Table of Content

24 April 2006, Volume 22 Issue 2

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Articles

Genetic Structure Analysis of Human Remains from Khitan Noble Necropolis

XU Yue, ZHANG Xiao-lei, CUI Yin-qiu, ZHANG Quan-chao, ZHOU Hui, ZHU Hong

2006, 22(2):  123-128. 

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Ancient DNA was extracted from 13 skeletal remains from the burial groups of Khitan nobles, which were excavated in northeast China.The hypervariable segment I sequences (HVS Ⅰ) of the mitochondrial DNA control region, in the 13 individuals, were used as genetic markers to determine the genetic relationships between the individuals and the genetic affinity to other interrelated populations by using the known database of mtDNA.Based on the phylogenetic analysis of these ancient DNA sequences, the genetic structures of two Khitan noble kindreds were obtained, including the Yel Yuzhi's kindred and the Xiao He's kindred.Furthermore, the relationships between the Khitan nobles and some modern interrelated populations were analyzed.On the basis of the result of the analysis, the gene flows of the ancient Khitans and their demographic expansion in history was deduced.

Expression Optimization and Characterization of the Catalytic Domain of Human MT3-MMP

SHI Xiu-juan, JIN Feng-hai, WANG Hui-ling, YANG Jin-gang, WANG Zhi-yong, FANG Xue-xun

2006, 22(2):  129-133. 

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Matrix metalloproteinases(MMPs) are a family of proteases that are required for many biological processes and are also elevated in many pathological conditions.MMP inhibitors (MMPIs) may therefore be useful as therapeutic agents in treating a number of diseases including cancer, cardiovascular diseases and arthritis.Attempts have been made to develop MMPIs.Recombinant MMPs have been used to screening MMPs in vitro assays.In this work, we report the expression of MMP-16 in E.coli and the characterization of the recombinant MMP-16 with a commonly used MMP substrate DQ-gelatin.

Statistical Optimization of Medium Components for Improved Product Ion of Recombinant Hyperthermophilic Esterase

REN Xiao-dong, YU Da-wei, HAN Si-ping, FENG Yan

2006, 22(2):  134-138. 

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The optimization of nutrient levels for the production of recombinant hyperthermophilic esterase by E.coli was carried out with response surface methodology(RSM) based on the central composite rotatable design(CCRD).A 2⁴central composite rotatable design was used to study the combined effect of the nutritional constituents like yeast extract, peptone, mineral salt and trace metals.The P-value of the coefficient for the linear effect of peptone concentration was 0.0081 and trace metals solution was less than 0.0001, suggesting that these were the principal variables with significant effect on the hyperthermophilic esterase production.The predicted optimal hyperthermophilic esterase yield was 269.17 U/mL, whereas an actual experimental value of 284.58 U/mL was obtained.

Effect of Notoginsenoside-Rg₁ on the Expression of Several Proteins in the Striatum of Rat Models with Parkinson's Disease

ZHANG Lin-bo, LIU Xiao-hua, JIANG Yuan, GUO Ping, SHA Li-jin, LI Yu

2006, 22(2):  139-144. 

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After establishing hemi-Parkinsonian rat models, the relationships between neuron death and the expression of several proteins, such as c-Fos, GFAP, GDNF, NF-κB and some cytokines were determined.Therapeutics experiments with notoginsenoside-Rg₁ were carried out.The research results show that the expressions of GFAP, NF-κB and c-Fos will obviously increase in the lesion side of the striatum and the expression of GDNF will decrease, which implies that the signal transduction pathway may participate in the apoptosis in neurons.The levels of some cytokines such as TNF-α, IL-1β in the striatum of PD rat models increased compared to those of normal rats.The results of the therapeutics experiments show that notoginsenoside-Rg₁ may repress the immune inflammation response and regulate the immune function through the neuro-immune molecular network.Therefore, notoginsenoside-Rg₁ can be used as an effective drug for anti-oxidation and anti-inflammation, and can be used in the therapy of Parkinson's disease(PD).

cDNA Cloning, Prokaryotic and Eukaryotic Expression and Characterization of Porcine Leukemia Inhibitory Factor

LI Ming-tang, JIANG Yong, WANG Shu-mei, LI Yong-ming, WANG Fang, HOU Xia, YANG Hong, MA Tong-hui

2006, 22(2):  145-149. 

