A Metal-Repressed Promoter From Gram-Positive Bacillus subtilis Is Highly Active and Metal-Induced in Gram-Negative Cupriavidus metallidurans

A synthetic version of the metal-regulated gene A (mrgA) promoter from Bacillus subtilis, which in this Gram-positive bacterium is negatively regulated by manganese, iron, cobalt, or copper turned out to promote high level of basal gene expression that is further enhanced by Co(II), Cd(II), Mn(II), Zn(II), Cu(II), or Ni(II), when cloned in the Gram-negative bacterium Cupriavidus metallidurans. Promoter activity was monitored by expression of the reporter gene coding for the enhanced green fluorescent protein (EGFP), and cellular intensity fluorescence was quantified by flow cytometry. Expression levels in C. metallidurans driven by the heterologous promoter, here called pan, ranged from 20- to 53-fold the expression level driven by the Escherichia coli lac promoter (which is constitutively expressed in C. metallidurans), whether in the absence or presence of metal ions, respectively. The pan promoter did also function in E. coli in a constitutive pattern, regardless of the presence of Mn(II) or Fe(II). In conclusion, the pan promoter proved to be a powerful tool to express heterologous proteins in Gram-negative bacteria, especially in C. metallidurans grown upon high levels of toxic metals, with potential applications in bioremediation. Biotechnol. Bioeng. 2010; 107: 469-477. (C) 2010 Wiley Periodicals, Inc.

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.

Effects of metal-induced oxidative stress on endothelial cells in vitro

Aseptic loosening of metal implants is mainly attributed to the formation of metal degradation products. These include particulate debris and corrosion products, such as metal ions (anodic half-reaction) and ROS (cathodic half-reaction). While numerous clinical studies describe various adverse effects of metal degradation products, detailed knowledge of metal-induced cellular reactions, which might be important for possible therapeutic intervention, is not comprehensive. Since endothelial cells are involved in inflammation and angiogenesis, two processes which are critical for wound healing and integration of metal implants, the effects of different metal alloys and their degradation products on these cells were investigated. Endothelial cells on Ti6Al4V alloy showed signs of oxidative stress, which was similar to the response of endothelial cells to cathodic partial reaction of corrosion induced directly on Ti6Al4V surfaces. Furthermore, oxidative stress on Ti6Al4V alloy reduced the pro-inflammatory stimulation of endothelial cells by TNF-α and LPS. Oxidative stress and other stress-related responses were observed in endothelial cells in contact with Co28Cr6Mo alloy. Importantly, these features could be reduced by coating Co28Cr6Mo with a TiO2 layer, thus favouring the use of such surface modification in the development of medical devices for orthopaedic surgery. The reaction of endothelial cells to Co28Cr6Mo alloy was partially similar to the effects exerted by Co2+, which is known to be released from metal implants. Co2+ also induced ROS formation and DNA damage in endothelial cells. This correlated with p53 and p21 up-regulation, indicating the possibility of cell cycle arrest. Since CoCl2 is used as an hypoxia-mimicking agent, HIF-1α-dependence of cellular responses to Co2+ was studied in comparison to anoxia-induced effects. Although important HIF-1α-dependent genes were identified, a more detailed analysis of microarray data will be required to provide additional information about the mechanisms of Co2+ action. All these reactions of endothelial cells to metal degradation products might play their role in the complex processes taking place in the body following metal device implantation. In the worst case this can lead to aseptic loosening of the implant and requirement for revision surgery. Knowledge of molecular mechanisms of metal-induced responses will hopefully provide the possibility to interfere with undesirable processes at the implant/tissue interface, thus extending the life-time of the implant and the overall success of metal implant applications.

Metallomics and chemical speciation: towards a better understanding of metal-induced stress in plants

Most metal ions are toxic to plants, even at low concentrations, despite the fact that some are essential for growth and play key roles in metabolism. The majority of metals induce the formation of reactive oxygen species, which require the synthesis of additional antoxidant compounds and enzymes for their removal. New techniques that have greatly improved the identification, localisation and quantification of metals within plant tissues have led to the science of metallomics. This advancement in knowledge should eventually allow the characterisation of plants used in the process of phytoremediation of soils contaminated with toxic metals.

SV40-induced expression of calretinin protects mesothelial cells from asbestos cytotoxicity and may be a key factor contributing to mesothelioma pathogenesis.

