Expression of bioactive human interferon-gamma in transgenic rice cell suspension cultures

We investigated the possibility of producing the therapeutic recombinant cytokine, Interferon-gamma (IFN-), in transgenic rice cell (Oryza sativa, cultivar TNG67) suspension cultures. We tested expression of two vector constructs, each harboring an Amy3 leader peptide and a C-terminus His 6 tag fused to a human IFN- cDNA, one driven by a sucrose-starvation inducible promoter (rice Amy3 promoter) and the other by a constitutive maize ubiquitin promoter, in rice cell suspensions, introduced via Agrobacterium tumefaciens. There was a significant difference in the amounts of recombinant IFN- protein produced by the Ups and Amy cell lines, as cytosolic and secretory proteins respectively. Immunological analysis of IFN- recombinant protein conferred a dose-dependent anti-dengue virus activity in human A549 cells, similar to the commercial product. We discuss the attractive attributes of using rice cell suspension system for the expression of therapeutic recombinant IFN-.     

Establishment and therapeutic use of human embryonic stem cell lines

Embryonic stem (ES) cell lines, which are derived from the inner cell mass of blastocysts, proliferate indefinitely in vitro, retaining their potency to differentiate into various cell types derived from all of the three embryonic germ layers: the ectoderm, mesoderm and endoderm. Establishment of human ES cell lines in 1998 has indicated the great potential of ES cells for applications in medical research and other purposes such as cell transplantation therapy. Careful assessment of safety and effectiveness using proper animal models is required before such therapies can be attempted on human patients. Monkey ES cell lines provide valuable models for such research.     

Immortalized renal proximal and collecting duct cell lines derived from transgenic mice harboring L-type pyruvate kinase promoters as tools for pharmacological and toxicological studies

Targeted oncogenesis in transgenic mice, where an oncogene is placed under the control of the regulatory sequences of a cell-specific gene, has been used to derive lines of differentiated kidney epithelial cells derived from proximal or distal tubules or from the collecting duct. These renal cell lines were obtained from kidneys of transgenic mice harboring the large-T and little-t antigens placed under the control of regulatory sequences of the L-type pyruvate kinase gene. This review summarizes the main properties of these differentiated cell lines, which are usefulex vivo cell systems for pharmacological and toxicological studies. This revised version was published online in August 2006 with corrections to the Cover Date.     

Selective cytotoxicity of marine algae extracts to several human leukemic cell lines

Extracts from 8 species of marine algae which showed selective cytotoxicity in our previous screening program, were further examined for cytotoxic spectra to five human leukemic cell lines. The extract from a red alga, Amphiroa zonata exhibited strong cytotoxicity to all human leukemic cell lines tested and murine leukemic cells L1210 at the final concentrations from 15 to 375 μg ml−¹. Then the cytotoxicity was not found in normal human fibroblast HDF and murine normal cells NIH-3T3. The active extract fraction from this alga was soluble in higher polar organic solvents and water and heat-stable. The extract from a brown alga Dilophus okamurae with weak selective cytotoxic activity to L1210 cells exhibited not only strong cytotoxicity to L1210, but also to human leukemic cells, HL60 and MOLT-4 at 50 μg ml-¹. While, the extract from a green alga, Cladophoropsis vaucheriaeformis with most selective cytotoxic activity, did not show cytotoxicity to any human leukemic cell lines tested at 50 μg ml-¹. However, this extract showed strong cytotoxicity to two human leukemic cell lines and NIH-3T3 at 100 μg ml−¹. Thus, it was considered that a red alga, Amphiroa zonata might be suitable natural source for development of anti-cancer agents without side-effect. This revised version was published online in June 2006 with corrections to the Cover Date.     

The H-2KbtsA58 transgenic mouse: A new tool for the rapid generation of novel cell lines

The ability to generate expanded populations of individual cell types able to undergo normal differentiationin vitro andin vivo is of critical importance in the investigation of the mechanisms that underly differentiation and in studies on the use of cell transplantation to repair damaged tissues. This review discusses the development of a strain of transgenic mice that allows the direct derivation of conditionally immortal cell lines from a variety of tissues, simply by dissociation of the tissue of interest and growth of cells in appropriate conditions. In these mice the tsA58 mutant of SV40 large T antigen is controlled by the interferon-inducible Class I antigen promoter. Cells can be grown for extended periodsin vitro simply by growing them at 33°C in the presence of interferon, while still retaining the capacity to undergo normal differentiationin vivo andin vitro. In addition, it appears that cell lines expressing mutant phenotypes can readily be generated by preparing cultures from appropriate offspring of matings between H-2K^btsA58 transgenic mice and mutant mice of interest.     