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Molecular cloning of the porcine leukemia inhibitor factor(pLIF) has not been reported.A full-length cDNA encoding pLIF was cloned, expressed and characterized.The full-length porcine LIF cDNA encodes a 202 amino acid protein that has an 84% sequence identity to mouse LIF and 86% sequence identity to human LIF.The deduced amino acid sequence of a pLIF protein contains six conserved consensus N-linked glycosylation sites and six cysteine groups to form potential disulfide bonds.The pLIF was expressed in E coli, as a mature form, and in CHO cells as a secreted form.Both the forms of the recombinant pLIFs can maintain murine embryonic stem cells in an undifferentiated state in a culture.The recombinant pLIFs will be useful in establishing a long-term culture of stable pluripotent porcine embryonic stem cells for further manipulation.

Reconstruction of Rabbit Corneal Layer Composed of Corneal Fibroblasts and Corneal Epithelium on the Lyophilized Amniotic Membrane

AHN Jae-il, JANG In-keun, YOON Mun-young, SEO Young-kwon, YOON Hee-hun, KIN Jae-chan, SONG Kye-yong, YANG Eun-kyung, PARK Jung-keug

2006, 22(2):  150-156. 

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Many researchers have employed the cryopreserved amniotic membrane(CAM) and corneal epithelial cells in the treatment of a severely damaged burned cornea, with corneal epithelial cells cultured on an amniotic membrane (AM).The lyophilized amniotic membrane (LAM) has a higher graft take and a longer shelf life; it is easier to store and safer because of gamma irradiation.Two Teflon rings(Ahn's supporter) were made for culturing the cells on the LAM, and were then used to support the LAM.To reconstruct a corneal layer composed of corneal fibroblasts and epithelium, the corneal fibroblasts were first cultivated on the stromal side of LAM for five days, followed by epithelial cells culture on the epithelial side, by using the air-liquid interface culture.The reconstructed corneal layer composed of corneal fibroblasts and corneal epithelial cells has a much healthier basal layer of corneal epithelium than the reconstructed corneal epithelium, which was got by using only corneal epithelial cells, and resembles the epithelium of normal corneas, without the horny layer.Thus, the reconstruction of the corneal layer by using a LAM is considered to be a good in vitro model, not only for its application in toxicological test kits, but also for transplantation in patients with a severely damaged cornea.

Expression and Characterization of Human Heart Type Fatty Acid Binding Protein in Pichia Pastoris

HOU Wei, TAN Yan, XU Shu-fen, YANG Xiao-hong, ZHANG Shu-hua, LIU Ling-li, CHE Yuan-yuan, LIU Li-hua

2006, 22(2):  157-161. 

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H-FABP is regarded as a tissue-specific protein existing only in myocardial cells.It is released from the cardiac tissue and gets into the plasma when a heart attack occurs; the myocardial infarction is a good case in point.As a result, the detection of H-FABP will be an early and important biomarker for the disease concerned.The objective of the study is to prepare the recombinant H-FABP by aeukaryotic expression system, pichia, to produce the protein mimicking natural H-FABP, as an immunogen for the production of the specific antibody.A gene fragment encoding H-FABP was cloned in the expressing vector pPICZα, after sequencing.The recombinant plasmid was transformed into the competent cells of the X-33 strain by means of electroporation.The expression of the target peptide indueed by methanol was screened by means of Western blotting, with the available MAb(Clone 6B6).Highly expressive engineer strains were obtained.The production of recombinant H-FABP under induction was about 0.7 g/L, with an M_r of 14.5 kDa and recognized by a commercially available MAb (Clone 6B6).The recombinant vector was successfully constructed.Following this, H-FABP was expressed in X-33, and it would become the source of the preparation of specific antibodies, to develop diagnostic kits.

Extraction and Purification of Depigmenting Agents from Chinese Plants

JIN Yin-zhe, LI Guang-hua, AHN So-young, ROW Kyung-ho, KIM Eun-ki

2006, 22(2):  162-167. 

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Depigmenting agents were solvent-extracted and purified by preparative and analytical HPLC from three Chinese plants; Chrysanthemum morifolium Ramat(Xizang Caijuhua), Rhodiola sachalinensis, and Terminalia chebula Retzius.Four fractions obtained from the ethyl ether layer of C.m.Rama and two fractions from the ethyl acetate layer of Rhodiola salientness show depigmenting effects.At δ 200, the ethyl acetate layers of Chrysanthemum morifolium Ramat, Rhodiola sachalinensis and the methanol extract of Terminalia chebula Retzius, can inhibit the melanin production of mouse B16 melanoma cells by 92%, 60% and 90%, respectively, whereas 46% inhibition was observed by commercially available depigmenting agents(arbutin).These results show the potential of these three Chinese plants as a novel resource for depigmenting agents in the cosmetic industry.

