Mouse Fibroblast-like Cells Research Articles

C5a–C5AR1 axis as a potential trigger of the rupture of intracranial aneurysms

Recent studies have indicated the involvement of neutrophil-mediated inflammatory responses in the process leading to intracranial aneurysm (IA) rupture. Receptors mediating neutrophil recruitment could thus be therapeutic targets of unruptured IAs. In this study, complement C5a receptor 1 (C5AR1) was picked up as a candidate that may cause neutrophil-dependent inflammation in IA lesions from comprehensive gene expression profile data acquired from rat and human samples. The induction of C5AR1 in IA lesions was confirmed by immunohistochemistry; the up-regulations of C5AR1/C5ar1 stemmed from infiltrated neutrophils, which physiologically express C5AR1/C5ar1, and adventitial fibroblasts that induce C5AR1/C5ar1 in human/rat IA lesions. In in vitro experiments using NIH/3T3, a mouse fibroblast-like cell line, induction of C5ar1 was demonstrated by starvation or pharmacological inhibition of mTOR signaling by Torin1. Immunohistochemistry and an experiment in a cell-free system using recombinant C5 protein and recombinant Plasmin indicated that the ligand of C5AR1, C5a, could be produced through the enzymatic digestion by Plasmin in IA lesions. In conclusion, we have identified a potential contribution of the C5a–C5AR1 axis to neutrophil infiltration as well as inflammatory responses in inflammatory cells and fibroblasts of IA lesions. This cascade may become a therapeutic target to prevent the rupture of IAs.

Bidirectional effect of vitamin D on brown adipogenesis of C3H10T1/2 fibroblast-like cells.

Brown adipose tissue (BAT) dissipates caloric energy as heat and plays a role in glucose and lipid metabolism. Therefore, augmentation and activation of BAT are the focus of new treatment strategies against obesity, a primary risk factor of metabolic syndrome. The vitamin D system plays a crucial role in mineral homeostasis, bone metabolism, and cell proliferation and differentiation. In this study, we investigated the effects of vitamin D3 [1,25(OH)2D3] on brown adipocyte differentiation. The mouse fibroblast-like cell line C3H10T1/2 was differentiated into brown adipocytes in the presence of 1,25(OH)2D3. The effect of 1,25(OH)2D3 on brown adipocyte differentiation was assessed by measuring lipid accumulation, the expression of related genes, and cytotoxicity. The viability of C3H10T1/2 cells was measured using the Cell Counting Kit-8 assay. Gene expression was investigated using quantitative reverse transcription-polymerase chain reaction. Protein expression was estimated using western blotting. 1,25(OH)2D3 inhibited adipocyte differentiation and exerted a cytotoxic effect at 1 nM. However, in the physiological concentration range (50-250 pM), 1,25(OH)2D3 promoted uncoupling protein 1 (UCP1) expression in C3H10T1/2 cells. This effect was not observed when 1,25(OH)2D3 was added 48 h after the initiation of differentiation, suggesting that the vitamin D system acts in the early phase of the differentiation program. We showed that 1,25(OH)2D3 increased the expression of two key regulators of brown adipogenesis, PR domain containing 16 (Prdm16) and peroxisome proliferator-activated receptor γ coactivator-1α (Pgc1α ). Furthermore, 1,25(OH)2D3 increased Ucp1 expression in 3T3-L1 beige adipogenesis in a dose-dependent manner. These data indicate the potential of vitamin D and its analogs as therapeutics for the treatment of obesity and related metabolic diseases.

About 4-Day Rhythm of Proliferative Activity of L-929 Cells in Culture Correlates with the Intensity of Secondary Cosmic Radiation Fluctuations.

