A novel peptide,selected from phage display library of random peptides,can efficiently target into human breast cancer cell

To develop a targeting vector for breast cancer biotherapy, MDA-MB-231 cell, a human breast cancer cell line, was co-cultured with pC89 （9 aa） phage display library of random peptides. In multiple inde-pendent peptide-presenting phage screening trials, subtilisin was used as a protease to inactivate extra-cellular phages. The internalized phages were collected by cell lysising and amplified in E. coli XLI-Blue. Through five rounds of selection, the pepUde-presenting phages which could be internalized in MDA-MB-231 cells were isolated. A comparison was made between internalization capacities of peptide-presenting phages isolated from MDA-MB-231 cells and RGD-integrin binding phage by coculturing them with other human tumor cell lines and normal cells. The nucleoUde sequences of isolated peptide-presenting phages were then determined by DNA sequencing. To uncover whether phage coat protein or amino acid order was required for the character of the pepUde to MDA-MB-231 cells, three peptides were synthesized. They are CASPSGALRSC, ASPSGALRS and CGVIFDHSVPC （the shifted sequence of CASPSGALRSC）, and after coculturing them with different cell lines, their targeting capacities to MDA-MB-231 cells were detected. These data suggested that the internalization process was highly selective, and capable of capturing a specific peptide from parent peptide variants. Moreover, the targeting internalization event of pepUdes was an amino acid sequence dependent manner. The results demonstrated the feasibility of using phage display library of random peptides to develop new targeting system for intracellular delivery of macromolecules, and the peptide we obtained might be modified as a targeting vector for breast cancer gene therapy.     

Physiological responses of osteoblasts to cyclic stretching and the change of intracellular calcium concentration

The development of bone tissues is regulated by mechanical stimulation.Cyclic stretching was applied to the osteoblasts that were delivered from rat calvarie,The results showed that stretching at 500με increased the cell proliferation while loading at 1000με and 1500με inhabited cell growth ,Loading also increased the adhesive force between cells and substrate as well as spreading areas of osteobalsts.Furthermore,the fluorescence probe Fluo-3/AM was used to investigate the effect of stretching stimulation on the intracellular calcium concentration of osteoblasts.The intracellular calcium concentration of osteoblasts that were stretched at 500 με for 5 min was 92.9% higher than the control ,After being treated with the panax ontoginseng saponins,the streteched osteoblasts still expressed 28.6% higher intracellular calcium concentration than that of the control ,which proved that both the influx of extracellualr calcium and the release of intracellular calcium store were involved in the increase of intracellular calcium concentration when osteoblasts responded to the cyclic stretching And the influx of extracellular calcium through transmembrance channel played a main role.     

JAK3 mediatesc-myc gene expression induced by interleukin-2

C myc gene expression can be rapidly induced by IL 2 through intracellular signal transduction which is triggered by the interaction between IL 2 and its receptor (IL 2R). JAK3 which associates to the intracellular domain of IL_2R γ may play a critical role in this process. To reveal the action of JAK3 in c myc induction, a chimeric receptor gene IL_2R α/γ/Δ NJAK3 is constructed which consists of extracellular domain derived from IL 2R α subunit (IL_2R α), the transmembrane sequence derived from IL_2R γ and the cytoplasmic domain derived from the catalytic domain of JAK3 (Δ NJAK3), and then transfected this chimeric gene into mouse fibroblast cell line L929 β which had been transfected with IL_2R β gene and stably expressed IL_2R β in high level. In the transfectants coexpressing IL_2R β and α/γ/Δ NJAK3, the stimulation of IL_2 could intensively induce c myc gene expression. Because the whole cytoplasmic domain of IL_2R γ which could recruit signaling molecules was replaced by JAK3 and the c myc could be still induced by IL_2 in this situation, the results here gave the direct evidence demonstrating that JAK3 plays an important role in c myc gene expression induced by IL_2.     