Denaturing Effects of Urea and Guanidine Hydrochloride on Hyperthermophilic Esterase from Aeropyrum pernix K1

GAO Ren-jun, XIE Gui-qiu, ZHOU Jun, FENG Yan, CAO Shu-gui

2006, 22(2):  168-172. 

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The changes in the activity and the conformation of the hyperthermophilic esterase derived from aerobic thermophilic Aeropyrumpernix K1 (APE1547) were studied during denaturation by guanidine hydrochloride (GdnHCl)and urea.The denaturation course of APE1547 was followed by the steady-state and time resolved fluorescence methods.An increase in the denaturant concentration in the denatured system can significantly enhance the inactivation and unfolding of APE1547.The enzyme can be completely inactivated with a urea concentration of 2.7 mol/L or a GdnHCl concentration of 7.5 mol/L.The fluorescence emission maximum of the enzyme protein red shifts in magnitude to a maximum value(355 nm) when the concentration of GdnHCl is 5.1 mol/L.The experimental results indicate that APE1547 has a high resistance to urea.Unfolding of APE1547 in GdnHCl(4.2-6.0 mol/L) was shown to be an irreversible process.The present results indicate that the ion pairs in this protein may be a key factor for the stability of this esterase.

Growth Inhibition Effect of Parthenolide on Human Hepatocellular Carcinomacell Line BEL-7402

SONG Xiao-kai, ZHANG Wen

2006, 22(2):  173-176. 

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To study the effects of parthenolide on human hepatocellular carcinoma cell line BEL-7402 and explore its possible mechanism, human hepatocellular carcinoma cell line BEL-7402 was cultured in vitro and treated with parthenolide of different concentrations.Cells without addition of parthenolide were used as control.The growth of inhibition of cells induced by various concentrations was analyzed by using the MTT assay.The morphologic changes of apoptosis was observed under an inversion microscope by Giemsa staining.The expression levels of PCNA albumen were measured by means of immunohistochemical methods.Parthenolide can inhibit the proliferation of BEL-7402 cells in vitro in adose-dependent manner.Apoptosis with nuclear chromatin condensation, fragmentation and cell shrinkage were found by means of inversion microscopy.Formation of apoptotic bodies was observed by Giemsa staining.The immunohistochemical results show that the expression of PCNA has been decreased.Parthenolide can inhibit the tumor growing of BEL-7402 cells, and induce the apoptosis of cells.The mechanisms of those functions may be via inhibiting the expression of PCNA.

Phylogenetic Analysis of mtDNA from the Ancient Human of Yuan Dynasty in Inner Mongolia in China

FU Yu-qin, XU Xue-lian, ZHANG Xiao-lei, ZHANG Quan-chao, ZHOU Hui, ZHU Hong

2006, 22(2):  177-180. 

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A study of the genetic structure of an ancient human excavated from the Yikeshu site of Yuanshangdu ancient city in Inner Mongolia and the relationships between the ancient population and the extant populations was carried out.Sequences of the control region and coding region of mtDNA from the ancient human were analyzed by using direct sequencing and restriction-fragment length polymorphism (RFLP) methods.Phylogenetic analysis and multidimensional scaling analysis were also performed on the mtDNA data of the ancient population and 12 extant populations.These results show that the ancient individuals of Yikeshu site can be assigned to D, G, B and Z haplogroups that are prevalent in Duars and Mongolians from Inner Mongolia.The ancient population is also closer to Duar and Mongolian populations in genetic distance than other compared populations.This study reveals that the ancient population from Yikeshu site in the Yuan Dynasty shares a common ancestor with Mongolic-speaking Daur and Mongolian tribes.

NHS Mediated CdTe Quantum Dots/Albumin Conjugates and Labeling C.Elegans

MA Hui-lian, WANG Chun-lei, LIU Han-zhi, LI Wei, XU Shu-kun, WANG Li-ping

2006, 22(2):  181-184. 

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Luminescent quantum dots(QDs) are a promising alternative to organic dyes for biomedical assays and imaging.A new conjugation method, NHS mediated conjugating, for QDs and BSA was introduced.The QDs-BSA conjugates were confirmed, and their stability has been proved.Caenorhabditis elegans (C.elegans) were used as animal models, and the imaging of QDs in the organism was studied.

Real-time Quantitative RT-PCR for CT9 Level in Human Cancer

JIN Xiang-qun, ZHANG Jing-min, XU Hui, ZHOU Yan, WANG Guang-shu, ZHAO Yan-qiu, ZHANG Han-qi

2006, 22(2):  185-188. 

