B→O blood conversion

To maintain a constant supply of “universal blood type”,or group O red blood cells,has benefit in specialized transfusion condition.In this study, α-galactosidase cDNA was cloned from Catimor coffee bean grown in Hainan island,China,by the RT-PCR method.We have constructed a vector for α-galactosidase cDNA expression and transferred α-galactosidase cDNA into Pichia pastoris GS115 cells by electroporation.The recombinant α-galactosidase(r-α-GalE)was purified by cation ion exchange chromatography.After studying the biochemical characters of r-α-GalE we have succeeded in converting human erythrocytes from group B to group O.The animal experiment showed that transfusion of enzymetically converted group O red blood cells(ECORBC)was perfectly safe.     

Rudimentary study on humanization of porcine red blood cells:Enzymatic removal of galactose-α1,3-galactose antigen from porcine red blood cell

The serological and biochemical characterization of porcine red blood cells (pRBCs) are similar to human red blood cells,Porcine erythrocytes are considered as an alternative source for human blood transfusion.But there exist galactose-α1,3-galactose antigens (Galα1,3Galβ1,4galNAc-R,abbreviated αGal antigen ) on pRBCs,which can induce anti-αGai antibodies in human serum ,The αGal epitopes are the major antigen responsible for hyperacute rejection in exnotransfusion .In this study ,recombined soybean α-galactosidase (rSα-GalE) was used to remove the αGal antigens from pPRCs for humanization .The results showed that αGal antigen was eleared by rSα-GalE and the structure and function of rSα-GalE treated pRBC were normal.     

Expression of PHGPx in mammalian cells and its antiviral effect against coxsackievirus group

To express human phospholipid hydroperoxide glutathione peroxidase (PHGPx) in eukaryotic cells and to study its antiviral effect against Coxsackievirus B3m (CVB3m) in vitro， PHGPx cDNA was amplified from a human testis library using specific primers and cloned into expression vector pcDNA3.1His. Expression of PHGPx was performed in COS-1 cells. The antiviral effect was studied by the treatment of HeLa cells with the recombinant PHGPx. Results showed that the activity of PHGPx expressed in COS-1 cells was 5-fold higher than that in control group, and it inhibited the cytopathic effect on HeLa cells caused by CVB3m. It can be concluded that recombinant PHGPx expressed in COS-1 cells has antiviral effect against CVB3m in vitro.     

Construction and characteristics of a transformed lepidopteran cell clone expressing baculovirus p35

A transformed cell line was constructed from Mythimna separata cells Ms7311 by lipofection method. TMs7311 cells were generated using a double selection technique involving a selection in the antibiotic Zeocin, followed by a second round of selection by exhibiting cell characterization. A cell clone expressing p35 was obtained with high level of AcMNPV and recombinant proteins. Compared with wild type Ms7311 cells, the cell clone showed increased resistance to Actinamycin D-induced apoptosis and a profound resistance to nutrient development （PBS）. When the cell clone was infected with recombinant baculoviruses expressing secreted alkaline phosphatase （SEAP） and β-galactosidase, expression of the recombinant proteins from TMs7311 cells exceeded that from parental Ms7311 cells. Production of budded virus and occlusion body was significantly higher than that from parental ceils Ms7311.     

CLINICAL USE OF HEAMONETICS TYPES V50BOD LO CELL SEPARATOL AND ITS INFLUENCE ON THE HUMAN BODY

Published in J. of Haematology.1985; 6/9:532-533. Hemonetics Type v50 blood cell separator has been used by us to perform simple collectetion of blood elememts and plasma exchange to ensure the special need for blood clinically. Such as infection or bleeding due to agranulocytop-enia or thrombocytopenia from various clinical causes which repoads satisfactority to elememt blood transfusion.     

Expression of PHGPx in mammalian cells and its antiviral effect against coxsackievirus group B

To express human phospholipid hydroperoxide glutathione peroxidase (PHGPx) in eukaryotic cells and to study its antiviral effect against Coxsackievirus B3m (CVB3m) in vitro, PHGPx cDNA was amplified from a human testis library using specific primers and cloned into expression vector pcDNA3. I/His. Expression of PHGPx was performed in COS-1 cells. The antiviral effect was studied by the treatment of HeLa cells with the recombinant PHGPx. Results showed that the activity of PHGPx expressed in COS-1 cells was 5-fold higher than that in control group, and it inhibited the cytopathic effect on HeLa cells caused by CVB3m. It can be concluded that recombinant PHGPx expressed in COS-1 cells has antiviral effect against CVB3m in vitro.     

