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( S )-1-(2-fluorophenyl) ethylamine plays a crucial role as a chiral building block in pharmaceutical synthesis. ω-transaminases are widely recognized as environmentally friendly and efficient catalysts for the preparation of chiral amines. In this study, we isolated a novel ω-transaminase, Pf TA, from Pseudogulbenkiania ferrooxidans through gene mining in the NCBI database. By employing semi-rational design, we obtained a Y168R/R416Q variant with enhanced enzyme activity. This variant exhibited the ability to catalyze the synthesis of ( S )-1-(2-fluorophenyl) ethylamine from 2-fluorophenone, achieving a yield of 83.58% and an enantioselectivity exceeding 99% after a 10 h reaction. Compared to the wild type, the specific enzyme activity of the Y168R/R416Q variant reached 47.04 U/mg, which represents an increase of 11.65 times. Additionally, the catalytic efficiency, as measured by k cat / K m , was increased by 20.9 times. Molecular docking and structural simulation analysis revealed that the primary factor contributing to the improved catalytic efficiency is the expansion of the enzyme's active pocket and the alleviation of steric hindrance.

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Objective: The docking and superantigen activity sites of staphylococcal enterotoxin-like W (SElW) and T cell receptor (TCR) were predicted, and its SElW was cloned, expressed and purified. Methods: AlphaFold was used to predict the 3D structure of SElW protein monomers, and the protein models were evaluated with the help of the SAVES online server from ERRAT, Ramachandran plot, and Verify_3D. The ZDOCK server simulates the docking conformation of SElW and TCR, and the amino acid sequences of SElW and other serotype enterotoxins were aligned. The primers were designed to amplify selw , and the fragment was recombined into the pMD18-T vector and sequenced. Then recombinant plasmid pMD18-T was digested with BamH Ⅰand Hind Ⅲ. The target fragment was recombined into the expression plasmid pET-28a(+). After identification of the recombinant plasmid, the protein expression was induced by isopropyl-beta-D- thiogalactopyranoside. The SElW expressed in the supernatant was purified by affinity chromatography and quantified by the BCA method. Results: The predicted three-dimensional structure showed that the SElW protein was composed of two domains, the amino-terminal and the carboxy-terminal. The amino-terminal domain was composed of 3 α-helices and 6 β-sheets, and the carboxy-terminal domain included 2 α-helices and 7 antiparallel β-sheets composition. The overall quality factor score of the SElW protein model was 98.08, with 93.24% of the amino acids having a Verify_3D score ≥0.2 and no amino acids located in disallowed regions. The docking conformation with the highest score (1 521.328) was selected as the analysis object, and the 19 hydrogen bonds between the corresponding amino acid residues of SElW and TCR were analyzed by PyMOL. Combined with sequence alignment and the published data, this study predicted and found five important superantigen active sites, namely Y18, N19, W55, C88, and C98. The highly purified soluble recombinant protein SElW was obtained with cloning, expression, and protein purification. Conclusions: The study found five superantigen active sites in SElW protein that need special attention and successfully constructed and expressed the SElW protein, which laid the foundation for further exploration of the immune recognition mechanism of SElW.

Telomeres are located at the ends of eukaryotic chromosomes DNA repeat sequence. The capacity of cell proliferation will gradully lose result from telomeres deletion and result in cellular senescence and death. Telomerase has the ability to catalyze the synthesis of telomeric DNA, which can help maintain telomere in length. Human telomerase catalytic subunit (hTERT) can activate telomerase activity, extend the ends of chromosomes DNA, maintain the stability and integrity of the genome thus to extend the cell ageing. Studies have confirmed the existence of a close relationship among estrogen, telomeres, telomerase and body aging, and estrogen can activate telomerase through various mechanisms to delay aging and influence the promoter activity of hTERT. In this paper, the mechanism of estrogen regulated telomerase activity anti-ageing were been summarized. [ABSTRACT FROM AUTHOR]

Covalently anchoring of a ligand/metal via polar amino acid side chain(s) is often observed in metalloenzyme, while the substitutability of metal-binding sites remains elusive. In this study, we utilized a zinc-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) as a model enzyme, analyzed the sequence conservation of the three residues Cys37, His59, and Asp150 that bind the zinc ion, and constructed the mutant library. After experimental validation, three out of 224 clones, which showed comparative conversion and ee values as the wild-type enzyme in the asymmetric reduction of the model substrate tetrahydrofuran-3-one, were screened out. The results reveal that the metal-binding sites in TbSADH are substitutable without tradeoff in activity and stereoselectivity, which lay a foundation for designing ADH-catalyzed new reactions via metal ion replacement.

Objective: To investigate the effects of glucose-6-phosphatase catalytic subunit (G6PC) recombinant adenovirus on proliferation and cell cycle regulation of liver cancer cells. Methods: Recombinant adenovirus AdG6PC was constructed. Huh7 cells and SK-Hep1 cells were set as Mock, AdGFP and AdG6PC group. Cell proliferation and clone formation assay were used to observe the proliferation of liver cancer cells. Transwell and scratch assay were used to observe the invasion and migration of liver cancer cells. Cell cycle flow cytometry assay was used to analyze the effect of G6PC overexpression on the proliferation cycle of liver cancer cells. Western blot was used to detect the effect of G6PC overexpression on the cell-cycle protein expression in liver cancer cells. Results: The recombinant adenovirus AdG6PC was successfully constructed. Huh7 and SK-Hep1 cells proliferation assay showed that the number of proliferating cells in the AdG6PC group was significantly lower than the other two groups ( P < 0.05). Clone formation assay showed that the number of clones was significantly lower in AdG6PC than the other two groups ( P < 0.05), suggesting that G6PC overexpression could significantly inhibit the proliferation of liver cancer cells. Transwell assay showed that the number of cell migration was significantly lower in AdG6PC than the other two groups ( P < 0.05). Scratch repair rate was significantly lower in AdG6PC than the other two groups ( P < 0.05), suggesting that G6PC overexpression can significantly inhibit the invasion and migration of liver cancer cells. Cell cycle flow cytometry showed that G6PC overexpression had significantly inhibited the Huh7 cells G(1)/S phase transition. Western blot result showed that G6PC overexpression had down-regulated the proliferation in cell-cycle related proteins expression. Conclusion: G6PC inhibits the proliferation, cell-cycle related expression, and migration of liver cancer cells by inhibiting the G(1)/S phase transition.