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CT9 is a recently cloned cancer-testis antigen, which is a member of the bromodomain and extraterminal family.Each member of this protein family contains two N-terminal bromodomain motifs.We investigated the distribution of CT9 in different tissues and the possibility for it to be used as a potential therapeutic target in cancer treament.By using the real-time RT-PCR method and 18SrRNA as an internal standard, we analyzed the CT9 expression in several normal human tissues and in the tissues of patients suffering from cancer.The result of this study shows that the highest level of mRNA is only present in testis tissue because the CT9 expression has not been detected in other normal tissues.In 6 of 10 cases of gastric adenocarcinoma, in 3 of 10 cases of esophageal squamous cell carcinoma, in 2 of 9 cases of endometrial carcinoma and only in 1 of 12 cases of brain cancer, the low level expression of CT9 was detected.In none of the 12 cases of cervical squamous cell carcinoma, the expression of CT9 was detected.Since the high level expression of CT9 is only found in the normal testis tissue, but the low expression in cancer tissues, for example tissues of cervical squamous cell carcinoma, brain cancer, endometrial adenocarcinoma, esophageal squamous cell carcinoma, we conclude that CT9 cannot be used as a cancer therapeutic target molecule for cervical squamous cell carcinoma, brain cancer, endometrial adenocarcinoma, esophageal squamous cell carcinoma.

6-O-Sulfated Modification of Natural Glycoalkaloids Chaconine and Solanine

ZHAO Ji-min, LI Sheng-yu, ZHOU Yi-fa, ZHANG Li-ping, ZHOU Dao-wei

2006, 22(2):  189-192. 

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Glycoalkaloids(GAS) have important biological and pharmaceutical activities.In order to study the relationship between the structures and the activities of carbohydrate chains, two natural glycoalkaloids, chaconine(compound 1)and solanine(compound 2) , were isolated from potato stems and leaves(Solanum tuberosum L.).The selective sulfation to the 6-hydroxy groups of chaconine and solanine was carried out in a strategy by the use of protective groups.The 6-hydroxyl groups of the sugar chains in chaconine and solanine were protected with 4,4'-dimethoxytrityl(DMT)while the other hydroxyl groups were acetylated.The protective group DMT was removed by using 0.5% TFA in dichloromethane.The free 6-hydroxyl groups were sulfated by chlorosulfonic acid pyridine to give 6-O-sulfated products.After the acetyl groups were removed, the final products obtained were sulfated chaconine and sulfated solanine.¹³C NMR spectra confirmed that chaconine and solanine were sulfated at O6 of the carbohydrate moiety.

Inhibitory Effect of Anti-HER-2 Anti-CD3 Bi-specific Antibody on the Growth of Gastric Carcinoma

FANG Xue-dong, REN Hui, ZHANG Yan, WANG Guan-jun

2006, 22(2):  193-196. 

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To evaluate the effect of anti-HER-2 × anti-CD3 bi-specific antibody (BsAb) on the growth of HER-2/neu-expressing human gastric carcinoma in vitro and in vivo, an MTT assay was carried out to test the inhibitive rates of herceptin, anti-CD3 and BsAb antibodies on SGC-7901 gastric carcinoma cells.Immunocytochemistry methods were used to test the HER-2 level of SGC-7901.Nude mice models were employed to test the effect of HER-2 CD3 BsAb combined with effector cells(peripheral blood lymphatic cells of healthy human beings) on the growth of tumors in animals.Compared with that of the untreated control group, the tumor cell growth rates in vitro and in vivo will both be significantly inhibited when treated with effector cells combined with anti-CD3 McAb, herceptin or HER2 CD3 BsAb (p <0.05), and the growth inhibition is the most remarkable in the group treated with HER2 CD3 BsAb combined with effector cells.The growth of tumor xenografts will also be significantly inhibited in the group treated with HER2CD3 BsAb combined with effector cells when compared with that in the group treated with anti-CD3 McAb or the group treated with herceptin combined with effector cells(p < 0.05).We can conclude that HER-2/neu is possibly a useful target for immunotherapy of gastric carcinoma, and anti-HER2 × anti-CD3 BsAb has evident anti-tumor efficacy both, in vitro and in vivo.

A Repressor in 5'-UTR of hKv4.3 Gene

LI Hao, JIANG Chun-lai, YU Xiang-hui, WU Yong-ge, LI Wei, KONG Wei

2006, 22(2):  197-200. 

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The effect of repressors on ion channel gene expression was studied.The hKv4.3 promoter and the sequence (+ 2-+ 160, S160) of 5'-UTR of the hKv4.3 gene was cloned into the pβ-gal-Basic vector.The transient expression of the pβ-gal vector and the analysis of the relative activity of β-galactosidase were carried out.The analysis of the mRNA level was carried out with the RT-PCR method.S160 could intensively repress the expression of the hKv4.3 gene with position-specificity.The level of mRNA did not alter obviously.A repressor(S160), in 5'-UTR of the hKv4.3 gene was found and its repression to gene expression may play a role in the post-transcription process.