Extraction of gold,palladium, and platinum from acidic media with cyclic sulfoxide derivative

The extraction of gold (Ⅲ), palladium (Ⅱ), and platinum (Ⅳ) from the acidic media with the cyclic sulfoxide derivative of α-dodecyl-tetrahydrothiophene 1-oxide (dtmso) was investigated. Gold (Ⅲ), palladium (Ⅱ), and platinum (Ⅳ) could be separated from the acidic media with suitable sulfoxide concentration and acidity. The extraction reaction of gold (Ⅲ), palladium (Ⅱ) or platinum (Ⅳ) is exothermic when dtmso is used as an extracting reagent. The coordination number was studied by the slope method. The results indicate that, in high acidity, the dtmso coordination number for extracting gold (Ⅲ) or palladium (Ⅱ) is 3, and that for platinum (Ⅳ) is 2. UV and FT-IR spectra were used to analyze the structure of the complex. Gold (Ⅲ) is coordinated with the oxygen atom in S=O group in dtmso, and palladium (Ⅱ) or platinum (Ⅳ) is coordinated with the sulfur atom in S=O group in dtmso.     

Specific anti-tumor effect induced by attenuated Salmonella typhimurium vaccine expressing extracellular region of vascular endothelial growth factor receptor 2

The purposes of this research were to study the stable expression of exogenous gene encoding therapeutic protein in attenuated Salmonella typhimurium, observe the metabolism of oral gene vaccine carried by attenuated Salmonella typhimurium in BALB/c mouse, and investigate the feasibility of prevention and treatment of tumors by the recombinant bacteria. Recombinant plasmid pcDNA3.1 VEGFR2(n1-7) was transformed into competent attenuated Salmonella typhimurium SL3261 to develop oral DNA vaccine SL3261-pcDNA3.1 VEGFR2(n1-7). To observe whether the exogenous gene can be expressed in the recombinant bacteria, PCR was performed to amplify the CMV promoter of the eukaryotic expression vector as the proof of stable expression of exogenous protein; transmission elec- tron microscopy (TEM) was applied to observe the morphology of the recombinant bacteria to confirm that the exogenous gene has no impact on the growth of the bacteria, and then BALB/c mice were immunized with the gene vaccine. After inoculation of the gene vaccine, the recombinant bacteria SL3261 could be detected in the tissues such as small intestine, colon, liver and spleen. And then, mice in each group were challenged with tumor cells. The results of animal experiment showed that tumor growth of the mice in experimental group was inhibited and survival time of immunized mice was prolonged compared with control groups. A higher lymphocyte infiltration in tumors from animals treated with DNA vaccine was observed. Immunohistochemical analysis of tumor samples revealed an en- hanced accumulation of CD8 cytotoxic T lymphocytes, as well as an increase in CD4 cells in the tumors of animals treated with the oral gene vaccine compared to tumors from control group mice. Ultrastructure of the tumor tissue showed that tumor cells in the samples of the immunized mice were well-differentiated. Our research confirmed that the exogenous gene can be stably expressed in the attenuated Salmonella typhimurium and has no impact on the growth of the recombinant bacteria; the exogenous gene can de delivered to the host by attenuated Salmonella typhimurium to produce anti-tumor effect with no obvious cytotoxity to the host. In this study, it is established that attenuated Salmonella typhimurium could be used as a vector for oral gene vaccine, and our study provided a theoretical basis for the body distribution and the metabolism of the recombinant bacteria. This strategy may provide a simple, safe and effective way for the prevention and treatment of tumors.     

