Abstract:Non-coding mRNA is a kind of newly discovered mRNA. Though spliced and tailed,it does not have any typical ORF. Up to date, several such genes have been cloned: H19 gene, XIST gene, XLSIRT gene, His-1 gene, bic gene, rox1 and rox2 genes. Non-coding mRNA play important roles in the development of embryos, in the inactivation of X chromosome and in the tumorigenesis.

Abstract:Progress of chromosome microdissection and microcloning technology were reviewed. Some main technology problems such as chromosome identification, microdissection and microcloning were disscussed. Some progress in plants was reviewed about chromosome microdissection and microcloning.

Abstract:Tumor necrosis factor related apoptosis-inducing ligand (TRAIL), which called also Apo-2L, is a new member of the tumor necrosis factor superfamily. TRAIL cDNA was isolated via searching homology to Fas/Apo-1 ligand and TNF in an expressed sequenced tag (EST) data base. It consists of 281 amino acids with a calculated molecular mass of 32.5 ku. Transfected TRAIL is expressed at the cell surface with its C-terminus exposed, indicating a type Ⅱ transmembrane protein topology. Like Fas/Apo-1 ligand and TNF, its C-terminal exhibits a homotrimeric subunit structure. Soluble TRAIL induces extensive apoptosis in most tumor cell lines, but the effect of TRAIL is not inhibited by soluble Fas/Apo-1 and TNF receptors. So far, three receptors of TRAIL have been identified, i.e. DR4, DR5 and TRID. These discoveries suggest that the mechanism of TRAIL is different from TNF and FasL. TRAIL may likely be elaborated as a new anti-cancer drug.

Abstract:Recent years the structural biology have been developing very fast and growing into a new phase. The rate of determination of biomacromolecular structure has been reached to 5.1/day and goes up as an exponential curve. To the date of April '98, the released coordinates from PDB have been 7 454. Meanwhile the structure research combines with the functional research to gain insight into mechanisms of many biological process. The application of the third generation synchrotron radiation has greatly impact on X-ray crystal structure determination. With very bright X-ray from the synchrotron, one can start from very small crystals and gain a big structure, and also detect the instantaneous change of structure in time scale of 10^－9 s. It therefore may open the door for the structure determination of membrane proteins and large complex and assembly as nucleosome core particle. The molecular weight of solution structure determined by NMR has beyond 35 000. Now the structural biology is marrying with the genomics to produce a new “big science”, the structural genomics, which will provide a large step towards the understanding of life in the postgenomic ear.

Abstract:The ability of tumor cell to adhere and migrate is closely related to metastasis. Cell adhesion molecules, such as selectins, integrins, Ig superfamily and cadherins mediate cell-to-cell and cell-to-matrix interactions. Cell adhesiveness changed by the different expression or distribution of cell adhesion molecules on tumor cell surfaces affects the metastatic potential directly or indirectly, so it is very important for tumor cells to separate from primary lesion and lodgement. Tumor cell migration is thought to be a major limiting step in metastasis. In general, the ability of tumor cells to migrate in vivo or in vitro is positively correlated with their metastatic potential. Once stimulated by motile factor, tumor cells can promote their migration by themselves through chemotaxis and haptotaxis, the molecular mechanism of which is unclear at present yet.

Abstract:Phenanthroline-Cu is a complex that have nuclease activity, it can induce several kinds of DNA damage, including base modifications, abasic sites, strand breaks and DNA protein crosslinks. There is a considerable interest on this complex in recent years in the area of free radical biology and medicine as well as nucleic acids chemistry, for the role of phenanthroline-Cu as a model of generating reactive oxygen species and as a chemical nuclease.

Abstract:The interaction between leukocytes and endothelial cells is mediated by cell adhesion molecules. There are three families of adhesion molecules which play important roles in cell adhesion. They are integrin family, immunoglobulin superfamily and selectin family. The initial stage of adhesion is mediated by selectins and the firm adhesion is formed thereafter by the binding of CD11/CD18 complex with ICAM-1. Certain cytokines and inflammatory factors can induce cell adhesion mentioned above. The interactions among different adhesion molecules are very complicated. Antibodies directed at adhesion molecules, soluable adhesion molecules, oligopeptides with high affinity for the binding sides of the adhesion molecules, and some drugs can block the adhesion.

