Modification with a phosphorylation tag of PKA in the TraT-based display vector of Escherichia coli

We have previously developed the TraT display system to express the preS1 peptide of human hepatitis B virus (HBV) and the snake venom rhodostomin (RHO) on the surface of Escherichia coli. In this study, we modified the pT2 vector by adding a thrombin cutting site and a phosphorylation tag of protein kinase A before the multiple restriction enzyme sites. The modified vector allowed us to label the TraT fusion protein (TraT-RHO) with [32P] and to increase the detection sensitivity of TraT-RHO expression bacteria binding to and being internalized into BHK-21 cells. After the thrombin cleavage, the isotope labeled RHO could be detected in a free form. We therefore suggest that the new version of pT2 vector, pT2-KL, will facilitate to identify the counterpart of displayed peptide.     

Cell surface staining of recombinant factor VIII is reduced in apoptosis resistant BHK-21 cells

We have recently demonstrated an increase in recombinant factor VIII (rFVIII) secretion from BHK-21 cells (rBHK-21(host)) following an over-expression of the chaperone protein heat shock protein 70 (Hsp70) (rBHK-21(Hsp70)) due to an inhibition of apoptotic cell death and an increased cellular expression of rFVIII [Ishaque, A., Thrift, J., Murphy, J.E., Konstantin, K., 2007. Over-expression of Hsp70 in BHK-21 cells engineered to produce recombinant factor VIII promotes resistance to apoptosis and enhances secretion. Biotechnol. Bioeng. Biotech. Bioeng. 97, 144-155]. In the present study we investigated the difference in adherence of rFVIII to the cell membrane surface by comparing changes in cell viability and extent of phosphatidylersine (PS) exposure in apoptosis between rBHK-21(host), rBHK-21(Hsp70), and parental BHK-21 cells devoid of rFVIII expression (BHK-21(native)) during batch cell culture experiments. The Zenon technique was used to double stain for cell surface and intracellular rFVIII using flow cytometric Guava PCA analysis. By this quantitative analysis intracellular rFVIII was shown to decrease in rBHK-21(host) cells as the cell viability declined while the rFVIII cell surface staining increased. Conversely, rBHK-21(Hsp70) cell cultures displayed higher cell viability and intracellular rFVIII with less cell surface rFVIII staining. Time dependent increases of rFVIII adherence to the surface of rBHK-21(host) cells and its reduction on the surface of rBHK-21(Hsp70) cells was also confirmed by fluorescence microscopy. Furthermore, greater rFVIII cell surface staining correlated with an increase in detectable PS exposure on the surface of BHK-21(native) batch cell cultures. However, PS exposure could not be identified to the same extent on rBHK-21(host) cells despite a similar decline in cell viability between rBHK-21(host) and BHK-21(native) batch cultures. Any exposed PS on rBHK-21(host) cells was most likely masked by secreted rFVIII, mimicking the effect on activated platelets where the externalization of PS also occurs, and serves as a ligand for FVIII activation in the blood coagulation cascade. Taken together we have identified that rFVIII sequestration on the membrane surface is another potential limitation to rFVIII productivity and one which can also be alleviated by reduction of apoptosis in a clone expressing human HSP70.     

The dissociation of the surface architecture described by enhanced lectin agglutinability and the transformed phenotype expressed as anchorage independence.

Using a series of cold-sensitive variants of chemically transformed BHK-21 cells, revertants to the normal phenotype derived from a dimethyl-nitrosamine transformed clone of BHK-21 as well as revertants to the normal phenotype derived from polyoma transformed BHK-21 cells we have demonstrated that the surface phenotype described by enhanced agglutinability with Con A and WGA can be dissociated from the transformed phenotype described by anchorage independence (growth in semisolid medium). Specifically we have demonstrated that the surface characteristic of enhanced agglutinability may be found in a variety of cell lines which fail to display to grow in agar. Our work clearly shows that the two phenotypes described are not concomitantly controlled and tends to suggest that the phenotype of enhanced lectin agglutinability may be dissociated from the transformed phenotype.     