Objective To explore the effect of glucose-6-phsophatase, catalytic subunit (G6PC) on the proliferation, migration and invasion of cervical cancer HeLa cells and the possible molecular mechanism. Methods RNA interfering (RNAi) was used to knockdown the expression of G6PC in HeLa cells, and the silencing effect of protein was confirmed by Western blotting. MTT assay and plate clony formation assay were performed to detect the effect of G6PC knockdown on the proliferation of HeLa cells; scratch healing assay and Transwell TM chamber assay were applied to observe the effect of G6PC knockdown on the invasion and migration abilities of HeLa cells; the tube-formation assay was used to detect the effect of G6PC knockdown on the angiogenesis ability of HeLa cells; the expression levels of epithelial-mesenchymal transition (EMT)-related proteins and AKT/mTOR signaling pathway-related proteins were determined by Western blotting. Results The expression of G6PC was effectively silenced by RNAi technology. G6PC knockdown obviously inhibited the proliferation, migration and angiogenesis of HeLa cells. Meanwhile, G6PC knockdown suppressed the EMT process, the phosphorylation of AKT and mTOR proteins. Conclusion G6PC knockdown can effectively inhibit the proliferation, migration, angiogenesis and EMT process of HeLa cells, which is related to the blocked AKT/mTOR signaling pathway.

Proteases are widely found in organisms participating in the decomposition of proteins to maintain the organisms' normal life activities. Protease inhibitors regulate the activities of target proteases by binding to their active sites, thereby affecting protein metabolism. The key amino acid mutations in proteases and protease inhibitors can affect their physiological functions, stability, catalytic activity, and inhibition specificity. More active, stable, specific, environmentally friendly and cheap proteases and protease inhibitors might be obtained by excavating various natural mutants of proteases and protease inhibitors, analyzing their key active sites by using protein engineering methods. Here, we review the studies on proteases' key active sites and protease inhibitors to deepen the understanding of the active mechanism of proteases and their inhibitors.

Objective To explore whether the downregulation of protein phosphatase 2A catalytic subunit(PP2Ac)involved in the pathogenesis of mitochondria fission/fusion dynamics and functional imbalance induced by human tau accumulation. Methods After cotransfection with mito-dsRed plasmids and pIRES-eGFP-tau40 plasmids 48 hours,the rat primary hippocampal neurons were observed with a laser scanning confocal microscope for their changes in shape and distribution of mitochondria.The expressions of mitochondria fission/fusion protein and PP2Ac and PP2Ab were detected by Western blotting.Furthermore,the shape and distribution of mitochondria of rat primary hippocampal neuron and wild type 293wt cells were assayed 48 hours after co-transfection with siPP2Ac-EGFP plasmids and mito-DsRed plasmids,and the fission/fusion dynamics of 293wt cells was captured with live cell time-lapse imaging after co-transfection with siPP2Ac plasmids and mito-Dendra2 plasmids.After transfection with siPP2Ac plasmids,the relative level of mitochondria fission/fusion protein of 293wt cells was assayed by Western blotting,and mitochondria membrane potential was detected by JC-1 staining,and the cellular viability was measured by CCK8 assay.Finally,the shape and distribution and membrane potential of mitochondria of HEK293 cells with stable transfection of htau40(293htau)were detected after co-transfection with PP2Ac and mito-dsRed plasmids. Results Human tau40 expression decreased distribution of mitochondria and significantly lowered PP2Ac level in primary hippocampal neuron( t =4.814, P =0.0086).Down-regulation of PP2Ac caused mitochondria elongation and perinuclear accumulation in primary hippocampal neuron and 293wt cells;in addition,down-regulation of PP2Ac in 293wt cells significantly increased mitochondria fusion rate( t =2.857, P =0.0074)and the levels of mitochondria fusion protein mitofusin(MFN)1( t =6.768, P =0.0025),MFN2( t =3.121, P =0.0035),and optic atrophy 1( t =3.775, P =0.0199);however,the levels of dynamin-like protein-1 and Fis1 remained unchanged.The down-regulation of PP2Ac in 293wt cells led to the significant decrease in mitochondria membrane potential( t =2.300, P =0.0270)and cell viability( t =6.249, P <0.0001).Finally,up-regulation of PP2Ac attenuated the abnormalities in the shape,distribution and function of mitochondria in the 293htau cells. Conclusion Down-regulation of PP2Ac is involved in the abnormal shape and distribution of mitochondria and its dysfunction induced by human tau40 in rat primary hippocampal neurons and HEK293 cells.

Pectin methylesterase (PME) is an important pectinase that hydrolyzes methyl esters in pectin to release methanol and reduce the degree of methylation of pectin. At present, it has broad application prospects in food processing, tea beverage, paper making and other production processes. With the in-depth study of PME, the crystal structures with different sources have been reported. Analysis of these resolved crystal structures reveals that PME belongs to the right-hand parallel β-helix structure, and its catalytic residues are two aspartic acids and a glutamine, which play the role of general acid-base, nucleophile and stable intermediate, in the catalytic process. At the same time, the substrate specificity is analyzed to understand the recognition mechanism of the substrate and active sites. This paper systematically reviews these related aspects.

Objective: To study the effect of hyperoxic exposure on the dynamic expression of heme oxygenase-1 (HO-1) and glutamate-L-cysteine ligase catalytic subunit (GCLC) in the lung tissue of preterm neonatal rats., Methods: Cesarean section was performed for rats on day 21 of gestation to obtain 80 preterm rats, which were randomly divided into air group and hyperoxia group after one day of feeding. The rats in the air group were housed in room air under atmospheric pressure, and those in the hyperoxia group were placed in an atmospheric oxygen tank (oxygen concentration 85%-95%) in the same room. Eight rats each were selected from each group on days 1, 4, 7, 10, and 14, and lung tissue samples were collected. Hematoxylin and eosin staining was used to observe the pathological changes of lung tissue at different time points after air or hyperoxic exposure. Western blot and RT-qPCR were used to measure the protein and mRNA expression of HO-1 and GCLC in the lung tissue of preterm rats at different time points after air or hyperoxic exposure., Results: Compared with the air group, the hyperoxia group had a significant reduction in the body weight (P<0.05). Compared with the air group, the hyperoxia group had structural disorder, widening of alveolar septa, a reduction in the number of alveoli, and simplification of the alveoli on the pathological section of lung tissue. Compared with the air group, the hyperoxia group had significantly lower relative mRNA expression of HO-1 in the lung tissue on day 7 and significantly higher expression on days 10 and 14 (P<0.05). Compared with the air group, the hyperoxia group had significantly lower mRNA expression of GCLC in the lung tissue on days 1, 4, and 7 and significantly higher expression on day 10 (P<0.05). Compared with the air group, the hyperoxia group had significantly higher protein expression of HO-1 in the lung tissue on all days, and the protein expression of GCLC had same results as HO-1, except on day 1 (P<0.05)., Conclusions: Hyperoxia exposure may lead to growth retardation and lung developmental retardation in preterm rats. Changes in the protein and mRNA expression of HO-1 and GCLC in the lung tissue of preterm rats may be associated with the pathogenesis of hyperoxia-induced lung injury in preterm rats.

