Generation and characterization of C305, a murine neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA

B-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) family and a key regulator of B cell response. Neutralizing single-chain fragment variable (scFv) antibody against BLyS binding to its receptor BCMA has the potential to play a prominent role in autoimmune disease therapy. A phage display scFv library constructed on pill protein of MI 3 filamentous phage was screened using BLyS.After five rounds of panning, their binding activity was characterized by phage-ELISA. Nucleotide sequencing revealed that at least two different scFv gene fragments (C305 and D416) were obtained. The two different scFv gene fragments were expressed to obtain the soluble scFv antibodies, then the soluble scFv antibodies were characterized by means of competitive ELISA and in vitro neutralization assay. The results indicated that C305 is the neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA.     

Selection of substrate recognition sequence of protein kinase with ferric chelation affinity chromatography

Protein kinase substrate phage (PKS phage) was constructed by fusing the substrate recognition consensus sequence of cAMP-dependent protein kinase (cAPK) with bacteriophage minor coat protein g3p and by dis-playing it on the surface of filamentous bacteriophage fd. Phosphorylation in vitro by cAPK showed a unique labelled band of approximately 60 ku, which was consistent with the molecular weight of the PKS-g3p fusion protein. Some weakly phosphorylated bands for both PKS phage and wild-type phage were also observed. Phage display random 15-mer peptide library phosphorylated by cAPK was selected with ferric (Fe3 ) chelalion affinity resin. After 4 rounds of screening, phage clones were picked out to determine the displayed peptide sequences by DNA sequencing. The results showed that 5 of 14 sequenced phages displayed the cAPK recognition sequence motif (R)RXS/T. Their in vitro phosphorylation analyses revealed the specific labelled bands corresponding to the positive PKS phages with and without the typ     

Radiolabeling and biodistribution of a nasopharyngeal carcinoma-targeting peptide identified by in vivo phage display

A dodecapeptide EDIKPKTSLAFR ligand targeting CEN- 1 human nasopharyngeal carcinoma （NPC） was identified by in vivo phage display. Two tridecapeptides and their derivatives, named YR13 （YEDIKPKTSLAFR）, EY 1 3 （EDIKPKTSLAFRY）, EY 1 3-NH2 （EDIKPKTSLAFRY-NH2） and Fmoc-YR 1 3 （Fmoc-YEDIKPKTSLAFR）, were synthesized and radiolabeled with ^[3]I. The stability in vitro, biodistribution and tissue distribution of selected phage particles in mice bearing NPC tumor were determined, and plasma metabolites analysis of radiolabeled peptides was carried out. Although Fmoc and NH2 groups could protect the peptide from deiodination, only Fmoc group inhibited the binding of Fmoc-YR13 to NPC tumors. The compound EY13-NH2, the C-terminal amide of peptide EY13, had the greatest serum stability, the least deiodination, and showed favorable tumor/blood ratios. The selected phage particles （phage 3 or phage 5） were more concentrated in NPC tumors than the control phage （initial phage display peptide library）. EY13 could also inhibit the binding of selected phage particles to tumors. The results indicated that EDIKPKTSLAFR was a good candidate in diagnostic and therapeutic NPC.     

The interaction of laminin and its membrane receptor on mouse macrophage membrane studied by STM and FRAP

The variation of membrane surface and lateral diffusion of membrane protein was studied after the interaction of laminin with its membrane receptor in mouse macrophages. A pattern of membrane surface which showed smaller and bigger peaks was obtained by scanning tunneling microscope(STM), looking like the domains of lipid groups and proteins in the model of fluid mosaic biomembrane. Some even more higher and wider peaks projected out from the membrane surface in STM image after the interacting of laminin with membrane receptor were, probably, the complexes of laminin and membrane receptor. Furthermore, the decreased lateral diffusion coefficient value (D) was obtained by fluorescence recovery after photobleaching (FRAP) after the laminin was reacted with membrane receptor. This phenomenon provides an evidence that the complexes of laminin and its membrane receptor were located on the membrane of macrophages. So we could consider that the laminin is combined with membrane receptor leading to the variation in the properties of membrane surface.     

