Vol 6 No.1 1996

Construction of normal human IgE phage antibody library and the screedning of the anti-trichosanthin IgE

ZAN HONG, MING YEH
Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China

ABSTRACT
Allergen specific IgE response is the major cause of immediate hypersensitively. However the number of IgE-priducing B cells and the amount of IgE , especially the soecific IgE ,are not low, it greatly impedes the study of the allergic-specific antibody responses. Here we report the construction of a normal huamn IgE combinatorical libarary . The repetorie of IgE
VH genes and of k gene were separately amplified from normal human peripheral blood lymphocytes fhrough RT-PCR,and were then constructed to form the phage surface display humanFab(IgEVH) library. A plant protein allergen, trichosanthin(TCS), was used to affinity-enrich and to secrren the anti-TCS phage HuFab clones from the library. Human IgE(Fab) to TCS were detected.

Key words: Phageantibody library , human IgE(Fab), human anti-trichosanthin IgE

Assemly of lamins in vitro
MIN GUNG WEI,XIANG JUN TONG, BIN CHEN, BO ZHANG,ZHONG FAN LIU MING XIAODING,ZHONG HE ZHAI
College of Life and Molecular Engineering, Peking University Beijing 100871,China

ABSTRACT
After lamins A,B and C were isolated and pirofoed from rat liver, their assembly properties were examined by electron microscopy and scanning tunneling microscopy using negative staining and the plycerol coating method, respectively. By varying the assembly time or the ionic conditions under shich polymerization takes place , we have observed different stages of lamin assembly, which may provide clues on the structure of the 10 nm lamin filaments. At the first level of stucture organization , two lamin polypeptides associate laterally into dimers with the two domains being paralled and in register ,. At the second level of structure organization , two dimers associate in a half-staggered and antiparallel fashion to form a tetramer 75 nm in length . At the third level of structural organization , 4-10 lamin tetramers associate laterally in register to form 75 nm long 10 nm filaments , which in turn combine head to head into long , fully assmenled lamin filaments. The assembled lamin filaments are nonpolar.

Key words: Nuclear lamina , lamins, scanning tunneling microscopy, assembly.

Correlation between inhibition of calcium -dependent apoptosis by cyclosporin A and calcium transportation in HL-60 cells

HUANG QI QING, MING FANG, HONG QING ZHANG, SHAO BAI XUE
Department of Biology, Beijing Normal University, Beijing 100875 , China.

ABSTRACT
Both calcium ionophore A23187 and endoplasmic reticulum Ca2+- ATPase inhibitionr thapsigargin (TG) could increase intracellular free calcium concentration and induce apoptosis in some cell lines . In the present study, we found thar HL-60 cells treated with A23187 (1μg/ml) for 4 h or with TG (0.5 ug/ml) for 2 h showed typical characteristics of apoptosis. Pretreatment with nontoxic concentration of cyclosporin A(CsA) (1ug/ml) could block these effects. Flow cytometric analysis of intracellular Ca2+ after staining with fluo-3 AM showed that CsA did not prevent the increase of intracellular calcium induced by A23187 or Tg, but it could maintain the high level of intancellular Ca2+ for a long time . These results suggest that CsA may prevent calcium- induced apoptosis by blocking the transportation of Ca2+ in HL-60cells.

Key words: Cyclosporin A, calcium ionophore A23187, thapsigargin, apoptosis, intracellular calcium.

Effect of osmotic shock on the redos system in plasma membrane of Dunaliella salina
CHEN SI XUE , CHI CHIONG YEN,XIN ZHI JIAO
Institute of Plant Physiology, Chinese Academy of Sciences, Shanghai 200031, China