ω-Transaminase catalyzes the asymmetric reductive amination of carbonyl compounds, and has great application prospect in the preparation of chiral amines. The application in synthesis of bulky chiral amines is limited by the special structure of substrate binding region in the wild-type enzyme. Moreover, there are also some drawbacks in the stereoselectivity and stability of ω-transaminase. So far, -tωransaminase satisfying the industrial requirements is still rare. In this review, we first introduce the structure and catalytic mechanism of ω-transaminase, and then discuss the structural differences between S-selective and R-selective enzymes. Molecular modification of ω-transaminase was introduced in detail, by focusing on the structure and mechanism-based molecular modification, including substrate specificity, stereoselectivity, and stability.

Objective To investigate the role of demethylation of protein phosphatase 2A catalytic subunit (PP2Ac) on M1 macrophage polarization. Methods THP-1 cells were induced to differentiate into M0 macrophages with phorbol ester (PMA) for 24 hours, and then stimulated to differentiate into M1 macrophages induced by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) for 48 hours. The administration group and the solvent control group were respectively cultured with 0.5 μmol/L ABL127 and the same volume of DMSO for 48 hours on M1 macrophage polarization models. Inverted microscope was used to observe the morphological changes of macrophages. The mRNA expression levels of cyclooxygenase 2 (COX-2), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and C-X-C motif chemokine ligand 10 (CXCL10) in M1 macrophages were detected by quantitative real-time PCR. M1 macrophage phagocytosis was determined by neutral red uptake assay. Western blot analysis was applied to test the level of PP2Ac demethylation. Results In the differentiation of M0 into M1 macrophages, the cells were elongated and became spindle-shaped. Furthermore, the expression of M1 macrophage markers including COX-2, TNF-α, IL-6 and CXCL10 significantly increased and phagocytosis was enhanced, but PP2Ac demethylation was decreased. Compared with the DMSO group, PP2Ac demethylation decreased while spindle cells, phagocytosis, and the mRNA levels of COX-2, TNF-α, IL-6 and CXCL10 significantly increased in the administration group with 0.5 μmol/L ABL127. Conclusion The inhibition of PP2Ac demethylation promotes the differentiation of M1 macrophages, and increases the expression of pro-inflammatory cytokines and the phagocytosis.

Chiral amines are important building blocks for the synthesis of pharmaceutical products and fine chemicals. Highly stereoselective synthesis of chiral amines compounds through asymmetric amination has attracted more and more attention. ω-transaminases (ω-TAs) are a promising class of natural biocatalysts which provide an efficient and environment-friendly access to production of chiral amines with stringent enantioselectivity and excellent catalytic efficiency. Compared with (S)-ω-TA, the research focused on (R)-ω-TA was relatively less. However, increasing demand for chiral (R)-amines as pharmaceutical intermediates has rendered industrial applications of (R)-ω-TA more attractive. Improving the thermostability of (R)-ω-TA with potential biotechnological application will facilitate the preparation of chiral amines. In this study, the dynamic surface loop with higher B-factor from Aspergillus terreus (R)-ω-TA was predicted by two computer softwares (PyMOL and YASARA). Then mutant enzymes were obtained by deleting amino acid residues of a dynamic surface loop using site-directed mutagenesis. The results showed that the best two mutants R131del and P132-E133del improved thermostability by 2.6 ℃ and 0.9 ℃ in T₅₀¹⁰ (41.1 ℃ and 39.4 ℃, respectively), and 2.2-fold and 1.5-fold in half-life (t1/2) at 40 ℃ (15.0 min and 10.0 min, respectively), compared to that of wild type. Furtherly, the thermostability mechanism of the mutant enzymes was investigated by molecular dynamics (MD) simulation and intermolecular interaction analysis. R131del in the loop region has lower root mean square fluctuation (RMSF) than the wild type at 400 K for 10 ns, and mutant enzyme P132-E133del increases four hydrogen bonds in the loop region. In this study, we obtain two stability-increased mutants of (R)-ω-TA from A. terreus by deleting its dynamic surface loop and also provide methodological guidance for the use of rational design to enhance the thermal stability of other enzymes.

Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.

Objective: To construct the expression vector of apolipoprotein B mRNA editing enzyme catalytic subunit 3A( APOBEC3A),express APOBEC3 A in eukaryotic cells and identify its cytosine deaminase activity., Methods: The APOBEC3 A gene was obtained by PCR and inserted into the eukaryotic expression vector pc DNA3. 0( +). The recombinant vector pc DNA3. 0-APOBEC3 A was then transfected into HEK293 T and Hep G2 cells after confirmed by DNA sequencing. The recombinant protein was purified by Ni-NTA His Bind affinity column. Western blot analysis was used to detect the expression of APOBEC3 A protein. The localization of APOBEC3 A protein in HEK293 T and Hep G2 cel s was identified by immunofluorescence cytochemistry. The deaminase activity of APOBEC3 A protein was characterized by fluorescence polarization., Results: DNA sequencing confirmed that APOBEC3 A gene( 600 bp) was inserted into pc DNA3. 0-APOBEC3 A,which was expressed in HEK293 T and Hep G2 cells successfully. APOBEC3 A protein was mainly expressed in cytoplasm of HEK293 T cells and cytoplasm and nuclei of Hep G2 cells. APOBEC3 A protein showed cytosine deaminase activity on the TTCA sequence in single-stranded DNA., Conclusion: The study constructed successfully APOBEC3 A eukaryotic expression vector,identified the differential expression of APOBEC3 A protein in HEK293 T and Hep G2 cells,and confirmed that the APOBEC3 A protein had cytosine deaminase activity.

