Effect of P15INK4b/MTS2 on the proliferation of human hepatoma cells SMMC-7721

The full length cDNA coding for P15 INK4b, which is a cyclin-dependent kinase inhibitor, was cloned to plasmid PXJ41-neo (Eco RⅠ/XhoⅠ site) and the new constructed plasmid pXJp15 was obtained. pXJp15 was transferred into the human hepatoma SMMC-7721 cells by lipofectine reagent. After G418 selection, a series of cell lines stably expressing high levels of P15 (named SHT) and the clone containing vector PXJ41-neo only (named SVXJ) were obtained by Northern and Western analysis. The results showed that the proliferation of SHT cells is inhibited compared with that of SVXJ cells. Cell cycle analysis indicated that overexpressing of P15 inhibited the growth of SHT cells by decreasing progrssion of cells from G1 to S and G2 to M phases. The levels of c-Myc and c-Fos were obviously decreased in SHT cells compared with control cells by Western blotting. The decreased expression of oncogene may be one of the molecular mechanisms of the effect of P15 on the proliferation of in SHT cells.     

Effects ofPKCα antisense RNA on human breast cancer cell proliferation and expression ofcyclinD1 andCDK4

The role of PKCα in human breast cancer cell proliferation and expression ofcyclinD1 andCDK4 has been investigated using inhibition ofPKCα expression by its antisense RNA. WhenPKCα expression was inhibited the rate of cell proliferation decreased apparently and the levels ofcyclinD1 andCDK4 mRNA were lower than the control. The results showed thatPKCα, a key member of signal transduction system, played an important role in human breast cancer cell proliferation and had a close relationship with expression ofcyclinD1 andCDK4 which control start of cell cycle.     

Construction and expression of retroviruses encoding dual drug resistance genes in human umbilical cord blood CD34+ cells

A series of retro viral vectors encoding humanmdr1 gene alone as wetl as in combination with either humanmgmt gene or human mutantSer ³¹-dhfr gene are engineered. The resultant retroviruses are used to transduce human umbilical cord blood CD34⁺ cetls. It has been shown that expression of dual drug resistance genes in transduced cetls confers a broad range of resistance to both kinds of corresponding drugs. These data suggest a rationale for the use of such double chemoresistance gene constructs in anin vivo model in which transduced hematopoietic cetls will acquire multiple protection against the cytotoxic side effects of combination chemotherapy and may have future application in chemoprotection of normal tissues, thus killing tumor cetls more effectivety.     

Construction of the glucose isomerase deficient strain ofStreptomyces M1033 by homologous recombination

After the establishment of the transformation conditions ofStreptomyces diastaticus No.7 Strain M1033, the integration plasmid pXW for homologous recombination, which contains a 600 bp fragment of incompleteGI (G138P. G247D) gene, has been constructed in order to realize the stable overexpression of theGI (G138P. G247D) which is valuable for large-scale industrial production. The Gigene’s disruption has been realized by pXW’s integration into M1033 chromosomes via homologous recombination andGI deficient strain ofStreptomyces M1033 has been obtained. The reliability of introduction of mutation has been proved by analysis of recombinant fragment and affirmance of existence of the mutation, as well as detection of the stability of the deficient strain.     

cdk2反义RNA对乳腺癌细胞增殖及致瘤性的抑制作用

为了研究cdk2对乳腺癌细胞生长及cyclinA,cyclinB1和ckdl（cdc2)mRNA表达水平的影响,利用直接表达载体p XJ41-neo构建了表达cdk2反义RNA的重组载体,并用此载体转染了人乳腺癌细胞系Bcap37,获得了ckd2受到抑制的细胞模型Bcap37-CDK2AS,然后将Bcap37-CDK2AS细胞的生长能力及cyclinA,cyclinB1和cdk1 mRNA折水平与转入空载体的对照细胞进行了对比分析,结果显示,cdk2表达受到抑制时,细胞生长速率下降,根据测定出的细胞生长曲线,细胞培养至第7天时,细胞生长抑制率为64％,在流式细胞术的分析结果中,G1期细胞中的百分比从39％增加到47％,S期细胞由51％下降到39％,裸鼠接种的实验表明,Bcap37－CDK2AS的致瘤性明显减弱,在对cyclinA,cyclinB1和cdk1mRNA折分析中发现,Bcap37－CDK2AS中这3种基因的mRAN水平均有不同程度的下降,依据这些结果可以推测,ckd2反义RNA可使乳腺癌细胞生长及致瘤性受到抑制,并且cdk2表达的抑制将cyclinA,cyclinB和cdk14的表达水平。     

