cells cultured in the serum-free medium with methylparaben, and this tendency was suggested to be related to the contents of BAD, a pro-apoptotic protein. Butylparaben did not show any tendency to enhance apoptosis.

Effects of styrene exposure on middle latency auditory-evoked potentials (MAEPs), which reflect the central auditory function, were investigated. As significant delay in latencies of No, Po, Na, and Pa components and increases in the peak to peak intervals of MAEPs, but not amplitudes, were observed in a dose-dependent manner. The present and our previous studies demonstrate that electrophysiological alterations occur before neuronal cell damage induced by styrene exposure. The present results suggested that styrene has a marked effect on the processing of auditory information related to MAEPs.

This study was undertaken to investigate the effect of taurine on the infiltration of macrophages and the progression of renal interstitial fibrosis in the kidneys of rats treated with cisplatin (CDDP). Male rats in different groups were treated as follows: (1) saline as control, (2) CDDP and (3) CDDP plus taurine in drinking water. At weeks 2, 4 and 6 after the fifth CDDP injection, we examined platinum content, the kinetics of macrophages and the areas of fibrosis. In the histologic analyses, fibrotic areas were found to have developed around dilated or atrophic tubules in the corticomedullary junction in the kidneys of CDDP-treated rats, while CDDP-plus-taurine-treated rats showed a reduction of the extent and magnitude of damage. These findings reflect the fact that the percentage of fibrotic areas in the kidney of CDDP-plus-taurine-treated rats was significantly lower than in that of CDDP-injected rats throughout the recovery period ( P<0.05). Compared with normal rats, the macrophages in CDDP-injected rats showed significant increases in number at weeks 2, 4 and 6 ( P<0.05). In contrast, the number of macrophages in CDDP-plus-taurine-treated rats was significantly smaller than that of CDDP-injected rats. These results suggested that taurine suppressed the increase of infiltrating macrophages, and led to the attenuation of renal interstitial fibrosis ( P<0.05).

ABSTRACT In order to investigate the neuropathological effects on the central nervous system of the rat by styrene exposure, identification of glial fibrillary acidic protein (GFAP) was observed using immunohistochemical staining and confocal laser scanning microscopy in the rat treated with styrene at 400 mg/kg and 800 mg/kg for 2 weeks. Both styrene-treated groups showed a marked increase of GFAP expression in cerebral cortex and hippocampus compared with the control group. These changes showed a dose- and time-dependent manner. Increased expression of GFAP in cerebral cortex and hippocampus provided the effects of styrene on the structüre of cerebral cortex and hippocampus objectively.

We examined synaptic plasticity in the optic tectum of rainbow trout by extracellular recordings. We found that the field-excitatory postsynaptic potential in the retinotectal synapses was potentiated by repetitive stimuli of 1.0 Hz for 20 s to the retinotectal afferents. The long-term potentiation (LTP) developed slowly, and was maintained for at least 2 h. Applications of an antagonist for N-methyl-D-aspartic acid (NMDA) receptors or Mg2+ -free saline showed that activation of NMDA receptors was required to form the LTP beyond the induction period. The present findings indicate that presynaptic stimulation in the retinotectal synapses causes LTP mediated by NMDA receptors in the optic tectum of rainbow trout.

Spatio-temporal patterns of neuronal activity before and after the induction of long-term potentiation in mouse hippocampal slices were studied using a real-time high-resolution optical recording system. After staining the slices with voltage-sensitive dye, transmitted light images and extracellular field potentials were recorded in response to stimuli applied to CA1 stratum radiatum. Optical and electrical signals in response to single test pulses were enhanced for at least 30 minutes after brief high-frequency stimulation at the same site. In two-pathway experiments, potentiation was restricted to the tetanized pathway. The optical signals demonstrated that both the amplitude and area of the synaptic response were increased, in patterns not predictable from the initial, pretetanus, pattern of activation. Optical signals will be useful for investigating spatio-temporal patterns of synaptic enhancement underlying information storage in the brain.

In the laboratory, using a PC12 cell system, studies have been conducted on the effects of various chemicals on apoptosis, as it is considered to be an essential part of normal development, maintenance, and defense in organisms. Stevioside is a natural sweetener extracted from the leaves of Stevia rebaudiana. Since it is widely used as a sugar replacement, it was decided to evaluate the toxicological effects of low concentrations of stevioside on apoptosis induced by serum deprivation using the PC12 cell system. It was found that based on data from DNA electrophoresis and TUNEL signal assays stevioside enhanced apoptosis induced by serum deprivation. This enhancement was caused by increased expression of Bax and of cytochrome c released into the cytosol. These findings suggest that stevioside affects the regulation of the normal apoptotic condition. Further investigation will be needed to clarify the detailed mechanism of the enhancement due to the treatment with stevioside.