Objective: We isolated bacterial strains with chitin-degrading activity from the digesta of large yellow croakers (Pseudosciaena crocea) fed with chitin-enriched trash fish, and characterized potential chitinases thereof., Methods: Chitin-degrading strains were screened with colloidal chitin agar from the digesta of P. crocea fed with trash fish. The chitinase gene (chi-X) was cloned and expressed in Escherichia coli, and the enzymatic properties of the chitinase (CHI-X) were characterized., Results: A Citrobacter freundii strain with chitin-degrading activity was isolated. The chitinase gene encodes a protein containing 493 amino acid residues, with a proposed glycoside hydrolase family-18 catalytic domain. CHI-X could hydrolyze colloidal chitin. The optimal pH for CHI-X was 4.0 at optimal temperature (60 ℃). CHI-X was active over a broad pH range, with around 90% of the activity maintained after incubation at pH between 3.0 and 11 for 1 h. The enzymatic activity of CHI-X was stimulated by Mn2+, Li+, and K+, but inhibited by Ag+. The enzyme was stable after treatment by proteases and grouper intestinal juice. CHI-X hydrolyzes colloidal chitin into GlcNAc and (GlcNAc)2. Furthermore, an synergic effect was observed between CHIX and ChiB565 (a chitinase from Aeromonas veronii B565) on colloidal chitin., Conclusion: CHI-X from intestinal bacterium may be potentially used as feed additive enzyme for warm water marine fish.

The chalcone synthase (CHS) superfamily of the type III polyketide synthases (PKSs) generates backbones of a variety of plant secondary metabolites. Benzalacetone synthase (BAS) catalyzes a condensation reaction of decarboxylation between the substrates of 4-coumaric coenzyme A and malonyl coenzyme A to generate benzylidene acetone, whose derivatives are series of compounds with various biological activities. A BAS gene Pcpks2 and a bifunctional CHS/BAS PcPKSI were isolated from medicinal plant P. cuspidatum. Crystallographic and structure-based mutagenesis studies indicate that the functional diversity of the CHS-superfamily enzymes is principally derived from small modifications of the active site architecture. In order to obtain an understanding of the biosynthesis of polyketides in P. cuspidatum, which has been poorly described, as well as of its activation mechanism, PcPKS2 was overexpressed in Escherichia coli as a C-terminally poly-His-tagged fusion protein, purified to homogeneity and crystallized, which is helpful for the clarification of the catalytic mechanism of the enzyme and lays the foundation for its genetic engineering manipulation.

Objective: To study the active sites of N-acetylhexosamine 1-kinase (NahK) from Bifidobacterium longum JCM12 17., Methods: We obtained expression strains of 10 single-mutants at 4 sites of NahK by site-directed mutagenesis, and expressed and purified both wild-type (WT) and mutant enzymes. Then, their optimum pH and optimum concentration of Mg²⁺ were determined by DNS assay and NADH-coupled microplate photometric assay, and their kinetic parameters were measured., Results: Four mutants (D208A, D208N, D208E and I24A) lost most part of the catalytic activity. The optimum pH of mutants H31A, H31V, F247A and I24V switched from pH 7.5 (for WT) to pH 7.0, and the optimum concentration of Mg²⁺ of mutants H31A and F247A increased to 10 mmol/L from 5 mmol/L (for WT). The kinetic parameters of WT and mutants indicate that mutant F247Y had higher enzymatic activity toward GlcNAc, GalNAc and ATP than WT., Conclusion: The key amino acids that affect the catalytic activity of NahK were determined by site-directed mutagenesis, and together with the mutant that has higher catalytic efficiency, this has laid a foundation for further modification and evolution of NahK.

Objective: DamH is a bifunctional hydrolase that hydrolyzes the amide and ester bonds. Previous studies demonstrated that mutagenesis of non-catalytic residues shows a negative impact on the soluble expression and specific activity of DamH. Therefore, we studied the catalytic triad of DamH and the effect of non-catalytic mutagenesis on the soluble expression and specific activity of DamH., Methods: We performed site-directed mutation experiment of the 3 possible catalytic sites: S149, E244 and H274 and the non-active sites by using overlapping PCR. In the whole cell catalytic experiments, 2'-methyl-6'-ethyl-2-chloroacetanilide (CMEPA) hydrolase activities of the three mutants (S149A, E244A and H274A) were assayed. Mutants of D165 and N192 were purified by affinity chromatography of Ni²⁺-NTA. At the same time the hydrolase activities of mutants were compared with that of the wild-type strain., Results: S149-E244-H274 was the catalytic triad of DamH. Kinetics shows that the CMEPA hydrolase activity of mutant S149A declined to 5% of the wild-type strain and none of E244A and H274A mutants showed any CMEPA hydrolase activity. Mutations of D165 and N192 would affect the soluble expression of recombinant enzyme. The soluble expression levels of D165P and N192P were 28.2% and 20.8% of the wild-type strain, respectively. Furthermore, hydrolase activities of N192P and D165P were only 55.5% and 49.7% of the wild-type enzyme, respectively., Conclusion: DamH uses the same active site to hydrolyze esters and amides.

Metacaspases are cysteine-dependent proteases found in protozoa, fungi and plants and are distantly related to metazoan caspases. Most of MCPs activation are the calcium dependent, but the mechanisms are still unknown. Based on the techniques of CD spectroscopy, fluorescence spectroscopy, and Terbium Stains-all probe, we selected three purified recombinant proteins from key residues mutated in tomato metacaspase (LeMCA1), including conserved catalytic site (C139A) mutant, N-sequenced cleaved site (K223G) mutant and the predicted Ca2+ binding sites (D116A/D117A) mutant, to explore the interaction mechanism of LeMCA1 and Ca2+. CD spectroscopy and Stains-all probe results suggested that the intense binding does not exist between LeMCA1 and Ca2+ as well as Ca2+ has little effect on the secondary structure of LeMCA1. However, fluorescence spectroscopy and Tb3+ probe results showed that Ca(2+)-induced the changes occur in the tertiary structure of LeMCA1, which contributes to the activation of zymogen. In addition, predicted Ca2+ binding residues, Asp-116 and Asp117, are the key sites resporisible for the Ca2+ interaction with LeMCA1, and the loss of these two residues resulted in decreased interaction. Our data firstly provided insight on the mechanism of the interaction between Ca2+ and recombinant purified Solanaceae type II metacaspase by spectroscopy and molecular probe techniques. Combined the results we got before from sequence-alignment and sites-mutation, the key residues Asp-116 and Asp117 affect the Ca(2+)-induced the changes of LeMCA1 tertiary structure. Our data provided information for the further biochemical and crystal assays of LeMCA1.