Characterization of γ-Fe2O3 nanoparticles prepared by transfor- mation of α-FeOOH

γ-Fe2O3 nanoparticles were successfully synthesized by a chemically induced transformation of α-FeOOH.In this method,the precursor(α-FeOOH)was prepared by chemical precipitation,and then treated with a mixed FeCl2/NaOH solution to produce the nanoparticles.X-ray diffraction indicated that when the precursor was treated with FeCl2(0.22 mol/L)and NaOH(0.19 mol/L),pure γ-Fe2O3 nanoparticles were obtained.However,when the concentration of FeCl2 was<0.22 mol/L or the concentration of NaOH was<0.19 mol/L,α-FeOOH and γ-Fe2O3 phases co-existed in the nanoparticles.Transmission electron microscopy observations showed that in the samples with co-existing phases,the nanoparticles did not have identical morphologies.The pure γ-Fe2O3 nanoparticles were polygonal rather than spherical.The volume ratio of α-FeOOH and γ-Fe2O3 was estimated for the two-phase samples from magnetization data obtained from a vibrating sample magnetometer.This chemically induced transformation is novel,and could provide an effective route for the synthesis of other metal oxide nanocrystallites.     

Quantifying cell binding kinetics mediated by surface-bound blood type B antigen to immobilized antibodies

Cell adhesion is crucial to many biological processes, such as inflammatory responses, tumor metastasis and thrombosis formation. Recently a commercial surface plasmon resonance （SPR）-based BIAcore biosensor has been extended to determine cell binding mediated by surface-bound bio- molecular interactions. How such cell binding is quantitatively governed by kinetic rates and regulating factors, however, has been poorly understood. Here we developed a novel assay to determine the binding kinetics of surface-bound biomolecular interactions using a commercial BIAcore 3000 bio- sensor. Human red blood cells （RBCs） presenting blood group B antigen and CM5 chip bearing immo- bilized anti-B monoclonal antibody （mAb） were used to obtain the time courses of response unit, or sensorgrams, when flowing RBCs over the chip surface. A cellular kinetic model was proposed to correlate the sensorgrams with kinetic rates. Impacts of regulating factors, such as cell concentration, flow duration and rate, antibody-presenting level, as well as pH value and osmotic pressure of sus- pending medium were tested systematically, which imparted the confidence that the approach can be applied to kinetic measurements of cell adhesion mediated by surface-bound biomolecular interactions. These results provided a new insight into quantifying cell binding using a commercial SPR-based BIAcore biosensor.     

2,3-Dihydroxybiphenyl dioxygenase gene was first discovered in Arthrobacter sp. strain PJ3

Bacterium strain PJ3,isolated from wastewater and identified as Arthrobacter sp. bacterium based on its 16S rDNA gene,could use carbazole as the sole carbon,nitrogen and energy source. The genomic library of strain PJ3 was constructed and a positive clone JM109(pUCW402) was screened out for the expression of dioxygenase by the ability to form yellow ring-fission product. A 2,3-dihydroxybiphenyl dioxygenase(23DHBD) gene of 933 bp was found in the 3360 bp exogenous fragment of pUCW402 by GenSCAN software and BLAST analysis. The phylogenetic analysis showed that 23DHBD from strain PJ3 formed a deep branch separate from a cluster containing most known 23DHBD in GenBank. Southern hybridization confirmed for the first time that the 23DHBD gene was from the genomic DNA of Arthrobacter sp. PJ3. In order to test the gene function,recombinant bacterium BL21(pETW-8) was constructed to express 23DHBD. The expression level in BL21(pETW-8) was highest compared with the recombinant bacteria JM109(pUCW402) and strain PJ3. We observed that 23DHBD was not absolute specific. The enzyme activity was higher with 2,3-dihydroxybiphenyl as a substrate than with catechol. The substrate specificity assay suggested that 23DHBD was essential for cleavage of bi-cyclic aromatic compounds during the course of aromatic compound biodegradation in Arthrobacter sp. strain PJ3.     

Gene transfer into primary cultures of fetal neural stem cells by a recombinant adenovirus carrying the gene for green fluorescent protein

Objective： To evaluate the transduction efficiency of a recombinant adenovirus carrying the gene for green fluorescent protein （Ad-GFP） into the primary cultures of fetal neural stem cells （NSCs） by the expression of GFP. Methods： The Ad-GFP was constructed by homologous recombination in bacteria with the AdEasy system; NSCs were isolated from rat fetal hippocampus and cultured as neurosphere suspensions. After infection with the recombinant Ad-GFP, NSCs were examined with a fluorescent microscopy and a flow cytometry for their expression of GFP. Results： After the viral infection, flow cytometry analysis revealed that the percentage of GFP-positive cells was as high as 97.05%. The infected NSCs sustained the GFP expression for above 4 weeks. After differentiated into astrocytes or neurons, they continued to express GFP efficiently. Conclusion： We have success- fully constructed a viral vector Ad-GFP that can efficiently infect the primary NSCs. The reporter gene was showed fully and sustained expression in the infected cells as well as their differentiated progenies.     

