Abstract:Interleukin 18 (IL-18) is a recently identified cytokine, originally called interferon gamma inducing factor. Due to its capacity to induce interferon gamma production in Th1 type cells. It has pleiotropic biological functions. IL-18 belongs structurally to the IL-1 cytokine family and shares biological properties with IL-12. Consequently, IL-18 is a potential regulator of antitumor immune response. IL-18 plays an important role in autoimmune disease and endotoxin-induced injury. So, future work will focus on its application as an immunotherapeutic agent for a variety of diseases.

Abstract:Proteins take center stage in directing the working of living cells. Every conceivable role within human bodies is played by proteins, from catalysis chemical reactions to defence against alien attack. A variety of quality control mechanisms that operate in the endoplasmic reticulum in downstream compart-ments of the secretary pathway to ensure the fidelity and regulation of protein expression during cell life and differentiation were introduced. The posttrans-lational quality control and its relation with protein misfolding were discussed in details. A number of diseases related with protein misfolding were introduced and the principles of therapy were discussed.

Abstract:With the coming of the post-genome era, proteome research, which was sponsored by National Natural Science Foundation of China in our country, has increasingly caught many biochemists’ attention. The fundamental technologies of proteome research are briefly introduced, including sample preparation, protein separation, protein spot detection, gel image analysis, protein identification, database construction, et al. The commonly used identification methods are amino acid analysis, protein sequence tags and peptide mass fingerprinting. The flow chat of proteome analysis is shown, and in particular the latest proteome technologies are summarized.

Abstract:The recently discovered coagulation factor Ⅸ/factor Ⅹ-binding protein family is widely present in the venom of viperidae snake, which is an unique subfamily in the C-type animal lectin superfamily. The proteins of this family are non-enzymatic anticoagulants that bind to the Gla-domain regions of factor Ⅸ or factor Ⅹ and form 1∶1 complexes with factor Ⅸ or factor Ⅹ in the presence of Ca²⁺ ions. They are heterodimeric proteins consisting of two highly homologous peptide chains linked by a single disulfide bridge. Each chain contains one Ca²⁺-binding site and an intrachain disulfide-bonding pattern similar to those of C-type carbohydrate recognition domain. The amino acid sequences of this family exhibit high homology amid them. The crystal structure of habu coagulation factors Ⅸ/Ⅹ-binding protein has been determined. It is an intertwined dimer with a central loop projecting into the adjoining chain. Excluding this loop, each chain has a fold similar to rat mannose-binding protein.

Abstract:The two-component system is a signal transduction pathway in both of the prokaryotes and eukaryotes. Since there are only two components, histidine protein kinase(HPK) and response-regulation protein(RR), in the signal transduction pathway in prokaryotes, therefore, it is named the two-component system. However, further research, especially with the extensive identification of this system in eukaryotes, established that this system is composed of more than two components besides HPK and RR, showing diversity in its composition and signaling pathway. Usually, the two-component system is divided into several processes such as signal input, HPK auto-phosphorylation, RR phosphorylation, and signal output. The mechanism for the signal transduction of the two-component system and its role in the perception and transduction of osmotic stress are reviewed.

Abstract:To actively transport many of its proteins to extracytoplasmic compartments such as the periplasm and outer membrane, E.coli employs a collection of Sec(secretion) proteins that catalyze the translocation of various polypeptides through the inner membrane. The targeting of periplamic proteins after the cleavage of signal peptide is commonly regarded as a default process, while the final destination of outer-membrane proteins needs the help of other factors and is well accepted as a periplasmic intermediate process.

Abstract:Following the importances of telomere and telomerase in senescence and tumor were recognized, the pathway of telomerase activation is becoming the focus of this area. By now, MYC was found to play key role in telomerase activation. It could activate telomerase directly by binding to E-box in promoter region of TERT gene. At the same time, MYC might mediate other molecules to activate telomerase, such as HPV-E6 and estrogen. Estrogen could activate telomerase directly by forming estrogen/estrogen receptor complex binding to imperfect palindromic degenerate estrogen-responding element in promoter region of TERT gene. APC, p53 and protein phosphorylation might also involve in telomerase activation.

