Abstract:Attributing to the progress in the Genome Project, more and more gene sequences, most of which are not known any information about function, have been accumulated in database. So analysing the function of those genes will become the main goal after the Genome Project is finished. Some strategies and procedures for large-scale functional analyses of genes, such as gene disruptions, serial analysis of gene expression,high density cDNA hybridization and proteome analyses, are introduced. Furthermore, the prospects of large-scale gene functional analyses are also discussed.

Abstract:Gene targeting is an experimental technique which could alter the genetic message of lives following the anticipated pattern through homologous recombination. Gene targeting combined with the system of the culture of the mouse embryonic stem cell facilitate introducing various mutations into living mouse, so that the expression, regulation and physiological function of higher eukaryotic genes could be studied as a whole. Recent progress in studying on gene targeting in mouse embryonic stem cell was introduced briefly.

Abstract:Matrix-attachment regions (MARs) has been identified to buffer the effects of flanking chromatin in stably transfected cell lines. This gives new area in transgenic animal gene expression. Some researchs on MARs in transgenic animal and possible mechanism are reviewed. It suggests that MARs can establish independent genetic domains and have important significance on increaseing gene expression in transgenic animal.

Abstract:Most recent researches have concentrated on the rapid development of enzymesensors, immunosensors and microbial biosensors, whereas the studies about the DNA biosensor are relatively scarce. The principles of DNA sensor based on the principle of nucleic acid hybridization are reviewed. Several kinds of DNA sensors such as electrochemical, optical, acoustic and piezoelectric are introduced and compared, the high specificity and low responsibility of DNA sensor are also discussed. Generally speaking, DNA biosensor has a more prospective future.

Abstract:The number of insect neuropeptides identified chemically grows rapidly and many important neuropeptides have already been characterized. After multi-year efforts, diapause hormone and pheromone biosynthesis activating neuropeptide have recently been isolated and sequenced, respectively. New approaches to search for new insect neuropeptides have been carried out by some groups of workers, which have succeeded in identifying several unique peptides. Cloning of cDNA or genomic DNA for insect neuropeptides showed new information about structure and function. It was found that two functionally distinct neuropeptides, Bombyx diapause hormone and pheromone biosynthesis activating neuropeptide, are encoded in a single gene.

Abstract:At present, more and more people are interested in studying the interaction between the receptors and their RGD-containing ligands. It is generally thought that the interaction between integrins and adhesive proteins is mediated through the RGD sequence within the adhesive proteins, which may participate in many physiologic process. A major description is given on the interaction between RGD-containing peptide and GpⅡb /Ⅲa and its inhibition on platelet aggregation. Also presented is the perspective of RGD-containing peptides or proteins in the study of the relationship of the structure and function of protein and in the pharmaceutical field.

Abstract:E.coli is still the first choice of host when foreign gene is expressed. Heterologous proteins could be expressed directly in the cytoplasm, secreted into the periplasmic space or extracellular medium. Recent developments on E.coli expression system are covered, according to the possible destiny of foreign proteins.

Abstract:Photorelease technique of Ca²⁺ can manipulate intracellular calcium concentation (［Ca²⁺］_i) by photolyzing specific photosensitive Ca²⁺ chelator in the cell to change its affinity with Ca²⁺. This technique is especially useful in elucidating the intracellular messenger role of Ca²⁺ in cell functions such as electrical excitability, muscle contraction and secretion, etc.

Abstract:Recombinant human Fab(rh Fab) with a hexahistidine tail attacked to the carboxyl end of Fd could be easily purified by immobilized metal ion affinity chromatography (IMAC). IMAC with different immobilized metal ions(Zn²⁺ and Cu²⁺) and different elution strategies (pH and imidazole gradient) were compared. The results showed that Cu²⁺ were more effective than Zn²⁺ in retaining the His tagged Fab protein. pH gradient showed better consistency in Fab recovery than imidazole gradient. Using Cu²⁺ IMAC more than 95% pure rh Fab could be obtained by one step purification.