Abstract:Since the discovery of structural and functional similarities between the product of the nematode cell-death gene Ced-3 and mammalian interleukin-1β-converting enzyme（ICE）, an expanding family of cysteine proteases genes have been coloned and shown to play a key role in execution stage of mammalian cell apoptosis. Several substrates are found to be cleaved by the active ICE/CED-3 proteases（because of cysteine protease with aspartates specificity, also called caspase）,and subsequent cytological effect is induced.

Abstract:Cell-free translation:the in vitro synthesis of protein using cellular extracts has been a routine technique in molecular biology labs for twenty years. But it has seldom studied in our country. The characteristics including types, preparation, basics, functions and progress of cell-free systems are introduced. Its advantages and disadvantages are also discussed. It is beneficial for the application and understanding of the technique by our researchers in applied biochemistry and molecular biology assays.

Abstract:Na^＋/H^＋ exchangers (NHE) are vital transmembrane transporters involved in multiple cellular functions including the regulation of intracellular pH, the control of cell volume and ion transport. Five isoforms of Na^＋/H^＋ exchangers have been cloned and characterized to date; they constitute a new gene family of vertebrate transporters. And these isoforms are highly regulated by a remarkably wide variety of stimuli which can modulate their expression level and activity. Among the isoforms, the increased expression level and activity of human NHE-1 have been found to play a role in some critical diseases, such as tumor, hypertension and diabetes. Thus it may provide a new way of diagnosing and treating for these diseases by investigating on the transcription modulation of NHE-1 gene and the activity regulation of the exchanger.

Abstract:Thrombi-targeted plasminogen activator has an important effect on thrombolysis . Some good results have been obtained by using monoclonal antibody or its fragments specific for fibrin or platelet to direct the plasminogen activator. Bispecific antibody and some peptides with properties of antithrombin or anticoagulation give new angle of view for targeted plasminogen activator.

Abstract:Auxilin is a molecular chaperon which induces binding of Hsp70c to clathrin, playing an important role in uncoating of coated vesicle isolated from brain. Through integrated analysis of public database such as dbEST, dbSTS, partial cDNA sequence of human auxilin was identified and mapped to 1p31, between marker D1S515 and D1S198. Totally 26 ESTs were found to be part of human auxilin and used to construct five contigs, which make totally 2.3 kb sequence and contains 501 bp coding sequence. All the sequences obtained and the corresponding putative translation showed high homologous to bovine auxilin. At the same time, EST data indicate that human auxilin expressed in several tissues at fetal stage, also in brain and melanocyte at adult stage.

Abstract:By use of the AR-CM-MIC system and other methods, the relationship between cellular viability of alveolar macrophages and the change of intracellular Ca²⁺ concentration(［Ca²⁺］_i) after the action of verapamil was investigated under different extracellular calcium concentrations. And the research about the effect of verapamil on experimental silicosis rats were conducted. The results showed: the ［Ca²⁺］_i rise induced by α-quartz was caused by enormous extracellular Ca²⁺ influx and the overloading of the intracellular Ca²⁺, which directly led to the death of alveolar macrophages and further the formation of silicosis. Verapamil could inhibit the ［Ca²⁺］_i rise, decline the death of cells. The results of the research about experimental silicosis indicated: verapamil has apparent inhibitory effect on the starting of silicosis. It suggested that verapamil appears to be a prevention and cure drug of silicosis.

Abstract:A replication-defective recombinant adenovirus vector containing HSV-TK gene under the control of CEA promoter was constructed (AdCEATK). Titer of the purified recombinant adenovirus is about 10¹²pfu/ml. HeLa cell (CEA-negative cell) infected with AdCMVTK became sensitive to GCV, while HeLa cell infected with AdCEATK was not. On the contrary, the LoVo cell infected with both AdCMVTK and AdCEATK were sensitive to GCV. It was shown that CEA promoter has a good tumor-specificity. Significant bystander effect was observed in the AdCEATK/GCV system too. This system should be useful for tumor-specificity suicide gene therapy of CEA-positive tumors.