P15——a new tumor suppressor gene

In addition to the tumor suppressor genes such as Rb and p53, it has been found that some molecules of the same class named CKI (cyclin-dependent kinase inhibitor) also play an important role in the inhibition of tumorigenesis and the tumor progression. In the KIP and INK4 families of CKIs, p15 shares extensive homology with p16. Findings in many tumors and their cell lines show that the inactivation of p15 (deletion, mutation, rearrangement, etc.) is very frequent, and inactive p15 is involved in the progress of some tumors. These studies provide evidence that the p15 is a new tumor suppressor gene. Furthermore, the research on the molecular mechanism of p15 in regulation of cell proliferation shows that p15 can inhibit the growth of some kinds of tumor cells, and p15 is the mediator of TGF-β-induced cell arrest. Investigations on p15 in cell differentiation suggest that increased p15 is related to the change of malignant phenotype. These results supply clues for further interpretation about the molecular mechnism of cell cycle control and cell tumorigenesis. And they may provide theoretical and experimental basis for application of p15 to clinical therapy of tumors.     

Construction and characterization of partiallyntrC-deleted mutants inAlcaligenes faecalis

To study the effect of ntrC gene product on the expression and regulation of other important nitrogen-fixing genes in Alcaligenes faecalis, partially ntrC-deleted mutants of A. faecalis have been generated. To start with, the ntrC gene of A. faecalis was cloned into a suicide plasmid pSUP202 to create a recombinant plasmid pSUM1. The ntrC gene in pSUM1 was then replaced by a lacZ-Km^r fragment resulted in the generation of a plasmid pSUM2. The lacZ fragment in pSUM2 was further removed and a plasmid pSUM3 produced. As a second step, the plasmid pSUM2 or pSUM3 was introduced into the wild type of A. faecalis A1501 by conjugation and two partially ntrC-deleted mutants A15CM1 (ntrC∷lacZ) and A15CM2 (ntrC ^- ) were obtained. To understand the regulatory effect of the NtrC on the expression of nifH and nifA, a nifH-lacZ gene or a nifH-lacZ gene was introduced into the ntrC^- mutant by conjugation. The results indicated that: (ⅰ) although the ntrC^-mutant was nif ⁺ , its nitrogen fixation activity was only 20% that of the wild type; (ⅱ) the ntrC^- mutant failed to grow on the medium containing nitrate as a sole nitrogen source; (ⅲ) the regulation of ntrC gene expression did not require its own product; (ⅳ) the expression of nifH in A . faecalis was positively regulated by the ntrC. Deletion of the ntrC resulted in the reduction of nifH expression or even totally inactivated nitrogen fixation; (ⅴ) there was no obvious influence on the expression of nifA in A. faecalis if the ntrC gene was deleted.     

Expression of soluble human tumor necrosis factor receptor Ⅰ in Aspergillus niger

The cDNA of soluble human tumor necrosis factor receptorⅠ(sTNFRI) was inserted into fusion-protein expression plasmid pIGF of A. niger to construct fusion expression vector pHBC containing a KEX2 like protein processing site designed on the fusion position. Extracellular protease-deficient strain of A. niger 3.795-1-23 was transformed with pHBC. Positive clone was estimated by Southern hybridization. SDS-PAGE for protein produced by recombinant strain showed the distinctive expression band. Western blotting indicated that the secreted protein had immunoactivity of sTNFRI.     

Improving DNA damage repair ability ofE. coli to UV-radiosensitivity by DNA-mediated gene transfer of low energy ion beam

Using calcium chloride method of transfer gene as control, a new technique of transferring gene by low energy ion beam has been applied to the study of improving DNA damage repair ability ofE. coli to UV-radiosensitivity. The genome DNA pieces ofDeinococcus radiodurans, as “foreign” genetic materials, were introduced into the UV-radiosensitive strains ofE. coli by implantation of 20 keV Ar⁺ at doses ranging from 1 × 10¹⁵ to 2 × 10¹⁵ ions/cm². Results show that the transfected strains present higher UV-radioresistance than that of un-transfected ones and start ones. The survival rate of transfected strains and their unscheduled DNA synthesis (UDS) ability is increased, indicating that the transfer gene is a success.     

Breeding of T. aestivum-Ag. intermedium translocation lines with T. timopheevii cytoplasm and characterization by GISH

A male-sterileT. aestivum-Ag. intermedium partial amphiploid with cytoplasm ofT. timopheevii as a female parent was crossed to common wheat. The hybrid was backcrossed to the male parent several times continually and setf-crossed at last. Two stable lines with common wheat phenotype, H96269-2 and H96278, have been obtained. The chromosome numbers of the two lines are all2n = 42 in somatic cetls. By inoculation test, the two lines show a high levet of resistance to yetlow rust. Through genomicin situ hybridization (GISH) withAg. intermedium total genomic DNA as a probe, it is demonstrated that the two stable lines are all small segmental translocation lines, and the translocated chromosome segments fromAg. intermedium are located on the short arm terminals of wheat chromosomes. Genetics analysis suggests that the yetlow rust resistance gene(s) are probably located on the translocated chromosome segments ofAg. intermedium.     

