• Environmental Analysis Health and Toxicology
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• v.18 no.2
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• pp.89-94
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• 2003

Tributyltin (TBT) used world-wide in antifouling paints for ships is a widespread environmental pollutant and cause reproductive organs atrophy in rodents. At low doses, antiproliferative modes of action have been shown to be involved, whereas at higher doses apoptosis seems to be the mechanism of toxicity in reproductive organs by TBT. In this study, we investigated that the mechanisms underlying DNA fragmentation induced by TBT in the rat leyding cell line, R2C. Effects of TBT on intracellular Ca$\^$2+/ level and reactive oxygen species (ROS) were investigated in R2C cells by fluorescence detector. TBT significantly induced intracellular Ca$\^$2+/ level in a time-dependent manner. The rise in intracellular Ca$\^$2+/ level was followed by a time-dependent generation of reactive oxygen species (ROS) at the cytosol level. Simultaneously, TBT induced the release of cytochrome c from the mitochondrial membrane into the cytosol. Furthermore, ROS production and the release of cytochrome c were reduced by BAPTA, an intracellular Ca$\^$2+/ chelator, indicating the important role of Ca$\^$2+/ in R2C during these early intracellular events. In addition, Z-DEVD FMK, a caspase-3 inhibitor, decreased apoptosis by TBT. Taken together, the present results indicated that the apoptotic pathway by TBT might start with an increase in intracellular Ca$\^$2+/ level, continues with release of ROS and cytochrome c from mitochondria, activation of caspases,and finally results in DNA fragmentation.

• Applied Microscopy
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• v.30 no.4
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• pp.357-365
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• 2000

Paneth cells have been suggested to contribute to the elimination of excess metals into the intestinal lumen. The purpose of this study wat to investigate the changes of the zinc pools in rats subjected to functional loading with zinc salt by mean of both light and electron microscopical autometallography (AMG). Wistar rats 4 were administrated with zinc chloride (20 mg/kg body weight) intraperitoneally dissolved in 1 ml distilled water. The control group received 1 ml saline IP. After further one hour the animals were transcardially perfused with 0.4% sodium sulphide dissolved in 0.1 M PB fellowed by 3% glutaraldehyde solution for 10 minutes. Pieces of ileum were frozen with solid $CO_2$ and sectioned on a cryostat. The sections $(20{\mu}m)$ were autometallographically developed. Sections selected for EM were reembedded on top of a blank Epon block, from which ultrathin sections (100 nm) were cut. The ultrathin sections were double stained with uranyl acetate (30 min) and lead citrate (5 min), then examined under electron microscope. Studies of comparable sections from control and zinc loaded animals with the AMG selenium method gave quite different results. The control animals demonstrated a weakly positive staining in the cytoplasm of the Paneth cells. In the electron microscope the AMG silver grains were found to be located in the cytoplasm, while the electron dense secretary granules and other cell organelles were void of staining. Few AMG grains were located at the apical surface of the Paneth cells. In sections from zinc loaded rats, the AMG grains were seen in abundance in the lumen of the Lieberkuhn crypts at light microscopic levels. At EM levels the zinc revealing silver grains were located in the cytoplasm as in the controls, but much more AMG grains were shifted into the secretary granules. Furthermore, profound AMG grains were found in the lumen of the crypts and surrounding vessels. And a few grains were seen in the endothelium. The AMG technique demonstrated a pattern of AMG grains in the Paneth cells that strongly suggests a transport of zinc ions through these cells.

• Applied Microscopy
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• v.30 no.4
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• pp.347-355
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• 2000

Zinc is one of the most abundant oligoelements in the living cell. It appears tightly bound to some metalloproteins and nucleic acids, loosely bound to some metallothioneins or even as free ion. Small amounts of zinc ions (in the nanomolar range) regulate a plentitude of enzymatic proteins, receptors and transcription factors, thus rolls need accurate homeostasis of zinc ions. Zinc is an essential catalytic or structural element of many proteins, and a signaling messenger that is released by neural activity at many central excitatory synapses. Growing evidences suggest that zinc may also be a key mediator and modulator of the neuronal death associated with transient global ischemia and sustained seizures, as well as perhaps other neurological disease stoles. Some neurons have developed mechanisms to accumulate zinc in specific membrane compartment ('vesicular zinc') which can be evidenced using histochemical techniques. Substances giving a bright colour or emitting fluorescence when in contact with divalent metal ions are currently used to detect them inside cells; their use leads to the so called 'direct' methods. The fixation and precipitation of metal ions as insoluble salt precipitates, their maintenance along the histological process and, finally, their demonstration after autometallographic development are essential steps for other methods, the so called 'indirect methods'. This study is a short report on the autometallograhical approaches for zinc detection in the central nervous system (CNS) by means of a modified selenium method.