Abstract:Due to the lack of efficient cell culture systems, animal models, the low amounts of viral antigens and RNA in infected tissues, knowledge about the replication mechanisms,especially the RNA-dependent RNA polymerase(RdRp) of hepatitis C virus(HCV) is poor.Based on analysis of amino acid sequence of HCV polyprotein and analogy to the closely related flaviviruses and pestviruses,it is assumed that NS5B may be the RdRp of HCV.By Baculovirus and E.coli expression system and in vitro RNA replication system,it was demonstrated that the de novo synthesis of RNA could be catalyzed by NS5B,which resembles other viral RdRp.The biochemical properties and the mutation-function relationships of RdRp of HCV were comprehensively reviewed,and the potential of NS5B as an important new target for antiviral therapy was also discussed.

Abstract:There are two kinds of molecular containing histone deacetylase activity: One is homology with yeast RPD3，and another is not homology with yeast RPD3. These deacetylases have different sources ,exit in different complex ,and catalyze different histone or other proteins deacetylation; There are close relationship between deacetylases and the regulation of gene transcription, especially repression of gene transcription.

Abstract:Flagellar motor is a molecular rotary motor,it plays central role on the structures and functions of bacterial flagella.The structure of flagellar motor was clarified on the whole,its stator and rotor were composed of five proteins:Mot A,Mot B,FliG,FliM and FliN.The driving force comes from transmembrane H⁺ or Na⁺ flux. At present its rotary dynamics and torque-generating mechanisms were understood preliminarily.As a perfect research model for molecular rotary motor,the further study of flagellar motor would be very helpful for realizing the mechanisms of bioenergetic conversion and cell motility,thus its study is of widespread importance for biology.

Abstract:The change of intracellular free Ca²⁺ in A₅₄₉ cells sensitive and A₅₄₉/DDP cells resistant to the cis-dichlorodiammine platinum (cisplatin) were measured by Fura-2/AM, the proliferation ability and cell cycle were measured by propidium iodide(PI) labeling cellular nuclear DNA. The results indicated that the concentration of intracellular free calcium of the sensitive A₅₄₉ cells was 2 times higher than that of the resistant A₅₄₉/DDP cells; the proliferation ability of the latter increased significantly than that of the former, the cell cycle also shortened. The proliferation ability and cell cycle of the two cell lines also clearly showed difference by decreasing or increasing their intracellular free calcium concentration if the cells were treated with BAPTA-AM or EGTA and A₂₃₁₈₇ or Thapsigargin. All of the results demonstrated that the concentration decrease of intracellular free calcium in the A₅₄₉/DDP cells resistant to cisplatin may affect the cellular proliferation, shorten the cellular cycle, which would be helpful to remain the multidrug resistance characteristics of A₅₄₉/DDP cells by specially modulating the cellular decisive G1 of cell cycle.

Abstract:R-phycoerythrin(R-PE) is one of important proteins involved in capturing light during photosynthesis in red algae, and it is highly fluorescent, and water-soluble chromophores. In vivo, it can transfer the light energy into photosynthetic center, however, it can deliver the captured light energy captured to the surrounding oxygen in vitro and produce reactive oxygen species such as singlet oxygen, which is toxic to tumor cells. R-PE was added to the culture medium of tumor cells, subsequently with irradiation of 488 nm, Argon laser of 25.6 J/cm². The result by MTT assay showed that the survival rate decreased with the increase of R-PE concentration from 1 to 100 mg/L. The result from ³H-TdR incorporation demonstrated that the synthesis of DNA reduced when the concentration of R-PE increased from 0.01 to 0.32 mg/L. Besides, pUC18 DNA showed a conversion from supercoiled into linear conformation. The conclusion comes that R-PE mediated PDT can influence the conformation of DNA, and it may be one of the mechanisms of R-PE mediated photodynamic therapy .

Abstract:Soybean vacuolar H⁺-ATPase is one of the ATPases and play an important role in the growing period of the plant. Hypocrellin B and KI quench the intrinsic fluorescence of outside and inside membrane domain respectively. This two quench probes have been used to quench the protein’s intrinsic fluorescence under different pH and temperature. The relationship between hydrolysis activity and folding condition of V-ATPase has been preliminarily studied. The K_SV of outside and inside membrane domain under different pH and temperature had been compared. It shows that the intrinsic fluorescence of the protein and K_SV of outside and inside membrane domain all dropped with the deviation of pH and temperature from the optimum condition and the activity of the enzyme dropped too. This illustrates that the folding condition had been changed with the dropping of the enzyme’s activity. The changing of the folding condition of the protein plays an important role in the inactivation mechanism.