High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) has been previously produced in tobacco cell suspension cultures. However, the amount of hGM-CSF accumulated in the culture medium dropped quickly from its maximum of 150 microg/L at 5 d after incubation. To overcome this problem, we sought an expression system in which heterologous gene expression could be induced at high levels. We selected a rice amylase expression system in which the promoter Ramy3D is induced to express recombinant protein by sucrose starvation. This induction system was found to give good yield of recombinant hGM-CSF in transgenic rice cell suspension culture and protease activity of this culture medium was low compared to that of tobacco culture system.     

氯吡格雷对大鼠肝药物酶CYP3A4 和CYP1A2 表达的影响

目的：氯吡格雷主要由CYP3A4 催化使其激活,CYP1A2 也参与氯吡格雷活化。关于氯吡格雷对肝微粒体酶的影响国内外 文献报道不多,因此本实验通过检测肝细胞色素氧化酶CYP3A4 和CYP1A2 的表达,探讨氯吡格雷对大鼠肝药物酶的影 响。方法：生理盐水为对照组,氯吡格雷设高、中、低三个剂量组（27,13.5,6.75mg/kg/d）,雄性健康大鼠连续灌胃给药7天,脱臼处 死,取肝组织,通过western blot法检测大鼠肝脏CYP3A4 和CYP1A2 蛋白表达情况。结果：1）、氯吡格雷抑制大鼠CYP3A4 蛋白 表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYP3A4 蛋白表达量降低（P<0.05）;氯吡格雷低中高剂量组间进行比较,大 鼠CYP3A4 蛋白表达量呈梯度减少（P<0.05）;2）、氯吡格雷抑制大鼠CYP1A2 蛋白表达,氯吡格雷高中低剂量组分别比生理盐水 组大鼠CYP1A2 蛋白表达量降低（P<0.05）,氯吡格雷低中高剂量组间进行比较,大鼠CYP1A2 蛋白表达量呈梯度减少（P<0.05）。 结论：氯吡格雷使肝细胞色素氧化酶CYP3A4 和CYP1A2 的表达量减少,因此氯吡格雷高、中、低3 个剂量组均不同程度的抑制 大鼠肝脏CYP3A4 和CYP1A2 的表达,提示当氯吡格雷与某些主要经CYP3A4 和CYP1A2 代谢的药物合用时,发生代谢性相关 作用的可能性大。     

氯吡格雷对大鼠肝药物酶CYP3A4和CYP1A2表达的影响

目的：氯吡格雷主要由CYP3A4催化使其激活,CYPlA2也参与氯吡格雷活化。关于氯吡格雷对肝微粒体酶的影响国内外文献报道不多,因此本实验通过检测肝细胞色素氧化酶CYP3A4和CYPlA2的表达,探讨氯吡格雷对大鼠肝药物酶的影响。方法：生理盐水为对照组,氯吡格雷设高、中、低三个剂量组（27,13．5,6．75mg／kg／d）,雄性健康大鼠连续灌胃给药7天,脱臼处死,取肝组织,通过westernblot法检测大鼠肝脏CYP3A4和CYPlA2蛋白表达情况。结果：1）、氯吡格雷抑制大鼠CYP3A4蛋白表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYP3A4蛋白表达量降低（P〈0．05）;氯吡格雷低中高剂量组间进行比较,大鼠CYP3A4蛋白表达量呈梯度减少（P〈0．05）;2）、氯吡格雷抑制大鼠CYPlA2蛋白表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYPlA2蛋白表达量降低（P〈0．05）,氯吡格雷低中高剂量组间进行比较,大鼠CYPlA2蛋白表达量呈梯度减少（P〈0．05）。结论：氯吡格雷使肝细胞色素氧化酶CYP3A4和CYPlA2的表达量减少,因此氯吡格雷高、中、低3个剂量组均不同程度的抑制大鼠肝脏CYP3A4和CYPlA2的表达,提示当氯吡格雷与某些主要经CYP3A4和CYPlA2代谢的药物合用时,发生代谢性相关作用的可能性大。     

The human apoE7 and apoE4 transgenic mice models

To scrutinize the disorders caused by human mutant apoE7/apoE4, human apoE4 and E7 transgenic mice were established with microinjection technique to examine molecular genetic phenomenain vivo. The integration and expression of h-apoE mutant genes in transgenic mice were determined with Southern blot, Northern blot and ELISA. The current studies indicated that the transgenes and the phenotypes regarding expression of transgenes could be transmitted stably in transgenic lines. The levels of serum lipid in transgenic mice showed the characteristics of hyperlipidemia. Besides, behavior tests demonstrated the degeneration of learning and memory in transgenic mice. Short life span was observed in 2 transgenic lines. After fed with high lipid food high serum lipid was found both in normal and transgenic mice, but their mechanism regulating lipid metabolism was different. It was also verified that the human apoE mutants located at either N-terminal or C-terminal had the same pathogenesis regarding disorders of lipid metabolism in murine.     

