L-arginine-dependent nitric oxide production of a murine macrophage-like RAW 264.7 cell line stimulated with Porphyromonas gingivalis lipopolysaccharide

The aim of this study was to determine nitric oxide (NO) production of a murine macrophage cell line (RAW 264.7 cells) when stimulated with Porphyromonas gingivalis lipopolysaccharides (Pg-LPS). RAW264.7 cells were incubated with i) various concentrations of Pg-LPS or Salmonella typhosa LPS (St-LPS), ii) Pg-LPS with or without L-arginine and/or N-G-monomethyl-L-arginine (NMMA), an arginine analog or iii) Pg-LPS and interferon-gamma (IFN-gamma) with or without anti-IFN-gamma antibodies or interleukin-10 (IL-10). Tissue culture supernatants were assayed for NO levels after 24 h in culture. NO was not observed in tissue culture supernatants of RAW 264.7 cells following stimulation with Pg-LPS, but was observed after stimulation with St-LPS. Exogenous L-arginine restored the ability of Pg-LPS to induce NO production; however, the increase in NO levels of cells stimulated with Pg-LPS with exogenous L-arginine was abolished by NMMA. IFN-gamma induced independent NO production by Pg-LPS-stimulated macrophages and this stimulatory effect of IFN-gamma could be completely suppressed by anti-IFN-gamma antibodies and IL-10. These results suggest that Pg-LPS is able to stimulate NO production in the RAW264.7 macrophage cell model in an L-arginine-dependent mechanism which is itself independent of the action of IFN-gamma.

Arginase activity in a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans

Aims: The aim of the present study was to determine the role of cyclic adenosine monophosphate (cAMP) on arginase activity in a murine macrophage cell line (RAW264.7 cells) stimulated with lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans. Materials and methods: The cells were treated with A. actinomycetemcomitans LPS for 24 h. The effects of SQ22536 (an adenylyl cyclase inhibitor), ODQ (a guanylyl cyclase inhibitor), dibutyryl cAMP (a cAMP analog), 8-bromo cyclic guanosine monophosphate (a cGMP analog), forskolin (an adenylyl cylase activator), and cycloheximide (a protein synthesis inhibitor) on arginase activity in A. actinomycetemcomitans LPS-stimulated RAW264.7 cells were also determined. Arginase activity was assessed in LPS-stimulated cells in the presence of 3-isobutyl-1-methylxanthine (IBMX), siguazodan and rolipram [phosphodiesterase (PDE) inhibitors] as well as KT5720 [a protein kinase A (PKA) inhibitor]. Results: Arginase activity in A. actinomycetemcomitans LPS-stimulated RAW264.7 cells was suppressed by SQ22536 but not ODQ. Enhancement of arginase activity was observed in the presence of cAMP analog or forskolin but not cGMP analog. Cycloheximide blocked arginase activity in the cells in the presence of cAMP analog or forskolin with or without A. actinomycetemcomitans LPS. IBMX augmented arginase activity in A. actinomycetemcomitans LPS-stimulated cells. Rolipram (a PDE4 inhibitor) increased the levels of arginase activity higher than siguazodan (a PDE3 inhibitor) in the antigen-stimulated cells. The effect of cAMP analog or forskolin on arginase activity in the presence or absence of A. actinomycetemcomitans LPS was blocked by the PKA inhibitor (KT5720). Conclusion: The results of the present study suggest that A. actinomycetemcomitans LPS may stimulate arginase activity in murine macrophages (RAW264.7 cells) in a cAMP-PKA-dependent pathway.

Processing of mutated human proinsulin to mature insulin in the non-endocrine cell line, CHO

Heterologous genes encoding proproteins, including proinsulin, generally produce mature protein when expressed in endocrine cells while unprocessed or partially processed protein is produced in non-endocrine cells. Proproteins, which are normally processed in the regulated pathway restricted to endocrine cells, do not always contain the recognition sequence for cleavage by furin, the endoprotease specific to the constitutive pathway, the principal protein processing pathway in non-endocrine cells. Human proinsulin consists of B-Chain-C-peptide-A-Chain and cleavage at the B/C and C/A junctions is required for processing. The B/C, but not the C/A junction, is recognised and cleaved in the constitutive pathway. We expressed a human proinsulin and a mutated proinsulin gene with an engineered furin recognition sequence at the C/A junction and compared the processing efficiency of the mutant and native proinsulin in Chinese Hamster Ovary cells. The processing efficiency of the mutant proinsulin was 56% relative to 0.7% for native proinsulin. However, despite similar levels of mRNA being expressed in both cell lines, the absolute levels of immunoreactive insulin, normalized against mRNA levels, were 18-fold lower in the mutant proinsulin-expressing cells. As a result, there was only a marginal increase in absolute levels of insulin produced by these cells. This unexpected finding may result from preferential degradation of insulin in non-endocrine cells which lack the protection offered by the secretory granules found in endocrine cells.