Design of a novel plasminogen activator based on the structure of hirudin

Using a phage library, seven peptide sequences with high affinity to human microplasminogen were obtained. Caseinolytic assay indicated that only the synthesized peptide P07 had slight fibrinolytic activity. To enhance its plasminogen activation ability, peptide P07 was fused into loop 32-35 of hirudin. In vitro assay demonstrated that this hirudin-like fusion protein can activate human plasminogen and retain the function of thrombin inhibition. Fusing the sequence ＂SPDASRL＂ into hirudin generated a plasminogen activation activity 100 times higher than peptide P07 in chromogenic and radial caseinolytic assay. This significant functional improvement might originate from a more specific active structure due to the hirudin scaffold.     

Influence of pH on Cd and Cu uptake, distribution and their effect on nitrate reductase activity in cucumber (Cucumis sativus L.) seedling roots

Influence of different pH solutions (5.0 and 7.0) on Cu²⁺ and Cd²⁺ absorption and distribution in root cells as well as effects of these metals on nitrate reductase activity (NR) in roots of cucumber seedlings were estimated. The absorption of Cu and Cd by roots measured as metal depletion in uptake solution was similar, both metal absorption was independent of the pH of solution. However, after rinsing of roots in distilled water (30 minutes), more Cu than Cd was found in protoplasts of root cells. More Cu was measured in all cell fractions when Cu was uptaken from pH 5.0 than from 7.0. The nitrate reductase activity after one hour of metal treatments was drastically decreased by Cu. The strongest reduction of enzyme activity was observed in roots treated with Cu in buffer with pH 5.0. Influence of Cd on the enzyme activity was weaker and was independent of the pH of solution. Lower concentration of Cd in solution (20 μM) increased NR activity. The data obtained prove the higher mobility of Cu than Cd into the cells of root. The mobility of Cu depends on pH of solution. Cu ions, but not Cd, influenced membrane permeability (K leakage). Cu acted more drasticly than Cd on NR activity.     

PSMA mimotope isolated from phage displayed peptide library can induce PSMA specific immune response

Prostate-specific membrane antigen (PSMA) is a cell surface glycoprotein expressed predominantly in prostate secretory acinar epithelium and prostate cancer cells as well as in several extraprostatic tissues.Mouse monoclonal antibody 4G5 specific to the extracellular domain of PSMA was used to screen two phage displayed peptide libraries (9aa linear and 9aa cys library).Three 4G5-reactive phagotopes were identified.Sequence analysis of isolated clones demonstrated that the interaction motif“VDPA/SK” has high homology to 719-725aa on PSMA.Immunohistochemical staining of the prostate cancer sample with the psma-mimic phagotope(mimotope) immunized serum antibodies demonstrate that the mimotope isolated from the phage displayed peptide libraries can induce PSMA specific immune response in vivo.     

Substitutions of the Conserved Gly47 Affect the CF1 Inhibitor and Proton Gate Functions of the Chloroplast ATP Synthase ε Subunit

The conserved residue Gly47 of the chloroplast ATP synthase ε subunit was substituted with Leu, Arg, Ala and Glu by site-directed mutagenesis. This process generated the mutants εG47L, εG47R, εG47A and εG47E, respectively. All the ε variants showed lower inhibitory effects on the soluble CF1(-ε) Ca^2 -ATPase compared with wild-type ε. In reduced conditions, εG47E and εG47R had a lower inhibitory effect on the oxidized CF1(-ε) Ca^2 -ATPase compared with wild-type ε. In contrast, εG47L and εG47Aincreased the Ca^2 -ATPase activity of soluble oxidized CF1(-ε). The replacement of Gly47 significantly impaired the interaction between the subunit ε and γ in an in vitro binding assay. Further study showed that all ε variants were more effective in blocking proton leakage from the thylakoid membranes. This enhanced ATP synthesis of the chloroplast and restored ATP synthesis activity of the reconstituted membranes to a level that was more efficient than that achieved by wild-type ε. These results indicate that the conserved Gly47 residue of the ε subunit is very important for maintaining the structure and function of the ε subunitand may affect the interaction between the ε subunit, β subunit of CF1 and subunit Ⅲ of CF0, therebyregulating the ATP hydrolysis and synthesis, as well as the proton translocation role of the subunit Ⅲ of CF0.     