Human Soluble TRAIL Protein Inducing Apoptosis in Osteosarcoma Cell

This study is to examine the effect of human recombinant soluble TRAIL （TNF-related apoptosis-inducing ligand） protein inducing apoptosis in MG-63 human osteosarcoma cells. The inhibitive rates of TRAIL to MG-63 cells were detected by MTT assay. The apoptosis induced by TRAIL in MG-63 human osteosarcoma cells was analyzed with FACS and TUNEL and the apoptotic bodies were observed by transmission electron microscope. MTT assay showed that the inhibitive rates of 500, 1 000, 2 000 and 4 000 ng/mL TRAIL for 24 h were 10.1%, 24.3%, 50.6% and 97.7% respectively. Flow cytometric analysis showed that after MG-63 cells were treated with 2 gg/mL TRAIL for 6 h, obvious apoptotic peak would immediately appear before diploid peak. Human soluble TRAIL protein can quickly kill MG-63 osteosarcoma cells selectively, and may have potential value for clinical treatment of osteosarcoma.     

A study on poly (N-vinyl-2-pyrrolidone) covalently bonded NiTi surface for inhibiting protein adsorption

Near equiatomic NiTi alloys have been extensively applied as biomaterials owing to its unique shape memory effect, superelasticity and biocompatibility. It has been demonstrated that surfaces capable of preventing plasma protein adsorption could reduce the reactivity of biomaterials with human blood. This motivated a lot of researches on the surface modification of NiTi alloy. In the present work, following heat and alkaline treatment and silanization by trichlorovinylsilane (TCVS), coating of poly (N-vinyl-2-pyrrolidone) (PVP) was produced on the NiTi alloy by gamma ray induced chemical bonding. The structures and properties of modified NiTi were characterized and in vitro biocompatibility of plasma protein adsorption was investigated. The results indicated that heat treatment at 823 K for 1 h could result in the formation of a protective TiO2 layer with “Ni-free” zone on NiTi surface. It was found that PVP was covalently bonded on NiTi surface to create a hydrophilic layer for inhibiting protein adsorption on the surface. The present work offers a green approach to introduce a bioorganic surface on metal and other polymeric or inorganic substrates by gamma irradiation.     

Crystallization and preliminary X-ray crystallographic analysis of human geranylgeranyl pyrophosphate synthase

Human geranylgeranyl pyrophosphate synthase (GGPS) is an enzyme that catalyzes the synthesis of geranylgeranyl pyrophosphate (GGPP) from farnesyldiphosphate and isopentenyldiphosphate. Recombinant human GGPS was crystallized by the hanging-drop vapor diffusion method. Crystals were grown at 18℃ using PEG 4000 as precipitant. Diffraction data were obtained to a resolution of 2.8 ? from a single frozen crystal belonging to space group P1, with unit-cell parameters: a=68.9 ?, b=107.7 ?, c=137.4 ?, α=99.6°, β=97.6°, γ=97.8°.     

Specific anti-tumor effect induced by attenuated <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> vaccine expressing extracellular region of vascular endothelial growth factor receptor 2