Construction of High Expression Plasmid of Human Augmenter of Liver Regeneration(hALR), Expression and Purification of hALR

SUN Tian-xu, WU Yong-ge, YU Xiang-hui, JIANG Chun-lai, JIN Ying-hua, CHENG Yue, KONG Wei

2006, 22(2):  201-204. 

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Experimental evidence has been presented to suggest that the human augmenter of liver regeneration (hALR)serves as a hepatotrophic growth factor during liver regeneration and as a generalized growth factor during pancreas transplant/regeneration.A prokaryotic expression plasmid, pRSET/6his-c-myc-hALR was constructed, by cloning synthesized hALR cDNA into pRSET/6his-c-myc that was improved on the basis of pRSET B by the group.As a result, the protein was highly expressed in E.coli BL21.The recombinant hALR was over 60% of the total protein in E.coli.Its validity was confirmed by meansof Western Blotting.The protein was purified by Ni-NTA affinity chromatography and this FAD-dependent sulfhydryl oxidase activity was measured.

A Study on Anti-oxidative Activity of Soybean Peptides with Linoleic Acid Peroxidation Systems

XU Li, LI Hong-mei, HUANG Yi-bing, WANG Hua, NIE Guang-jun, ZHANG Xue-zhong

2006, 22(2):  205-208. 

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Soybean bioactive peptides(SBPs) were prepared from the isolated soybean protein by proteolysis with an alkaline protease, alcalase, at 50 ℃ and pH = 8.0.The dependence of hydrolysis time on hydrolysis degree and molecular weight distribution were examined.The hydrolysate was fractionated on a Sephadex G-25 column and the anti-oxidative activities of the fractions were detected by the method of pyrogallol auto-oxidation.The average chain length of soybean peptides that have anti-oxidative activity was estimated to be about 7.The anti-oxidative properties of the soybean peptide were also studied by using linoleic acid peroxidation systems.The optimal condition of the peroxidation system was set up, Vc/Cu²⁺ as the inducer at pH = 7.4 and 25 ℃.In addition, soybean peptides show higher antioxidative activity compared with GSH.

Impact of Arsenic Trioxide on LS-174T Cell Growth in vitro and the Activity of Telomerase

WANG Xi-shan, WANG Gui-yu, XU Hai-tao, DONG De-li, FU Song-bin, ZHANG Qi-fan, YANG Bao-feng

2006, 22(2):  209-212. 

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The therapeutic action of arsenic trioxide(As₂O₃) on solid tumors has aroused widespread interest among scholars.To study the impact of As2 O3 on human colorectal carcinoma cells(LS-174T cell) and the activity of telomerase,the methods of PCR-ELISA, flow cytometry (FCM) and MTT assay in vitro were utilized.The results show that (1) with an increase in the concentration of As2 O3, the ratio of living the cells to dead cells decreases significantly,and the IC50 value is 5.23 μg/mL; (2) the cells of the experimental groups can endure a series of morphological changes similar to the features of apoptosis; (3) the apoptotic curves of FCM pictures appear after 24 h, and the cells show the apoptosis in a time-dependent manner; (4) As₂O₃ can inhibit the activity of telomerase of the cell extraction obviously in a concentration-dependent and time-dependent manner after 24 h.It can be concluded from the experiment results in vitro that As₂O₃ can induce the apoptosis of LS-174T cells and inhibit the telomerase activity.Therefore, it has been proposed, for the first time, that these two factors(the apoptosis of LS-174T cells and the inhibition to the telomerase activity) are important causes of the LS-174T cell death caused by As₂O₃.

Construction of Recombinant Modified Vaccinia Ankara (MVA) Expressing Hepatitis B Virus Surface Antigen

CHEN Yu, YANG Xiao-song, YU Xiang-hui, JIANG Chun-lai, WU Yong-ge, JIN Ying-hua, CHEN Yue, CHEN Yan, LI Wei, KONG Wei

2006, 22(2):  213-216. 

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The T lymphocyte response has been shown to be the determinant in the clearance of many viral infections.Hence, therapeutic vaccine candidates against HBV are designed to enhance this response of the immune system.Vaccinia virus vector-based vaccines have been proposed as excellent candidates to elicit long-term and strong T lymphocyte mediated immune responses.In this study, the recombinant MVA expressing HBV surface antigen has been constructed, which can elicit a potent T cell mediated response.The ELISA results for the surface protein in the medium of the recombinant MVA, strongly indicate that the recombinant virus has been successfully obtained.