Growth limitation in hybridoma cell cultures: The role of inhibitory or toxic metabolites

Hybridoma cells usually grow to fairly low cell densities in batch cultures (1–3×10⁶ cells/ml). The reason for this is either that essential nutritional components of the medium are consumed, or that the cells produce some kind of inhibitory or toxic metabolite. This investigation presents evidence for the latter. Spent medium from cultures of hybridoma cells did not support growth of cells at lower cell densities (1–3×10⁵ cells/ml). The ability to support cell growth could not be restored by adding additional serum, energy sources (glucose, pyruvate) or L-glutamine. Furthermore, the consumption of amino acids could not account for this growth inhibition. On the contrary, the spent medium contained a substance that inhibited cell growth. This substance or metabolite was found in a fraction eluted from a gel filtration column when spent medium was applied to the column. This substance was found in the spent medium from all hybridoma and myeloma cell lines that were tested. The molecular weight of the substance was about 5 kD. Spent medium from two hybridoma cell lines also contained a substance that was eluted in the same fraction as albumin (67 kD). It is likely that this (or these) substance(s) is responsible for the growth limitation in hybridoma cell cultures.Abbreviations PBS phosphate buffered saline     

The effect of cell synchronization upon the detection of T and B lymphoid cell receptors on two continuous lymphoid cell lines

Summary In the present study, the effect of the cell synchronization on the detection of T and B cell surface markers of two continuous lines of lymphoid cells (FL-74 and CT45-S) was examined. Suspension cultures were synchronized by deprivation of isoleucine and surface markers were quantitated by T rosette formation with guinea pig erythrocytes (E) and B rosette formation with an erythrocyte-antibody-complement (EAC) complex. After 24 hr, cells were resuspended in complete culture medium. Virtually 100% of FL-74 cells expressed the T cell marker at time 0, with a progressive decline to 80% at saturation density. A bell-shaped curve for expression of the EAC marker on CT45-S cells was seen with maximum expression in the logarithmic phase of the growth cycle. Spent culture medium was examined for the presence of free soluble receptor. Preincubation of E and EAC in appropriate old medium resulted in 42% inhibition of E rosettes and 42% inhibition of EAC rosettes with FL-74 and CT45-S cells, respectively. Thus quantitation of lymphocyte subpopulations as B, T or null cells with these cellular markers may be influenced by the age of the cell examined, phase of the cell cycle and the amount of free receptor present in the surrounding medium. This research was supported in part by contract NO1 CP 5-3571 with the Virus Cancer Program of the NCI, NIH, PHS grant no. 2 RO1 A1-09022-07, Allergy and Infectious Diseases NIH, PHS and The State of Ohio Canine Research Funds.     

The human apoE7 and apoE4 transgenic mice models

To scrutinize the disorders caused by human mutant apoE7/apoE4, human apoE4 and E7 transgenic mice were established with microinjection technique to examine molecular genetic phenomena in vivo. The integration and expression of h-apoE mutant genes in transgenic mice were determined with Southern blot, Northern blot and ELISA. The current studies indicated that the transgenes and the phenotypes regarding expression of transgenes could be transmitted stably in transgenic lines. The levels of serum lipid in transgenic mice showed the characteristics of hyperlipidemia. Besides, behavior tests demonstrated the degeneration of learning and memory in transgenic mice. Short life span was observed in 2 transgenic lines. After fed with high lipid food high serum lipid was found both in normal and transgenic mice, but their mechanism regulating lipid metabolism was different. It was also verified that the human apoE mutants located at either N-terminal or C-terminal had the same pathogenesis regarding disorders of     

Effects of Oral Anorexiant Sibutramine on the Expression of Cytochromes P450s in Human Hepatocytes and Cancer Cell Lines

Sibutramine is a serotonin–norepine‐phrine reuptake inhibitor that was used for weight‐loss management in obese patients. Even though it was officially withdrawn from the market in 2010, it is still present in some tainted weight‐loss pills (as reported by US Food and Drug Administration). Thus, it is still reasonable to study the effects of this compound. The aim of this work was to investigate the potential of sibutramine to induce CYP1A1/CY3A4 in human cancer cell lines and CYP1A1/2, CYP2A6, CYP2B6, and CYP3A4 in human hepatocytes, a competent model of metabolically active cells. The levels of mRNA and protein of CYP1A1/1A2/3A4/2A6/2B6 were compared with the typical inducers, 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) and rifampicin (RIF) for CYP1A1/2 and for other CYPs, respectively. The mRNA and protein levels of all genes in either cancer cell lines or human hepatocytes were induced when treated with typical inducers but not with sibutramine.     