Developmental characteristics and response to iron toxicity of root border cells in rice seedlings

To investigate the Fe^2＋ effects on root tips in rice plant, experiments were carried out using border cells in vitro. The border cells were pre-planted in aeroponic culture and detached from root tips. Most border cells have a long elliptical shape. The number and the viability of border cells in situ reached the maxima of 1600 and 97.5%, respectively, at 20---25 mm root length. This mortality was more pronounced at the first 1-12 h exposure to 250 mg/L Fe^2＋ than at the last 12-36 h. After 36 h, the cell viability exposed to 250 mg/L Fe^2＋ decreased to nought, whereas it was 46.5% at 0 mg/L Fe^2＋. Increased Fe^2＋ dosage stimulated the death of detached border cells from rice cultivars. After 4 h Fe^2＋ treatment, the cell viabilities were _〉80% at 0 and 50 mg/L Fe^2＋ treatment and were 〈62% at 150, 250 and 350 mg/L Fe^2＋ treatment; The viability of border cells decreased by 10% when the Fe^2＋ concentration increased by 100 mg/L. After 24 h Fe^2＋ treatment, the viabilities of border cells at all the Fe^2＋ levels were 〈65%; The viability of border cells decreased by 20% when the Fee＋ concentration increased by 100 mg/L. The decreased viabilities of border cells indicated that Fe^2＋ dosage and treatment time would cause deadly effect on the border cells. The increased cell death could protect the root tips from toxic harm. Therefore, it may protect root from the damage caused by harmful iron toxicity.     

Regulation of mouse blastocyst adhesion,outgrowth and matrix metalloproteinase—2 by focal adhesion kinase

The interaction of extracellular matrix-integrin markedly influences the adhesion,outgrowth,differentiation and expression of serine proteinases by the blastocyst,so it is regarded as a vital factor in blastocyst implantation.Although the mechanism of extracellular interactions between extracellular matrix and integrins has been well elucidated,the roles of the signaling molecules in the extracellular matrix-integrin signal transduction pathway in blastocyst implantation are unknown.This limits the understanding of blastocyst implantation and ECM-integrin signal transduction pathway.In the present study,in vitro blastocyst culture and indirect immunocytochemistry,matrix metalloproteinases(MMPs) zymography and antisense oligodeoxynucleotide(ODN) were used to investigate the expression of a fundamental molecule of integrin-dependent signal transduction pathways,focal adhesion kinase(FAK),in mouse blastocysts and its influence on mouse blastocyst adhesion,outgrowth and MMP-2.The results showed that mouse blastocysts expressed FAK.FAK protein was clustered in the peripheral migrating trophoblast cells and dispersed in the central area of blastocyst outgrowth.Fibronectin triggered pro-MMP-2 and 64kD MMP-2 activities.The antisense ODN to FAK attnuated pro-MMP-2 and 64kD MMP-2 activites which decreased abruptly and tended to disappear with increasting concentrations of the antisense ODN.Both mouse blastocyst adhesion and outgrowth on fibronectin were also influenced by the antisense ODN.Up to 20μg/mL of the antisense ODN concentration,the adhesion and out-growth rates were decreased in a dose-dependent manner.The results indicated that FAK influenced mouse blastocyst adhesion,outgrowth and MMP-2 activity by intracellular signal transduction.In other words,FAK regulates mouse implantation in terms of blastocyst adhesive and invasive abilities.     