Abstract:Four kinds of different hammerhead ribozymes (ribozymeA, ribozymeB,ribozymeC₁,ribozymeC₂) were designed and synthesized according to the secondary structure of HCV-RNA 5′-untranslated region and part of the neighbour C-region. Firstly, the cleavage of the four Rzs were tested in vitro, and only the ribozyme with GTA↓ motif at-11nt site of HCV-RNA showed cleavage activity. RzA-RNA and the combinated pCl-neo-luciferase in which a luciferase gene were ligated downstream the target sequence were then co-transfected into HepG₂ cell lines with lipofectine. The cleavage of RzA-RNA was tested by determined the expression of luciferase gene. Therefore, the gene of RzA was ligated into expression vector pCl-neo. This pCl-neo-RzA and the vector pCl-neo-luciferase were co-transfected into HepG₂ cell lines again with lipofectine. Since the pCl-neo-RzA was more stable than RzA-RNA in vivo and could produce RzA-RNA continously, it showed better cleavage activity.

Abstract:As the expanding of application of ES cell gene targeting technique, cloning and structural analysis of genomic DNA for phage library for homologous fragments in targeting vector are becoming more and more important. An effective strategy has been developed, termed as “separating/combining” strategy, to make restriction mapping of large genomic insert in recombinant phage vector. In this strategy, a set of subclone with commonly used plasmid vector, such as pBluescript^TM series, was generated as first step, restriction mapping of each subclone was analyzed and then, digested the whole length phage DNA and analyzed the restriction site combined with the mapping of subclones. An accurate restriction mapping of a large insert of a phage clone, which contain mouse coagulation factor IX gene, was generated successfully with this strategy.

Abstract:By combination of laser scanning confocal microscopy(LSCM) and the highly sensitive fluorescent dyes BODIPY FL-LDL and DiI-AcLDL, it is possible that activities of both LDL and scavenger receptors in a single cell can be measured simultaneously and quantitatively. For example, in C57BL/6J macrophage, it was found that the cells were incubated for 5 h at 37℃ with 5 mg/L DiI-AcLDL and 5 mg/L BODIPY FL-LDL resulted in excellent color imaging under LSCM, the LDL receptor with green and scavenger receptor with red. The high selectivity and visulization of this method provide detailed information on the localization and activity of both receptors in a single macrophage. The rapidity and accuracy of this assay allows its application for studying receptor-mediated lipoprotein uptake.

Abstract:A method is described for staining lactate dehydrogenase(LDH) and esterase (EST) isozymes on the same slab gel. By utilizing the noninterference of two kinds of isozymes in their staining reaction and the difference in their colour, LDH and then EST were stained successively, resulting in distinct bands of both isoenzymes on the same slab gel. The band patterns of either enzyme on the same slab gel were completely the same as those on separate slab gels. Being able to save reagents,time and cost necessary for isozymic analysis,and convenient for the identification and comparison of different samples, this is an economical and efficient staining method, which can also be applied to staining malate dehydrogenenase (MDH) and EST isozymes on the same slab gel.

Abstract:When the thylakoid membranes of blue-green algae broken over a longer time than usual by sonication were solubilized with low concentration of LDS and subjected to discontinuous SDS-PAGE improved at 4℃,15 containing chl bands for the first time was resolves. CPa1-CPa6 of them had similar absorption spectra. The fluorescence spectra of CPas at 77 K were very similar too,all having a emission peaks at 685 nm. It is concluded that 6 CPas were chl a-protein complexes of PSⅡ. The resolved capability to PSⅡ of the new system were 3 times of the popular ones.

Abstract:Human lipopolysaccharide binding protein (LBP) gene was cloned from a human liver cDNA library by PCR. Sequencing result showed that the amino acid sequence coded by this gene is identical to that of the latest reported human LBP.

Abstract:A method for RT-PCR amplifying long fragments cDNA was established and the 7.4 kb full-length cDNA of Hepatitis A virus(HAV) H₂ strain was amplifyed by reverse transcription and polymerase chain reaction (RT-PCR).HAV H₂ was precipitated by anti-H₂ serum specifically, then the RNA of HAV H₂ was isolated from this precipitation by acid guanidinium hydrochloride-phenal-chloroform extraction. The first-strand of HAV H₂ cDNA was synthesised by reverse transcriptase without RNase H activity,then was ampliflied by PCR using the 32mer primer and the Taq DNA polymerase with Deep Vent DNA polymerase. For obtaining longer PCR products,it is necessary to prepare high quality RNA and employ the longer primers and special Taq DNA polymerase.