S67Abstracts / Cell Biology International 32 (2008) S1eS67

CpdB on the PVN neurons may be mediated by the phosphorylation of GA-

BAA receptors through CaM-KII and this process was initiated by Ca2+ influx

through T-type calcium channels. We found that oral administration of

ECBRC-AG and CpdB reduced plasma corticosterone levels in rats during

light period, which indicates the down regulation of HPA axis functions.

Our data suggested that the HPA axis is involved in the sedative effects of

ECBRC-AG and CpdB, and T-type calcium channels may play important roles

in mediating the inhibitory effects of CpdB on the PVN neurons.

ASTROCYTES EXPRESS FUNCTIONAL N-METHYL-D-ASPARTATE RECEPTORSXiong Zhang, Chu ChenNeuroscience Institute, Zhejiang University School of Medicine,Hangzhou, China 310058Increasing evidence indicates that astrocytes can respond to the chemical

transmitter glutamate, since they express the ionotropic and the metabotropic

glutamate receptors. However, the N-methyl-D-aspartate (NMDA) receptor,

one of the ionotropic glutamate receptors broadly expressed in neurons, has

still not been identified in astrocytes in vivo. Using hippocampal slices from

adult GFAP-GFP transgenic mice, we found that a stimulus-evoked current

in astrocytes could be inhibited 38.1 � 5.1 % and 82.7 � 2.0% by the selective

NMDA receptor antagonist D-AP5 in Mg2+-containing and in Mg2+-free extra-

cellular solution, respectively. Furthermore, the mRNAs of the NMDA recep-

tor subunits NR1, NR2A and NR2B were detected in astrocytes using single

cell rt-PCR analysis. Finally, to investigate the functional significance of these

NMDA receptors, we subjected astrocytes to tetanic stimulation and found, as

in neurons, tetanic stimulation induced LTP-like responses in astrocytes,

which were blocked by D-AP5, but not by the selective AMPA receptor antag-

onist DNQX or by a K+ channel inhibitor Cs+. These results suggest that func-

tional NMDA receptors are expressed in hippocampal astrocytes of adult mice.

And astrocytic NMDA receptors may contribute to enhanced astrocytic re-

sponses to neuron activity. Supported by National Science Foundation of

China (Grant No.30600169).

EFFECT OF ESTROGEN ON OXYGEN-GLUCOSEDEPRIVED INJURY OF ASTROCYTESYan Zhang, Li Xiang Wu, Fang He, Li Juan Yang, Bo Sheng Huang,Xiao Lu DengDepartment of Physiology, Xiangya School of Medicine, CentralSouth University, Changsha, ChinaThe aim of this study was to explore the effect of estrogen on oxygen-glucose

deprived injury and apoptosis of astrocytes. Primary culture of the rat cerebral

cortical astrocytes with a cell damage model was induced by oxygen-glucose

deprivation. Astrocytes were pretreated with estrogen at various final concen-

tration of 1-100nmol/L. The change of cell morphology was observed by

Giemsas staining. The cell damage and viability were evaluated by 3- [4,5-di-

mehyl-2-thiazolyl] -2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction

assay and lactate dehydrogenase (LDH) released rate, and the detection of ap-

optotic cells was determined by the flow cytometry. Oxygen-glucose depriva-

tion decreased the cell viability, increased the LDH release rate and increased

the number of apoptotic astrocytes. While with the estrogen pretreatment at the

same condition, the cell viability increased, the LDH release rate decreased,

and the percentage of apoptotic cells decreased (p<0.05). The maximal pro-

tective dose was 20 nmol/L. Our results show that estrogen could protect pri-

mary cultured astrocytes from oxygen-glucose deprivation injury, and

attenuate the apoptosis of astrocytes in a dose-dependent manner.