Monitoring p21 mRNA expression in living cell based on molecular beacon fluorescence increasing rate

Studying the expression level of mRNA in living cells will offer tremendous opportunities for advancement in cell biology research, disease diagnostics, and drug discovery. In this paper, a molecular beacon （MB） specific for the important tumor suppressor gene p21 has been designed and synthesized. The fluorescence signal was detected in real-time after the MB entered the cytoplasm of nasopharyngeal carcinoma cells. After injecting the p21MB into nasopharyngeal carcinoma cell and p33-transfected nasopharyngeal carcinoma cell, the consistent increase of fluorescent signal intensity was detected in both cell lines, and maximum fluorescence intensity achieved in about 15 min. In about 4 min following microinjection, the fluorescence increasing rate was significantly different between these two cell lines, which indicate the different p21 mRNA expression levels. The results obtained in the real-time detection were also validated by RT-PCR. Analysis of the initial fluorescence increasing rate can efficiently reduce the side effect of enzyme and improve the accuracy in living cell mRNA detection.     

Examination of Subunit Composition of Bombyx Mori Silk Fibroin

Main subunits of the silk fibroin were separated by GFC(gel filtration chromatography)technigue.The nativesilk fibroin and α,c subunits were measured by gel elec-trophoresis.The aminoacid compositions of the nativesilk fibroin and α,c subunits were analyzed by means ofaminoacid measurement.Some properties of silk was inter-preted in view of subunit composition of silk fibroin.     

Effect of serum on PEGylated quantum dots: Cellular uptake and intracellular distribution

Protein adsorption is closely related with the interactions between nanoparticles and physiological systems,and further influences the cellular uptake and distribution of nanoparticles in cells.Although polyethylene glycol(PEG)ylation can largely reduce specific protein adsorption,some protein components in whole serum still interact with nanoparticles.In this work,PEGylated quantum dots(QDs) were used for investigating the quantitative and qualitative relationships of fetal bovine serum(FBS) and the cellular uptake/intracellular distribution in human hepatoma(HepG2) cell line.Nondenaturing polyacrylamide gel electrophoresis and two dimensional electrophoresis were used to analyze the adsorption of protein by PEGylated QDs.Quantitative cellular uptake of PEGylated QDs was determined by fluorescence activated cell sorting(FACS) with different FBS concentrations and incubating durations.The intracellular location of PEGylated QDs in HepG2 cells was observed using a confocal laser scanning microscope(CLSM) and a transmission electron microscope(TEM).This work will be helpful to understand the interaction between nanoparticles and cells with serum.     

Study on the interaction between Jak3 and IL-2R γ using the yeast two-hybrid system

Both interleukin-2 (IL-2) receptor γ subunit and non-receptor tyrosine kinase Jak3 play important roles in IL-2 physiological functions. Jak3 has been known to bind IL-2Rγ and the interaction is very important for IL-2 signaling. In order to find the domains directly involved in the interaction between IL-2Rγ and Jak3, various deletion mutants have been constructed and their interaction has been studied using the yeast two-hybrid system. Results show that the JH3-JH7 region of Jak3 N-terminal can bind to intracellular domain of IL-2Ry directly and the intact structure of JH3-JH7 region is necessary for this combination. In addition, the region from 305 to 317 amino acid residues near the C-terminal of IL-2Ry plays a critical role in this interaction .     

A novel element (NIRS) participates in the regulation ofinterleukin 2 receptorαgene expression

A novel element at -153/- 143 bp in the interleukin 2 receptor α(IL-2Rα) gene has been coined as NRE-inverse repeat sequence (NIRS) due to its inversely repeated to the known negative regulatory element (NRE) further upstream of the gene. In order to explore the role of NIRS in the expression of IL-2Rαgene,luciferase reporter plasmids driven by 4 individually deleted IL-2Rα genes promoter regions were constructed. Transfection of the reporter plasmids into Jurkat cells and HeLa cells respectively, we found that both NIRS and NRE were critical for repressing the constitutive expression of IL-2Rα gene and were also necessary for promoter activity induced by PHA. EMSA results showed that double-stranded NRE- and NIRS-binding proteins existed in both HeLa cells and Jurkat cells. However, single-stranded NIRS- and NRE-binding protein was only found in HeLa cells. Interestingly, the supershift band showed up in EMSA system with Jurkat cells (no matter whether activated or not) adding to the cell lysate of HeLa cells. UV-crosslinking showed a double stranded NRE- and NIRS-binding protein p83 in both Jurkat cells and HeLa cells. Our results suggest that trans-acting factors play a key role in regulating promoter activity of IL-2Rα gene by interacting with double or single stranded NRE and/or NIRS selectively in different cells.     