• Journal of Nutrition and Health
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• v.35 no.3
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• pp.296-302
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• 2002

This study was designed to investigate the effects of a butanol extract of pine needles (Pinus densiflora Sieb et Zucc) on lipid metabolism and oxidative stress in rats. Twenty-eight male Sprague-Dawley (SD) rats were divided into four groups over a 45 days study period: the control group on a basic diet, and three experimental groups on three different dietary levels of the butanol fraction, specifically 25 mg (BuOH-25), 50 mg (BuOH-50), and 100 mg (BuOH-100) butanol fraction/kg body weight/day, thereby 0.025%, 0.05%, 0.1% of butanol extract of pine needles was added to basic diet respectively. At the end of the experimental period, body weights and food intakes were food intakes were not different among the four groups. Total and LDL-cholesterol levels were markedly decreased in the BuOH-25, BuOH-50, and BuOH-100 groups, respectively, as follows: 12.8%, 19.1% and 21.6% reductions in total cholesterol; and 10.2%, 15.6% and 23.7% reductions in LDL-cholesterol. However, HDL-cholesterol levels were significantly increased (by approximately 20%) in the serum of the BuOH-100 group only, compared with the control and other experimental groups. Atherogenic indices were also markedly decreased in the three experimental groups, by 24.8%, 30.4% and 36.2%, for each of the BuOH-25, BuOH-50, and BuOH-100 groups, respectively, compared with the control group. The levels of the hydroxyl radical (·OH) and of lipid peroxide (LPO) in the serum of the three experimental groups were significantly reduced, by 9.8%, 19.7% and 21.2%; and by 13.3%, 13.3% and 16.7%, for the BuOH-25, BuOH-50, and BuOH-100 groups, respectively Significant increases in serum superoxide dismutase (SOD) were observed in the BuOH-50 and BuOH-100 groups; specifically, 12.1% in the BuOH-50 group and 23.3% in the BuOH-100 group, compared with the control group. Significant increases in catalase (CAT) avtivities, of 24.7% in the BuOH-50 group and 29.2% in the BuOH-100 group, were also observed, compared to the control group. These results suggest that a butanol extract of pine needles could inhibit chronic degenerative disease through improving lipid metabolism, and could also effectively modulate the aging process attenuating oxidative stress.

• Journal of Life Science
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• v.18 no.8
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• pp.1053-1058
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• 2008

Glutathione is a well known chemotherapeutic agent for liver disease and is a popular nutritional supplement in the United States. Previous our studies reported the suppressive effects of glutathione-enriched Saccharomyces cerevisiae FF-8 strain (FF-8GY) on carbon tetrachloride- and alcohol-induced hepatotoxicity. The primary objective of this study was to investigate the comparative effects of FF-8GY and commercially available glutathione-enriched yeast extract (GYE) against the oxidative stress in alcohol-induced fatty liver of rats. The lipid peroxidative index (thiobarbituric acid-reactive substances, TBARS) and antioxidant status (reduced glutathione level) were used to monitor those protective roles of FF-8GY or GYE treatment. When the rat was treated alcohol, the TBARS levels in the whole liver and the subfractions of microsomal and mitochondria were significantly increased but these were significantly decreased by FF-8GY treatment and tended to be lowered by GYE treatment. The concentration of hepatic glutathione is known to be closely associated with antioxidant system and this was slightly deplete in the alcohol-induced rats, but this was recovered by treating with FF-8GY. However, the glutathione concentration was more significantly decreased in the GYE supplementation in alcohol feeding rats. Alcohol treatment also negatively affected the serum total protein and albumin, but these were significantly increased near normal levels in FF-8GY coadministered rats. These results suggest that glutathione-enriched Saccharomyces cerevisiae FF-8 strain may have positively mediate the alcohol-induced oxidative stress, and this effect was more pronounced in FF-8GY compared to GYE.

• Journal of Preventive Medicine and Public Health
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• v.38 no.3
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• pp.330-336
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• 2005