THE INFLUNCE OF THE STRUCTURE OF RIBOZYME,M1GS, ON ITS ACTIVITYYong Xia Zheng, Xin Mei Zhou, Huang Xu, Bo DongDepartment of Biochemistry, Jiaxing University School of Medicine,Jiaxing, ChinaTo investigate the rule of the varying structure of M1RNA with guide sequence

(M1GS) on its activity in vitro, we constructed a specific ribozyme, M1GS-T7,

which targeted the mRNA segment of deoxyribonucleic acid polymerase gene,

unique long 54 gene (UL54), in human cytomegalovirus (HCMV). We simu-

lated the secondary structure of M1GS-T7 under different temperature by

Vienna RNA Package. Meanwhile, we tested its activity in relevant tempera-

ture. The simulation showed that if the change of temperature resulted in mi-

nor diversification, then the activity varied but existed; if that led to failing in

formation of catalytic center, meanwhile the activity lose. We made the con-

clusion that the formation of ribozyme’s catalytic center was necessary for

its activity, while flexibility of catalytic center led to the change of its activity

degree.

AN ENHANCED METHOD FOR OPTIMAL MMPASESTAINING ON PARAFFIN EMBEDDED OF AS SECTIONSJun Zhou 1, Ai Fujimoto 2, Jagat Narula 2

1 The Third Xiang-ya Hospital, Central South University Xiang-YaSchool of Medicine, Changsha, Hunan Province, China2 Division of Cardiology, University of California, Irvine School ofMedicine, Irvine, California, USAMatrix metalloproteinase (MMPs) are a group of>20 zinc containing endopep-

tidases that are either secreted or expressed at the cell surface of all of the main

vascular cell types. Intervention studies in animal models have been used to in-

vestigate the potential roles of MMPs in cardiovascular disease. In standard im-

munohistochemical procedures, We incubated formalin-fixed and paraffin-

embedded atherosclerotic lesion sections of rabbit with a spectrum of routinely

employed MMP-1, 2, 3, 7, 8 and 9 antibodies and used unmask the antigens

methods within incubation heating. But the immunohistochemical staining of

these MMPase showed weak signal in section of formalin fixation and paraffin

embedded. Tyramide signal amplification is an enzyme-mediated detection

method that utilizes the catalytic activity of horseradish peroxidase to generate

high-density labeling of target protein. The yield in terms of target amplifica-

tion has allowed three to five thousands fold dilution of primary antibodies.

This study evaluated a Tryamide-enhanced immunoassaying method on athero-

sclerotic lesions sections of paraffin embedded. Results, compared with the

standard immunoassaying technique, Tyramide-enhanced immunostaining,

showed superior signal intensity on these tissue specimens. This method is

highly sensitive and obtained optimal staining of MMPase without a loss of

specificity or increment of background staining. Tyramide signal amplification

provides an excellent morphology result and a powerful tool.

THE CORRELATION OF INTEGRIN BETA 4 EXPRESSIONAND ASTHMAXiao Yan Zhou, Yu Rong Tan, Yang Xiang, Hui Jun Liu, Fei Qu,Chi Liu, Xiao Ling Zhu, Xiao Qun Qin*

Department of Physiology, Xiangya School of Medicine, CentralSouth University, Changsha, 410078, ChinaAdhesion molecules expressed on leukocytes play a vital role in airway hyper-

responsiveness or airway inflammation. In order to probe the relationship be-

tween imbalance of expressions of adhesion molecules and asthma

pathogenicity, we designed experiments to screen differential expression adhe-

sion molecules by using cDNA microarray. The results showed that there were

various adhesive molecules with abnormal expressions and expressions of in-

tegrin b4 were down-regulated in all 6 asthma patients, which was further ver-

ified by real-time PCR. In order to verify the correlation of integrin beta 4

expression and asthma, the 5’flanking region of integrin b 4 from 100 asthma

patients, age from 50 to 60 with COPD and 20 normal adults were amplified

and sequenced. The expression of integrin beta 4 was also assayed by real-time

PCR. The result showed that there were three variation sites in 5’flanking re-

gion of integrin b4 at e1106, -1143 and -1162 from A to G in asthma patients

which were correlated with downregulated expression of integrin beta 4. This

present study demonstrated that Integrin b4 was highly correlated with asthma.

Down-regulated integrin b4 expression was associated with the variation in

5’flanking region.