• Biomolecules & Therapeutics
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• 제8권2호
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• pp.153-159
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• 2000

A novel serine/threonine protein phosphatase with EF-hand motif, which belongs to PPEF family was partially cloned from rat brain cDNA by employing RT-PCR method. The size of the amplified clone was 1.6kbp. The amplified DNA was subcloned into pGEM-T-Easy vector and the resulting plasmid was maned as pGEM-rPPEF2. The nucleuotide sequence is shared by 88% with that of mouse PPEF-2 cDNA, and the deduced amino acid sequence reveal 92% homology with that of mouse PPEF-2 cDNA. The N-terminal region of the cloned rat brain PPEF contains a putative phosphatase catalytic domain (PP domain) and the C-terminal region contains multiple $Ca^{2+}$ binding sites (EF region). The putative catalytic domin (PP) and the EF-hand motif (EF) regions were subcloned into pGEX4T-1 and were overexpressed in E. coli DH5 as glutathione-S-transferase (GST) fusion proteins. Expression of the desired fusion protein was identified by SDS-PAGE and also by immunoblot analysis using monoclonal antibody against GST. The recombinant proteins were purified by glutathione-agarose chromatography. This report is first to demonstrate the cloning of PPEF family from rat brain tissues. The clone reported here would be invaluable for the investigation of the role of this new type-phosphatase in rat brain.

• Animal cells and systems
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• 제3권3호
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• pp.311-317
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• 1999

Prolactin (PRL) was reported to be locally synthesized in many brain areas including the hypothalamus, thalamus (TH) and hippocampus (HIP). In the pituitary lactotrophs, PRL synthesis is dependent upon a pituitary-specific transcription factor, Pit-1. In the present study, we attempted to identify Pit-1 or Pit-1-like protein in brain areas known as the synthetic sites of PRL. Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis showed the same Pit-1 transcripts in brain areas such as the medial basal hypothalamus (MBH), preoptic area (POA), TH, and HIP with the Pit-1 transcripts in the anterior pituitary (AP). Electrophoretic mobility shift assay (EMSA) was run with nuclear protein extracts from brain tissues using a double strand oligomer probe containing a putative Pit-1 binding domain. Shifted bands were found in EMSA results with nuclear proteins from MBH, POA, TH and HIP. Specific binding of the Pit-1-like protein was further confirmed by competition with an unlabeled cold probe. Antisense Pit-1 oligodeoxynucleotide (Pit-1 ODN), which was designed to bind to the Pit-1 translation initiation site and block Pit-1 biosynthesis, was used to test Pit-1 dependent brain PRL transcription. Two nmol of Pit-1 ODN was introduced into the lateral ventricle of a 60-day old male rat brain. RNA blot hybridization and in situ hybridization indicated a decrease of PRL mRNA signals by the treatment of Pit-1 ODN. Taken together, the present study suggests that Pit-1 may play an important role in the transcriptional regulation of local PRL synthesis in the brain.

• 대한약리학회지
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• 제23권2호
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• pp.101-111
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• 1987

