Abstract:SINEs (short interspersed elements) are mobile elements of approximately 100～500 bp that are often present as more than 10⁵ copies per genome. They can be divided into families and subfamilies according to the mutational or diagnostic sequence loci. SINEs are widespread in eukaryotic genomes and create additional sequence combinations through dispersal and exchange of genetic information. So, they are believed to be of major importance in creating genetic diversity, gene inactivity, new gene, and especially in gene expression and gene regulation. Almost all SINEs reported to date are derived from tRNAs, with the exception of the primate Alu and the rodent B1 families, which are derived from 7SL RNA. The tRNA-derived SINEs have a composite structure, with a region homologous to a tRNA, a middle tRNA-unrelated region, and a terminal AT-rich region. Each SINE contains an internal promoter for RNA polymerase Ⅲ and lacks open reading frames. “Master source gene model”, “multiple source gene model”, “transposon model (parasitism model)”, and “horizontal transfer model”have been postulated to explain the origin and evolution of SINEs. The observation of their molecular phylogeny was made with examples of salmons, whales and artiodactyls，hominoid primates，and western Palearctic water frogs. Their application to genetic profiles and antitumor technique was also introduced.

Abstract:The physiological actitivities of organ and cell are affected significantly by mechanical stimulation. Although cells have different responses to each kind of mechanical stimulus, the mechanism of mechanosense are almost similar. First, the mechanical stimulus was sensed by cell membrane or integrin-cytoskeleton, then G protein and other signal molecules were activated, and finally some physiological or pathological effects were caused. So G protein may play a central role in the process of mechanical energy transforming to chemical energy.

Abstract:Osteoclastogenesis inhibitory factor or osteoprotegerin(OPG/OCIF) is a novel secreted glycoprotein involved in the regulation of bone density. It is a novel member of the TNF receptor superfamily. Mature OPG/OCIF contains 7 domains and can be divided into 3 regions(TNF receptor cysteine-rich region; death domain region and heperin binding region). The OPG/OCIF gene is a single-copy gene consisting of five exons and four introns. It is located in 8q23～24. The factors involved in the regulation of bone formation and resoption(eg. TGF-β1,1,25(OH)₂VD₃ and TNF-α)regulate the expression of OPG/OCIF gene. The mechanisms by which OPG/OCIF inhibits bone resoption can be concluded that: 1) OPG/OCIF inhibits survival of osteoclasts and induces apoptosis of osteoclasts; 2) OPG/OCIF inhibits formation of osteoclasts.

Abstract:In recent years the research on the roles of eukaryotic mRNA 3′UTR in suppression of malignant phenotype of tumor cells has achieved very exciting advances. It has been found that the 3′UTR is involved in regulation of function of known oncogenes and antioncogenes; that the inactivation of some antioncogenes came from the structural changes of their 3′UTR; and that the 3′UTR of several genes exerted antioncogene activity after transfection into malignant cells. Apart from these, further studies have also been performed on the roles of 3′UTR in regulation of mRNA expression. Recent advances of part of studies in this field are reviewed.

Abstract:Three-dimensional structure of insecticidal crystal proteins of Bacillus thuringiensis has been revealed to be three distinct domains. It has been found that different Cry toxins share similar structures. Domain Ⅰ, consisting of a bundle of α-helices in which a hydrophobic helix 5 is surrounded by 6～7 amphipathic helices, plays a unique role in pore formation. Domain Ⅱ, consisting of three antiparallel β-sheets with a loop at each apex, is responsible for receptor binding. Domain Ⅲ consists of two twisted, antiparallel β-sheets forming aβ-sandwich with a “jelly roll” topology, it might prevent the activated toxin from excessive degradation.

Abstract:Neural stem cells (NSCs) maintain the potential of proliferation and differentiation in nerve system. The research and application of NSCs have developed into a frontier of neuroscience in recent years. The establishment of NSCs culture in vitro offers an efficient way for study on them. Now the focus is on the origin and location of NSCs in brains and their transplantation for therapy of CNS(central nerve system) diseases.