Development of a real-time RT-PCR assay for plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA levels in a human breast epithelial cell line.

The plasma membrane Ca2+ pump is a key regulator of cytosolic free Ca2+. Recent studies have demonstrated the dynamic expression of the plasma membrane Ca2+ pump in a variety of cell types. Furthermore, alterations in plasma membrane calcium pump activity have now been implicated in human disease. In this study, the development of a technique to quantitatively assess mRNA expression of the human plasma membrane Ca2+ ATPase (PMCA1) isoform of the plasma membrane Ca2+ pump, using a real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay in a human breast epithelial cell line (MCF-7) is described. The sequences of the PMCA1 primers and probe for real-time RT-PCR are presented. The results also indicate that PMCA1 mRNA can be normalized to both 18S ribosomal RNA (18S rRNA) and human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) in MCF-7 cells. Real-time RT-PCR will be most useful in assessing PMCA1 mRNA expression in cases where only low amounts of RNA are available and/or when numerous samples must be assessed simultaneously. (C) 2001 Elsevier Science Inc. All rights reserved.

Tetracycline-Inducible packaging cell line for production of Flavivirus replicon particles.

We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 x 10(9) VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 1010 VLPs per 106 transfected cells. Passaging of VLPs on Vero cells or intracerebral injection into 2- to 4-day-old suckling mice illustrated the complete absence of any infectious Kunjin virus. tetKUNCprME cells were also capable of packaging replicon RNA from closely and distantly related flaviviruses, West Nile virus and dengue virus type 2, respectively. The utility of high-titer KUN replicon VLPs was demonstrated by showing increasing CD8(+)-T-cell responses to encoded foreign protein with increasing doses of KUN VLPs. A single dose of 2.5 x 10(7) VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 10(6) splenocytes in an ex vivo gamma interferon enzyme-linked immunospot assay. The packaging cell line thus represents a significant advance in the development of the noncytopathic Kunjin virus replicon-based gene expression system and may be widely applicable to the basic studies of flavivirus RNA packaging and virus assembly as well as to the development of gene expression systems based on replicons from different flaviviruses.

Ratiometric and nonratiometric Ca2+ indicators for the assessment of intracellular free Ca2+ in a breast cancer cell line using a fluorescence microplate reader

Transporters of Ca2+ are potential drug targets and Ca2+ is a useful signal in the assessment of G-protein-coupled receptor activation. Assays involving the assessment of intracellular Ca2+ using microplate readers most often use Ca2+ indicators which do not exhibit a spectra shift on Ca2+ binding (e.g. fluo-3). Indicators that do exhibit a spectral shift upon Ca2+ binding (e.g. fura-2) offer potential advantages for the calibration of intracellular Ca2+ levels. However, experimental limitations may limit the use of ratiometric dyes in microplate readers capable of screening. In this study, we compared the assessment of intracellular Ca2+ in adherent breast cancer cells using ratiometric and nonratiometric Ca2+ indicators. Our results demonstrate that both fluo-3 and fura-2 detect ATP dose-dependent increases in intracellular Ca2+ in the MCF-7 breast cancer cell line and that some of the limitations in the use of fura-2 appear to be overcome by the use of glass bottom microplates. The calibrated intracellular Ca2+ levels derived using fura-2 are consistent with those from microscopy and cuvette-based studies. Fura-2 may be useful in microplate studies, where cell lines with different properties are compared or where screening treatments lead to differences in the number of cells or dye loading. (C) 2003 Elsevier B.V. All rights reserved.