Objectives : The aim of this study was to investigate the effects of bisphenol A (BPA), an estrogen-like environmental endocrine disrupter, on the placental function and reproduction in rats. The mRNA levels of the placental prolactin-growth hormone(PRL-GH) gene family, placental trophoblast cell frequency and reproductive data were analyzed. Methods : The pregnancies of F344 Fisher rats ($160g{\pm}20g$) were detected by the presence of the copulatory plug or sperm in the vaginal smear, which marked Day 0 of pregnancy. Pregnant rats were divided into three groups. The control group was intraperitoneally injected with a sesame oil vehicle. The two remaining groups were injected with 50 or 500 mg/kg B.W/day of BPA, resuspended in sesame oil, on either days 7 to 11 or 16 to 20 of pregnancy, with the rats sacrificed on either day 11 or 20, respectively. The mRNA levels of PRL-GH and Pit-1a and b isotype genes were analyzed by Northern blot hybridization and reverse transcription-polymerase chain reaction. The hormone concentrations were analyzed by radioimmunoassay, and the frequency of the placental trophoblast cells observed by a histochemical study. Reproductive data, such as the placental weight and litter size, were surveyed on day 20. The fetal weight was surveyed for 4 weeks after birth. A statistical analysis was carried out using the SAS program (version 8.1). Results : The mRNA levels of the PRL-GH gene family, such as placental lactogen I, Iv and II, prolactin like protein A, C and Cv, and decidual prolactin-related protein were significantly reduced due to BPA exposure. The mRNA levels of the Pit-1a and b isotype genes, which induce the expression of the PRL-GH gene family in the rat placenta, were also reduced due to BPA exposure. The PL-Iv and PL-II concentrations were reduced in the BPA exposed group. During the middle to last stage of pregnancy (Days 11-20), a high dose of BPA exposure reduced the frequency of spongiotrophoblast cells, which are responsible for the secretion of the PRL-GH hormones. Reproductive data, such as the placental and fetal weights and the litter size, were reduced, but that of the pregnancy period was extended in the BPA exposed compared to the control group. Conclusions : BPA disrupts the placental functions in rats, which leads to reproductive disorders.

• The Korean Journal of Pharmacology
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• v.26 no.2
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• pp.193-207
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• 1990

Transforming growth factor ${\beta}(TGF-{\beta})$ is a multifunctional polypeptide with diverse effects on the proliferation, differentiation and other functions in many cell types. $TGF-{\beta}$ is highly abundant in bone matrix and induces divergent responses in many aspects of bone cell metabolism . Several lines of investigation indicate that matrix-associated $TGF-{\beta}$ is the products of bone cells themselves. However, exact bone cell type reponsible for the production of $TGF-{\beta}$ is still in controversy, The present study was undertaken to determine the cellular origin of matrix-associated $TGF-{\beta}$ and to assess how different bone cells respond to $TGF-{\beta}$. As a prerequisite for this, 5 bone cell populations of distinct phenotype were isolated from fetal calvaria with sequential enzyme digestion protocol and biochemical characterization. Calvarial cell populations released in early stage showed fibroblastic features whereas populations relesed later was enriched with osteoblast-like cell as judged by their acid and alkaline phosphatase activities, cAMP responsiveness to parathyroid hormone, calcitonin and prostaglandin $E_2$ and collagen synthesis rate. By polyacylamide gel and immunoblot analysis of bone and calvarial cell extracts, presence of $TGF-{\beta}$ in bone tissues and production of $TGF-{\beta}$ by bone cells were confirmed again. Subsequent analysis of calvarial cell extracts prepared as individual population revealed that all calvarial cell populations synthesize $TGF-{\beta}$. Exogenously added $TGF-{\beta}$ induced biphasic response upon bone cell proliferation under serum-free condition. In osteoblastic cell populations, it was stimulatory whereas inhibitory in fibroblastic cell populations. In contrast, collagen and noncollagen protein synthesis of all calvarial cell populations were stimulated by $TGF-{\beta}$. Enhancement of protein synthesis was found to be more general rather than specific for collagen synthesis. In addition, effects of $TGF-{\beta}$ on protein synthesis were independent to its effects on cell proliferation. In summary, production of $TGF-{\beta}$ by bone cells and differential actions on various cell populations observed in this study suggest that $TGF-{\beta}$ may play an important role in the regulation of bone metabolism by modulating the specific cellular functions in autocrine and paracrine fashion.

• The Korean Journal of Pharmacology
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• v.26 no.2
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• pp.101-111
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• 1990