Biochemical analysis of a papain-like protease isolated from the latex of Asclepias curassavica L.

Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase （asclepain c-II） has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. （Asclepiadaceae）. Asclepain c-II was the minor proteolytic component in the latex, but showed higher specific activity than asclepain c-I, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography （FPLC）. Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain c-II displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain c-II is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantrollne, phenyimethanesulfonyl fluoride, and pepstatine. The N-terminal sequence （LPSFVDWRQKGVVFPIRNQGQ CGSCWTFSA） showed a high similarity with those of other plant cysteine proteinases. When assayed on N-α-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-α-CBZ-L-Gln-p-nitrophenyl ester as substrate： Km = 0.1634 mM, kcat = 121.48 s^-1, and kcat/Km = 7.4 ×10^5 s^-1/mM.     

In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (＞1013 different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents.     

Effect of amino acid residue and oligosaccharide chain chemical modifications on spectral and hemagglutinating activity of Millettia dielsiana Harms. ex Diels. lectin

The effects of modifying the carbohydrate chain and amino acids on the conformation and activity of Millettia dielsiana Harms. ex Diels. lectin (MDL) were studied by hemagglutination, fluorescence and circular dichroism analysis. The modification of tryptophan residues led to a compete loss of hemagglutinating activity; however, the addition of mannose was able to prevent this loss of activity. The results indicate that two tryptophan residues are involved in the carbohydrate-binding site. Modifications of the carboxyl group residues produced an 80% loss of activity, but the presence of mannose protected against the modification. The results suggest that the carboxyl groups of aspartic and glutamic acids are involved in the carbohydrate-binding site of the lectin. However, oxidation of the carbohydrate chain and modification of the histidine and arginine residues did not affect the hemagglutinating activity of MDL. Fluorescence studies of MDL indicate that tryptophan residues are present in a relatively hydrophobic region, and the binding of mannose to MDL could quench tryptophan fluorescence without any change in λmax. The circular dichroism spectrum showed that all of these modifications affected the conformation of the MDL molecule to different extents, except the modification of arginine residues. Fluorescence quenching showed that acrylamide and iodoacetic acids are able to quench 77% and 98% of the fluorescence of tryptophan in MDL, respectively. However, KI produced a barely perceptible effect on the fluorescence of MDL, even when the concentration of I^- was 0.15M. This demonstrates that most of tryptophan residues are located in relatively hydrophobic or negatively charged areas near the surface of the MDL molecule.     

弱氧化醋杆菌阿拉伯糖醇脱氢酶基因克隆及其在大肠杆菌中的表达

The partial genomic library of Acetobacter suboxydans was constructed using Yeast\| E.coli shuttle plasmid YEp352 as vector.Two positive transformants,designated as DH5α(pAD91) and DH5α(pAD98),were obtained by screening the growth of transformants on the agar plate in which D\|arabitol was used as the sole carbon source.The results of Southern blot and restriction endonuclease analysis showed that the two recombinants are identical.The insert is about 2.3kb.Arabitol dehydrogenase activity assay indic…     

Human immune suppression is inducible by trichosanthin via CD8 cell—mediated pathway

Trichosanthin(Tk),a polypeptide with 249 amino acid residues isolated and purified from a Chinese medicinal herb,showed the capability of inducing abortion and was able to inhibit tumor growth and HIV replication.Owing to sequence homology of the peptide with a ribosomeinactivating protein,the downward activity of Tk was suggested to be related to its cytotoxic property.We report here,however,that Tk could exert potent inhibitory effects on human lymphoproliferative responses in vitro to allogeneic,mitogenic and soluble antigens with 50% inhibition doses ranged between 0.05 and 0.5μg／ml.The lowresponsivenesss caused by Tk was not due to toxic cytolysis.Rather,evidences suggested that,in the dose range adopted,the Tk-induced inhibition was attributable,at least in part,to immune suppression,in view of (1) Tk was more effective in the early stage of alloreactivity;(2)Suppression also occurred if responder cells were pulsetreated with Tk rather than cocultured;(3)Irradiated Tk-pulsed cells were capable of inducing suppression in a Tk-free culture;(4)Suppression could also be transferred by the supernatants of Tk-pulsed cultured cells;(5)Tk-induced immune suppression was diminished by depletion of CD8^ cells from the culture,and,finally;(6)Adding CD8^ cells back to the culture could restore the suppres sion.Thus the possibility that Tk might function as a down-regulator by immunological mechanisms in human immune responses is discussed.     