Abstract:It had been a long time to study the effect of mitochondria in the regulation of cytosolic calcium signal. Recently, following the development of new method and technology, it is found that mitochondria plays an important role in calcium signaling. Mitochondria can sense the existence of surrounding calcium microdomains and uptake Ca²⁺. It can also release Ca²⁺ through 2Na⁺/Ca²⁺ exchanger and mitochondrial permeablize transition pore. Therefore, the time-spatio characteristic of cytosolic calcium signal can be regulated and related cellular function can be affected by mitochondria. However, due to the limitation of technique used now, confused and contradictory results are often obtained and further exploration is needed.

Abstract:Neuronal growth inhibitory factor(GIF), a brain-specific member of metallothionein family named metallothionein-Ⅲ (MT-Ⅲ), is first validated to be capable of inhibiting the growth of neuronal cell in nervous system. GIF’s amino acid sequence, structure and metal-binding properties are like other metallothioneins’, and its gene shows strikingly high homology to other metallothionein-encoding genes, but they adopt different gene-regulation approaches. With its β-domain CPCP-loop, GIF may bind to some correlative factors that lie in brain extracts to display its specific physiological function. It is considered that GIF is markedly reduced in the brain of Alzheimer’s disease (AD) patients and in several other neurodegenerative disease.

Abstract:How transcription factors regulate gene transcription on chromatin is the central question of the study of gene expression regulation. Recent investigations showed that regulatory factors form different complex in nucleus such as RNA polymerase Ⅱ holoenzyme, chromatin remodeling complexes, nucleosome and enhanceosome, which participate each step of gene transcription and assemble into active transcription complex. Formation of these complexes integrates lots of information of transcription regulation and increase the efficiency of transcription, which is the basis of orderly efficient expression of genes. On the other hand, it is a challenge to study these transcription complexes and development of new technology is important.

Abstract:It is very important to produce recombinant proteins for therapy in biophar-maceutical. Chinese hamster ovary cells(CHO cells) have become one of the best eukaryotic expression systems in recent years. The factors influencing the high-level expression of foreign protein in CHO cells and the core strategies of overexpression of recombinant proteins in CHO cells were discussed.

Abstract:Luteinizing hormone-releasing hormone(LHRH) agonists as antitumor agents for hormone-dependent tumor have been on the market for about ten years, while the LHRH antagonists that have been developed furthest are still in clinical trial. Significant progress has been made in searching for LHRH antagonists with high activity, low histamine releasing, good aqueous solubility. Smaller linear or cyclic peptides and peptidomimetics showed antagonistic activity both in vitro and in vivo. The βⅡ-turn in the central tetrapeptide and the N-terminal tripeptide segment of decapeptide LHRH antagonists play an important role in receptor binding.

Abstract:Monoamine oxidase (MAO) catalyzes the oxidative deamination of a number of biogenic amines in the brain and peripheral tissues by the production of hydrogen peroxide(H₂O₂). The cloning of MAO A and B genes has demonstrated that the enzymes are made of different polypeptides. MAO A and B genes are located on the X-chromosome (Xp11.23) and consist of 15 exons with identical intron-exon organization, which suggests that they are derived from the same ancestral gene. MAO A and B exhibit distinct differences in the substrate selectivity and inhibitor sensitivity and play different role in the neurotrasmitter metabolism and behavior.

Abstract:The study on mitochondrial genome is one hot field in recent molecular biology. The progress of mtDNA research now concentrate on the expression, control and heteroplasmy of mtDNA, and the relationship between the mitochondrial genome and the nuclear genome. However, the discovery in the paternal inheritance of mtDNA brings new sight on traditional inheritance of mtDNA. With the widely using of mtDNA as a genetic marker in population and evolutionary biology, the neutral evolution of mtDNA with respect to fitness and the nuclear DNA has been challenged by nonneutral evolution behavior of mtDNA.