The dynamics of proliferative activity of the L-929 cell culture of mouse fibroblast-like cells in the phase of logarithmic growth was compared with some heliogeophysical parameters (Ap and ULF indexes of geomagnetic activity, vertical component of the interplanetary magnetic field, and intensity of fluctuations of secondary cosmic radiation estimated by the neutron monitoring near the Earth's surface). Among the considered heliogeophysical parameters, only the magnitude of fluctuations of minute-to-minute changes in the neutron monitor indicator reliably and negatively correlates with the rate of cell culture reproduction. Considering that the amplitude of secondary cosmic fluctuations is about 5%, which is 0.1% of the total ray flux, and proliferative activity varies in the range of 30-50%, the probability of a direct biotrophic effect of this physical factor is extremely low. It seems likely that proliferative activity of L-929 cell culture is directly affected by another environmental factor, the marker of which is the intensity of neutron counting rate fluctuations.

Expression of Ascorbate Peroxidase Derived from Cyanidioschyzon merolae in Mammalian Cells.

Ascorbate peroxidase (APX) derived from Cyanidioschyzon merolae, a primitive red alga living in high temperature and acidic environments, has greater anti-oxidative capacity than similar peroxidases occurring in other plants. In the present study, we examined whether expression of Cyanidioschyzon merolae-derived APX (cAPX) in mammalian cells increases cellular anti-oxidative capacity. The cAPX gene was introduced into the mouse fibroblast-like cell line C3H10T1/2. Production of reactive oxygen species (ROS) and/or cell viability was assessed after heat, H2O2 and acid stimulation. Heat and H2O2 stimulation resulted in ROS production. cAPX-expressing cells were more tolerant to oxidative stress induced by heat, H2O2 and acid stimulations than control cells lacking cAPX. Introduction of cAPX increases the anti-oxidative capacity in mammalian cells.

Evaluation of the Cytotoxicity and Immune and Subacute Toxicity of Camptothecin-loaded Nanoparticles

CPT-loaded NPs with polyethylene glycol shells can increase its solubility in water and remain in circulation for a long time. This experiment has evaluated cytotoxicity and immune and subacute toxicity of CPT NPs. Camptothecin (CPT) was incorporated into biotin-F127-PLA or F127-PLA polymeric nanoparticles; a dialysis method was used for non-targeted CPT NPs and anti-CA125 antibodies for targeted CPT NPs. A cytotoxicity test was also conducted based on the growth inhibition of the mouse fibroblast-like L-929 cell line. The effects of the two CPT-loaded NPs on immunity were also determined through a carbon particle clearance rate assay. Each mouse was intraperitoneally injected with 0.4 ml/20 g.bw nanoparticles for 28 consecutive days in the subacute toxicity test. Results have showed that CPT NPs have reduced the toxicity of free CPT on the L-919 cells. The CPT NPs did not induce phagocytosis in the normal mice. At the end of subacute toxicity study no difference was found between the CPT NP and control groups in blood parameter analysis and main organ weight visceral coefficients. These results suggested that the new CPT NPs might elicit low toxic effects at cellular and organism levels.

Hyperoxia causes diffuse alveolar damage through mechanisms involving upregulation of c-Myc/Bax and enhanced production of reactive oxygen species.

Hyperoxia is a known cause of diffuse alveolar damage (DAD). We previously reported the transcript profiling of DAD induced by hyperoxia exposure in mouse lungs and showed that the gene expression of myelocytomatosis oncogene (c-Myc) was significantly upregulated whereas that of surfactant-associated protein (SP)-C was downregulated. However, the mechanism underlying hyperoxia-induced DAD is not well understood. The hyperoxia-induced changes in SP-A/B/C/D, c-Myc, B-cell chronic lymphocytic leukemia/lymphoma (Bcl)-2, and Bcl-2-associated X protein (Bax) expression in mouse lungs were examined by cDNA microarray analysis. The expression levels of the above mentioned genes, cell viability, caspase activity, and reactive oxygen species (ROS) production were also examined in the human lung adenocarcinoma cell line A549 and mouse fibroblast-like cell line NIH/3T3. Hyperoxia induced a decrease in SP-C/A expression in mouse lungs, and SP-C downregulation was also confirmed in A549 cells. In addition to enhanced c-Myc expression, Bax expression also increased following exposure of the mice to hyperoxia. In vitro analysis showed that expression of these genes is regulated in a cell-type-dependent manner, i.e., upregulation of c-Myc in NIH/3T3 cells and Bax in A549 cells occurred regardless of whether there was a similar decrease in cell viability and increase in caspase-3/7 activation in response to hyperoxia. ROS production and caspase-8 activation were also observed in both cells. We concluded that hyperoxia induces ROS production and cell death in lung tissues through a cell-type specific mechanism involving the upregulation of c-Myc/Bax, and caspase-8 and -3/7 activation-dependent pathways, thereby leading to the development of DAD.