The Response Difference of Mitochondria in Recalcitrant Antiaris toxicaria Axes and Orthodox Zea mays Embryos to Dehydration Injury

Long-term preservation of recalcitrant seeds is very difficult because the physiological basis on their desiccation sensitivity is poorly understood. Survival of Antiaris toxicaria axes rapidly decreased and that of immature maize embryos very slowly decreased with dehydration. To understand their different responses to dehydration, we examined the changes in mitochondria activity during dehydration. Although activities of cytochrome （Cyt） c oxidase and malate dehydrogenase of the A. toxicaria axis and maize embryo mitochondria decreased with dehydration, the parameters of maize embryo mitochondria were much higher than those of A. toxicaria, showing that the damage was more severe for the A. toxicaria axis mitochondria than for those of maize embryo. The state I and III respiration of the A. toxicaria axis mitochondria were higher than those of maize embryo, the former rapidly decreased, and the latter slowly decreased with dehydration. The proportion of Cyt c pathway to state III respiration for the A. toxicaria axis mitochondria was low and rapidly decreased with dehydration, and the proportion of alternative oxidase pathway was high and slightly increased with dehydration. In contrast, the proportion of Cyt c pathway for maize embryo mitochondria was high, and that of alternative oxidase pathway was low. Both pathways decreased slowly with dehydration.     

Effects of Phosphorus Nutrient on the Hydraulic Conductivity of Sorghum （Sorghum vulgare Pers.） Seedling Roots Under Water Deficiency

Hydroponic experiments were conducted in a growth chamber and changes in the hydraulic conductivity of sorghum (Sorghum vulgare Pers.) roots (Lpr) at the three-leaf stage were measured using the pressure chamber method. Water deficiency was imposed with polyethylene glycol (PEG) 6000 and the phosphorus (P) levels were controlled by complete Hoagland solution with and without P nutrient. The objective of this study was to investigate the effect of P nutrition on root Lpr under water deficiency. The results showed that the Lpr in P deficiency treatments decreased markedly, but the Lpr recovered to the same value as that of control when sufficient P was supplied for 4-24 h. Water deficiency decreased Lpr, but the hydraulic conductivity of the roots with sufficient P supply was still higher than that of plants without P supply. When resuming water supply, the Lpr of the water-deficient plants under P supply recovered faster than that of plants without P supply, which indicates that plants with sufficient P nutrient are more drought tolerant and have a greater ability to recover after drought. The treatment of HgCl2 indicated that P nutrient could regulate the Lpr by affecting the activity and the expression levels of aquaporins.     

Distribution of α-D-mannose residue on zona pellucida and its role(s) in fertilization in pigs

The present study aims to identify the distribution of α-D-mannose residues on zona pellucida (ZP) and their role(s) in fertilization in pigs. In experiment 1, in vitro matured pig oocytes were freed from cu- mulus cells and treated with fluorescein isothiocyanate-labelled Lens culinaris (FITC-LCA), a D-mannose specific binding lectin. After 30 min of treatment, LCA bound evenly throughout the ZP with strong fluorescence. In experiment 2, when LCA-treated oocytes were used for in vitro fertilization, the number of sperm bound to ZP was significantly decreased, and sperm penetration was almost com- pletely blocked. In experiment 3, polysaccharide mannan was added to the in vitro fertilization medium as a competitive inhibitor. Both the number of sperm bound to ZP and the rate of fertilized oocytes were significantly reduced in the mannan-treated group compared with the control group. In experiment 4, spermatozoa were incubated with mannan in vitro. The number of acrosome-reacted spermatozoa was evidently increased in a time-dependent manner during the incubation. These results suggest that α-D-mannose residues presenting on pig ZP might be an important component of sperm receptor and might induce sperm acrosome reaction and thus facilitate the sperm penetration into the ZP.     