Biology Department, East China Normal University, Shanghai 200062, China

ABSTRACT
The unicellular halotolerant alga Dunaliella salina had the ability to oxidize NADH and reduce Fe(CN)63-. The redox reactions were to some extent stimulated by slight hyperosomtic shock (2.0 mol/L NaCl), but markably inhibited by abtupt hyperosomotic shock(2.0 mol/L->3.5 mol/L NaCl ) and hypoosomtic shock (2.0 mol/L->1.0 mol/L NaCL; 2.0 mol/L->0.67 mol/L NaCl). With the adaptation of algal cells to osmotic shock by accumulating or degrading intracellular glycerol, the plasmalemma redox activities were also restored . The O2 uptake stimulated by NADH could be promoted by FA and SHAM. Hypoosmotic shock increase the bassl respiration rate of alga cells , but weakened the stimulating effects of NADH,FA and SHAM on O2 uptake. On the other hand , hyperosomotic shock reduced the basal respiration reate, but relatively enhanced the above effects of NADH, FA and SHAM. H+extrusion of alga cells was inhibited by NADH and stimulated by Fe(CN)63-. Vanadate and DES could inhibit H+ efflux, but had little effect in the presence of NADH and Fe(CN)63-. Both hyperand hypoosmotic shock stimulated H+ extrusion. This effect could be totally inhitited by vanadate and DES, but almost unaffected by 8-hydroxyuinoline . It was suggested that H+ ATPase probably played a more important role in H+ extrusion and osmoregulation under the conditions of osmotic shock.

Key words: Osmotic shock, Dunaliella salina, Plasmalemma redox system.

Chromosomal lacalization of a tandemly repeated DNA sequence in Trifolium repens L.

ZHU JM, NW ELLISON, RE ROWLAND

ABSTRACT
A karyotype of Trifolium repens constructed from mitotic cells revealed 13 paris of metacentric and 3 paris of submetacentric chromosomes including a pair of satellites located at the end of the short arm of chromosome 16. C-bands were identified around the centromeric regions of 8 paris of chromosomes. A 350 bp tandemlyrepeated DNA sequence from T . repens labeled with digoxygenin hybridized to the proximal centromeric regions of 12 chromosome paris. Some correlation between the distribution of the repeat sequence and the distribution of C-banding was demonstrated.

Key words: Tandem repeat, in situ hybridization, digoxygenin-labelled probe,Cbanding, karyotype.

Effect of captopril or verapamil on intracellular sodium in cultured vascular smooth muscle cells.
QI JIAN HUA, LU ZHUANG, JUN WANG, MIN LU, XIN MING WANG, ZHENG JUN JIN
Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China
Department of Parmacology , Shanghai Second Medical University, Shanghai 200025, China.

ABSTRACT
The effects of captopril (Cap) and verapamil (Ver) alone and in combination on intracellular Na+ concentration([Na+]i) in cultured aortic smooth muscle cells (ASMC) of rabbits was evaluated by a direct measurement of [Na+] I with fluorescent dye sodium-binding benzofuran isophthalate (SBFI) combined with digital. [Na+]I in resulting cells was found to be 11.9±0.7 mmol/L. Angiotensin II (Ang-II, 0.1-10 umol/L) induced an increase of [Na+]I in concentration-dependent manner. Ver(0.1-10 μmol/L) inhibited Ang-II (1μmol/L)-induced increase in [Na+]I while Cap enhanced Ang-II-induced increase in [Na+]I at 10μmol/L but not at 0.1-1μmol/L. Ver(0.1-1μmol/L) abolished enhancement of Ang-II-induced increase in [Na+]I by Cap . Thus , the inhibition of Cap-enhanced[Na+]I byVer may provide a new hypothesis for the underlying molecular mechanism of synergistic effect of the combination of Ca2+ antagonists and angiotensin coverting enzyme inhibitors in controlling blood pressure.

Key words: Cultured aortic smooth muscle cells , angiotensin II, captopril, verapamil,sodium-binding benzofuran isophthalate.