Protective effects of Scutellaria lindbergii root extract against oxidative-induced cell and DNA damage in mouse fibroblast-like cells

Context: Scutellaria lindbergii Rech. f. (Lamiaceae) is an Iranian species of Scutellaria which has been shown to exert antimicrobial, antioxidant and cytotoxic effects. Objective: The protective properties of total methanol extract (TME) of S. lindbergii and its fractions (defatted and CH2Cl2) were investigated against cytotoxic and genotoxic effects of H2O2 in NIH 3T3 cell line as non-malignant cells. Materials and methods: The cells were incubated with different concentrations of S. lindbergii root extracts [TME (15–250 μg ml−1), defatted fraction (15–500 μg ml−1) and CH2Cl2 fraction (5–40 μg ml−1)] and toxic concentration of H2O2 (200 µM) at 37 °C for 2 h concurrently and Cell viability was quantitated by MTT assay. The antigenotoxic effect of extracts was investigated using comet assay. The cells were incubated with extracts [TME (25–250 μg ml−1), defatted fraction (25–500 μg ml−1) and CH2Cl2 fraction (5–40 μg ml−1)] and H2O2 (25 µM) at 4 °C for 20 min, then the comet assay was performed. DNA damage was expressed as percentage tail DNA. Results: Total methanol extract of S. lindbergii and its fractions had a significant inhibitory effect on DNA damage. The IC50 values of TME, defatted fraction and CH2Cl2 fraction against DNA damage were determined as 48, 138 and 8 μg ml−1, respectively. Conclusion: S. lindbergii extracts can prevent oxidative DNA damage, which is likely due to its flavonoids and phenolic compounds as antioxidant constituents.

Bio-inspired Aloe vera sponges for biomedical applications

Chemical composition and biological properties of Aloe vera (AV), a tropical plant, explain its potential use for cosmetic, nutritional and biomedical applications. AV gel present in AV leaves is rich in several compounds, nutrients and polysaccharides. This work proposes using AV gel complex structure and chemical composition, associated with freeze-drying, to produce sponges. To increase the structures stability in aqueous media, a thin coating of gellan gum (GG), was applied onto AV gel. AV-based sponges showed a heterogeneous porous formation, interconnected pores and good porosity (72–77%). The coating with a GG layer onto AV influenced the stability, swelling behavior and mechanical properties of the resulting sponges. Moreover, sponges provided the sustained release of BSA-FTIC, used as a model protein, over 3 weeks. Also, in vitro cell culture studies evidenced that sponges are not cytotoxic for a mouse fibroblast-like cell line. Therefore, developed AV-based sponges have potential use in biomedical applications.

Cell Proliferation Ability of Mouse Fibroblast-Like Cells and Osteoblast-Like Cells on a Ti-6Al-4V Alloy Film Produced by Selective Laser Melting

Successful regeneration of tissues and organs relies on the application of suitable substrates or scaffolds in scaffold-based regenerative medicine. In this study, Ti-6Al-4V alloy films (Ti alloy film) were produced using a three-dimensional printing technique called Selective Laser Melting (SLM), which is one of the metal additive manufacturing techniques. The thickness of produced Ti alloy film was approximately 250 μm. The laser-irradiated surface of Ti alloy film had a relatively smooth yet porous surface. The non-irradiated surface was also porous but also retained a lot of partially melted Ti-6Al-4V powder. Cell proliferation ability of mouse fibroblast-like cells (L929 cells) and mouse osteoblast-like cells (MC3T3-E1 cells) on both the surfaces of Ti alloy film was examined using WST assay. Both L929 and MC3T3-E1 cells underwent cell proliferation during the culture period. These results indicate that selective laser melting is suitable for producing a cell-compatible Ti-6Al-4V alloy film for biomaterials applications.