중추신경계 특히 뇌내(腦內)의 무수카린성 콜린 수용체 (mAchR)에 대한 수용체 특성의 연구의 하나로, 물리 화학적 성상에 다른 두 종류의 콜린길항제를 사용하여 서로 다른 두 형태의 조직에서 약물의 작용양상 및 다른 약물과의 상호작용을 정초하였다. 실험동물로는 흰쥐를 일정기간 규정사료를 사육하였고, 사용한 Radioactive ligands는 $(^3H)$ QNB와 $(^3H)$ NMS였으며 그외에 다른 수종의 길항제 또는 효능제와의 치환작용을 brain homogenates와 intact brain cell aggregates에서 관찰하여 다음과 같은 결과를 얻었다. 1. $(^3H)$ QNB와 $(^3H)$ NMS는 모두 질량작용의 법칙에 비례하여 수용체와의 결합에서 높은 친화력과 포화를 보였으며 또한 높은 결합 능력을 나타내었다. 더욱이 homogenates 제제와 intact cell aggregates제제에서의 결과 사이에는 유사한 점이 많았다. 2. Homogenates제제를 사용한 실험에서, 제 3 급아민콜린길항제인 QNB, atropine과 scopolamine 또는 제 4 급 암모늄골린 길항제인 methylatropine과 methylscopolamine을 사용하여 위의 radioactive ligands와의 치환작용을 검토하였다. $(^3H)$ NMS 실험군에서는 제 3 급아민 및 제 4 급 암모늄길항제 모두가 구조의 구별없이 질량작용의 법칙에 따라 치환되었으나 $(^3H)$ QNB 실험군에서는 제 4급 암모늄콜린 길항제들을 단일성(unity)이 아닌 높고 낮은 두 종류의 친화도를 가진 결합부위의 양상을 나타내었다. 또 비특이성 콜린길항제인 pirenzepine을 사용한 실험군에서는 두 ligands을 모두 치환시켰고 서로 다른 결합부위가 있음을 보였다. 3. Intact cell aggregates 제제를 사용한 실험에서, $(^3H)$ NMS와 $(^3H)$ QNB 모두 homogenates 제제에서와 같은 양상의 반응을 보였다. 또 $(^3H)$ NMS를 radioligand로 하여 수종의 콜린길항제와 수종의 콜린 효능제를 사용하여 약물 상호작용으로 수용체의 성질을 검토하였다. 그 결과 콜린 길항제들은 질량작용의 법칙에 따라 치환되었으나 콜린 효능제 투여군에서는 높고 낮은 두 종류의 다른 친화력의 결항부위를 나타내었다. 4. 위의 실험의 결과로,(a) 친유성콜린 길항제인 $(^3H)$ QNB는 친수성 콜린길항제인 $(^3H)$ NMS보다 훨씬 높은 결합능력을 보였으며 이것으로 수용체 특히 mAchR의 존재 장소 또는 mAchR의 형상의 일부는 세포막 표면 뿐 아니라 세포막내의 어떤 부위와도 관계가 되는 것으로 간주되는데 이것이 $(^3H)$ QNB가 $(^3H)$ NMS보다 높은 최대 결합능력 $(B_{max})$을 나타낼 이유이다. (b) 두 종류의 다른 제제에서 우리는 같은 양상의 결과를 관찰하었기에 결점이 많은 homogenates 제제보다는 intact cell aggregates 제제를 수용체 연구에 대한 새로운 실험모형(experiment model)으로 사용할 수 있는 가능성을 제시하고자 한다.

• 동의신경정신과학회지
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• 제9궈1호
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• pp.59-71
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• 1998

The effect of Samyonjihwangtang(SJT) on th level of brain antioxidants was examined in aged rat. Samyongjihwangtang(SJT) is assed Cervi Pantiri-chum Cornu, Ginseng Radix to Yukmijihwangtang. The experimental groups were divided into three groups and treated as follows ; normal group(NC), Vt.E administrated group(PC), SJT administrated Group(SJT). From the purified microsome of brain tissue, those were measures the amounts of oxidant materials like malonaldehyde(MDA) and H_2O_2, then activities of antioxidants enxymes like Superoxide dismutase, Catalase, NADPH-cytochrome P-450 reductase.The results were as follows;1. In TBA reaction to measure the amount of MDA, oxidant material of brain tissue of aged rat, both treated groups showed significant decrease.2. In the formation of Hydrogen peroxide, the treated group(SJT) showed a little decrease.3. The activity of Superoxide dismutase was increased significantly in both treated groups than normal group.4. the activity of Catalase was increased significantly in both treated groups than normal group. 5. The activity of NADPH-cytochrome P-450 reductase in the treated group(SJT) showed a little increase.According to the above results, it is suggested that Samyongjihwangtag(SJT) has some antioxidant effects on the tissue of brain.

• 생약학회지
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• 제21권1호
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• pp.92-99
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• 1990

Effects of the methanol extract of Bupleuri Radix on primary cultured chicken embryonic brain cells, dorsal root ganglia (DRG) and rat hepatocytes were studied. The methanol extract of Bupleuri Radix at the concentration ranging from $10{\;}{\mu}g/ml\;to\;100{\;}{\mu}g/ml$ significantly recovered the cytotoxicity of rat hepatocytes induced by the treatment of galactosamine; at the concentration of $100\;{\mu}g/ml$, values of GOT and GPT in the culture medium were reduced by the 60% and 75%, respectively of those in the absence of the methanol extract of Bupleuri Radix. The addition of the methanol extract of Bupleuri Radix. into chicken embryonic brain cells which were cultured with a deficient medium significantly increased the number of cells promoting the neurite outgrowth. However, the methanol extract of Bupleuri Radix showed no effect on the activities of PDHC and acetylcholinesterase in primary cultured brain cells.