Abstract:The effect of N-LDL, AC-LDL and OX-LDL on the production of nitric oxide (NO) and expression of nitric oxide synthase (NOS) in mouse peritoneal macrophages stimulated by LPS was studied by measuring nitrite in media and mRNA for NOS in macrophages. The results showed that OX-LDL could inhibit NO production in macrophages stimulated by LPS, but N-LDL and AC-LDL could not. The inhibitory effect of OX-LDL was enhanced with the increase of the degree of oxidation of LDL and was time and concentration dependent. Results obtained from slot hybridization showed that OX-LDL could decrease the content of mRNA for NOS in macrophages induced by LPS, which implies that inhibition of NO production of OX-LDL was taken place at the level of transcription.

Abstract:Dynamics in countercurrent chromatography, a kind of liquid-liquid chromatography without solid matrix, is very different to that in other liquid-liquid chromatography. Dynamics in countercurrent chromatography was analyzed and the rate equation that fits countercurrent chromatography was derived out. Some experiments were done to measure H(plate height) of two kinds of CCPC(centrifuge countercurrent partition chromatography) columns. The results of both theoretical deriving and experiments reveal that the diameter of column, capacity factor, mass transfer coefficient, the average diameter and velocity of mobile phase droplets are main factors that determines H. Mathematics analysis is given.

Abstract:Purified recombinant Fusarium oxysporum cytochrome P-450nor (rF·P-450nor) was used for spectroscopic and kinetic studies. K_m and V_max were determined. K_m(NO)and K_m(NADH) were 0.128 mmol/L and 0.208 mmol/L respectively. V_max for N₂O was 11 363 min^-1. The spectroscopic study indicated that: rF·P-450nor has the typical characteristics of a heme protein, and has the maximum absorbance at 413 nm. After added Na₂S₂O₄，the maximum absorption peak was moved to 405 nm. After added CO and Na₂S₂O₄, the absorption peak was at 450 nm. When combined with NO, the absorption peak was moved to 430 nm. These spectrum characteristics were the same as those of the native F·P-450nor.

Abstract:Digestion rate markedly increased as exonuclease Ⅲ digesting linear DNA with double chains which had reacted with Bleomycin A₅-Ce(Ⅲ) ［BLMA₅-Ce(Ⅲ)］. In additon, there were other products to the four mono-nucleotides (5-dAMP, 5-dTMP, 5-dCMP, 5-dGMP). It is suggested that BLMA₅-Ce(Ⅲ) cleaves phosphooryl diester bonds along the direction of 5′～3′ at specific sides of double-chain DNA forming more exposed 3′-OH ends.

Abstract:Shark cartilage preparation (SCP) was extracted from shark cartilage by the method of guanidine hydrochloride extraction, acetone fractional precipitation, and ultrafiltration. The effect of SCP on the cytoskeleton of endothelial cell was investigated by the whole cell scanning electron microscopy. The effects of SCP on endothelial cell migration and angiogenesis were measured by the cell migration experiment and the chicken embryo chorioallantoic membrane experiment, respectively. The results showed that the cytoskeleton of endothelial cell was wrinkled, and SCP inhibited the migration of endothelial cell, the inhibitory rate was concentration dependent. SCP also inhibited the angiogenesis in the chorioallantoic membrane of chicken embryos significantly. These results suggest that SCP has the inhibitory effect on angiogenesis, and the mechanism may be that SCP inhibits the cytoskeleton of endothelial cell, so inhibits the cell's migration, and so inhibits angiogenesis.

Abstract:The effect of antibacterial peptide CM4 component act on the K562 cancer cells in vitro is reported. The tests showed that: the purified antibacterial peptide CM4 component can kill human myelogenous leukemia cancer (K562). The observation of ultrastructure of the cancer treated with the antibacterial peptides by using the scanning and transmission electron microscope and the microfault picture analyses of laser confocal microscope. The results showed that the pruified antibacterial peptides brought about a series of pathological changes on cancer cells. As a result, the cells were high swelling, plasmolysis and disorders in subcellular and membrane structrue occurred, outer coat of cell dissolved, thus appearing irregular holes opening in a utensil and serious broken of skeleton structure of the cells. The membrane structure of some areas was broken and injured and intracellular substances leaked out. As a result, the cells disintegrated into small fragmants and died.