Cryopreserved human hepatocytes as alternative in vitro model for cytochrome P450 induction studies

Induction of cytochrome P450 (CYP) by drugs is one of major concerns for drug-drug interactions. Thus, the assessment of CYP induction by novel compounds is a vital component in the drug discovery and development processes. Primary human hepatocytes are the preferred in vitro model for predicting CYP induction in vivo. However, their use is hampered by the erratic supply of human tissue and donor-to-donor variability. Although cryopreserved hepatocytes have been recommended for short-term applications in suspension, their use in studies on induction of enzyme activity has been limited because of poor attachment and response to enzyme inducers. In this study, we report culture conditions that allowed the attachment of cryopreserved human hepatocytes and responsiveness to CYP inducers. We evaluated the inducibility of CYP1A1/2 and CYP3A4 enzymes in cryopreserved hepatocytes from three human donors. Cryopreserved human hepatocytes were cultured in serum-free medium for 4 d. They exhibited normal morphology and measurable viability as evaluated by the reduction of tetrazolium salts (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) by cellular dehydrogenases. Treatment with beta-naphthoflavone (10 microM) for 3 d increased ethoxyresorufin-O-deethylase activity (CYP1A1/2) by 6- to 11-fold over untreated cultures and increased CYP1A2 messenger ribonucleic acid (mRNA) expression by three- to eightfold. Similarly, treatment of cryopreserved human hepatocytes with rifampicin (25 microM) for 3 d increased testosterone 6 beta-hydroxylase activity (CYP3A4) by five- to eightfold over untreated cultures and increased CYP3A4 mRNA expression by four- to eightfold. The results suggest that cryopreserved human hepatocytes can be a suitable in vitro model for evaluating xenobiotics as inducers of CYP1A1/2 and CYP3A4 enzymes.     

Development of an oxygenation culture method for activating the liver-specific functions of HepG2 cells utilizing a collagen vitrigel membrane chamber

We recently developed a collagen vitrigel membrane (CVM) chamber possessing a scaffold composed of high-density collagen fibrils. In this study, we first confirmed that the advantage of CVM chamber in comparison to the traditional culture chamber with porous polyethylene terephthalate membrane is to preserve a culture medium poured in its inside even though the under side is not a liquid phase but solid and gas phases. Subsequently, we designed three different culture systems to grow HepG2 cells in a culture medium (liquid phase) on the CVM which the under side is a culture medium, a plastic surface (solid phase) or 5 % CO₂ in air (gas phase) and aimed to develop a brief culture method useful for activating the liver-specific functions and analyzing the pharmacokinetics of fluorescein diacetate. HepG2 cells cultured for 2 days on the liquid–solid interface and subsequently for 1 day on the liquid–gas interface represented excellent cell viability and morphology in comparison to the others, and remarkably improved albumin secretion and urea synthesis to almost the same level of freshly isolated human hepatocytes and CYP3A4 activity to about half the level of differentiated HepaRG cells. Also, the cells rapidly absorbed fluorescein diacetate, distributed it in cytosol, metabolized it into fluorescein, and speedily excreted fluorescein into both bile canaliculus-like networks and extracellular solution. These data suggest that hepatic structure and functions of monolayered HepG2 cells can be induced within a day after the oxygenation from beneath the CVM.     

High expression of a human lactoferrin in transgenic tobacco cell cultures

Transgenic Nicotiana tabacum cell lines were developed expressing the human lactoferrin gene driven by the oxidative stress-inducible peroxidase (SWPA2) promoter. Western blot analysis showed the accumulation of both the full-length human lactoferrin protein as well as a immuno-reactive truncated fragment. Accumulation of human lactoferrin as monitored by ELISA increased proportionally to cell growth and reached a maximal (up to 4.3% of total soluble proteins) at the stationary phase of growth. Protein extracts from transgenic tobacco cells exhibited antibacterial activity.