Isolation and Characterization of Proteins Interacting with Activin Type H Receptors

LIU Biao, BAO Yong-li, WEI Zhuang, WU Yin, MENG Xiang-ying, LI Yu-xin, YIN Wei-tian

2006, 22(2):  217-220. 

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Regulation of the number of activin receptors that are present in the cell membrane plays a key role in the modulation of cellular responses to activin.In order to find the regulators, a novel protein ARIPzip, interacting with activin type Ⅱ receptors, was searched and identified by using yeast two-hybrid screening.ARIPzip is a splicing variant of ARIP2.This has been discussed previously.ARIPzip can specifically interact with ActR Ⅱ A, and is widely distributed in mouse tissues.Overexpression of ARIPzip can cause the activin-induced transcriptional activities to increase in a dose-dependent manner while the overexpression of ARIP2 can decrease these activities.These data suggest that the C-terminal regions of ARIP2 and ARIPzip are involved in the regulation of activin signaling.

Kinetics of Phosphatase of Regenerating Liver-3 (PRL-3) Inhibition by Small-molecular Inhibitors

SHEN Xing-gui, SUN Li-wen, JIAO Ming, LI Zhao-fa, ZHAO Zhi-zhuang, LI Qing-shan, FU Xue-qi, HUANG Zhong-xiu

2006, 22(2):  221-224. 

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Phosphatase of Regenerating Liver-3 (PRL-3) is a newly identified colorectal cancer metastasis-related protein,which isa 22 kDa non-classical protein tyrosine phosphatase with a C-terminal prenylation motif.In this study, the inhibition kinetics of protein tyrosine phosphatases (PTPs) by a fluorescent substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) was evaluated.PRL-3 exhibits classical Michaelis-Menten kinetics with a v_max value of 11.3 -μmol· L^-1 · m in^-1.Analysis o f PRL-3 by aM ichae lis-M enten plot and a doub le-rec iprocal plot ind ica ted that the inhibitor magnolol can cause K_m to increase, but does not alter the v_max value, which suggests the competitive inhibition of PRL-3.At the same time, it was found that DiFMUP is a more sensitive substrate for PRL-3 than para-nitrophenyl phosphate(pNPP) that is more frequently used at present.Furthermore, the method of screening for PTPs by the use of DiFMUP was developed, which studied the acceptance of DiFMUP by other PTPs.

Design and Synthesis of a Novel Peptidomimetic Inhibitor of Caspase-3

ZHANG Jin-liang, LIU Lin-hua, GUO Yang-hong, YANG Jin-gang, LI Wei, ZHONG Da-fang, WANG Ji-dong

2006, 22(2):  225-228. 

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Caspases, a family of cysteine proteases, comprise of highly homologous enzymes that play an important role in apoptotic cell death.Caspase-3 shows key functions in apoptosis, mediating apoptotic cascade from the intrinsic and extrinsic activation pathways.Therefore, caspase-3 is an attractive target for therapeutic intervention.For instance,inhibitors of caspase-3 have been described as promising cardioprotectants, neuroprotectants and antiarthritic agents.A novel peptidomimetic inhibitor of caspase-3, has been designed, which still has the properties of a reversible inhibitor, while the P1 site at the C-terminal remains, and only L-amino acid has been replaced by D-amino acid.Also presented here is the synthesis of the inhibitor and its inhibitory activity against caspase-3, which was tested by the fluorescent activity assay.

Synergistic Effects of Activin A and Fibroblast Growth Factor 2 in the Modulation of Insulin Expression

BAO Yong-li, JIANG Hong-yu, JI Shou-xian, WU Yin, MENG Xiang-ying, LI Yu-xin

2006, 22(2):  229-232. 

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Diabetes is the most prevalent and serious metabolic disease, and the number of diabetic patients worldwide is increasing.The reduction of insulin biosynthes is in pancreatic β-cells is closely associated with the onset and progression of diabetes, therefore, it is important to search for ways to induce insulin-producing cells in non-β-cells.In the present study, it has been reported that activin A and a basic fibroblast growth factor 2 (FGF2), can synergistically increase the insulin mRNA level, in both mouse E14 striatal primary cell cultures and the hippocampal neuronal cell line HT22.Activin A and FGF2 can jointly stimulate the nuclear translocation of Smad3 and specifically activate ERK1/2.It is interesting to note that a specific inhibitor for MEK, U0126, can efficiently block the induction of an insulin promoter activity by activin A and FGF2.This indicates that activin A collaborates with FGF2, giving a signal to induce the insulin gene through selective activation of the ERK-type MAP kinase and Smad3 in mouse striatal and HT22 cells.These data suggest that activin A may act in concert with FGF2 for the development of insulin -positive neurons.