Can iverexpression of TCG-β1 gene change the sex ratio in transgenic mice

TSUNG HSIAO CHEN, CHIEN,JIE XU,LU XIA XU,XIU LAN LI,WEI KANG SHI, ZHEN YAO
Shanghai Institute of Cell Biology, Chinese Academy of Sciences , Shanghai 200031,China

ABSTRACT
Mouse TGF-β1 gene was microinjected into male pronuclei of F2 Hybrid fertilized eggs obtained by mating CSJLF1 and C57BL/6J inbred strains to generate transgenic mice with over-expressed TGF--β1 gene. The rate of found production is 31% and Southern blot analysis of founder mice tail DNAs gave an integrated to the chromosomes of transgenix mice and transmitted to their progeny at a rate of 33% in the second generation. Dot blot analysis of tail RNA of some transgenic mice indicated a moderate expression of the transgene. The most interesting finding of the present work is the striking deviation from the normal male: female sex ration in transgenic mice, with an average ration of 6.7:1. The possible nature of the predominance of male sex in transgenic mice overexpressing TGF-β1 is discussed.

Key words: Transgenix mice, over-expressioned TGF-β1 gene, MIS, sex ration.

Molecular cloning and primary sequence analysis of a gene encoding a putative chitinase gene in Brassica oleracea var . capitata

TANG GUO QING, YONG YAN BAI,SHI WEI LOO
Shanghai Institute of Plant Physiology, Chinese Academy of Sciences , 300 Frenglin Road Shanghai, China 200032.

ABSTRACT
Chitinase which catalyzes the hydrolysis of the β-1, 4-acetyl-D-glucosamine linkages of the fungal cell wall polymer chitin , is involved in inducible plants defense system. By construction of cabbage (Brassica oleracea var. capitata) genomic library and screening the library with pRCH8,a probe of rice chitinase gene fragment, a chitinase genomic sequence was isolated . the complete nucleotide sequence of the putative cabbage chittnase gene (cabch29) was determined, with its longest open reading frame (ORF) encoding a polypeptide of 413 aa . This polypeptide consists of a 21 aa N-terminal signal peptide, two chitin-binding domains different from those of other classes of plant chitinases , anda catalytic domain. Homology analysis illustrated that this cabch29 gene has 58.8% identity at the nucleotide level with the pRCH8 ORF probe and has 50% identity at the amino acid level with the catalytic domain of chitinase from bean, maize and sugar beet. Meanwhile several kinds of cis-elements, such as TATA box,CAAT box, GATA motif, ASF-1 binding site, would-response elements and AATAAA,have also been discovered in the flanking region of cabch29 gene.

Key words:
Cloning, sequence, cabbage, chitinase gene.

Functional properties of a chitinase promoter from cabbage (Brassica oleracea var. capital)
TANG GUO QING, YONG YAN BAI,SHI WEI LOO
Shanghai Institute of Plant Physiology, Chinese Academy of Sciences , 300 Frenglin Road Shanghai, China 200032.

ABSTRACT
The 5’-region of the chitinase gene cabch29,derived from Brassica oleracea var. capitata, has been sequenced and analyzed for cis-acting elements important in controlling gene expression in transgenic tobacco plants . Different 5’-deletion fragments were linked to reporter gene β-glucuronidase (GUS) as translational fusions, and the expression of the expression of these chimeric genes was analyzed in vegetative organs and tissues. Sequences up to-651 showed some basal GUS activity woth nearly equal levels in wounded and intact tissues. The addition of further upstream sequences (-651 to –1284) enhanced expression level , and the expression driven by this fragment was inducible by a factor of two to three-fold by wounding . Histochemical analysis of different tissue from transgenic plants that contain cabch29 promoter-GUS fusion gene demonstrated wound-inducible and tissue-specific cabch29 promoter activity in plants containing the 1308 base pair fragment. The location of GUS activity appears to be cells of stem, leaf and roots. Meanwhile , the temporal and spatial expression of cabch29-GUS fusion gene has been investigated . Among the different vegetative organs, a high level of GUS activity was observed in stem and a moderate one on roots; whereas , wounding stress led to a high level of GUS in wtem and moderate one in leaf.

Key words: Cabbage chitinase gene, promoter, cis-element, would-response.