Alteration of the specificity ofPst I restriction endonuclease

The influence of factors on the substrate-specificity ofPst I restriction endonuclease has been studied with the method of electrophoresis. The results show that, the specificity ofPst I almost can not be influenced by the single alteration of the concentration of Tris·HCl, Mg²⁺ or Na⁺ in the reaction system, but it can be altered by the reduction of any two of them. The specificity can not be altered by the single alteration of pH or the replacement of Mg²⁺ with Mn²⁺. The addition of glycerol or dimethylsulphoxide (DM-SO) to the reaction system results in the relaxation of the substrate-specificity ofPst I, but dimethyl-methylformide, glycol and ethyl alcohol can not bring about the alteration ofPst I specificity. Through the method of cloning and sequencing, the nucleotides of No. 1 and 6 in the recognition sequence ofPst I have changed (1C→A or 6G→T). Used with the enzyme analysis of an artificially synthetic DNA segment containing a special sequence, the nucleotides of No. 1 and 6 have both changed (1C→A and 6G→T). The recognition sequence ofPst I is speculated to be changed from CTGCA→G to TGCA→. Foundation item: Supported by the Research Fund for the Doctoral Program of Higher Education. Biography: Zou Guo-lin (1947-), male, professor. research direction, biochemistry.     

Cloning and functional research of renal cell carcinoma related novel gene—— GYLZ-RCC18

After the renal cell carcinoma related novel gene fragmentGYLZ-RCC18 was cloned by using suppression subtractive hybridization (SSH), we used the SMART RACE technology to clone the full length ofGYLZ-RCC18 and performed chromosome location by the FISH method. RT-PCR was used to detect the expression of the first reading frame ofGYLZ-RCC18 in different stages and grades of renal cell carcinoma tissue and other tissues. Also we transfected the antisense oligonucleotide ofGYLZ-RCC18 to renal cell carcinoma cell line GRC-1, and analyzed the proliferation activity, growth speed, apoptosis and mortality changes in GRC-1. The results show that the full length ofGYLZ-RCC18 (GenBank accession No.: BE825133) cDNA is about 3.5 kb long which is located at No. 14 chromosome.GYLZ-RCC18 has a higher expression in higher grades and stages of renal cell carcinoma than in the lower ones. The expression ofGYLZ-RCC18 in renal cell carcinoma was much higher than that in normal kidney and other tissues. After transfection ofGYLZ-RCC18 antisense oligonucleotide, the mortality of GRC-1 increases evidently, the proliferation activity and growth speed were inhibited remarkably at the same time. Also the antisense oligonucleotide can induce the apoptosis of GRC-1 all through the observation time. Our results indicated thatGYLZ-RCC18 is an important novel gene related to renal cell carcinoma. Its overexpression would stimulate the growth and proliferation activity and plays an antidead and antiapoptosis effect in renal cell carcinoma. Transfection of antisense oligonucleotide could inhibit the generation and development of renal cell carcinoma. The study provides a new clue for the research of renal cell carcinoma, and also provides an instruction for special genetic diagnosis and the therapy of renal cell carcinoma.     

槲皮素镧配合物对S.Pombe细胞生长代谢热动力学的影响

采用八通道微量热法探讨槲皮素镧配合物对粟酒裂殖酵母细胞生长代谢热动力学的影响。测定在槲皮素镧配合物作用下S.Pombe细胞的生长代谢产热曲线,并研究其生长速率常数(k)、最大发热功率(pmax)、达最大发热功率时间(tmax)和抑制率(I)等热动力学参数。微量热结果表明:随着槲皮素镧配合物浓度c的增大,最大发热功率pmax和速率常数k减小;达最大发热功率时间tmax和抑制率I增大。研究表明槲皮素镧配合物对粟酒裂殖酵母细胞生长代谢产生了抑制作用。     

A Note on the Prime Spectrums of Character Rings of Finite Groups

0Introduction LetGbeafinitegroupoforder|G|=g,andletR(G)denotethecharacterringofG,whichisgeneratedbyalltheirreduciblecomplexcharactersofG.LetZbetherationalintegerringandNthesetofnatural numbers,andletZ[ω]betheintegralextensiongeneratedbyaprimitiveg throotωofunity.SupposethatSisasubringof thealgebraicnumberfieldsuchthatZ[ω]S.πisasetofra tionalprimenumbersdefinedasfollowsπ={p｜pisarationalprimenumbersuchthatp-1S}.Definition1WecallthataconjugacyclassCofthefinite groupGisaπregularconju…     