Microspectroscopic Study of Liposome-to-cell Interaction Revealed by Förster Resonance Energy Transfer

We report the Förster resonance energy transfer (FRET)-labeling of liposomal vesicles as an effective approach to study in dynamics the interaction of liposomes with living cells of different types (rat hepatocytes, rat bone marrow, mouse fibroblast-like cells and human breast cancer cells) and cell organelles (hepatocyte nuclei). The in vitro experiments were performed using fluorescent microspectroscopic technique. Two fluorescent dyes (DiO as the energy donor and DiI as an acceptor) were preloaded in lipid bilayers of phosphatidylcholine liposomes that ensures the necessary distance between the dyes for effective FRET. The change in time of the donor and acceptor relative fluorescence intensities was used to visualize and trace the liposome-to-cell interaction. We show that FRET-labeling of liposome vesicles allows one to reveal the differences in efficiency and dynamics of these interactions, which are associated with composition, fluidity, and metabolic activity of cell plasma membranes.

P40 The up-regulation role of H2S in 3T3L1 on adipogenesis

The endogenous gas, hydrogen sulfide (H2S), plays an important role in regulating cardiovascular function and inflammation. It also contributes to the pathogenesis of insulin resistance and glucose metabolism [1] , [2] , [3] . Recently, it has been reported that large amounts of H2S are formed from L-cysteine by cystathionine gamma lyase (CSE) in fat tissues and that H2S reduces glucose uptake in rat adipose tissue [3] . However, the biological roles of H2S in adipocytes and its contribution to adipogenesis are not yet clear. Here, we investigate whether H2S-synthesizing enzymes; cystathionine beta-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), are endogenously expressed in adipocytes and play a role in adipogenesis. In the present experiments, we stimulated the mouse fibroblast-like cell line, 3T3L1, to differentiate into adipocytes for up to one week as a model of adipogenesis in vitro. Over 7 days differentiation, whole cell lysate at day 0, 1, 3, 5 and 7 were collected for Western blotting of CBS, CSE and 3-MST. Endogenous CBS and 3-MST in adipocytes peaked at day 5 of differentiation whereas CSE protein expression was up-regulated in a time-dependent manner and maximally expressed at day 7 (n = 3, P [4] , [5] , [6] , but that these enzymes are also present in large amounts in adipose tissue and that their up-regulation during adipogenesis promotes adipocyte maturation and prevent lipid degradation. Thus, it is possible that modulation of the H2S system may be useful for obesity treatment.

Hybrid biodegradable membranes of silane-treated chitosan/soy protein for biomedical applications

In recent years, progress in the field of hybrid materials has been accelerated through use of the sol–gel process for creating materials and devices, which benefit from the incorporation of both inorganic and organic components. In this work, organic–inorganic hybrid membranes were prepared from tetraethoxysilane and a blend system composed of chitosan and soy protein. By introducing a small amount of siloxane bond into the chitosan/soy protein system, the chitosan/soy protein hybrid membranes were improved in terms of structure, topography and mechanical properties. It appears that the chitosan/soy protein hybrid membranes were formed by discrete inorganic moieties entrapped in the chitosan/soy protein blend, which improved the stability and mechanical performance assessed by the dynamic mechanical analysis as compared to chitosan/soy protein membrane. Also, in vitro cell culture studies evidenced that the chitosan/soy protein hybrid membranes are non-cytotoxic over a mouse fibroblast-like cell line. The hybrid membranes of silane-treated chitosan/soy protein developed in this work have potential in biomedical applications, including tissue engineering.