Glycopeptides prepared from mouse cerebrum inhibit protein synthesis and cell division in baby hamster kidney cells, but not in their polyoma virus-transformed analogs

Glycopeptides isolated from mouse cerebral cortex cell surfaces (BCSG) were shown to inhibit cell growth and protein synthesis in baby hamster kidney (BHK)-21 cells, whereas polyoma virus-transformed BHK-21 cells (pyBHK-21) were refractory to the inhibitory activity of the glycopeptides. Growth inhibition was shown to be reversible and non-lethal to BHK-21 cells. Despite that difference in sensitivity to the action of the glycopeptides, both cell lines could bind the inhibitor in a saturable fashion and in similar quantities. After trypsinization, BHK-21 cells appeared refractory to the inhibitor, whereas pyBHK-21 cells became sensitive. The data suggested the presence of a receptor for BCSG on the cell surface of both cell lines. Incubating BCSG with conditioned medium from pyBHK-21 cells resulted in loss of the glycopeptide's inhibitory activity. In contrast, medium conditioned by BHK-21 cells had no effect on the inhibitory activity of BCSG. We hypothesize that the refractoriness of pyBHK-21 cells to BCSG is related to their autonomous growth characteristics and failure to respond to topo-inhibitory growth control. BCSG may be a naturally occurring growth regulator whose function can be explored by use of the BHK-21/ pyBHK-21 model system.     

Membrane Permeabilization by Small Hydrophobic Nonstructural Proteins of Japanese Encephalitis Virus

Infection with Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, may cause acute encephalitis in humans and induce severe cytopathic effects in various types of cultured cells. We observed that JEV replication rendered infected baby hamster kidney (BHK-21) cells sensitive to the translational inhibitor hygromycin B or alpha-sarcine, to which mock-infected cells were insensitive. However, little is known about whether any JEV nonstructural (NS) proteins contribute to virus-induced changes in membrane permeability. Using an inducible Escherichia coli system, we investigated which parts of JEV NS1 to NS4 are capable of modifying membrane penetrability. We found that overexpression of NS2B-NS3, the JEV protease, permeabilized bacterial cells to hygromycin B whereas NS1 expression failed to do so. When expressed separately, NS2B alone, but not NS3, was sufficient to alter bacterial membrane permeability. Similarly, expression of NS4A or NS4B also rendered bacteria susceptible to hygromycin B inhibition. Examination of the effect of NS1 to NS4 expression on bacterial growth rate showed that NS2B exhibited the greatest inhibitory capability, followed by a modest repression from NS2A and NS4A, whereas NS1, NS3, and NS4B had only trivial influence with respect to the vector control. Furthermore, when cotransfected with a reporter gene luciferase or beta-galactosidase, transient expression of NS2A, NS2B, and NS4B markedly reduced the reporter activity in BHK-21 cells. Together, our results suggest that upon JEV infection, these four small hydrophobic NS proteins have various modification effects on host cell membrane permeability, thereby contributing in part to virus-induced cytopathic effects in infected cells.     

In vivo detection of membrane protein expression using surface plasmon enhanced fluorescence spectroscopy (SPFS)

Surface plasmon enhanced fluorescence spectroscopy (SPFS) was applied for the detection of expression and functional incorporation of integral membrane proteins into plasma membranes of living cells in real time. A vesicular stomatitis virus (VSV) tagged mutant of photoreceptor bovine rhodopsin was generated for high level expression with the semliki forest virus (SFV) system. Adherent baby hamster kidney (BHK-21) cells were cultivated on fibronectin-coated gold surfaces and infected with genetically engineered virus driving the expression of rhodopsin. Using premixed fluorescently (Alexa Fluor 647) labeled anti-mouse secondary antibody and monoclonal anti-VSV primary antibody, expression of rhodopsin in BHK-21 cells was monitored by SPFS. Fluorescence enhancement by surface plasmons occurs exclusively in the close vicinity of the gold surface. Thus, only the Alexa Fluor 647 labeled antibodies binding to the VSV-tag at rhodopsin molecules exposed on the cell surface experienced fluorescence enhancement, whereas, unbound antibody molecules in the bulk solution were negligibly excited. With this novel technique, we successfully recorded an increase of fluorescence with proceeding rhodopsin expression. Thus, we were able to observe the incorporation of heterologously expressed rhodopsin in the plasma membrane of living cells in real time using a relatively simple and rapid method. We confirmed our results by comparison with conventional wide field fluorescence microscopy.     

Evidence that TraT interacts with OmpA of Escherichia coli

The OmpA protein is one of the major outer membrane proteins of Escherichia coli. Among other functions the protein serves as a receptor for several phages and increases the efficiency of F-mediated conjugation when present in recipient cells. TraT is an F-factor-coded outer membrane lipoprotein involved in surface exclusion, the mechanism by which E. coli strains carrying F-factors become poor recipients in conjugation. To determine a possible interaction of TraT with OmpA, the influence of TraT on phage binding to cells was measured. Because TraT inhibits inactivation of OmpA-specific phages it is suggested that TraT interacts directly with OmpA. Sequence homology of TraT with proteins 38, the phage proteins recognizing outer membrane proteins, supports this finding. A model of protein interactions is discussed.     

Antigen 43 from Escherichia coli induces inter- and intraspecies cell aggregation and changes in colony morphology of Pseudomonas fluorescens

Antigen 43 (Ag43) is a surface-displayed autotransporter protein of Escherichia coli. By virtue of its self-association characteristics, this protein is able to mediate autoaggregation and flocculation of E. coli cells in static cultures. Additionally, surface display of Ag43 is associated with a distinct frizzy colony morphology in E. coli. Here we show that Ag43 can be expressed in a functional form on the surface of the environmentally important Pseudomonas fluorescens strain SBW25 with ensuing cell aggregation and frizzy colony types. Using green fluorescence protein-tagged cells, we demonstrate that Ag43 can be used as a tool to provide interspecies cell aggregation between E. coli and P. fluorescens. Furthermore, Ag43 expression enhances biofilm formation in P. fluorescens to glass surfaces. The versatility of this protein was also reflected in Ag43 surface display in a variety of other gram-negative bacteria. Display of heterologous Ag43 in selected bacteria might offer opportunities for rational design of multispecies consortia where the concerted action of several bacterial species is required, e.g., waste treatment and degradation of pollutants.     

中国首次分离的辛德毕斯病毒XJ-160病毒株感染性全基因组cDNA克隆的构建与分析

报告了中国首次分离的辛德毕斯病毒XJ-160株的感染性全基因组cDNA克隆的构建与鉴定。利用RT—PCR方法获得覆盖病毒全长基因组的cDNA片段,以低拷贝质粒pBR322作为骨架,将基因组cDNA置于SP6RNA聚合酶启动子之后,基因组3’末端带有35个连续的A,通过DNA重组技术组装成病毒基因组全长cDNA克隆。该克隆可在大肠杆菌DH5a中稳定扩增。经体外转录,RNA转录体转染BHK-21细胞,细胞发生病变,恢复病毒滴度达到10^7～10^8PFU／ml。全基因组cDNA克隆构建过程中引入的沉默突变(8453位核苷酸由C变为T)产生XbaⅠ酶切位点作为遗传标记,在子代恢复病毒的基因组中稳定存在。从细胞病变的特征、BHK-21细胞的空斑形态、病毒的抗原性、病毒在细胞中的生长动力学特征以及对乳鼠的致病性等方面比较,恢复病毒和亲本病毒XJ-160没有显著区别,提示获得了具有感染性的XJ-160病毒全长cDNA克隆。该病毒感染性全基因组cDNA克隆可以作为反向遗传学系统,为进一步研究病毒复制和致病机制,以及开发相应的载体表达系统提供分子生物学工具。     

Evaluation of apoptosis markers in different cell lines infected with equine arteritis virus

Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation, but the DNA ladder was detected at different times post-infection depending on the cell line, i.e., 48, 72 and 96 h post-infection in RK-13, Vero and BHK-21 cells, respectively. Measurement of viral titers obtained with each cell line indicated that apoptosis causes interference with viral replication and therefore decreased viral titers. As an unequivocal marker of apoptosis, we measured the expression of caspase-3 and caspases-8 and -9 as extrinsic and intrinsic markers of apoptosis pathways, respectively. Caspase-8 in BHK-21 cells was the only protease that was not detected at any of the times assayed. We found that Bucyrus EAV strain exhibited a distinctive apoptosis pathway depending on the cell line.     

Cell surface display of hepatitis B virus surface antigen by using Pseudomonas syringae ice nucleation protein

A new system designed for cell surface display of recombinant proteins on Escherichia coli was evaluated for expression of eukaryotic viral antigens. The major surface antigen of hepatitis B virus (HBsAg) was fused to the ice nucleation protein (INP), an outer membrane protein of Pseudomonas syringae. Western blotting, immunofluorescence microscopy, whole-cell ELISA, and ice nucleation activity assay confirmed expression of recombinant proteins on the surface of Escherichia coli. This study indicated that INP-based cell surface display can be used for epitope mapping and recombinant bacteria expressing hepatitis viral antigens may be used for developing live vaccines.     

Expression of random peptide fused to invasin on bacterial cell surface for selection of cell-targeting peptides

The protein invasin expressed on the cell surface of the pathogenic bacteria Yersinia pseudotuberculosis mediates the entry of this bacterium into cultured mammalian cells. We have developed a system for expression of random peptides on the cell surface of Escherichia coli (E. coli) by creation of a fusion hybrid between a peptide and the invasin protein. The fusion protein constructs consist of part of the outer membrane domain of the invasin protein, six proline spacers, and a decamer of random peptides flanked by cysteine residues (CX(10)C). Peptides were constitutively expressed on the cell surface in the resulting random decamer peptide library, which we designated as ESPEL (E. coli Surface Peptide Expression Library). The ESPEL was systematically screened for its binding affinity toward human cultured cells. Several bacterial clones were identified whose binding to human cells was mediated by peptides expressed on the bacterial cell surface. Flow cytometric analysis showed that both the identified bacterial clones and these corresponding chemically synthesized peptides bound to human cells specifically. The techniques described provide a new method that uses E. coli random peptide library to select targeting peptides for mammalian cells without any knowledge of the human cellular receptors.     

Further characterization of a CD99-related 21-kDa transmembrane protein (VAP21) expressed in Syrian hamster cells and its possible involvement in vesicular stomatitis virus production

The VAP21, a CD99-related 21-kDa transmembrane protein, was first detected in the enveloped virions that were grown in a Syrian hamster-derived cell line, BHK-21 (Sagara et al., 1997; Yamamoto et al., 1999). We further tried to elucidate the nature and properties of VAP21. The VAP21 was detected in various organs of the Syrian hamster as well as in the Syrian hamster-derived cell lines (BHK-21 and HmLu-1). We could not detect the VAP21 antigen in other cell lines derived from other animal species we examined, including a Chinese hamster (CHO-K1), mouse (neuroblastoma C1300, clone NA), dog (MDCK), monkey (COS-7), and human (HeLa, HepG2). We tried to introduce the VAP21 gene into VAP21-negative cell lines using a tetracycline-regulated gene expression system. All of our trials, however, resulted in failure to establish stably positive inducible cell lines. To the contrary, we could easily establish the VAP21-overexpressing cell lines from the Syrian hamster cell lines, which were successfully grown and maintained without any loss of VAP21 expression even under the induced culture conditions. In such VAP21-overexpressing cells, production of the vesicular stomatitis virus (VSV) was increased several-fold, while suppression of the VAP21 expression resulted in reducing the VSV yields. From these results, we conclude that the VAP21 is a physiologically active cell membrane component of some animal species including the Syrian hamster, and might positively be involved in the VSV replication.     

风疹病毒包膜糖蛋白E1中二硫键对其细胞融合活性的影响

为了探讨风疹病毒包膜糖蛋白E1中二硫键对风疹病毒细胞融合活性的影响,在构建重组载体pBSK-SPE2E1的基础上,利用PCR定点突变与体内同源重组相结合的方法,构建了11个突变体,分别将E1外功能区的11个半胱氨酸残基突变为其它氨基酸残基,从而去除一个二硫键,利用Giemsa染色法定性检测由此引起的细胞融合情况,流式细胞术检测导入的外源DNA在细胞表面的表达效率,血吸附检测重组表达的突变体蛋白的受体识别活性。结果表明E1外功能区的10个二硫键对RV的细胞融合活性都有重要影响,任何一个二硫键的去除均导致E1的细胞融合活性丧失;其中第5和第8个半胱氨酸残基所形成的二硫键与E2和E1的相互作用有关,第3、第4和第13个半胱氨酸残基所形成的二硫键可能直接影响E1的细胞融合功能。     

Evaluation of various serum and animal protein free media for the production of a veterinary rabies vaccine in BHK-21 cells

We have carried out the adaptation of BHK-21 cells to two serum free (Ex Cell 520 and HyQ PF CHO) and three animal protein free media: Ex Cell 302, HyQ PF CHO MPS and Rencyte BHK. After a direct switch or a gradual adaptation, we have achieved BHK-21 cells growth in the following media: HyQ PF CHO, HyQ PF CHO MPS, Rencyte BHK and Ex Cell 302. The most suitable media for BHK-21 cells growth, with respect to cell density and specific growth rate, were HyQ PF CHO and HyQ PF CHO MPS. Hence we have selected these media to study cell growth and the production of rabies virus. Kinetic studies of cell growth in spinner flasks using the selected media have shown that a maximal cell density of 2x10(6) cells x ml(-1) was reached in both media. For rabies virus production, the viral titer obtained was 1.7x10(6) FFU x ml(-1) in HyQ PF CHO as well as in HyQ PF CHO MPS medium. The optimization of rabies virus production by BHK-21 cells grown in a 2 l bioreactor using the selected media, pointed to the following parameters: culture mode, perfusion rate and multiplicity of infection (MOI), as being the critical factors for achieving a good virus yield. When tested in mice, the activity of the experimental vaccines prepared on HyQ PF CHO MPS medium has shown a protective activity that meets WHO requirements.     

Adenocarcinoma cells exposed in vitro to Navelbine or Taxol increase Ep-CAM expression through a novel mechanism

Ep-CAM antigen expression was shown to vary by phase across the cell cycle. Following pretreatment of various adenocarcinoma cells in culture with clinically relevant concentrations of vinorelbine tartrate (Navelbine) or paclitaxel (Taxol), cell surface expression of Ep-CAM antigen increased by two- to ten-fold compared to that of untreated control cells and was associated with arrest of cell cycle progression and accumulation of cells in the S and G2/M phases. We demonstrated that increases in cell surface antigen expression resulted in improved biological effectiveness of the targeting antibody as measured in vitro by antibody-dependent cellular cytotoxicity and in vivo by enhanced antibody targeting to Ep-CAM-expressing xenografts in mice pretreated with Navelbine. No effect on cell cycle progression or Ep-CAM antigen expression was seen with human interferon-alpha and interferon-gamma, agents that increase gene expression of various tumor and normal antigens and may upregulate some antigens. Thus, the upregulation of cell surface Ep-CAM expression following pretreatment with G2/M blockers is through a novel mechanism involving residence time of the antigen on the cell surface. This significant increase in Ep-CAM expression appears to be tumor-specific since we saw no increase in antigen expression on normal epithelial cells. Studies to reveal relative internalization rates suggest that the increase in cell surface expression of Ep-CAM following pretreatment with G2/M blockers is a consequence of an inhibition of normal cycles of antigen endocytosis and expression on the cell surface. The present work provides a mechanism for the improved clinical efficacy of therapeutic antibodies used in combination with traditional cell cycle-specific chemotherapeutic drugs.     