Adrenomedullin inhibits the endothelin production induced by urotensin II in rat vascular smooth muscle cells

The aim of this study was to observe the effects of adrenomedullin (ADM) on endothelin (ET) production induced by urotensin Ⅱ (UⅡ) in rat vascular smooth muscle cells (VSMCs). Cultured VSMCs which were incubated with UⅡ (10^-8 mol/L) and with various concentrations of ADM were used to measure the VSMCs ³H-TdR incorpora- tion, the activity of extracellular signal-regulated kinase (ERK), the amount of ET mRNA and ET production in VSMCs. In this work we found that incubation with UⅡ(10^-8 mol/L) increased obviously the amount of ET mRNA in VSMCs and ET production in medium, however, coincubation with ADM (10^-10—10^-8 mol/L) and UⅡ(10^-8 mol/L) reduced the amount of ET mRNA by 15%, 24% and 45% (P< 0.01) respectively, compared with UⅡ alone. The content of ET in medium was 14.13, 11.38 and 11.00 pg/mL. ADM alone (10^-8 mol/L) had no effect on ET production in VSMCs. UⅡ (10^-8 mol/L) promoted the 3H-TdR incorpo- ration and activity of ERK in VSMCs. ADM inhibited VSMCs 3H-TdR incorporation and activation of ERK in a concentration-dependent manner. Compared with UⅡ group, after coincubation with ADM (10^-10—10^-8 mol/L) and UⅡ (10^-8 mol/L) the VSMCs 3H-TdR incorporation was decreased by 7% (P > 0.05), 32% (P < 0.05) and 41% (P < 0.01), respectively, and the activity of ERK was decreased by 24% (P > 0.05), 32% (P < 0.05) and 36% (P < 0.05), re- spectively, in a concentration-dependent manner. The results show that in cultured VSMCs ADM inhibits ET mRNA expression, ET production and proliferation stimulated by UⅡ, and that inhibitory effect of ADM on UⅡ bioaction could be mediated through inhibiting MAPK pathway.     

Integration and inheritance stability of foreignBt toxin gene in the bivalent insect-resistant transgenic cotton plants

Genetic and expressional stability of Bt toxin gene is crucial for the breeding of insect-resistant transgenic cotton varieties and their commercialization. Genomic Southern blot analysis of R₃, R₄ and R₅ generations of bivalent transgenic insect-resistant cotton plants was done in order to determine the integration, the copy number and the inheritance stability of Bt toxin gene in the transgenic cotton plants. The results indicated that there was a 4.7 kb positive band in the Southern blot when the genomic DNA of the bivalent transgenic insect-resistant cotton plants and the positive control (the plasmid) were digested with HindⅢ respectively. This result proved that the Bt toxin gene had been integrated into the genome of the cotton in full length. There is only one XhoⅠ restriction site in the Bt toxin gene. Southern blot analysis indicated that many copies of Bt toxin gene had been integrated into the genome of the cotton when the genomic DNA of transgenic plants was digested with XhoⅠ. Among them, there were four copies (about 17.7, 8, 5.5 and 4.7 kb in size) existing in all the tested plants of ₃, R₄ and R₅ generations. The preliminary conclusion was that there were more than four copies of Bt toxin gene integrated into the genome of the cotton, among them, more than one copy can express and inherit steadily. This result provides a scientific basis for the breeding of the bivalent insect-resis- tant transgenic cotton plants and its commercialization.     

A tumor suppressor genefhit prokaryotic expression and identification

The recombinant expression vector pGEMD-fhit which contains full encoding region offhit gene was constructed. The recombinant was introduced into the BL21 (DE3) strain ofE. coli and induced by 1 mmol/L IPTG to express a 29×10³ polypeptide offhit fusion protein. And the 29×10³ protein was sensitive and specific in reaction with anti-fhit antibody in Western blot. Foundation item: Supported by the National Natural Science Foundation of China (39770373) Biography: SUN Yan (1975−), female, Master of science, Research direction: gene engineering     

Transformation of ginkgo hairy root and establishment of its suspension culture clone

Tender leaves, buds and stems ofginkgo biloba L. were transformed successfully byAgrobacterium rhizogenes with Ri plasmid, and its suspension cultured clone was eastablished. The result of detection of opines suggested thatA. rhizogenes had integrated the T-DNA of the Ri plasmid into genomic DNA of ginkgo cells. A new way is offered to exploit ginkgo resources with biological technology. Liu Shunan: born in 1972, Postgraduate