Abstract:Native type Ⅳ collagen (CⅣ) was isolated and purified from human placenta by pepsin limited digestion, fractional salt precipitation, reduction and alkylation, and cellulose chromatography. The two constituent α peptide chains of the purified CⅣ were identified by SDS-PAGE. Antiserum was prepared by immunizing rabbits with the purified CⅣ. The obtained antiserum against human CⅣ showed high titer and specificity.

Abstract:The Principle and method of a new kind of stereogram, Periodical random-dot stereogram(PRDS) was described. An example of a circle of PRDS and flowchart were given. The effect of the value of Period on depth perception was discussed.

Abstract:D2-04 RNA was isolated magnetically from cultured C6/36 cells. With this RNA as template, the cDNA both 5′ and 3′-tumini were amplified by RT-PCR respectively. After inserted into pGEM-3Z plasmid, the cDNAs were sequenced by dye-primer and dye terminator PCR sequencing strategy, and the primary and stable secondary RNA structure were analyzed by computer software on VAX-11-780 computer.

Abstract:An improved enzymatic end-point method for determination of plasma ammonia was reported. Semi-micromethod were used with a total incubation volume 1 165 μl. Mix 125 μl EDTA Na₂-anticoagulation plasma and 1 000 μl PBS-substrate-solution containing 1.9 kU/L LDH (pH 8.0), then add 40 μl GLDH (≥12×10⁴ U/L) to start the reaction. Incubation periods were shortened to 5 min. The limited detection for ammonia in plasma is 4 μmol/L, and this method is linear from 6 to 200 μmol/L. The method is accurate and reliable. The analytical recovery is from 90% to 105%. The within-run CV% was≤5%. The routine condition variance of the method ranged from 5% to 10%. It was found that 1.9 kU/L LDH was sufficient to eliminate the decreace of NADH by endogenouse pyruvate and LDH in plasma. The normal fasting ammonia in plasma is 10～70 μmol/L.

Abstract:CHO-EPO C2 cells were cultured in the Packed Bed Bioreactor with free-fetal bovine serum medium, and the supertant contained 2 000～3 000 U/ml of recombinant Human EPO(rHuEPO). The SDS-PAGE result of the final products from the reported purification schemes was a single band, which purity was over 98% with measurement of UV scanning. The specific activity was 1.5×10⁵ U/mg protein. Reserch showed that the molecular weight was about 35～40 ku and the pI was about 3.75～4.15.The product possessed the antigenity of native HuEPO. The peptide electrophoresis result was in accord with theory deducation. 15 amino acid sequence of rHuEPO N terminal was as the same as native HuEPO. All these results described above revealed that the final product were high purity rHuEPO and the purification procedure was rapid and efficient, which can be used to produce clinic rHuEPO in large scale.

Abstract:Several superoxide dismutases (SODs), such as porcine and bovine erythrocyte Cu/Zn-SODs and N.tabacum Mn-SOD, were found to exhibit the activity to cleave supercoiled DNA in vitro. They converted supercoiled DNA into nicked and further into linear form. They did not act on linear double-stranded DNA. Activity assays after they were inactivated by H₂O₂ or guanidine and hydrolyzed by proteases suggested that these two activities of dismuting O^·₂ and cleaving supercoiled-DNA involved in different sites in SODs.

Abstract:A thrombin-like enzyme was purified from the venom of the snake Agkistrodon acutus. It was shown to be a single band both in SDS-PAGE and IEF. Its molecular weight is about 38 ku and isoelectric point is near 4.0. Also, its 15 amino acid sequence of N-terminal was obtained. It is VIGGVECDINEHRFL. It has high homology with thrombin-like enzymes from other snake venoms.

Abstract:Solubilized rat liver mitochondrial choline dehydrogenase was further purified by the SDS-PAGE and electroblotting methods. Both the lipids and detergents etc. were removed during the process of purification. The amino terminal sequence of the purified CDH was determined, it was: VAAAAGGGKD. Although the sequence showed high homology with mouse CO5 protein, rat adenylyl cyclase and human oxysterol-binding protein, there was no similarity with CDH of E.coli.

Abstract:BIA Technology now is widely used in all kind of Bio-research. It will be found how BIA have been used for ligand fishing to detect and purify the unknown ligand. The other application is to detect the affinity and kinetic information of monoclonal antibody directly from hybridoma supernatant.