Abstract:By “interfacial affinity chromatography” on GDX101 column, commercial Candida rugosa lipase (CRL) is fractionated into four fractions containing three isoenzymes (CRL-1, CRL-2 and CRL-3). They have different enantioselectivity for the asymmetric hydrolysis of (R, S)-Naproxen methyl ester in the aqueous-organic solvent biphase system. As analyzed on SDS -polyacrylamide gel electrophoresis, isoelectric focusing and organic solvent treatment, slight structural difference among these isoenzymes has been found. CRL-1 and CRL-2 are associated noncovalently with low molecular mass acidic components in different degree, CRL-3 is disassociated with the low molecular mass acidic components. Because of the perhaps hamper of the low molecular mass acidic components, the open extent of hydrophobic pocket around active site on CRL-3 is bigger than on CRL-1 and CRL-2. According to the slight structural difference, isoenzymes with different enantioselectivity for hydrolysis of Naproxen ester have been immobilized selectively on different hydrophobic supports (GDX101 and YWG-NH₂). Via a sample and easily performed selectivity adsorption step, the purification can be combined with the immobilization. This new method seems to be very suitable for an easily separation of such isoenzymes with slightly different structure.

Abstract:Anticoagulation factor Ⅱ (ACFⅡ) from the venom of Agkistrodon acutus forms a 1∶1 complex with activated coagulation factor Ⅹ (FⅩa) in the presence of Ca²⁺ ions. The result that ACFⅡ fails to form a complex with FⅩa in the absence of Ca²⁺ ions indicates that the binding is in the Ca²⁺-dependent manner. ACFⅡ is a new member of the Ⅸ/Ⅹ-bp family, and has a similar amino acid composition to the other members of this family. It is composed of 251 amino acid residues with a relative molecular mass of 29 468.1, determined by MALDI-TOF-MS. ACFⅡ prolongs plasma prothrombin time (PPT) in the dose-dependent fashion and exhibits marked anticoagulant activity only at the concentration higher than the critical concentration (12 nmol/L) of ACFⅡ.

Abstract:Vitronectin was purified from human plasma by heparin affinity chromatography and rough plasma protein containing fibrinogen was isolated from human plasma by (NH₄)₂SO₄ precipitation. Protein gel was generated by addition of thrombin to the complex composed of plasma protein, bovine fetal serum and DMEM.Bovine aortic endothelial cell(BAEC) was plated on the gel matrix and the growth，vascular formation of endothelial cell in the gel were observed. The results demonstrated that BAEC can adhere to the gel surface and grow normally as grow on the culture plate, while under the induction of bFGF, BAEC can migrate into the gel and form vascular-like structure in the gel matrix. Many vascular-like structures can link to each other to form capilary net structure.

Abstract:The system of the photoelectric response of Bacteriorhodopsin membrane (SPRBM) is an important system for molecular-electric research. Based on the Hong’s concept of chemical capacitance, a functional block diagram model of SPRBM was developed. It consists of C-capacitance and its current source; N-capacitance and its current source; proton transfer channel and back channel .By using this model ,a pulse response function and potential distribution of the “Fe/Br/Gel/Cu” photo-cell and its equivalent circuit were derived.

Abstract:T-lymphocyte depletion of the marrow graft can prevent graft versus-host disease(GVHD) effectively. T-lymphocytes of mice were transfected with high-titer (1.5×10⁵ CFU/ml) retrovirus supernatant, and by selection with G418 positive cell clones (T/pCD₂) were obtained. PCR and RT-PCR showed that CD gene was transferred into the T-lymphocyte and expressed successfully. T-lymphocyte and T/pCD₂ cells were exposed to different doses of 5-FCyt for various hours，and were then observed under light microscope and the viability of these cells was determined by MTT colorimetric cell proliferation assay. The results showed that retrovirus-mediated CD gene transferred to T/ pCD₂ cells could confer them high sensitivity to 5-FCyt, and T/ pCD₂ cells exposed to 5-FCyt (＞1 μmol/L) can be killed. But 5-FCyt was not harmful to normal T-lymphocyte cells. The survival time of T/ pCD₂ cells in the presence of 5-FCyt(3～5 d) was significantly shorter than that of T-lymphocyte(＞14 d).