An improved terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labeling method for the quantification of DNA damage in tissues and cultured cells was developed. Many reports have revealed that histochemistry of DNA damage can be visualized using immunohistochemistry for the terminal deoxynucleotidyl transferase reaction in tissue sections. However, few reports have described quantification of DNA damage in tissues or cells. In this study, to estimate the degree of DNA damage, the confirmed method for histochemistry using biotinylated dUTP and deoxynucleotidyl transferase was applied to label the cleaved DNA ends caused by DNA damage in tissues or cells. After end-labeling, avidin-conjugated peroxidase was reacted. A significant correlation was observed between numbers of cleaved DNA ends and peroxidase activity after the reaction. The obtained signals for presented method showed higher than those for ordinary method, and correlate with degree of DNA damage caused by serum deprivation and chemical dose. In addition, DNA damage caused by apoptosis in cells treated with 6-hydroxydopamine or Cu and in the tissues of rats administered a diet containing no Zn could be evaluated quantitatively using the present method.

Using confocal microscopy in conjunction with microdrop application of Dil, we have imaged and measured individual dendritic spines of living hippocampal CA1 pyramidal neurons in acute brain slices, before and approximately 3 hr after induction of long-term potentiation by chemical means. Statistical analysis of changes in the length of individual spines, and comparison with results of Monte Carlo simulations, suggests that two forms of structural change occur in chemically induced long-term potentiation: growth of a subpopulation of small spines, and angular displacement of spines. These changes could provide a structural basis for the expression of long-term potentiation.

Macrophage migration inhibitory factor (MIF) is a molecule known to regulate macrophage accumulation at sites of inflammation. To elucidate the role of MIF in progression of liver fibrosis, the immunohistochemical localization of MIF and macrophages in the liver were examined. Male Wistar rats received thioacetamide (TA) injections (200 mg/kg, i.p.) for 1 or 6 weeks. In biochemical and histological tests, it was confirmed that liver fibrosis was induced. In immunohistochemical analyses, the expression of MIF protein was seen in hepatocytes in the areas extending out from the central veins to the portal tracts. In particular, at 6 weeks, immunoreactivity was detected in degenerated hepatocytes adjacent to the fibrotic areas but hardly observed in the fibrotic areas. On the other hand, a number of exudate macrophages stained by antibody ED1 were seen in the areas from the central veins to the portal tracts at 1 week and in the fibrotic areas at 6 weeks. Macrophages also showed a signi...

The development of a surface water evaluation index is a critical factor in the assessment, restoration and protection of stream water quality. Quantifying water pollution in specific grade using dominant parameters is important, as this can explain the current state of water pollution with accuracy. As a result, an integrated multi-parameter water quality index has been developed. It is based on the 10 most prevailing parameters (pH, conductivity, nitrate ions, phosphate ions, Escherichia coli number, cadmium, chromium, lead, copper and manganese) with a scale of 1 to 4, wherein the grades are classified into 1: good; 1.1-1.5: slightly polluted; 1.6-2.0: moderately polluted; 2.1-2.9: heavily polluted and 3.0-4.0: gravely polluted. The measuring stick used was according to the 2011 background values of the World Health Organization (WHO) in which a value of 0.1 was attributed for each, so that the final grade can be calculated. Water quality data were successfully fitted in an integ...

The high-speed camera has become to be able to take sequential two-dimensional images with 0.6 msec per one frame. The images of voltage change in the mouse hippocampal slice were recorded with high-speed camera system (Deltaron; Fujifilm, Tokyo, Japan) and data processing was executed with homemade software. It shows the electrophysiological pathway in the mouse brain using voltage sensitive dye. However the images include so many points (128 x128) and it was difficult to image the actual signal transmission with such huge data. We developed new analysis method that shows the pathway with only two pictures. One picture shows the maximum value of each point and shows the distribution of signals in the slice. The other shows the time when the maximum value was recorded at each point and shows the time with colors. Each color shows the time point of the maximum value was recorded. This picture shows the actual signal transmission at a glance. Moreover it suggests a hardware which can ...