The interaction of laminin and its membrane receptor on mouse macrophage membrane studied by STM and FRAP

The variation of membrane surface and lateral diffusion of membrane protein was studied after the interaction of laminin with its membrane receptor in mouse macrophages. A pattern of membrane surface which showed smaller and bigger peaks was obtained by scanning tunneling microscope(STM), looking like the domains of lipid groups and proteins in the model of fluid mosaic biomembraoe. Some even more higher and wider peaks projected out from the membrane surface in STM im-age after the interacting of laminin with membrane receptor were probably, tile complexes of laminin and membraue receptor. Furthermore. the deeceasad lateral diffusion coofficeent value(D^_) was obtained by Huorescence recovery after photobleaching (FRAP) after the laminin was reacted with membrane receptor. This phenomenon provides an evidence that the complexes of laminin and its membrane receptor were located on the membrane of macrophages. So we could consider that the laminin is combined with membrane receptor leading to the variation in the properties of membrane surface.     

Effect of EGF on initiation of primordial follicle growth in ovary of newborn rat

The present study was designed to look at the effect of epidermal growth factor (EGF) and tomcie-stimulating hormone (FSH) on initiation of primordial follicle growth and differentiation in the ovary of newborn rat with a sensitive marker of proliferating cell nuclear antigen (PCNA). The results showed that more cuboidal granulosa cells (GC) were found in the ovary two days after injection of EGF. More proliferative GC were observed on D4. No such action of FSH on primordial follicles was demonstrated. Using in situ hybridization, inhibin a mRNA expression in GC was detected from D5, while FSH receptor (FSHR) mRNA expression started from D6 after birth. Both mRNAs increased following further development of the follicles. These results suggest that it is EGF, but not FSH, that may play a certain role in initiation of primordial follicle growth. FSH may be involved in further differentiation and growth of the early developmental follicles.     

Structural analysis and molecular modeling of two antitrichosanthin IgE clones from phage antibody library

Recently we constructed a murine IgE phage surface display library and screened out two IgE (Fab) clones with specific binding activity to Trichosanthin (TCS).In this work,the Vε and Vκ genes of the two clones were sequenced and their putative germline gene usages were studied.On the basis of the known 3D structure of Trichosanthin and antibody,molecular modeling was carried out to study the antigen-antibody interaction.The possible antigenic determinant sites on the surface of TCS recognized by both the clones were analyzed,and the reaction forces between TCS and two Fab fragments were also analyzed respectively.     

Preparation and evaluation of a new releasable PEGylated tumor necrosis factor-α(TNF-α) conjugate for therapeutic application

To design a releasable PEGylated TNF-α(rPEG-TNF-α ), a cathepsin B-sensitive dipeptide (Val-Cit moiety) was inserted into conventional PEG-modified TNF- (PEG-TNF- ), facilitating its clinical use for anti-tumor therapy. Comparative pharmaco- kinetic and pharmacodynamic studies showed that the half-lives of both PEGylated forms of TNF-α were ~60-fold greater than that of unmodified TNF-α . In addition, the in vitro bioactivity of rPEG-TNF-α was greater than that of PEG-TNF-α with the same degree of PEG modification. Release of TNF-α from rPEG-TNF-α in vitro was dependent on the presence of cathepsin B and was inhibited by a cathepsin B inhibitor. Despite the potent cytotoxicity of unmodified TNF-α against normal cells, its PEGylated forms at higher TNF-α concentrations showed low cytotoxic activity against these cells. In contrast, both forms of PEGylated TNF-α showed potent cytotoxic activity against the B16 and L929 cell lines, with rPEG-TNF-α being 5- and 9- fold more potent, respectively, than PEG-TNF-α . Moreover, rPEG-TNF-α was a more potent in vivo antitumor agent than PEG-TNF-α .