Abstract:To quantitate DNA breaks, a method based on saturation labeling 3′-ends of DNA fragments with α-³²P dCTP in the presence of 2′, 3′-dideoxy-cytidine-5′-triphosphate (ddCTP) by terminal deoxynucleotidyl transferase (TdT) was developed. The saturation labeling of 3′-ends of DNA fragments was performed by adding different concentrations of α-³²P dCTP to a DNA sample, from which a maximal labeling (L_max) and a kinetic parameter(K_m) of the TdT reaction were calculated. Results were confirmed by agarose gel electrophoresis, fluorescein-dUTP and exogenous teminal deoxynucleotidyl transferase(TUNEL), flow cytometric analysis (FCA). The method mentioned above requires as little as 5 ng of DNA, increases in the sensitivity of DNA fragments detection by at least 200-fold relative to the widely used agarose gel electrophoresis, and the linearity of the assay is about 5～5 000 ng DNA. The application of the method in the apoptosis study showed that(1) a time- and dose-dependent increase in the number of DNA strand breaks in apoptotic Raji lymphoma lymphocytes induced by dexamethasone, and (2) age-dependent increase in the number of DNA strand breaks occurred in the cardiac tissues of spontaneously hypertensive rats (SHR) compared with that of normal control rats (WKY). Results of the assay were confirmed by the DNA ladder pattern exhibited after electrophoresis, fluorescein-dUTP and exogenous terminal deoxynucleotidyl transferase(TUNEL), flow cytometric analysis(FCA)(r＞0.98). It is a quantitative, simple, sensitive, specific and useful assay for assessing DNA degradation in molecular and cell biology especially in apoptosis research.

Abstract:In order to investigate the effects of cholecystokinin octapeptide(CCK₈) on calmodulin activity in rat cerebral cortex，the cerebral cortex neurocytes was used as a model. CCK₈ stimulated the activation of CaM in a time-dependent manner. After 15 minutes of treatment of 1 μmol/L CCK₈，the CaM activity reached the maximum increase. CaM activity was increased in a dose-dependent manner by CCK₈ (10^－12～10^－7 mol/L). The CCK_B-specific receptor antagonist L-365，260 and with a weaker efficiency，the CCK_A-specific receptor antagonist L-364，718，were able to block a maximal effect of CCK₈-induced CaM activation，suggesting that CCK_B receptor may regulate CaM activity in cerebral cortex.

Abstract:By transmission electron microscopy, DNA agarose gel electrophoresis, flow cytometry and TUNEL staining, changes of the murine peritoneal macrophages treated with high dose of dexamethasone were observed. The results clearly showed that, treated with high dose of dexamethasone, the macrophages presented various changes in ultrastructure: chromatin condensed and abutted sharply against the nuclear membrane, and cytoplasm condensed. Half hour after dexamethasone treatment, DNA ladder was visualized by agarose gel electrophoresis. The macrophages displayed TUNEL positive. The characteristic apoptosis peak of DNA was showed by flow cytometry. The results indicated that the macrophages treated with high dose of dexamesathone may develop apoptosis rapidly and efficiently.

Abstract:The high effective antibody of interleukin-6 was obtained by immunizing rabbits and guinea pigs with recombinant IL-6 many times. The IL-6 was labeled by ¹²⁵I with chloramines-T methods and purified by the Sephadex-G25 chromatograph column. The reaction between antigen and antibody was carried out by one step balance method and incubated in 4℃ for 24 hours, then separated bond and free antigen by PR reagent. The detection range of this method was about 0.1～3.2 μg/L, the lowest detection level was 0.1 μg/L, error within batches and between batches was less than 6.4% and 10% respectively. The serum IL-6 concentration in normal male was (0.270±0.13) μg/L (n=115), and in female was (0.260±0.10) μg/L (n=101), there was no difference in male and female group. Otherwise, the level of IL-6 in serum of rabbits was significantly higher than that of self-control at 24 hours after hemorrhagic shock and reperfusion. IL-6 in lymph fluid of rat after hemorrhagic shock was significantly elevated, then it was descended by the treatment of anisodamime (1 mg/kg). The liberation of IL-6 also was promoted and obviously higher than that of the control when fibre cells around tooth were cultured with endotoxin (10 mg/L) at different time in vitro.