The calcium-binding protein calretinin has emerged as a useful marker for the identification of mesotheliomas of the epithelioid and mixed types, but its putative role in tumor development has not been addressed previously. Although exposure to asbestos fibers is considered the main cause of mesothelioma, undoubtedly, not all mesothelioma patients have a history of asbestos exposure. The question as to whether the SV40 virus is involved as a possible co-factor is still highly debated. Here we show that increased expression of SV40 early gene products in the mesothelial cell line MeT-5A induces the expression of calretinin and that elevated calretinin levels strongly correlate with increased resistance to asbestos cytotoxicity. Calretinin alone mediates a significant part of this protective effect because cells stably transfected with calretinin cDNA were clearly more resistant to the toxic effects of crocidolite than mock-transfected control cells. Down-regulation of calretinin by antisense methods restored the sensitivity to asbestos toxicity to a large degree. The protective effect observed in clones with higher calretinin expression levels could be eliminated by phosphatidylinositol 3-kinase (PI3K) inhibitors, implying an important role for the PI3K/AKT signaling (survival) pathway in mediating the protective effect. Up-regulation of calretinin, resulting from either asbestos exposure or SV40 oncoproteins, may be a common denominator that leads to increased resistance to asbestos cytotoxicity and thereby contributes to mesothelioma carcinogenesis.

9-cis-retinoic acid enhances fatty acid-induced expression of the liver fatty acid-binding protein gene.

The role of retinoic acids (RA) on liver fatty acid-binding protein (L-FABP) expression was investigated in the well differentiated FAO rat hepatoma cell line. 9-cis-Retinoic acid (9-cis-RA) specifically enhanced L-FABP mRNA levels in a time- and dose-dependent manner. The higher induction was found 6 h after addition of 10(-6) M 9-cis-RA in the medium. RA also enhanced further both L-FABP mRNA levels and cytosolic L-FABP protein content induced by oleic acid. The retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR), which are known to be activated, respectively, by 9-cis-RA and long chain fatty acid (LCFA), co-operated to bind specifically the peroxisome proliferator-responsive element (PPRE) found upstream of the L-FABP gene. Our result suggest that the PPAR-RXR complex is the molecular target by which 9-cis-RA and LCFA regulate the L-FABP gene.

Temporal quantification of MAPK induced expression in single yeast cells.

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood

Lipopolysaccharide (LPS) activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R) and smooth (S) forms signal through Toll-like receptor 4 (TLR4), but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS) and nitric oxide (NO) generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.

Effects of propofol on lipopolysaccharide-induced expression and release of HMGB1 in macrophages

This study aimed to determine the effects of different concentrations of propofol (2,6-diisopropylphenol) on lipopolysaccharide (LPS)-induced expression and release of high-mobility group box 1 protein (HMGB1) in mouse macrophages. Mouse macrophage cell line RAW264.7 cells were randomly divided into 5 treatment groups. Expression levels of HMGB1 mRNA were detected using RT-PCR, and cell culture supernatant HMGB1 protein levels were detected using enzyme-linked immunosorbent assay (ELISA). Translocation of HMGB1 from the nucleus to the cytoplasm in macrophages was observed by Western blotting and activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus was detected using ELISA. HMGB1 mRNA expression levels increased significantly in the cell culture supernatant and in cells after 24 h of stimulating RAW264.7 cells with LPS (500 ng/mL). However, HMGB1 mRNA expression levels in the P2 and P3 groups, which received 500 ng/mL LPS with 25 or 50 μmol/mL propofol, respectively, were significantly lower than those in the group receiving LPS stimulation (P<0.05). After stimulation by LPS, HMGB1 protein levels were reduced significantly in the nucleus but were increased in the cytoplasm (P<0.05). Simultaneously, the activity of NF-κB was enhanced significantly (P<0.05). After propofol intervention, HMGB1 translocation from the nucleus to the cytoplasm and NF-κB activity were inhibited significantly (each P<0.05). Thus, propofol can inhibit the LPS-induced expression and release of HMGB1 by inhibiting HMGB1 translocation and NF-κB activity in RAW264.7 cells, suggesting propofol may be protective in patients with sepsis.