Induction of embryonic stem cells to hematopoietic cellsin vitro

In order to get hematopoietic cells from embryonic stem (ES) cells and to study development mechanisms of hematopoietic cells, the method of inducing embryonic stem cells to hematopoietic cells was explored by differenciating mouse ES cells and human embryonic cells in three stages. The differentiated cells were identified by flow cytometry, immunohistochemistry and Wright’s staining. The results showed that embryoid bodies (EBs) could form when ES cells were cultured in the medium with 2-mercaptoethanol (2-ME). However, cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukin-6 (IL-6), erythropoietin (EPO) and granular colony stimulating factor (G-CSF), were not helpful for forming EBs. SCF, TPO and embryonic cell conditional medium were useful for the differentiation of mouse EBs to hematopoietic progenitors. Eighty-six percent of these cells were CD34⁺ after 6-d culture. Hematopoietic progenitors differentiated to B lymphocytes when they were cocultured with primary bone marrow stroma cells in the DMEM medium with SCF and IL-6. 14 d later, most of the cells were CD34^－CD38⁺. Wright’s staining and immunohistochemistry showed that 80% of these cells were plasma-like morphologically and immunoglubolin positive. The study of hematopoietic cells from human embryonic cells showed that human embryonic cell differentiation was very similar to that of mouse ES cells. They could form EBs in the first stage and the CD34 positive cells account for about 48.5% in the second stage.     

Subunit Structure and Conformation of Bombyx Mori Silk Proteins

Molecular weights of the silk fibroin were determined by polyacrylamide gel electrophoresis in the presence of so-didium dodecyl sulfate (SDS - PAGE): The silk fibroin molecule consisted of subunits a, b and c with molecular weights of 280 kD, 230 kD and 25 kD respectively, of which the b subunit was composed of two subunits e and f with molecular weights of 130 kD and 125 kD, respec-tively, connected by disulfide bonds. The conformation of silk fibroin and subunits was determinated by Raman spectroscopy and Large angle X - ray diffraction) LAXS. The native silk fibroin only contained a - helix and random coil, but there were three conformation such as random - coil.a - helix and β - sheet in the silk fibroin dissolved in KSCN solution and frozen at - 20 °C. This suggested that KSCN solution and - 20°C freezing action could lead to the conformational transi-tion from random - coil and a - helix to P - sheet. The a subunit mainly existed in β - sheet conformation, in con-trast, the c subunit was chie     

A suppressed gene in integument cells of a fiberless seed mutant in upland cotton

A fiberless seed mutant(fl) was identified in a commercial cotton(Gossypium hirsutum L.) variety Xu-Zhou 142(FL).THis phenotype is associated with lack of fiber cell initiation in the outer integument of the ovule,as was characterized by analysis of genes related to fiber differentiation and development.Two genes,fl-E6 and FL-E6,were cloned from fl-integument cells and FL-fiber or integument cells,respectively.Compared with FL-E6,fl-E6 showed a dramatic change in nucleotide sequence:(1) FL-E6 contained a tandem repetitive sequence in which GGCTCA( Gly-Ser) is repeated five times between the 82nd and the 93rd codon from the first ATG codon,while in fl-E6 the same sequence is repeated four times;(2) The fl-E6 gene encodes a polypeptide of 241 amino acids but lacks two codons between the 90th and 93rd codon and three between the 171st and 174th relative to FL-E6;(3) There are also 12 nucleotide substitutions which would result in 7 amino acid differences between fl-E6 and FL-E6.Analysis of RT-PCR and Northern Blot showed that expression of the fl-E6 gene is suppressed in the fl-integument cells.but highly expressed in FL-fiber cells.The difference between fl-E6 and FL-E6 may be associated with lower expression of fl-E6 in the fli-inbackbones of two arabinogalactan-proteins(AGPs),one from the filtrate of suspension-cultured cells of Pyrus communis (AGPPc2) and the other from Nicotiana alata(AGPNa2),Although the function of the FL-E6 protein in differentation and development of cotton fiber cells is not known,the data indicate that the mutation of fl-E6 gene from FL-E6 gene may inhibit the fiber cell initiation from epidermal cells of the outer integument of the ovule.     