Hybridization between Gossypium herbaceum and Gossypium stochsii through embryo rescue
GILL MANJEET S, YPS BAJAJ
Punjab Agricultureal University, Ludhiana, India

ABSTRACT
Natrient media and culture conditions have been defined for ovules 3 and 5 d after pollination and embryos of Gossypium herbaceum respectively. The technique was then used to produce interspecific hybrids between a cultivated Gossypium herbaceum and a wild species. G. shocksii. The hybrid plants were transferred to field and they existed most of the characters of the pollen parent i.e. G.stocksii.

Key words: Cotton, biotechnology, embryo culture, wide hybridization.

Vol 6 No.2 1996

Predicton of antigenic determinants of trichosanthin by molecular modeling

HE YONG NING ZONG XIANG XIA, YIN WANG YONG YONG JI,MING YEN
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
Shanghhai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China

ABSTRACT
The antigenic determinants of trichosanthin were predicted by modeling. First, the three-dimensional structure model of the antigen-binding franment of anti-trichosanthin immunoglobulin E was built on the basis of its amino-acid sequence and the known three-dimensional structure of an antibody with similar sequence. Secondly, the preferable antigen-antibody interactions obtained based on the known three-dimensional structure of trichosanthin and of the hypervariable regions of anti-trichosanthin immunoglobulin E. Two regions in the molecular surface of trichosanthin were found to form form extensive interactions with the hypervariable regions of the antibody and have been predicted to be the possible antogenic determinants: one is composed of two polypeptide segments, Ile201-Glu210 and Ile225-Asp229, which are close to each other in the three-dimensional structure; and the other is the segment Lys173-Thr178. The former region seems to be the more reasonable antigenic determinant than the latter one.

Key words: Antigenic determinants, trichosanthin, anti-trichosanthin immunoglobulin E, molecular modeling.

Syudys on DNA-protein interactions in the upstream regulatory region of the human ε-globin gene promoter

YAN ZHI JIANG, YA DI CHEN,RUO LAN QIAN
Shanghai Institute of Cell Biology, Chinese Academy of Sciences, 320 yueyang Road, Shanghai 200031, China

ABSTRACT
The erythroid –and developmental stage-specific expression of the humanε-globin gene is controlled, in part, by the 5’flanking DNA sequence of this gene. In the present study, we have used DNA sequence of this gene. In the present study, we have used DNA-pretdin binding assays to identify trans-acting factors which regulate the temporal expression of the huamanε-globin gene durind development. Using gel mobility shift assays and DNasel footprinting assays , a nuclear protein factor (termedε-SSF1 ) in the nuclear extracts from mouse hasmatopoietic tissues at d 11 and d 13 of gestation was identified . It could specifically bind to the positive control region (between -535 and -453bp) of the humanε-globin gene. We speculated that theε-SSF1 might be an erythroid –and developmental stage-specific activator. In addition, we found another nuclear protein factor (termedε-R1) in the nuclear extract from mouse fetal liber at d 18 of gestation, which could strongly bind to the silencer region (between-392 and -177bp) of this gene.) there fore , we speculated that theε-R1 might be an erythroid- and developmental stage-specific repressor. Our data suggest that bothε-SSF1 andε-R1 might play important reles in developmental regulation of the humanε-globin gene expression during the early embryonic life. On the other hand, we observed that the binding patterns of nuclear proteins from three cell lines (K562, HEL and Raji ) to these regulatory regions were partially different. These results suggest that different trans-acting factors in K562, HEL and Raji cells might be responsible for activating or silencing the humanε-globin gene in three different cell lines.

Key words : Humanε-globin gene, positive control region, silencer, trans-acting factor.