Abstract:Neonatal rat cardiomyocytes were cultured and cytotoxicity in cultured cardiomyocytes was induced by H₂O₂ over a wide concentration range (0.05～50 mmol/L) to assess dynamically the effect of H₂O₂ on cardiomyocytes. The results showed that application of ＜0.1 mmol/L H₂O₂ to cardiomyocytes caused accumulation of lipid peroxide(MDA) at 24 h, and phase changes of cellular proliferation cycle at 2h, which represented early biochemical changes. Exposure of cardiomyocytes to the increasing concentrations of H₂O₂ (1～5 mmol/L) induced progressively biochemical injury; the levels of LDH and MDA were significantly higher in the cardiomyocytes exposed to H₂O₂ than in control, with concomitant morphologic changes. When exposed to high concentrations (＞10 mmol/L), a large number of cardiomyocytes were found dead by MTT assay, and morphologic examination by HE-staining showed that cardiomyocytes contracted extremely, forming a large dropping areas of cardiomyocytes, concurrently with the marked increase of membrane permeability which caused LDH substantial leakage from myocytes to H₂O₂. It is proposed that H₂O₂ accumulation can induce cardiomyocyte cytotoxicity in a dose-and time-dependent manner. Treatment with low concentrations of H₂O₂ causes cardiomyocytes early slight biochemical changes which represent pre-apototic injurious features. High concentrations of H₂O₂ can progressively induce lipid peroxidation, which cause the severe damage of the cell membrane. With exposure of cardiomyocytes to H₂O₂, the magnitude of the cytotoxicity is modulated by horseradish peroxidase (HRP). It is suggested that HRP may protect cardiomyocytes against reactive oxygen species.

Abstract:Y2 receptor, one of the two early recognized major receptor subtypes of neuropeptide Y (NPY) was considered to be involved in multiplicate biological and pathological functions induced by NPY. In order to make antibody against Y2 receptor and study the distribution of Y2 receptor, NPY Y2 receptor gene was amplified by RT-PCR from the total RNA of rat hippocampus. Then the C-terminal fragment of Y2 receptor was amplified by PCR and cloned into expression vector. The expression vector of C-terminal peptide of NPY Y2 receptor was transformed in E.coli, and the product of expression was purified.

Abstract:The plasma membrane from suspended-cultured Populus euphratica(PE) cells had been isolated and purified by two-phase partition composed of Dextran T-500 and PEG 3350. Effect of different concentrations of the polymer (5.5%，5.7%，5.9%，6.1%，6.3%，6.5%) and KCl (0，5，10，15 mmol/L) on the purification were examined. The results indicated that two-phase system composed of 5.9% polymer without salt resulted in plasma membrane of PE with higher H^＋-ATPase activity. The activity of the membrane H^＋-ATPase has been increased 8-fold (from 4.23 to 32.63 μmol/mg·h) with better orientation and most of the membrane H^＋-ATPases are right side-out. These results may provide better basis and availability for further studies of the purified H^＋-ATPase from PE plasma membrane.

Abstract:A random decapeptide library was constructed by using phage-surface display. The oligonucleotide sequence (NNK) was digested with SfiⅠ and NotⅠ and ligated into the phagemid pCANTAB5E. The recombinant DNA was introduced into E.coli TG1 by electroporation, and 5.3×10⁷ phage was harvested. The insert was present in 66.7% of phage,thus the random deca-peptide library had a complexity of 3.53×10⁷.The titer of phage supernatant was 4.8×10¹¹ after the helper phage M13KO7 super-infection. This library was screened using angiogenin protein. 26 ANG-binding clones were indentified from 94 enriched individual phagemid clones after two rounds of panning, The nucleotide sequences encoding peptide recombined in 12 positive phagemid clones were determined. ELISA showed that all of them could specifically bind to ANG.