• Journal of Microbiology and Biotechnology
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• 제18권9호
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• pp.1590-1598
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• 2008

Cell proliferation and differentiation are critical processes in a developing fetal rat brain, during which programmed cell death (PCD) also plays an important role. One of the decisive factors for PCD is Bcl-2 family proteins, where Bax induces cell death, whereas Bcl-2 acts as an inhibitor of PCD. As maternal drinking is known to cause fetal alcohol syndrome (FAS) or malformation of the fetal brain during pregnancy, the objective of the present study was to investigate whether maternal ethanol exposure alters the PCD-related Bax and Bcl-2 protein expression during fetal brain development. Pregnant female rats were orally treated with 10% ethanol and the subsequent expressions of the Bax and Bcl-2 proteins examined in the fetal brain, including the forebrain, midbrain, and hindbrain, from gestational day (GD) 15.5 to GD 19.5, using Western blots, in situ hybridization, and immunohistochemistry. With regard to the ratio of Bcl-2 to Bax proteins (Bcl-2/Bax), the Bax protein was dominant in the forebrain and midbrain of the control GD 15.5 fetuses, except for the hindbrain, when compared with the respective ethanol-treated groups. Moreover, Bcl-2 became dominant in the midbrain of the control GD 17.5 fetuses when compared with the ethanol-treated group, representing an alternation of the natural PCD process by ethanol. Furthermore, a differential expression of the Bcl-2 and Bax proteins was found in the differentiating and migrating zones of the cortex, hippocampus, thalamus, and cerebellum. Thus, when taken together, the present results suggest that ethanol affects PCD in the cell differentiation and migration zones of the prenatal rat brain by modulating Bax and Bcl-2 expression in an age- and area-dependent manner. Therefore, this is the first evidence that ethanol may alter FAS-associated embryonic brain development through the alteration of Bax and Bc1-2 expression.

• Journal of Nutrition and Health
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• 제26권6호
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• pp.661-671
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• 1993

The change in fatty acid composition in brain tissue of the second generation rats(Sprague-Dawley strain) was studied using four different fat diets(Corn oil=CO, Soybean oil=SO, Perilla oil=PO, Fish oil=FO, 10% by Wt). The experimental diets were started from pregnancy in four different groups, each consisting of 9 rats. The seound generation rats were fed the same diet as their mothers. Animals were anesthetized with ether at 0, 3, 9 & 16 weeks of age. Whole brains were dissected out, brain tissues were, then, homogenized and lipids were extracted from brain tissues. The fatty acid compositions were measured after methylation by gas-liquid chromatography at 0, 3, 9 and 16 weeks of age of offspring. The changes in the relative concentrations of polyunsaturated fatty acids(PUFA) or more specifically docosahexaenoic acid(22 : 6, $\omega$3, DHA), the major $\omega$3 fatty acid component in rat brain at different age were similar to changes in the amount of DNA in brain tissue showing the maximum value during the lactation. The changes in saturated fatty acid(SFA) content showed a contrasting patten to those of PUFA, while monounsaturated fatty acid(MUFA) increased steadily throughout the experimental period. At birth, the relative concentrations of $\omega$3 series fatty acids the relative concentrations of PUFA, MUFA and SFA converged to very similar values respectively regardless of the dietary fatty acid compositions. In brain tissue, it is of value to note that while changes in relative concentrations of linoleic acid (18 : 2, $\omega$6, LA) and arachidonic acid(20 : 4, $\omega$6, AA) showed a precursor-product-like relationship, $\alpha$-linolenic acid(18 : 3, $\omega$3, $\alpha$-LnA) and DHA showed a different pattern. Even when the $\omega$3 fatty acid content in very low in maternal diet(CO), the second generation rat brain tissues appeared to secure DHA content, suggesting an essential role of this fatty acid in the brain. The fact that a large amount of $\alpha$-LnA in the maternal diet did not have a significant effect on the second generation rat brain $\alpha$-LnA content, indicated that DHA seemed essential component for the brain development in our experimental condition. In all groups, the relative content of $\alpha$-LnA in the brain tissues remained relatively constant throughout the experimental period at the very low level. The study of the specific concentrations and essential role(s) of DHA in each parts of brain tissue is needed in more details.