Revealing the potential of squid chitosan-based structures for biomedical applications

In recent years, much attention has been given to different marine organisms, namely as potential sources of valuable materials with a vast range of properties and characteristics. In this work, β-chitin was isolated from the endoskeleton of the giant squid Dosidicus gigas and further deacetylated to produce chitosan. Then, the squid chitosan was processed into membranes and scaffolds using solvent casting and freeze-drying, respectively, to assess their potential biomedical application. The developed membranes have shown to be stiffer and less hydrophobic than those obtained with commercial chitosan. On the other hand, the morphological characterization of the developed scaffolds, by SEM and micro-computed tomography, revealed that the matrices were formed with a lamellar structure. The findings also indicated that the treatment with ethanol prior to neutralization with sodium hydroxide caused the formation of larger pores and loss of some lamellar features. The in vitro cell culture study has shown that all chitosan scaffolds exhibited a non-cytotoxic effect over the mouse fibroblast-like cell line, L929 cells. Thus, chitosan produced from the endoskeletons of the giant squid Dosidicus gigas has proven to be a valuable alternative to existing commercial materials when considering its use as biomaterial.

Poly(acrylic acid)-block-poly(vinyl alcohol) anchored maghemite nanoparticles designed for multi-stimuli triggered drug release

Original core/corona nanoparticles composed of a maghemite core and a stimuli-responsive polymer coating made of poly(acrylic acid)-block-poly(vinyl alcohol) macromolecules were fabricated for drug delivery system (DDS) application. This kind of DDS aims to combine the advantage of stimuli-responsive polymer coating, in order to regulate the drug release behaviours under different conditions and furthermore, improve the biocompatibility and in vivo circulation half-time of the maghemite nanoparticles. Drug loading capacity was evaluated with methylene blue (MB), a cationic model drug. The triggered release of MB was studied under various stimuli such as pH, ionic strength and temperature. Local heating generated under alternating magnetic field (AMF) application was studied, and remotely AMF-triggered release was also confirmed, while a mild heating-up of the release medium was observed. Furthermore, their potential application as magnetic resonance imaging (MRI) contrast agents was explored via relaxivity measurements and acquisition of T2-weighted images. Preliminary studies on the cytotoxicity against mouse fibroblast-like L929 cell line and also their cellular uptake within human melanoma MEL-5 cell line were carried out. In conclusion, this kind of stimuli-responsive nanoparticles appears to be promising carriers for delivering drugs to some tumour sites or into cellular compartments with an acidic environment.

Evaluation of antioxidant and cytoprotective activities of Arnica montana L. and Artemisia absinthium L. ethanolic extracts.

BackgroundArnica montana L. and Artemisia absinthium L. (Asteraceae) are medicinal plants native to temperate regions of Europe, including Romania, traditionally used for treatment of skin wounds, bruises and contusions. In the present study, A. montana and A. absinthium ethanolic extracts were evaluated for their chemical composition, antioxidant activity and protective effect against H2O2-induced oxidative stress in a mouse fibroblast-like NCTC cell line.ResultsA. absinthium extract showed a higher antioxidant capacity than A. montana extract as Trolox equivalent antioxidant capacity, Oxygen radical absorbance capacity and 2,2-diphenyl-1-picrylhydrazyl free radical-scavenging activity, in correlation with its flavonoids and phenolic acids content. Both plant extracts had significant effects on the growth of NCTC cells in the range of 10–100 mg/L A. montana and 10–500 mg/L A. absinthium. They also protected fibroblast cells against hydrogen peroxide-induced oxidative damage, at the same doses. The best protection was observed in cell pre-treatment with 10 mg/L A. montana and 10–300 mg/L A. absinthium, respectively, as determined by Neutral red and lactate dehydrogenase assays. In addition, cell pre-treatment with plant extracts, at these concentrations, prevented morphological changes induced by hydrogen peroxide. Flow-cytometry analysis showed that pre-treatment with A. montana and A. absinthium extracts restored the proportion of cells in each phase of the cell cycle.ConclusionsA. montana and A. absinthium extracts, rich in flavonoids and phenolic acids, showed a good antioxidant activity and cytoprotective effect against oxidative damage in fibroblast-like cells. These results provide scientific support for the traditional use of A. montana and A. absinthium in treatment of skin disorders.