In vitro cultivation of Wolbachia pipientis in an Aedes albopictus cell line

A continuous cell line, Aa23, was established from eggs of a strain of the Asian tiger mosquito, Aedes albopictus, naturally infected with the intracellular symbiont Wolbachia pipientis. The resulting cell line was shown to be persistently infected with the bacterial endosymbiont. Treatment with antibiotics cured the cells of the infection. In the course of establishing this cell line it was noticed that RFLPs in the PCR products of two Wolbachia genes from the parental mosquitoes were fixed in the infected cell line. This indicates that the mosquito host was naturally superinfected with different Wolbachia strains, whereas the infected cell line derived from these mosquitoes only contained one of the original Wolbachia strains. The development of anin vitroculture system for this fastidious microorganism should facilitate molecular analysis of the reproduction distorting phenotypes it induces in natural arthropod hosts.

Identification of cellular changes associated with increased production of human growth hormone in a recombinant Chinese hamster ovary cell line

A proteomics approach was used to identify the proteins potentially implicated in the cellular response concomitant with elevated production levels of human growth hormone in a recombinant Chinese hamster ovary (CHO) cell line following exposure to 0.5 mM butyrate and 80 muM zinc sulphate in the production media. This involved incorporation of two-dimensional (2-D) gel electrophoresis and protein identification by a combination of N-terminal sequencing, matrix-assisted laser desorption/ionisation-time of flight mass spectrometry, amino acid analysis and cross species database matching. From these identifications a CHO 2-D reference,map and annotated database have been established. Metabolic labelling and subsequent autoradiography showed the induction of a number of cellular proteins in response to the media additives butyrate and zinc sulphate. These were identified as GRP75, enolase and thioredoxin. The chaperone proteins GRP78, HSP90, GRP94 and HSP70 were not up-regulated under these conditions.

Process development for a recombinant Chinese Hamster Ovary (CHO) cell line utilizing a metal induced and amplified metallothionein expression system

The suspension Chinese Hamster Ovary cell line, 13-10-302, utilizing the metallothionein (MT) expression system producing recombinant human growth hormone (hGH) was studied in a serum-free and cadmium-free medium at different fermentation scales and modes of operation. Initial experiments were carried out to optimize the concentration of metal addition to induce the MT promoter. Subsequently, the cultivation of the 13-10-302 cell line was scaled up from spinner flasks into bioreactors, and the cultivation duration was extended with fed-batch and perfusion strategies utilizing 180 muM zinc to induce the promoter controlling expression of recombinant hGH. It was shown that a fed-batch process could increase the maximum cell numbers twofold, from 3.3 to 6.3 x 10(6) cell/mL, over those obtained in normal batch fermentations, and this coupled with extended fermentation times resulted in a fourfold increase in final hGH titer, from 135 +/- 15 to 670 +/- 70 mg/L at a specific productivity q(hGH) value of 12 pg cell(-1)d(-1). The addition of sodium butyrate increased the specific productivity of hGH in cells to a value of approximately 48 pg cell(-1)d(-1), resulting in a final hGH titer of over a gram per liter during fed-batch runs. A BioSep acoustic cell recycler was used to retain the cells in the bioreactor during perfusion operation. It was necessary to maintain the specific feeding rates (SFR) above a value of 0.2 vvd/(10(6) cell/mL) to maintain the viability and productivity of the 13-10-302 cells; under these conditions the viable cell number increased to over 107 cell/mL and resulted in a volumetric productivity of over 120 mg(hGH) L(-1)d(-1). Process development described in this work demonstrates cultivation at various scales and sustained high levels of productivity under cadmium free condition in a CHO cell line utilizing an inducible metallothionein expression system. (C) 2004 Wiley Periodicals, Inc.

Super-CHO - A cell line capable of autocrine growth under fully defined protein-free conditions

Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cell line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in protein-free medium, DNA coding for insulin and transferrin production was transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/10(6)cells/24h. Insulin was poorly expressed, with rates peaking at 5 ng/10(6)cells/24h. Characterisation of the secreted insulin indicated that the CHO cells were incompletely processing the insulin molecule. Site-directed mutagenesis was used to introduce a furin (prohormone converting enzyme) recognition sequence into the insulin molecule, allowing the production of active insulin. However, the levels were still too low to support autocrine growth. Further investigations revealed insulin degrading activity (presumably due to the presence of insulin degrading enzymes) in the cytoplasm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was completely processed and expressed at rates greater than 500 ng/10(6)cells/24h. In this paper we report autonomous growth of the transfected CHO-K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and final cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplemented serum-free media.