Human nectin-like 1, a novel membrane protein interacting with protein 4.1 R

Human nectin-like 1 (NECL1) full-length cDNA was cloned by bioinformatics method when searching for candidate membrane proteins interacting with members of protein 4.1 family. The cytoplasmic and extracellular regions of NECL1 were expressed in and purified from E. coli, and the polyclonal antibody was produced. Interaction between the cytoplasmic region of NECL1 and the 30 kD membrane binding domain of protein 4.1 on red blood cell (4. 1R) was demonstrated by IAsys-biosensor system and GST pull-down experiment. Results of biotin-labeled peptide ELISA further demonstrated the key amino acids for the binding. The interaction research of NECL1's cytoplasmic domain provides basis for further study of the functions of NECL1 in nervous system.     

Insights into the subunit interactions of the chloroplast ATP synthase

Subunit interactions of the chloroplast F0F1- ATP synthase were studied using the yeast two-hybrid system. The coding sequences of all the nine subunits of spinach chloroplast ATP synthase were cloned in two-hybrid vectors. The vectors were transformed into the yeast strains HF7c and SFY526 by various pairwise combinations, and the protein interactions were analyzed by measuring the yeast growth on minimal SD medium without serine, lucine and histidine. Interactions of γ Subunit with wild type or two truncated mutants of γ sununit, △εN21 and △εC45, which lose their abilities to inhibit the ATP hydrolysis, were also detected by in vitro and in vivo binding assay. The present results are largely accordant to the common structure model of F0F1-ATP synthase. Different from that in the E. Coli F0F1-ATP synthase, the δ subunit of chloroplast ATP syn- thase could interact with β,γ,ε and all the CF0 subunits in the two-hybrid system. These results suggested that though the chloroplast ATP synthase shares the similar structure and composition of subunits with the enzyme from E. Coli, it may be different in the subunit interactions and con- formational change during catalysis between these two sources of ATP synthase. Based on the present results and our knowledge of structure model of E. Coli ATP synthase, a deduced structure model of chloroplast ATP synthase was proposed.     

Rational design of glycerol dehydratase： Swapping the genes encoding the subunits of glycerol dehydratase to improve enzymatic properties

1,3-propanediol (1,3-PD) is an important material for chemical industry,and there has been always much interest in the production of 1,3-PD using all possible routes. The genes encoding glyc-erol dehydratase (GDHt) from Citrobacter freundii,Klebsiella pneumoniae and metagenome were cloned and expressed in E. coli. All glycerol dehy-dratases but the one from metagenome could be detected to show enzyme activities. In order to im-prove the enzymatic properties of GDHts,the genes encoding α and β-γ subunits were cloned,and the enzyme characteristics were evolved by rational de-sign based on their 3D structures which were con-structed by homology modeling. Six heteroenzymes were obtained by swapping the α subunit genes of these three different-source-derived GDHts. The pH,thermal stability and Vmax of some heteroenzymes were dramatically improved by 2―5 times compared with the wild one (GDHtKP). The GDHt cloned from metagenome,originally proved to be with no enzyme activity,was converted into active enzyme by swap-ping its subunits with other different GDHts. In addi-tion,the effect of subtle 3D structural changes on the properties of the enzyme was also observed.