The effect of physostigmine (PS), which has been shown to act on the muscarinic receptors in the brains of the rat, dog and cat, on the arterial blood pressure (BP) was investigated in urethane-anesthetized rabbits. Intravenous (iv) PS, $25{\sim}300\;{\mu}g/kg$, caused little change in BP. However, after treatment of rabbits with either of chlorisondamine (CS), hexamethonium, intracerebroventricular (icv) clonidine, icv xylazine and icy reserpine iv PS produced a pressor response. Spinalization of the rabbit also caused iv PS to increase BP. The pressor effect of iv PS in CS-treated rabbits was markedly reduced after prazosin or pirenzepine. Iv PS inhibited the pressor response to McN-A-343 in CS-treated and in spinal rabbits; alternately during the infusion of McN-A-343 iv PS failed to produce the pressor response. The pressor response to DMPP was not affected by iv PS. Icv PS, $12{\sim}200\;{\mu}g/kg$, produced a pressor response which was accentuated after CS-treatment. This pressor effect was inhibited, though not complete, by prazosin or by pirenzepine. A simultaneous treatment of rabbits with both $[Sar^{1},\;Ala^{8}]-angiotensin$ II, an angiotensin II antagonist, and prazosin or pirenzepine almost completely abolished the pressor effect of icv PS, whereas the angiotensin II antagonist did not enhance the inhibitory effect of pirenzepine and prazosin on the pressor response to iv PS . Icv pirenzepine blocked the pressor response to icv PS without affecting that to iv PS. The present results show that the pressor response to iv PS in CS-treated and in spinal rabbits arises from stimulation of the muscarinic receptors in the sympathetic ganglia, whereas the pressor response by icv PS via activation of the muscarinic receptors in the brain which causes an enhancement in the outflow of sympathetic discharge and angiotensin. The results also suggest that iv PS is unable to produce a pressor response in the rabbit unless the sensitivity of the gangionic muscarinic receptors is altered by ganglionic nicotinic blockade, by the decrease of central sympathetic outflow on the sympathetic ganglia or by spinalization.

• Journal of Preventive Medicine and Public Health
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• v.32 no.1
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• pp.88-94
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• 1999

Objectives. In order to gain a better understanding of the mechanism of DMF toxicity, recent studies have focused on hepatic drug metabolizing enzymes. In this study, we investigated the effects of DMF on the induction of P450 and the activities of other related enzymes in rat liver microsomes. Methods. DMF was administered to male Sprague Daweley rats by intraperitoneal injection at 0(control), 450(D1), 900(D2), 1,800(D3) mg DMF/kg body weight in olive oil once a day for three days. Hepatic P450 was measured by method of Omura and Sato. We evaluated selective assays for the three drug metabolizing cytochrome P450 isoenzymes 1A1, 2B1 and 2E1. Results. The content of microsomal protein, P450 and b5 were tended to be decreased in DMF treated group, but they were not statistically significant. The activity of NADPH-cytochrome P450 reductase was significantly increased dose dependently(p<0.01), but the activity of NADH-b5 reductase was decreased in the treated group(p<0.01). The activities of PROD and EROD were not significant between control and treated group. The activities of pNPH in the DMF treated groups were higher than that of the control group(p<0.01). When Western immunoblottings were carried out utilizing three monoclonal antibodies which were specific against P4501A1/1, P4502B1/2 and P4502E1, the strong density band corresponding to P4502E1 was observed with the microsomes obtained from the rats treated with DMF. But there were no significant increased in the P4501A1/2 and P4502B1/2 band densities in immunoblotting. Conclusions. These result suggested that P4502E1 was inducible by DMF and P4502E1 isozyme might be responsible for the hydroxylation of DMF to HMMF.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.299-308
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• 2002

In horse, a single gene encodes both eCG and eLH $\beta$ subunits. The difference between eCG and eLH lies in the structure of their glycoresidues, which are both sialylated and sulfated in LH and sialylated in CG eCG consists of highly glycosyiated $\alpha$- and $\beta$-subunits and is an unique member of the gonadotropin family because it elicits response characteristics of both FSH and LH in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of gonadotropin structure-function relationships and the understanding of the molecular bases of the specific interactions of these hormones with their receptors. Thus, eCG is a dintinct molecule from the view points of its biological function and glycoresidue structures. The oligosaccharide at Asn 56 of the $\alpha$-subunit plays an indispensable role, whereas the carboxyl-terminal extension of the eCG $\beta$-subunit with its associated O-linked oligosaccharides is not improtant for, the in vitro LH-like activity of eCG. In contrast, both N- and O-linked oligosaccharides play important roles for FSH-like activity and increase FSH-like activity by removal of N- and O-linked oligosaccharides. Therefore, the dual LH- and FSH-like activities of eCG can be clearly separated by removal of either the N-linked oligosaccharide on the $\alpha$-subunit or CTP-associated O-linked oligosaccharides from its $\beta$-subunit. The glycoresidues seem to play crucial roles fer biological activities. The tethered-eCG was effciently secreted and showed similar LH-like activity to the dimeric eCG $\alpha$/ $\beta$ and native eCG. FSH-like activity of the tethered-eCG was also shown similarly in comparison with the native and wild type eCG $\alpha$/ $\beta$. Our data for the first time suggest that the tethered-eCG can be expressed efficiently and the produced product by the CHO-Kl cells is fully LH- and FSH-like activities in rat in vitro bioassay system. Our results also suggest that this molecular can imply particular models ot FSH-like activity not LH-like activity in the eCG. Taken together, these data indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion.