Use of Vero cell line to verify the biodetoxification efficiency of castor bean waste

Ricin is a toxic protein present in castor bean seeds (Ricinus communis). A toxic residue named castor bean waste is generated during biodiesel production process, such as that developed by PETROBRAS (the national petroleum company of Brazil). Solid-state fermentation (SSF) was used to detoxify castor bean waste through the Penicillium simplicissimum growth. After 24 h of fungal growth, the ricin was no longer identified by Sephadex G-50 gel chromatography. In order to verify the biological activity of ricin after several treatment stages, an in vitro assay using Vero cell line was carried out. Through this methodology, it was verified that after 24 and 48 h of treatment, the cell culture showed slightly growth inhibition. The waste was completely detoxified only after 72 h of fungal growth. This fact shows that an in vitro assay is important to verify the real efficiency of detoxification. Moreover, a relationship between the fungal protease production and the waste detoxification was observed.     

稳定表达貉犬瘟热病毒细胞受体SLAM的Vero细胞系的建立及应用

[目的]信号淋巴激活分子(SLAM又称CD150)为犬瘟热病毒(Canine distemper virus,CDV)的细胞受体.本研究目的在于建立稳定表达犬瘟热易感动物貉SLAM基因的Vero细胞系,用于犬瘟热诊断及CDV强毒株的快速分离.[方法]从重组质粒pMD-18-T-rSLAM扩增rSLAM基因编码区,通过分子克隆技术构建真核表达质粒pIRES2-EGFP-rSLAMhis.将该质粒转染Vero细胞后,经EGFP荧光观察、G418抗性压力和单细胞克隆化及RT-PCR筛选表达rSLAM阳性细胞系.应用获得细胞系对临床犬瘟热病例进行病毒分离,对分离得到CDV进行RT-PCR鉴定及动物回归试验.[结果]经过RT-PCR和免疫组化试验证实,本实验筛选获得一株能稳定表达rSLAM的Vero细胞系——Vero-rSLAM.该细胞系接种CDV阳性样品36-48h即可产生典型CDV致细胞融合病变,而其亲本Vero细胞接种至6d均无可见细胞病变出现.应用Vero-rSLAM细胞系从狐狸、貉犬瘟热阳性病料中分离获得了3株犬瘟热病毒.其中犬瘟热病毒LN(10) f1株对易感狐狸、貉均可产生致死性感染.[结论]成功建立了稳定表达rSLAM的Vero细胞系,用它分离的CDV对本动物保持较强的毒力.     

Secretory production system of bionanocapsules using a stably transfected insect cell line

Bionanocapsules (BNCs) are hollow nanoscale particles composed of L protein of the hepatitis B virus surface antigen that represent specific affinity for human hepatocytes. BNCs can transfer genes and drugs into human hepatocytes efficiently and specifically. BNC can be expressed in yeast cells. In this study, we developed a new L particle production system using a stably transfected insect cell line. For this purpose, we established a host–vector system using the Trichoplusia ni insect cell line. L particles were efficiently secreted by the overexpression of the L protein, which was fused to the secretion signal peptide. The concentration of L particles was reached approximately 1.7 μg/ml in 5 days during cultivation in a serum-free medium without antibiotic selective pressure. The production of L particles was maintained for at least 75 days. The secretory production of L particles facilitated their easy purification by chromatography. Furthermore, it was demonstrated that purified L particles can transfect only human hepatocytes. Therefore, an insect cell expression system is an attractive tool for the production of BNC.     

Use of BRL-conditioned medium in combination with feeder layers to isolate a diploid embryonal stem cell line

Summary The derivation of a karyotypically normal embryonal stem (ES) cell line, E14, from inner cell masses (ICMs) isolated by immunosurgery from 129/Ola late mouse blastocysts is described. Disaggregated ICMs were cultured on mitotically-arrested fibroblast feeder layers in droplets of medium conditioned with Buffalo rat liver (BRL) cells under oil. BRL-conditioned medium inhibits the differentiation of established embryonal carcinoma (EC) and ES cell lines which can be maintained indefinitely in the complete absence of feeder cells (Smith and Hooper 1987). At clonal densities, however, a combination of BRL-conditioned medium and a feeder layer was most effective in preventing the differentiation of E14 cells. This effect was less pronounced at higher passage suggesting it may be particularly important to use a combination in the early stages of isolation. Once established, E14 has been maintained in BRL-conditioned medium alone. In non-conditioned medium on agarose, E14 cells formed embryoid bodies which when allowed to reattach differentiated into a wide variety of tissues. An HPRT-deficient sub line of E14, E14TG2a, has been demonstrated to form germline chimaeras with high efficiency after injection into blastocysts (Hooper et al. 1987). The modifications to the ES cell isolation procedure described here may improve the efficiency with which karyotypically normal lines can be derived.     

Prediction of naphthalene bioaccumulation using an adipocyte cell line model

A long-term goal of this research is to develop an in vitro model to study the metabolism, distribution, and fate of chemicals or pharmaceuticals in animals and humans. An important component of such a system is an in vitro model to study bioaccumulation of specific chemicals in adipose tissue. Due to the difficulties in maintaining primary adipocytes in culture and conducting reproducible experiments, transformed adipocyte cell lines have been used as an alternative. In this paper, several rodent preadipocyte cell lines (3T3-L1, 3T3-F442A, and TA1 cells) that differentiate into adipocytes when exposed to the appropriate stimuli are tested as an investigative tool to study naphthalene accumulation. The in vitro model is tested by comparison of its performance to that of primary adipocytes. All the experimental evidence supports the hypothesis that naphthalene accumulation is primarily dependent on the level of intracellular lipid. Furthermore, the level of naphthalene bioaccumulation is linearly correlated with the amount of triglyceride content with the slope of 37.7 +/- 0.5 microg of naphthalene/(mg of triglyceride). Indomethacin/dexamethasone/insulin are shown to be more effective in promoting preadipocyte differentiation than methylisobutylxanthine/dexamethasone/insulin. Additionally, external factors, such as the presence of albumin and serum in the medium, affect the cellular naphthalene uptake by decreasing the amount of naphthalene transported into fat cells. Among the three cell lines tested, 3T3-L1 adipocytes accumulated the highest intracellular lipid and, hence, yielded the highest level of naphthalene accumulation. Its ability to accumulate naphthalene is comparable to that of primary adipocytes. The 3T3-L1 adipocyte model is appropriate for studying the bioaccumulation of xenobiotics that are aromatic hydrocarbons.     

Checking graphite and stainless anodes with an experimental model of marine microbial fuel cell

A procedure was proposed to mimic marine microbial fuel cell (MFC) in liquid phase. A graphite anode and a stainless steel cathode which have been proven, separately, to be efficient in MFC were investigated. A closed anodic compartment was inoculated with sediments, filled with deoxygenated seawater and fed with milk to recover the sediment's sulphide concentration. A stainless steel cathode, immersed in aerated seawater, used the marine biofilm formed on its surface to catalyze oxygen reduction. The cell implemented with a 0.02m(2)-graphite anode supplied around 0.10W/m(2) for 45 days. A power of 0.02W/m(2) was obtained after the anode replacement by a 0.06m(2)-stainless steel electrode. The cell lost its capacity to make a motor turn after one day of operation, but recovered its full efficiency after a few days in open circuit. The evolution of the kinetic properties of stainless steel was identified as responsible for the power limitation.     

Expression and regulation of Q8 b in a transfected cell line

RNase protection experiments showed that Q8 ^b was actively transcribed in a stably transfected cell line. Moreover, Q8 ^b responded to interferon- (IFN-) treatment with increased levels of mRNA expression. Thus Q8 ^b demonstrates a regulatory response to IFN- characteristic of many other class I genes. Cell surface expression of a Q8^b product could also be detected by flow cytometric analysis with the Qa-2-specific monoclonal antibody D3.262. The expression of the Q8^b cell surface product increased only slightly after cells were treated with IFN-. The Q8^b cell surface product was not sensitive to cleavage by phosphatidylinositol-phospholipase C. These results suggest that the Q8^b product, unlike the predominant forms of Qa-2-bearing molecules, is not anchored via phosphatidylinositol to the cell membrane. These results also suggest that Q8 ^b has the potential to contribute to the Qa-2 phenotype in vivo. Address correspondence and offprint requests to: L. Flaherty.     

In silico prediction and experimental validation of siRNAs targeting ORF1ab of MERS-CoV in Vero cell line

The Middle East Respiratory Syndrome Coronavirus is well known to cause respiratory syndrome and this virus was identified and isolated for the first time from Jeddah, Saudi Arabia in 2012 from infected patient. In this report, we have conducted the in-silico prediction, designing and evaluation of siRNAs targeting Middle East Respiratory Syndrome Coronavirus orf1ab gene to inhibit the virus replication. By using bioinformatics software, total twenty-one functional, off-target reduced siRNA were selected from four hundred and sixty-two siRNAs based on their greater potency and specificity. We have evaluated only seven siRNAs to analyze their performance and efficacy as antivirals by reverse transfection approach in Vero cells. There was no cytotoxicity of siRNAs at various concentrations was observed in Vero cells. Based on the real-time PCR results, better inhibition of viral replication was observed in the siRNA-1 and 4 as compared to other siRNAs. The results generated from this work provided suitable information about the efficacy of siRNAs which encouraged us to further evaluate the remaining siRNAs to determine their inhibitory effect on the virus replication. We concluded that the insilico prediction and designing resulted in the screening of potential siRNAs with better efficiency, and strength. This can be used to develop oligonucleotide-based antiviral therapeutics against MERS-CoV in the near future.     

Establishment of a lipid accumulation model in an insect cell line

The study of adipocyte differentiation and lipid accumulation in insects has been limited by the lack of a system suitable for analysis of molecular mechanisms. Here, we describe the establishment of a model system of lipid accumulation in BmN4 cells, which are derived from silkworm ovary. In BmN4 cells, dexamethasone treatment induced accumulation of lipid, suppressed cellular proliferation, and caused the cells to form aggregates. We isolated the Bombyx mori fatty acid binding protein 1 gene (BmFABP1), which is the silkworm homologue of mouse Fabp4 (aP2), a marker of adipocyte differentiation in mammals. BmFABP1 expression was increased by dexamethasone treatment. We also isolated the BmFABP1 promoter, and found that it was activated by a combination of drugs that included dexamethasone. The demonstration of dexamethasone-stimulated lipid accumulation and BmFABP1 expression in BmN4 cells provides a useful model of inducible adipogenesis. This system should be valuable for investigation of the molecular mechanisms of fat body formation, adipocyte differentiation, and lipid accumulation in the silkworm and other Lepidopteran insects.     

Comparative susceptibility of a canine cell line and bluetongue virus susceptible cell lines to a bluetongue virus isolate pathogenic for dogs

Summary Recently, bluetongue virus (BLU) serotype 11 was detected in diseased dogs that had been inoculated with live attenuated vaccine contaminated with this serotype of bluetongue virus (Akita et al., 1994). For various laboratory tests, BLU can be propagated in different cell cultures. No information was found in the literature about the possibility of propagating this virus in canine cells. To determine whether the BLU isolate from the contaminated canine vaccine (BLU-vac) is unique in its ability to replicate in canine cells, this virus was studied in parallel with U.S. prototype strains of BLU (serotypes 2, 10, 11, 13, and 17), in hamster lung (HmLu-1) and canine kidney (MDCK) cell cultures. In HmLu-1 cell cultures, the BLU-vac produced cytopathic effect (CPE) of the same type as the U.S. prototype BLU strains by 4 to 6 d postinoculation. In MDCK cell cultures, all of the BLU strains tested were able to replicate but did not produce CPE. The BLU-inoculated MDCK cells became persistently infected, and these cultures continued to produce infectious BLU even after six serial passages over 2 1/2 mo. In none of these cultures was CPE observed. In mixed cultures containing both HmLu-1 and MDCK cells, CPE first affected the HmLu-1 islands; subsequently, CPE spread also to the areas with MDCK cells. The silent persistent infection of the MDCK cells with BLU indicates that more stringent screening of the cells used in the production of live vaccines for various contaminating viruses is necessary.     

Use of an s-triazine nitrogen source to select for and isolate a recombinant chlorobenzoate-degrading Pseudomonas

Abstract A novel mating system, which uses the s -triazine nitrogen source (cyanuric acid, CA) as a selective nutritional marker, was developed to evaluate mobility and facilitate identification of chlorobenzoate (CBA) degradation genes in Pseudomonas aeruginosa strain JB2. Matings were done between strain JB2, which is unable to grow with CA, and a CA-utilizer Pseudomonas sp. strain D on 2-CBA-CA and 3-CBA-CA media. Isolates were recovered only from the 2-CBA-CA and were all identified as strain D derivatives. Subtractive hybridization analysis of genomic DNAs from the parental strains and a selected isolate, Pseudomonas sp. strain JPL, was used to determine that the latter organism had acquired approx. 16.1 kb of DNA from strain JB2.     

Use of ATP to characterize biomass viability in freely suspended and immobilized cell bioreactors

This work describes investigations into the viability of cells growing on 3,4-dichloroaniline (34DCA). Two bioreactors are employed for microbial growth, a continuous stirred tank (CST) bioreactor with a 2-L working volume, and a three-phase air lift (TPAL) bioreactor with a 3-L working volume. Experiments have been performed at several dilution rates between 0.027 and 0.115 h(-1) in the CST bioreactor and between 0.111 and 0.500 h(-1) in the TPAL bioreactor. The specific ATP concentration was calculated at each dilution rate in the suspended biomass in both bioreactors as well as in the immobilized biomass in the TPAL bioreactor. The ATP was extracted from the cells using boiling tris-EDTA buffer (pH 7.75), and the quantity determined using a firefly (bioluminescence) technique. The cultures were inspected under an electron microscope to monitor compositional changes. Results from the CST bioreactor showed that the biomass-specific ATP concentration increases from 0.44 to 1.86 mg ATP g(-1) dry weight (dw) as dilution rate increases from 0.027 to 0.115 h(-1). At this upper dilution rate the cells were washed out. The specific ATP concentration reached a limiting average value of 1.73 mg ATP g(-1) dw, which is assumed to be the quantity of ATP in 100% viable biomass. In the TPAL bioreactor, the ATP level increased with dilution rate in both the immobilized and suspended biomass. The specific ATP concentration in the immobilized biomass increased from approximately 0.051 mg ATP g(-1) dw at dilution rates between 0.111 and 0.200 h(-1) to approximately 0.119 mg ATP g(-1) dw at dilution rates between 0.300 and 0.500 h(-1). This indicates that the immobilized biomass contained a viable cell fraction of around 5%. Based on these results, kinetic data for freely suspended cells should not be applied to the modeling of immobilized cell systems on the assumption that immobilized biomass is 100% viable. (c) 1993 John Wiley & Sons, Inc.     

Use of a new strategy to isolate and characterize 436 Drosophila cDNA clones corresponding to RNAs detected in adult heads but not in early embryos

We describe a new strategy for producing tissue-specific cDNA libraries and subsequently identifying tissue-specific clones. This method was used to screen for cDNA clones corresponding to RNAs expressed in the Drosophila head that cannot be detected in the early embryo. RNA blots were used to assess the spatial and temporal patterns of expression of these RNAs. The ensemble of 436 head-not-embryo clones identified roughly 700 distinct RNAs that are differentially expressed in the Drosophila head. The RNA expression patterns can be classified into five major categories. it is argued that this ensemble of clones represents a large fraction of all genes differentially expressed in the adult head, but not detected in the early embryo. Many of these genes are likely to encode eye- and nervous system-specific products.     