Millimeter wave absorption spectra of biological samples

A solid-state computer-controlled system has been used to make swept-frequency measurements of absorption of biological specimens from 26.5 to 90.0 GHz. A wide range of samples was used, including solutions of DNA and RNA, and suspensions of BHK-21/C13 cells, Candida albicans, C krusei, and Escherichia coli. Sharp spectra reported by other workers were not observed. The strong absorbance of water (10--30 dB/mm) caused the absorbance of all aqueous preparations that we examined to have a water-like dependence on frequency. Reduction of incident power (to below 1.0 microW), elimination of modulation, and control of temperature to assure cell viability were not found to significantly alter the water-dominated absorbance. Frozen samples of BHK-21/C13 cells tested at dry ice and liquid nitrogen temperatures were found to have average insertion loss reduced to 0.2 dB/cm but still showed no reproducible peaks that could be attributed to absorption spectra. It is concluded that the special resonances reported by others are likely to be in error.     

CD46 (membrane cofactor protein) acts as a human epithelial cell receptor for internalization of opsonized uropathogenic Escherichia coli

Escherichia coli is a common urinary pathogen whose uptake into epithelial cells is mediated by attachment through type 1 fimbriae. In this study, we show by using using human urinary tract epithelial cells that maximal internalization of E. coli is achieved only when bacteria are opsonized with complement. The concentrations of complement proteins in the urine rise sufficiently during infection to allow bacterial opsonization. The complement regulatory protein, CD46 (membrane cofactor protein), acts in cohort with fimbrial adhesion to promote the uptake of pathogenic E. coli. This uptake is inhibited by RNA interference to lower the expression of CD46 and by soluble CD46 that will competitively inhibit opsonized bacteria binding to cell surface CD46. We propose that efficient internalization of uropathogenic E. coli by the human urinary tract depends on cooperation between fimbrial-mediated adhesion and C3 receptor (CD46)-ligand interaction. Complement receptor-ligand interaction could pose a new target for interrupting the cycle of reinfection due to intracellular bacteria.     

Neuroblastoma Cell Membranes: Specificity for Cell Fusion Mediated by a Temperature-Sensitive Mutant of Vesicular Stomatitis Virus

Abstract: Temperature-sensitive mutant G3 1 of vesicular stomatitis virus induces mouse neuroblastoma N-18 cells to fuse during infections that are nonpermissive for virus replication, but BHK-21 cells do not undergo the viral glycoprotein-mediated cell fusion. The viral glycoprotein was expressed at the cell surface of both N-18 and BHK-21 cells; therefore, the host cell specificity did not stem from an absence of the viral glycoprotein at the surface of BHK-21 cells. Cell fusion readily occurred between infected and uninfected N-18 cells in mixed cultures, demonstrating that the viral glycoprotein was interacting with an uninfected cell for the initial cell-cell interaction of the cell fusion. Mixing infected BHK-21 cells with uninfected N-18 cells resulted in cell fusion initiated by BHK-21 cell-synthesized viral glycoprotein, but 88% of the nucleiin polykaryocytes were N-18 nuclei. The N-18 cell fusion specificity was readily apparent when infected N-18 cells were mixed with uninfected BHK-21 cells; 98% of the nuclei in polykaryocytes were N-18 nuclei. Similar results also were obtained with mixed cultures of N-18 cells and primary astroglial cells. Thus, the viral glycoprotein synthesized in any of the cell types could initiate cell fusion, but the properties of plasma membranes of neuroblastoma cells appeared to be much more suitable for cell-cell fusion.