Low temperature induces oocytes P34cdc2 synthesis and accumulation – the acquisition of competence to resume meiosis in toad oocytes

LU JI NING,ZHENG GU,HONG SHENG,CHUANG JU LIU,JIA KE TSO
Shanghai Institute of Cell Biology, Chinese Academy of Sciences, 320 yueyang Road, Shanghai 200031, China

ABSTRACT
Full grown oocytes derived from Bufo Bufo gargarizans rearing at high temperture environment (24’C). never underwent GVBD after progesterone treatment . No P34cdc2 H1 kinase activity was detected in the oocytes after progesterone stimulation or OA microinjection;Western blotting analysis showed that the level of P34cdc2 and P33 in the oocytes are significantly lower than those in the oocytes derived from the hibernating toads (between 10 ‘C). 35 S-Met incorporation analysis showed that when the oocytes derived from the hibernating toads (below 10’C). 35S-Met incorportation analysis showed that when the oocytes were incubated at 6’C, synthesis of about thirty defferent polypetides was promoted or induced , including P34cdc2 and some other P13suc1-binding proteins. All these results indicated that a low temperature environment is essential for the oocytes of Bufo Bufo gargarizans to express and store some cell cycle drivers and its regulators , and to gain the maturation competence. These results will also provide a new clue for explaining the molecular mechanisms why gametogenesis of some organisms depends on a relative low temperature how to maintain the geographical distributin of some animals.
Key words: Maturation competence, P34cdc2,low temperature environment.

Isolation of T-DNA flanking plant DNA from T-DNA insertional embryo-lethal mutants of Arabidopsis thaliana by plasmid resced technique

YAO XIAO LI,JIAN GE SUN,ZHI PING ZHU
Chinese National Laboratory of Plant Molecular Genetics
Shanghai Institute of plant Physiology , Chinese Academy of Sciences, 320 yueyang Road, Shanghai 200031, China
ABSTRACT
Three T-DNA insertional embryonic lethal mutants from NASC( The Nottingham Arabidopsis Stock Center) were first checked with their segreation ration of abortive and normal seeds and the copy number of T-DNA insertion . The N4081 mutant has a segreation ration of 1:3:04 in average and one T-DNA insertion for further analysis
To isolate the joint fragment of T-DNA ,which contained pBR322 was selected from left border of T-DNA,which contained pBR322 was selected from ampicillin plate . The T-DNA fragment of pEL –7 was checked by restriction enzyme analysis and Southern Blot . Restriction analysis confirmed the presence of known sites of EcoRI,PstI and PvuII on it . For confirming the presence of flanking plant DNA in this plasmid ,pEL –7 DNA was labeled and hypridized with wild type and mutant plant DNA . The Southern Blot indicated the hybridization band in both of them .
Furthermore , the junction of T-DNA/plant DNA was subcloned into bluescript SK + and sequenced by Applied Plasmid rescue and cloning of T-DNA /Plant DNA junction

Key words :arabidopsis thaliana,embryo –lethal mutant , plasmid rescue ,T-DNA insertion flanking plant DNA.

Induction of apoptosis and change of bel-2 expression in macrophage Ana-1 cells by all-trans retinoic aicd

YIN DE LING,XIU HAI REN,SHI ZHONG BU,YA LAN WU,LI ZHEN JIANG,ZHI JIANG WU,WEI HU,GANG PEI
Shanghhai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China

ABSTRACT
Macrophage cells play an important role in the initiation and regulation of the immune response . All-transretinoic acid (ATRA) and its natural and synthetic analogs (retinoids) affect a large number of biologyal processes. Recently,retinoids have been shown promise in the therapy and prevention of various cancers. However,many interesting questions related to the activities of retinoids remain to be answered: (I) Molecular mechanisms by which retinoids exert their effects; (II) shy the clinical uses of varying severity with a higher frequency of blood system symptoms ;(III) little is known for its impacts on macrophage cells etc. we set up this experiment ,therefore, to examine the apoptosis of ATRA on macrophage Ana-1 cell line .Apoptosis of the cells was quantitated after staining cells with propidium iodide (PI) ,by both accounting nuclear condensation and flow cytometry. When the cells were treated with ATRA at or higher htan 1 μM for more than 24h ,sighificant amount of the apoptotic cells was observed . Induction of apotosis of Ana-1 cells by ATRA was in time –and dose –dependent manners,exhibiting the similar pattern as the apoptosis induced by actinomycin D (ACTD) . ATRA treatment of Ana-a cells also caused the changes of the mRNA levels of ATRA- induced apoptosis and change of bcl-2 in Ana-1 cells