Expression of RNA Editing Deaminase on Human Glioma Cell Lines

TIAN Yu, PAN Yu-zuo, GAO Yu-fei, LI Gui-lin, ZHAO Xing-li, GAO Jun, LI Gui-ying, ZHANG Xing-yi, WANG Ren-zhi

2006, 22(2):  233-235. 

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To study the expression of RNA editing deaminases ADAR2 and ADAR3 in different malignant glioma cell lines and the effect of phenylacetate on the expression of these genes, the primarily glial cells of human brain tissue were isolated and cultured.The human glioma SHG-44, U-251, BT-325 cell lines were maintained in culture.The expressions of ADAR2 and ADAR3 mRNA were detected by the semiquantitative reverse transcription-polymerase chain reaction(RT-PCR).The changes in ADAR2 mRNA expression before and after phenylacetate treatment were detected by RT-PCR and image analysis.The level of ADAR gene expression is expressed as the ratio expression rate(RER)of ADAR gene to β-actin according to computer image analysis.ADAR2 displays moderate expression in glial cells,low expression in low-grade malignant glioma SHG-44 cells, and high level expression in high-grade malignant glioma U-251and BT-325 cells.The expression of ADAR2 can be decreased by phenylacetate treatment in glioma U-251cells.ADAR3 is not expressed in normal brain glial cells, or glioma SHG-44, U-251 and BT-325 cells before and after phenylacetate treatment.The enhanced expression of ADAR2 may be involved in the tumor progression of malignant glioma.Phenylacetate can decrease the expression of ADAR2 in glioma cells, suggesting that it may act on the RNA editing process in glioma.

Cloning and Expression of Rat Liver S-Adenosylmethionine Synthetase

CUI Wei, HUANG Lei, LI Wei, WANG Li-ping

2006, 22(2):  236-238. 

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The S-adenosylmethionine synthetase (SAM synthetase) is responsible for in vivo synthesis of S-adenosylmethionine(SAM), which is a kind of biologically active molecules distributed in all body tissues and fluids and involved in a number of biochemical reactions.In this study, a cDNA containing the coding sequence for rat liver SAM synthetase was cloned into the prokaryotic expression vector pQE30 and expressed in E.coli M15.A major band corresponding to a protein of 48 kDa was detected on SDS-PAGE.The protein was distributed in both the soluble fraction and the insoluble fraction.In soluble fractions the protein was fully active.

Effects of Phenylacetate on Cell Proliferation and Homeobox Genes Expression in the HCT-8 Colorectal Carcinoma Cell Line

ZHANG Yan, REN Hui, FANG Xue-dong, TIAN Yu

2006, 22(2):  239-241. 

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To study the effects of phenylacetate (PA) on cell proliferation and homeobox (HOX) genes expression in the colorectal carcinoma HCT-8 cell line, HCT-8 cells were grown in the presence or absence of PA.The cellular proliferation inhibition was evaluated by the MTT assay.Twenty-two HOX genes were divided into three groups (P1, P2,P3) according to their primer sequences, and the samples of cells were analyzed for the HOX genes' mRNA expression by means of the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).The level of the HOX genes' expression was expressed as the ratio expression rate of HOX gene to theβ-actin.HCT-8 cells were treated with 1.0-5.0 mmol/L PA for 24-72 h.With the increase of the PA concentration or the prolongation of the treating time, the cell proliferation is inhibited in a dose- and time-dependent manner.The P1 group mRNA * expression(0.5781 ± 0.0836) is significantly lower than that of the untreated group (0.7701 ± 0.0883) in HCT-8 cells (p<0.001).Both the mRNA expressions of groups P2(0.3941 ±0.0819) and P3(0.5601 ±0.0736) in the PA treated group are significantly higher than those of the untreated groups P2 (0.1221 ± 0.0782) and P3 (0.1806 ±0.0811) in HCT-8 cells (p< 0.001).PA could effectively inhibit cell proliferation by regulating the HOX genes expression and the mechanisms of the PA action are correlated with the transcription process in HCT-8 cells.

Binding Mode of Insulin Receptor and Agonist Peptide

WANG Song, WANG Li-ping, SHAN Ya-ming, WANG Yu-hong, LI Wei, SUN Chia-chung

2006, 22(2):  242-244. 