Uridine phosphorylase (UPP) is a key enzyme of pyrimidine salvage pathways, catalyzing the reversible phosphorolysis of ribosides of uracil to nucleobases and ribose 1-phosphate. UPP plays an important role in the regulation of uridine homeostasis. Although UPP from a variety of organisms have many similarities in their functions, there are differences in many other aspects, such as physical and chemical properties, structure characteristics, active sites, and substrate binding sites. Therefore, UPP has broad application prospects in the design and development of antibacterial, antiparasitic drugs. This article summarizes the physico-chemical property and research progress of UPP from a variety of organisms, in order to integrate information of UPP, provide theoretic basis for further study of Toxoplasma gondii UPP protein as a feasible target antigen for toxoplasmosis vaccination.

Protein kinase D (PKD) is a novel family of serine/threonine kinases and diacylglycerol (DAG) receptors and has been documented in a variety of cellular processes. To get high purity catalytic domain of PKD1 (PKD1-cat) for crystallography study, the GST-tagged PKD1-cat gene was cloned into a baculovirus transfer vector pFastBac1 (donor plasmid). When the recombinant plasmid was transformed into DH10Bac competent Escherichia coli, which contains a baculovirus shuttle vector (bacmid), transposition occurs to generate a recombinant bacmid containing the gene of interest (GST-PKD1-cat). The recombinant bacmid DNA was transfected into Spodoptera frugiperda Sf9 insect cells to generate recombinant baculovirus, which was then amplified through multiple rounds of infection in Sf9 cells. After that, Trichoplusia ni insect cells in suspension culture were infected with baculoviral stock at a multiplicity of infection (MOI) of 5 PFU/cell. SDS-PAGE and Western blotting analysis confirmed the detection of a 68 kDa protein by the glutathione S-transferase (GST) monoclonal antibody. The recombination protein was purified by Glutathione sepharose affinity chromatography and cleaved by PreScission Protease to remove GST tag, and a highly pure 42 kDa protein which was consistent with the molecular weight of the expected PKD1-cat protein was detected on SDS-PAGE. The activity of purified PKD1-cat protein was determined by in vitro PKD kinase assay. Our data showed that the kinase activity increased with the concentration of purified PKD1-cat protein. These results showed that the truncated recombinant PKD1-cat protein was highly active and pure, and could potentially be used for solving 3D structure of this protein by Nuclear Magnetic Resonance (NMR) or crystallography.

Rat brain kinesin is a conventional kinesin that uses the energy from ATP hydrolysis to walk along the microtubule progressively. Studying how the chemical energy in ATP is utilized for mechanical movement is important to understand this moving function. The monomeric motor domain, rK354, was crystallized. An ATP analog, AMPPNP, was soaked in the active site. Comparing the complex structure of rK354 x AMPPNP and that of rK354ADP, a hypothesis is proposed that Glu237 in the Switch II region sensors the presence of gamma-phosphate and transfers the signal to the microtubule binding region.

Anthocyanins are a ubiquitous group of water-soluble plant pigments of the flavonoid family, with anticancer property through HER-2 signaling pathway. Nowadays, molecular docking plays an important role in exposing the active sites and obtaining the bioactive conformation involving protein-ligand interactions. According to the crystal structure of HER-2 kinase domain and 12 main antitumor compounds of anthocyanins as well as ATP, a molecular docking study was performed by MVD program. All 12 compounds could bind to the same cavity of HER-2 kinase domain by high affinity (MolDock Score < -105 kJ/mol for anthocyanidins, < -130 kJ/mol for anthocyanidins-glc), where hydrophobic force and hydrogen bond played key roles. Additionally, this cavity overlapped with ATP binding (MolDock Score = -161 kJ/mol) domain; the binding of anthocyanins presumably interfered the H bond formation between ATP and HER-2. These results indicate that anthocyanins may competitively bind to ATP binding site in HER-2 kinase domain by suppressing HER-2 activation and downstream signaling cascade. This may provide useful theoretical instruction for the molecular mechanism of HER-2 kinase activity inhibition by anthocyanins in cancer prevention and treatment.

Objective: To investigate the interaction of body mass index(BMI)and a single nucleotide polymorphism (SNP, rs17883901) in catalytic subunit of glutamate-cysteine ligase (GCLC) on breast cancer risk., Methods: A total of 839 women with incident breast cancer and 863 age-matched controls without cancer were recruited at the same period in three affiliated hospitals of Sun Yat-sen University in Guangzhou from October 2008 to June 2010. GCLC rs17883901 was genotyped by MALDI-TOF-MS. Binary unconditional logistic regression was applied to calculate odds ratios and 95% confidence intervals., Results: The difference of present BMI and BMI at age 20 was not statistically significant between cases and controls, either as the genotypes of GCLC. No association was found between BMI at present and premenopausal or postmenopausal breast cancer risk. But we found that women who had a BMI at age 20 of 18.5 to 22.9 had a marginally decreased risk of premenopausal breast cancer [OR and 95%CI:0.69(0.48, 1.00)]. Among women with CT/TT genotypes, whose present BMI was greater than 25 had a increased risk [OR and 95%CI:1.91(1.09, 3.36)] of breast cancer and a decreased risk [OR and 95%CI:0.56 (0.31,0.99)] with a BMI at age 20 of 18.5 to 22.9. There was a interaction between GCLC gene(rs17883901)and BMI at present in breast cancer risk (P = 0.043), which was not found between rs17883901 and BMI at age 20., Conclusion: Our findings indicate BMI at age 20 may be a protective factor of premenopausal breast cancer, while no association appears between GCLC(rs17883901) and breast cancer. Obesity at present may significantly increase the risk of breast cancer among women with CT/TT genotypes of GCLC (rs17883901).