The purposes of this research were to study the stable expression of exogenous gene encoding therapeutic protein in attenuated Salmonella typhimurium, observe the metabolism of oral gene vaccine carried by attenuated Salmonella typhimurium in BALB/c mouse, and investigate the feasibility of prevention and treatment of tumors by the recombinant bacteria. Recombinant plasmid pcDNA3.1＋ VEGFR2（n1-7） was transformed into competent attenuated Salmonella typhimuriurn SL3261 to develop oral DNA vaccine SL3261-pcDNA3.1＋VEGFR2（n1-7）. To observe whether the exogenous gene can be expressed in the recombinant bacteria, PCR was performed to amplify the CMV promoter of the eukaryotic expression vector as the proof of stable expression of exogenous protein; transmission elec- tron microscopy （TEM） was applied to observe the morphology of the recombinant bacteria to confirm that the exogenous gene has no impact on the growth of the bacteria, and then BALB/c mice were immunized with the gene vaccine. After inoculation of the gene vaccine, the recombinant bacteria SL3261 could be detected in the tissues such as small intestine, colon, liver and spleen. And then, mice in each group were challenged with tumor cells. The results of animal experiment showed that tumor growth of the mice in experimental group was inhibited and survival time of immunized mice was prolonged compared with control groups. A higher lymphocyte infiltration in tumors from animals treated with DNA vaccine was observed. Immunohistochemical analysis of tumor samples revealed an enhanced accumulation of CD8^＋ cytotoxic T lymphocytes, as well as an increase in CD4^＋ cells in the tumore of animals treated with the oral gene vaccine compared to tumors from control group mice. UI- trestructure of the tumor tissue showed that tumor cells in the samples of the immunized mice were well-differentiated. Our research confirmed that the exogenous gene can be stably expressed in the attenuated Salmonella typhimurium and has no impact on the growth of the r     

JWA protein binds to α-tubulin in PC12 cells

Our previous study elucidated that JWA protein was a newly identified microtubule-associated protein (MAP), which combined to and co-localized with α-tubulin. In the present study, we designed a series of experiments to explore if any interactions between JWA protein and α-tubulin existed and how JWA protein would functionally link to α-tubulin, especially in cell mitosis. Results of coimmnnoprecipitation, gene transfection and immnnoflnores-cence microscopy from PC12 and HEK293 cells provided strong evidence for a linkage between JWA protein and α-tubulin. Our data showed that JWA protein bound to α-tubulin stably no matter whether α-tubulin was polymerized or not. In addition, by using antisense oligonncleotides, cell cycle blocking agents and hypothermia disposal techniques, we also found the interaction between JWA protein and α-tubulin. The further analysis using flow cytometry and confocal microscopy showed that both proteins co-existed in PC12 cells and were independent on the cell cycle. In conclusion, JWA protein is a newly identified microtnbnle-associated protein, binds to α-tubulin, and probably plays an important role in regulation of microtnbnlar stability.     

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.     

Repeated epitope in the recombinant epitope-peptide could enhance ELDKWA-epitope-specific antibody response

Based on the hypothesis suggested by us that epitope-vaccine may be a new strategy against HIV mutation, we have studied several neutralizing epitopes on HIV envelope proteins. However we do not know whether a repeated epitope in a recombinant epitope-peptide can enhance epitope-specific antibody response or not. ELDKWA-epitope (aa669-674) on the C-domain of HIV-1 gp41 is a neutralizing epitope defined by the monoclonal antibody (mAb) 2F5 with broad neutralizing activity. In this study, we designed and prepared a series of the recombinant epitope-peptides bearing 1, 4 and 8 copies of ELDKWA-epitope respectively. In the comparison of the antisera induced by the three recombinant antigens, an obviously increased titre of ELDKWA-epitope-specific antibody was observed in the case of four and eight repeated epitopes. In flow cytometry analysis, the epitope-specific antibodies in both antisera showed stronger activity to bind the transfected CHO-WT cells that stably express HIV-1 envelope glycoprotein on the cell surfaces. These experimental results indicated that repeated epitope in the recombinant epitope-peptide could enhance ELDKWA-epitope-specific antibody response, which could contribute to designing an effective recombinant epitope-vaccine.     

Isolation of plasma from whole blood using a microfludic chip in a continuous cross-flow

A novel microfluidic chip is developed for crossflow filtration plasma from the whole blood which is carried out in a continuous manner. This microfluidic chip was made of a silicon substrate sealed with a compound cover. The silicon substrate fabricated by micro-electro-mechanical system (MEMS) technology consisted of microposts array, microchannels and reservoirs. Then the silicon substrate was characterized by Scaning Electron Microscopy (SEM). The performance of the microfluidic chip was valued by the experiments of plasma isolation. During more than one hour of continuous blood infusion through the chip, there were no problems of jamming or clogging, and the plasma selectivity of 97.78％ was achieved. Due to the chip’s simple structure and control mechanism with a continuous, real time operating manner, this microfluidic chip is easily expected to be integrated into micro total analytical system (μTAS) which will create a microanalysis system for point-of-care diagnostics.