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Insulin is a protein hormone secreted by pancreatic β cells.One of its main functions is to keep the balance of glucose inside the body by regulating the absorption and metabolism of glucose in the periphery tissue, as well as the production and storage of hepatic glycogen.The insulin receptor is a transmembrane glycoprotein in which two α subunits with a molecular weight of 135 kD and twoβ subunits with a molecular weight of 95 kD are joined by a disulfide bond to form a β-α-α-β structure.The extracellular α subunit, especially, its three domains near the N-terminal are partially responsible for signal transduction or ligand-binding, as indicated by the experiments.The extracellular α subunits are involved in binding the ligands.The experimental results indicate that the three domains of the N-terminal of the α subunits are the main determinative parts of the insulin receptor to bind the insulin or mimetic peptide.We employed the extracellular domain (PDBID: 1IGR) of the insulin-like growth factor-1 receptor (IGF-1 R) as the template to simulate and optimize the spatial structures of the three domains in the extracellular domain of the insulin receptor, which includes 468 residues.The work was accomplished by making use of the homology program in the Insight Ⅱ package on an Origin3800 server.The docking calculations of the insulin receptor obtained by homology with hexapeptides were carried out by means of the program Affinity.The analysis indicated that there were hydrogen bonding, and electrostatic and hydrophobic effects in the docking complex of the insulin receptor with hexapeptides.Moreover, we described the spatial orientation of a mimetic peptide with agonist activity in the docking complex.We obtained a rough model of binding of DLAPSQ or STIVYS with the insulin receptor, which provides the powerful theoretical support for designing the minimal insulin mimetic peptide with agonist activity, making it possible to develop oral small molecular hypoglycemic drugs.

Inhibition of Metalloproteinase Activity by Chinese Formulations Used to Treat Inflammatory Diseases

JIN Feng-hai, WANG Hui-ling, ZHAO Shu-hua, YANG Jin-gang, FANG Xue-xun

2006, 22(2):  245-247. 

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Matrix metalloproteinases (MMPs) are known to be involved in a number of pathological processes including cancer, atherosclerosis, arthritis and neurodegenerative disease among others.The drive to develop MMP inhibitors as therapeutics has been put forward for years by pharmaceutical companies as well as academicians.In an attempt to screen for MMP inhibitors from Traditional Chinese Medicines (TCMs), a number of Chinese formulations used to treat inflammatory diseases such as nephritis and hepatitis have been studied.Strong inhibitory effects of three Chinese formulations toward the activity of MMP-16 have been discovered.These results suggest that these anti-inflammatory medicines contain some unknown MMP inhibitory compound(s) and provide reasonable molecular mechanisms for their therapeutic effects.

A Global Minimization Algorithm for Empirical Contact Potential Functions

WANG Yu-hong, LI Wei

2006, 22(2):  248-250. 

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Global minimization algorithm is indispensable for solving protein folding problems based on thermodynamic hypothesis.A contact difference (CD) based on pseudo potential function, for simulating empirical contact potential functions and testing global minimization algorithm was proposed.The present article describes a conformational sampling and global minimization algorithm, which is called WL, based on Monte Carlo simulation and simulated annealing.Itcan be used to locate CD's globe minimum and refold extended protein structures, as small as 0.03 nm, from the native structures, back to ones with root mean square distance(RMSD).These results demonstrate that the global minimization problems for empirical contact potential functions may be solvable.

Research Notes

Structure-function Relationships in Human Hypoxanthine-guanine Phosphoribosyltransferase (HGPRT) by Random Mutagenesis

ZHENG Yuan-zhi, Luke Guddat, Anthony Farlow, John de Jersey

2006, 22(2):  251-252. 

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Recent Progress of Thioester Method

Saburo Aimoto, Kenta Teruya, Koki Hasegawa, Takeshi Sato, Kenichi Akaji, Toru Kawakami

2006, 22(2):  253-257. 

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Dimethyltyrosine, the Viagra of Opioids

Lawrence H.Lazarus, Sharon D.Bryant, Severo Salvadori, Remo Geurrini, Gianfranco Balboni, Yuko Tsuda, Yoshio Okada

2006, 22(2):  258-262. 

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Mammalian-like Purple Acid Phosphatases in Plants

John de Jersey, ZHENG Yuan-zhi, FAN Hong-kuan, Gary Schenk, Luke Guddat, Susan Hamilton

2006, 22(2):  263-264. 

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Synthetic Channel-forming Peptides and Ion Selectivity

GUO Li-li, WANG Dong-xia, Roger W.Roeske

2006, 22(2):  265-266. 

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Biosynthesis of Monoterpenoid Indole Alkaloid Ajmaline Catalyzed by Novel Reductases

GAO Shu-juan, Gerald von Schumann, Joachim Stöckigt

2006, 22(2):  267-269. 

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