• Journal of Nutrition and Health
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• 제28권7호
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• pp.602-611
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• 1995

This study was designed to find out the effects of $\omega$-3 and $\omega$-6 polyunsaturated and saturated fatty acid from prenatal to growing period on the brain growth and behavioral development of rats. Rats(Sprague-Dawley strain) were fed experimental diets-fish oil, corn oil or beef tallow-with different contents of $\omega$-3 and $\omega$-6 fatty acids throughout the prenatal and lactational period and up to 10 weeks of age. DNA and RNA concentration of rat brain were determined at 0, 3, 6 weeks of age and choline and acetylcholine concentrations were analyzed at 10 weeks of age. When the rats were 7 weeks of age, position reversional test in a Y-shaped water maze for 4 weeks was measured. The experimental results obtained are summarized as follows. Food intakes were significantly lower in fish oil group and body weight gain was low in the group fed beef tallow and the groups fed fish oil and corn oil were somewhat good. Food efficiency ratio was not significantly different among the groups. Brain weight was not affected by the fatty acid composition of experimental diets and DNA and RNA concentration of the rat brain were consistently maintained at the same level. It was not different significantly among the dietary groups in the DNA and RNA concentrations of the rat brain during the experimental period. The acetylcholine concentration in the fish oil group was somewhat higher than the other groups. The position reversional test in a Y-shaped water maze showed a significant difference the score of test among the experimental groups. The score of the rats fed the fish oil diet was significantly higher than the other groups and the concentration of acetylcholine in brain were too. Therefore the correlatin between the Y-shaped water maze test score and the acetylcholine concentratin in the brain was found. Above finding support the content that dietary fatty acid composition does not affect to the brain cell number and cell size but the behavior development is influenced. Therefore, the improvement of behavior development is required the effective usage of finny tribe.

• Biomolecules & Therapeutics
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• 제10권2호
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• pp.99-103
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• 2002

Taurine has a neuroprotective action from oxidative stress in neural cell. In the present study, we studied taurine transport under basal and stressed conditions in conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13) in vitro. The uptake of[$^3{H}$]taurine in the TR-BBB13 was increased by time-dependently and dependent on both Na$^{+}$ and Cl/ sup -/. Furthermore, $\beta$-alanine strongly inhibited the uptake of [TEX>$^3{H}$]taurine in the TR-BBB13. To study the effcts of oxidative stress on taurine transport, we used diethyl maleate (DEM) and lipopolysccharide (LPS). Diethyl maleate (DEM, $300\Mu\textrm{M}$) significantly reduced uptake of [TEX>$^3{H}$]taurine by time-dependently until 8 hr exposure in TR-BBB 13. But, the [TEX>$^3{H}$]taurine uptake was not changed by lipopolysccharide (LPS, 10 ng/ml) in TR-BBB13.3.

• Journal of Korean Neurosurgical Society
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• 제65권2호
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• pp.180-185
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• 2022

Objective : Drug-eluting stents and balloons are occasionally used to reduce restenosis in medically intractable intracranial atherosclerotic stenosis. The authors aimed to determine whether such drugs can cause neurotoxicity due to local effects in a rat model. Methods : Intra-arterial catheters were placed in the right common carotid artery of rats. Mannitol was injected to transiently open the brain-blood barrier (BBB), followed by high-dose drug (paclitaxel and rapamycin) injection. The optimal time interval of transient BBB opening for maximal drug penetration was determined to be 10 minutes. Paclitaxel and rapamycin were intra-arterially administered in various doses. All the rats were neurologically evaluated, and their brain tissues were histologically examined. Results : Neither neurological deficits nor histological abnormalities were observed in all the rats. Conclusion : Paclitaxel and rapamycin did not cause neurotoxicity in a rat model with transient BBB opening.