A series of tacrine-methoxybenzene hybrids (5a-5i) were designed, synthesized and evaluated as inhibitors of cholinesterases (ChEs). All the compounds had better ChEs inhibitory activities than tacrine with IC50 values at the nanomolar range. Compound 5h exhibited the strongest inhibition on acetylcholinesterase (AChE) with an IC50 value of 6.74 nmol x L(-1) and compound 5f showed the most potent inhibition on butyrylcholinesterase with IC50 value of 3.83 nmol x L(-1). Kinetic and molecular modeling studies showed that these hybrids targeted both the catalytic active site and the peripheral anionic site of AChE.

We cloned the lipoxygenase gene (ana-LOX) from Anabaena sp. PCC 7120 and expressed it in Escherichia coli BL21 (DE3) pLysS. We determined the active site of the recombinant ana-LOX through site-directed gene mutagenesis and obtained the shortest length of the functional gene. Meanwhile, we studied the properties of recombinant ana-LOX after purification. The C-terminal of the Aos (allene oxide synthase)-LOX fusion gene in Anabaena sp. PCC 7120 genome was found belonging to LOXs family by bioinformatics analysis. Further results of site-directed gene mutagenesis confirmed that the active sites of ana-LOX were His197, His202, His369, Asn373and Ile455. The shortest length of functional gene was identified to be 1 254 bp based on the strategy of shortening the gene length gradually. The highest activity of recombinant ana-LOX of 6 750 U/mL could be achieved when constructed to pET-32a vector and expressed at low temperature 16 degrees C. We purified the enzyme by Ni-NTA chelating affinity chromatography, with 60.89% yield and specific activity of 11.4 x 10(4) U/mg. The optimum reaction temperature and pH for ana-LOX were 45 degrees C and 6.0, respectively. Furthermore, the obtained ana-LOX was stable at room temperature. The effect of metal ions on ana-LOX was determined also. Fe2+, Mg2+ Ca2+ could markedly promote the activity of this enzyme whereas Fe3+ and Cu2+ had a strong inhibitory effect on it. Finally, the ana-LOX could improve the microscopical structure of dough. The results of this study will provide a basis for future improvements and food industrial applications of ana-LOX.

Objective: We investigated the structure model and function of xylitol dehydrogenase from Aspergillus oryzae., Methods: Xylitol dehydrogenase (XDH) gene from Aspergillus oryzae was cloned and sequenced. We constructed four tertiary structure models of XDH by homology modeling with Swiss-MODEL and Modeller and obtained the best quality model by evaluation of PROCHECK and Prosa2003. The dockings of NAD+, Zn2+ and xylitol with XDH were performed by Molsoft program., Results: Structure analysis suggested that XDH was a member of medium-chain dehydrogenase/reductase (MDR) family. This was supported by the presence of the zinc-containing alcohol dehydrogenase signature and a typical alcohol dehydrogenase Rossmann fold pattern composed by NAD+ binding domain present in MDR superfamily. The molecular docking indicated that amino acid residues Asp206, Arg211, Ser255, Ser301 and Arg303 in XDH binding domain had hydrogen bonding with NAD+, His72 and Glu73 in catalytic domain had hydrogen bonding with Zn2+, Ile46, Ile349, Lys350 and Thr351 in catalytic domain had hydrogen bonding with xylitol., Conclusion: These key amino acid residues might play a vital role in the XDH catalytic reaction and can instruct the further directed modification of XDH.

Background and Objective: DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays an important role in repairing irradiation-induced DNA double-strand break (DSB), and affects the radiosensitivity of tissue cells. This study was to detect the expression of DNA-PKcs in different non-small cell lung cancer (NSCLC) cell lines and evaluate its correlation to radiosensitivity., Methods: The content and activity of DNA-PKcs in five NSCLC cell lines A549, H1299, L78, PGCL3 and H460 were measured by Western blot and the DNA-PK activity assay. Cell survival was analyzed using clonogenic formation assay., Results: The radiosensitivities of five NSCLC cell lines were different. The values of survival fraction at 2 Gy (SF2) were 0.74 in A549 cells, 0.25 in H1299 cells, 0.21 in H460 cells, 0.48 in PGCL3 cells, and 0.58 in L78 cells. The protein levels of DNA-PKcs were 3.26+/-0.98 in A549 cells, 0.51+/-0.07 in L78 cells, 0.51+/-0.11 in H1299 cells, 0.86+/-0.23 in H460 cells, and 2.60+/-0.76 in PGCL3 cells. The activity values of DNA-PKcs were 8.30+/-1.03 in A549 cells, 2.45+/-0.52 in H1299 cells, 0.11+/-0.02 in H460 cells, 4.13+/-0.87 in PGCL3 cells, and 0.42+/-0.07 in L78 cells. In adenocarcinoma and large cell carcinoma cell lines, SF2 were correlated to DNA-PKcs content (P<0.05, r=0.95) and activity (P=0.03, r=0.98)., Conclusion: DNA-PKcs is an important factor to predict the radiosensitivity in adenocarcinoma and large cell lung cancer cell lines.

We cloned genes of four sentrin-specific protease (SENP), three small ubiquitin-like modifiers (SUMO), enhanced cyan fluorescent protein (ECFP) and yellow fluorescent protein (EYFP) by two-step PCR. Then we constructed expression vector B28 for SENP and B13 for ECFP-SUMO-EYFP. The recombinant plasmids were transformed into Escherichia coli BL21 and expression was induced by isopropyl beta-D-thiogalactoside, then recombinant proteins were purified by Ni-NTA agarose column ion-exchange chromatography. The proteins were analyzed with SDS-PAGE and identified with Western blotting. Except that SENP3 catalytic domain (SENP3C) truncated in the C termini and SENP5C expressed in inclusion body, others were expressed as soluble proteins. SDS-PAGE analysis showed that the relative molecular mass of these fusion proteins were consistent with theoretical ones, and the specificity of the fusion proteins were confirmed with Western blotting. The fusion proteins of SENP and ECFP-SUMO-EYFP can be successfully expressed in prokaryotic expression system. It lays the foundation for the fluorescence resonance energy transfer analysis.