Tenascin-W is found in malignant mammary tumors, promotes alpha8 integrin-dependent motility and requires p38MAPK activity for BMP-2 and TNF-alpha induced expression in vitro

Tenascins represent a family of extracellular matrix glycoproteins with distinctive expression patterns. Here we have analyzed the most recently described member, tenascin-W, in breast cancer. Mammary tumors isolated from transgenic mice expressing hormone-induced oncogenes reveal tenascin-W in the stroma around lesions with a high likelihood of metastasis. The presence of tenascin-W was correlated with the expression of its putative receptor, alpha8 integrin. HC11 cells derived from normal mammary epithelium do not express alpha8 integrin and fail to cross tenascin-W-coated filters. However, 4T1 mammary carcinoma cells do express alpha8 integrin and their migration is stimulated by tenascin-W. The expression of tenascin-W is induced by BMP-2 but not by TGF-beta1, though the latter is a potent inducer of tenascin-C. The expression of tenascin-W is dependent on p38MAPK and JNK signaling pathways. Since preinflammatory cytokines also act through p38MAPK and JNK signaling pathways, the possible role of TNF-alpha in tenascin-W expression was also examined. TNF-alpha induced the expression of both tenascin-W and tenascin-C, and this induction was p38MAPK- and cyclooxygenase-dependent. Our results show that tenascin-W may be a useful diagnostic marker for breast malignancies, and that the induction of tenascin-W in the tumor stroma may contribute to the invasive behavior of tumor cells.

Antibiotic-induced expression of a cryptic cpb2 gene in equine beta2-toxigenic Clostridium perfringens

The cpb2 gene of beta2-toxigenic Clostridium perfringens isolated from horses, cattle, sheep, human and pigs was sequenced. The cpb2 gene of equine and other non-porcine isolates differed from porcine isolates by the absence of an adenine in a poly A tract immediately downstream of the start codon in all non-porcine C. perfringens strains. This deletion involved formation of a cryptic gene harbouring a premature stop codon after only nine amino acid codons, while the full beta2-toxin protein consists of 265 amino acids. Immunoblots carried out with antibodies directed against a recombinant beta2-toxin showed the absence of expression of the beta2-toxin in equine and the other non-porcine strains under standard culture conditions. However, treatment of C. perfringens with the aminoglycosides gentamicin or streptomycin was able to induce expression of the cpb2 gene in a representative equine strain of this group, presumably by frameshifting. The presence of the beta2-toxin was revealed by immunohistology in tissue samples of small and large intestine from horses with severe typhlocolitis that had been treated before with gentamicin. This result may explain the finding that antibiotic treatment of horses affected by beta2-toxigenic C. perfringens leads to a more accentuated and fatal progression of equine typhlocolitis. Clinical observations show a reduced appearance of strong typhlocolitis in horses with intestinal complications admitted to hospital care since the standard use of gentamicin has been abandoned. This is the first report on expression of a bacterial toxin gene by antibiotic-induced ribosomal frameshifting.

Light-induced expression of fatty acid desaturase genes

In cyanobacterial cells, fatty acid desaturation is one of the crucial steps in the acclimation processes to low-temperature conditions. The expression of all the four acyl lipid desaturase genes of Synechocystis PCC 6803 was studied as a function of temperature and separately as a function of light. We used cells grown at 25°C in light-activated heterotrophic growth conditions. In these cells, the production of α-linolenic acid and 18:4 fatty acids was negligible and the synthesis of γ-linolenic acid was remarkably suppressed compared with those of the cells grown photoautotrophically. The cells grown in the light in the presence of glucose showed no difference in fatty acid composition compared with cells grown photoautotrophically. The level of desC mRNA for Δ9 desaturase was not affected by either the temperature or the light. It was constitutively expressed at 25°C with and without illumination. The level of desB transcripts was negligible in the dark-grown cells and was enhanced about 10-fold by exposure of the cells to light. The maximum level of expression occurred within 15 min. The level of desA and desD mRNAs was higher in dark-grown cells than that of desB mRNA for ω3 desaturase. However, the induction of both desA and desD mRNAs for Δ12 and Δ6 desaturases, respectively, was enhanced by light about 10-fold. Rifampicin, chloramphenicol, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea completely blocked the induction of the expression of desA, desB, and desD. Consequently, we suggest the regulatory role of light via photosynthetic processes in the induction of the expression of acyl lipid desaturases.

Crystal structures of the copper and nickel complexes of RNase A: Metal-induced interprotein interactions and identification of a novel copper binding motif

We report the crystal structures of the copper and nickel complexes of RNase A. The overall topology of these two complexes is similar to that of other RNase A structures. However, there are significant differences in the mode of binding of copper and nickel. There are two copper ions per molecule of the protein, but there is only one nickel ion per molecule of the protein. Significant changes occur in the interprotein interactions as a result of differences in the coordinating groups at the common binding site around His-105. Consequently, the copper- and nickel-ion-bound dimers of RNase A act as nucleation sites for generating different crystal lattices for the two complexes. A second copper ion is present at an active site residue His-119 for which all the ligands are from one molecule of the protein. At this second site, His-119 adopts an inactive conformation (B) induced by the copper. We have identified a novel copper binding motif involving the α-amino group and the N-terminal residues.