Use of inhibitors to characterize intermediates in the processing of N-glycans synthesized by insect cells: a metabolic study with Sf9 cell line.

The most frequent type of N-glycan synthesized by lepidopteran Sf9 cells appears to be fucosylated Man3GlcNAc2,and this has been a limitation for a large scale production and utilization of therapeutic glycoproteins in cultured insect cells. The current knowledge of the protein glycosylation pathway derived from structural studies on recombinant glyco-proteins expressed by using baculovirus vectors. In this work we provide more direct evidence for the sequential events occurring in the processing of endogenous N-glycoproteins of noninfected Sf9 cells. By metabolic labeling with radioactive mannose, we characterized the glycan structures which accumulated in the presence of processing inhibitors (castanospermine and swainsonine) and in the presence of an intracellular trafficking inhibitor (monensin). We thus demonstrated that from the glycan precursor Glc3Man9GlcNAc2 to GlcNAcMan5(Fuc)GlcNAc2 intermediate, the processing pathway in Sf9 cells paralleled the one demonstrated in mammalian cells. By using monensin, we demonstrated the formation of Man3(Fuc)GlcNAc2 from GlcNAcMan3(Fuc)GlcNAc2, a reaction which has not been described in mammalian cells. Our results support the idea that the hexosaminidase activity is of physiological relevance to the glycosylation pathway and is Golgi located.     

Optimization of an electroporation protocol using the K562 cell line as a model: role of cell cycle phase and cytoplasmic DNAses

The improvement of gene therapy protocols is intimately related to the establishment of efficient gene transfer methods. Electroporation has been increasingly employed in in vitro and in vivo protocols, and much attention has been given to increasing its transfection potential. The method is based on the application of an electric field of short duration and high voltage to the cells, forming reversible pores through which molecules can enter the cell. In this work, we describe the optimization of a protocol for the electroporation of K562 cells involving the combination of electric field, resistance and capacitance values. Using RPMI 1640 as pulsing buffer and 30 μg of pEGFP-N1 plasmid, 875 V cm⁻¹, 500 μF and infinite resistance, we achieved transfection rates of 82.41 ± 3.03%, with 62.89 ± 2.93% cell viability, values higher than those reported in the literature. Analyzing cell cycle after electroporation, with three different electric field conditions, we observed that in a heterogeneous population of cells, viability of G₁ cells is less affected by electroporation than that of cells in late S and G₂/M phases. We also observed that efficiency of electroporation can be improved using the DNAse inhibitor Zn, immediately after the pulse. These results can represent a significant improvement of current methods of electroporation of animal and plant cells.     

Determination of HSV-1 UL5 and UL29 gene copy numbers in an HSV complementing Vero cell line

The genetic stability of transgenes is a critical characteristic used to assess constructed cell lines used for vaccine production. The evaluation of gene copy numbers by a qPCR method, is one of the most common approaches used to assess the consistency of transgenes in a constructed cell line. The cell line AV529-19 is a Vero-based cell line specifically engineered to express the HSV-1 UL5 and UL29 open reading frames. AV529-19 is used to support the replication of a defective HSV-2 viral candidate vaccine called HSV529. To assess the genetic stability of the UL5 and UL29 transgenes in AV529-19 cells, a digital PCR-based approach was developed. During characterization of the test method, the specificity, accuracy, and intermediate precision of the assay was investigated based on regulatory guidelines. The developed assay was used to monitor the stability of the transgenes in the manufactured AV529-19 cell lines by comparison of transgene copy numbers in the master cell bank (MCB) with their copy numbers in the extended cell bank (ECB). Results showed that the UL29 and UL5 transgenes are stable in that there are one and three copies of the UL29 and UL5 genes, respectively, per cell in both the AV529-19 MCB and ECB.