Key words: All-trans retinoic acid ,apoptosis,Ana-1 cells bcl-2

Antisense EGFR sequence enhances apoptosis in a human hepatoma cell line BEL-7404

FU TAO,HE LIU,FAN LIU JUNGU,WAN LU JIANG,YONG HUA XU
Laboratory of Cellualr and Molecular Oncology ,Shanghai Institute of Cell Biology ,Chinese Academy of Sciences, Shanghai 200031
Shanghai Bureau of Hygiene
Laboratory Molecular Biology ,Naval Medical Research Institute ,Shanghai 200433,China

ABSTRACT
Effects of antisense epidermal growth factor receptor (EGFR) sequence on apoptotic cell death were examined in a human hepatoma cell line BEL –7404 stably transfected with antisense EGFR vector (Cell Research ,2:75,1993) ,an enhanced rate (9.5%) of spontaneous apoptosis was detected by flow cytometry, whereas the rates of spontaneous apoptosis in JX-0 cells ,a sub-clone of BEL –7404 transfected by control vector ,and the parent BEL-7404 cells were almost equal and about 1.7%. Serum-starvation for 72h increased the rate of apoptosis of JX –1 cells up tp 33.7% ,while JX –0 and BEL –7404 cells ,under the same condition , produced less than 5% of apoptotic cells .Observation with electron microscope demonstrated that condensation and fragmentation of chromatin and formation of apoptotic bodies often occurred in JX-1 cells ,especially during serumstarvation . These results ,combined with the data of DNA fragmentation Elisa test, suggested that antisense EGFR sequence enhances apoptosis in the human hepatoma cells.
Antisebse EGFR ebgabces apoptosis in epatoma cells

Key words: Antisense EGFR ,hepatoma cells ,apoptosis.

Resersion of malignancy in human gastric cancer MKN –45 cells through the transfection of transforming growth factor-βtype II receptor gene

SUN HONG,WEI KANG SHI, ZHEN YAO
Shanghhai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China

ABSTRACT
Human gastric cancer MKN-45 cells which are resisrant to TGR-βtype I and type III receptors ,but not type II receptors ,have been used as model system to reconstitute these cancer cells with TGR-βRII cDNA . The results of these experiments indicated that the reexpression of TGF-βRII gene in MKN –45 cells can restore their sensitivity to TGF-β growth inhibition ,decrease their growth rate, reduce their cloning efficiency in soft agar and tumorigenicity in nude mice in stable transfectants, in comparison with their control MKN-45 cells ,Among different RII transfectants, dheir defference in the changes of these parameters , as a result of the regain of autocrine negative growth control by TGF-β, is roughly proportional to their level of expression of transfected RII mRNA . From these data , it is concleded that the inactivation of TGF-β RII gene is related to the excape of growth control by TGF-β in MKN-45 cells . The importance of the study of the interplay of TGF-β and tis receptor system in the nagative growth control of gastric cancer , and possibly also of other cancers , is discussed.

Key words: Gastric cancer cell, TGF-β RII gene reconstitution ,growth inhibation , reduced tumorigenicity.

The fertilization-indeced Ca2+ oscillation in mouse oocytes is cytoplasmic maturation dependent