Abstract:Two-dimensional gel electrophoresis (2-DE) is a key technique for proteomics. To analyze the proteome of PC12 cells and rat central nerve tissues, including brain and spinal cord, 2-DE technique is established. Due to much lipid and other non-protein interfering constituents, protein extraction is much more difficult for nerve tissues than for other tissues. Two different methods, i.e., precipitation with tricholoroacetic acid/acetone and ultra-centrifugation were employed to extract protein from rat brain and spinal cord for 2-DE. Other factors such as method and volume of loading sample, choice of IPG gels, concentration of SDS gels, preset of electric parameters, protocol for staining and drying the gels were also improved. Using the proper method described above, satisfactory 2-DE maps of PC12 cells, rat brain and spinal cord were obtained.

Abstract:Determination of lipid signaling pathways and the production of some intracellular signaling molecules has been an focus of some research work in the field of signal transduction research. For example, the determination of the regulation of different phospholipase activity and the formation of intracellular second messengers and other lipid bioactive molecules from different sources is an important component in the studies on the biological effects and their mechanisms of growth factors and other molecules. Here, a method was introduced for selectively labeling different phospholipids, which has been widely used in the study of lipid signaling pathways. Experimental evidence was presented to demonstrate how to determine the different signaling pathway and the generation as well as the alteration of relevant signaling molecules. The application of this method is flexible and has a high reproducibility. It can be used to pinpoint some signaling pathways and the formation of different signaling molecules. A brief introduction about lipid signaling and its significance in the area of signal transduction was also given.

Abstract:A facile DNA shuffling protocol was introduced. Two genes of about 1 700 bp were obtained by PCR from two different individual templates, separately, which shared over 93% homology. After mixed with equimolar of each, these two genes were further cut randomly by DNaseⅠ into small fragments of 10～50 bp under the existence of Mg²⁺. These small fragments were successfully re-assembled to form full genes with original size by one round of PCR without any external primers and two other rounds of normal PCR amplification. This shuffling protocol may help to construct chimera genes from a family of genes with high sequence homology.

Abstract:A very significant linear relationship was found between the capacity of scavenging DPPH free radical and concentrations of six antioxidants (r=0.898～0.994)determined by spectrophotometry.There was an obvious difference in the capacity of scavenging DPPH free radical among different antioxidants. Both scavenging DPPH and inhibiting the oxidation of adrenalin were closely related with the concentration of ascorbic acid. The change of DPPH levels is more sensitive than that of adrenalin in the presence of ascorbate. The antioxidative ability in leaves extracts of two woody plants grown under different light intensities was measured by either scavenging DPPH or inhibiting the oxidation of linoleic acid. The same conclusion was drawn through these two assays. It is suggested that scavenging DPPH free radical is a rapid,simple,sensitive and practical assay for the evaluation of antioxidative capacity in plants.

Abstract:A new strategy of cloning uncompatible DNA ends was reported here with the example of cloning of pC/C and C gene of hepatitis B virus. When the terminals of DNA fragments digested by restriction enzymes are blunt ends, they can be formed into tails with a single 3′ adenosine overhanged by modified with template-independent terminal transferase activity of Taq polymerase in reaction buffer containing dATP. Furthermore, if the DNA fragments have 5′-protruding sticky ends, these terminus can be filled into blunt ends with 5′ to 3′ Taq polymerase activity in the existence of dNTP, and then again added a single adenosine at their 3′ ends by the transferase activity of Taq polymerase. The fragments modified as such above-mentioned can be easily subcloned into T vector.

Abstract:It has been many years to diagnose acute myocardial infarction （AMI） by evaluating the activity of serum enzymes such as asartate aminotransferase （AST）, lactate dehydrogenase （LD）, creatine kinase （CK） and their isoenzymes. In recent year, some protein markers, such as CK-MB mass, myoglobin, troponinT and troponin I have been used in clinical diagnosis. Myoglobin is a good marker to rule out AMI because of its high negative predictive value (NPV), troponin is a good marker to confirm diagnosis because of its high clinical specificity. The analysis property is better than that of CK-MB. Protein markers can be used to do risk stratification and monitor treatment as well. Due to the low price of analysis, serum enzymes are still effective markers for assistance of AMI diagnosis. Sampling time is very important for the evaluation of all these markers.