The osteogenesis of bacterial cellulose scaffold loaded with bone morphogenetic protein-2

Bacterial cellulose (BC) is a nanofibrous biological material with attractive physicochemical properties and biocompatibility. Its fiber is similar to the collagenous fiber of bone. To explore if BC could be utilized as a localized delivery system to increase the local concentration of cytokines for tissue engineering, we prepared the BC scaffold from Acetobacter xylinum X-2 (A. xylinum X-2) and investigated the osteogenic potential of the BC scaffold coated with bone morphogenetic protein-2 (BMP-2). The data showed that BC had a good biocompatibility and induced differentiation of mouse fibroblast-like C2C12 cells into osteoblasts in the presence of BMP-2 in vitro, as demonstrated by alkaline phosphatase (ALP) activity assays. Within a certain range (0 ∼ 3 μg/scaffold), the osteogenic activity of induced osteoblasts was positively correlated to the concentrations of BMP-2. In in vivo subcutaneous implantation studies, BC scaffolds carrying BMP-2 showed more bone formation and higher calcium concentration than the BC scaffolds alone at 2 and 4 weeks, respectively. The ALP activity assay and the measurement of calcium concentration of BC scaffolds also showed that more new bone was developed in the BC scaffolds carrying BMP-2 than in the BC scaffolds alone. Our studies suggest that BC is a good localized delivery system for BMPs and would be a potential candidate in bone tissue engineering.

In Vitro Cytotoxic Effect of Glyphosate Mixture Containing Surfactants

We investigated whether glyphosate influences the cellular toxicity of the surfactants TN-20 and LN-10 on the mouse fibroblast-like cells, alveolar epithelial cells, and a heart cell line. The cytotoxicity of TN-20 and LN-10 (0.4-100 µM), in the presence or absence of glyphosate was determined by assessing membrane integrity. TN-20 toxicity was significantly lower in the presence of 50 µM glyphosate for the fibroblast-like cell (6.25 µM; 3.9% ± 3.4% vs -4.8% ± 0.7%), for the alveolar cells (0.78 µM; 5.7% ± 0.9% vs 0.1% ± 0.6%), and for the heart cell line (25.0 µM; 7.9% ± 3.0% vs 19.4% ± 0.7%) compared to that of TN-20 alone. The cellular toxicity of LN-10 towards the fibroblast-like cells was found to be increased in the presence of 50 µM glyphosate when LN-10 concentrations of 50 µM (31.3% ± 3.9% vs 19.2% ± 0.9%) and 100 µM (62.1% ± 3.4% vs 39.0% ± 0.7%) were compared to that of LN-10 alone. These results suggest that the mixture toxicity may be a factor in glyphosate-surfactant toxicity in patients with acute glyphosate herbicide intoxication.

Modulation of hyaluronan production by CD44 positive glioma cells.

This study examines the functional relationship between glioma cell production of hyaluronan (HA), known to play a role in glioma invasion, expression of its CD44 receptor, and glioma cell viability. Production of HA by CD44 positive mouse G26 and human U373 glioma cell lines was evaluated and compared to that of a CD44 positive mouse fibroblast-like L929 cell line. We found that both G26 and U373 MG glioma cells, but not L929 fibroblast-like cells, synthesized HA. The synthesis of HA by glioma cells was found during the proliferative phase as well as post-confluency, as detected by fluorophore-assisted carbohydrate electrophoresis. Eighty to ninety percent of the HA synthesized was secreted into the medium and 10-20% remained associated with the cells. To examine a possible mechanistic link between the CD44-HA interaction and endogenous HA production, glioma cells were treated with either anti-CD44 antibodies (clones KM201 or IM7) or HA oligosaccharides (hexamer oligoHA-6 or decamer oligoHA-10). We found that oligoHA-10, which was previously shown to compete effectively with the CD44-HA interaction, enhanced glioma HA synthesis by approximately 1.5-fold, without affecting cell viability. IM7 treatment of human U373 glioma cells resulted in over 50% decrease of HA production, which was associated with changes in cell size and apoptosis. Taken together, these data show that CD44 specific ligands, such as the IM7 antibody or oligoHA-10 could down-regulate or up-regulate glioma HA production, respectively. Our results suggest that interference with CD44/HA may lead to the discovery and development of new treatment modalities for glioma.