To investigate the antitumor mechanism of artemisninin, a flexible docking analysis was used to score all kinds of functions of 11 Qinghaosu derivatives and transferrin with different resolutions. The distances of Asp-63, Tyr-188, His-249, Arg-124 and Lys-296 with Qinghaosu were less than 0.5 nm, separately. Meanwhile, the higher is the activity of Qinghaosu derivatives the higher is the score. Our model explains that Fe2+ is more feasible to react with Qinghaosu, and not involved in other metabolism in presence of transferrin. Docking results unveil that Iron(II)-transferrin increased the cytotoxicity of Qinghaosu derivatives and provide a rational basis for further design and synthesis of novel Qinghaosu derivatives.

In order to express and purify the catalytic domain of SHP-1/SHP-2 (named as D1C and D2C respectively) and determine their kinetics, the constructed D1C and D2C plasmids were transformed into Escherichia coli BL21 and the expression was induced with IPTG. The harvested cells were suspended in extraction buffer. After sonication, the solution was applied to HPLC and the results were confirmed by SDS-PAGE. The purified peptides were further subjected to kinetic specificity study using synthetic phosphotyrosine (pY) as substrate by malachite green method and analyzed by Lineweaver-Burk plot calculation. From this study, we found D1C and D2C were expressed successfully in soluble state in Escherichia coli BL21 and purified efficiently with HPLC system. The molecular weight of D1C was 34.6 kD, and its Michaelis constant (K(m)) was 2.04 mmol catalytic constant (K(cat)) was 44.98 s(-1), specific constant (K(cat)/K(m)) was 22.05 L/(mmol x s); the molecular weigh of D2C was 35.3 kD, and its Michaelis constant (K(m)) was 2.47 mmol, catalytic constant (K(cat)) was 27.45 s(-1), specific constant (K(cat)/K(m)) was L/(mmol x s). The enzyme activity of D1C is stronger than that of D2C.

Trehalose synthase inverts maltose into trehalose, and plays a very important role in trehalose industrial production. Here we reviewed the recent progress on studies of molecular biology including the cloning of trehalose synthase gene, gene engineering application, structure and catalytic mechanism and the role of the enzyme in vivo.

Genistein is a high specific and noncompetitive inhibitor of epidermal growth factor receptor tyramine kinase domain (EGFR-TK). In the paper, a molecular docking between genistein and EGFR-TK was studied to explore the mechanism of their interaction and antitumor mechanism of genistein by AUTODOCK 3.05 program. The results indicated that genistein located in the active cavity of EGFR-TK by high affinity (deltaG = -31.2 kJ/mol), and genistein inhibited EGFR-TK by interfering with forming of Lys721/Glu738 ion pair. The inhibition belonged to noncompetitive interaction, in which hydrophobic force and hydrogen bond played key roles.

Objective: To investigate the expression of DNA dependent protein kinase catalytic subunit (DNA-PKcs) and P16 (multiple tumor suppressor 1) genes in colorectal carcinoma and clinical significance thereof., Methods: 359 specimens of colorectal carcinoma obtained during operation and 35 specimens of normal mucosa beside the carcinoma underwent immunohistochemical examination and Western blotting to detect the expression of DNA-PKcs and P16., Results: The positive expression rates of DNA-PKcs and P16 of the colorectal carcinoma specimens were 63.5% (228/359) and 53.2% (191/359) respectively, both significantly higher than those of the para-cancer normal tissues (both P < 0.05). DNA-PKcs and P16 expression were both related to the clinical stage, lymphatic invasion, and distant metastasis (all P < 0.05). The 5-year survival rates of the patients positive in DNA-PKcs and P16 expression were significantly lower than those of the patients negative in DNA-PKcs and P16 expression (both P < 0.05). The DNA-PKcs positive rate of the P16-positive patients was 78.6%, significantly higher than that of the P16-negative patients (55.3%, r = 0.149, P = 0.036)., Conclusion: DNA-PKcs and P16 play a significant role in the carcinogenesis and evolution of colorectal carcinoma and may be used as one of the useful indicators for clinical assessment of tumor biological behavior and prognosis of colorectal carcinoma.

Objective: To predict the structure and function of SjLDH using bioinformatics method., Methods: By online analysis at bioinformatics websites such as NCBI (http://www.ncbi.nlm.nih.gov/) and Expasy (http://cn.expasy. org/), and employing software packages such as Vector NTI suite and PCgene to do multi-sequence homological alignment, phylogenetic analysis, secondary structure and topological prediction, homology modeling of tertiary structure, antigenic epitope analysis, etc., Results: Same conservative sites and key catalytic sites existed among SjLDH and LDHs from other species. Similarity of SjLDH compared to CsLDH, TvLDH and HsLDH was 75%, 17%, 58%-60% respectively. Phylogenetic analysis demonstrated that the evolution relation between SjLDH and DmLDH was closer than the relation between SjLDH and CeLDH, the relationship between SjLDH and HsLDH-B, -C was closer than HsLDH-A. Three transmembrane regions were found, the region of 98aa-106aa in three hydrophilic regions located outside of membrane was inferred as the major antigen epitope. This antigen epitope had significant difference with LDHs from protozoon (Pf., Tg., Tv.) and had 1-3 amino acid residue difference with MmLDH, HsLDH-A, -B, -C, and was the same with CsLDH. One of the key catalytic residues and substrate (pyruvate) binding loop were located in this region. Tertiary structure demonstrated that 98aa-106aa was on the surface of the protein and formed a substrate binding loop, other two key catalytic sites were at the position near the loop., Conclusion: The prediction implied that LDH was an ideal molecule for phylogenetic analysis; SjLDH might be a potential molecular target for immunodiagnosis, anti-schistosome drug and vaccine development.

Objective: To clone and express a new protein adenosine deaminase of Schistosoma japonicum (SjADA)., Method: Specific primers were designed according to the EST sequence and used for amplification of the encoding sequence from the cDNA clone containing SjADA. The gene was sub-cloned into pET32 plasmid and expressed in E.coli BL31 (DE) pLys strain and the recombinant proteins were purified by chelating sepharose FF. The structure and phylifunctions of this protein were analyzed by MrBayes and GeneAtlas., Result: The full length sequence of SjADA gene was obtained and the recombinant protein was cloned, expressed and purified successfully. The bioinformatics analysis showed that the identity of SjADA and SmADA gene sequence was only 25% and they belonged to different subfamily. The structure of SjADA had 41% identity with it's PDB template 1A4M., Conclusion: The recombinant protein of SjADA has been obtained. The purine salvage pathway of S. japonicum may be different with that of S. mansoni.