DENG MAN QI ,FANG ZHEN SUN

Laboratory Molecular Developmental Biology ,Institute of Developmental Biology, Chinese Academy of Sciences, Beijing ,100080, China
ABSTRACT
Mature eggs( at metaphase II stage)produce a series of Ca2+ oscillation at fertilization . To define whether the fertilization –induced Ca2+ oscillation os restrict to the metaphase II eggs and cell cycle dependent, mouse oocytes at prophase I (arrested at germinal vesicle stage), metaphase I ,metaphase II , as well as the pronuclear embryos at interphase of the first mitotic division derived from dertiliation or parthenogenetic activation were inseminated after removal of zona pellucida. The results show that the fertilization –induced Ca2+ oscillationis not specific to metaphase II eggs. This is supported by the fact that immature oocytes generated the Ca2+ oscillation at fertilization regardless of their nuclear nuclear progression from prophase I to metaphase I (in vitro matured) stage . More interestingly, it was first found that pronuclear embryos at interphase derived from parthenogenetic activation showed Ca2+ oscillation in response to fertilization while the zygotes at interphase did not after reinsemination or intracytoplasmic at interphase did not after reinsemination or intracytoplasmic injection of sperm extracts which induce Ca2+ oscillation in MII eggs. This suggests that the ability of oocytes to generate Ca2+ oscillation in response to sperm penetration is not regulated in a cell cycle dependent manner but dependent on the cypoplasmic maturation.
Key words: Fertilization-induced Ca2+ oscillation, mouse oocyte, pronuclear embryo, parthenogenetic activation ,cell cycle.

Selective proliferation of human γδ T cells in vitro

CHEN SONG HUA, AKINORI OKI
TADAO OHNO,SATOKO ISHIKAWA,MASATAKA MOCHIZHKI,YU FANG CHE,HUI MING DAI , XI RUI GE
Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China
Richen Cell Bank, Koyadai,Tsukuba Sciences City, Japan
Department of Gynecology, Tsukuba University, Amakubo ,Tsukuba Science City, Japan
Kyoritsu College of Pharmacy ,Shibakoen, Minato-ku , Tokyo, Japan

ABSTRACT
The effect of monoethylphosphate (MEP, commercial available or synthesized ) together with IL –2 on the selective proliferation of humanγδ T cells in vitro from peripheral blood mononuclear cells (PBMC) of healthy donors and of cancer patients was investigated . Theγδ T cells were stimulated by MEP to proliferate in a dose –dependent manner . The effect of synthesized MEP was 10 times greater than that of commercial MEP . When the total cell number increased about 1000-3000 fold ; and the ration ofγδ T cells reached to 70%-80%. The selective expansion ofγδ T cells depended on the synergic action of MEP and IL-2. The bulk culturedγδ T cells cells exhibited obvious cytotoxic activities against allogenic tumor cells .The culture system described here not only offers a simple method for obtaining a large number ofγδ T cells which may become a new effector in the adoptive immunotherapy, but also provides a useful model for the further studies of the structure and function ofγδ T cells in vitro.

Key words: γδ T cells proliferation, monoethylphosphate, cytotoxicity.

Abrupt decrease of c-myc expression by antisense transcripts induced terminal differerntiation and apoptosis in human promyelocytic leukemuia HL-60 cells
HAO XIU JUAN,PEI HSIEN TANG, XIU SEN LI FEI ZI JIANG, YONG ZHI XI, NING MAO ,DE LIU DU , MIN WU
Institute of Basis Medical Sciences ,Beijing 100850
National Laboratory of Molecular Oncology, Institute of Cancer Research Beijing, Chinese Academy of Medical Sciences, Beijing 100021

ABSTRACT
This study was designed using c-myc antisense transcripts to evaluate how alteration of c-myc expression in human myeloid leukemic HL-60 cells could influence the myelomonocytic differentiation and induction of apoptosis. The recombinant plasmid pDACx expressing antisense transcripts to c-myc fragment containing a part of inron 1 and 137 nt exon 2 was constructed. PDACx was transfected into HL –60 cell line by lipofectin , peroxidase and α-NAE as well as detection of CD13 and CD33 antigens by flow cytometric analysis indicated occurrence of myelomonocytic differentiation in cells expression antisense transcripts to c-myc. DNA degradation measured by DNA gel electrophoresis and typical morphological changes observed under electron microscope proved the switch –on of apoptosis in terminally differentatin HL –60 cells.

Key words: c-myc oncogene, antisense transcripts , differentiation ,apoptosis.

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