Transport of bile acids in multidrug-resistance-protein 3-overexpressing cells co-transfected with the ileal Na+-dependent bile-acid transporter.

Many of the transporters involved in the transport of bile acids in the enterohepatic circulation have been characterized. The basolateral bile-acid transporter of ileocytes and cholangiocytes remains an exception. It has been suggested that rat multidrug resistance protein 3 (Mrp3) fulfills this function. Here we analyse bile-salt transport by human MRP3. Membrane vesicles from insect ( Spodoptera frugiperda ) cells expressing MRP3 show time-dependent uptake of glycocholate and taurocholate. Furthermore, sulphated bile salts were high-affinity competitive inhibitors of etoposide glucuronide transport by MRP3 (IC50 approximately 10 microM). Taurochenodeoxycholate, taurocholate and glycocholate inhibited transport at higher concentrations (IC50 approximately 100, 250 and 500 microM respectively). We used mouse fibroblast-like cell lines derived from mice with disrupted Mdr1a, Mdr1b and Mrp1 genes to generate transfectants that express the murine apical Na+-dependent bile-salt transporter (Asbt) and MRP3. Uptake of glycocholate by these cells is Na+-dependent, with a K(m) and V(max) of 29+/-7 microM and 660 +/- 63 pmol/min per mg of protein respectively and is inhibited by several organic-aniontransport inhibitors. Expression of MRP3 in these cells limits the accumulation of glycocholate and increases the efflux from cells preloaded with taurocholate or glycocholate. In conclusion, we find that MRP3 transports both taurocholate and glycocholate, albeit with low affinity, in contrast with the high-affinity transport by rat Mrp3. Our results suggest that MRP3 is unlikely to be the principal basolateral bile-acid transporter of ileocytes and cholangiocytes, but that it may have a role in the removal of bile acids from the liver in cholestasis.

Characterization of mouse melanoma cell lines by their mortal malignancy using an experimental metastatic model

We characterized the metastatic ability and mortality of four different mouse melanoma cell lines, B16-F0, -F1, -F10 and -BL6. B16-F0 is the parent cell line. B16-F1 was obtained by a one-time selective procedure and B16-F10 by a ten-time selective procedure using Fidler's method. B16-BL6 derived from B16-F10 has much more invasive activity than B16-F10. To investigate the difference in mortal malignancy among B16-F0, -F1, -F10 and -BL6, we examined the survival time of syngeneic C57BL/6Cr mice intravenously inoculated with these cells. As a control, we used the C57BL/6J-embryo mouse fibroblast-like semi-normal cell line. The ability to form lung metastatic nodules in mice gradually increased in the order: B16-F0, -F1, and -F10 (=-BL6). C57BL/6J-embryo cell (1×10 5/mouse)-inoculated mice survived for over 46 days. B16-F0, -F1, -F10 and -BL6 (1×10 5/mouse)-inoculated mice survived 31.4±4.4 (7), 25.7±2.8 (7), 23.6±1.5 (7) and 25.3±2.3 (7) days [mean±S. D. (number of mice)], respectively. According to the Mann-Whitney test, the B16-F0 inoculated group versus -F1 inoculated group (P<0.05), -F0 inoculated group versus -BL6 inoculated group (P<0.05), and -F0 inoculated group versus -F10 inoculated group (P<0.01) were significantly different, but the B16-F1 group versus -F10 group, -F1 group versus -BL6 group, and -F10 group versus -BL6 group were not. These results suggest that mortal malignancy is not necessarily correlated with lung-colonizing potential and even only one-time selected B16-F0 mouse melanoma cells are useful as an experimental metastatic model in vivo.