Aim: To screen and identify the short-peptide which specifically bind to human CD59 so as to design short-peptide clamp with counteracting tumor escape activity., Methods: Using CHO cells which high express human CD59 as target cells, the phage 12 peptide library was screened for 5 rounds by competitive binding test. After five rounds of screening and competitive binding test, positive phage clones were screened by ELISA and sequenced., Results: 8 out of 16 phage clones which were chosen randomly were identified to have high binding power to CD59. After sequencing, 3 high homologous amino acids were obtained. DNAstar analysis showed that 3 sequences had some homology with the sequence of human CD2 published by PubMed., Conclusion: The obtained sequence HxAxxxxxxPxx is helpful for design of short-peptide clamp of active sites of CD59 related to tumor escape.

Hydrogenases are enzymes that catalyse the oxidation of hydrogen and the reduction of protons. It plays an important role in the process of biohydrogen production. According to the metal atoms within hydrogenase, it can be classified as NiFe-hydrogenase, Fe-hydrogenase and metal-free hydrogenase. The overwhelming majority of hydrogenases are metalloenzymes. The metal atoms are involved in the forming of active site and [Fe-S] clusters. The active site directly catalyzes the reduction of protons and the oxidation of hydrogen. The [Fe-S] clusters are involved in the transport of electrons between the H2-activating site and the redox partners of hydrogenase. Presently, the crystal structures of NiFe-hydrogenase and Fe-hydrogenase from a few kinds of microorganism have been revealed. The metal-free hydrogenase, characterized by the absence of [Fe-S] cluster and the presence of an iron-containing cofactor, shows a great diversity comparing with those of NiFe-hydrogenases and Fe-hydrogenases. Recent progress have also indicated the mechanisms of activation.

Objective: To investigate the direct effects of recombinant matrix metalloproteinase-14 (MMP-14) catalytic domain (RMC) on cultured human osteoblastic SaOS-2 cells., Methods: Western Blotting was used to analyze the expression of MMP-14 in cultured media. The effect of RMC on activation of proMMP-2 was measured by gelatin zymograms analysis, and the influence of RMC on cells adhesion was analyzed as well. The apoptosis was detected by acidine orange/ethidium bromide staining and ELISA., Results: RMC activated proMMP-2 secreted into media by SaOS-2 cells, and this process was blocked by EDTA treatment. RMC inhibited the adhesion of SaOS-2 cells to immobilized Type I collagen in a dose-dependent manner, which was also abolished by EDTA. RMC induced SaOS-2 cells apoptosis in a dose-dependent manner, and apoptosis-inducing activity of MMP-14 catalytic domain was blocked by EDTA., Conclusion: Since adhesion of cells to extracellular matrix serves as a survival mechanism in osteoblasts, the catalytic activity of recombinant MMP-14 catalytic domain on matrix proteins contributes to its apoptosis-inducing activity.

Objective: To study the effects of antisense oligodeoxynucleotides (ODN) of hEST2 (AODN) on telomerase activity and proliferation in ovarian cancer cell lines SKOV3 and COC1., Methods: Antisense and sense human telomerse catalytic sub-unit (hEST2) phosphorothioate (SODN) and random ODN were designed, synthesized and transfected into SKOV3 and COC1 cells by lipofectamine. The expression of hEST2 mRNA and telomerase activity in SKOV3 and COC1 were tested by reverse transcription-polymerase chain reaction and telomeric repeat amplification protocol before and after transfection. The proliferation and growth in SKOV3 and COC1 were also investigated by methyl thiazolyl tetrazolium and growth curve before and after transfection., Results: AODN could down-regulate the expression of hEST2 mRNA, inhibit telomerase activity and proliferation of ovarian cell lines. The efficiency depends on dose and period of administration. At 48 h, 30 micromol/L AODN had the highest activity. The expression of hEST2 mRNA were declined 54.6% and 44.6% in SKOV3 and COC1 respectively. And also the inhibition of telomerase activity were 47.9% and 42.7% respectively in the two cell lines., Conclusions: AODN of hEST2 clearly inhibited the proliferation of ovarian cancer cell lines. hEST2 may thus be a new target of gene therapy in ovarian carcinoma.

Objective: To develop monoclonal antibodies against the catalytic subunit of human telomerase hTERT for its expression detection of human tumors., Methods: A dominant epitope in hTERT (peptide hTERT(9))was automatically synthesized based on Fmoc method, and was used to immunize BALB/c mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, and the characterization of which were performed by Western blotting and immunohistochemical staining., Results: Antigenic peptide hTERT(9) was synthesized and confirmed by MALDI-TOF-MS and HPLC analysis. Three hybridoma cell lines secreting anti-hTERT(9) antibodies designated as H4, G8 and A11 were established after primary screening and consequent three rounds of limited dilution. Both of H4 and G8 were IgM, while A11 was IgG1 in isotyping. The competitive assay showed that the antibodies were hTERT(9) specific, and the affinity of G8 was stronger than that of H4 and A11 assayed by affinity ranking. However, in Western blotting, both of H4 and G8 stained an about 123 000 protein band with HeLa and 293 cell extracts but not with normal 2BS cells. Besides, positive staining presented in the nucleus of HeLa, while 2BS was non-reactive immunohistochemically. The sections from paraffin-embedded blocks of 127 cases of human cancer, 40 of precancerous and 19 of benign tumors were in situ stained by G8 antibody, the results showed that the human cancer tissues were 80.31% (102/127) positive in specific nuclear reaction, on the contrary, only a minority of precancerous lesions present weak positive (17.5%, 7/40), and negative in benign tumors (0/19)., Conclusions: The monoclonal antibodies developed against synthetic peptide were hTERT-specific and could recognize both the native and the denatured form. Thus their use in immunoblotting or immunohistochemistry for detecting the telomerase hTERT expression of cancer cell and tissues was promising.

The direct interaction of metal ion in active center of copper-zinc superoxide dismutase (Cu2Zn2SOD) with inorganic cobalt chloride (CoCl2) in buffer has been first studied by visible spectroscopy. The effects of increasing amounts of CoCl2, changing pH,different acid salt and equilibrium time on the interaction have been discussed. It was found that the Co(II) of CoCl2 makes an exchanging interaction with Zn(II) in Cu2Zn2SOD and thus the partial Cu2Co2SOD is formed.