Abstract:A method for preparing biospecific affinity chromatography agarose for angiotensin-converting enzyme is reported. Bisoxiranes, 1,4-butanediol diglycidyl ether, has been used for introducing reactive oxirane groups into Sepharose CL-4B, and coupling to ACE selective inhibitor——lisinopril in anion conditions including NaBH as deoxidizer. 0.79 mg ACE protein was purified from 200 g hog lung homogenate by 1.6～2.6 mol/L ammonium sulfate fractionation, dialysis, buffer balance and affinity chromatography. The recovery of ACE activity was 11.9% with specific activity of 38.8 U/mg protein and the enzyme was purified 808 fold in comparison with the homogenate supernatant of hog lungs, specially, only one step of affinity chromatography, the enzyme was purified 264 fold. Purified ACE showed a single band after migrated identically in sodium dodecyl sulphate-polyacrylamide gel electrophoresis and given an apparent molecular mass about 180 ku.

Abstract:Levels of hydrogen peroxide in plant extracts were overestimated by the method of only using titanium(Ⅳ) because of the interference of pigments and other materials while underestimated by the method of adding activated charcoal(A.C.) in 5% trichloroacetic acid extracts to remove pigments. These problems were avoided by a developed method of extraction, which could not only remove the pigments in acetone extracts conveniently but also get a high recovery more than 95%. Hydrogen peroxide was determined by its reaction with the complex of titanium(Ⅳ)and 4-(2-pyridylazo) resorcinol against references catalase-treated. The minimum concentration of hydrogen peroxide determinated in this essay was 0.25 μmol·L^－1. Levels of hydrogen peroxide in leaves of some plant species ranged from 0.1～0.8 μmol·g^－1.

Abstract:Thalli from a brown alga Undaria pinnatifida were soaked by CaCl₂ solution with different concentration and time at 4℃, the effect of CaCl₂ solution on efficiency and influorescence emission spectra of chloroplasts were examined. The results show that the efficiency of collected chloroplasts is increased markedly after soaking in CaCl₂ solution. According to the results of collected efficiency and characteristic of influorescence emission spectra at room temperature of chloroplasts, it was suggested that soaking in the 0.2 mol/L CaCl₂ solution for 10 min is optimum. Under this condition, the efficiency of collected chloroplasts is as 5 fold as control group, and the characteristics of chloroplasts obtained by CaCl₂ soaking are similar to that of traditional method.

Abstract:To detect trace amount of DNA point mutation in mammalian cell pool, a semi-nest allele specific PCR was optimized by adding 5% formamide and decreasing all the components’ amount of the polymerase chain reaction to eliminate the nonspecific amplification. This modification was so effective and highly specific that it can be used to detect the point mutation which is lower than 0.025% in mammalian cell pool.

Abstract:The culture of callus and cell suspension induced from leaves of Rhodiola algida Var.tangutica collected from Qinghai plateau(altitude:4 000 m),on solid and in liquid culture medium(MS+BA₂+NAA_0.2)respectivelly,was successful.After cold (4～5℃)acclimation or ABA treatment for 10 or 14 days, the freezing resistance of suspension cells was increased. Proteins from the liquid suspension cell culture medium exhibited antifreeze activity. Treatment of the antifreeze proteins(AFPs) with boiling or trypsin caused inactivation,proving the protein characteristics of AFPs.Periodate and borate inhibited the antifreeze activity of AFPs,suggesting the presence of carbohydrate. Of the four treatments(25℃;25℃+ABA;5℃;5℃+ABA),2～3 polypeptides(29～50 ku) could be detected from the one dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）. PAS(periodic acid-schift’s reagent) reaction of SDS-PAGE showed the presence of carbohydrate in the polypeptides.

Abstract:A simple method of protein staining with Coomassie Brilliant Blue G250 is introduced, which had almost no background,needed less time and only two reagents. The working concentration of Coomassie Brilliant Blue G250 in the staining system with diluted hydrochloric acid was 0.0015%, and the sensitivity was 0.02 μg protein per electrophoresis zone.

Abstract:To evaluate the direct measurement of low density lipoprotein cholesterol(LDL-C) and improve the technology, a comparison study between 2 reagents kits for the application, which are commercially available now, is hereby reported. It shows the advantage of direct measurement is simple, rapid, accurate and suitable for auto-analysis. Both of the 2 reagent kits could meet the currently established analytical performance goals. Analytical CV＜4%, significant correlation (r=0.994, y=1.0572x－0.1052, S_y/x=0.1913), with similar results of many tests, the 2 kits could be used for routine applications according to the user’s real situation.